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Sample records for chondrogenesis

  1. MicroRNAs regulate osteogenesis and chondrogenesis

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    Dong, Shiwu, E-mail: shiwudong@gmail.com [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China); Yang, Bo; Guo, Hongfeng; Kang, Fei [Laboratory of Biomechanics, Department of Anatomy, The Third Military Medical University, Chongqing (China)

    2012-02-24

    Highlights: Black-Right-Pointing-Pointer To focus on the role of miRNAs in chondrogenesis and osteogenesis. Black-Right-Pointing-Pointer Involved in the regulation of miRNAs in osteoarthritis. Black-Right-Pointing-Pointer To speculate some therapeutic targets for bone diseases. -- Abstract: MicroRNAs (miRNAs) are a class of small molecules and non-coding single strand RNAs that regulate gene expression at the post-transcriptional level by binding to specific sequences within target genes. miRNAs have been recognized as important regulatory factors in organism development and disease expression. Some miRNAs regulate the proliferation and differentiation of osteoblasts, osteoclasts and chondrocytes, eventually influencing metabolism and bone formation. miRNAs are expected to provide potential gene therapy targets for the clinical treatment of metabolic bone diseases and bone injuries. Here, we review the recent research progress on the regulation of miRNAs in bone biology, with a particular focus on the miRNA-mediated control mechanisms of bone and cartilage formation.

  2. Extracellular matrix mediates epithelial effects on chondrogenesis in vitro.

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    Solursh, M; Jensen, K L; Zanetti, N C; Linsenmayer, T F; Reiter, R S

    1984-10-01

    It has been previously observed that single chick embryonic limb mesenchymal cells can differentiate into chondrocytes without cell-cell interactions when cultured in collagen or agarose gels. In the present study, limb ectoderm, but not dermis, inhibits chondrogenesis when placed on such collagen gel cultures. The inhibitory influence can be transmitted extensive distances in the gel, even when the ectoderm is placed on a porous filter. Collagen gels, preconditioned with limb ectoderms, are also inhibitory to chondrogenesis. On the other hand, chondrogenesis is less inhibited by ectoderm when the mesenchymal cells are placed in agarose. These results suggest that the antichondrogenic effect of limb ectoderm is mediated through alterations of the collagenous extracellular matrix and support the idea that the extracellular matrix must be considered as an organized, functional unit capable of regulating cell differentiation.

  3. Thyroid hormones regulate fibroblast growth factor receptor signaling during chondrogenesis.

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    Barnard, Joanna C; Williams, Allan J; Rabier, Bénédicte; Chassande, Olivier; Samarut, Jacques; Cheng, Sheue-Yann; Bassett, J H Duncan; Williams, Graham R

    2005-12-01

    Childhood hypothyroidism causes growth arrest with delayed ossification and growth-plate dysgenesis, whereas thyrotoxicosis accelerates ossification and growth. Thyroid hormone (T(3)) regulates chondrocyte proliferation and is essential for hypertrophic differentiation. Fibroblast growth factors (FGFs) are also important regulators of chondrocyte proliferation and differentiation, and activating mutations of FGF receptor-3 (FGFR3) cause achondroplasia. We investigated the hypothesis that T(3) regulates chondrogenesis via FGFR3 in ATDC5 cells, which undergo a defined program of chondrogenesis. ATDC5 cells expressed two FGFR1, four FGFR2, and one FGFR3 mRNA splice variants throughout chondrogenesis, and expression of each isoform was stimulated by T(3) during the first 6-12 d of culture, when T(3) inhibited proliferation by 50%. FGFR3 expression was also increased in cells treated with T(3) for 21 d, when T(3) induced an earlier onset of hypertrophic differentiation and collagen X expression. FGFR3 expression was reduced in growth plates from T(3) receptor alpha-null mice, which exhibit skeletal hypothyroidism, but was increased in T(3) receptor beta(PV/PV) mice, which display skeletal thyrotoxicosis. These findings indicate that FGFR3 is a T(3)-target gene in chondrocytes. In further experiments, T(3) enhanced FGF2 and FGF18 activation of the MAPK-signaling pathway but inhibited their activation of signal transducer and activator of transcription-1. FGF9 did not activate MAPK or signal transducer and activator of transcription-1 pathways in the absence or presence of T(3). Thus, T(3) exerted differing effects on FGFR activation during chondrogenesis depending on which FGF ligand stimulated the FGFR and which downstream signaling pathway was activated. These studies identify novel interactions between T(3) and FGFs that regulate chondrocyte proliferation and differentiation during chondrogenesis.

  4. Gene Transfer Strategies to Promote Chondrogenesis and Cartilage Regeneration.

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    Im, Gun-Il

    2016-04-01

    Gene transfer has been used experimentally to promote chondrogenesis and cartilage regeneration. While it is controversial to apply gene therapy for nonlethal conditions such as cartilage defect, there is a possibility that the transfer of therapeutic transgenes may dramatically increase the effectiveness of cell therapy and reduce the quantity of cells that are needed to regenerate cartilage. Single or combination of growth factors and transcription factors has been transferred to mesenchymal stem cells or articular chondrocytes using both nonviral and viral approaches. The current challenge for the clinical applications of genetically modified cells is ensuring the safety of gene therapy while guaranteeing effectiveness. Viral gene delivery methods have been mainstays currently with enhanced safety features being recently refined. On the other hand, efficiency has been greatly improved in nonviral delivery. This review summarizes the history and recent update on the gene transfer to enhance chondrogenesis from stem cells or articular chondrocytes.

  5. Induction of mesenchymal stem cell chondrogenesis by polyacrylate substrates.

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    Glennon-Alty, Laurence; Williams, Rachel; Dixon, Simon; Murray, Patricia

    2013-04-01

    Mesenchymal stem cells (MSCs) can generate chondrocytes in vitro, but typically need to be cultured as aggregates in the presence of transforming growth factor beta (TGF-β), which makes scale-up difficult. Here we investigated if polyacrylate substrates modelled on the functional group composition and distribution of the Arg-Gly-Asp (RGD) integrin-binding site could induce MSCs to undergo chondrogenesis in the absence of exogenous TGF-β. Within a few days of culture on the biomimetic polyacrylates, both mouse and human MSCs, and a mesenchymal-like mouse-kidney-derived stem cell line, began to form multi-layered aggregates and started to express the chondrocyte-specific markers, Sox9, collagen II and aggrecan. Moreover, collagen II tended to be expressed in the centre of the aggregates, similarly to developing limb buds in vivo. Surface analysis of the substrates indicated that those with the highest surface amine content were most effective at promoting MSC chondrogenesis. These results highlight the importance of surface group functionality and the distribution of those groups in the design of substrates to induce MSC chondrogenesis.

  6. Hydrophilic polyurethane matrix promotes chondrogenesis of mesenchymal stem cells.

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    Nalluri, Sandeep M; Krishnan, G Rajesh; Cheah, Calvin; Arzumand, Ayesha; Yuan, Yuan; Richardson, Caley A; Yang, Shuying; Sarkar, Debanjan

    2015-09-01

    Segmental polyurethanes exhibit biphasic morphology and can control cell fate by providing distinct matrix guided signals to increase the chondrogenic potential of mesenchymal stem cells (MSCs). Polyethylene glycol (PEG) based hydrophilic polyurethanes can deliver differential signals to MSCs through their matrix phases where hard segments are cell-interactive domains and PEG based soft segments are minimally interactive with cells. These coordinated communications can modulate cell-matrix interactions to control cell shape and size for chondrogenesis. Biphasic character and hydrophilicity of polyurethanes with gel like architecture provide a synthetic matrix conducive for chondrogenesis of MSCs, as evidenced by deposition of cartilage-associated extracellular matrix. Compared to monophasic hydrogels, presence of cell interactive domains in hydrophilic polyurethanes gels can balance cell-cell and cell-matrix interactions. These results demonstrate the correlation between lineage commitment and the changes in cell shape, cell-matrix interaction, and cell-cell adhesion during chondrogenic differentiation which is regulated by polyurethane phase morphology, and thus, represent hydrophilic polyurethanes as promising synthetic matrices for cartilage regeneration.

  7. Optimizing a novel method for low intensity ultrasound in chondrogenesis induction

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    Hajar Shafaei

    2013-01-01

    Conclusion: Using LIUS resulted in early chondrogenesis in comparison with terminally differentiated chondrocytes by TGFβ. Therefore, LIUS might provide an applicable, safe, efficient, and cheap tool for chondrogenic differentiation of ASCs in cartilage tissue engineering.

  8. Chondroblastoma and chondromyxoid fibroma : disentangling the neoplastic chondrogenesis of two rare cartilaginous tumours

    NARCIS (Netherlands)

    Romeo, Salvatore

    2010-01-01

    The scope of this study was to disentangle neoplastic chondrogenesis in two rare cartilaginous tumours: chondroblastoma and chondromyxoid fibroma. It was addressed: 1 The spectrum of phenotypic differentiation in chondroblastoma and chondromyxoid fibroma, 2 The signalling pathways driving chondrobla

  9. Roles of DMP1 processing in osteogenesis, dentinogenesis and chondrogenesis.

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    Sun, Yao; Chen, Li; Ma, Su; Zhou, Jin; Zhang, Hua; Feng, Jian Q; Qin, Chunlin

    2011-01-01

    Dentin matrix protein 1 (DMP1) is an acidic protein that plays critical roles in osteogenesis and dentinogenesis. Protein chemistry studies have demonstrated that DMP1 primarily exists as processed NH₂⁻ and COOH-terminal fragments in the extracellular matrix of bone and dentin. Our earlier work showed that the substitution of Asp²¹³ (a residue at a cleavage site) by Ala²¹³ blocks the processing of mouse DMP1 in vitro. Recently, we generated transgenic mice expressing this mutant DMP1 (designated 'D213A-DMP1'). By crossbreeding these transgenic mice with Dmp1-knockout (Dmp1-KO) mice, we obtained mice expressing the D213A-DMP1 transgene in the Dmp1-null background (named 'Dmp1-KO/D213A-Tg' mice). In this study, we analyzed the long bone, mandible, dentin, and cartilage of Dmp1-KO/D213A-Tg mice in comparison with wild-type, Dmp1-KO, and Dmp1-KO mice expressing the normal DMP1 transgene (Dmp1-KO/normal-Tg). Our results showed that D213A-DMP1 was barely cleaved in the dentin matrix of Dmp1-KO/D213A-Tg mice and the expression of D213A-DMP1 failed to rescue the developmental defects in Dmp1-null mice. Interestingly, enlarged growth plates and condylar cartilages were observed in Dmp1-KO/D213A-Tg mice, indicating a potential role of the full-length form of DMP1 in chondrogenesis.

  10. Forkhead transcription factor foxe1 regulates chondrogenesis in zebrafish.

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    Nakada, Chisako; Iida, Atsumi; Tabata, Yoko; Watanabe, Sumiko

    2009-12-15

    Forkhead transcription factor (Fox) e1 is a causative gene for Bamforth-Lazarus syndrome, which is characterized by hypothyroidism and cleft palate. Applying degenerate polymerase chain reaction using primers specific for the conserved forkhead domain, we identified zebrafish foxe1 (foxe1). Foxe1 is expressed in the thyroid, pharynx, and pharyngeal skeleton during development; strongly expressed in the gill and weakly expressed in the brain, eye, and heart in adult zebrafish. A loss of function of foxe1 by morpholino antisense oligo (MO) exhibited abnormal craniofacial development, shortening of Meckel's cartilage and the ceratohyals, and suppressed chondrycytic proliferation. However, at 27 hr post fertilization, the foxe1 MO-injected embryos showed normal dlx2, hoxa2, and hoxb2 expression, suggesting that the initial steps of pharyngeal skeletal development, including neural crest migration and specification of the pharyngeal arch occurred normally. In contrast, at 2 dpf, a severe reduction in the expression of sox9a, colIIaI, and runx2b, which play roles in chondrocytic proliferation and differentiation, was observed. Interestingly, fgfr2 was strongly upregulated in the branchial arches of the foxe1 MO-injected embryos. Unlike Foxe1-null mice, normal thyroid development in terms of morphology and thyroid-specific marker expression was observed in foxe1 MO-injected zebrafish embryos. Taken together, our results indicate that Foxe1 plays an important role in chondrogenesis during development of the pharyngeal skeleton in zebrafish, probably through regulation of fgfr2 expression. Furthermore, the roles reported for FOXE1 in mammalian thyroid development may have been acquired during evolution.

  11. Cytomegalovirus induces abnormal chondrogenesis and osteogenesis during embryonic mandibular development

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    Bringas Pablo

    2008-03-01

    Full Text Available Abstract Background Human clinical studies and mouse models clearly demonstrate that cytomegalovirus (CMV disrupts normal organ and tissue development. Although CMV is one of the most common causes of major birth defects in humans, little is presently known about the mechanism(s underlying CMV-induced congenital malformations. Our prior studies have demonstrated that CMV infection of first branchial arch derivatives (salivary glands and teeth induced severely abnormal phenotypes and that CMV has a particular tropism for neural crest-derived mesenchyme (NCM. Since early embryos are barely susceptible to CMV infection, and the extant evidence suggests that the differentiation program needs to be well underway for embryonic tissues to be susceptible to viral infection and viral-induced pathology, the aim of this study was to determine if first branchial arch NCM cells are susceptible to mCMV infection prior to differentiation of NCM derivatives. Results E11 mouse mandibular processes (MANs were infected with mouse CMV (mCMV for up to 16 days in vitro. mCMV infection of undifferentiated embryonic mouse MANs induced micrognathia consequent to decreased Meckel's cartilage chondrogenesis and mandibular osteogenesis. Specifically, mCMV infection resulted in aberrant stromal cellularity, a smaller, misshapen Meckel's cartilage, and mandibular bone and condylar dysmorphogenesis. Analysis of viral distribution indicates that mCMV primarily infects NCM cells and derivatives. Initial localization studies indicate that mCMV infection changed the cell-specific expression of FN, NF-κB2, RelA, RelB, and Shh and Smad7 proteins. Conclusion Our results indicate that mCMV dysregulation of key signaling pathways in primarily NCM cells and their derivatives severely disrupts mandibular morphogenesis and skeletogenesis. The pathogenesis appears to be centered around the canonical and noncanonical NF-κB pathways, and there is unusual juxtaposition of abnormal stromal

  12. Quantitative transcriptional profiling of ATDC5 mouse progenitor cells during chondrogenesis

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    Chen, Li; Fink, Trine; Zhang, Xiao-Yan;

    2005-01-01

    During the differentiation of a mouse chondroprogenitor cell line, ATDC5, an analysis of the transcription cartilage-related genes was carried out using real-time RT-PCR in a semiquantitative fashion. A total number of 104 genes both previously linked to chondrogenesis and hitherto not associated...

  13. Hypoxic treatment inhibits insulin-induced chondrogenesis of ATDC5 cells despite upregulation of DEC1

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    Chen, Li; Fink, Trine; Ebbesen, Peter;

    2006-01-01

    Chondrogenesis occurs in vivo in a hypoxic environment, in which the hypoxia inducible factor 1, HIF-1, plays a regulatory role, possibly mediated through the transcription factor DEC1. We have analyzed the effect of hypoxia (1% oxygen) alone and in combination with insulin on the chondrogenic di...

  14. 17{beta}-Estradiol inhibits chondrogenesis in the skull development of zebrafish embryos

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    Fushimi, Shigeko, E-mail: fushimi@med.kawasaki-m.ac.jp [Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Wada, Naoyuki, E-mail: wada@med.kawasaki-m.ac.jp [Department of Molecular and Developmental Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Nohno, Tsutomu, E-mail: nohno@bcc.kawasaki-m.ac.jp [Department of Molecular and Developmental Biology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Tomita, Masafumi, E-mail: toxicology@med.kawasaki-m.ac.jp [Department of Medical Toxicology, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan); Saijoh, Kiyofumi, E-mail: saijohk@med.kanazawa-u.ac.jp [Department of Hygiene, Kanazawa University School of Medicine, 13-1 Takaramachi, Kanazawa 920-8564 (Japan); Sunami, Shigeo, E-mail: ssunami@med.kawasaki-m.ac.jp [Department of Clinical Nutrition, Kawasaki University of Medical Welfare, 288 Matsushima, Kurashiki 701-0193 (Japan); Katsuyama, Hironobu, E-mail: katsu@med.kawasaki-m.ac.jp [Department of Public Health, Kawasaki Medical School, 577 Matsushima, Kurashiki 701-0192 (Japan)

    2009-12-13

    17{beta}-Estradiol (E2) plays important roles in the development and differentiation of the gonad and central nervous systems, but little is known regarding the effects of exogenous E2 on chondrogenesis in skeletal development. In the present study, we found that treatment with E2 1-5 days post-fertilization (dpf) at concentrations above 1.5 x 10{sup -5} M increased the mortality rate in zebrafish embryos. Morphological analysis showed that treatment with E2 1-5 dpf caused abnormal cartilage formation in a dose-dependent manner at concentrations above 5 x 10{sup -6} M. E2 1-5 dpf at 1.5 x 10{sup -5} M caused defects of the ethmoid plate, parallel cleft of the trabecular cartilage, and hypoplasia of Meckel's cartilage and the ceratohyal cartilage. The sensitivity of embryos to E2 depended on the developmental stage. In early chondrogenesis (1-2 dpf), the embryos were highly sensitive to E2, leading to hypoplasia of the cartilage. In situ hybridization studies showed that expression levels of patched1 (ptc1) and patched2 (ptc2) receptor mRNAs were markedly decreased by exposure to 2 x 10{sup -5} M E2 1-2 dpf. However, the expression levels of sonic hedgehog (shh) and tiggywinkle hedgehog (twhh) mRNAs were constant in the E2-treated embryos. In addition, the estrogen receptor antagonist ICI 182,780 did not completely abolish the effects of E2, suggesting that E2 may not inhibit chondrogenesis through its nuclear estrogen receptor. These results suggest that exposure to exogenous E2 possibly inhibits chondrogenesis via inhibition of the hedgehog (Hh) signal transduction system.

  15. The role of transforming growth factor alpha in rat craniofacial development and chondrogenesis.

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    Huang, L; Solursh, M; Sandra, A

    1996-08-01

    To explore the possible role of transforming growth factor alpha (TGF-alpha) in craniofacial development, its expression in the craniofacial region of rat embryos from embryonic day (d) 9 to d 20 was examined by in situ hybridisation and immunostaining. The TGF-alpha transcripts were first detected in the neural fold of embryonic d 9 and 10 embryos. In the craniofacial region, the TGF-alpha transcripts were not detected until embryonic d 16 in mesenchyme surrounding the olfactory bulb, within the olfactory bulb, the nasal capsule, vomeronasal organ, and vibrissal follicle. In addition, TGF-alpha message was detected in mesenchyme in the vicinity of Meckel's cartilage, and in the dental epithelium and lamina. This expression pattern of TGF-alpha transcripts persisted until embryonic d 17 but disappeared by d 18. The presence of TGF-alpha protein largely coincided with TGF-alpha message although, unlike the message, it persisted throughout later embryogenesis in the craniofacial region. The possible function of TGF-alpha in chondrogenesis was explored by employing the micromass culture technique. Cartilage nodule formation in mesenchymal cells cultured from rat mandibles in the presence of TGF-alpha was significantly inhibited. This inhibitory effect of TGF-alpha on chondrogenesis was reversed by addition of antibody against the EGF receptor, which crossreacts with the TGF-alpha receptor. The inhibitory effect of TGF-alpha on chondrogenesis in vitro was further confirmed by micromass culture using mesenchymal cells from rat embryonic limb bud. Taken together, these results demonstrate the involvement of TGF-alpha in chondrogenesis during embryonic development, possibly by way of a specific inhibition of cartilage formation from mesenchymal precursor cells.

  16. Effect of Fibroblast Growth Factor 2 on Equine Synovial Fluid Chondroprogenitor Expansion and Chondrogenesis

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    Marta Bianchessi

    2016-01-01

    Full Text Available Mesenchymal stem cells have been identified in the synovial fluid of several species. This study was conducted to characterize chondroprogenitor (CP cells in equine synovial fluid (SF and to determine the effect of fibroblast growth factor 2 (FGF-2 on SF-CP monolayer proliferation and subsequent chondrogenesis. We hypothesized that FGF-2 would stimulate SF-CP proliferation and postexpansion chondrogenesis. SF aspirates were collected from adult equine joints. Colony-forming unit (CFU assays were performed during primary cultures. At first passage, SF-cells were seeded at low density, with or without FGF-2. Following monolayer expansion and serial immunophenotyping, cells were transferred to chondrogenic pellet cultures. Pellets were analyzed for chondrogenic mRNA expression and cartilage matrix secretion. There was a mean of 59.2 CFU/mL of SF. FGF-2 increased the number of population doublings during two monolayer passages and halved the population doubling times. FGF-2 did not alter the immunophenotype of SF-CPs during monolayer expansion, nor did FGF-2 compromise chondrogenesis. Hypertrophic phenotypic markers were not expressed in control or FGF-2 groups. FGF-2 did prevent the development of a “fibroblastic” cell layer around pellet periphery. FGF-2 significantly accelerates in vitro SF-CP expansion, the major hurdle to clinical application of this cell population, without detrimentally affecting subsequent chondrogenic capacity.

  17. Microenvironment is involved in cellular response to hydrostatic pressures during chondrogenesis of mesenchymal stem cells.

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    Ye, Rui; Hao, Jin; Song, Jinlin; Zhao, Zhihe; Fang, Shanbao; Wang, Yating; Li, Juan

    2014-06-01

    Chondrocytes integrate numerous microenvironmental cues to mount physiologically relevant differentiation responses, and the regulation of mechanical signaling in chondrogenic differentiation is now coming into intensive focus. To facilitate tissue-engineered chondrogenesis by mechanical strategy, a thorough understanding about the interactional roles of chemical factors under mechanical stimuli in regulating chondrogenesis is in great need. Therefore, this study attempts to investigate the interaction of rat MSCs with their microenvironment by imposing dynamic and static hydrostatic pressure through modulating gaseous tension above the culture medium. Under dynamic pressure, chemical parameters (pH, pO2, and pCO2) were kept in homeostasis. In contrast, pH was remarkably reduced due to increased pCO2 under static pressure. MSCs under the dynamically pressured microenvironment exhibited a strong accumulation of GAG within and outside the alginate beads, while cells under the statically pressured environment lost newly synthesized GAG into the medium with a speed higher than its production. In addition, the synergic influence on expression of chondrogenic genes was more persistent under dynamic pressure than that under static pressure. This temporal contrast was similar to that of activation of endogenous TGF-β1. Taken altogether, it indicates that a loading strategy which can keep a homeostatic chemical microenvironment is preferred, since it might sustain the stimulatory effects of mechanical stimuli on chondrogenesis via activation of endogenous TGF-β1.

  18. Inhibitory and stimulatory effects of limb ectoderm on in vitro chondrogenesis.

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    Solursh, M; Reiter, R S

    1988-11-01

    Previous studies have indicated possible dual effects of the limb ectoderm in cartilage differentiation. On one hand, explants from early (stage 15) wing buds are dependent on contact with the limb ectoderm for cartilage differentiation (Gumpel-Pinot, J. Embryol. Exp. Morph. 59:157-173, 1980). On the other hand, limb ectoderm from stage 23/24 wing buds inhibits cartilage differentiation by cultured limb mesenchyme cells even without direct contact (Solursh et al., Dev. Biol. 86:471-482, 1981). In the present study, ectoderms from both stage 15/16 and stage 23/24 wings are cultured under the same conditions, and ectoderms from each source are shown to have two effects. Each stimulates chondrogenesis in stage 15 wing bud mesenchyme, and each inhibits chondrogenesis in older wing mesenchyme. The results suggest that the limb ectoderm has at least dual effects on cartilage differentiation, depending on the stage of the mesenchyme. One effect involves an early mesenchymal dependence on the ectoderm. This effect requires contact between the ectoderm and mesoderm (Gumpel-Pinot, J. Embryol. Exp. Morphol. 59:157-173, 1980) but also can be observed at a distance from the ectoderm. Later, the ectoderm can act without direct contact between the ectoderm and mesoderm to inhibit chondrogenesis over some distance.

  19. Differential effects of dexamethasone on the chondrogenesis of mesenchymal stromal cells: Influence of microenvironment, tissue origin and growth factor

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    N Shintani

    2011-11-01

    Full Text Available nchymal stromal cells (MSCs, which reside within various tissues, are utilized in the engineering of cartilage tissue. Dexamethasone (DEX – a synthetic glucocorticoid – is almost invariably applied to potentiate the growth-factor-induced chondrogenesis of MSCs in vitro, albeit that this effect has been experimentally demonstrated only for transforming-growth-factor-beta (TGF-β-stimulated bone-marrow-derived MSCs. Clinically, systemic glucocorticoid therapy is associated with untoward side effects (e.g., bone loss and increased susceptibility to infection. Hence, the use of these agents should be avoided or limited. We hypothesize that the influence of DEX on the chondrogenesis of MSCs depends upon their tissue origin and microenvironment [absence or presence of an extracellular matrix (ECM], as well as upon the nature of the growth factor. We investigated its effects upon the TGF-β1- and bone-morphogenetic-protein 2 (BMP-2-induced chondrogenesis of MSCs as a function of tissue source (bone marrow vs. synovium and microenvironment [cell aggregates (no ECM vs. explants (presence of a natural ECM]. In aggregates of bone-marrow-derived MSCs, DEX enhanced TGF-β1-induced chondrogenesis by an up-regulation of cartilaginous genes, but had little influence on the BMP-2-induced response. In aggregates of synovial MSCs, DEX exerted no remarkable effect on either TGF-β1- or BMP-2-induced chondrogenesis. In synovial explants, DEX inhibited BMP-2-induced chondrogenesis almost completely, but had little impact on the TGF-β1-induced response. Our data reveal that steroids are not indispensable for the chondrogenesis of MSCs in vitro. Their influence is context dependent (tissue source of the MSCs, their microenvironment and the nature of the growth-factor. This finding has important implications for MSC based approaches to cartilage repair.

  20. Proteome analysis during chondrocyte differentiation in a new chondrogenesis model using human umbilical cord stroma mesenchymal stem cells.

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    De la Fuente, Alexandre; Mateos, Jesús; Lesende-Rodríguez, Iván; Calamia, Valentina; Fuentes-Boquete, Isaac; de Toro, Francisco J; Arufe, Maria C; Blanco, Francisco J

    2012-02-01

    Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies.

  1. Proteome Analysis During Chondrocyte Differentiation in a New Chondrogenesis Model Using Human Umbilical Cord Stroma Mesenchymal Stem Cells*

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    De la Fuente, Alexandre; Mateos, Jesús; Lesende-Rodríguez, Iván; Calamia, Valentina; Fuentes-Boquete, Isaac; de Toro, Francisco J.; Arufe, Maria C.; Blanco, Francisco J.

    2012-01-01

    Umbilical cord stroma mesenchymal stem cells were differentiated toward chondrocyte-like cells using a new in vitro model that consists of the random formation of spheroids in a medium supplemented with fetal bovine serum on a nonadherent surface. The medium was changed after 2 days to one specific for the induction of chondrocyte differentiation. We assessed this model using reverse transcriptase-polymerase chain reaction, flow cytometry, immunohistochemistry, and secretome analyses. The purpose of this study was to determine which proteins were differentially expressed during chondrogenesis. Differential gel electrophoresis analysis was performed, followed by matrix-assisted laser desorption/ionization mass spectrometry protein identification. A total of 97 spots were modulated during the chondrogenesis process, 54 of these spots were identified as 39 different proteins and 15 were isoforms. Of the 39 different proteins identified 15 were down-regulated, 21 were up-regulated, and 3 were up- and down-regulated during the chondrogenesis process. Using Pathway Studio 7.0 software, our results showed that the major cell functions modulated during chondrogenesis were cellular differentiation, proliferation, and migration. Five proteins involved in cartilage extracellular matrix metabolism found during the differential gel electrophoresis study were confirmed using Western blot. The results indicate that our in vitro chondrogenesis model is an efficient and rapid technique for obtaining cells similar to chondrocytes that express proteins characteristic of the cartilage extracellular matrix. These chondrocyte-like cells could prove useful for future cell therapy treatment of cartilage pathologies. PMID:22008206

  2. Incorporation of hyaluronic acid into collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis

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    Tang Shunqing [Department of Biomedical Engineering, Jinan University, Guangzhou 510632 (China); Spector, Myron [Tissue Engineering, VA Boston Healthcare System, Boston, MA 02130 (United States)

    2007-09-15

    Hyaluronic acid (HA), a principal matrix molecule in many tissues, is present in high amounts in articular cartilage. HA contributes in unique ways to the physical behavior of the tissue, and has been shown to have beneficial effects on chondrocyte activity. The goal of this study was to incorporate graduated amounts of HA into type I collagen scaffolds for the control of chondrocyte-mediated contraction and chondrogenesis in vitro. The results demonstrated that the amount of contraction of HA/collagen scaffolds by adult canine articular chondrocytes increased with the HA content of the scaffolds. The greatest amount of chondrogenesis after two weeks was found in the scaffolds which had undergone the most contraction. HA can play a useful role in adjusting the mechanical behavior of tissue engineering scaffolds and chondrogenesis in chondrocyte-seeded scaffolds.

  3. Optimized chondrogenesis of ATCD5 cells through sequential regulation of oxygen conditions

    DEFF Research Database (Denmark)

    Chen, Li; Fink, Trine; Ebbesen, Peter;

    2006-01-01

    , chondrocyte-specific extracellular matrix (ECM) production was monitored. Furthermore, the transcription of collagen II, an early-phase marker, and collagen X, a marker of hypertrophic conversion, was followed by real-time RT-PCR. Low oxygen concentrations between 1 and 9% inhibited chondrogenic conversion......, as evidenced by reduced glycosaminoglycan deposition in the ECM in a manner proportional to the degree of hypoxia. Cells cultured at oxygen concentrations of 12 and 15% underwent a faster and higher degree of early-phase chondrogenesis when compared to control cells cultured at ambient air (21% O2...

  4. Cell-based semiquantitative assay for sulfated glycosaminoglycans facilitating the identification of chondrogenesis.

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    Yen, Ching-Yu; Wu, Yu-Wei; Hsiung, Chao-Nan; Yeh, Min-I; Lin, Yi-Ming; Lee, Sheng-Yang

    2015-10-01

    Glycosaminoglycans (GAGs), in particular chondroitin sulfate, are an accepted marker of chondrogenic cells. In this study, a cell-based sulfated GAG assay for identifying the chondrogenesis of mesenchymal stem cells was developed. Based on fluorescent staining using safranin O and 4',6-diamidino-2-phenylindole (DAPI), this method was highly sensitive. The results were both qualitative and quantitative. The method is suitable for identifying the chondrogenic process and also for screening compounds. The method may be helpful for discovering novel bioactive compounds for cartilage regeneration.

  5. Kaempferol induces chondrogenesis in ATDC5 cells through activation of ERK/BMP-2 signaling pathway.

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    Nepal, Manoj; Li, Liang; Cho, Hyoung Kwon; Park, Jong Kun; Soh, Yunjo

    2013-12-01

    Endochondral bone formation occurs when mesenchymal cells condense to differentiate into chondrocytes, the primary cell types of cartilage. The aim of the present study was to identify novel factors regulating chondrogenesis. We investigated whether kaempferol induces chondrogenic differentiation in clonal mouse chondrogenic ATDC5 cells. Kaempferol treatment stimulated the accumulation of cartilage nodules in a dose-dependent manner. Kaempferol-treated ATDC5 cells stained more intensely with alcian blue staining than control cells, suggesting greater synthesis of matrix proteoglycans in the kaempferol-treated cells. Similarly, kaempferol induced greater activation of alkaline phosphatase activity than control cells, and it enhanced the expression of chondrogenic marker genes, such as collagen type I, collagen type X, OCN, Runx2, and Sox9. Kaempferol induced an acute activation of extracellular signal-regulated kinase (ERK) but not c-jun N-terminal kinase or p38 MAP kinase. PD98059, an inhibitor of MAPK/ERK, decreased in stained cells treated with kaempferol. Furthermore, kaempferol greatly expressed the protein and mRNA levels of BMP-2, suggesting chondrogenesis was stimulated via a BMP-2 pathway. Taken together, our results suggest that kaempferol has chondromodulating effects via an ERK/BMP-2 signaling pathway and could potentially be used as a therapeutic agent for bone growth disorders.

  6. Chondrogenesis of infrapatellar fat pad derived adipose stem cells in 3D printed chitosan scaffold.

    Directory of Open Access Journals (Sweden)

    Ken Ye

    Full Text Available Infrapatellar fat pad adipose stem cells (IPFP-ASCs have been shown to harbor chondrogenic potential. When combined with 3D polymeric structures, the stem cells provide a source of stem cells to engineer 3D tissues for cartilage repair. In this study, we have shown human IPFP-ASCs seeded onto 3D printed chitosan scaffolds can undergo chondrogenesis using TGFβ3 and BMP6. By week 4, a pearlescent, cartilage-like matrix had formed that penetrated the top layers of the chitosan scaffold forming a 'cap' on the scaffold. Chondrocytic morphology showed typical cells encased in extracellular matrix which stained positively with toluidine blue. Immunohistochemistry demonstrated positive staining for collagen type II and cartilage proteoglycans, as well as collagen type I. Real time PCR analysis showed up-regulation of collagen type II, aggrecan and SOX9 genes when IPFP-ASCs were stimulated by TGFβ3 and BMP6. Thus, IPFP-ASCs can successfully undergo chondrogenesis using TGFβ3 and BMP6 and the cartilage-like tissue that forms on the surface of 3D-printed chitosan scaffold may prove useful as an osteochondral graft.

  7. In vitro chondrogenesis with lysozyme susceptible bacterial cellulose as a scaffold.

    Science.gov (United States)

    Yadav, Vikas; Sun, Lin; Panilaitis, Bruce; Kaplan, David L

    2015-12-01

    A current focus of tissue engineering is the use of adult human mesenchymal stem cells (hMSCs) as an alternative to autologous chondrocytes for cartilage repair. Several natural and synthetic polymers (including cellulose) have been explored as a biomaterial scaffold for cartilage tissue engineering. While bacterial cellulose (BC) has been used in tissue engineering, its lack of degradability in vivo and high crystallinity restricts widespread applications in the field. Recently we reported the formation of a novel bacterial cellulose that is lysozyme-susceptible and -degradable in vivo from metabolically engineered Gluconacetobacter xylinus. Here we report the use of this modified bacterial cellulose (MBC) for cartilage tissue engineering using hMSCs. MBC's glucosaminoglycan-like chemistry, combined with in vivo degradability, suggested opportunities to exploit this novel polymer in cartilage tissue engineering. We have observed that, like BC, MBC scaffolds support cell attachment and proliferation. Chondrogenesis of hMSCs in the MBC scaffolds was demonstrated by real-time RT-PCR analysis for cartilage-specific extracellular matrix (ECM) markers (collagen type II, aggrecan and SOX9) as well as histological and immunohistochemical evaluations of cartilage-specific ECM markers. Further, the attachment, proliferation, and differentiation of hMSCs in MBC showed unique characteristics. For example, after 4 weeks of cultivation, the spatial cell arrangement and collagen type-II and ACAN distribution resembled those in native articular cartilage tissue, suggesting promise for these novel in vivo degradable scaffolds for chondrogenesis.

  8. Biomimetic alginate/polyacrylamide porous scaffold supports human mesenchymal stem cell proliferation and chondrogenesis

    Energy Technology Data Exchange (ETDEWEB)

    Guo, Peng [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Yuan, Yasheng, E-mail: yuanyasheng@163.com [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China); Eaton-Peabody Laboratory, Massachusetts Eye and Ear Infirmary, Harvard Medical School, Boston, MA 02114 (United States); Chi, Fanglu [Department of ENT-Head and Neck Surgery, EENT Hospital, Shanghai 200031 (China); Shanghai Medical School, Fudan University, 210029 (China)

    2014-09-01

    We describe the development of alginate/polyacrylamide (ALG/PAAm) porous hydrogels based on interpenetrating polymer network structure for human mesenchymal stem cell proliferation and chondrogenesis. Three ALG/PAAm hydrogels at molar ratios of 10/90, 20/80, and 30/70 were prepared and characterized with enhanced elastic and rubbery mechanical properties, which are similar to native human cartilage tissues. Their elasticity and swelling properties were also studied under different physiological pH conditions. Finally, in vitro tests demonstrated that human mesenchymal stem cells could proliferate on the as-synthesized hydrogels with improved alkaline phosphatase activities. These results suggest that ALG/PAAm hydrogels may be a promising biomaterial for cartilage tissue engineering. - Highlights: • ALG/PAAm hydrogels were prepared at different molar ratios for cartilage tissue engineering. • ALG/PAAm hydrogels feature an interpenetrating polymer network structure. • ALG/PAAm hydrogels demonstrate strengthened elastic and rubbery mechanical properties. • hMSCs could be cultured on the ALG/PAAm hydrogels for proliferation and chondrogenesis.

  9. Increase in levels of cyclic AMP during avian limb chondrogenesis in vitro.

    Science.gov (United States)

    Solursh, M; Reiter, R; Ahrens, P B; Pratt, R M

    1979-01-01

    In the present study the level of cAMP was measured during in vitro chondrogenesis of wing mesenchyme of stage 24 chick embryos and was found to increase significantly from 6.3 pmol/mg protein at the end of the first day of culture to 9.7 pmol/mg protein on the second day, when chondrogenic expression is first detected by the appearance of an Alcian blue staining extracellular matrix. Nonchondrogenic cultures derived from wings of stage 19 embryos had a lower level of cAMP (4.4 +/- 0.07 pmol/mg protein). The level of cAMP in intact wings was 4.5 +/- 0.4 pmol/mg protein and did not change between stages 19 through 25. The correlatin between increased levels of cAMP and the onset of chondrogenesis is consistent with a role of cAMP in the expression of differentiated functions in chondrocytes, as well as in some other cell types.

  10. The Cross-talk Between TGF-β1 and Dlk1 Mediates Early Chondrogenesis During Embryonic Endochondral Ossification

    DEFF Research Database (Denmark)

    Taipaleenmaki, Hanna; M, Linda; Chen, Li

    2012-01-01

    Dlkl/Pref-1/FA1 (delta like-1/preadipocyte factor-1/Fetal Antigen-1) is a novel surface marker for embryonic chondroprogenitor cells undergoing lineage progression from proliferation to prehypertrophic stages. However, mechanisms mediating control of its expression during chondrogenesis...

  11. Effects of FGF-2 on human adipose tissue derived adult stem cells morphology and chondrogenesis enhancement in Transwell culture

    Energy Technology Data Exchange (ETDEWEB)

    Kabiri, Azadeh, E-mail: z_kabiri@resident.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Esfandiari, Ebrahim, E-mail: esfandiari@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Hashemibeni, Batool, E-mail: hashemibeni@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Kazemi, Mohammad, E-mail: m_kazemi@med.mui.ac.i [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Mardani, Mohammad, E-mail: mardani@med.mui.ac.ir [Department of Anatomical Sciences and Molecular Biology, Faculty of Medicine, Isfahan University of Medical Sciences (Iran, Islamic Republic of); Esmaeili, Abolghasem, E-mail: abesmaeili@yahoo.com [Cell, Molecular and Developmental Biology Division, Department of Biology, Faculty of Sciences, University of Isfahan, Isfahan (Iran, Islamic Republic of)

    2012-07-27

    Highlights: Black-Right-Pointing-Pointer We investigated effects of FGF-2 on hADSCs. Black-Right-Pointing-Pointer We examine changes in the level of gene expressions of SOX-9, aggrecan and collagen type II and type X. Black-Right-Pointing-Pointer FGF-2 induces chondrogenesis in hADSCs, which Bullet Increasing information will decrease quality if hospital costs are very different. Black-Right-Pointing-Pointer The result of this study may be beneficial in cartilage tissue engineering. -- Abstract: Injured cartilage is difficult to repair due to its poor vascularisation. Cell based therapies may serve as tools to more effectively regenerate defective cartilage. Both adult mesenchymal stem cells (MSCs) and human adipose derived stem cells (hADSCs) are regarded as potential stem cell sources able to generate functional cartilage for cell transplantation. Growth factors, in particular the TGF-b superfamily, influence many processes during cartilage formation, including cell proliferation, extracellular matrix synthesis, maintenance of the differentiated phenotype, and induction of MSCs towards chondrogenesis. In the current study, we investigated the effects of FGF-2 on hADSC morphology and chondrogenesis in Transwell culture. hADSCs were obtained from patients undergoing elective surgery, and then cultured in expansion medium alone or in the presence of FGF-2 (10 ng/ml). mRNA expression levels of SOX-9, aggrecan and collagen type II and type X were quantified by real-time polymerase chain reaction. The morphology, doubling time, trypsinization time and chondrogenesis of hADSCs were also studied. Expression levels of SOX-9, collagen type II, and aggrecan were all significantly increased in hADSCs expanded in presence of FGF-2. Furthermore FGF-2 induced a slender morphology, whereas doubling time and trypsinization time decreased. Our results suggest that FGF-2 induces hADSCs chondrogenesis in Transwell culture, which may be beneficial in cartilage tissue engineering.

  12. Gene Modification of Mesenchymal Stem Cells and Articular Chondrocytes to Enhance Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Saliya Gurusinghe

    2014-01-01

    Full Text Available Current cell based treatment for articular cartilage and osteochondral defects are hampered by issues such as cellular dedifferentiation and hypertrophy of the resident or transplanted cells. The reduced expression of chondrogenic signalling molecules and transcription factors is a major contributing factor to changes in cell phenotype. Gene modification of chondrocytes may be one approach to redirect cells to their primary phenotype and recent advances in nonviral and viral gene delivery technologies have enabled the expression of these lost factors at high efficiency and specificity to regain chondrocyte function. This review focuses on the various candidate genes that encode signalling molecules and transcription factors that are specific for the enhancement of the chondrogenic phenotype and also how epigenetic regulators of chondrogenesis in the form of microRNA may also play an important role.

  13. In Vivo Chondrogenesis in 3D Bioprinted Human Cell-laden Hydrogel Constructs

    Science.gov (United States)

    Möller, Thomas; Hägg, Daniel; Brantsing, Camilla; Rotter, Nicole; Apelgren, Peter; Lindahl, Anders; Kölby, Lars; Gatenholm, Paul

    2017-01-01

    Background: The three-dimensional (3D) bioprinting technology allows creation of 3D constructs in a layer-by-layer fashion utilizing biologically relevant materials such as biopolymers and cells. The aim of this study is to investigate the use of 3D bioprinting in a clinically relevant setting to evaluate the potential of this technique for in vivo chondrogenesis. Methods: Thirty-six nude mice (Balb-C, female) received a 5- × 5- × 1-mm piece of bioprinted cell-laden nanofibrillated cellulose/alginate construct in a subcutaneous pocket. Four groups of printed constructs were used: (1) human (male) nasal chondrocytes (hNCs), (2) human (female) bone marrow–derived mesenchymal stem cells (hBMSCs), (3) coculture of hNCs and hBMSCs in a 20/80 ratio, and (4) Cell-free scaffolds (blank). After 14, 30, and 60 days, the scaffolds were harvested for histological, immunohistochemical, and mechanical analysis. Results: The constructs had good mechanical properties and keep their structural integrity after 60 days of implantation. For both the hNC constructs and the cocultured constructs, a gradual increase of glycosaminoglycan production and hNC proliferation was observed. However, the cocultured group showed a more pronounced cell proliferation and enhanced deposition of human collagen II demonstrated by immunohistochemical analysis. Conclusions: In vivo chondrogenesis in a 3D bioprinted human cell-laden hydrogel construct has been demonstrated. The trophic role of the hBMSCs in stimulating hNC proliferation and matrix deposition in the coculture group suggests the potential of 3D bioprinting of human cartilage for future application in reconstructive surgery. PMID:28280669

  14. Feasibility of arthroscopic placement of autologous matrix-induced chondrogenesis grafts in the cadaver hip joint

    Directory of Open Access Journals (Sweden)

    Fritz Thorey

    2013-09-01

    Full Text Available An assortment of clinical trials have been done presenting the effectiveness of autologous matrix-induced chondrogenesis (AMIC for the regeneration of chondral leasions. The purpose of the study was to underline the accessability of the acetabulum and the femoral head through the known portals and prove i the feasibility of placing the AMIC in the different zones of the hip joint and ii check for dislocation after joint movement. Six human cadavers underwent hip arthroscopy on both hips. Two chondral lesions were set on each femoral head and two in the acetabulum to evaluate a total of 48 defects. After microfracturing an autologous matrix-induced chondrogenesis graft was placed on these lesions arthroscopically. After repeated joint movement the dislocation of the graft was checked. It was possible to place the AMIC graft in all 48 chondral lesions. The time needed for placing the graft was 8±2.9 minutes. A trend of time reduction could be detected throughout this study as the surgeon gained more experience. For the femoral head, after twenty cycles of joint movement 18/24 spots showed no displacement, 4/24 showed minor displacement (<3 mm and 2/24 showed major displacement (>3 mm. None showed total displacement. For the acetabulum 22/24 spots showed no displacement and 2/24 showed minor displacement. A combined microfracturing and placing of an AMIC graft of focal chondral lesions of the hip joint can be done arthroscopically. Prospective randomized in vivo studies should compare the results of arthroscopilally placed AMIC grafts with microfracturing and microfracturing alone.

  15. Small Molecule-BIO Accelerates and Enhances Marrow-Derived Mesenchymal Stem Cell in Vitro Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2014-03-01

    Full Text Available Background: Hyaline cartilage defects exhibit a major challenge in the field of orthopedic surgery owing to its limited repair capacity. On the other hand, mesenchymal stem cells (MSCs are regarded as potent cells with a property of cartilage regeneration. We aimed to optimize marrow-derived MSC chondrogenic culture using a small bioactive molecule referred to as BIO. Methods: MSCs from the marrow of NMRI mice were extracted, culture-expanded, and characterized. Micro-mass culture was then established for chondrogenic differentiation (control group. The cultures of MSC in chondrogenic medium supplemented with 0.01, 0.05, 0.1, and 1 µM BIO were taken as the experimental groups. Cartilage differentiation was examined by both histological sections and real-time PCR for Sox9, aggrecan, and collagen II at different time points. Moreover, the involvement of the Wnt pathway was investigated. Results: Based on histological sections, there was seemingly more intense metachromatic matrix produced in the cultures with 0.01 µM BIO. In this experimental group, cartilage-specific genes tended to be upregulated at day 14 compared to day 21 of the control group, indicating the accelerating effect of BIO on cartilage differentiation. Overall, there was statistically a significant increase (P=0.01 in the expression level of cartilage-specific genes in cultures with 0.01 µM BIO (enhancing effects. These upregulations appeared to be mediated through the Wnt pathway evident from the significant upregulation of T-cell factor and beta-catenin molecules (P=0.01. Conclusion: Taken together, BIO at 0.01 µM could accelerate and enhance in vitro chondrogenesis of mouse marrow-derived MSCs. Please cite this article as: Baghaban Eslaminejad MR, Fallah N. Small Molecule-BIO Accelerates and Enhances Marrow-Derived Mesenchymal Stem Cell in Vitro Chondrogenesis. Iran J Med Sci. 2014;39(2:107-116.

  16. Midkine-deficiency delays chondrogenesis during the early phase of fracture healing in mice.

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    Melanie Haffner-Luntzer

    Full Text Available The growth and differentiation factor midkine (Mdk plays an important role in bone development and remodeling. Mdk-deficient mice display a high bone mass phenotype when aged 12 and 18 months. Furthermore, Mdk has been identified as a negative regulator of mechanically induced bone formation and it induces pro-chondrogenic, pro-angiogenic and pro-inflammatory effects. Together with the finding that Mdk is expressed in chondrocytes during fracture healing, we hypothesized that Mdk could play a complex role in endochondral ossification during the bone healing process. Femoral osteotomies stabilized using an external fixator were created in wildtype and Mdk-deficient mice. Fracture healing was evaluated 4, 10, 21 and 28 days after surgery using 3-point-bending, micro-computed tomography, histology and immunohistology. We demonstrated that Mdk-deficient mice displayed delayed chondrogenesis during the early phase of fracture healing as well as significantly decreased flexural rigidity and moment of inertia of the fracture callus 21 days after fracture. Mdk-deficiency diminished beta-catenin expression in chondrocytes and delayed presence of macrophages during early fracture healing. We also investigated the impact of Mdk knockdown using siRNA on ATDC5 chondroprogenitor cells in vitro. Knockdown of Mdk expression resulted in a decrease of beta-catenin and chondrogenic differentiation-related matrix proteins, suggesting that delayed chondrogenesis during fracture healing in Mdk-deficient mice may be due to a cell-autonomous mechanism involving reduced beta-catenin signaling. Our results demonstrated that Mdk plays a crucial role in the early inflammation phase and during the development of cartilaginous callus in the fracture healing process.

  17. Ewing sarcoma ewsa protein regulates chondrogenesis of Meckel's cartilage through modulation of Sox9 in zebrafish.

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    Chris Merkes

    Full Text Available Ewing sarcoma is the second most common skeletal (bone and cartilage cancer in adolescents, and it is characterized by the expression of the aberrant chimeric fusion gene EWS/FLI1. Wild-type EWS has been proposed to play a role in mitosis, splicing and transcription. We have previously shown that EWS/FLI1 interacts with EWS, and it inhibits EWS activity in a dominant manner. Ewing sarcoma is a cancer that specifically develops in skeletal tissues, and although the above data suggests the significance of EWS, its role in chondrogenesis/skeletogenesis is not understood. To elucidate the function of EWS in skeletal development, we generated and analyzed a maternal zygotic (MZ ewsa/ewsa line because the ewsa/wt and ewsa/ewsa zebrafish appeared to be normal and fertile. Compared with wt/wt, the Meckel's cartilage of MZ ewsa/ewsa mutants had a higher number of craniofacial prehypertrophic chondrocytes that failed to mature into hypertrophic chondrocytes at 4 days post-fertilization (dpf. Ewsa interacted with Sox9, which is the master transcription factor for chondrogenesis. Sox9 target genes were either upregulated (ctgfa, ctgfb, col2a1a, and col2a1b or downregulated (sox5, nog1, nog2, and bmp4 in MZ ewsa/ewsa embryos compared with the wt/wt zebrafish embryos. Among these Sox9 target genes, the chromatin immunoprecipitation (ChIP experiment demonstrated that Ewsa directly binds to ctgfa and ctgfb loci. Consistently, immunohistochemistry showed that the Ctgf protein is upregulated in the Meckel's cartilage of MZ ewsa/ewsa mutants. Together, we propose that Ewsa promotes the differentiation from prehypertrophic chondrocytes to hypertrophic chondrocytes of Meckel's cartilage through inhibiting Sox9 binding site of the ctgf gene promoter. Because Ewing sarcoma specifically develops in skeletal tissue that is originating from chondrocytes, this new role of EWS may provide a potential molecular basis of its pathogenesis.

  18. Non-destructive label-free continuous monitoring of in vitro chondrogenesis via electrical conductivity and its anisotropy.

    Science.gov (United States)

    Oh, Tong In; Kim, Changhwan; Karki, Bishal; Son, Youngsook; Lee, EunAh; Woo, Eung Je

    2015-02-01

    Non-destructive label-free continuous monitoring of in vitro tissue culture is an unmet demand in tissue engineering. Noting that different compositions of cartilage lead to different electrical tissue properties, we propose a new method to measure the electrical conductivity and its anisotropy during in vitro chondrogenesis. We used a conductivity tensor probe with 17 electrodes and a bio-impedance spectroscopy (BIS) device to measure the conductivity values and the anisotropy ratios at the bottom and top surfaces of the tissue samples during the culture period of 6 weeks. Clearly distinguishing glycosaminoglycans (GAGs), collagen, and also various mixtures of them, the measured conductivity value and the estimated tissue anisotropy provide diagnostic information of the depth-dependent tissue structure and compositions. Continuously monitoring the individual tissue during the entire chondrogenesis process without any adverse effect, the proposed method may significantly increase the productivity of cartilage tissue engineering.

  19. Molecular Study of a Hoxa2 Gain-of-Function in Chondrogenesis: A Model of Idiopathic Proportionate Short Stature.

    OpenAIRE

    Catherine Nyssen-Behets; René Rezsöhazy; Lengelé, Benoît G.; Deprez, Pierre M L; Nichane, Miloud G

    2013-01-01

    In a previous study using transgenic mice ectopically expressing Hoxa2 during chondrogenesis, we associated the animal phenotype to human idiopathic proportionate short stature. Our analysis showed that this overall size reduction was correlated with a negative influence of Hoxa2 at the first step of endochondral ossification. However, the molecular pathways leading to such phenotype are still unknown. Using protein immunodetection and histological techniques comparing transgenic mice to cont...

  20. The immediate early gene product EGR1 and polycomb group proteins interact in epigenetic programming during chondrogenesis.

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    Frank Spaapen

    Full Text Available Initiation of and progression through chondrogenesis is driven by changes in the cellular microenvironment. At the onset of chondrogenesis, resting mesenchymal stem cells are mobilized in vivo and a complex, step-wise chondrogenic differentiation program is initiated. Differentiation requires coordinated transcriptomic reprogramming and increased progenitor proliferation; both processes require chromatin remodeling. The nature of early molecular responses that relay differentiation signals to chromatin is poorly understood. We here show that immediate early genes are rapidly and transiently induced in response to differentiation stimuli in vitro. Functional ablation of the immediate early factor EGR1 severely deregulates expression of key chondrogenic control genes at the onset of differentiation. In addition, differentiating cells accumulate DNA damage, activate a DNA damage response and undergo a cell cycle arrest and prevent differentiation associated hyper-proliferation. Failed differentiation in the absence of EGR1 affects global acetylation and terminates in overall histone hypermethylation. We report novel molecular connections between EGR1 and Polycomb Group function: Polycomb associated histone H3 lysine27 trimethylation (H3K27me3 blocks chromatin access of EGR1. In addition, EGR1 ablation results in abnormal Ezh2 and Bmi1 expression. Consistent with this functional interaction, we identify a number of co-regulated targets genes in a chondrogenic gene network. We here describe an important role for EGR1 in early chondrogenic epigenetic programming to accommodate early gene-environment interactions in chondrogenesis.

  1. Cdc42 is required for chondrogenesis and interdigital programmed cell death during limb development.

    Science.gov (United States)

    Aizawa, Ryo; Yamada, Atsushi; Suzuki, Dai; Iimura, Tadahiro; Kassai, Hidetoshi; Harada, Takeshi; Tsukasaki, Masayuki; Yamamoto, Gou; Tachikawa, Tetsuhiko; Nakao, Kazuki; Yamamoto, Matsuo; Yamaguchi, Akira; Aiba, Atsu; Kamijo, Ryutaro

    2012-01-01

    Cdc42, a member of the Rho subfamily of small GTPases, is known to be a regulator of multiple cellular functions, including cytoskeletal organization, cell migration, proliferation, and apoptosis. However, its tissue-specific roles, especially in mammalian limb development, remain unclear. To investigate the physiological function of Cdc42 during limb development, we generated limb bud mesenchyme-specific inactivated Cdc42 (Cdc42(fl/fl); Prx1-Cre) mice. Cdc42(fl/fl); Prx1-Cre mice demonstrated short limbs and body, abnormal calcification of the cranium, cleft palate, disruption of the xiphoid process, and syndactyly. Severe defects were also found in long bone growth plate cartilage, characterized by loss of columnar organization of chondrocytes, and thickening and massive accumulation of hypertrophic chondrocytes, resulting in delayed endochondral bone formation associated with reduced bone growth. In situ hybridization analysis revealed that expressions of Col10 and Mmp13 were reduced in non-resorbed hypertrophic cartilage, indicating that deletion of Cdc42 inhibited their terminal differentiation. Syndactyly in Cdc42(fl/fl); Prx1-Cre mice was caused by fusion of metacarpals and a failure of interdigital programmed cell death (ID-PCD). Whole mount in situ hybridization analysis of limb buds showed that the expression patterns of Sox9 were ectopic, while those of Bmp2, Msx1, and Msx2, known to promote apoptosis in the interdigital mesenchyme, were down-regulated. These results demonstrate that Cdc42 is essential for chondrogenesis and ID-PCD during limb development.

  2. Alpha-smooth muscle actin expression and structure integrity in chondrogenesis of human mesenchymal stem cells.

    Science.gov (United States)

    Hung, Shih-Chieh; Kuo, Pei-Yin; Chang, Ching-Fang; Chen, Tain-Hsiung; Ho, Larry Low-Tone

    2006-06-01

    The expression of alpha-smooth muscle actin (SMA) by human mesenchymal stem cells (hMSCs) during chondrogenesis was investigated by the use of pellet culture. Undifferentiated hMSCs expressed low but detectable amounts of SMA and the addition of transforming growth factor beta1 (TGF-beta1) to the culture medium increased SMA expression in a dose-dependent manner. Differentiation in pellet culture was rapidly induced in the presence of TGF-beta1 and was accompanied by the development of annular layers at the surface of the pellet. These peripheral layers lacked expression of glycosaminoglycan and type II collagen during early differentiation. Progress in differentiation increased the synthesis of glycosaminoglycan and type II collagen and the expression of SMA in these layers. Double-staining for type II collagen and SMA by immunofluorescence demonstrated the differentiation of hMSCs into cells positive for these two proteins. The addition of cytochalasin D, a potent inhibitor of the polymerization of actin microfilaments, caused damage to the structural integrity and surface smoothness of the chondrogenic pellets. The SMA-positive cells in the peripheral layers of the chondrogenic pellets mimic those within the superficial layer of articular cartilage and are speculated to play a major role in cartilage development and maintenance.

  3. Human Umbilical Cord Blood-Derived Mesenchymal Stem Cells Contribute to Chondrogenesis in Coculture with Chondrocytes

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    Xingfu Li

    2016-01-01

    Full Text Available Human umbilical cord blood-derived mesenchymal stem cells (hUCB-MSCs have been shown as the most potential stem cell source for articular cartilage repair. In this study, we aimed to develop a method for long-term coculture of human articular chondrocytes (hACs and hUCB-MSCs at low density in vitro to determine if the low density of hACs could enhance the hUCB-MSC chondrogenic differentiation as well as to determine the optimal ratio of the two cell types. Also, we compared the difference between direct coculture and indirect coculture at low density. Monolayer cultures of hUCB-MSCs and hACs were investigated at different ratios, at direct cell-cell contact groups for 21 days. Compared to direct coculture, hUCB-MSCs and hACs indirect contact culture significantly increased type II collagen (COL2 and decreased type I collagen (COL1 protein expression levels. SRY-box 9 (SOX9 mRNA levels and protein expression were highest in indirect coculture. Overall, these results indicate that low density direct coculture induces fibrocartilage. However, indirect coculture in conditioned chondrocyte cell culture medium can increase expression of chondrogenic markers and induce hUCB-MSCs differentiation into mature chondrocytes. This work demonstrates that it is possible to promote chondrogenesis of hUCB-MSCs in combination with hACs, further supporting the concept of novel coculture strategies for tissue engineering.

  4. Effects of the mesonephros and insulin-like growth factor I on chondrogenesis of limb explants.

    Science.gov (United States)

    Geduspan, J S; Solursh, M

    1993-04-01

    The mesonephros has been shown to have a growth-promoting influence in vivo on limb outgrowth. This influence has been studied in detail using an organ culture system. The results show that in the presence of the mesonephros limb explants formed larger cartilages than cultures without mesonephros. Furthermore, with mesonephros, morphology of the cartilages is comparable to that of skeletal elements in vivo while cartilages formed in cultures lacking mesonephros were amorphous. The mesonephric influence also promoted the formation of a well-organized extracellular matrix in the cartilage while cartilage in cultures without mesonephros formed an abnormal appearing matrix. Cartilage matrices in cultures with or without mesonephros were immunoreactive to type IX and type II collagens, cartilage proteoglycan PGH, and link protein although cultures lacking mesonephros had a very restricted distribution of type IX collagen immunoreactivity. Despite the different distribution of type IX collagen, long-form-type IX collagen transcripts appeared similar in both types of culture based on in situ hybridization. The mesonephric effect on limb explants could be partially duplicated by the addition of insulin-like growth factor I (IGF-I) to cultures without mesonephros. Furthermore, the mesonephric influence on cartilage growth and morphological differentiation could be blocked by the addition of a blocking antibody to IGF-I to cultures with mesonephros. The results support the hypothesis that IGF-I is one of the growth factors produced by the mesonephros which may play a role in early limb development and chondrogenesis.

  5. Role of Nuclear Factor Kappa B (NF-κB) in Growth Plate Chondrogenesis.

    Science.gov (United States)

    De Luca, Francesco

    2016-06-01

    Abstract Nuclear Factor kappa B (NF-κB) is a group of seven transcription factors. Upon activation by a variety of stimuli, NF-κB translocates to the nucleus and modulates the expression of target genes involved in cell growth, survival, and death. Previous evidence indicates that NF-κB regulates bone growth and development. We have shown that the NF-κB p65 is expressed in the growth plate and facilitates longitudinal bone growth by inducing chondrocyte proliferation and differentiation and by preventing apoptosis. Furthermore, we have demonstrated in rodents that NF-κB expressed in growth plate chondrocytes mediates the promoting effects of GH and IGF-1 on longitudinal bone growth and growth plate chondrogenesis. Lastly, functional studies carried out in two children with growth failure and GH insensitivity, and affected by two different mutations impairing NF-κB activation, indicate that NF-κB also mediates the growth-promoting effects of GH in humans.

  6. Chemical modification of extracellular matrix by cold atmospheric plasma-generated reactive species affects chondrogenesis and bone formation.

    Science.gov (United States)

    Eisenhauer, Peter; Chernets, Natalie; Song, You; Dobrynin, Danil; Pleshko, Nancy; Steinbeck, Marla J; Freeman, Theresa A

    2016-09-01

    The goal of this study was to investigate whether cold plasma generated by dielectric barrier discharge (DBD) modifies extracellular matrices (ECM) to influence chondrogenesis and endochondral ossification. Replacement of cartilage by bone during endochondral ossification is essential in fetal skeletal development, bone growth and fracture healing. Regulation of this process by the ECM occurs through matrix remodelling, involving a variety of cell attachment molecules and growth factors, which influence cell morphology and protein expression. The commercially available ECM, Matrigel, was treated with microsecond or nanosecond pulsed (μsp or nsp, respectively) DBD frequencies conditions at the equivalent frequencies (1 kHz) or power (~1 W). Recombinant human bone morphogenetic protein-2 was added and the mixture subcutaneously injected into mice to simulate ectopic endochondral ossification. Two weeks later, the masses were extracted and analysed by microcomputed tomography. A significant increase in bone formation was observed in Matrigel treated with μsp DBD compared with control, while a significant decrease in bone formation was observed for both nsp treatments. Histological and immunohistochemical analysis showed Matrigel treated with μsp plasma increased the number of invading cells, the amount of vascular endothelial growth factor and chondrogenesis while the opposite was true for Matrigel treated with nsp plasma. In support of the in vivo Matrigel study, 10 T1/2 cells cultured in vitro on μsp DBD-treated type I collagen showed increased expression of adhesion proteins and activation of survival pathways, which decreased with nsp plasma treatments. These results indicate DBD modification of ECM can influence cellular behaviours to accelerate or inhibit chondrogenesis and endochondral ossification. Copyright © 2016 John Wiley & Sons, Ltd.

  7. Differential co-expression of long and short form type IX collagen transcripts during avian limb chondrogenesis in ovo.

    Science.gov (United States)

    Swiderski, R E; Solursh, M

    1992-05-01

    Using RNA blot analysis of developmentally staged avian limb buds, we demonstrate that transcripts of several cartilage marker genes appear in limb tissue prior to overt chondrogenesis. Type II collagen mRNA, cartilage proteoglycan core protein mRNA, alpha 2(IX) collagen mRNA, and transcripts of the short form alpha 1(IX) collagen chain derived from the downstream promoter are co-expressed in limb tissue approximately 24-36 hours before the appearance of the respective polypeptides in differentiating cartilagenous tissue. Transcripts of the long form alpha 1(IX) collagen chain derived from the upstream promoter appear somewhat later in development; nearly coincident with the immunolocalization of type IX collagen in the cartilage elements of the limb. The spatial distribution of type II and type IX collagen transcripts was analyzed by in situ hybridization. Type II collagen and the long form alpha 1(IX) collagen transcripts co-localized in the chondrogenic elements of the developing forelimb. In contrast, short form alpha 1(IX) collagen transcripts which lack the 5' region encoding the NC4 globular amino-terminal domain were distributed throughout the non-chondrogenic, non-myogenic mesenchymal regions of the limb and were not detectable above background levels in the limb chondrogenic elements. The precocious appearance of several cartilage marker gene transcripts prior to chondrogenesis suggests that multiple levels of gene regulation including alternative promoter use, alternative RNA splicing, alternative polyadenylation, and other post-transcriptional as well as translational mechanisms are active prior to, and during avian limb chondrogenesis.

  8. High Field Sodium MRI Assessment of Stem Cell Chondrogenesis in a Tissue-Engineered Matrix.

    Science.gov (United States)

    Majumdar, Shreyan; Pothirajan, Padmabharathi; Dorcemus, Deborah; Nukavarapu, Syam; Kotecha, Mrignayani

    2016-04-01

    The development of non-invasive assessment techniques in vitro and in vivo is essential for monitoring and evaluating the growth of engineered cartilage tissues. Magnetic resonance imaging (MRI) is the leading non-invasive imaging modality used for assessing engineered cartilage. Typical MRI uses water proton relaxation times (T1 and T2) and apparent diffusion coefficient (ADC) to assess tissue growth. These techniques, while excellent in providing the first assurance of tissue growth, are unspecific to monitor the progress of engineered cartilage extracellular matrix components. In the current article, we present high field (11.7 T, (1)H freq. = 500 MHz) sodium MRI assessment of tissue-engineered cartilage at the early stage of tissue growth in vitro. We observed the chondrogenesis of human bone marrow derived stromal cells seeded in a gradient polymer-hydrogel matrix made out of poly(85 lactide-co-15 glycolide)--PuraMatrix™ for 4 weeks. We calculated the sodium concentration in the engineered constructs using a model of sodium MRI voxels that takes into account scaffold volume, cell density and amount of glycosaminoglycan (GAG). The sodium concentration was then converted to the fixed charge density (FCD) and compared with FCD derived from biochemical GAG analysis. Despite the small amount of GAG present in the engineered constructs, the sodium MRI derived FCD is found to be correlated (Pearson correlation coefficient R = 0.79) with the FCD derived from biochemical analysis. We conclude that sodium MRI could prove to be an invaluable tool in assessing engineered cartilage quantitatively during the repair or regeneration of cartilage defects.

  9. Conditional expression of Wnt4 during chondrogenesis leads to dwarfism in mice.

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    Hu-Hui Lee

    Full Text Available Wnts are expressed in the forming long bones, suggesting roles in skeletogenesis. To examine the action of Wnts in skeleton formation, we developed a genetic system to conditionally express Wnt4 in chondrogenic tissues of the mouse. A mouse Wnt4 cDNA was introduced into the ubiquitously expressed Rosa26 (R26 locus by gene targeting in embryonic stem (ES cells. The expression of Wnt4 from the R26 locus was blocked by a neomycin selection cassette flanked by loxP sites (floxneo that was positioned between the Rosa26 promoter and the Wnt4 cDNA, creating the allele designated R26(floxneoWnt4. Wnt4 expression was activated during chondrogenesis using Col2a1-Cre transgenic mice that express Cre recombinase in differentiating chondrocytes. R26(floxneoWnt4; Col2a1-Cre double heterozygous mice exhibited a growth deficiency, beginning approximately 7 to 10 days after birth, that resulted in dwarfism. In addition, they also had craniofacial abnormalities, and delayed ossification of the lumbar vertebrae and pelvic bones. Histological analysis revealed a disruption in the organization of the growth plates and a delay in the onset of the primary and secondary ossification centers. Molecular studies showed that Wnt4 overexpression caused decreased proliferation and altered maturation of chondrocytes. In addition, R26(floxneoWnt4; Col2a1-Cre mice had decreased expression of vascular endothelial growth factor (VEGF. These studies demonstrate that Wnt4 overexpression leads to dwarfism in mice. The data indicate that Wnt4 levels must be regulated in chondrocytes for normal growth plate development and skeletogenesis. Decreased VEGF expression suggests that defects in vascularization may contribute to the dwarf phenotype.

  10. Chondrogenesis of human bone marrow mesenchymal stromal cells in highly porous alginate-foams supplemented with chondroitin sulfate

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    Huang, Zhao [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Nooeaid, Patcharakamon [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Kohl, Benjamin [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Roether, Judith A.; Schubert, Dirk W. [Institute of Polymer Materials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Meier, Carola [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Boccaccini, Aldo R. [Institute of Biomaterials, Department of Materials Science and Engineering, University of Erlangen-Nuremberg (Germany); Godkin, Owen; Ertel, Wolfgang; Arens, Stephan [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Schulze-Tanzil, Gundula, E-mail: gundula.schulze@pmu.ac.at [Department of Orthopaedic, Trauma and Reconstructive Surgery, Charité-Universitätsmedizin-Berlin Campus Benjamin Franklin, Berlin (Germany); Institute of Anatomy, Paracelsus Medical University, Nuremberg (Germany)

    2015-05-01

    To overcome the limited intrinsic cartilage repair, autologous chondrocyte or bone-marrow-derived mesenchymal stromal cell (BM-MSC) was implanted into cartilage defects. For this purpose suitable biocompatible scaffolds are needed to provide cell retention, chondrogenesis and initial mechanical stability. The present study should indicate whether a recently developed highly porous alginate (Alg) foam scaffold supplemented with chondroitin sulfate (CS) allows the attachment, survival and chondrogenesis of BM-MSCs and articular chondrocytes. The foams were prepared using a freeze-drying method; some of them were supplemented with CS and subsequently characterized for porosity, biodegradation and mechanical profile. BM-MSCs were cultured for 1–2 weeks on the scaffold either under chondrogenic or maintenance conditions. Cell vitality assays, histology, glycosaminoglycan (sGAG) assay, and type II and I collagen immunolabelings were performed to monitor cell growth and extracellular matrix (ECM) synthesis in the scaffolds. Scaffolds had a high porosity ~ 93–95% with a mean pore sizes of 237 ± 48 μm (Alg) and 197 ± 61 μm (Alg/CS). Incorporation of CS increased mechanical strength of the foams providing gradually CS release over 7 days. Most of the cells survived in the scaffolds. BM-MSCs and articular chondrocytes formed rounded clusters within the scaffold pores. The BM-MSCs, irrespective of whether cultured under non/chondrogenic conditions and chondrocytes produced an ECM containing sGAGs, and types II and I collagen. Total collagen and sGAG contents were higher in differentiated BM-MSC cultures supplemented with CS than in CS-free foams after 14 days. The cell cluster formation induced by the scaffolds might stimulate chondrogenesis via initial intense cell–cell contacts. - Highlights: • Alginate foam scaffolds revealed a high porosity and mean pore size of 197–237 μm. • Chondroitin sulfate was released over 14 days by the scaffolds. • Chondrocytes

  11. The Influence of IL-10 and TNFα on Chondrogenesis of Human Mesenchymal Stromal Cells in Three-Dimensional Cultures

    Directory of Open Access Journals (Sweden)

    Michal Jagielski

    2014-09-01

    Full Text Available Chondrogenic differentiated mesenchymal stromal cells (MSCs are a promising cell source for articular cartilage repair. This study was undertaken to determine the effectiveness of two three-dimensional (3D culture systems for chondrogenic MSC differentiation in comparison to primary chondrocytes and to assess the effect of Interleukin (IL-10 and Tumor Necrosis Factor (TNFα on chondrogenesis by MSCs in 3D high-density (H-D culture. MSCs were isolated from femur spongiosa, characterized using a set of typical markers and introduced in scaffold-free H-D cultures or non-woven polyglycolic acid (PGA scaffolds for chondrogenic differentiation. H-D cultures were stimulated with recombinant IL-10, TNFα, TNFα + IL-10 or remained untreated. Gene and protein expression of type II collagen, aggrecan, sox9 and TNFα were examined. MSCs expressed typical cell surface markers and revealed multipotency. Chondrogenic differentiated cells expressed cartilage-specific markers in both culture systems but to a lower extent when compared with articular chondrocytes. Chondrogenesis was more pronounced in PGA compared with H-D culture. IL-10 and/or TNFα did not impair the chondrogenic differentiation of MSCs. Moreover, in most of the investigated samples, despite not reaching significance level, IL-10 had a stimulatory effect on the type II collagen, aggrecan and TNFα expression when compared with the respective controls.

  12. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System

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    Hyuck Joon Kwon

    2015-01-01

    Full Text Available Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  13. Dual Monitoring of Secretion and ATP Levels during Chondrogenesis Using Perfusion Culture-Combined Bioluminescence Monitoring System.

    Science.gov (United States)

    Kwon, Hyuck Joon; Han, Youngbae

    2015-01-01

    Skeletal pattern formation in limb development depends on prechondrogenic condensation which prefigures the cartilage template. However, although morphogens such as TGF-βs and BMPs have been known to play essential roles in skeletal patterning, how the morphogens induce prechondrogenic cells to aggregate and determine patterns of cartilage elements has remained unclear. Our previous study reported that ATP oscillations are induced during chondrogenesis. This result suggests the possibility that ATP oscillations lead to the oscillatory secretion of morphogens, due to the fact that secretion process requires ATP. To examine the correlation between ATP oscillations and secretion levels of morphogens, we have developed perfusion culture-combined bioluminescence monitoring system to simultaneously monitor intracellular ATP levels and secretion levels. Using this system, we found that secretory activity oscillates in phase with ATP oscillations and that secretion levels of TGF-β1 and BMP2 oscillate during chondrogenesis. The oscillatory secretion of the morphogens would contribute to amplifying the fluctuation of the morphogens, underlie the spatial patterning of morphogens, and consequently lead to skeletal pattern formation.

  14. Silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid effectively enhances in vitro chondrogenesis of bone marrow mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Sawatjui, Nopporn [Biomedical Sciences, Graduate School, Khon Kaen University, Khon Kaen 40002 (Thailand); Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Damrongrungruang, Teerasak [Department of Oral Diagnosis, Faculty of Dentistry, Khon Kaen University, Khon Kaen 40002 (Thailand); Leeanansaksiri, Wilairat [Stem Cell Therapy and Transplantation Research Group, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); School of Microbiology, Suranaree University of Technology, Nakhon Ratchasima 30000 (Thailand); Jearanaikoon, Patcharee [Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand); Hongeng, Suradej [Department of Pediatrics, Faculty of Medicine, Ramathibodi Hospital, Mahidol University, Bangkok 10400 (Thailand); Limpaiboon, Temduang, E-mail: temduang@kku.ac.th [Centre for Research and Development of Medical Diagnostic Laboratories, Faculty of Associated Medical Sciences, Khon Kaen University, Khon Kaen 40002 (Thailand)

    2015-07-01

    Tissue engineering is becoming promising for cartilage repair due to the limited self-repair capacity of cartilage tissue. We previously fabricated and characterized a three-dimensional silk fibroin/gelatin–chondroitin sulfate–hyaluronic acid (SF–GCH) scaffold and showed that it could promote proliferation of human bone marrow mesenchymal stem cells (BM-MSCs). This study aimed to evaluate its biological performance as a new biomimetic material for chondrogenic induction of BM-MSCs in comparison to an SF scaffold and conventional pellet culture. We found that the SF–GCH scaffold significantly enhanced the proliferation and chondrogenic differentiation of BM-MSCs compared to the SF scaffold and pellet culture in which the production of sulfated glycoaminoglycan was increased in concordance with the up-regulation of chondrogenic-specific gene markers. Our findings indicate the significant role of SF–GCH by providing a supportive structure and the mimetic cartilage environment for chondrogenesis which enables cartilage regeneration. Thus, our fabricated SF–GCH scaffold may serve as a potential biomimetic material for cartilage tissue engineering. - Highlights: • SF–GCH scaffold enhances proliferation and chondrogenic differentiation of BM-MSCs. • SF–GCH acts as a supportive and biomimetic material for BM-MSC chondrogenesis. • SF–GCH is a potential biomimetic scaffold suitable for cartilage tissue engineering.

  15. Enamel Matrix Derivative Stimulates Osteogenesis-and Chondrogenesis-related Transcription Factors in C3H10T1/2 Cells

    Institute of Scientific and Technical Information of China (English)

    Masataka NARUKAWA; Naoto SUZUKI; Tadahiro TAKAYAMA; Yasuhiko YAMASHITA; Kichibee OTSUKA; Koichi ITO

    2007-01-01

    Our purpose was to determine how enamel matrix derivative (EMD) affects the expression of osteogenesis-and chondrogenesis-related transcription factors in undifferentiated mesenchymal cells.C3H10T1/2 cell line, a typical pluripotential mesenchymal cell line, was cultured with or without EMD for up to 7 d. Expression of mRNAs encoding osteogenesis-and chondrogenesis-related transcription factors(Runx2, Osterix, AJ18, Dlx5, Msx2, Sox5, Sox9 and Zfp60) was measured using real-time polymerase chain reaction. Runx2 and Sox9 protein expression and the presence of bone morphogenetic protein (BMP)-6-like molecules in EMD were determined by Western blotting. EMD substantially increased mRNA levels of osteogenesis-and chondrogenesis-related transcription factors. EMD also induced Runx2 and Sox9 protein expression. Western blotting analysis of EMD using anti-BMP-6 antibody revealed immunoreactive bands corresponding to about 14 kDa and 60 kDa. These results suggest that EMD stimulates osteogenesis-and chondrogenesis-related transcription factors, and these activities may be mediated, at least in part, by BMP-6in EMD.

  16. Regulating Chondrogenesis of Human Mesenchymal Stromal Cells with a Retinoic Acid Receptor-Beta Inhibitor: Differential Sensitivity of Chondral Versus Osteochondral Development

    Directory of Open Access Journals (Sweden)

    Solvig Diederichs

    2014-05-01

    Full Text Available Aim: Main objective was to investigate whether the synthetic retinoic acid receptor (RAR-β antagonist LE135 is able to drive in vitro chondrogenesis of human mesenchymal stromal cells (MSCs or improve differentiation by suppressing hypertrophic chondrocyte development. Methods: Chondrogenesis of human bone marrow and adipose tissue-derived MSCs was induced in micromass pellet culture for six weeks. Effects of LE135 alone and in combinatorial treatment with TGF-β on deposition of cartilaginous matrix including collagen type II and glycosaminoglycans, on deposition of non-hyaline cartilage collagens type I and X, and on hypertrophy markers including alkaline phosphatase (ALP, indian hedghehog (IHH and matrix metalloproteinase (MMP-13 were assessed. Results: LE135 was no inducer of chondrogenesis and failed to stimulate deposition of collagen type II and glycosaminoglycans. Moreover, addition of LE135 to TGF-β-treated pellets inhibited cartilaginous matrix deposition and gene expression of COL2A1. In contrast, non-hyaline cartilage collagens were less sensitive to LE135 and hypertrophy markers remained unaffected. Conclusion: This demonstrates a differential sensitivity of chondral versus endochondral differentiation pathways to RARβ signaling; however, opposite to the desired direction. The relevance of trans-activating versus trans-repressing RAR signaling, including effects on activator protein (AP-1 is discussed and implications for overcoming current limits of hMSC chondrogenesis are considered.

  17. Three-dimensional scaffold-free fusion culture: the way to enhance chondrogenesis of in vitro propagated human articular chondrocytes

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    M. Lehmann

    2013-11-01

    Full Text Available Cartilage regeneration based on isolated and culture-expanded chondrocytes has been studied in various in vitro models, but the quality varies with respect to the morphology and the physiology of the synthesized tissues. The aim of our study was to promote in vitro chondrogenesis of human articular chondrocytes using a novel three-dimensional (3-D cultivation system in combination with the chondrogenic differentiation factors transforming growth factor beta 2 (TGF-b2 and L-ascorbic acid. Articular chondrocytes isolated from six elderly patients were expanded in monolayer culture. A single-cell suspension of the dedifferentiated chondrocytes was then added to agar-coated dishes without using any scaffold material, in the presence, or absence of TGF-b2 and/or L-ascorbic acid. Three-dimensional cartilage-like constructs, called single spheroids, and microtissues consisting of several spheroids fused together, named as fusions, were formed. Generated tissues were mainly characterized using histological and immunohistochemical techniques. The morphology of the in vitro tissues shared some similarities to native hyaline cartilage in regard to differentiated S100-positive chondrocytes within a cartilaginous matrix, with strong collagen type II expression and increased synthesis of proteoglycans. Finally, our innovative scaffold-free fusion culture technique supported enhanced chondrogenesis of human articular chondrocytes in vitro. These 3-D hyaline cartilage-like microtissues will be useful for in vitro studies of cartilage differentiation and regeneration, enabling optimization of functional tissue engineering and possibly contributing to the development of new approaches to treat traumatic cartilage defects or osteoarthritis.

  18. Hedgehog signaling is required for cranial neural crest morphogenesis and chondrogenesis at the midline in the zebrafish skull.

    Science.gov (United States)

    Wada, Naoyuki; Javidan, Yashar; Nelson, Sarah; Carney, Thomas J; Kelsh, Robert N; Schilling, Thomas F

    2005-09-01

    Neural crest cells that form the vertebrate head skeleton migrate and interact with surrounding tissues to shape the skull, and defects in these processes underlie many human craniofacial syndromes. Signals at the midline play a crucial role in the development of the anterior neurocranium, which forms the ventral braincase and palate, and here we explore the role of Hedgehog (Hh) signaling in this process. Using sox10:egfp transgenics to follow neural crest cell movements in the living embryo, and vital dye labeling to generate a fate map, we show that distinct populations of neural crest form the two main cartilage elements of the larval anterior neurocranium: the paired trabeculae and the midline ethmoid. By analyzing zebrafish mutants that disrupt sonic hedgehog (shh) expression, we demonstrate that shh is required to specify the movements of progenitors of these elements at the midline, and to induce them to form cartilage. Treatments with cyclopamine, to block Hh signaling at different stages, suggest that although requirements in morphogenesis occur during neural crest migration beneath the brain, requirements in chondrogenesis occur later, as cells form separate trabecular and ethmoid condensations. Cell transplantations indicate that these also reflect different sources of Shh, one from the ventral neural tube that controls trabecular morphogenesis and one from the oral ectoderm that promotes chondrogenesis. Our results suggest a novel role for Shh in the movements of neural crest cells at the midline, as well as in their differentiation into cartilage, and help to explain why both skeletal fusions and palatal clefting are associated with the loss of Hh signaling in holoprosencephalic humans.

  19. Nicotine-induced retardation of chondrogenesis through down-regulation of IGF-1 signaling pathway to inhibit matrix synthesis of growth plate chondrocytes in fetal rats

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    Deng, Yu; Cao, Hong; Cu, Fenglong [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Xu, Dan [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Lei, Youying [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Tan, Yang [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China); Magdalou, Jacques [UMR 7561 CNRS-Nancy Université, Faculté de Médicine, Vandoeuvre-lès-Nancy (France); Wang, Hui [Department of Pharmacology, Basic Medical School of Wuhan University, Wuhan 430071 (China); Research Center of Food and Drug Evaluation, Wuhan University, Wuhan 430071 (China); Chen, Liaobin, E-mail: lbchen@whu.edu.cn [Department of Orthopedic Surgery, Zhongnan Hospital of Wuhan University, Wuhan 430071 (China)

    2013-05-15

    Previous studies have confirmed that maternal tobacco smoking causes intrauterine growth retardation (IUGR) and skeletal growth retardation. Among a multitude of chemicals associated with cigarette smoking, nicotine is one of the leading candidates for causing low birth weights. However, the possible mechanism of delayed chondrogenesis by prenatal nicotine exposure remains unclear. We investigated the effects of nicotine on fetal growth plate chondrocytes in vivo and in vitro. Rats were given 2.0 mg/kg·d of nicotine subcutaneously from gestational days 11 to 20. Prenatal nicotine exposure increased the levels of fetal blood corticosterone and resulted in fetal skeletal growth retardation. Moreover, nicotine exposure induced the inhibition of matrix synthesis and down-regulation of insulin-like growth factor 1 (IGF-1) signaling in fetal growth plates. The effects of nicotine on growth plates were studied in vitro by exposing fetal growth plate chondrocytes to 0, 1, 10, or 100 μM of nicotine for 10 days. Nicotine inhibited matrix synthesis and down-regulated IGF-1 signaling in chondrocytes in a concentration-dependent manner. These results suggest that prenatal nicotine exposure induces delayed chondrogenesis and that the mechanism may involve the down-regulation of IGF-1 signaling and the inhibition of matrix synthesis by growth plate chondrocytes. The present study aids in the characterization of delayed chondrogenesis caused by prenatal nicotine exposure, which might suggest a candidate mechanism for intrauterine origins of osteoporosis and osteoarthritis. - Highlights: ► Prenatal nicotine-exposure could induce delayed chondrogenesis in fetal rats. ► Nicotine inhibits matrix synthesis of fetal growth plate chondrocytes. ► Nicotine inhibits IGF-1 signaling pathway in fetal growth plate chondrocytes.

  20. Fibrin and poly(lactic-co-glycolic acid hybrid scaffold promotes early chondrogenesis of articular chondrocytes: an in vitro study

    Directory of Open Access Journals (Sweden)

    Idrus Ruszymah BH

    2008-04-01

    Full Text Available Abstract Background Synthetic- and naturally derived- biodegradable polymers have been widely used to construct scaffolds for cartilage tissue engineering. Poly(lactic-co-glycolic acid (PLGA are bioresorbable and biocompatible, rendering them as a promising tool for clinical application. To minimize cells lost during the seeding procedure, we used the natural polymer fibrin to immobilize cells and to provide homogenous cells distribution in PLGA scaffolds. We evaluated in vitro chondrogenesis of rabbit articular chondrocytes in PLGA scaffolds using fibrin as cell transplantation matrix. Methods PLGA scaffolds were soaked in chondrocytes-fibrin suspension (1 × 106cells/scaffold and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes was used as control. All constructs were cultured for a maximum of 21 days. Cell proliferation activity was measured at 1, 3, 7, 14 and 21 days in vitro using 3-(4,5-dimethylthiazole-2-yl-2-, 5-diphenyltetrazolium-bromide (MTT assay. Morphological observation, histology, immunohistochemistry (IHC, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 3 weeks to elucidate in vitro cartilage development and deposition of cartilage-specific extracellular matrix (ECM. Results Cell proliferation activity was gradually increased from day-1 until day-14 and declined by day-21. A significant cartilaginous tissue formation was detected as early as 2-week in fibrin/PLGA hybrid construct as confirmed by the presence of cartilage-isolated cells and lacunae embedded within basophilic ECM. Cartilage formation was remarkably evidenced after 3 weeks. Presence of cartilage-specific proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs were confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrix. Chondrogenic properties were further

  1. Gpr177, a novel locus for bone mineral density and osteoporosis, regulates osteogenesis and chondrogenesis in skeletal development.

    Science.gov (United States)

    Maruyama, Takamitsu; Jiang, Ming; Hsu, Wei

    2013-05-01

    Human genetic analysis has recently identified Gpr177 as a susceptibility locus for bone mineral density and osteoporosis. Determining the unknown function of this gene is therefore extremely important to furthering our knowledge base of skeletal development and disease. The protein encoded by Gpr177 exhibits an ability to modulate the trafficking of Wnt, similar to the Drosophila Wls/Evi/Srt. Because it plays a critical role in Wnt regulation, Gpr177 might be required for several key steps of skeletogenesis. To overcome the early lethality associated with the inactivation of Gpr177 in mice, conditional gene deletion is used to assess its functionality. Here we report the generation of four different mouse models with Gpr177 deficiency in various skeletogenic cell types. The loss of Gpr177 severely impairs development of the craniofacial and body skeletons, demonstrating its requirement for intramembranous and endochondral ossifications, respectively. Defects in the expansion of skeletal precursors and their differentiation into osteoblasts and chondrocytes suggest that Wnt production and signaling mediated by Gpr177 cannot be substituted. Because the Gpr177 ablation impairs Wnt secretion, we therefore identify the sources of Wnt proteins essential for osteogenesis and chondrogenesis. The intercross of Wnt signaling between distinct cell types is carefully orchestrated and necessary for skeletogenesis. Our findings lead to a proposed mechanism by which Gpr177 controls skeletal development through modulation of autocrine and paracrine Wnt signals in a lineage-specific fashion.

  2. All-Arthroscopic Autologous Matrix-Induced Chondrogenesis for the Treatment of Osteochondral Lesions of the Talus

    Science.gov (United States)

    Usuelli, Federico Giuseppe; de Girolamo, Laura; Grassi, Miriam; D'Ambrosi, Riccardo; Montrasio, Umberto Alfieri; Boga, Michele

    2015-01-01

    Several surgical techniques have been described for the treatment of talar chondral lesions. Among them, microfracture is well established. Autologous matrix-induced chondrogenesis (AMIC), using microfracture and biomaterials, has shown promising results for the treatment of knee osteochondral lesions and has been proposed for the ankle as an open technique. We describe an all-arthroscopic AMIC technique. The benefits of an all-arthroscopic procedure include smaller incisions with less soft-tissue dissection, better visualization of the joint, and a quicker recovery compared with open surgery. The use of matrix to support cartilage regeneration promotes good-quality cartilage tissue with satisfactory long-term outcomes. Our all-arthroscopic AMIC technique uses a type I–type III porcine collagen matrix (Chondro-Gide; Geistlich Pharma, Wolhusen, Switzerland) and is characterized by 2 different arthroscopic surgical phases. First, adequate exposure is achieved through use of a Hintermann spreader (Integra LifeSciences, Plainsboro, NJ) with sufficient joint distraction and wet lesion preparation. The second surgical step is performed dry, involving matrix placement and fixation. The all-arthroscopic AMIC technique for the treatment of osteochondral lesions of the talus allows a very precise reconstruction in the case of cartilage defects and avoids the need for a more invasive operation associated with higher morbidity and a longer surgical time. PMID:26258040

  3. Molecular Study of a Hoxa2 Gain-of-Function in Chondrogenesis: A Model of Idiopathic Proportionate Short Stature

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    Catherine Nyssen-Behets

    2013-10-01

    Full Text Available In a previous study using transgenic mice ectopically expressing Hoxa2 during chondrogenesis, we associated the animal phenotype to human idiopathic proportionate short stature. Our analysis showed that this overall size reduction was correlated with a negative influence of Hoxa2 at the first step of endochondral ossification. However, the molecular pathways leading to such phenotype are still unknown. Using protein immunodetection and histological techniques comparing transgenic mice to controls, we show here that the persistent expression of Hoxa2 in chondrogenic territories provokes a general down-regulation of the main factors controlling the differentiation cascade, such as Bapx1, Bmp7, Bmpr1a, Ihh, Msx1, Pax9, Sox6, Sox9 and Wnt5a. These data confirm the impairment of chondrogenic differentiation by Hoxa2 overexpression. They also show a selective effect of Hoxa2 on endochondral ossification processes since Gdf5 and Gdf10, and Bmp4 or PthrP were up-regulated and unmodified, respectively. Since Hoxa2 deregulation in mice induces a proportionate short stature phenotype mimicking human idiopathic conditions, our results give an insight into understanding proportionate short stature pathogenesis by highlighting molecular factors whose combined deregulation may be involved in such a disease.

  4. Fibroblast growth factor receptors in in vitro and in vivo chondrogenesis: relating tissue engineering using adult mesenchymal stem cells to embryonic development.

    Science.gov (United States)

    Hellingman, Catharine A; Koevoet, Wendy; Kops, Nicole; Farrell, Eric; Jahr, Holger; Liu, Wei; Baatenburg de Jong, Robert J; Frenz, Dorothy A; van Osch, Gerjo J V M

    2010-02-01

    Adult mesenchymal stem cells (MSCs) are considered promising candidate cells for therapeutic cartilage and bone regeneration. Because tissue regeneration and embryonic development may involve similar pathways, understanding common pathways may lead to advances in regenerative medicine. In embryonic limb development, fibroblast growth factor receptors (FGFRs) play a role in chondrogenic differentiation. The aim of this study was to investigate and compare FGFR expression in in vivo embryonic limb development and in vitro chondrogenesis of MSCs. Our study showed that in in vitro chondrogenesis of MSCs three sequential stages can be found, as in embryonic limb development. A mesenchymal condensation (indicated by N-cadherin) is followed by chondrogenic differentiation (indicated by collagen II), and hypertrophy (indicated by collagen X). FGFR1-3 are expressed in a stage-dependent pattern during in vitro differentiation and in vivo embryonic limb development. In both models FGFR2 is clearly expressed by cells in the condensation phase. No FGFR expression was observed in differentiating and mature hyaline chondrocytes, whereas hypertrophic chondrocytes stained strongly for all FGFRs. To evaluate whether stage-specific modulation of chondrogenic differentiation in MSCs is possible with different subtypes of FGF, FGF2 and FGF9 were added to the chondrogenic medium during different stages in the culture process (early or late). FGF2 and FGF9 differentially affected the amount of cartilage formed by MSCs depending on the stage in which they were added. These results will help us understand the role of FGF signaling in chondrogenesis and find new tools to monitor and control chondrogenic differentiation.

  5. Study of Carbon Nano-Tubes Effects on the Chondrogenesis of Human Adipose Derived Stem Cells in Alginate Scaffold

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    Ali Valiani

    2014-01-01

    Full Text Available Background: Osteoarthritis is one of the most common diseases in middle-aged populations in the World and could become the fourth principal cause of disability by the year 2020. One of the critical properties for cartilage tissue engineering (TE is the ability of scaffolds to closely mimic the extracellular matrix and bond to the host tissue. Therefore, TE has been presented as a technique to introduce the best combination of cells and biomaterial scaffold and to stimulate growth factors to produce a cartilage tissue resembling natural articular cartilage. The aim of study is to improve differentiation of adipose derived stem cells (ADSCs into chondrocytes in order to provide a safe and modern treatment for patients suffering from cartilage damages. Methods: After functionalization, dispersions and sterilizing carbon nano-tubes (CNTs, a new type of nanocomposite gel was prepared from water-soluble CNTs and alginate. ADSCs seeded in 1.5% alginate scaffold and cultured in chondrogenic media with and without transforming growth factor-β1 (TGF-β1 for 7 and 14 days. The genes expression of sex determining region Y-box 9 (SOX9, types II and X collagens was assessed by real-time polymerase chain reaction and the amount of aggrecan (AGC and type I collagen was measured by ELISA. Results: Our findings showed that the expression of essential cartilage markers, SOX9, type II collagen and AGC, in differentiated ADSCs at the concentration of 1 μg/ml CNTs in the presence of TGF-β1 were significantly increased in comparison with the control group (P < 0.001. Meanwhile, type X collagen expression and also type I collagen production were significantly decreased (P < 0.001. Conclusions: The results showed that utilized three-dimensional scaffold had a brilliant effect in promoting gene expression of chondrogenesis.

  6. Bone morphogenetic protein 2 promotes transforming growth factor β3-induced chondrogenesis of human osteoarthritic synovium-derived stem cells

    Institute of Scientific and Technical Information of China (English)

    RUI Yun-feng; DU Lin; WANG You; WANG Yang; LUI Pauline po-yee; TANG Ting-ting; CHAN Kai-ming; DAI Ke-rong

    2010-01-01

    Background Synovium-derived stem cells (SDSCs) with higher chondrogenic potential are attracting considerable attention as a cell source for cartilage regeneration. We investigated the effect of bone morphogenetic protein 2 (BMP-2) on transforming growth factor beta3 (TGF-β3)-induced chondrogenesis of SDSCs isolated from human osteoarthritic synovium in a pellet culture system. Methods The clonogenicity, stem cell marker expression and multi-differentiation potential of isolated SDSCs were determined by colony forming unit assay, flow cytometry and specific staining including alizarin red S, Oil red O and alcian blue staining, respectively. SDSCs pellet was cultured in chondrogenic medium with or without TGF-β3 or/and BMP-2. At day 21, the diameter and the weight of the pellets were measured. Chondrogenic differentiation of SDSCs was evaluated by Safranin O staining, immunohistochemical staining of collagen type Ⅱ, sulfated glycosaminoglycan (sGAG) synthesis and mRNA expression of collagen type Ⅱ, aggrecan, SOX9, link-protein, collagen type X and BMP receptor Ⅱ. Results Cells isolated under the optimized culturing density (104/60 cm2) showed clonogenicity and multi-differentiation potential. These cells were positive (>99%) for CD44, CD90, CD105 and negative (<10%) for CD34 and CD71. SDSCs differentiated to a chondrocytic phenotype in chondrogenic medium containing TGF-β3 with or without BMP-2. Safranin O staining of the extracellular matrix was positive and the expression of collagen type Ⅱ was detected. Cell pellets treated with TGF-β3 and BMP-2 were larger in diameter and weight, produced more sGAGs, and expressed higher levels of collagen type Ⅱ and other chondrogenic markers, except COL10A1, than medium with TGF-β3 alone. Conclusions SDSCs could be isolated from human osteoarthritic synovium. Supplementation with BMP-2 significantly promoted the in vitro TGF-β3-induced chondrogenic differentiation of SDSCs.

  7. Genipin-crosslinked cartilage-derived matrix as a scaffold for human adipose-derived stem cell chondrogenesis.

    Science.gov (United States)

    Cheng, Nai-Chen; Estes, Bradley T; Young, Tai-Horng; Guilak, Farshid

    2013-02-01

    Autologous cell-based tissue engineering using three-dimensional scaffolds holds much promise for the repair of cartilage defects. Previously, we reported on the development of a porous scaffold derived solely from native articular cartilage, which can induce human adipose-derived stem cells (ASCs) to differentiate into a chondrogenic phenotype without exogenous growth factors. However, this ASC-seeded cartilage-derived matrix (CDM) contracts over time in culture, which may limit certain clinical applications. The present study aimed to investigate the ability of chemical crosslinking using a natural biologic crosslinker, genipin, to prevent scaffold contraction while preserving the chondrogenic potential of CDM. CDM scaffolds were crosslinked in various genipin concentrations, seeded with ASCs, and then cultured for 4 weeks to evaluate the influence of chemical crosslinking on scaffold contraction and ASC chondrogenesis. At the highest crosslinking degree of 89%, most cells failed to attach to the scaffolds and resulted in poor formation of a new extracellular matrix. Scaffolds with a low crosslinking density of 4% experienced cell-mediated contraction similar to our original report on noncrosslinked CDM. Using a 0.05% genipin solution, a crosslinking degree of 50% was achieved, and the ASC-seeded constructs exhibited no significant contraction during the culture period. Moreover, expression of cartilage-specific genes, synthesis, and accumulation of cartilage-related macromolecules and the development of mechanical properties were comparable to the original CDM. These findings support the potential use of a moderately (i.e., approximately one-half of the available lysine or hydroxylysine residues being crosslinked) crosslinked CDM as a contraction-free biomaterial for cartilage tissue engineering.

  8. The effect of estrogen on the expression of cartilage-specific genes in the chondrogenesis process of adipose-derived stem cells

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    Farzaneh Sadeghi

    2015-01-01

    Full Text Available Background: During adolescence, sex hormones play an important role in regulating proliferation, differentiation, maturation, and the scheduled death of chondrocytes. Although some studies have reported the regulatory role of estrogen in the development and progression of cartilage, some of the mechanisms still remain unclear, including the role of estrogen in the expression of cartilage-specific genes in chondrogenesis process, which we cover in this study. Materials and Methods: In the present study, we used adipose-derived stem cells (ADSCs to differentiate into cartilage. Differentiated cartilage cells were used in the control (without estrogen E2 in the culture medium and experimental (with estrogen in the culture medium groups to evaluate the expression of type II collagen and aggrecan as chondrogenic genes markers, with -real-time polymerase chain reaction technique. Results: Our results indicated that estrogen leads to inhibition of type II collagen gene expression and reduction of aggrecan gene expression. Conclusion: Therefore, estrogen probably has negative effects on chondrogenesis process of ADSCs.

  9. Master regulator for chondrogenesis, Sox9, regulates transcriptional activation of the endoplasmic reticulum stress transducer BBF2H7/CREB3L2 in chondrocytes.

    Science.gov (United States)

    Hino, Kenta; Saito, Atsushi; Kido, Miori; Kanemoto, Soshi; Asada, Rie; Takai, Tomoko; Cui, Min; Cui, Xiang; Imaizumi, Kazunori

    2014-05-16

    The endoplasmic reticulum (ER) stress transducer, box B-binding factor 2 human homolog on chromosome 7 (BBF2H7), is a basic leucine zipper (bZIP) transmembrane transcription factor. This molecule is activated in response to ER stress during chondrogenesis. The activated BBF2H7 accelerates cartilage matrix protein secretion through the up-regulation of Sec23a, which is responsible for protein transport from the ER to the Golgi apparatus and is a target of BBF2H7. In the present study, we elucidated the mechanisms of the transcriptional activation of Bbf2h7 in chondrocytes. The transcription of Bbf2h7 is regulated by Sex determining region Y-related high-mobility group box 9 (Sox9), a critical factor for chondrocyte differentiation that facilitates the expression of one of the major cartilage matrix proteins Type II collagen (Col2), through binding to the Sox DNA-binding motif in the Bbf2h7 promoter. BBF2H7 is activated as a transcription factor in response to physiological ER stress caused by abundant synthesis of cartilage matrix proteins, and consequently regulates the secretion of cartilage matrix proteins. Taken together, our findings demonstrate novel regulatory mechanisms of Sox9 for controlling the secretion of cartilage matrix proteins through the activation of BBF2H7-Sec23a signaling during chondrogenesis.

  10. TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants and arrests downstream differentiation at an early stage of hypertrophy.

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    Nahoko Shintani

    Full Text Available BACKGROUND: Synovial explants furnish an in-situ population of mesenchymal stem cells for the repair of articular cartilage. Although bone morphogenetic protein 2 (BMP-2 induces the chondrogenesis of bovine synovial explants, the cartilage formed is neither homogeneously distributed nor of an exclusively hyaline type. Furthermore, the downstream differentiation of chondrocytes proceeds to the stage of terminal hypertrophy, which is inextricably coupled with undesired matrix mineralization. With a view to optimizing BMP-2-induced chondrogenesis, the modulating influences of fibroblast growth factor 2 (FGF-2 and transforming growth factor beta 1 (TGF-ß1 were investigated. METHODOLOGY/PRINCIPAL FINDINGS: Explants of bovine calf metacarpal synovium were exposed to BMP-2 (200 ng/ml for 4 (or 6 weeks. FGF-2 (10 ng/ml or TGF-ß1 (10 ng/ml was introduced at the onset of incubation and was present either during the first week of culturing alone or throughout its entire course. FGF-2 enhanced the BMP-2-induced increase in metachromatic staining for glycosaminoglycans (GAGs only when it was present during the first week of culturing alone. TGF-ß1 enhanced not only the BMP-2-induced increase in metachromasia (to a greater degree than FGF-2, but also the biochemically-assayed accumulation of GAGs, when it was present throughout the entire culturing period; in addition, it arrested the downstream differentiation of cells at an early stage of hypertrophy. These findings were corroborated by an analysis of the gene- and protein-expression levels of key cartilaginous markers and by an estimation of individual cell volume. CONCLUSIONS/SIGNIFICANCE: TGF-ß1 enhances the BMP-2-induced chondrogenesis of bovine synovial explants, improves the hyaline-like properties of the neocartilage, and arrests the downstream differentiation of cells at an early stage of hypertrophy. With the prospect of engineering a mature, truly articular type of cartilage in the context of

  11. 3, 3', 5-triiodo-L-thyronine Increases In Vitro Chondrogenesis of Mesenchymal Stem Cells From Human Umbilical Cord Stroma Through SRC2.

    Science.gov (United States)

    Fernández-Pernas, Pablo; Fafián-Labora, Juan; Lesende-Rodriguez, Iván; Mateos, Jesús; De la Fuente, Alexandre; Fuentes, Isaac; De Toro Santos, Javier; Blanco García, Fco; Arufe, María C

    2016-09-01

    Our group focuses on the study of mesenchymal stem cells (MSCs) from human umbilical cord stroma or Warthońs jelly and their directed differentiation toward chondrocyte-like cells capable of regenerating damaged cartilage when transplanted into an injured joint. This study aimed to determine whether lactogenic hormone prolactin (PRL) or 3, 3', 5-triiodo-L-thyronine (T3), the active thyroid hormone, modulates chondrogenesis in our in vitro model of directed chondrogenic differentiation, and whether Wnt signalling is involved in this modulation. MSCs from human umbilical cord stroma underwent directed differentiation toward chondrocyte-like cells by spheroid formation. The addition of T3 to the chondrogenic medium increased the expression of genes linked to chondrogenesis like collagen type 2, integrin alpha 10 beta 1, and Sox9 measured by quantitative real time polymerase chain reaction (qRT-PCR) analysis. Levels of collagen type 2 and aggrecane analyzed by immunohistochemistry, and staining by Safranin O were increased after 14 days in spheroid culture with T3 compared to those without T3 or only with PRL. B-catenin, Frizzled, and GSK-3β gene expressions were significantly higher in spheroids cultured with chondrogenic medium (CM) plus T3 compared to CM alone after 14 days in culture. The increase of chondrogenic differentiation was inhibited when the cells were treated with T3 plus ML151, an inhibitor of the T3 steroid receptor. This work demonstrates, for first time, that T3 promotes differentiation towards chondrocytes-like cells in our in vitro model, that this differentiation is mediated by steroid receptor co-activator 2 (SRC2) and does not induce hypertrophy. J. Cell. Biochem. 117: 2097-2108, 2016. © 2016 Wiley Periodicals, Inc.

  12. Chondrogenesis of periodontal ligament stem cells by transforming growth factor-β3 and bone morphogenetic protein-6 in a normal healthy impacted third molar

    Institute of Scientific and Technical Information of China (English)

    Sunyoung Choi; Tae-Jun Cho; Soon-Keun Kwon; Gene Lee; Jaejin Cho

    2013-01-01

    The periodontal ligament-derived mesenchymal stem cell is regarded as a source of adult stem cells due to its multipotency. However, the proof of chondrogenic potential of the cells is scarce. Therefore, we investigated the chondrogenic differentiation capacity of periodontal ligament derived mesenchymal stem cells induced by transforming growth factor (TGF)-β3 and bone morphogenetic protein (BMP)-6. After isolation of periodontal ligament stem cells (PDLSCs) from human periodontal ligament, the cells were cultured in Dulbecco's modified Eagle's medium (DMEM) with 20% fetal bovine serum (FBS). A mechanical force initiated chondrogenic differentiation of the cells. For chondrogenic differentiation, 10 μg ·L-1 TGF-β3 or 100 μg ·L-1 BMP-6 and the combination treating group for synergistic effect of the growth factors. We analyzed the PDLSCs by fluorescence-activated cell sorting and chondrogenesis were evaluated by glycosaminoglycans assay, histology, immunohistochemistry and genetic analysis. PDLSCs showed mesenchymal stem cell properties proved by FACS analysis. Glycosaminoglycans contents were increased 217% by TGF-β3 and 220% by BMP-6. The synergetic effect of TGF-β3 and BMP-6 were shown up to 281% compared to control. The combination treatment increased Sox9, aggrecan and collagen II expression compared with not only controls, but also TGF-β3 or BMP-6 single treatment dramatically. The histological analysis also indicated the chondrogenic differentiation of PDLSCs in our conditions. The results of the present study demonstrate the potential of the dental stem cell as a valuable cell source for chondrogenesis, which may be applicable for regeneration of cartilage and bone fracture in the field of cell therapy.

  13. Treatment of osteochondral lesions of the talus with autologous collagen-induced chondrogenesis: clinical and magnetic resonance evaluation at one-year follow-up

    Science.gov (United States)

    USUELLI, FEDERICO GIUSEPPE; GRASSI, MIRIAM; MANZI, LUIGI; GUARRELLA, VINCENZO; BOGA, MICHELE; DE GIROLAMO, LAURA

    2016-01-01

    Purpose the aim of this study is to report the clinical and imaging results recorded by a series of patients in whom osteochondral lesions of the talus (OLTs) were repaired using the autologous collagen-induced chondrogenesis (ACIC) technique with a completely arthroscopic approach. Methods nine patients (mean age 37.4±10 years) affected by OLTs (lesion size 2.1±0.9 cm2) were treated with the ACIC technique. The patients were evaluated clinically both preoperatively and at 12 months after surgery using the American Orthopaedic Foot and Ankle Society Ankle-Hindfoot Scale (AOFAS) and a visual analog scale (VAS). For morphological evaluation, the magnetic resonance observation of cartilage repair tissue (MOCART) score was used. Results the AOFAS score improved from 51.4±11.6 preoperatively to 71.8±20.6 postoperatively, while the VAS value decreased from 6.9±1.8 to 3.2±1.9. The mean MOCART score was 51.7±16.6 at 12 months of follow-up; these scores did not directly correlate with the clinical results. Conclusion use of the ACIC technique for arthroscopic repair of OLTs allowed satisfactory clinical results to be obtained in most of the patients as soon as one year after surgery, with no major complications or delayed revision surgery. ACIC is a valid and low-invasive surgical technique for the treatment of chondral and osteochondral defects of the talus. Level of evidence therapeutic case series, level IV. PMID:27602347

  14. Crystallization and X-ray diffraction analysis of the HMG domain of the chondrogenesis master regulator Sox9 in complex with a ChIP-Seq-identified DNA element.

    Science.gov (United States)

    Vivekanandan, Saravanan; Moovarkumudalvan, Balasubramanian; Lescar, Julien; Kolatkar, Prasanna R

    2015-11-01

    Sox9 is a fundamental sex-determining gene and the master regulator of chondrogenesis, and is involved in the development of various vital organs such as testes, kidney, heart and brain, and in skeletal development. Similar to other known Sox transcription factors, Sox9 recognizes and binds DNA with the consensus sequence C(T/A)TTG(T/A)(T/A) through the highly conserved HMG domain. Nonetheless, the molecular basis of the functional specificity of Sox9 in key developmental processes is still unclear. As an initial step towards a mechanistic understanding of Sox9 transcriptional regulation, the current work describes the details of the purification of the mouse Sox9 HMG domain (mSox9HMG), its crystallization in complex with a ChIP-Seq-identified FOXP2 promoter DNA element and the X-ray diffraction data analysis of this complex. The mSox9HMG-FOXP2 promoter DNA complex was crystallized by the hanging-drop vapour-diffusion method using 20% PEG 3350 in 200 mM sodium/potassium phosphate with 100 mM bis-tris propane at pH 8.5. The crystals diffracted to 2.7 Å resolution and the complex crystallized in the tetragonal space group P41212, with unit-cell parameters a = b = 99.49, c = 45.89 Å. Crystal-packing parameters revealed that asymmetric unit contained one mSox9HMG-FOXP2 promoter DNA complex with an estimated solvent content of 64%.

  15. Hedgehog信号调控骨髓间充质干细胞成软骨细胞分化:调控方式及其串话机制尚待研究%Hedgehog signal regulates the chondrogenesis from bone marrow mesenchymal stem cells:controlling methods and cross-talking relationship with other signals need further studies

    Institute of Scientific and Technical Information of China (English)

    刘宽; 吴兴

    2014-01-01

    背景:Hedgehog 信号通路在骨髓间充质干细胞成软骨细胞分化过程中发挥重要的调控作用,但具体的调控机制,以及与其他信号通路的串话机制仍需进一步的研究,是近来研究的热点。目的:介绍hedgehog信号在调控骨髓间充质干细胞成软骨分化过程中信号转导机制的研究现状与发展趋势,以及与其他信号通路的相互串话。方法:通过搜索CNKI,PubMed及Google Scholar等数据库,以“hedgehog,骨髓间充质干细胞,软骨形成,软骨细胞”和“hedgehog,IHH,SHH,bone marrow mesenchymal stem cel ,chondrogenesis, cartilage, chondrocyte”为检索词,查阅有关hedgehog信号与骨髓间充质干细胞分化相关的文献,最终共纳入36篇文献进行综述。结果与结论:骨髓间充质干细胞是目前公认的组织工程种子细胞来源,hedgehog信号通路是运动系统发育过程中重要的信号分子。Hedgehog信号蛋白IHH和SHH参与调控骨髓间充质干细胞的增殖与成软骨分化,以及软骨形成后的表型维持,且与其他信号通路发挥协同作用。然而,具体的调控方式以及与其他信号的串话机制,仍需进一步的研究,是这一领域未来研究的方向。%BACKGROUND:The hedgehog pathway has paid an important role in the progress of chondrogenesis from bone marrow mesenchymal stem cells. However, the definite signal transduction pathway and cross-talking relationship with other common signal pathways are stil poorly understood and the researches related to this field is to continue as a hotspot in the future study. OBJECTIVE:To investigate the research progress of hedgehog signal pathway on the regulation of the chondrogenesis from bone marrow mesenchymal stem cells and the relationship between hedgehog and other signal pathways in the process. METHODS:A computer-based online search in CNKI, PubMed and Google Scholar databases was performed using key words

  16. Ectopic chondrogenesis of bone marrow mesenchymal stem cells combined with platelet-rich fibrin in vivo%骨髓间充质干细胞复合富血小板纤维蛋白异位成软骨的实验研究

    Institute of Scientific and Technical Information of China (English)

    李轶杰; 陈慧; 刘鹏; 张旻; 陈永进

    2013-01-01

    AIM: To explore the ectopic chondrogenesis effects of double membrane transplant constructed by homologous rabbit platelet-rich fibrin (PRF) and bone marrow mesenchymal stem cell (BMSC) sheet in cartilage microenvironment in vivo. METHODS: BMSCs was obtained from New Zealand rabbits by density gradient centrifuga-tion. The cells were identified by flow cytomety, osteogenic and adipogenic induction assays and then used to form BMSC sheet cultured by standard medium with Vitamin C. PRF membrane was prepared by centrifuging the whole blood from the rabbit auricle arterial and then squeezing out the serum from the clot. The PRF membrane was co-cultured with BMSC sheet. Afterwards, the BMSCs/PRF compound membranes enwraping auricle cartilage fragments were implanted into the dorsal subcutaneous area of 15 nude mice. Only BMSC sheets enwraping auricle cartilage fragments were implanted into another 15 nude mice as the controls. 4, 8 and 12 weeks after implantation, cartilage formation was observed by HE staining and toluidine blue staining. IPP 6.0 was used for image analysis and SPSS 13.0 was used for data analysis. RESULTS: HE staining showed chondrogenesis in ectopic sites 2, 4 and 8 weeks after implantation in experimental group. In contrast, only a few chondrocytes were observed in control group at 8 weeks. IPP data analy- sis showed the average optical density value of experimental group increased gradually and was significantly higher than that of the control at the same time points (P <0. 05). CONCLUSION: The new autotransplant constructed by the BMSC sheet and PRF can provide an effective way for chondrogenesis.%目的:探讨富血小板纤维蛋白(platelet-rich fibrin,PRF)复合兔骨髓间充质干细胞膜片在软骨微环境下异位成软骨的可行性.方法:取3~4月龄雄性新西兰兔骨髓,体外分离、培养骨髓间充质干细胞;经来源鉴定后,取第3代细胞诱导培养形成细胞膜片;抽取同一只兔动脉血制备PRF后与

  17. Chondrogenesis of synovial mesenchymal stem cells co-cultured with chondrocytes on the three-dimensional scaffold%滑膜间充质干细胞与软骨细胞三维条件下混合培养向软骨细胞的分化

    Institute of Scientific and Technical Information of China (English)

    宁晓婷; 胡露露; 邵博; 龚忠诚; 刘慧; 凌彬; 克热木•阿巴斯; 林兆全; 杨萌; 尹小朋

    2014-01-01

    BACKGROUND:Articular chondrocytes with the ability of autocrine and paracrine can provide the growth factors and microenvironment for synovial mesenchymal stem cels differentiating into the chondrocyte. The three-dimensional scaffold could provide space for cels adhesion, proliferation and differentiation. OBJECTIVE: To study the ability of chondrogenesis by co-culturing synovial mesenchymal stem cels and chondrocytes under the three-dimensional condition. METHODS:The synovial membrane and articular cartilage were harvested from rat knee joint. The synovial mesenchymal stem cels and chondrocytes were obtained through the method of enzyme digestion. The passage 3 synovial mesenchymal stem cels and passage 2 chondrocytes were co-cultured in the chitosan/I colagen composite scaffolds at the ratio of 1:2. Then, the cels/scaffold composite was harvested to be examined morphologicaly, histologicaly and immunohistochemicaly after being cultured 21 days. The confocal laser was also employed to detect the cels distribution in the scaffold. RESULTS AND CONCLUSION: After being cultured 72 hours, it could be observed from the cels/scaffold composite examined through the scanning electron microscope that the cels adhered on the surface of the scaffold and extracelular matrix surrounding the cels was seen on the scaffold. After being cultured 21 days, it could be found through the confocal laser scanning that the cels were wel-distributed on the scaffold, and cels decreased gradualy. Type II colagen was positive in the extracelular matrix immunohistochamicaly. It suggested from this study that the synovial mesenchymal stem cels could be co-cultured with chondrocytes in the chitosan/I colagen composite scaffolds and have the ability of chondrogenesis differentiation.%背景:软骨细胞通过自分泌及旁分泌的作用可以为滑膜间充质干细胞向软骨细胞分化提供所需的生长因子及微环境,三维条件下更有利于细胞的黏附增殖与分化

  18. CHONDROGENESIS OF ADIPOSE DERIVED STEM CELLS INDUCED BY MISSHAPEN AURICULAR CHONDROCYTES FROM MICROTIA IN VITRO%残耳软骨细胞诱导脂肪来源干细胞体外软骨形成实验研究

    Institute of Scientific and Technical Information of China (English)

    蔡震; 潘博; 林琳; 蒋海越; 庄洪兴; 游晓波; 傅荣

    2013-01-01

    Objective To investigate the effects of the misshapen auricular chondrocytes from microtia in inducing chondrogenesis of human adipose derived stem cells (ADSCs) in vitro. Methods Human ADSCs at passage 3 and misshapen auricular chondrocytes at passage 2 were harvested and mixed at a ratio of 7 '. 3 as experimental group (group A, 1.0 ×106 mixed cells). Misshapen auricular chondrocytes or ADSCs at the same cell number served as control groups (groups B and C, respectively). All samples were incubated in the centrifuge tubes. At 28 days after incubation, the morphological examination was done and the wet weight was measured; the content of glycosaminoglycan (GAG) was detected by Alcian blue colorimetry; the expressions of collagen type Ⅱ and Aggrecan were determined with RT-PCR; and HE staining, toluidine blue staining, Safranin O staining of GAG, and collagen type Ⅱ immunohistochemical staining were used for histological and immunohistochemical observations. Results At 28 days after incubation, all specimens formed disc tissue that was translucent and white with smooth surface and good elasticity in groups A and B; the specimens shrank into yellow spherical tissue without elasticity in group C. The wet weight and GAG content of specimens in groups A and B were significantly higher than those in group C (P < 0.05), but no significant difference was found between groups A and B in the wet weight (t=1.820 3, P=0.068 7) and in GAG content (t= 1.861 4, P=0.062 7). In groups A and B, obvious expressions of collagen type Ⅱ and Aggrecan mRNA could be detected by RT-PCR, but no obvious expressions were observed in group C; the expressions in groups A and B were significantly higher than those in group C (P < 0.05), but no significant difference was found between groups A and B in collagen type Ⅱ mRNA expression (t=1.457 6, P=0.144 9) and Aggrecan mRNA expression (t=1.519 5, P=0.128 6). Mature cartilage lacunas and different degrees of dyeing for the extracellular

  19. Collagen Type II Enhances Chondrogenesis in Adipose Tissue-Derived Stem Cells by Affecting Cell Shape

    NARCIS (Netherlands)

    Lu, ZuFu; Doulabi, Behrouz Zandieh; Huang, ChunLing; Bank, Ruud A.; Helder, Marco N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

  20. Collagen type II enhances chondrogenesis in adipose tissue-derived stem cells by affecting cell shape

    NARCIS (Netherlands)

    Lu, Z.; Doulabi, B.Z.; Huang, C.; Bank, R.A.; Helder, M.N.

    2010-01-01

    Ideally, biomaterials have inductive properties, favoring specific lineage differentiation. For chondrogenic induction, these properties have been attributed to collagen type II. However, the underlying mechanisms are largely unknown. This study aimed to investigate whether collagen type II favors c

  1. Identification of SCAN domain zinc-finger gene ZNF449 as a novel factor of chondrogenesis.

    Directory of Open Access Journals (Sweden)

    Keita Okada

    Full Text Available Transcription factors SOX9, SOX5 and SOX6 are indispensable for generation and differentiation of chondrocytes. However, molecular mechanisms to induce the SOX genes are poorly understood. To address this issue, we previously determined the human embryonic enhancer of SOX6 by 5'RACE analysis, and identified the 46-bp core enhancer region (CES6. We initially performed yeast one-hybrid assay for screening other chondrogenic factors using CES6 as bait, and identified a zinc finger protein ZNF449. ZNF449 and Zfp449, a counterpart in mouse, transactivated enhancers or promoters of SOX6, SOX9 and COL2A1. Zfp449 was expressed in mesenchyme-derived tissues including cartilage, calvaria, muscle and tendon, as well as in other tissues including brain, lung and kidney. In limb cartilage of mouse embryo, Zfp449 protein was abundantly located in periarticular chondrocytes, and decreased in accordance with the differentiation. Zfp449 protein was also detected in articular cartilage of an adult mouse. During chondrogenic differentiation of human mesenchymal stem cells, ZNF449 was increased at an early stage, and its overexpression enhanced SOX9 and SOX6 only at the initial stage of the differentiation. We further generated Zfp449 knockout mice to examine the in vivo roles; however, no obvious abnormality was observed in skeletal development or articular cartilage homeostasis. ZNF449 may regulate chondrogenic differentiation from mesenchymal progenitor cells, although the underlying mechanisms are still unknown.

  2. Chondrogenesis of adipose-derived adult stem cells in a poly-lactide-co-glycolide scaffold

    DEFF Research Database (Denmark)

    Mehlhorn, Alexander T; Zwingmann, Jorn; Finkenzeller, Guenter

    2009-01-01

    combined immunodeficiency mice and harvested after 8 weeks. Chondrospecific messenger RNA (mRNA) expression was analyzed using reverse transcriptase polymerase chain reaction. Corresponding extracellular matrix (ECM) synthesis was demonstrated using immunohistochemical staining. Chondrospecific marker...... implantation in a subcutaneous pocket of nude mice. Human ASCs were seeded into PLGA (polylactic acid:polyglycolic acid = 90:10) scaffolds and cultured in transforming growth factor beta 1 (TGF-beta1)-containing medium for 3 weeks in vitro. Then specimens were implanted into a subcutaneous pocket of severe...

  3. Enhanced chondrogenesis of human nasal septum derived progenitors on nanofibrous scaffolds

    Energy Technology Data Exchange (ETDEWEB)

    Shafiee, Abbas [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Institute of Health and Biomedical Innovation, Queensland University of Technology (QUT), Brisbane, QLD (Australia); Seyedjafari, Ehsan [Department of Biotechnology, College of Science, University of Tehran, Tehran (Iran, Islamic Republic of); Sadat Taherzadeh, Elham [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); Dinarvand, Peyman [Stem Cell biology and Tissue Engineering Departments, Stem Cell Technology Research Center, Tehran (Iran, Islamic Republic of); The Edward A. Doisy Department of Biochemistry and Molecular Biology, Saint Louis University School of Medicine, Saint Louis, MO (United States); Soleimani, Masoud [Hematology Department, Faculty of Medical Science, Tarbiat Modares University, Tehran (Iran, Islamic Republic of); Ai, Jafar, E-mail: jafar_ai@tums.ac.ir [Department of Tissue Engineering, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of); Brain and Spinal Injury Research Center, Imam Hospital, Tehran University of Medical Sciences, Tehran (Iran, Islamic Republic of)

    2014-07-01

    Topographical cues can be exploited to regulate stem cell attachment, proliferation, differentiation and function in vitro and in vivo. In this study, we aimed to investigate the influence of different nanofibrous topographies on the chondrogenic differentiation potential of nasal septum derived progenitors (NSP) in vitro. Aligned and randomly oriented Ploy (L-lactide) (PLLA)/Polycaprolactone (PCL) hybrid scaffolds were fabricated via electrospinning. First, scaffolds were fully characterized, and then NSP were seeded on them to study their capacity to support stem cell attachment, proliferation and chondrogenic differentiation. Compared to randomly oriented nanofibers, aligned scaffolds showed a high degree of nanofiber alignment with much better tensile strength properties. Both scaffolds supported NSP adhesion, proliferation and chondrogenic differentiation. Despite the higher rate of cell proliferation on random scaffolds, a better chondrogenic differentiation was observed on aligned nanofibers as deduced from higher expression of chondrogenic markers such as collagen type II and aggrecan on aligned scaffolds. These findings demonstrate that electrospun constructs maintain NSP proliferation and differentiation, and that the aligned nanofibrous scaffolds can significantly enhance chondrogenic differentiation of nasal septum derived progenitors. - Highlights: • Electrospun nanofiber scaffolds with different topographies were fabricated. • Aligned nanofiber scaffolds had better tensile strength properties. • Nasal septum derived progenitors were cultured on nanofibrous scaffolds. • Both topographies support proliferation and chondrogenic differentiation. • Better chondrogenic differentiation was observed on aligned nanofibers.

  4. Catabolic factors and osteoarthritis-conditioned medium inhibit chondrogenesis of human mesenchymal stem cells.

    NARCIS (Netherlands)

    Heldens, G.T.H.; Blaney Davidson, E.N.; Vitters, E.L.; Schreurs, B.W.; Piek, E.; Berg, W.B. van den; Kraan, P.M. van der

    2012-01-01

    Articular cartilage has a very limited intrinsic repair capacity leading to progressive joint damage. Therapies involving tissue engineering depend on chondrogenic differentiation of progenitor cells. This chondrogenic differentiation will have to survive in a diseased joint. We postulate that catab

  5. Autologous Membrane Induced Chondrogenesis (AMIC) for the treatment of acetabular chondral defect

    Science.gov (United States)

    Fontana, Andrea

    2016-01-01

    Summary Background Acetabular chondral defect are very frequently associated to FAI. Treatment options are still questionable. Methods Between 2008 and 2014, 201 patients over 583 have been arthroscopically treated with the AMIC procedure for grade III and/or IV acetabular chondral lesions. Patients age was between 18 and 50 years; acetabular chondral lesion size was between 2 and 4 cm2; radiological Tönnis degree of osteoarthritis was ≤ 2. Results The mean follow up of the entire group of 201 patients was 5 years (from 8 to 2). Significant improvement, as measured by the mHHS, was observed at 6 months in comparison to preoperative levels (80.3 ± 8.3) (prepair medium-sized chondral defects on the acetabular side of the hip found during treatment of FAI and lead to long-term favourable outcomes. Level of evidence IV. PMID:28066742

  6. The role of transforming growth factor alpha in rat craniofacial development and chondrogenesis.

    OpenAIRE

    Huang, L; Solursh, M; Sandra, A

    1996-01-01

    To explore the possible role of transforming growth factor alpha (TGF-alpha) in craniofacial development, its expression in the craniofacial region of rat embryos from embryonic day (d) 9 to d 20 was examined by in situ hybridisation and immunostaining. The TGF-alpha transcripts were first detected in the neural fold of embryonic d 9 and 10 embryos. In the craniofacial region, the TGF-alpha transcripts were not detected until embryonic d 16 in mesenchyme surrounding the olfactory bulb, within...

  7. A combinatorial approach towards the design of nanofibrous scaffolds for chondrogenesis

    Science.gov (United States)

    Ahmed, Maqsood; Ramos, Tiago André Da Silva; Damanik, Febriyani; Quang Le, Bach; Wieringa, Paul; Bennink, Martin; van Blitterswijk, Clemens; de Boer, Jan; Moroni, Lorenzo

    2015-10-01

    The extracellular matrix (ECM) is a three-dimensional (3D) structure composed of proteinaceous fibres that provide physical and biological cues to direct cell behaviour. Here, we build a library of hybrid collagen-polymer fibrous scaffolds with nanoscale dimensions and screen them for their ability to grow chondrocytes for cartilage repair. Poly(lactic acid) and poly (lactic-co-glycolic acid) at two different monomer ratios (85:15 and 50:50) were incrementally blended with collagen. Physical properties (wettability and stiffness) of the scaffolds were characterized and related to biological performance (proliferation, ECM production, and gene expression) and structure-function relationships were developed. We found that soft scaffolds with an intermediate wettability composed of the highly biodegradable PLGA50:50 and collagen, in two ratios (40:60 and 60:40), were optimal for chondrogenic differentiation of ATDC5 cells as determined by increased ECM production and enhanced cartilage specific gene expression. Long-term cultures indicated a stable phenotype with minimal de-differentiation or hypertrophy. The combinatorial methodology applied herein is a promising approach for the design and development of scaffolds for regenerative medicine.

  8. Effect of Collagen Type I or Type II on Chondrogenesis by Cultured Human Articular Chondrocytes

    NARCIS (Netherlands)

    Rutgers, M.; Saris, D.B.F.; Vonk, L.A.; Rijen, van M.H.P.; Akrum, V.; Langeveld, D.; Boxtel, van A.; Dhert, W.J.A.; Creemers, L.B.

    2013-01-01

    Introduction: Current cartilage repair procedures using autologous chondrocytes rely on a variety of carriers for implantation. Collagen types I and II are frequently used and valuable properties of both were shown earlier in vitro, although a preference for either was not demonstrated. Recently, ho

  9. Differential expression pattern of extracellular matrix molecules during chondrogenesis of mesenchymal stem cells from bone marrow and adipose tissue

    DEFF Research Database (Denmark)

    Mehlhorn, A T; Niemeyer, P; Kaiser, S;

    2006-01-01

    Adipose-derived adult stem cells (ADASCs) or bone marrow-derived mesenchymal stem cells (BMSCs) are considered as alternative cell sources for cell-based cartilage repair due to their ability to produce cartilage-specific matrix. This article addresses the differential expression pattern of extra......Adipose-derived adult stem cells (ADASCs) or bone marrow-derived mesenchymal stem cells (BMSCs) are considered as alternative cell sources for cell-based cartilage repair due to their ability to produce cartilage-specific matrix. This article addresses the differential expression pattern...... mRNA was monitored via reverse transcriptase-polymerase chain reaction (RT-PCR). Corresponding ECM synthesis was demonstrated using immunohistochemistry. After chondroinduction, expression of collagen type II, type X, COMP and aggrecan mRNA was 3-15-fold higher than in ADASCs. The type IIA splicing...... form of alpha(1)-procollagen type II was expressed in both populations, and the type IIB splicing form was exclusively detected in BMSCs. In response to TGF-beta, collagen type II and X were secreted more strongly by BMSCs than by ADASCs. BMSCs express a more mature phenotype than ADASCs after...

  10. Effect of a synthetic folic acid analogue, 9-methyl-pteroylglutamic acid, on fetal chondrogenesis: ultrastructural observations.

    Science.gov (United States)

    Schmidt, R R; Slobodian, S A; Chepenik, K P; Cotler, J M

    1983-01-01

    Maternally administered folic acid antagonists (x-methyl-PGA and 9-methyl-PGA) are known to produce various skeletal malformations in the neonate. These defects are thought to be due in part to abnormal metabolism and/or deposition of various extracellular matrix components, i.e., collagen and glycosaminoglycans. Experimental reduction of glycosaminoglycan biosynthesis in vitro has been shown previously to alter the spatial orientation and normal pattern of collagen fibrillogenesis. Furthermore, dietary withdrawal of folic acid concomitant with maternal administration of 9-methyl-PGA has been shown to result in abnormal collagen, uronic acid, and hexosamine metabolism by fetal limbs. In the present study pregnant rats were exposed to a transitory folic acid deficiency from day 11 to 14 of gestation and fetal tibias (mid-diaphyseal region) were examined with the electron microscope on day 18 of gestation. Although we were unable to ascertain any aberrant patterns of fibrillogenesis and orientation with respect to collagen, this particular teratogenic regimen resulted in an altered pattern of chondrocyte development when observed at the ultrastructural level.

  11. Chondrogenesis of Precartilaginous Stem Cells in KLD-12 Self-assembling Peptide Nanofiber Scaffold Loading TGF-β3 Gene

    Institute of Scientific and Technical Information of China (English)

    YOU Hongbo; CHEN Anmin; SUN Kai; LIU Tie

    2011-01-01

    The effect of culture in KLD-12 self-assembling peptide nanofiber scaffold containing TGF-β3 gene on differentiation of precartilaginous stem cells(PSCs)into chondrocytes was studied.KLD-12 was synthesized by solid-state method.After TGF-β3 plasmid was loaded into KLD-12 self-assembling peptide nanofiber scaffold,DNA release ability was investigated.PSCs and hTGF-β3 gene were loaded into KLD-12 3-D scaffold,and MTT assay was performed to investigate the cell proliferation,and ELASA assay was used to investigate the expression of TGF-β3.Specific cartilage matrix was examined by quantitative real-time PCR,immunohistochemistry and Alcian Blue staining.Compared with control group,DNA synthesis level of PSCs reached the peak within 3 days when PSCs were cultured in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,and maintained this high level within 2 weeks.MTT results showed that the proliferation ability of experimental group was statistically higher than that in control group(P<0.05).Quantitative realtime PCR suggested that the percentage of TGF-β3 positive PSCs in experimental group was higher than that in control group(P<0.01).ELISA assay showed that the TGF-β3 protein level increased in supernatant of experimental group's PSCs,reached the peak after 72 h and then declined a little to the plateau phase.Compared with the control group,the specific gene of chondrocyte typical extracellular matrix significantly up-regulated (P<0.01).The results showed that PSCs differentiated into chondrocytes in self-assembling peptide nanofiber scaffold loading TGF-β3 plasmid,which provided a flesh approach to cartilage tissue engineering.

  12. Gpr177, a novel locus for bone-mineral-density and osteoporosis, regulates osteogenesis and chondrogenesis in skeletal development

    OpenAIRE

    Maruyama, Takamitsu; Jiang, Ming; Hsu, Wei

    2013-01-01

    Human genetic analysis has recently identified Gpr177 as a susceptibility locus for bone-mineral-density and osteoporosis. Determining the unknown function of this gene is therefore extremely important to further our knowledge base of skeletal development and disease. The protein encoded by Gpr177 exhibits an ability to modulate the trafficking of Wnt similar to the Drosophila Wls/Evi/Srt. Because of a critical role in Wnt regulation, Gpr177 might be required for several key steps of skeletog...

  13. The Role of Cyclic AMP and Its Relationship to Parathyroid Hormone Response in an In Vitro Model of Chondrogenesis.

    Science.gov (United States)

    1992-06-01

    meaningful and complete information. When a report is prepared in more than one volume , repeat the primary title, add volume number, and include subtitle...et al. (1990) found human PTH (hPTH) 1-34 inhibited ALPase activity and stimulated mitogenesis in mandibular condylar cartilage in vitro. Conversely...properties of cartilage cells in vivo. Copray et al. (1988) observed prominent differences in the response to PTH by rat secondary mandibular condylar and

  14. Designer Nodal/BMP2 Chimeras Mimic Nodal Signaling, Promote Chondrogenesis, and Reveal a BMP2-like Structure

    Science.gov (United States)

    Esquivies, Luis; Blackler, Alissa; Peran, Macarena; Rodriguez-Esteban, Concepcion; Izpisua Belmonte, Juan Carlos; Booker, Evan; Gray, Peter C.; Ahn, Chihoon; Kwiatkowski, Witek; Choe, Senyon

    2014-01-01

    Nodal, a member of the TGF-β superfamily, plays an important role in vertebrate and invertebrate early development. The biochemical study of Nodal and its signaling pathway has been a challenge, mainly because of difficulties in producing the protein in sufficient quantities. We have developed a library of stable, chemically refoldable Nodal/BMP2 chimeric ligands (NB2 library). Three chimeras, named NB250, NB260, and NB264, show Nodal-like signaling properties including dependence on the co-receptor Cripto and activation of the Smad2 pathway. NB250, like Nodal, alters heart looping during the establishment of embryonic left-right asymmetry, and both NB250 and NB260, as well as Nodal, induce chondrogenic differentiation of human adipose-derived stem cells. This Nodal-induced differentiation is shown to be more efficient than BPM2-induced differentiation. Interestingly, the crystal structure of NB250 shows a backbone scaffold similar to that of BMP2. Our results show that these chimeric ligands may have therapeutic implications in cartilage injuries. PMID:24311780

  15. Transcriptional signature of human adipose tissue-derived stem cells (hASCs) preconditioned for chondrogenesis in hypoxic conditions

    Energy Technology Data Exchange (ETDEWEB)

    Pilgaard, L.; Lund, P.; Duroux, M. [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark); Lockstone, H.; Taylor, J. [Bioinformatics and Statistical Genetics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN (United Kingdom); Emmersen, J.; Fink, T. [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark); Ragoussis, J. [Genomics, Wellcome Trust Centre for Human Genetics, Oxford University, Roosevelt Drive, Oxford, OX3 7BN (United Kingdom); Zachar, V., E-mail: vlaz@hst.aau.dk [Laboratory for Stem Cell Research, Aalborg University, Fredrik Bajers Vej 3B, 9220 Aalborg (Denmark)

    2009-07-01

    Hypoxia is an important factor involved in the control of stem cells. To obtain a better insight into the phenotypical changes brought about by hypoxic preconditioning prior to chondrogenic differentiation; we have investigated growth, colony-forming and chondrogenic capacity, and global transcriptional responses of six adipose tissue-derived stem cell lines expanded at oxygen concentrations ranging from ambient to 1%. The assessment of cell proliferation and colony-forming potential revealed that the hypoxic conditions corresponding to 1% oxygen played a major role. The chondrogenic inducibility, examined by high-density pellet model, however, did not improve on hypoxic preconditioning. While the microarray analysis revealed a distinctive inter-donor variability, the exposure to 1% hypoxia superseded the biological variability and produced a specific expression profile with 2581 significantly regulated genes and substantial functional enrichment in the pathways of cell proliferation and apoptosis. Additionally, exposure to 1% oxygen resulted in upregulation of factors related to angiogenesis and cell growth. In particular, leptin (LEP), the key regulator of body weight and food intake was found to be highly upregulated. In conclusion, the results of this investigation demonstrate the significance of donor demographics and the importance of further studies into the use of regulated oxygen tension as a tool for preparation of ASCs in order to exploit their full potential.

  16. Composite poly(l-lactic-acid)/silk fibroin scaffold prepared by electrospinning promotes chondrogenesis for cartilage tissue engineering.

    Science.gov (United States)

    Li, Zhengqiang; Liu, Peng; Yang, Ting; Sun, Ying; You, Qi; Li, Jiale; Wang, Zilin; Han, Bing

    2016-05-01

    Nanofibrous materials produced by electrospinning have attracted considerable attention from researchers in regenerative medicine. A combination of nanofibrous scaffold and chondrocytes is considered promising for repair of cartilage defect or damage. In the present study, we fabricated a poly(l-lactic-acid) (PLLA)/silk fibroin (SF) nanofibrous scaffold by electrospinning and evaluated its chondrogenic potential. The PLLA/SF nanofibers were characterized for diameter, surface wettability, swelling ratio, and tensile strength. Throughin vitroexperiments, PLLA/SF scaffold-chondrocyte interactions were investigated relative to the unmodified PLLA scaffold with regard to cellular adhesion, spreading, and proliferation by scanning electron microscopy and confocal laser scanning microscopy, and through analyses of DNA, sulfated glycosaminoglycan, and collagen. In addition, hematoxylin-eosin and Alcian blue-nuclear fast red staining were used to observe growth of chondrocytes, and secretion and distribution of cartilage-specific extracellular matrices in the scaffolds. Expressions of cartilage-related genes (collagen II, aggrecan, sox9, collagen I, and collagen X) were detected by real-time quantitative PCR. The PLLA/SF scaffold had better hydrophilicity, and could support chondrocytes adhesion and spreading more effectively than the unmodified PLLA scaffold. Chondrocytes secreted more cartilage-specific extracellular matrices and maintained their phenotype on the PLLA/SF scaffold. So it is concluded that the PLLA/SF scaffold is more conducive toin vitroformation of cartilage-like new tissues than the unmodified PLLA scaffold, and may be a promising material in cartilage tissue engineering.

  17. Effects of intermittent hydrostatic pressure magnitude on the chondrogenesis of MSCs without biochemical agents under 3D co-culture.

    Science.gov (United States)

    Jeong, Jae Young; Park, So Hee; Shin, Ji Won; Kang, Yun Gyeong; Han, Ki-Ho; Shin, Jung-Woog

    2012-11-01

    Without using biochemical agents, in this study, we sought to investigate the potential of controlling the differentiation of mesenchymal stem cells (MSCs) into a specific cell type through the use of 3D co-culturing and mechanical stimuli. MSCs and primary cultured chondrocytes were separately encapsulated into alginate beads, and the two types of beads were separated by a membrane. For the investigation a computer-controllable bioreactor was designed and used to engage intermittent hydrostatic pressure (IHP). Five different magnitudes (0.20, 0.10, 0.05, 0.02 MPa and no stimulation) of IHP were applied. The stimulation pattern was the same for all groups: 2 h/day for 7 days starting at 24 h after seeding; 2 and 15 min cycles of stimulating and resting, respectively. Biochemical (DNA and GAG contents), histological (Alcian blue), and RT-PCR (Col II, SOX9, AGC) analyses were performed on days 1, 5, 10, and 20. The results from these analyses showed that stimulation with higher magnitudes of IHP (≥0.10 MPa) were more effective on the proliferation and differentiation of co-cultured MSCs. Together, these data demonstrate the potential of using mechanical stimulation and co-culturing for the proliferation and differentiation of MSCs, even without biochemical agents.

  18. Diminished Chondrogenesis and Enhanced Osteoclastogenesis in Leptin-Deficient Diabetic Mice (ob/ob) Impair Pathologic, Trauma-Induced Heterotopic Ossification

    Science.gov (United States)

    Agarwal, Shailesh; Loder, Shawn; Li, John; Brownley, Cameron; Peterson, Jonathan R.; Oluwatobi, Eboda; Drake, James; Cholok, David; Ranganathan, Kavitha; Sung, Hsiao Hsin; Goulet, James; Li, Shuli

    2015-01-01

    Diabetic trauma patients exhibit delayed postsurgical wound, bony healing, and dysregulated bone development. However, the impact of diabetes on the pathologic development of ectopic bone or heterotopic ossification (HO) following trauma is unknown. In this study, we use leptin-deficient mice as a model for type 2 diabetes to understand how post-traumatic HO development may be affected by this disease process. Male leptin-deficient (ob/ob) or wild-type (C57BL/6 background) mice aged 6–8 weeks underwent 30% total body surface area burn injury with left hind limb Achilles tenotomy. Micro-CT (μCT) imaging showed significantly lower HO volumes in diabetic mice compared with wild-type controls (0.70 vs. 7.02 mm3, P < 0.01) 9 weeks after trauma. Ob/ob mice showed evidence of HO resorption between weeks 5 and 9. Quantitative real time PCR (qRT-PCR) demonstrated high Vegfa levels in ob/ob mice, which was followed by disorganized vessel growth at 7 weeks. We noted diminished chondrogenic gene expression (SOX9) and diminished cartilage formation at 5 days and 3 weeks, respectively. Tartrate-resistant acid phosphatase stain showed increased osteoclast presence in normal native bone and pathologic ectopic bone in ob/ob mice. Our findings suggest that early diminished HO in ob/ob mice is related to diminished chondrogenic differentiation, while later bone resorption is related to osteoclast presence. PMID:26413838

  19. A Simplified Method for the Aspiration of Bone Marrow from Patients Undergoing Hip and Knee Joint Replacement for Isolating Mesenchymal Stem Cells and In Vitro Chondrogenesis

    Directory of Open Access Journals (Sweden)

    Subhash C. Juneja

    2016-01-01

    Full Text Available The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA or total hip arthroplasty (THA may vary from an OR (operating room to OR based on the surgeon’s skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and characterization of MSCs from the aspirated bone marrow specimens and show that the bone marrow presented no clotting or exhibited minimal clotting. This represents an economical source and convenient source of MSCs from bone marrow for use in regenerative medicine. Also, we presented the detailed protocol and showed that the MSCs derived from such bone marrow specimens exhibited MSCs characteristics and generated micromass cartilages, the recipe for regenerative medicine for osteoarthritis. The protocols we presented can be used as standard operating procedures (SOPs by researchers and clinicians.

  20. A Simplified Method for the Aspiration of Bone Marrow from Patients Undergoing Hip and Knee Joint Replacement for Isolating Mesenchymal Stem Cells and In Vitro Chondrogenesis.

    Science.gov (United States)

    Juneja, Subhash C; Viswanathan, Sowmya; Ganguly, Milan; Veillette, Christian

    2016-01-01

    The procedure for aspiration of bone marrow from the femur of patients undergoing total knee arthroplasty (TKA) or total hip arthroplasty (THA) may vary from an OR (operating room) to OR based on the surgeon's skill and may lead to varied extent of clotting of the marrow and this, in turn, presents difficulty in the isolation of mesenchymal stem cells (MSCs) from such clotted bone marrow. We present a simple detailed protocol for aspirating bone marrow from such patients, isolation, and characterization of MSCs from the aspirated bone marrow specimens and show that the bone marrow presented no clotting or exhibited minimal clotting. This represents an economical source and convenient source of MSCs from bone marrow for use in regenerative medicine. Also, we presented the detailed protocol and showed that the MSCs derived from such bone marrow specimens exhibited MSCs characteristics and generated micromass cartilages, the recipe for regenerative medicine for osteoarthritis. The protocols we presented can be used as standard operating procedures (SOPs) by researchers and clinicians.

  1. Feasibility and limitations of the round robin test for assessment of in vitro chondrogenesis evaluation protocol in a tissue-engineered medical product.

    Science.gov (United States)

    Yokoi, Masako; Hattori, Koji; Narikawa, Koichi; Ohgushi, Hajime; Tadokoro, Mika; Hoshi, Kazuto; Takato, Tsuyoshi; Myoui, Akira; Nanno, Katsuhiko; Kato, Yukio; Kanawa, Masami; Sugawara, Katsura; Kobo, Tomoko; Ushida, Takashi

    2012-07-01

    Tissue-engineered medical products (TEMPs) should be evaluated before implantation. Therefore, it is indispensable to establish evaluation protocols in regenerative medicine. Whether or not such evaluation protocols are reasonable is generally verified through a 'round robin' test. However, the round robin test for TEMPs intrinsically includes a deficiency, because 'identical' specimens can not be prepared for TEMPs. The aim of the study was to assess the feasibility and limitations of the round robin test for TEMPs by using a prepared evaluation protocol. We adopted tissue-engineered cartilage constructs as delivered specimens and a protocol of measuring sGAG content as an evaluation protocol proposed to ISO TC150/SC7, which is an invasive, but usually applied, method, although non-invasive methods are keenly required in evaluating TEMPs. The results showed that: (a) the coefficient of variation (CV) of the measured sGAG contents in intralaboratory tests was ~5% at most; (b) the CV of sGAG content in the scheme where each participating laboratory measured different constructs was comparable with that in the scheme where each participating laboratory measured one half of a construct along with the organizing laboratory; (c) the CV caused by factors other than the specimen was ~15%, comparable to that in reproducible experiments in biomedical fields. Based on these results, the study concludes that a round robin test for a TEMP could be valuable, under the condition that the delivered TEMPs are sufficiently reproducible so that the CV of the measured values is < 5% in the organizing laboratory.

  2. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis.

    Science.gov (United States)

    Iohara, Koichiro; Zheng, Li; Ito, Masataka; Tomokiyo, Atsushi; Matsushita, Kenji; Nakashima, Misako

    2006-11-01

    Dental pulp has the potential to form dentin as a regenerative response to caries. This regeneration is mediated by stem/progenitor cells. Thus, stem cell therapy might be of potential utility in induction of reparative dentin. We isolated side population (SP) cells from dental pulp based on the exclusion of the DNA binding dye Hoechst 33342 by flow cytometry and compared its self-renewal capacities and multipotency with non-SP cells and primary pulp cells. The cumulative cell number of the SP cells was greater than the non-SP cells and primary pulp cells. Bmi1 was continuously expressed in SP cells, suggesting longer proliferative lifespan and self-renewal capacity of SP cells. Next, the maintenance of the multilineage differentiation potential of pulp SP cells was investigated. Expression of type II collagen and aggrecan confirmed chondrogenic conversion (30%) of SP cells. SP cells expressed peroxisome proliferator-activated receptor gamma and adaptor protein 2, showing adipogenic conversion. Expression of mRNA and proteins of neurofilament and neuromodulin confirmed neurogenic conversion (90%). These results demonstrate that pulp SP cells maintain multilineage differentiation potential. We further examined whether bone morphogenetic protein 2 (BMP2) could induce differentiation of pulp SP cells into odontoblasts. BMP2 stimulated the expression of dentin sialophosphoprotein (Dspp) and enamelysin in three-dimensional pellet cultures. Autogenous transplantation of the Bmp2-supplemented SP cells on the amputated pulp stimulated the reparative dentin formation. Thus, adult pulp contains SP cells, which are enriched for stem cell properties and useful for cell therapy with BMP2 for dentin regeneration.

  3. Ascorbate-dependent impact on cell-derived matrix in modulation of stiffness and rejuvenation of infrapatellar fat derived stem cells toward chondrogenesis.

    Science.gov (United States)

    Pizzute, Tyler; Zhang, Ying; He, Fan; Pei, Ming

    2016-08-10

    Developing an in vitro microenvironment using cell-derived decellularized extracellular matrix (dECM) is a promising approach to efficiently expand adult stem cells for cartilage engineering and regeneration. Ascorbic acid serves as a critical stimulus for cells to synthesize collagens, which constitute the major component of dECM. In this study, we hypothesized that optimization of ascorbate treatment would maximize the rejuvenation effect of dECM on expanded stem cells from human infrapatellar fat pad in both proliferation and chondrogenic differentiation. In the duration regimen study, we found that dECM without L-ascorbic acid phosphate (AA) treatment, exhibiting lower stiffness measured by atomic force microscopy, yielded expanded cells with higher proliferation capacity but lower chondrogenic potential when compared to those with varied durations of AA treatment. dECM with 250 µM of AA treatment for 10 d had better rejuvenation in chondrogenic capacity if the deposited cells were from passage 2 rather than passage 5, despite no significant difference in matrix stiffness. In the dose regimen study, we found that dECMs deposited by varied concentrations of AA yielded expanded cells with higher proliferation capacity despite lower expression levels of stem cell related surface markers. Compared to cells expanded on tissue culture polystyrene, those on dECM exhibited greater chondrogenic potential, particularly for the dECMs with 50 µM and 250 µM of AA treatment. With the supplementation of ethyl-3,4-dihydroxybenzoate (EDHB), an inhibitor targeting procollagen synthesis, the dECM with 50 µM of AA treatment exhibited a dramatic decrease in the rejuvenation effect of expanded cell chondrogenic potential at both mRNA and protein levels despite no significant difference in matrix stiffness. Defined AA treatments during matrix preparation will benefit dECM-mediated stem cell engineering and future treatments for cartilage defects.

  4. In vitro chondrogenesis of human adipose derived stem cells with microcarrier in RCSS%人脂肪干细胞结合微载体在生物反应器中向软骨细胞分化

    Institute of Scientific and Technical Information of China (English)

    康红军; 卢世璧; 许文静; 张莉; 孙明学; 袁玫; 赵斌; 郭全义

    2007-01-01

    [目的] 探索在旋转生物反应器内,应用微载体技术快速扩增并向软骨分化人脂肪干细胞.[方法] 将人脂肪干细胞结合Cytodex3微载体在旋转的生物反应器(RCSS)内进行动态培养,应用倒置显微镜和扫描电镜对微载体表面的脂肪干细胞进行动态观察,并对收获的脂肪干细胞进行Safranin-O、toluidine blue染色等组织化学染色及Ⅱ型胶原的免疫化学染色分析.[结果] 脂肪干细胞于24 h内贴附于Cytodex3微载体表面,细胞形态为短梭形,随时间的延长,贴附于微载体的细胞逐渐增多,到培养后期,细胞密度可达最初接种的19倍左右,在微载体上收获的细胞进行番红花O、阿利新蓝染色呈阳性,Ⅱ型胶原染色阳性,均强于对照组.[结论] 利用微载体细胞培养技术可简便快速地在体外扩增脂肪干细胞,并成功实现向软骨细胞分化.

  5. The Study on the Role of the Cell-cell Contact in Inducing Bone Marrow Stromal Cells Chondrogenesis%细胞间直接接触诱导BMSC软骨分化的初步研究

    Institute of Scientific and Technical Information of China (English)

    刘霞; 周广东; 刘伟; 曹谊林

    2010-01-01

    目的 探讨细胞间直接接触是否能够单独诱导骨髓基质干细胞(Bone Marrow Stromal Cells,BMSC)软骨分化.方法 体外培养扩增猪BMSC与关节软骨细胞,将1.0×106的细胞总量以2 000 r/min离心8 min,制成细胞团块,即Pellet培养.实验分为5组,实验组:经0.5%多聚甲醛固定的软骨细胞与BMSC以1∶1混合;对照组1:同等数量的未经多聚甲醛固定的软骨细胞与BMSC 1∶1共培养制成Pellet;对照组2:同等数量的单纯软骨细胞制成Pellet;对照组3:同等数量的单纯BMSC制成Pellet;对照组4:同等数量的单纯经多聚甲醛固定的软骨细胞制成Pellet.每3天换液一次,每组3例.培养4周后,以大体观察、组织学、免疫组织化学、RT-PCR等方法对Pellet进行全面评价.结果 培养4周后,实验组形成的Pellet,明显缩小,形状略不规则,颜色灰暗,柔软且没有弹性,组织学检测提示主要为纤维性成分及死细胞样结构,Safranin O染色及Ⅱ型胶原免疫组化均为阴性,RT-PCR检测未表达Ⅱ型胶原.对照组1和对照组2均形成圆盘状组织块,表面光滑,触之有一定弹性,组织学检测软骨陷窝形态规则,Safranin O染色及Ⅱ型胶原免疫组化均有阳性表达,RT-PCR检测Ⅱ型胶原高表达.对照组3的组织块明显收缩,呈褐色,无弹性.对照组4,细胞松散,不能形成Pellet.对照组3和4的各种软骨特异性相关检测均为阴性.结论 细胞间直接接触不能够单独诱导BMSC向软骨分化,不是软骨细胞与BMSC混合共培养中发挥诱导作用的主要因素.

  6. 大鼠多能成体祖细胞体外成软骨的诱导和鉴定%Chondrogenesis of Rats’Bone Marrow-derived Multipotent Adult Progenitor Cells in vitro and Related Identification

    Institute of Scientific and Technical Information of China (English)

    徐海艇; 潘骏; 李永领; 朱烈烈

    2016-01-01

    [Objective] To explore chondrogenic differentiation of rats bone marrow-derived multipotent adult progenitor cells(MAPCs) in experimental culture medium.[Methods] MAPCs were isolated from rats bone marrow and expanded to passage 2. The morphology of MAPCs was observed in vitro. Then the cell groups were cultured in chondrogenic media of H-DMEM containing TGF-β3, BMP-6, IGF-1, the control one was made natural differentiation with routine fluid 10% FB5, and dexamethasone forming cell pellet and assessed by Safranin O and collagen Ⅱ staining. And Alcian blue chromatomery of glycosaminoglycan(GAG) was used to determine the differentiation. [Results] MAPCs demonstrated fibroblast-like morphology and showed good proliferation ability. After chondrogenic induction for 4 weeks, the pellets formed by MAPCs had a transparent white cartilage-like morphology in chondrogenic medium while a less shape in control medium. Histologically, the group showed a homogeneous cartilage-like structure with strong safranin O staining and strong collagen 2 staining. Alcian blue staining showed the content of GAG formatting were higher than the control group(P<0.05), with difference of statistical meaning. [Conclusion]MAPCs have high chondrogenic potential and could be an ideal seed in cartilage tissue engineering.%[目的]探讨大鼠多能成体祖细胞(multipotent adult progenitor cells,MAPCs)在特定培养条件下向软骨细胞定向分化的能力。[方法]分离培养大鼠MAPCs,扩增至第2代,观察其形态学特点,并将其离心成团后分为2组,诱导组应用转化生长因子-β3(transforming growth factor-β3,TGF-β3)、骨形态发生蛋白-6(bone morphogenetic protein-6,BMP-6)、胰岛素样生长因子-1(insulin-like growth factors-1,IGF-1)及地塞米松等进行成软骨诱导分化,对照组用含10%胎牛血清(fetal bovine serum, FBS)的高糖DMEM培养液(dulbecco's modifiedeagle medium high-glucose, H-DMEM)常规培养液自然分化。以Safranin O和Ⅱ型胶原免疫组化染色等进行鉴定,阿尔新蓝比色法测定糖胺聚糖(glycosaminoglycan,GAG)的含量。[结果]体外定向诱导后MAPCs表现出软骨细胞的特性。诱导组形成了透明软骨样外观的细胞团,而对照组体积小、松散,Ⅱ型胶原免疫组化染色显示诱导组可见成熟软骨陷窝形成,Safranin O染色显示诱导组软骨特异性细胞外基质沉积,诱导组GAG的含量高于对照组,差异有统计学意义(P<0.05)。[结论]MAPCs具有良好的成软骨潜能,可以成为软骨组织工程良好的种子细胞来源。

  7. Induction of chondrogenesis and expression of superficial zone protein (SZP)/lubricin by mesenchymal progenitors in the infrapatellar fat pad of the knee joint treated with TGF-beta1 and BMP-7.

    Science.gov (United States)

    Lee, Sang Yang; Nakagawa, Toshiyuki; Reddi, A Hari

    2008-11-01

    Superficial zone protein (SZP) is a key mediator of boundary lubrication of articular cartilage in joints. In this investigation, we made the unexpected discovery that SZP was expressed in infrapatellar fat pad (IFP) from bovine knee. Quantitative analysis of secreted proteins in the medium of the IFP stromal cells demonstrated a significant stimulation by TGF-beta1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-beta1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-beta1 and BMP-7 to synthesize and secrete SZP. SZP mRNA was significantly up-regulated by chondrogenic induction for 21 days. These findings indicate that the stimulation of SZP expression by TGF-beta and BMP-7 may lead to functional improvement of damaged intraarticular tissues and that IFP progenitor cells may be a potential useful source for inducing superficial zone of articular cartilage by tissue engineering for regeneration of damaged articular cartilage due to osteoarthritis.

  8. Activity of protein kinase C during the differentiation of chick limb bud mesenchymal cells.

    Science.gov (United States)

    Sonn, J K; Solursh, M

    1993-07-01

    To investigate the relationship between protein kinase C (PKC) and chondrogenesis, PKC activity was assayed in cultures of stage 23/24 chick limb bud mesenchymal cells under various conditions. PKC activities of cytosolic and particulate fractions were low in 1 day cultured cells. As chondrogenesis proceeds, cytosolic PKC activity increased more than twofold, while that of the particulate fraction increased only slightly. Three days' treatment of cultures with phorbol-12-myristate-13-acetate (PMA, 5 x 10(-8) M) inhibited chondrogenesis judged by the accumulation of Alcian blue bound to the extracellular matrix and depressed PKC activity in cytosolic fraction. When cells were grown for 3 days in control medium after 3 days' treatment with PMA, chondrogenesis resumed and PKC activity recovered to normal values. PKC activity in cultures plated at low density (5 x 10(6) cells/ml) where chondrogenesis is reduced was as low as that in 1 day cultured cells plated at high density (2 x 10(7) cells/ml) or that in PMA treated cells. On the other hand, staurosporine promoted chondrogenesis without affecting PKC activity. Furthermore, reversal of PMA's inhibitory effect on chondrogenesis by staurosporine was not accompanied by recovery of PKC activity. These data indicate that increases in PKC activity is closely related to chondrogenesis and that PMA inhibits chondrogenesis by depressing PKC. However, staurosporine's enhancing effect on chondrogenesis is not related to PKC activity.

  9. Cartilage stem cells: regulation of differentiation.

    Science.gov (United States)

    Solursh, M

    1989-01-01

    The developing limb bud is a useful source of cartilage stem cells for studies on the regulation of chondrogenesis. In high density cultures these cells can progress through all stages of chondrogenesis to produce mineralized hypertrophic cartilage. If the cells are maintained in a spherical shape, single stem cells can progress through a similar sequence. The actin cytoskeleton is implicated in the regulation of chondrogenesis since conditions that favor its disruption promote chondrogenesis and conditions that favor actin assembly inhibit chondrogenesis. Since a number of extracellular matrix receptors mediate effects of the extracellular matrix on cytoskeletal organization and some of these receptors are developmentally regulated, it is proposed that matrix receptor expression plays a central role in the divergence of connective tissue cells during development.

  10. Delta-like 1/fetal antigen-1 (Dlk1/FA1) is a novel regulator of chondrogenic cell differentiation via inhibition of the Akt kinase-dependent pathway

    DEFF Research Database (Denmark)

    Chen, Li; Qanie, Diyako; Jafari, Abbas

    2011-01-01

    Delta-like 1 (Dlk1, also known as fetal antigen-1, FA1) is a member of Notch/Delta family that inhibits adipocyte and osteoblast differentiation; however, its role in chondrogenesis is still not clear. Thus, we overexpressed Dlk1/FA1 in mouse embryonic ATDC5 cells and tested its effects...... interaction of Dlk1/FA1 and fibronectin in chondrogenic cells. Our results identify Dlk1/FA1 as a novel regulator of chondrogenesis and suggest Dlk1/FA1 acts as an inhibitor of the PI3K/Akt pathways that leads to its inhibitory effects on chondrogenesis....

  11. 力学刺激促进软骨细胞与骨髓基质干细胞共培养体外软骨分化%Mechanical stimulation to promote in vitro chondrogenesis by Co-culture of bone marrow stromal cells and chondrocytes

    Institute of Scientific and Technical Information of China (English)

    田甜; 章庆国

    2009-01-01

    目的:研究不同的应力刺激对软骨细胞与骨髓基质干细胞(BMSCs)共培养体外构建组织工程化软骨的影响.方法:分离、培养、扩传兔MSCs及软骨细胞,二者按7:3比例混和,以5.0 x 107/ml的细胞密度接种于聚羟基乙酸(PGA)支架上,一周后根据不同的施加力分为4组:离心组、摇床组、搅拌组,静止培养作为对照组.6周后取材行相关检测.结果:三受力组形成的细胞材料复合物基本保持原来的体积与外形.HE染色结果显示大量成熟软骨陷窝形成,细胞外基质沉积均匀;Safranin-0及甲苯胺兰染色显示有大量的GAG形成,免疫组化检测Ⅱ型胶原表达强阳性.三受力组标本组织湿重、体积,GAG含量等指标均优于对照组.结论:力学刺激有利于促进少量软骨细胞与BMSCs共培养体外软骨分化;并在三维支架材料上构建组织工程化软骨.

  12. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Directory of Open Access Journals (Sweden)

    Kazumasa Mitogawa

    Full Text Available Axolotls (Ambystoma mexicanum can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  13. Isolation of mesenchymal stem cells from equine umbilical cord blood

    DEFF Research Database (Denmark)

    Koch, Thomas Gadegaard; Heerkens, Tammy; Thomsen, Preben Dybdahl;

    2007-01-01

    staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis. Conclusion: We here report...

  14. Comparative Analysis of Cartilage Marker Gene Expression Patterns during Axolotl and Xenopus Limb Regeneration.

    Science.gov (United States)

    Mitogawa, Kazumasa; Makanae, Aki; Satoh, Ayano; Satoh, Akira

    2015-01-01

    Axolotls (Ambystoma mexicanum) can completely regenerate lost limbs, whereas Xenopus laevis frogs cannot. During limb regeneration, a blastema is first formed at the amputation plane. It is thought that this regeneration blastema forms a limb by mechanisms similar to those of a developing embryonic limb bud. Furthermore, Xenopus laevis frogs can form a blastema after amputation; however, the blastema results in a terminal cone-shaped cartilaginous structure called a "spike." The causes of this patterning defect in Xenopus frog limb regeneration were explored. We hypothesized that differences in chondrogenesis may underlie the patterning defect. Thus, we focused on chondrogenesis. Chondrogenesis marker genes, type I and type II collagen, were compared in regenerative and nonregenerative environments. There were marked differences between axolotls and Xenopus in the expression pattern of these chondrogenesis-associated genes. The relative deficit in the chondrogenic capacity of Xenopus blastema cells may account for the absence of total limb regenerative capacity.

  15. Pressureless mechanical induction of stem cell differentiation is dose and frequency dependent.

    Directory of Open Access Journals (Sweden)

    Roland Fuhrer

    Full Text Available Movement is a key characteristic of higher organisms. During mammalian embryogenesis fetal movements have been found critical to normal tissue development. On the single cell level, however, our current understanding of stem cell differentiation concentrates on inducing factors through cytokine mediated biochemical signaling. In this study, human mesenchymal stem cells and chondrogenesis were investigated as representative examples. We show that pressureless, soft mechanical stimulation precipitated by the cyclic deformation of soft, magnetic hydrogel scaffolds with an external magnetic field, can induce chondrogenesis in mesenchymal stem cells without any additional chondrogenesis transcription factors (TGF-β1 and dexamethasone. A systematic study on the role of movement frequency revealed a classical dose-response relationship for human mesenchymal stem cells differentiation towards cartilage using mere mechanical stimulation. This effect could even be synergistically amplified when exogenous chondrogenic factors and movement were combined.

  16. Subglottic stenosis in Shwachman-Diamond syndrome - is there a link?

    Science.gov (United States)

    Keereweer, S; Appel, I M; Hoeve, L J

    2012-10-01

    Clinically relevant cartilaginous subglottic stenosis was found in 2 patients with Shwachman-Diamond syndrome (SDS) for which tracheotomy was required in one case. Considering the pathogenesis of SDS, including deficient chondrogenesis, we hypothesise that subglottic stenosis may be a rare symptom of SDS. Otorhinolaryngologist and paediatricians should be aware of the risk of airway pathology in patients with SDS.

  17. Thyroid development in zebrafish lacking Taz

    NARCIS (Netherlands)

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, S.; Zannini, Mariastella; Sordino, Paolo

    2015-01-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated k

  18. Sliding Hydrogels with Mobile Molecular Ligands and Crosslinks as 3D Stem Cell Niche.

    Science.gov (United States)

    Tong, Xinming; Yang, Fan

    2016-09-01

    The development of a sliding hydrogel with mobile crosslinks and biochemical ligands as a 3D stem cell niche is reported. The molecular mobility of this sliding hydrogel allows stem cells to reorganize the surrounding ligands and change their morphology in 3D. Without changing matrix stiffness, sliding hydrogels support efficient stem cell differentiation toward multiple lineages including adipogenesis, chondrogenesis, and osteogenesis.

  19. The pericellular environment regulates cytoskeletal development and the differentiation of mesenchymal stem cells and determines their response to hydrostatic pressure

    Directory of Open Access Journals (Sweden)

    AJ Steward

    2013-02-01

    Full Text Available The objective of this study was to examine the interplay between matrix stiffness and hydrostatic pressure (HP in regulating chondrogenesis of mesenchymal stem cells (MSCs and to further elucidate the mechanotransductive roles of integrins and the cytoskeleton. MSCs were seeded into 1 %, 2 % or 4 % agarose hydrogels and exposed to cyclic hydrostatic pressure. In a permissive media, the stiffer hydrogels supported an osteogenic phenotype, with little evidence of chondrogenesis observed regardless of the matrix stiffness. In a chondrogenic media, the stiffer gels suppressed cartilage matrix production and gene expression, with the addition of RGDS (an integrin blocker found to return matrix synthesis to similar levels as in the softer gels. Vinculin, actin and vimentin organisation all adapted within stiffer hydrogels, with the addition of RGDS again preventing these changes. While the stiffer gels inhibited chondrogenesis, they enhanced mechanotransduction of HP. RGDS suppressed the mechanotransduction of HP, suggesting a role for integrin binding as a regulator of both matrix stiffness and HP. Intermediate filaments also appear to play a role in the mechanotransduction of HP, as only vimentin organisation adapted in response to this mechanical stimulus. To conclude, the results of this study demonstrate that matrix density and/or stiffness modulates the development of the pericellular matrix and consequently integrin binding and cytoskeletal structure. The study further suggests that physiological cues such as HP enhance chondrogenesis of MSCs as the pericellular environment matures and the cytoskeleton adapts, and points to a novel role for vimentin in the transduction of HP.

  20. Particulate cartilage under bioreactor-induced compression and shear

    DEFF Research Database (Denmark)

    Wang, Ning; Grad, Sibylle; Stoddart, Martin J

    2014-01-01

    PURPOSE: Our aim was to explore the effect of varying in vitro culture conditions on general chondrogenesis of minced cartilage (MC) fragments. METHODS: Minced, fibrin-associated, bovine articular cartilage fragments were cultured in vitro within polyurethane scaffold rings. Constructs were...

  1. microRNA-140 targets RALA and regulates chondrogenic differentiation of human mesenchymal stem cells by translational enhancement of SOX9 and ACAN.

    Science.gov (United States)

    Karlsen, Tommy A; Jakobsen, Rune B; Mikkelsen, Tarjei S; Brinchmann, Jan E

    2014-02-01

    Lesions of articular cartilage do not heal spontaneously. One treatment strategy would be to make cartilage in the laboratory by directed chondrogenic differentiation of mesenchymal stem cells (MSCs). To promote our understanding of the molecular control of chondrogenesis, we have compared the changes in microRNAs (miRNAs) during in vitro chondrogenesis of MSCs with those observed in uncultured and dedifferentiated articular chondrocytes (ACs). Several miRNAs showed a reciprocal relationship during the differentiation of MSCs and dedifferentiation of ACs. miR-140-5p and miR-140-3p changed the most during in vitro chondrogenesis, they were the miRNAs most highly expressed in tissue-engineered chondrocytes, and they were also among the miRNAs most highly expressed in uncultured ACs. There was a 57% overlap for the 100 most highly expressed miRNAs in differentiated MSCs and uncultured ACs, but for other miRNAs, the expression pattern was quite different. We transiently and stably inhibited and overexpressed miR-140-5p and miR-140-3p in differentiating MSCs and dedifferentiating ACs, respectively, to describe global effects and identify and validate new targets. Surprisingly, SOX9 and aggrecan proteins were found to be downregulated in anti-miR-140 transduced differentiating MSCs despite unchanged mRNA levels. This suggests that miR-140 stimulates in vitro chondrogenesis by the upregulation of these molecules at the protein level. RALA, a small GTPase, was identified as a miR-140 target and knockdown experiments showed that RALA regulated SOX9 at the protein level. These observations shed new light on the effect of miR-140 for chondrogenesis in vitro and in vivo.

  2. Chondrogenic differentiation of adipose-derived stromal cells in combinatorial hydrogels containing cartilage matrix proteins with decoupled mechanical stiffness.

    Science.gov (United States)

    Wang, Tianyi; Lai, Janice H; Han, Li-Hsin; Tong, Xinming; Yang, Fan

    2014-08-01

    Adipose-derived stromal cells (ADSCs) are attractive autologous cell sources for cartilage repair given their relative abundance and ease of isolation. Previous studies have demonstrated the potential of extracellular matrix (ECM) molecules as three-dimensional (3D) scaffolds for promoting chondrogenesis. However, few studies have compared the effects of varying types or doses of ECM molecules on chondrogenesis of ADSCs in 3D. Furthermore, increasing ECM molecule concentrations often result in simultaneous changes in the matrix stiffness, which makes it difficult to elucidate the relative contribution of biochemical cues or matrix stiffness on stem cell fate. Here we report the development of an ECM-containing hydrogel platform with largely decoupled biochemical and mechanical cues by modulating the degree of methacrylation of ECM molecules. Specifically, we incorporated three types of ECM molecules that are commonly found in the cartilage matrix, including chondroitin sulfate (CS), hyaluronic acid (HA), and heparan sulfate (HS). To elucidate the effects of interactive biochemical and mechanical signaling on chondrogenesis, ADSCs were encapsulated in 39 combinatorial hydrogel compositions with independently tunable ECM types (CS, HA, and HS), concentrations (0.5%, 1.25%, 2.5%, and 5% [w/v]), and matrix stiffness (3, 30, and 90 kPa). Our results show that the effect of ECM composition on chondrogenesis is dependent on the matrix stiffness of hydrogels, suggesting that matrix stiffness and biochemical cues interact in a nonlinear manner to regulate chondrogenesis of ADSCs in 3D. In soft hydrogels (~3 kPa), increasing HA concentrations resulted in substantial upregulation of aggrecan and collagen type II expression in a dose-dependent manner. This trend was reversed in HA-containing hydrogels with higher stiffness (~90 kPa). The platform reported herein could provide a useful tool for elucidating how ECM biochemical cues and matrix stiffness interact together to

  3. Microtiter micromass cultures of limb-bud mesenchymal cells.

    Science.gov (United States)

    Paulsen, D F; Solursh, M

    1988-02-01

    A method is described for growing high-density micromass cultures of chick and mouse limb mesenchyme cells in 96-well microtiter plates (microT microM cultures). Rapid quantitative estimates of chondrogenic expression were obtained by automated spectrophotometric analysis of Alcian-blue-stained cartilage matrix extracts performed in the wells in which the cells had been grown. Quantitative estimates of myogenic expression were obtained similarly using anti-sarcomere myosin monoclonal antibody and modified ELISA techniques. This microT microM-ELISA method may be adapted for use with other antigens for which specific antibodies are available. These methods were used to compare cartilage and muscle differentiation in 1 to 4 d microT microM cultures grown in serum-containing (SCM) and defined (DM) media. The DM contains minimal additives (insulin, hydrocortisone, and in some cases, ascorbate or transferrin) and supports both chondrogenesis and myogenesis. The colorimetric analyses agree well with the morphologic appraisal of chondrogenesis and myogenesis. Similar numbers of cartilage nodules formed in all cultures, but in DM the nodules failed to enlarge; explaining the reduced matrix synthesis in DM as compared with SCM, and suggesting that nodule enlargement is a discrete, serum-dependent step. Studies of selected additives to DM show that transferrin enhances myogenesis, ascorbic acid enhances chondrogenesis, and retinoic acid inhibits chondrogenesis. Together, the microT microM system, in situ colorimetric assays of chondrogenesis and myogenesis, and DM will allow rapid prescreening of teratogens and screening of various bioactive compounds (e.g., hormones, growth factors, vitamins, adhesion factors) for effects on limb mesenchymal cell differentiation.

  4. Dry arthroscopy with a retraction system for matrix-aided cartilage repair of patellar lesions.

    Science.gov (United States)

    Sadlik, Boguslaw; Wiewiorski, Martin

    2014-02-01

    Several commercially available cartilage repair techniques use a natural or synthetic matrix to aid cartilage regeneration (e.g., autologous matrix-induced chondrogenesis or matrix-induced cartilage implantation). However, the use of matrix-aided techniques during conventional knee joint arthroscopy under continuous irrigation is challenging. Insertion and fixation of the matrix can be complicated by the presence of fluid and the confined patellofemoral joint space with limited access to the lesion. To overcome these issues, we developed a novel arthroscopic approach for matrix-aided cartilage repair of patellar lesions. This technical note describes the use of dry arthroscopy assisted by a minimally invasive retraction system. An autologous matrix-induced chondrogenesis procedure is used to illustrate this novel approach.

  5. Expression of chondrogenic potential of mouse trunk neural crest cells by FGF2 treatment.

    Science.gov (United States)

    Ido, Atsushi; Ito, Kazuo

    2006-02-01

    There is a significant difference between the developmental patterns of cranial and trunk neural crest cells in the amniote. Thus, whereas cranial neural crest cells generate bone and cartilage, trunk neural crest cells do not contribute to skeletal derivatives. We examined whether mouse trunk neural crest cells can undergo chondrogenesis to analyze how the difference between the developmental patterns of cranial and trunk neural crest cells arises. Our present data demonstrate that mouse trunk neural crest cells have chondrogenic potential and that fibroblast growth factor (FGF) 2 is an inducing factor for their chondrogenesis in vitro. FGF2 altered the expression patterns of Hox9 genes and Id2, a cranial neural crest cell marker. These results suggest that environmental cues may play essential roles in generating the difference between developmental patterns of cranial and trunk neural crest cells.

  6. The ECM-Cell Interaction of Cartilage Extracellular Matrix on Chondrocytes

    Directory of Open Access Journals (Sweden)

    Yue Gao

    2014-01-01

    Full Text Available Cartilage extracellular matrix (ECM is composed primarily of the network type II collagen (COLII and an interlocking mesh of fibrous proteins and proteoglycans (PGs, hyaluronic acid (HA, and chondroitin sulfate (CS. Articular cartilage ECM plays a crucial role in regulating chondrocyte metabolism and functions, such as organized cytoskeleton through integrin-mediated signaling via cell-matrix interaction. Cell signaling through integrins regulates several chondrocyte functions, including differentiation, metabolism, matrix remodeling, responses to mechanical stimulation, and cell survival. The major signaling pathways that regulate chondrogenesis have been identified as wnt signal, nitric oxide (NO signal, protein kinase C (PKC, and retinoic acid (RA signal. Integrins are a large family of molecules that are central regulators in multicellular biology. They orchestrate cell-cell and cell-matrix adhesive interactions from embryonic development to mature tissue function. In this review, we emphasize the signaling molecule effect and the biomechanics effect of cartilage ECM on chondrogenesis.

  7. Dry Arthroscopy With a Retraction System for Matrix-Aided Cartilage Repair of Patellar Lesions

    OpenAIRE

    Sadlik, Boguslaw; Wiewiorski, Martin

    2014-01-01

    Several commercially available cartilage repair techniques use a natural or synthetic matrix to aid cartilage regeneration (e.g., autologous matrix–induced chondrogenesis or matrix-induced cartilage implantation). However, the use of matrix-aided techniques during conventional knee joint arthroscopy under continuous irrigation is challenging. Insertion and fixation of the matrix can be complicated by the presence of fluid and the confined patellofemoral joint space with limited access to the ...

  8. Electric Field Stimulation Enhances Healing of Post-Traumatic Osteoarthritic Cartilage

    Science.gov (United States)

    2015-10-01

    RESPONSIBLE PERSON USAMRMC a. REPORT U b. ABSTRACT U c. THIS PAGE U UU 19b. TELEPHONE NUMBER (include area code) Standard Form 298 (Rev. 8-98...in clumps such that there are areas of 120% the density of native cartilage, and some areas with practically 0% of the native density), b) finding...chondrogenesis are markedly different from those that act on juvenile bovine cells. 5. Products Nothing to report. 6. Participants & Other

  9. The trans-well coculture of human synovial mesenchymal stem cells with chondrocytes leads to self-organization, chondrogenic differentiation, and secretion of TGFβ

    DEFF Research Database (Denmark)

    Kubosch, Eva Johanna; Heidt, Emanuel; Bernstein, Anke

    2016-01-01

    BACKGROUND: Synovial mesenchymal stem cells (SMSC) possess a high chondrogenic differentiation potential, which possibly supports natural and surgically induced healing of cartilage lesions. We hypothesized enhanced chondrogenesis of SMSC caused by the vicinity of chondrocytes (CHDR). METHODS....... RESULTS: After 7 days, phase-contrast microscopy revealed cell aggregation of SMSC in coculture with CHDR. Afterwards, cells formed spheres and lost adherence. However, this phenomenon was not observed when culturing SMSC alone. Fluorescence labeling showed concurrent collagen type II expression. Addition...

  10. FOXO3A regulation by miRNA-29a Controls chondrogenic differentiation of mesenchymal stem cells and cartilage formation.

    Science.gov (United States)

    Guérit, David; Brondello, Jean-Marc; Chuchana, Paul; Philipot, Didier; Toupet, Karine; Bony, Claire; Jorgensen, Christian; Noël, Danièle

    2014-06-01

    Skeletal development and cartilage formation require stringent regulation of gene expression for mesenchymal stem cells (MSCs) to progress through stages of differentiation. Since microRNAs (miRNAs) regulate biological processes, the objective of the present study was to identify novel miRNAs involved in the modulation of chondrogenesis. We performed miRNA profiling and identify miR-29a as being one of the most down-regulated miRNAs during the chondrogenesis. Using chromatin immunoprecipitation, we showed that SOX9 down-regulates its transcription. Moreover, the over-expression of miR-29a strongly inhibited the expression of chondrocyte-specific markers during in vitro chondrogenic differentiation of MSCs. We identified FOXO3A as a direct target of miR-29a and showed a down- and up-regulation of FOXO3a protein levels after transfection of, respectively, premiR- and antagomiR-29a oligonucleotides. Finally, we showed that using the siRNA or premiR approach, chondrogenic differentiation was inhibited to a similar extent. Together, we demonstrate that the down-regulation of miR-29a, concomitantly with FOXO3A up-regulation, is essential for the differentiation of MSCs into chondrocytes and in vivo cartilage/bone formation. The delivery of miRNAs that modulate MSC chondrogenesis may be applicable for cartilage regeneration and deserves further investigation.

  11. Significance of soluble growth factors in the chondrogenic response of human umbilical cord matrix stem cells in a porous three dimensional scaffold

    Directory of Open Access Journals (Sweden)

    RS Nirmal

    2013-11-01

    Full Text Available Stem cell based tissue engineering has emerged as a promising strategy for articular cartilage regeneration. Foetal derived mesenchymal stem cells (MSCs with their ease of availability, pluripotency and high expansion potential have been demonstrated to be an attractive cell source over adult MSCs. However, there is a need for optimisation of chondrogenic signals to direct the differentiation of these multipotent MSCs to chondrogenic lineage. In this study we have demonstrated the in vitro chondrogenesis of human umbilical cord matrix MSCs in three dimensional PVA-PCL (polyvinyl alcohol-polycaprolactone scaffolds in the presence of the individual growth factors TGFβ1, TGFβ3, IGF, BMP2 and their combination with BMP2. Gene expression, histology and immunohistology were evaluated after 28 d culture. The induced cells showed the feature of chondrocytes in their morphology and expression of typical chondrogenic extracellular matrix molecules. Moreover, the real-time PCR assay has shown the expression of gene markers of chondrogenesis, SOX9, collagen type II and aggrecan. The expression of collagen type I and collagen type X was also evaluated. This study has demonstrated the successful chondrogenic induction of human umbilical cord MSCs in 3D scaffolds. Interestingly, the growth factor combination of TGF-β3 and BMP-2 was found to be more effective for chondrogenesis as shown by the real-time PCR studies. The findings of this study suggest the importance of using growth factor combinations for successful chondrogenic differentiation of umbilical cord MSCs.

  12. Simultaneous monitoring of intracellular ATP and oxygen levels in chondrogenic differentiation using a dual-color bioluminescence reporter.

    Science.gov (United States)

    Kwon, Hyuck Joon; Ohmiya, Yoshihiro; Yasuda, Kazunori

    2014-12-01

    A number of assay methods which measure cellular metabolic activity have only measured intracellular ATP levels because it has been speculated that ATP production and oxygen consumption are obligatorily coupled to each other under normal conditions. However, there exist many cases in which ATP production and oxygen consumption are uncoupled. Therefore, measurement of only intracellular ATP levels has a limit for understanding the overall metabolic states during various cellular functions. Here, we report a novel system for simultaneously monitoring intracellular ATP and oxygen levels using a red-emitting Phrixothrix hirtus luciferase (PxRe) and a blue-emitting Renilla luciferase (Rluc). Using this system, we monitored the dynamic changes in both intracellular ATP and oxygen levels during chondrogenesis. We found that the oxygen level oscillated at twice the frequency of ATP in chondrogenesis and the oxygen oscillations have an antiphase mode to the ATP oscillations; we also found an independent mode for the ATP oscillations. This result indicates that both mitochondrial and non-mitochondrial respiration oscillate and thus play a role in chondrogenesis. This dual-color monitoring system is useful for studying metabolic regulations that underlie diverse cellular processes.

  13. Induction of chondrogenic differentiation of human adipose-derived stem cells by low frequency electric field

    Science.gov (United States)

    Mardani, Mohammad; Roshankhah, Shiva; Hashemibeni, Batool; Salahshoor, Mohammadreza; Naghsh, Erfan; Esfandiari, Ebrahim

    2016-01-01

    Background: Since when the cartilage damage (e.g., with the osteoarthritis) it could not be repaired in the body, hence for its reconstruction needs cell therapy. For this purpose, adipose-derived stem cells (ADSCs) is one of the best cell sources because by the tissue engineering techniques it can be differentiated into chondrocytes. Chemical and physical inducers is required order to stem cells to chondrocytes differentiating. We have decided to define the role of electric field (EF) in inducing chondrogenesis process. Materials and Methods: A low frequency EF applied the ADSCs as a physical inducer for chondrogenesis in a 3D micromass culture system which ADSCs were extracted from subcutaneous abdominal adipose tissue. Also enzyme-linked immunosorbent assay, methyl thiazolyl tetrazolium, real time polymerase chain reaction and flowcytometry techniques were used for this study. Results: We found that the 20 minutes application of 1 kHz, 20 mv/cm EF leads to chondrogenesis in ADSCs. Although our results suggest that application of physical (EF) and chemical (transforming growth factor-β3) inducers at the same time, have best results in expression of collagen type II and SOX9 genes. It is also seen EF makes significant decreased expression of collagens type I and X genes. Conclusion: The low frequency EF can be a good motivator to promote chondrogenic differentiation of human ADSCs. PMID:27308269

  14. A comparative study of the chondrogenic potential between synthetic and natural scaffolds in an in vivo bioreactor

    Science.gov (United States)

    Huang, Jung-Ju; Yang, Shu-Rui; Chu, I.-Ming; Brey, Eric M.; Hsiao, Hui-Yi; Cheng, Ming-Huei

    2013-10-01

    The clinical demand for cartilage tissue engineering is potentially large for reconstruction defects resulting from congenital deformities or degenerative disease due to limited donor sites for autologous tissue and donor site morbidities. Cartilage tissue engineering has been successfully applied to the medical field: a scaffold pre-cultured with chondrocytes was used prior to implantation in an animal model. We have developed a surgical approach in which tissues are engineered by implantation with a vascular pedicle as an in vivo bioreactor in bone and adipose tissue engineering. Collagen type II, chitosan, poly(lactic-co-glycolic acid) (PLGA) and polycaprolactone (PCL) were four commonly applied scaffolds in cartilage tissue engineering. To expand the application of the same animal model in cartilage tissue engineering, these four scaffolds were selected and compared for their ability to generate cartilage with chondrocytes in the same model with an in vivo bioreactor. Gene expression and immunohistochemistry staining methods were used to evaluate the chondrogenesis and osteogenesis of specimens. The result showed that the PLGA and PCL scaffolds exhibited better chondrogenesis than chitosan and type II collagen in the in vivo bioreactor. Among these four scaffolds, the PCL scaffold presented the most significant result of chondrogenesis embedded around the vascular pedicle in the long-term culture incubation phase.

  15. The Synergistic Effects of Matrix Stiffness and Composition on the Response of Chondroprogenitor Cells in a 3D Precondensation Microenvironment.

    Science.gov (United States)

    Carrion, Bita; Souzanchi, Mohammad F; Wang, Victor T; Tiruchinapally, Gopinath; Shikanov, Ariella; Putnam, Andrew J; Coleman, Rhima M

    2016-05-01

    Improve functional quality of cartilage tissue engineered from stem cells requires a better understanding of the functional evolution of native cartilage tissue. Therefore, a biosynthetic hydrogel was developed containing RGD, hyaluronic acid and/or type-I collagen conjugated to poly(ethylene glycol) acrylate to recapitulate the precondensation microenvironment of the developing limb. Conjugation of any combination of the three ligands did not alter the shear moduli or diffusion properties of the PEG hydrogels; thus, the influence of ligand composition on chondrogenesis could be investigated in the context of varying matrix stiffness. Gene expression of ligand receptors (CD44 and the b1-integrin) as well as markers of condensation (cell clustering and N-cadherin gene expression) and chondrogenesis (Col2a1 gene expression and sGAG production) by chondroprogenitor cells in this system were modulated by both matrix stiffness and ligand composition, with the highest gene expression occurring in softer hydrogels containing all three ligands. Cell proliferation in these 3D matrices for 7 d prior to chondrogenic induction increased the rate of sGAG production in a stiffness-dependent manner. This biosynthetic hydrogel supports the features of early limb-bud condensation and chondrogenesis and is a novel platform in which the influence of the matrix physicochemical properties on these processes can be elucidated.

  16. Periodic heat shock accelerated the chondrogenic differentiation of human mesenchymal stem cells in pellet culture.

    Directory of Open Access Journals (Sweden)

    Jing Chen

    Full Text Available Osteoarthritis (OA is one of diseases that seriously affect elderly people's quality of life. Human mesenchymal stem cells (hMSCs offer a potential promise for the joint repair in OA patients. However, chondrogenic differentiation from hMSCs in vitro takes a long time (∼ 6 weeks and differentiated cells are still not as functionally mature as primary isolated chondrocytes, though chemical stimulations and mechanical loading have been intensively studied to enhance the hMSC differentiation. On the other hand, thermal stimulations of hMSC chondrogenesis have not been well explored. In this study, the direct effects of mild heat shock (HS on the differentiation of hMSCs into chondrocytes in 3D pellet culture were investigated. Periodic HS at 41 °C for 1 hr significantly increased sulfated glycosaminoglycan in 3D pellet culture at Day 10 of chondrogenesis. Immunohistochemical and Western Blot analyses revealed an increased expression of collagen type II and aggrecan in heat-shocked pellets than non heat-shocked pellets on Day 17 of chondrogenesis. In addition, HS also upregulated the expression of collagen type I and X as well as heat shock protein 70 on Day 17 and 24 of differentiation. These results demonstrate that HS accelerated the chondrogenic differentiation of hMSCs and induced an early maturation of chondrocytes differentiated from hMSCs. The results of this study will guide the design of future protocols using thermal treatments to facilitate cartilage regeneration with human mesenchymal stem cells.

  17. Development of the turtle plastron, the order-defining skeletal structure.

    Science.gov (United States)

    Rice, Ritva; Kallonen, Aki; Cebra-Thomas, Judith; Gilbert, Scott F

    2016-05-10

    The dorsal and ventral aspects of the turtle shell, the carapace and the plastron, are developmentally different entities. The carapace contains axial endochondral skeletal elements and exoskeletal dermal bones. The exoskeletal plastron is found in all extant and extinct species of crown turtles found to date and is synaptomorphic of the order Testudines. However, paleontological reconstructed transition forms lack a fully developed carapace and show a progression of bony elements ancestral to the plastron. To understand the evolutionary development of the plastron, it is essential to know how it has formed. Here we studied the molecular development and patterning of plastron bones in a cryptodire turtle Trachemys scripta We show that plastron development begins at developmental stage 15 when osteochondrogenic mesenchyme forms condensates for each plastron bone at the lateral edges of the ventral mesenchyme. These condensations commit to an osteogenic identity and suppress chondrogenesis. Their development overlaps with that of sternal cartilage development in chicks and mice. Thus, we suggest that in turtles, the sternal morphogenesis is prevented in the ventral mesenchyme by the concomitant induction of osteogenesis and the suppression of chondrogenesis. The osteogenic subroutines later direct the growth and patterning of plastron bones in an autonomous manner. The initiation of plastron bone development coincides with that of carapacial ridge formation, suggesting that the development of dorsal and ventral shells are coordinated from the start and that adopting an osteogenesis-inducing and chondrogenesis-suppressing cell fate in the ventral mesenchyme has permitted turtles to develop their order-specific ventral morphology.

  18. Activin Receptor-Like Kinase Receptors ALK5 and ALK1 Are Both Required for TGFβ-Induced Chondrogenic Differentiation of Human Bone Marrow-Derived Mesenchymal Stem Cells

    Science.gov (United States)

    de Kroon, Laurie M. G.; Narcisi, Roberto; Blaney Davidson, Esmeralda N.; Cleary, Mairéad A.; van Beuningen, Henk M.; Koevoet, Wendy J. L. M.; van Osch, Gerjo J. V. M.; van der Kraan, Peter M.

    2015-01-01

    Introduction Bone marrow-derived mesenchymal stem cells (BMSCs) are promising for cartilage regeneration because BMSCs can differentiate into cartilage tissue-producing chondrocytes. Transforming Growth Factor β (TGFβ) is crucial for inducing chondrogenic differentiation of BMSCs and is known to signal via Activin receptor-Like Kinase (ALK) receptors ALK5 and ALK1. Since the specific role of these two TGFβ receptors in chondrogenesis is unknown, we investigated whether ALK5 and ALK1 are expressed in BMSCs and whether both receptors are required for chondrogenic differentiation of BMSCs. Materials & Methods ALK5 and ALK1 gene expression in human BMSCs was determined with RT-qPCR. To induce chondrogenesis, human BMSCs were pellet-cultured in serum-free chondrogenic medium containing TGFβ1. Chondrogenesis was evaluated by aggrecan and collagen type IIα1 RT-qPCR analysis, and histological stainings of proteoglycans and collagen type II. To overexpress constitutively active (ca) receptors, BMSCs were transduced either with caALK5 or caALK1. Expression of ALK5 and ALK1 was downregulated by transducing BMSCs with shRNA against ALK5 or ALK1. Results ALK5 and ALK1 were expressed in in vitro-expanded as well as in pellet-cultured BMSCs from five donors, but mRNA levels of both TGFβ receptors did not clearly associate with chondrogenic induction. TGFβ increased ALK5 and decreased ALK1 gene expression in chondrogenically differentiating BMSC pellets. Neither caALK5 nor caALK1 overexpression induced cartilage matrix formation as efficient as that induced by TGFβ. Moreover, short hairpin-mediated downregulation of either ALK5 or ALK1 resulted in a strong inhibition of TGFβ-induced chondrogenesis. Conclusion ALK5 as well as ALK1 are required for TGFβ-induced chondrogenic differentiation of BMSCs, and TGFβ not only directly induces chondrogenesis, but also modulates ALK5 and ALK1 receptor signaling in BMSCs. These results imply that optimizing cartilage formation by

  19. Global comparative transcriptome analysis of cartilage formation in vivo

    Directory of Open Access Journals (Sweden)

    Brachvogel Bent

    2009-03-01

    Full Text Available Abstract Background During vertebrate embryogenesis the initial stages of bone formation by endochondral ossification involve the aggregation and proliferation of mesenchymal cells into condensations. Continued growth of the condensations and differentiation of the mesenchymal cells into chondrocytes results in the formation of cartilage templates, or anlagen, which prefigure the shape of the future bones. The chondrocytes in the anlagen further differentiate by undergoing a complex sequence of maturation and hypertrophy, and are eventually replaced by mineralized bone. Regulation of the onset of chondrogenesis is incompletely understood, and would be informed by comprehensive analyses of in vivo gene expression. Results Tibial and fibular pre-condensed mesenchyme was microdissected from mouse hind limbs at 11.5 dpc, and the corresponding condensations at 12.5 dpc and cartilage anlagen at 13.5 dpc. Total RNA was isolated, and cRNA generated by linear amplification was interrogated using mouse whole genome microarrays. Differential expression was validated by quantitative PCR for Agc1, Bmp8a, Col2a1, Fgfr4, Foxa3, Gdf5, Klf2, Klf4, Lepre1, Ncad, Sox11, and Trpv4. Further, independent validation of the microarray data was achieved by in situ hybridization to analyse the expression of Lepre1, Pcdh8, Sox11, and Trpv4 from 11.5 dpc to 13.5 dpc during mouse hind limb development. We found significant differential expression of 931 genes during these early stages of chondrogenesis. Of these, 380 genes were down-regulated and 551 up-regulated. Our studies characterized the expression pattern of gene families previously associated with chondrogenesis, such as adhesion molecules, secreted signalling molecules, transcription factors, and extracellular matrix components. Gene ontology approaches identified 892 differentially expressed genes not previously identified during the initiation of chondrogenesis. These included several Bmp, Gdf, Wnt, Sox and Fox

  20. MicroRNA Levels as Prognostic Markers for the Differentiation Potential of Human Mesenchymal Stromal Cell Donors.

    Science.gov (United States)

    Georgi, Nicole; Taipaleenmaki, Hanna; Raiss, Christian C; Groen, Nathalie; Portalska, Karolina Janaeczek; van Blitterswijk, Clemens; de Boer, Jan; Post, Janine N; van Wijnen, Andre J; Karperien, Marcel

    2015-08-15

    The ability of human mesenchymal stromal/stem cells (hMSCs) to differentiate into various mesenchymal cell lineages makes them a promising cell source for the use in tissue repair strategies. Since the differentiation potential of hMSCs differs between donors, it is necessary to establish biomarkers for the identification of donors with high differentiation potential. In this study, we show that microRNA (miRNA) expression levels are effective for distinguishing donors with high differentiation potential from low differentiation potential. Twenty hMSC donors were initially tested for marker expression and differentiation potential. In particular, the chondrogenic differentiation potential was evaluated on the basis of histological matrix formation, mRNA expression levels of chondrogenic marker genes, and quantitative glycosaminoglycan deposition. Three donors out of twenty were identified as donors with high chondrogenic potential, whereas nine showed moderate and eight showed low chondrogenic potential. Expression profiles of miRNAs involved in chondrogenesis and cartilage homeostasis were used for the distinction between high-performance hMSCs and low-performance hMSCs. Global mRNA expression profiles of the donors before the onset of chondrogenic differentiation revealed minor differences in gene expression between low and high chondrogenic performers. However, analysis of miRNA expression during a 7-day differentiation period identified miR-210 and miR-630 as positive regulators of chondrogenesis. In contrast, miR-181 and miR-34a, which are negative regulators of chondrogenesis, were upregulated during differentiation in low-performing donors. In conclusion, profiling of hMSC donors for a specific panel of miRNAs may have a prognostic value for selecting donors with high differentiation potential to improve hMSC-based strategies for tissue regeneration.

  1. Human immunodeficiency virus type 1 enhancer-binding protein 3 is essential for the expression of asparagine-linked glycosylation 2 in the regulation of osteoblast and chondrocyte differentiation.

    Science.gov (United States)

    Imamura, Katsuyuki; Maeda, Shingo; Kawamura, Ichiro; Matsuyama, Kanehiro; Shinohara, Naohiro; Yahiro, Yuhei; Nagano, Satoshi; Setoguchi, Takao; Yokouchi, Masahiro; Ishidou, Yasuhiro; Komiya, Setsuro

    2014-04-01

    Human immunodeficiency virus type 1 enhancer-binding protein 3 (Hivep3) suppresses osteoblast differentiation by inducing proteasomal degradation of the osteogenesis master regulator Runx2. In this study, we tested the possibility of cooperation of Hivep1, Hivep2, and Hivep3 in osteoblast and/or chondrocyte differentiation. Microarray analyses with ST-2 bone stroma cells demonstrated that expression of any known osteochondrogenesis-related genes was not commonly affected by the three Hivep siRNAs. Only Hivep3 siRNA promoted osteoblast differentiation in ST-2 cells, whereas all three siRNAs cooperatively suppressed differentiation in ATDC5 chondrocytes. We further used microarray analysis to identify genes commonly down-regulated in both MC3T3-E1 osteoblasts and ST-2 cells upon knockdown of Hivep3 and identified asparagine-linked glycosylation 2 (Alg2), which encodes a mannosyltransferase residing on the endoplasmic reticulum. The Hivep3 siRNA-mediated promotion of osteoblast differentiation was negated by forced Alg2 expression. Alg2 suppressed osteoblast differentiation and bone formation in cultured calvarial bone. Alg2 was immunoprecipitated with Runx2, whereas the combined transfection of Runx2 and Alg2 interfered with Runx2 nuclear localization, which resulted in suppression of Runx2 activity. Chondrocyte differentiation was promoted by Hivep3 overexpression, in concert with increased expression of Creb3l2, whose gene product is the endoplasmic reticulum stress transducer crucial for chondrogenesis. Alg2 silencing suppressed Creb3l2 expression and chondrogenesis of ATDC5 cells, whereas infection of Alg2-expressing virus promoted chondrocyte maturation in cultured cartilage rudiments. Thus, Alg2, as a downstream mediator of Hivep3, suppresses osteogenesis, whereas it promotes chondrogenesis. To our knowledge, this study is the first to link a mannosyltransferase gene to osteochondrogenesis.

  2. Hydrostatic pressure acts to stabilise a chondrogenic phenotype in porcine joint tissue derived stem cells

    Directory of Open Access Journals (Sweden)

    T Vinardell

    2012-02-01

    Full Text Available Hydrostatic pressure (HP is a key component of the in vivo joint environment and has been shown to enhance chondrogenesis of stem cells. The objective of this study was to investigate the interaction between HP and TGF-β3 on both the initiation and maintenance of a chondrogenic phenotype for joint tissue derived stem cells. Pellets generated from porcine chondrocytes (CCs, synovial membrane derived stem cells (SDSCs and infrapatellar fat pad derived stem cells (FPSCs were subjected to 10 MPa of cyclic HP (4 h/day and different concentrations of TGF-β3 (0, 1 and 10 ng/mL for 14 days. CCs and stem cells were observed to respond differentially to both HP and TGF-β3 stimulation. HP in the absence of TGF-β3 did not induce robust chondrogenic differentiation of stem cells. At low concentrations of TGF-β3 (1 ng/mL, HP acted to enhance chondrogenesis of both SDSCs and FPSCs, as evident by a 3-fold increase in Sox9 expression and a significant increase in glycosaminoglycan accumulation. In contrast, HP had no effect on cartilage-specific matrix synthesis at higher concentrations of TGF-β3 (10 ng/mL. Critically, HP appears to play a key role in the maintenance of a chondrogenic phenotype, as evident by a down-regulation of the hypertrophic markers type X collagen and Indian hedgehog in SDSCs irrespective of the cytokine concentration. In the context of stem cell based therapies for cartilage repair, this study demonstrates the importance of considering how joint specific environmental factors interact to regulate not only the initiation of chondrogenesis, but also the development of a stable hyaline-like repair tissue.

  3. Human umbilical cord Wharton's jelly stem cells undergo enhanced chondrogenic differentiation when grown on nanofibrous scaffolds and in a sequential two-stage culture medium environment.

    Science.gov (United States)

    Fong, Chui-Yee; Subramanian, Arjunan; Gauthaman, Kalamegam; Venugopal, Jayarama; Biswas, Arijit; Ramakrishna, Seeram; Bongso, Ariff

    2012-03-01

    The current treatments used for osteoarthritis from cartilage damage have their disadvantages of donor site morbidity, complicated surgical interventions and risks of infection and graft rejection. Recent advances in tissue engineering have offered much promise in cartilage repair but the best cell source and in vitro system have not as yet been optimised. Human bone marrow mesenchymal stem cells (hBMSCs) have thus far been the cell of choice. However, we derived a unique stem cell from the human umbilical cord Wharton's jelly (hWJSC) that has properties superior to hBMSCs in terms of ready availability, prolonged stemness characteristics in vitro, high proliferation rates, wide multipotency, non-tumorigenicity and tolerance in allogeneic transplantation. We observed enhanced cell attachment, cell proliferation and chondrogenesis of hWJSCs over hBMSCs when grown on PCL/Collagen nanoscaffolds in the presence of a two-stage sequential complex/chondrogenic medium for 21 days. Improvement of these three parameters were confirmed via inverted optics, field emission scanning electron microscopy (FESEM), MTT assay, pellet diameters, Alcian blue histology and staining, glycosaminglycans (GAG) and hyaluronic acid production and expression of key chondrogenic genes (SOX9, Collagen type II, COMP, FMOD) using immunohistochemistry and real-time polymerase chain reaction (qRT-PCR). In separate experiments we demonstrated that the 16 ng/ml of basic fibroblast growth factor (bFGF) present in the complex medium may have contributed to driving chondrogenesis. We conclude that hWJSCs are an attractive stem cell source for inducing chondrogenesis in vitro when grown on nanoscaffolds and exposed sequentially first to complex medium and then followed by chondrogenic medium.

  4. Irx3 and Bmp2 Regulate Mouse Mesenchymal Cell Chondrogenic Differentiation in Both a Sox9-Dependent and -Independent Manner.

    Science.gov (United States)

    Tamamura, Yoshihiro; Katsube, Kenichi; Mera, Hisashi; Itokazu, Maki; Wakitani, Shigeyuki

    2017-01-06

    Sox9, a master regulator of cartilage development, controls the cell fate decision to differentiate from mesenchymal to chondrogenic cells. In addition, Sox9 regulates the proliferation and differentiation of chondrocytes, as well as the production of cartilage-specific proteoglycans. The existence of Sox9-independent mechanisms in cartilage development remains to be determined. Here, we attempted to identify genes involved in such putative mechanisms via microarray analysis using a mouse chondrogenic cell line, N1511. We first focused on transcription factors that exhibited upregulated expression following Bmp2 treatment, which was not altered by subsequent treatment with Sox9 siRNA. Among these, we selected positive regulators for chondrogenesis and identified Iroquois-related homeobox 3 (Irx3) as one of the candidate genes. Irx3 expression gradually increased with chondrocyte terminal differentiation in a reciprocal manner to Sox9 expression, and promoted the chondrogenic differentiation of mesenchymal cells upon Bmp2 treatment. Furthermore, Irx3 partially rescued impaired chondrogenesis by upregulating the expression of epiphycan and lumican under reduced Sox9 expression. Finally, Irx3 was shown to act in concert with Bmp2 signaling to activate the p38 MAPK pathway, which in turn stimulated Sox9 expression, as well as the expression of epiphycan and lumican in a Sox9-independent manner. These results indicate that Irx3 represents a novel chondrogenic factor of mesenchymal cells, acts synergistically with Bmp2-mediated signaling, and regulates chondrogenesis independent of the transcriptional machinery associated with Sox9-mediated regulation. This article is protected by copyright. All rights reserved.

  5. Environmental regulation of type X collagen production by cultures of limb mesenchyme, mesectoderm, and sternal chondrocytes.

    Science.gov (United States)

    Solursh, M; Jensen, K L; Reiter, R S; Schmid, T M; Linsenmayer, T F

    1986-09-01

    We have examined whether the production of hypertrophic cartilage matrix reflecting a late stage in the development of chondrocytes which participate in endochondral bone formation, is the result of cell lineage, environmental influence, or both. We have compared the ability of cultured limb mesenchyme and mesectoderm to synthesize type X collagen, a marker highly selective for hypertrophic cartilage. High density cultures of limb mesenchyme from stage 23 and 24 chick embryos contain many cells that react positively for type II collagen by immunohistochemistry, but only a few of these initiate type X collagen synthesis. When limb mesenchyme cells are cultured in or on hydrated collagen gels or in agarose (conditions previously shown to promote chondrogenesis in low density cultures), almost all initiate synthesis of both collagen types. Similarly, collagen gel cultures of limb mesenchyme from stage 17 embryos synthesize type II collagen and with some additional delay type X collagen. However, cytochalasin D treatment of subconfluent cultures on plastic substrates, another treatment known to promote chondrogenesis, induces the production of type II collagen, but not type X collagen. These results demonstrate that the appearance of type X collagen in limb cartilage is environmentally regulated. Mesectodermal cells from the maxillary process of stages 24 and 28 chick embryos were cultured in or on hydrated collagen gels. Such cells initiate synthesis of type II collagen, and eventually type X collagen. Some cells contain only type II collagen and some contain both types II and X collagen. On the other hand, cultures of mandibular processes from stage 29 embryos contain chondrocytes with both collagen types and a larger overall number of chondrogenic foci than the maxillary process cultures. Since the maxillary process does not produce cartilage in situ and the mandibular process forms Meckel's cartilage which does not hypertrophy in situ, environmental influences

  6. Tissue engineering strategies to study cartilage development, degeneration and regeneration.

    Science.gov (United States)

    Bhattacharjee, Maumita; Coburn, Jeannine; Centola, Matteo; Murab, Sumit; Barbero, Andrea; Kaplan, David L; Martin, Ivan; Ghosh, Sourabh

    2015-04-01

    Cartilage tissue engineering has primarily focused on the generation of grafts to repair cartilage defects due to traumatic injury and disease. However engineered cartilage tissues have also a strong scientific value as advanced 3D culture models. Here we first describe key aspects of embryonic chondrogenesis and possible cell sources/culture systems for in vitro cartilage generation. We then review how a tissue engineering approach has been and could be further exploited to investigate different aspects of cartilage development and degeneration. The generated knowledge is expected to inform new cartilage regeneration strategies, beyond a classical tissue engineering paradigm.

  7. Potential benefits and limitations of utilizing chondroprogenitors in cell-based cartilage therapy.

    Science.gov (United States)

    Jayasuriya, Chathuraka T; Chen, Qian

    2015-01-01

    Chondroprogenitor cells are a subpopulation of multipotent progenitors that are primed for chondrogenesis. They are believed to have the biological repertoire to be ideal for cell-based cartilage therapy. In addition to summarizing recent advances in chondroprogenitor cell characterization, this review discusses the projected pros and cons of utilizing chondroprogenitors in regenerative medicine and compares them with that of pre-existing methods, including autologous chondrocyte implantation (ACI) and the utilization of bone marrow derived mesenchymal stem cells (MSCs) for the purpose of cartilage tissue repair.

  8. A Joint Less Ordinary: Intriguing Roles for Hedgehog Signalling in the Development of the Temporomandibular Synovial Joint

    Directory of Open Access Journals (Sweden)

    Malgorzata Kubiak

    2016-08-01

    Full Text Available This review highlights the essential role of Hedgehog (Hh signalling in the developmental steps of temporomandibular joint (TMJ formation. We review evidence for intra- and potentially inter-tissue Hh signaling as well as Glioma-Associated Oncogene Homolog (GLI dependent and independent functions. Morphogenesis and maturation of the TMJ’s individual components and the general landscape of Hh signalling is also covered. Comparison of the appendicular knee and axial TMJ also reveals interesting differences and similarities in their mechanisms of development, chondrogenesis and reliance on Hh signalling.

  9. Mucopolysaccharidosis IVA and glycosaminoglycans.

    Science.gov (United States)

    Khan, Shaukat; Alméciga-Díaz, Carlos J; Sawamoto, Kazuki; Mackenzie, William G; Theroux, Mary C; Pizarro, Christian; Mason, Robert W; Orii, Tadao; Tomatsu, Shunji

    Mucopolysaccharidosis IVA (MPS IVA; Morquio A: OMIM 253000) is a lysosomal storage disease with an autosomal recessive trait caused by the deficiency of N-acetylgalactosamine-6-sulfate sulfatase. Deficiency of this enzyme leads to accumulation of specific glycosaminoglycans (GAGs): chondroitin-6-sulfate (C6S) and keratan sulfate (KS). C6S and KS are mainly produced in the cartilage. Therefore, the undegraded substrates are stored primarily in cartilage and in its extracellular matrix (ECM), leading to a direct impact on cartilage and bone development, and successive systemic skeletal dysplasia. Chondrogenesis, the earliest phase of skeletal formation, is maintained by cellular interactions with the ECM, growth and differentiation factors, signaling pathways, and transcription factors in a temporal-spatial manner. In patients with MPS IVA, the cartilage is disrupted at birth as a consequence of abnormal chondrogenesis and/or endochondral ossification. The unique skeletal features are distinguished by a disproportional short stature, odontoid hypoplasia, spinal cord compression, tracheal obstruction, pectus carinatum, kyphoscoliosis, platyspondyly, coxa valga, genu valgum, waddling gait, and laxity of joints. In spite of many descriptions of these unique clinical features, delay of diagnosis still happens. The pathogenesis and treatment of systemic skeletal dysplasia in MPS IVA remains an unmet challenge. In this review article, we comprehensively describe historical aspect, property of GAGs, diagnosis, screening, pathogenesis, and current and future therapies of MPS IVA.

  10. Interleukin-6 inhibits early differentiation of ATDC5 chondrogenic progenitor cells.

    Science.gov (United States)

    Nakajima, Shoko; Naruto, Takuya; Miyamae, Takako; Imagawa, Tomoyuki; Mori, Masaaki; Nishimaki, Shigeru; Yokota, Shumpei

    2009-08-01

    Interleukin (IL)-6 is a causative agent of systemic juvenile idiopathic arthritis (sJIA), a chronic inflammatory disease complicated with severe growth impairment. Recent trials of anti-IL-6 receptor monoclonal antibody, tocilizumab, indicated that tocilizumab blocks IL-6/IL-6 receptor-mediated inflammation, and induces catch-up growth in children with sJIA. This study evaluates the effects of IL-6 on chondrogenesis by ATDC5 cells, a clonal murine chondrogenic cell line that provides an excellent model for studying endochondral ossification at growth plate. ATDC5 cells were examined for the expression of IL-6 receptor and gp130 by fluorescence-activated cell sorting analysis. Recombinant murine IL-6 was added to ATDC5 cultures to observe cell differentiation, using a quantitative RT-PCR for the chondrogenic differentiation markers type II collagen, aggrecan, and type X collagen. To block IL-6, the anti-mouse IL-6 receptor monoclonal antibody MR16-1 was added. As a result, the cells expressed IL-6 receptor and gp130. The expression of chondrogenic differentiation marker gene was reduced by IL-6, but this was abrogated by MR16-1. We conclude that IL-6 inhibits early chondrogenesis of ATDC5 cells suggesting that IL-6 may affect committed stem cells at a cellular level during chondrogenic differentiation of growth plate chondrocytes, and that IL-6 may be a cellular-level factor in growth impairment in sJIA.

  11. Human stem cells and articular cartilage regeneration.

    Science.gov (United States)

    Inui, Atsuyuki; Iwakura, Takashi; Reddi, A Hari

    2012-11-05

    The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES) cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS) cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  12. Cartilage tissue engineering application of injectable gelatin hydrogel with in situ visible-light-activated gelation capability in both air and aqueous solution.

    Science.gov (United States)

    Lin, Hang; Cheng, Anthony Wai-Ming; Alexander, Peter G; Beck, Angela M; Tuan, Rocky S

    2014-09-01

    Chondroprogenitor cells encapsulated in a chondrogenically supportive, three-dimensional hydrogel scaffold represents a promising, regenerative approach to articular cartilage repair. In this study, we have developed an injectable, biodegradable methacrylated gelatin (mGL)-based hydrogel capable of rapid gelation via visible light (VL)-activated crosslinking in air or aqueous solution. The mild photocrosslinking conditions permitted the incorporation of cells during the gelation process. Encapsulated human-bone-marrow-derived mesenchymal stem cells (hBMSCs) showed high, long-term viability (up to 90 days) throughout the scaffold. To assess the applicability of the mGL hydrogel for cartilage tissue engineering, we have evaluated the efficacy of chondrogenesis of the encapsulated hBMSCs, using hBMSCs seeded in agarose as control. The ability of hBMSC-laden mGL constructs to integrate with host tissues after implantation was further investigated utilizing an in vitro cartilage repair model. The results showed that the mGL hydrogel, which could be photopolymerized in air and aqueous solution, supports hBMSC growth and TGF-β3-induced chondrogenesis. Compared with agarose, mGL constructs laden with hBMSCs are mechanically stronger with time, and integrate well with native cartilage tissue upon implantation based on push-out mechanical testing. VL-photocrosslinked mGL scaffold thus represents a promising scaffold for cell-based repair and resurfacing of articular cartilage defects.

  13. Artificial ants deposit pheromone to search for regulatory DNA elements

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    Liu Yunlong

    2006-08-01

    Full Text Available Abstract Background Identification of transcription-factor binding motifs (DNA sequences can be formulated as a combinatorial problem, where an efficient algorithm is indispensable to predict the role of multiple binding motifs. An ant algorithm is a biology-inspired computational technique, through which a combinatorial problem is solved by mimicking the behavior of social insects such as ants. We developed a unique version of ant algorithms to select a set of binding motifs by considering a potential contribution of each of all random DNA sequences of 4- to 7-bp in length. Results Human chondrogenesis was used as a model system. The results revealed that the ant algorithm was able to identify biologically known binding motifs in chondrogenesis such as AP-1, NFκB, and sox9. Some of the predicted motifs were identical to those previously derived with the genetic algorithm. Unlike the genetic algorithm, however, the ant algorithm was able to evaluate a contribution of individual binding motifs as a spectrum of distributed information and predict core consensus motifs from a wider DNA pool. Conclusion The ant algorithm offers an efficient, reproducible procedure to predict a role of individual transcription-factor binding motifs using a unique definition of artificial ants.

  14. The role of aragonite matrix surface chemistry on the chondrogenic differentiation of mesenchymal stem cells.

    Science.gov (United States)

    Gross-Aviv, Talia; Vago, Razi

    2009-02-01

    In the present research we study the effects of surface chemistry of an aragonite crystalline biomatrix on the chondrogenesis of mesenchymal stem cells (MSCs). An aragonite matrix obtained from the coral Porites lutea and a gold-coated P. lutea matrix were seeded with MSCs, with and without the addition of growth factors (GFs). Scanning electron microscopy, histochemical staining, immunofluorescence, biochemical analyses and quantitative polymerase chain reaction showed that the chemistry of the matrix influenced the differentiation process of the MSCs. The calcium carbonate composition of the coral promoted osteogenesis, while impeding cell-material contact (by gold coating) altered the differentiation lineage of MSCs towards chondrogenic fate. Supplementation of the culture medium with GFs intensified the influence of the surface composition on the differentiation of MSCs, and the synergistic effect of the biomatrix surface composition and the GFs induced chondrogenesis and facilitated maintenance of the chondrocyte phenotype. Therefore, we suggest that scaffolding material candidates for tissue engineering should be examined for their effects on the MSCs differentiation process and their effect on signal transduction events in the cells.

  15. Histone Acetylation Influences the Activity of Sox9-related Transcriptional Complex

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    Furumatsu,Takayuki

    2010-12-01

    Full Text Available Chondrocyte differentiation is the fundamental process in skeletal development. From the mesenchymal condensation of chondroprogenitors to the hypertrophic maturation of chondrocytes, chondrogenesis is sequentially regulated by cross-talk among transcription factors, growth factors, and chromatin structure. The master transcription factor Sry-type HMG box (Sox 9 has an essential role in the expression of chondrogenic genes through the association with Sox9-binding sites on its target genes. Several transcription factors and coactivators, such as Scleraxis/E47 and p300, cooperatively modulate the Sox9-dependent transcription by interacting with Sox9. The Sox9-related transcriptional apparatus activates its target gene expression through p300-mediated histone acetylation on chromatin. The transforming growth factor (TGF-β superfamily also plays a key role in chondrocyte differentiation. The TGF-β-regulated Smad3/4 complex activates Sox9-dependent transcription on chromatin by associating with Sox9 itself, and by recruiting p300 onto Sox9. These findings suggest that the epigenetic status including histone modification and chromatin structure, directly influences Sox9-regulated chondrocyte differentiation. In this article, we review the regulators of Sox9 expression itself, modulators of posttranslational Sox9 function, and Sox9-associating factors in the Sox9-dependent epigenetic regulation during chondrogenesis.

  16. The role of calcium signalling in the chondrogenic response of mesenchymal stem cells to hydrostatic pressure

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    AJ Steward

    2014-10-01

    Full Text Available The object of this study was to elucidate the role of Ca++ signalling in the chondrogenic response of mesenchymal stem cells (MSCs to hydrostatic pressure (HP. MSCs were seeded into agarose hydrogels, subjected to HP or kept in free swelling conditions, and were cultured either with or without pharmacological inhibitors of Ca++ mobility and downstream targets. Chelating free Ca++, inhibiting voltage-gated calcium channels, and depleting intracellular calcium storessuppressed the beneficial effect of HP on chondrogenesis, indicating that Ca++ mobility may play an important role in the mechanotransduction of HP. However, inhibition of stretch-activated calcium channels in the current experiment yielded similar results to the control group, suggesting that mechanotransduction of HP is distinct from loads that generate cell deformations. Inhibition of the downstream targets calmodulin, calmodulin kinase II, and calcineurin all knocked down the effect of HP on chondrogenesis, implicating these targets in MSCs response to HP. All of the pharmacological inhibitors that abolished the chondrogenic response to HP also maintained a punctate vimentin organisation in the presence of HP, as opposed to the mechanoresponsive groups where the vimentin structure became more diffuse. These results suggest that Ca++ signalling may transduce HP via vimentin adaptation to loading.

  17. Transforming Growth Factors β Coordinate Cartilage and Tendon Differentiation in the Developing Limb Mesenchyme*

    Science.gov (United States)

    Lorda-Diez, Carlos I.; Montero, Juan A.; Martinez-Cue, Carmen; Garcia-Porrero, Juan A.; Hurle, Juan M.

    2009-01-01

    Transforming growth factor β (TGFβ) signaling has an increasing interest in regenerative medicine as a potential tool to repair cartilages, however the chondrogenic effect of this pathway in developing systems is controversial. Here we have analyzed the function of TGFβ signaling in the differentiation of the developing limb mesoderm in vivo and in high density micromass cultures. In these systems highest signaling activity corresponded with cells at stages preceding overt chondrocyte differentiation. Interestingly treatments with TGFβs shifted the differentiation outcome of the cultures from chondrogenesis to fibrogenesis. This phenotypic reprogramming involved down-regulation of Sox9 and Aggrecan and up-regulation of Scleraxis, and Tenomodulin through the Smad pathway. We further show that TGFβ signaling up-regulates Sox9 in the in vivo experimental model system in which TGFβ treatments induce ectopic chondrogenesis. Looking for clues explaining the dual role of TGFβ signaling, we found that TGFβs appear to be direct inducers of the chondrogenic gene Sox9, but the existence of transcriptional repressors of TGFβ signaling modulates this role. We identified TGF-interacting factor Tgif1 and SKI-like oncogene SnoN as potential candidates for this inhibitory function. Tgif1 gene regulation by TGFβ signaling correlated with the differential chondrogenic and fibrogenic effects of this pathway, and its expression pattern in the limb marks the developing tendons. In functional experiments we found that Tgif1 reproduces the profibrogenic effect of TGFβ treatments. PMID:19717568

  18. BMP-Smad4 signaling is required for precartilaginous mesenchymal condensation independent of Sox9 in the mouse.

    Science.gov (United States)

    Lim, Joohyun; Tu, Xiaolin; Choi, Kyunghee; Akiyama, Haruhiko; Mishina, Yuji; Long, Fanxin

    2015-04-01

    Bone morphogenetic proteins (BMPs) regulate multiple aspects of skeletal development in vertebrates. Although exogenously applied BMPs can induce chondrogenesis de novo, the role and mechanism of physiologic BMP signaling during precartilaginous mesenchymal condensation is not well understood. By deleting the type I BMP receptors or the transcription factor Smad4 in the limb bud mesenchyme, we find that loss of BMP-Smad signaling abolishes skeletal development due to a failure in mesenchymal condensation. In the absence of Smad4, expression of Sox9, an essential transcription factor for chondrogenesis, initiates normally in the proximal mesenchyme of the limb bud, but fails to maintain its level or expand to the more distal territory at the later stages. However, forced-expression of Sox9 does not restore cartilage formation in the Smad4-deficeint embryo. In vitro micromass cultures show that the Smad4-deficient cells fail to condense in a cell-autonomous manner, even though they express several cell adhesion molecules either normally or even at a higher level. Thus, BMP-Smad signaling critically controls mesenchymal condensation to initiate skeletal development likely through a Sox9-independent mechanism.

  19. The role of energy dissipation of polymeric scaffolds in the mechanobiological modulation of chondrogenic expression.

    Science.gov (United States)

    Abdel-Sayed, Philippe; Darwiche, Salim E; Kettenberger, Ulrike; Pioletti, Dominique P

    2014-02-01

    Mechanical stimulation has been proposed to induce chondrogenesis in cell-seeded scaffolds. However, the effects of mechanical stimuli on engineered cartilage may vary substantially between different scaffolds. This advocates for the need to identify an overarching mechanobiological variable. We hypothesize that energy dissipation of scaffolds subjected to dynamic loading may be used as a mechanobiology variable. The energy dissipation would furnish a general criterion to adjust the mechanical stimulation favoring chondrogenesis in scaffold. Epiphyseal chondro-progenitor cells were then subject to unconfined compression 2 h per day during four days in different scaffolds, which differ only by the level of dissipation they generated while keeping the same loading conditions. Scaffolds with higher dissipation levels upregulated the mRNA of chondrogenic markers. In contrast lower dissipation of scaffolds was associated with downregulation of chondrogenic markers. These results showed that energy dissipation could be considered as a mechanobiology variable in cartilage. This study also indicated that scaffolds with energy dissipation level close to the one of cartilage favors chondrogenic expression when dynamical loading is present.

  20. Chondrogenic potential of human mesenchymal stem cells and expression of Slug transcription factor.

    Science.gov (United States)

    Brini, Anna T; Niada, Stefania; Lambertini, Elisabetta; Torreggiani, Elena; Arrigoni, Elena; Lisignoli, Gina; Piva, Roberta

    2015-06-01

    The scientific literature rarely reports experimental failures or inconsistent outcomes in the induction of cell differentiation; however, researchers commonly experience poor or unsuccessful responses to differentiating agents when culturing stem cells. One way of investigating the underlying reasons for such responses is to look at the basal expression levels of specific genes in multipotent stem cells before the induction of differentiation. In addition to shedding light on the complex properties of stem cells and the molecular modulation of differentiation pathways, this strategy can also lead to the development of important time- and money-saving tools that aid the efficient selection of cellular specimens--in this case, stem cells that are more prone to differentiate towards specific lineages and are therefore more suitable for cell-based therapeutic protocols in regenerative medicine. To address this latter aspect, this study focused on understanding the reasons why some human mesenchymal stem cell (hMSC) samples are less efficient at differentiating towards chondrogenesis. This study shows that analysis of the basal expression levels of Slug, a negative regulator of chondrogenesis in hMSC, provides a rapid and simple tool for distinguishing stem cell samples with the potential to form a cartilage-like matrix, and that are therefore suitable for cartilage tissue engineering. It is shown that high basal levels of Slug prevent the chondrogenic differentiation of hMSCs, even in the presence of transforming growth factor-β and elevated levels of Sox9.

  1. Matrix dimensions, stiffness, and structural properties modulate spontaneous chondrogenic commitment of mouse embryonic fibroblasts.

    Science.gov (United States)

    Fernández-Muiños, Teresa; Suárez-Muñoz, Melva; Sanmartí-Espinal, Marta; Semino, Carlos E

    2014-04-01

    Experimental models for cartilage and bone development have been studied in order to understand the biomechanical and biological parameters that regulate skeletal tissue formation. We have previously described that when mouse embryonic fibroblasts (MEFs) were cultured in a three-dimensional (3D)-soft self-assembling peptide nanofiber, the system engaged in a spontaneous process of cartilage-like formation evidenced by the expression of Sox9, Collagen type II, and proteoglycans. In the present work, we studied the influence that matrix mechanical properties have in modulating lineage commitment in an in vitro model of chondrogenesis. This effect was observed only when MEFs were cultured at low elastic modulus values (∼ 0.1 kPa). Interestingly, under these conditions, the system expressed the chondrogenic inductor BMP4 and its antagonist Noggin. On the other hand, at higher elastic modulus values (∼ 5 kPa), the system expressed Noggin but not BMP4, and did not engage in chondrogenesis, which suggest that the balance between bone morphogenetic protein/Noggin could be implicated in the chondrogenic process. Finally, no evidence of hypertrophy was detected under the conditions tested (by assessing expression of Collagen type X and Runx2) unless we challenged the system by co-culturing it with endothelial cells. Importantly, under these new conditions, the system underwent spontaneous matrix calcium mineralization. These results suggest that the 3D-system described here is sensitive to respond to environmental changes such as biomechanical and biological cues.

  2. Yield stress determines bioprintability of hydrogels based on gelatin-methacryloyl and gellan gum for cartilage bioprinting.

    Science.gov (United States)

    Mouser, Vivian H M; Melchels, Ferry P W; Visser, Jetze; Dhert, Wouter J A; Gawlitta, Debby; Malda, Jos

    2016-07-19

    Bioprinting of chondrocyte-laden hydrogels facilitates the fabrication of constructs with controlled organization and shape e.g. for articular cartilage implants. Gelatin-methacryloyl (gelMA) supplemented with gellan gum is a promising bio-ink. However, the rheological properties governing the printing process, and the influence of gellan gum on the mechanical properties and chondrogenesis of the blend, are still unknown. Here, we investigated the suitability of gelMA/gellan for cartilage bioprinting. Multiple concentrations, ranging from 3% to 20% gelMA with 0%-1.5% gellan gum, were evaluated for their printability, defined as the ability to form filaments and to incorporate cells at 15 °C-37 °C. To support the printability assessment, yield stress and viscosity of the hydrogels were measured. Stiffness of UV-cured constructs, as well as cartilage-like tissue formation by embedded chondrocytes, were determined in vitro. A large range of gelMA/gellan concentrations were printable with inclusion of cells and formed the bioprinting window. The addition of gellan gum improved filament deposition by inducing yielding behavior, increased construct stiffness and supported chondrogenesis. High gellan gum concentrations, however, did compromise cartilage matrix production and distribution, and even higher concentrations resulted in too high yield stresses to allow cell encapsulation. This study demonstrates the high potential of gelMA/gellan blends for cartilage bioprinting and identifies yield stress as a dominant factor for bioprintability.

  3. First evidence of dinosaurian secondary cartilage in the post-hatching skull of Hypacrosaurus stebingeri (Dinosauria, Ornithischia.

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    Alida M Bailleul

    Full Text Available Bone and calcified cartilage can be fossilized and preserved for hundreds of millions of years. While primary cartilage is fairly well studied in extant and fossilized organisms, nothing is known about secondary cartilage in fossils. In extant birds, secondary cartilage arises after bone formation during embryonic life at articulations, sutures and muscular attachments in order to accommodate mechanical stress. Considering the phylogenetic inclusion of birds within the Dinosauria, we hypothesized a dinosaurian origin for this "avian" tissue. Therefore, histological thin sectioning was used to investigate secondary chondrogenesis in disarticulated craniofacial elements of several post-hatching specimens of the non-avian dinosaur Hypacrosaurus stebingeri (Ornithischia, Lambeosaurinae. Secondary cartilage was found on three membrane bones directly involved with masticatory function: (1 as nodules on the dorso-caudal face of a surangular; and (2 on the bucco-caudal face of a maxilla; and (3 between teeth as islets in the alveolar processes of a dentary. Secondary chondrogenesis at these sites is consistent with the locations of secondary cartilage in extant birds and with the induction of the cartilage by different mechanical factors - stress generated by the articulation of the quadrate, stress of a ligamentous or muscular insertion, and stress of tooth formation. Thus, our study reveals the first evidence of "avian" secondary cartilage in a non-avian dinosaur. It pushes the origin of this "avian" tissue deep into dinosaurian ancestry, suggesting the creation of the more appropriate term "dinosaurian" secondary cartilage.

  4. Alendronate Can Improve Bone Alterations in Experimental Diabetes by Preventing Antiosteogenic, Antichondrogenic, and Proadipocytic Effects of AGEs on Bone Marrow Progenitor Cells

    Science.gov (United States)

    2016-01-01

    Bisphosphonates such as alendronate are antiosteoporotic drugs that inhibit the activity of bone-resorbing osteoclasts and secondarily promote osteoblastic function. Diabetes increases bone-matrix-associated advanced glycation end products (AGEs) that impair bone marrow progenitor cell (BMPC) osteogenic potential and decrease bone quality. Here we investigated the in vitro effect of alendronate and/or AGEs on the osteoblastogenic, adipogenic, and chondrogenic potential of BMPC isolated from nondiabetic untreated rats. We also evaluated the in vivo effect of alendronate (administered orally to rats with insulin-deficient Diabetes) on long-bone microarchitecture and BMPC multilineage potential. In vitro, the osteogenesis (Runx2, alkaline phosphatase, type 1 collagen, and mineralization) and chondrogenesis (glycosaminoglycan production) of BMPC were both decreased by AGEs, while coincubation with alendronate prevented these effects. The adipogenesis of BMPC (PPARγ, intracellular triglycerides, and lipase) was increased by AGEs, and this was prevented by coincubation with alendronate. In vivo, experimental Diabetes (a) decreased femoral trabecular bone area, osteocyte density, and osteoclastic TRAP activity; (b) increased bone marrow adiposity; and (c) deregulated BMPC phenotypic potential (increasing adipogenesis and decreasing osteogenesis and chondrogenesis). Orally administered alendronate prevented all these Diabetes-induced effects on bone. Thus, alendronate could improve bone alterations in diabetic rats by preventing the antiosteogenic, antichondrogenic, and proadipocytic effects of AGEs on BMPC. PMID:27840829

  5. Extracellular matrix and cell surface as determinants of connective tissue differentiation.

    Science.gov (United States)

    Solursh, M

    1989-09-01

    This paper reviews in vitro studies, largely from the author's laboratory, concerning the conditions that are permissive for the differentiation of limb bud mesenchymal cells into chondrocytes. In high-density cell culture, even in a defined medium, the same normal sequence of events that is found in vivo in developing cartilage is also observed. This system can be used to study heritable disorders in model systems such as in mutant mouse embryos. In addition, single mesenchymal cells can differentiate into hypertrophic chondrocytes in hydrated collagen gel or agarose cultures. A rounded cell shape promotes chondrogenesis, while a flattened cell shape promotes fibroblast differentiation. The actin cytoskeleton is shown to play a central role in regulating connective tissue cell differentiation. By use of such cell culture manipulations, it is now possible to grow large numbers of fibroblastic cells from human biopsy material for storage and to carry out experimental studies after re-expression of chondrogenesis in gel cultures. It is suggested that cytoskeletal-extracellular matrix interactions play a fundamental role in connective tissue differentiation. Matrix receptors might be developmentally regulated and modify epithelial effects on mesenchymal cells. In this way mesenchymal cells differentiate in a highly organized manner in spatial and temporal terms.

  6. Fdp, a new fibrocyte-derived protein related to MIA/CD-RAP, has an in vitro effect on the early differentiation of the inner ear mesenchyme.

    Science.gov (United States)

    Cohen-Salmon, M; Frenz, D; Liu, W; Verpy, E; Voegeling, S; Petit, C

    2000-12-22

    During the course of a study aimed at isolating transcripts specifically or preferentially expressed in the inner ear, we identified a novel gene, encoding a fibrocyte-derived protein, that we named Fdp. Fdp is predicted to be a secreted 128-amino acid protein, which is highly homologous to the melanoma-inhibiting activity/cartilage-derived retinoic acid-sensitive protein (MIA/CD-RAP), a cartilage-specific protein also expressed in several tumors. Fdp and MIA/CD-RAP thus define a new family of proteins. Fdp is expressed from embryonic day 10.5 in the mesenchyme surrounding the otic epithelium. During development, these cells progressively aggregate, condense, and differentiate into cartilaginous cells forming the otic capsule, which no longer expresses Fdp, and into fibrocytes surrounding the epithelia, which strongly express Fdp. In order to address the function of Fdp, we developed an in vitro antisense oligonucleotide approach using microdissected periotic mesenchyme micromass cultures, and showed that Fdp antisense oligonucleotide treatment results in a significant reduction in chondrogenesis. Our results demonstrate that Fdp plays a role in the initiation of periotic mesenchyme chondrogenesis. Accordingly, Fdp and its human ortholog FDP, which map to chromosome 2 and band 20p11, respectively, could be candidate genes for forms of deafness associated with malformations of the otic capsule.

  7. Alendronate Can Improve Bone Alterations in Experimental Diabetes by Preventing Antiosteogenic, Antichondrogenic, and Proadipocytic Effects of AGEs on Bone Marrow Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Sara Rocío Chuguransky

    2016-01-01

    Full Text Available Bisphosphonates such as alendronate are antiosteoporotic drugs that inhibit the activity of bone-resorbing osteoclasts and secondarily promote osteoblastic function. Diabetes increases bone-matrix-associated advanced glycation end products (AGEs that impair bone marrow progenitor cell (BMPC osteogenic potential and decrease bone quality. Here we investigated the in vitro effect of alendronate and/or AGEs on the osteoblastogenic, adipogenic, and chondrogenic potential of BMPC isolated from nondiabetic untreated rats. We also evaluated the in vivo effect of alendronate (administered orally to rats with insulin-deficient Diabetes on long-bone microarchitecture and BMPC multilineage potential. In vitro, the osteogenesis (Runx2, alkaline phosphatase, type 1 collagen, and mineralization and chondrogenesis (glycosaminoglycan production of BMPC were both decreased by AGEs, while coincubation with alendronate prevented these effects. The adipogenesis of BMPC (PPARγ, intracellular triglycerides, and lipase was increased by AGEs, and this was prevented by coincubation with alendronate. In vivo, experimental Diabetes (a decreased femoral trabecular bone area, osteocyte density, and osteoclastic TRAP activity; (b increased bone marrow adiposity; and (c deregulated BMPC phenotypic potential (increasing adipogenesis and decreasing osteogenesis and chondrogenesis. Orally administered alendronate prevented all these Diabetes-induced effects on bone. Thus, alendronate could improve bone alterations in diabetic rats by preventing the antiosteogenic, antichondrogenic, and proadipocytic effects of AGEs on BMPC.

  8. Human Stem Cells and Articular Cartilage Regeneration

    Directory of Open Access Journals (Sweden)

    A. Hari Reddi

    2012-11-01

    Full Text Available  The regeneration of articular cartilage damaged due to trauma and posttraumatic osteoarthritis is an unmet medical need. Current approaches to regeneration and tissue engineering of articular cartilage include the use of chondrocytes, stem cells, scaffolds and signals, including morphogens and growth factors. Stem cells, as a source of cells for articular cartilage regeneration, are a critical factor for articular cartilage regeneration. This is because articular cartilage tissue has a low cell turnover and does not heal spontaneously. Adult stem cells have been isolated from various tissues, such as bone marrow, adipose, synovial tissue, muscle and periosteum. Signals of the transforming growth factor beta superfamily play critical roles in chondrogenesis. However, adult stem cells derived from various tissues tend to differ in their chondrogenic potential. Pluripotent stem cells have unlimited proliferative capacity compared to adult stem cells. Chondrogenesis from embryonic stem (ES cells has been studied for more than a decade. However, establishment of ES cells requires embryos and leads to ethical issues for clinical applications. Induced pluripotent stem (iPS cells are generated by cellular reprogramming of adult cells by transcription factors. Although iPS cells have chondrogenic potential, optimization, generation and differentiation toward articular chondrocytes are currently under intense investigation.

  9. Chondrogenic potential of subpopulations of cells expressing mesenchymal stem cell markers derived from human synovial membranes.

    Science.gov (United States)

    Arufe, M C; De la Fuente, A; Fuentes, I; de Toro, F J; Blanco, F J

    2010-11-01

    In this study we analyzed the chondrogenic potential of subpopulations of mesenchymal stem cells (MSCs) derived from human synovial membranes enriched for CD73, CD106, and CD271 markers. Subpopulations of human synovial membrane MSCs enriched for CD73, CD106, and CD271 markers were isolated using a cytometry sorter and characterized by flow cytometry for MSC markers. The expression of Sox9, Nanog, and Runx2 genes by these cells was measured by reverse transcriptase-polymerase chain reaction. The chondrogenesis of each subpopulation was assessed by culturing the cells in a defined medium to produce spontaneous spheroid formation and differentiation towards chondrocyte-like cells. The examination of the spheroids by histological and immunohistochemical analyses for collagen type II (COL2), aggrecan, collagen type I (COL1), metalloprotease 13 (MMP13), and collagen type X (COLX) levels were performed to assess their chondrogenesis capacity. The adipogenesis and osteogenesis potential of each subpopulation was determined using commercial media; the resulting cells were stained with oil red O or red alizarin to test the degree of differentiation. The subpopulations had different profiles of cells positive for the MSC markers CD44, CD69, CD73, CD90, and CD105 and showed different expression levels of the genes Sox9, Nanog, and Runx2 involved in chondrogenesis, undifferentiation, and osteoblastogenesis, respectively. Immunohistochemical analysis demonstrated that COL1, COL2, COLX, MMP13, and aggrecan were expressed in the spheroids as soon as 14 days of culture. The CD271(+) subpopulation expressed the highest levels of COL2 staining compared to the other subpopulations. CD105 and Runx2 were shown by immunohistochemistry and genetic analysis to have significantly higher expression CD271(+) subpopulation than the other subpopulations. Spheroids formed from CD271-enriched and CD73-enriched MSCs from normal human synovial membranes mimic the native cartilage extracellular

  10. Fibroblast growth factor expression in the postnatal growth plate.

    Science.gov (United States)

    Lazarus, Jacob E; Hegde, Anita; Andrade, Anenisia C; Nilsson, Ola; Baron, Jeffrey

    2007-03-01

    Fibroblast growth factor (FGF) signaling is essential for endochondral bone formation. Mutations cause skeletal dysplasias including achondroplasia, the most common human skeletal dysplasia. Most previous work in this area has focused on embryonic chondrogenesis. To explore the role of FGF signaling in the postnatal growth plate, we quantitated expression of FGFs and FGF receptors (FGFRs) and examined both their spatial and temporal regulation. Toward this aim, rat proximal tibial growth plates and surrounding tissues were microdissected, and specific mRNAs were quantitated by real-time RT-PCR. To assess the FGF system without bias, we first screened for expression of all known FGFs and major FGFR isoforms. Perichondrium expressed FGFs 1, 2, 6, 7, 9, and 18 and, at lower levels, FGFs 21 and 22. Growth plate expressed FGFs 2, 7, 18, and 22. Perichondrial expression was generally greater than growth plate expression, supporting the concept that perichondrial FGFs regulate growth plate chondrogenesis. Nevertheless, FGFs synthesized by growth plate chondrocytes may be physiologically important because of their proximity to target receptors. In growth plate, we found expression of FGFRs 1, 2, and 3, primarily, but not exclusively, the c isoforms. FGFRs 1 and 3, thought to negatively regulate chondrogenesis, were expressed at greater levels and at later stages of chondrocyte differentiation, with FGFR1 upregulated in the hypertrophic zone and FGFR3 upregulated in both proliferative and hypertrophic zones. In contrast, FGFRs 2 and 4, putative positive regulators, were expressed at earlier stages of differentiation, with FGFR2 upregulated in the resting zone and FGFR4 in the resting and proliferative zones. FGFRL1, a presumed decoy receptor, was expressed in the resting zone. With increasing age and decreasing growth velocity, FGFR2 and 4 expression was downregulated in proliferative zone. Perichondrial FGF1, FGF7, FGF18, and FGF22 were upregulated. In summary, we have

  11. Culture temperature affects human chondrocyte messenger RNA expression in monolayer and pellet culture systems.

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    Akira Ito

    Full Text Available Cell-based therapy has been explored for articular cartilage regeneration. Autologous chondrocyte implantation is a promising cell-based technique for repairing articular cartilage defects. However, there are several issues such as chondrocyte de-differentiation. While numerous studies have been designed to overcome some of these issues, only a few have focused on the thermal environment that can affect chondrocyte metabolism and phenotype. In this study, the effects of different culture temperatures on human chondrocyte metabolism- and phenotype-related gene expression were investigated in 2D and 3D environments. Human chondrocytes were cultured in a monolayer or in a pellet culture system at three different culture temperatures (32°C, 37°C, and 41°C for 3 days. The results showed that the total RNA level, normalized to the threshold cycle value of internal reference genes, was higher at lower temperatures in both culture systems. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH and citrate synthase (CS, which are involved in glycolysis and the citric acid cycle, respectively, were expressed at similar levels at 32°C and 37°C in pellet cultures, but the levels were significantly lower at 41°C. Expression of the chondrogenic markers, collagen type IIA1 (COL2A1 and aggrecan (ACAN, was higher at 37°C than at 32°C and 41°C in both culture systems. However, this phenomenon did not coincide with SRY (sex-determining region Y-box 9 (SOX9, which is a fundamental transcription factor for chondrogenesis, indicating that a SOX9-independent pathway might be involved in this phenomenon. In conclusion, the expression of chondrocyte metabolism-related genes at 32°C was maintained or enhanced compared to that at 37°C. However, chondrogenesis-related genes were further induced at 37°C in both culture systems. Therefore, manipulating the culture temperature may be an advantageous approach for regulating human chondrocyte metabolic activity and

  12. Zebrafish con/disp1 reveals multiple spatiotemporal requirements for Hedgehog-signaling in craniofacial development

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    Schwend Tyler

    2009-11-01

    Full Text Available Abstract Background The vertebrate head skeleton is derived largely from cranial neural crest cells (CNCC. Genetic studies in zebrafish and mice have established that the Hedgehog (Hh-signaling pathway plays a critical role in craniofacial development, partly due to the pathway's role in CNCC development. Disruption of the Hh-signaling pathway in humans can lead to the spectral disorder of Holoprosencephaly (HPE, which is often characterized by a variety of craniofacial defects including midline facial clefting and cyclopia 12. Previous work has uncovered a role for Hh-signaling in zebrafish dorsal neurocranium patterning and chondrogenesis, however Hh-signaling mutants have not been described with respect to the ventral pharyngeal arch (PA skeleton. Lipid-modified Hh-ligands require the transmembrane-spanning receptor Dispatched 1 (Disp1 for proper secretion from Hh-synthesizing cells to the extracellular field where they act on target cells. Here we study chameleon mutants, lacking a functional disp1(con/disp1. Results con/disp1 mutants display reduced and dysmorphic mandibular and hyoid arch cartilages and lack all ceratobranchial cartilage elements. CNCC specification and migration into the PA primorida occurs normally in con/disp1 mutants, however disp1 is necessary for post-migratory CNCC patterning and differentiation. We show that disp1 is required for post-migratory CNCC to become properly patterned within the first arch, while the gene is dispensable for CNCC condensation and patterning in more posterior arches. Upon residing in well-formed pharyngeal epithelium, neural crest condensations in the posterior PA fail to maintain expression of two transcription factors essential for chondrogenesis, sox9a and dlx2a, yet continue to robustly express other neural crest markers. Histology reveals that posterior arch residing-CNCC differentiate into fibrous-connective tissue, rather than becoming chondrocytes. Treatments with Cyclopamine, to

  13. Surface Markers for Chondrogenic Determination: A Highlight of Synovium-Derived Stem Cells

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    Douglas D. Campbell

    2012-11-01

    Full Text Available Cartilage tissue engineering is a promising field in regenerative medicine that can provide substantial relief to people suffering from degenerative cartilage disease. Current research shows the greatest chondrogenic potential for healthy articular cartilage growth with minimal hypertrophic differentiation to be from mesenchymal stem cells (MSCs of synovial origin. These stem cells have the capacity for differentiation into multiple cell lineages related to mesenchymal tissue; however, evidence exists for cell surface markers that specify a greater potential for chondrogenesis than other differentiation fates. This review will examine relevant literature to summarize the chondrogenic differentiation capacities of tested synovium-derived stem cell (SDSC surface markers, along with a discussion about various other markers that may hold potential, yet require further investigation. With this information, a potential clinical benefit exists to develop a screening system for SDSCs that will produce the healthiest articular cartilage possible.

  14. A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid.

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    Chopin, Nathalie; Sinquin, Corinne; Ratiskol, Jacqueline; Zykwinska, Agata; Weiss, Pierre; Cérantola, Stéphane; Le Bideau, Jean; Colliec-Jouault, Sylvia

    2015-01-01

    GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS) was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG-) like compound was modified in a classical solvent (N,N'-dimethylformamide). However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation) was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR) was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

  15. Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro

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    Achim Salamon

    2014-02-01

    Full Text Available Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.

  16. Analyzing the cellular contribution of bone marrow to fracture healing using bone marrow transplantation in mice.

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    Colnot, C; Huang, S; Helms, J

    2006-11-24

    The bone marrow is believed to play important roles during fracture healing such as providing progenitor cells for inflammation, matrix remodeling, and cartilage and bone formation. Given the complex nature of bone repair, it remains difficult to distinguish the contributions of various cell types. Here we describe a mouse model based on bone marrow transplantation and genetic labeling to track cells originating from bone marrow during fracture healing. Following lethal irradiation and engraftment of bone marrow expressing the LacZ transgene constitutively, wild type mice underwent tibial fracture. Donor bone marrow-derived cells, which originated from the hematopoietic compartment, did not participate in the chondrogenic and osteogenic lineages during fracture healing. Instead, the donor bone marrow contributed to inflammatory and bone resorbing cells. This model can be exploited in the future to investigate the role of inflammation and matrix remodeling during bone repair, independent from osteogenesis and chondrogenesis.

  17. REGENERATION OF ARTICULAR CARTILAGE UNDER THE IMPLANTATION OF BONE MATRIX

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    Yuri M. Iryanov, Nikolay A. Kiryanov, Olga V. Dyuriagina , Tatiana Yu. Karaseva, Evgenii A. Karasev

    2015-07-01

    Full Text Available Background: The damage or loss of articular cartilage is costly medical problem. The purpose of this work – morphological analysis of reparative chondrogenesis when implanted in the area of the knee joint cartilage of granulated mineralized bone matrix. Material and Methods: The characteristic features of the knee cartilage regeneration studied experimentally in pubertal Wistar rats after modeling a marginal perforated defect and implantation of granulated mineralized bone matrix obtained according to original technology without heat and demineralizing processing into the injury zone. Results: This biomaterial established to have pronounced chondro- and osteoinductive properties, and to provide prolonged activation of reparative process, accelerated organotypical remodeling and restoration of the articular cartilage injured. Conclusion: The data obtained demonstrate the efficacy of МВМ in clinical practice for the treatment of diseases and injuries of the articular cartilage.

  18. Genetic Influences on Temporomandibular Joint Development and Growth.

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    Hinton, Robert J; Jing, Junjun; Feng, Jian Q

    2015-01-01

    The temporomandibular joint (TMJ) is a small synovial joint at which the mandible articulates with the skull during movements involved in speaking and mastication. However, the secondary cartilage lining its joint surfaces is indicative of a very different developmental history than limb cartilages. This review summarizes our current knowledge of genes that regulate the formation of primary components of the TMJ, as well as genes that regulate postnatal growth of the TMJ. Although the TMJ is regulated by some of the same genes that are important in limb joints, others appear unique to the TMJ or have different actions. Runx2, Sox9, and members of the TGF-β/BMP family are critical drivers of chondrogenesis during condylar cartilage morphogenesis, and Indian hedgehog (Ihh) is important for formation of the articular disc and cavitation. Osterix (Osx) is a critical regulator of endochondral bone formation during postnatal TMJ growth.

  19. Temporal evolution of mechanical properties of skeletal tissue regeneration in rabbits. An experimental study

    CERN Document Server

    Mokoko, Didier; Chabrand, Patrick

    2007-01-01

    Various mathematical models represent the effects of local mechanical environment on the regulation of skeletal regeneration. Their relevance relies on an accurate description of the evolving mechanical properties of the regenerating tissue. The object of this study was to develop an experimental model which made it possible to characterize the temporal evolution of the structural and mechanical properties during unloaded enchondral osteogenesis in the New Zealand rabbit, a standard animal model for studies of osteogenesis and chondrogenesis. A 25mm segment of tibial diaphysis was removed sub-periosteally from rabbits. The defect was repaired by the preserved periosteum. An external fixator was applied to prevent mechanical loading during osteogenesis. The regenerated skeletal tissues were studied by CT scan, histology and mechanical tests. The traction tests between 7 to 21 days post-surgery were done on formaldehyde-fixated tissue allowing to obtain force/displacement curves. The viscoelastic properties of ...

  20. Indirect coculture of stem cells with fetal chondrons using PCL electrospun nanofiber scaffolds.

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    Nikpou, Parisa; Soleimani Rad, Jafar; Mohammad Nejad, Daryoush; Samadi, Nasser; Roshangar, Leila; Navali, Amir Mohammad; Shafaei, Hajar; Nozad Charoudeh, Hojjatollah; Danandeh Oskoei, Neda; Soleimani Rad, Sara

    2017-03-01

    In vitro coculture system provides a powerful tool for tissue engineering. In this study, we evaluated the gene expressions of human adipose-derived stem cells (ASCs) on polycaprolactone (PCL) scaffold in coculture model with fetal chondrons. Electrospun PCL scaffolds (900 nm fiber diameter) were created and human infrapatellar fat pad-adipose-derived stem cells (IPFP-ASCs) were seeded on these scaffolds. Scanning electron microscopy (SEM) showed attachment of human IPFP-ASCs to scaffold. IPFP-ASCs on scaffolds were cocultured with fetal chondrons in transwell. Gene expressions were investigated using real-time polymerase chain reaction (real-time PCR). In comparison with control group, the expression level of collagen type 2 and aggrecan were significantly decreased but Indian Hedgehog(IHH) significantly increased (P fetal chondrons are tending toward osteogenesis rather than chondrogenesis.

  1. Critical review on the physical and mechanical factors involved in tissue engineering of cartilage.

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    Gaut, Carrie; Sugaya, Kiminobu

    2015-01-01

    Articular cartilage defects often progress to osteoarthritis, which negatively impacts quality of life for millions of people worldwide and leads to high healthcare expenditures. Tissue engineering approaches to osteoarthritis have concentrated on proliferation and differentiation of stem cells by activation and suppression of signaling pathways, and by using a variety of scaffolding techniques. Recent studies indicate a key role of environmental factors in the differentiation of mesenchymal stem cells to mature cartilage-producing chondrocytes. Therapeutic approaches that consider environmental regulation could optimize chondrogenesis protocols for regeneration of articular cartilage. This review focuses on the effect of scaffold structure and composition, mechanical stress and hypoxia in modulating mesenchymal stem cell fate and the current use of these environmental factors in tissue engineering research.

  2. GEP, a Local Growth Factor, is Critical for Odontogenesis and Amelogenesis

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    Zhengguo Cao, Baichun Jiang, Yixia Xie, Chuan-ju Liu, Jian Q. Feng

    2010-01-01

    Full Text Available Granulin epithelin precursor (GEP is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic mice led to a reduction of dentin thickness, an increase in predentin thickness, and a reduction in mineral content in enamel. The in vitro application of recombinant GEP up-regulated molecular markers important for odontogenesis (DMP1, DSPP, and ALP and amelogenesis (ameloblastin, amelogenin and enamelin. In conclusion, both the in vivo and the in vivo data support an important role of GEP in tooth formation during postnatal development.

  3. GEP, a local growth factor, is critical for odontogenesis and amelogenesis.

    Science.gov (United States)

    Cao, Zhengguo; Jiang, Baichun; Xie, Yixia; Liu, Chuan-ju; Feng, Jian Q

    2010-11-25

    Granulin epithelin precursor (GEP) is a new growth factor that functions in brain development, chondrogenesis, tissue regeneration, tumorigenesis, and inflammation. The goal of this study was to study whether GEP was critical for odontogenesis and amelogenesis both in vivo and in vitro. The in situ hybridization and immunohistochemistry data showed that GEP was expressed in both odontoblast and ameloblast cells postnatally. Knockdown of GEP by crossing U6-ploxPneo-GEP and Sox2-Cre transgenic mice led to a reduction of dentin thickness, an increase in predentin thickness, and a reduction in mineral content in enamel. The in vitro application of recombinant GEP up-regulated molecular markers important for odontogenesis (DMP1, DSPP, and ALP) and amelogenesis (ameloblastin, amelogenin and enamelin). In conclusion, both the in vivo and the in vivo data support an important role of GEP in tooth formation during postnatal development.

  4. Multiscale dynamics of biological cells with chemotactic interactions: from a discrete stochastic model to a continuous description

    CERN Document Server

    Alber, M; Glimm, T; Lushnikov, P M; Alber, Mark; Chen, Nan; Glimm, Tilmann; Lushnikov, Pavel M.

    2006-01-01

    The Cellular Potts Model (CPM) has been used for simulating various biological phenomena such as differential adhesion, fruiting body formation of the slime mold Dictyostelium discoideum, angiogenesis, cancer invasion, chondrogenesis in embryonic vertebrate limbs, and many others. In this paper, we derive continuous limit of discrete one dimensional CPM with the chemotactic interactions between cells in the form of a Fokker-Planck equation for the evolution of the cell probability density function. This equation is then reduced to the classical macroscopic Keller-Segel model. In particular, all coefficients of the Keller-Segel model are obtained from parameters of the CPM. Theoretical results are verified numerically by comparing Monte Carlo simulations for the CPM with numerics for the Keller-Segel model.

  5. Effects of exposure to a DNA damaging agent on the hypoxia inducible factors in organogenesis stage mouse limbs.

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    Chunwei Huang

    Full Text Available Hypoxia plays a critical role in coordinating cell survival, differentiation and death in normal embryogenesis; during limb pattern formation, hypoxia affects two key processes, chondrogenesis and cell death. Hypoxia promotes chondrocyte differentiation and cartilage matrix synthesis and suppresses terminal differentiation. Depending on the context, hypoxia may induce cell cycle arrest, pro- or anti-apoptotic genes, or autophagy. The response to hypoxia is controlled by hypoxia inducible transcription factors, specifically Hif1a, Hif2a and Hif3a. Under normoxia, the hypoxia-inducible factors respond to a variety of stimuli that include several well established teratogens, such as retinoic acid, heavy metals and hyperglycemia. We hypothesize that teratogenic exposures disrupt limb development by altering the hypoxia signalling pathway. To test this hypothesis, we assessed the effects of a DNA damaging alkylating agent, 4-hydroperoxycyclophosphamide, on the hypoxia inducible factor (HIF transcription factors and on hypoxia in the murine limb bud culture system. 4-Hydroperoxycyclophosphamide exposure increased HIF1 DNA binding activity and HIF1A and HIF2A, but not HIF3A, protein concentrations. HIF1A and HIF2A immunoreactivities were detected in the apical ectodermal ridge and interdigital regions, where cell death sculpts the limb; 4-hydroperoxycyclophosphamide treatment enhanced their immunoreactivities, specifically in these regions. In contrast, hypoxia was localized to areas of chondrogenesis, the cartilaginous anlagen of the developing long bone and phalanges, and was not enhanced by drug exposure. Thus, the exposure of limb buds in vitro to a DNA damaging teratogen triggered a hypoxia signalling response that was associated with cell death. During limb development the HIFs have oxygen-independent functions.

  6. Ultra-structural changes and expression of chondrogenic and hypertrophic genes during chondrogenic differentiation of mesenchymal stromal cells in alginate beads

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    Havva Dashtdar

    2016-03-01

    Full Text Available Chondrogenic differentiation of mesenchymal stromal cells (MSCs in the form of pellet culture and encapsulation in alginate beads has been widely used as conventional model for in vitro chondrogenesis. However, comparative characterization between differentiation, hypertrophic markers, cell adhesion molecule and ultrastructural changes during alginate and pellet culture has not been described. Hence, the present study was conducted comparing MSCs cultured in pellet and alginate beads with monolayer culture. qPCR was performed to assess the expression of chondrogenic, hypertrophic, and cell adhesion molecule genes, whereas transmission electron microscopy (TEM was used to assess the ultrastructural changes. In addition, immunocytochemistry for Collagen type II and aggrecan and glycosaminoglycan (GAG analysis were performed. Our results indicate that pellet and alginate bead cultures were necessary for chondrogenic differentiation of MSC. It also indicates that cultures using alginate bead demonstrated significantly higher (p < 0.05 chondrogenic but lower hypertrophic (p < 0.05 gene expressions as compared with pellet cultures. N-cadherin and N-CAM1 expression were up-regulated in second and third weeks of culture and were comparable between the alginate bead and pellet culture groups, respectively. TEM images demonstrated ultrastructural changes resembling cell death in pellet cultures. Our results indicate that using alginate beads, MSCs express higher chondrogenic but lower hypertrophic gene expression. Enhanced production of extracellular matrix and cell adhesion molecules was also observed in this group. These findings suggest that alginate bead culture may serve as a superior chondrogenic model, whereas pellet culture is more appropriate as a hypertrophic model of chondrogenesis.

  7. Swim-training changes the spatio-temporal dynamics of skeletogenesis in zebrafish larvae (Danio rerio).

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    Fiaz, Ansa W; Léon-Kloosterziel, Karen M; Gort, Gerrit; Schulte-Merker, Stefan; van Leeuwen, Johan L; Kranenbarg, Sander

    2012-01-01

    Fish larvae experience many environmental challenges during development such as variation in water velocity, food availability and predation. The rapid development of structures involved in feeding, respiration and swimming increases the chance of survival. It has been hypothesized that mechanical loading induced by muscle forces plays a role in prioritizing the development of these structures. Mechanical loading by muscle forces has been shown to affect larval and embryonic bone development in vertebrates, but these investigations were limited to the appendicular skeleton. To explore the role of mechanical load during chondrogenesis and osteogenesis of the cranial, axial and appendicular skeleton, we subjected zebrafish larvae to swim-training, which increases physical exercise levels and presumably also mechanical loads, from 5 until 14 days post fertilization. Here we show that an increased swimming activity accelerated growth, chondrogenesis and osteogenesis during larval development in zebrafish. Interestingly, swim-training accelerated both perichondral and intramembranous ossification. Furthermore, swim-training prioritized the formation of cartilage and bone structures in the head and tail region as well as the formation of elements in the anal and dorsal fins. This suggests that an increased swimming activity prioritized the development of structures which play an important role in swimming and thereby increasing the chance of survival in an environment where water velocity increases. Our study is the first to show that already during early zebrafish larval development, skeletal tissue in the cranial, axial and appendicular skeleton is competent to respond to swim-training due to increased water velocities. It demonstrates that changes in water flow conditions can result into significant spatio-temporal changes in skeletogenesis.

  8. Fibroblast-Derived Extracellular Matrix Induces Chondrogenic Differentiation in Human Adipose-Derived Mesenchymal Stromal/Stem Cells in Vitro

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    Kevin Dzobo

    2016-08-01

    Full Text Available Mesenchymal stromal/stem cells (MSCs represent an area being intensively researched for tissue engineering and regenerative medicine applications. MSCs may provide the opportunity to treat diseases and injuries that currently have limited therapeutic options, as well as enhance present strategies for tissue repair. The cellular environment has a significant role in cellular development and differentiation through cell–matrix interactions. The aim of this study was to investigate the behavior of adipose-derived MSCs (ad-MSCs in the context of a cell-derived matrix so as to model the in vivo physiological microenvironment. The fibroblast-derived extracellular matrix (fd-ECM did not affect ad-MSC morphology, but reduced ad-MSC proliferation. Ad-MSCs cultured on fd-ECM displayed decreased expression of integrins α2 and β1 and subsequently lost their multipotency over time, as shown by the decrease in CD44, Octamer-binding transcription factor 4 (OCT4, SOX2, and NANOG gene expression. The fd-ECM induced chondrogenic differentiation in ad-MSCs compared to control ad-MSCs. Loss of function studies, through the use of siRNA and a mutant Notch1 construct, revealed that ECM-mediated ad-MSCs chondrogenesis requires Notch1 and β-catenin signaling. The fd-ECM also showed anti-senescence effects on ad-MSCs. The fd-ECM is a promising approach for inducing chondrogenesis in ad-MSCs and chondrogenic differentiated ad-MSCs could be used in stem cell therapy procedures.

  9. Chondrogenic differentiation of human subchondral progenitor cells is affected by synovial fluid from donors with osteoarthritis or rheumatoid arthritis

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    Krüger Jan

    2012-03-01

    Full Text Available Abstract Background Microfracture is a first-line treatment option for cartilage repair. In microfracture, subchondral mesenchymal cortico-spongious progenitor cells (CSP enter the defect and form cartilage repair tissue. The aim of our study was to investigate the effects of joint disease conditions on the in vitro chondrogenesis of human CSP. Methods CSP were harvested from the subchondral bone marrow. CSP characterization was performed by analysis of cell surface antigen pattern and by assessing the chondrogenic, osteogenic and adipogenic differentiation potential, histologically. To assess the effect of synovial fluid (SF on chondrogenesis of CSP, micro-masses were stimulated with SF from healthy (ND, osteoarthritis (OA and rheumatoid arthritis donors (RA without transforming growth factor beta 3. Results CSP showed the typical cell surface antigen pattern known from mesenchymal stem cells and were capable of osteogenic, adipogenic and chondrogenic differentiation. In micro-masses stimulated with SF, histological staining as well as gene expression analysis of typical chondrogenic marker genes showed that SF from ND and OA induced the chondrogenic marker genes aggrecan, types II and IX collagen, cartilage oligomeric matrix protein (COMP and link protein, compared to controls not treated with SF. In contrast, the supplementation with SF from RA donors decreased the expression of aggrecan, type II collagen, COMP and link protein, compared to CSP treated with SF from ND or OA. Conclusion These results suggest that in RA, SF may impair cartilage repair by subchondral mesenchymal progenitor cells in microfracture, while in OA, SF may has no negative, but a delaying effect on the cartilage matrix formation.

  10. Forelimb-hindlimb developmental timing changes across tetrapod phylogeny

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    Selwood Lynne

    2007-10-01

    Full Text Available Abstract Background Tetrapods exhibit great diversity in limb structures among species and also between forelimbs and hindlimbs within species, diversity which frequently correlates with locomotor modes and life history. We aim to examine the potential relation of changes in developmental timing (heterochrony to the origin of limb morphological diversity in an explicit comparative and quantitative framework. In particular, we studied the relative time sequence of development of the forelimbs versus the hindlimbs in 138 embryos of 14 tetrapod species spanning a diverse taxonomic, ecomorphological and life-history breadth. Whole-mounts and histological sections were used to code the appearance of 10 developmental events comprising landmarks of development from the early bud stage to late chondrogenesis in the forelimb and the corresponding serial homologues in the hindlimb. Results An overall pattern of change across tetrapods can be discerned and appears to be relatively clade-specific. In the primitive condition, as seen in Chondrichthyes and Osteichthyes, the forelimb/pectoral fin develops earlier than the hindlimb/pelvic fin. This pattern is either retained or re-evolved in eulipotyphlan insectivores (= shrews, moles, hedgehogs, and solenodons and taken to its extreme in marsupials. Although exceptions are known, the two anurans we examined reversed the pattern and displayed a significant advance in hindlimb development. All other species examined, including a bat with its greatly enlarged forelimbs modified as wings in the adult, showed near synchrony in the development of the fore and hindlimbs. Conclusion Major heterochronic changes in early limb development and chondrogenesis were absent within major clades except Lissamphibia, and their presence across vertebrate phylogeny are not easily correlated with adaptive phenomena related to morphological differences in the adult fore- and hindlimbs. The apparently conservative nature of this

  11. Purinergic Signaling Regulates the Transforming Growth Factor-β3-Induced Chondrogenic Response of Mesenchymal Stem Cells to Hydrostatic Pressure.

    Science.gov (United States)

    Steward, Andrew J; Kelly, Daniel J; Wagner, Diane R

    2016-06-01

    Although hydrostatic pressure (HP) is known to regulate chondrogenic differentiation of mesenchymal stromal/stem cells (MSCs), improved insight into the mechanotransduction of HP may form the basis for novel tissue engineering strategies. Previously, we demonstrated that matrix stiffness and calcium ion (Ca(++)) mobility regulate the mechanotransduction of HP; however, the mechanisms, by which these Ca(++) signaling pathways are initiated, are currently unknown. The purinergic pathway, in which adenosine triphosphate (ATP) is released and activates P-receptors to initiate Ca(++) signaling, plays a key role in the mechanotransduction of compression, but has yet to be investigated with regard to HP. Therefore, the objective of this study was to investigate the interplay between purinergic signaling, matrix stiffness, and the chondrogenic response of MSCs to HP. Porcine bone marrow-derived MSCs were seeded into soft or stiff agarose hydrogels and subjected to HP (10 MPa at 1 Hz for 4 h/d for 21 days) or kept in free swelling conditions. Stiff constructs were incubated with pharmacological inhibitors of extracellular ATP, P2 receptors, or hemichannels, or without any inhibitors as a control. As with other loading modalities, HP significantly increased ATP release in the control group; however, inhibition of hemichannels completely abrogated this response. The increase in sulfated glycosaminoglycan (sGAG) synthesis and vimentin reorganization observed in the control group in response to HP was suppressed in the presence of all three inhibitors, suggesting that purinergic signaling is involved in the mechanoresponse of MSCs to HP. Interestingly, ATP was released from both soft and stiff hydrogels in response to HP, but HP only enhanced chondrogenesis in the stiff hydrogels, indicating that matrix stiffness may act downstream of purinergic signaling to regulate the mechanoresponse of MSCs to HP. Addition of exogenous ATP did not replicate the effects of HP on

  12. Cartilage Tissue Engineering via Avocado/Soybean Unsaponifible and Human Adipose Derived Stem Cells on Poly (lactide-co–glycolide /Hyaluronic acid composite scaffold

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    Zynolabedin Sharifian

    2016-09-01

    Full Text Available Background: Growth factors and chemical stimulants have key role in stem cell to chondrocyte differentiation in cartilage tissue engineering, but this agents have adverse effects on cells as well as they are expensive and they have short half time. Todays there is great interest in the application of herbal agent for treatment of diseases.Avocado/soybean unsaponifiable (ASU with herbal components has chondroprotective, anti-inflammatory and pro-anabolic effects that it causes stimulate of deposition of extracellular matrix in chondrocytes and relief of osteoarthritis. The aim of this study was an investigation of the chondrogenic effect of ASU in human adipose derived stem cells (hADSCs on PLGA/HA scaffold. Materials and Methods: The 3-D scaffold of Poly lactide-co –glycolide acid (PLGA prepared via solvent/casting leaching method and impregnated with hyaluronic acid to produce composite scaffold. The characterizations of the scaffold, such as surfaces morphology were observed by scanning electron microscopy (SEM and the degradation behaviour of the composite scaffold were evaluated. hADSCs seeded in PLGA/HA scaffold and cultured in chondrogenic media with and without ASU. The expression of chondrogenic related genes (Sox9, type II collagen, Aggrecan and hypertrophic marker (type X collagen were quantified by real time PCR and viability of cells in different groups were assessed by MTT. Results: Our results showed that the expression of genes related chondrogenesis markers Sox9 and type II collagen and aggrecan in differentiated cells in the presence of ASU were significantly increased compared with the control groups (P<0.05, on the other hand, type X collagen expression was not significantly increased. Conclusions: Our results indicated that ASU could be as an appropriate inducer for chondrogenesis of hADSCs and cartilage tissue engineering.

  13. Identification and characterization of chondrogenic progenitor cells in the fascia of postnatal skeletal muscle

    Institute of Scientific and Technical Information of China (English)

    Guangheng Li; Bo Zheng; Laura B. Meszaros; Joseph B. Vella; Arvydas Usas; Tomoyuki Matsumoto; Johnny Huard

    2011-01-01

    Intramuscular injection of bone morphogenetic proteins (BMPs) has been shown to induce ectopic bone formation.A chondrogenic phase is typically observed in this process,which suggests that there may exist a chondrogenic subpopulation of cells residing in skeletal muscle.Two prospective cell populations were isolated from rat skeletal muscle:fascia-derived cells (FDCs),extracted from gluteus maximus muscle fascia (epimysium) and muscle-derived cells (MDCs) isolated from the muscle body.Both populations were investigated for their cell surface marker profiles (flowcytometry analysis),proliferation rates as well as their myogenic and chondrogenic potentials.The majority of FDCs expressed mesenchymai stromai cell markers but not endothelial cell markers.FDCs underwent chondrogenic differentiation after BMP4 treatment in vitro,but not myogenic differentiation.Although MDCs showed chondrogenic potential,they expressed the myogenic cell marker desmin and readily underwent myogenic differentiation in vitro; however,the chondrogenic potential of the MDCs is confounded by the presence of FDC-like cells residing in the muscle perimysium and endomysium.To clarify the role of the muscle-derived myogenic cells in chondrogenesis,mixed pellets with varying ratios of FDCs and L6 myoblasts were formed and studied for chondrogenic potential.Our results indicated that the chondrogenic potential of the mixed pellets decreased with the increased ratio of myogenic cells to FDCs supporting the role of FDCs in chondrogenesis.Taken together,our results suggest that non-myogenic cells residing in the fascia of skeletal muscle have a strong chondrogenic potential and may represent a novel donor cell source for cartilage regeneration and repair.

  14. Mangiferin reduces the inhibition of chondrogenic differentiation by IL-1β in mesenchymal stem cells from subchondral bone and targets multiple aspects of the Smad and SOX9 pathways.

    Science.gov (United States)

    Huh, Jeong-Eun; Koh, Pil-Seong; Seo, Byung-Kwan; Park, Yeon-Chul; Baek, Yong-Hyun; Lee, Jae-Dong; Park, Dong-Suk

    2014-09-11

    Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs) from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β). Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF)-β, bone morphogenetic protein (BMP)-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y-box (SRY-box) containing gene 9 (SOX9), type 2α1 collagen (Col2α1), cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP)-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5). Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.

  15. Mangiferin Reduces the Inhibition of Chondrogenic Differentiation by IL-1β in Mesenchymal Stem Cells from Subchondral Bone and Targets Multiple Aspects of the Smad and SOX9 Pathways

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    Jeong-Eun Huh

    2014-09-01

    Full Text Available Mangiferin is a natural immunomodulator found in plants including mango trees. The effects of mangiferin on chondrogenesis and cartilage repair have not yet been reported. This study was designed to determine the effect of mangiferin on chondrogenic differentiation in IL-1β-stimulated mesenchymal stem cells (MSCs from subchondral bone and to explore the mechanisms underlying these effects. MSCs were isolated from the subchondral bone of rabbit and treated with mangiferin alone and/or interleukin-1β (IL-1β. Mangiferin induced chondrogenic differentiation in MSCs by upregulating transforming growth factor (TGF-β, bone morphogenetic protein (BMP-2, and BMP-4 and several key markers of chondrogenesis, including sex-determining region Y–box (SRY-box containing gene 9 (SOX9, type 2α1 collagen (Col2α1, cartilage link protein, and aggrecan. In IL-1β-stimulated MSCs, mangiferin significantly reversed the production of TGF-β, BMP-2, BMP-4, SOX9, Col2α1, cartilage link protein, and aggrecan, as well as matrix metalloproteinase (MMP-1, MMP-13, and a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS5. Mangiferin upregulated the phosphorylation of Smad 2, Smad 3, Smad 1/5/8, and SOX9 in IL-1β-stimulated MSCs. In the presence of mangiferin, SOX9 siRNA suppressed the activation of Smad 2, Smad 3, Smad 1/5/8, aggrecan, and Col2α1 expression. In conclusion, mangiferin exhibits both chondrogenic and chondroprotective effects on damaged MSCs and mediates these effects by targeting multiple aspects of the Smad and SOX9 signaling pathways.

  16. Transient expression of a cell surface heparan sulfate proteoglycan (syndecan) during limb development.

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    Solursh, M; Reiter, R S; Jensen, K L; Kato, M; Bernfield, M

    1990-07-01

    Syndecan is an integral membrane proteoglycan that contains both heparan sulfate and chondroitin sulfate chains and that links the cytoskeleton to interstitial extracellular matrix components, including collagen and fibronectin. Immunohistochemistry with a monoclonal antibody directed to the core protein of the syndecan ectodomain has been used to analyze the distribution of this proteoglycan in the developing mouse limb bud and in high-density cultures of limb mesenchyme cells. By Day 9 of gestation when the limb buds are just apparent, syndecan is detected on cells throughout the limb region, including both ectodermal and mesenchymal components. This distribution does not change as the limb bud elongates along its proximodistal axis, except for its reduction in the apical ectodermal ridge. By Day 11, the intensity of immunofluorescence in the central core decreases relative to other regions. By Day 13 immunostaining is lost in the regions destined for chondrogenesis and myogenesis but persists in the limb ectoderm and peripheral and distal mesenchyme. In the limb mesenchyme cell cultures, syndecan is initially undetected, but is found throughout the culture by 24 hr. With further culture the antigen becomes reduced in chondrogenic foci and in association with myogenic cells. When chick limb ectoderm is placed on the high-density cultures, immunoreactivity in the mouse mesenchyme is enhanced suggesting that epithelial-mesenchymal interactions modulate syndecan expression in the limb bud. Based on analysis of 35S-labeled syndecan from the cultures, syndecan from limb mesenchyme cells contains more glycosaminoglycan chains and is larger in size than the previously described polymorphic forms of syndecan from various epithelia. The high affinity of syndecan for components of the extracellular matrix and its distribution in the early limb bud are consistent with a role in maintaining the morphologic integrity of the limb bud during the period of initiation and rapid

  17. Peptide-functionalized starPEG/heparin Hydrogels Direct Mitogenicity, Cell Morphology and Cartilage Matrix Distribution in vitro and in vivo.

    Science.gov (United States)

    Hesse, Eliane; Freudenberg, Uwe; Niemietz, Thomas; Greth, Carina; Weisser, Melanie; Hagmann, Sébastien; Binner, Marcus; Werner, Carsten; Richter, Wiltrud

    2017-01-13

    Cell-based tissue engineering is a promising approach for treating cartilage lesions, but available strategies still provide a distinct composition of the extracellular matrix and an inferior mechanical property compared to native cartilage. To achieve fully functional tissue replacement more rationally designed biomaterials may be needed, introducing bioactive molecules which modulate cell behavior and guide tissue regeneration. This study aimed at exploring the impact of cell instructive, adhesion (GCWGGRGDSP called RGD) and collagen-binding (CKLER/CWYRGRL) peptides, incorporated in a tunable, matrixmetalloprotease (MMP)-responsive multi-arm poly(ethylene glycol) (starPEG)/heparin hydrogel on cartilage regeneration parameters in vitro and in vivo. MMP-responsive-starPEG-conjugates with cysteine termini and heparin-maleimide, optionally pre-functionalized with RGD, CKLER, CWYRGRL or control peptides, were cross-linked by Michael type addition to embed and grow mesenchymal stromal cells (MSC) or chondrocytes. While starPEG/heparin-hydrogel strongly supported chondrogenesis of MSC according to COL2A1, BGN and ACAN induction, MMP-degradability enhanced cell viability and proliferation. RGD-modification of the gels promoted cell spreading with intense cell network formation without negative effects on chondrogenesis. However, CKLER and CWYRGRL were unable to enhance the collagen content of constructs. RGD-modification allowed more even collagen type II distribution by chondrocytes throughout the MMP-responsive constructs especially in vivo. Collectively, peptide-instruction via heparin-enriched MMP-degradable starPEG allowed adjustment of self-renewal, cell morphology and cartilage matrix distribution in order to guide MSC and chondrocyte-based cartilage regeneration towards an improved outcome.

  18. Overexpression of TGF-β1 enhances chondrogenic differentiation and proliferation of human synovium-derived stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Yong Il; Ryu, Jae-Sung; Yeo, Jee Eun; Choi, Yun Jin; Kim, Yong Sang [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of); Ko, Kinarm [Center for Stem Cell Research, Institute of Advanced Biomedical Science, Konkuk University, Seoul 143-701 (Korea, Republic of); Koh, Yong-Gon, E-mail: yonseranglab@daum.net [Center for Stem Cell and Arthritis Research, Department of Orthopedic Surgery, Yonsei Sarang Hospital, Seoul (Korea, Republic of)

    2014-08-08

    Highlights: • Continuous TGF-β1 overexpression in hSD-MSCs did not influence their phenotypes. • Retroviral-mediated transduction of TGFB1 in hSD-MSCs enhances cell proliferation. • TGF-β1 overexpression did not effect to adipo- or osteogenic potential of hSD-MSCs. • TGF-β1 overexpression in hSD-MSCs could stimulate and accelerate chondrogenesis. - Abstract: Transforming growth factor-beta (TGF-β) superfamily proteins play a critical role in proliferation, differentiation, and other functions of mesenchymal stem cells (MSCs). During chondrogenic differentiation of MSCs, TGF-β up-regulates chondrogenic gene expression by enhancing the expression of the transcription factor SRY (sex-determining region Y)-box9 (Sox9). In this study, we investigated the effect of continuous TGF-β1 overexpression in human synovium-derived MSCs (hSD-MSCs) on immunophenotype, differentiation potential, and proliferation rate. hSD-MSCs were transduced with recombinant retroviruses (rRV) encoding TGF-β1. The results revealed that continuous overexpression of TGF-β1 did not affect their phenotype as evidenced by flow cytometry and reverse transcriptase PCR (RT-PCR). In addition, continuous TGF-β1 overexpression strongly enhanced cell proliferation of hSD-MSCs compared to the control groups. Also, induction of chondrogenesis was more effective in rRV-TGFB-transduced hSD-MSCs as shown by RT-PCR for chondrogenic markers, toluidine blue staining and glycosaminoglycan (GAG)/DNA ratio. Our data suggest that overexpression of TGF-β1 positively enhances the proliferation and chondrogenic potential of hSD-MSCs.

  19. Effects of cyclophosphamide and acrolein in organoid cultures of mouse limb bud cells grown in the presence of adult rat hepatocytes.

    Science.gov (United States)

    Ghaida, J; Merker, H J

    1992-01-01

    The effects were evaluated of cyclophosphamide (CPA) and its metabolite, acrolein, on chondrogenesis in organoid cultures of mouse limb bud mesenchymal cells co-cultured with non-enzymatically isolated adult rat hepatocytes. The studies were conducted with or without the simultaneous addition of 2-mercaptoethanesulphonic acid sodium (mesna) or glutathione (GSH). Alcian blue binding assay and light and electron microscopic techniques were used. Increasing concentrations of the two compounds (bioactivated CPA, 18-180 mum; acrolein, 50-500 mum) led to a dose-dependent inhibition of chondrogenesis associated with cellular dedifferentiation and/or cytotoxicity. Addition of mesna (1 mm) or GSH (1 mm) partially protected the cultures against CPA and acrolein. However, the protective effect depended on the dose of CPA or acrolein used. A higher protection was observed with mesna than with GSH, and the effect was more pronounced with acrolein than with CPA. The morphological findings suggested that CPA and acrolein acted by different mechanisms. Bioactivated CPA primarily inhibited the differentiation process, whereas acrolein exhibited a high cytotoxic activity affecting particularly monolayer cells that normally grow on the periphery of the cultures. These findings suggest that acrolein possesses a specific mode of action directed towards this type of cell. This could be explained by the specific shape and/or behaviour of the cells (i.e. cytoskeletal arrangement, proliferation rate, migration activity, intercellular communication pattern, etc.). The results demonstrated that the cell system used was suitable for the performance of cytotoxicity and teratogenicity studies such as those conducted with CPA and acrolein.

  20. TiO2 coating promotes human mesenchymal stem cell proliferation without the loss of their capacity for chondrogenic differentiation.

    Science.gov (United States)

    Kaitainen, Salla; Mähönen, Anssi J; Lappalainen, Reijo; Kröger, Heikki; Lammi, Mikko J; Qu, Chengjuan

    2013-06-01

    Human mesenchymal stem cells (hMSCs) are used in applications, which may require a large amount of cells; therefore, efficient expansion of the cells is desired. We studied whether TiO2 coating on plastic cell culture dishes could promote proliferation of hMSCs without adverse effects in chondrogenic differentiation. TiO2-films were deposited on polystyrene dishes and glass coverslips using an ultrashort pulsed laser deposition technique. Human MSCs from three donors were expanded on them until 95% confluence, and the cells were evaluated by morphology, immunocytochemistry and quantitative RT-PCR (qRT-PCR). The chondrogenic differentiation in pellets was performed after cultivation on TiO2-coated dishes. Chondrogenesis was evaluated by histological staining of proteoglycans and type II collagen, and qRT-PCR. Human MSC-associated markers STRO-1, CD44, CD90 and CD146 did not change after expansion on TiO2-coated coverslips. However, the cell number after a 48h-culture period was significantly higher on TiO2-coated culture dishes. Importantly, TiO2 coating caused no significant differences in the proteoglycan and type II collagen staining of the pellets, or the expression of chondrocyte-specific genes in the chondrogenesis assay. Thus, the proliferation of hMSCs could be significantly increased when cultured on TiO2-coated dishes without weakening their chondrogenic differentiation capacity. The transparency of TiO2-films allows easy monitoring of the cell growth and morphology under a phase-contrast microscope.

  1. Novel bio-synthetic hybrid materials and coculture systems for musculoskeletal tissue engineering

    Science.gov (United States)

    Lee, Hyeseung Janice

    Tissue Engineering is a truly exciting field of this age, trying to regenerate and repair impaired tissues. Unlike the old artificial implants, tissue engineering aims at making a long-term functional biological replacement. One strategy for such tissue engineering requires the following three components: cells, scaffolds, and soluble factors. Cells are cultured in a three-dimensional (3D) scaffold with medium containing various soluble factors. Once a tissue is developed in vitro, then it is implanted in vivo. The overall goal of this thesis was to develop novel bio-synthetic hybrid scaffolds and coculture system for musculoskeletal tissue engineering. The most abundant cartilage extracellular matrix (ECM) components are collagen and glycosaminoglycan (GAG), which are the natural scaffold for chondrocytes. As two different peptides, collagen mimetic peptide (CMP) and hyaluronic acid binding peptide (HABPep) were previously shown to bind to collagen and hyaluronic acid (HA) of GAG, respectively, it was hypothesized that immobilizing CMP and HABP on 3D scaffold would results in an interaction between ECM components and synthetic scaffolds via peptide-ECM bindings. CMP or HABPep-conjugated photopolymerizable poly(ethylene oxide) diacrylate (PEODA) hydrogels were synthesized and shown to retain encapsulated collagen or HA, respectively. This result supported that conjugated CMP and HABPep can interact with collagen and HA, respectively, and can serve as biological linkers in 3D synthetic hydrogels. When chondrocytes or mesenchymal stem cells (MSCs) were seeded, cells in CMP-conjugated scaffolds produced significantly more amount of type II collagen and GAG, compared to those in control scaffolds. Moreover, MSCs cultured in CMP-conjugated scaffolds exhibited lower level of hypertrophic markers, cbfa-1 and type X collagen. These results demonstrated that enhanced interaction between collagen and scaffold via CMP improves chondrogenesis of chondrocytes and MSCs and

  2. Functional Characterization of the Osteoarthritis Susceptibility Mapping to CHST11-A Bioinformatics and Molecular Study.

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    Louise N Reynard

    Full Text Available The single nucleotide polymorphism (SNP rs835487 is associated with hip osteoarthritis (OA at the genome-wide significance level and is located within CHST11, which codes for carbohydrate sulfotransferase 11. This enzyme post-translationally modifies proteoglycan prior to its deposition in the cartilage extracellular matrix. Using bioinformatics and experimental analyses, our aims were to characterise the rs835487 association signal and to identify the causal functional variant/s. Database searches revealed that rs835487 resides within a linkage disequilibrium (LD block of only 2.7 kb and is in LD (r2 ≥ 0.8 with six other SNPs. These are all located within intron 2 of CHST11, in a region that has predicted enhancer activity and which shows a high degree of conservation in primates. Luciferase reporter assays revealed that of the seven SNPs, rs835487 and rs835488, which have a pairwise r2 of 0.962, are the top functional candidates; the haplotype composed of the OA-risk conferring G allele of rs835487 and the corresponding T allele of rs835488 (the G-T haplotype demonstrated significantly different enhancer activity relative to the haplotype composed of the non-risk A allele of rs835487 and the corresponding C allele of rs835488 (the A-C haplotype (p < 0.001. Electrophoretic mobility shift assays and supershifts identified several transcription factors that bind more strongly to the risk-conferring G and T alleles of the two SNPs, including SP1, SP3, YY1 and SUB1. CHST11 was found to be upregulated in OA versus non-OA cartilage (p < 0.001 and was expressed dynamically during chondrogenesis. Its expression in adult cartilage did not however correlate with rs835487 genotype. Our data demonstrate that the OA susceptibility is mediated by differential protein binding to the alleles of rs835487 and rs835488, which are located within an enhancer whose target may be CHST11 during chondrogenesis or an alternative gene.

  3. Differentiation of synovial CD-105(+) human mesenchymal stem cells into chondrocyte-like cells through spheroid formation.

    Science.gov (United States)

    Arufe, M C; De la Fuente, A; Fuentes-Boquete, I; De Toro, Francisco J; Blanco, Francisco J

    2009-09-01

    Mesenchymal stem cells (MSCs) have the capacity to differentiate into several cell lineages, some of which can generate bone, cartilage, or adipose tissue. The presence of MSCs in the synovial membrane was recently reported. Data from comparative studies of MSCs derived from various mesenchymal tissues suggest that MSCs from synovial membranes have a superior chondrogenesis capacity. Previous chondrogenic differentiation studies have used the total population of MSCs, including cells with several MSC markers, such as CD44, CD90, CD105, or CD73. However the chondrogenic capacity of an individual population of MSCs has not been examined. Our aim was to study the chondrogenic capacity of the cellular MSC subset, CD105(+), derived from synovial membrane tissues of patients with osteoarthritis (OA) and normal donors. The tissues were digested with a cocktail of collagenase/dispase and the isolated MSCs were seeded into plates. The subpopulation of CD105(+)-MSCs was separated using a magnetic separator. The MSCs were then differentiated towards chondrocyte-like cells using a specific medium to promote spheroid formation. Spheroids were collected after 14, 28, and 46 days in chondrogenic medium and stained with hematoxylin, eosin, Safranin O or Alcian blue to evaluate the extracellular matrix. Immunohistochemistry was performed to study collagen types I (COLI) and II (COLII) and aggrecan expression. Phenotypic characterization of the isolated CD105(+)-MSCs shows that these cells are also positive for CD90 and CD44, but negatives for CD34 and CD45. In addition, this cellular subset expressed Sox-9. Spheroids appeared after 7 days in culture in the presence of chondrogenic medium. Our studies show no differences between MSCs obtained from OA and normal synovial membranes during chondrogenesis. The morphological analysis of spheroids revealed characteristics typical of chondrocyte cells. The intensity of Safranin O, Alcian blue and aggrecan staining was positive and constant

  4. Connective-Tissue Growth Factor (CTGF/CCN2 Induces Astrogenesis and Fibronectin Expression of Embryonic Neural Cells In Vitro.

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    Fabio A Mendes

    Full Text Available Connective-tissue growth factor (CTGF is a modular secreted protein implicated in multiple cellular events such as chondrogenesis, skeletogenesis, angiogenesis and wound healing. CTGF contains four different structural modules. This modular organization is characteristic of members of the CCN family. The acronym was derived from the first three members discovered, cysteine-rich 61 (CYR61, CTGF and nephroblastoma overexpressed (NOV. CTGF is implicated as a mediator of important cell processes such as adhesion, migration, proliferation and differentiation. Extensive data have shown that CTGF interacts particularly with the TGFβ, WNT and MAPK signaling pathways. The capacity of CTGF to interact with different growth factors lends it an important role during early and late development, especially in the anterior region of the embryo. ctgf knockout mice have several cranio-facial defects, and the skeletal system is also greatly affected due to an impairment of the vascular-system development during chondrogenesis. This study, for the first time, indicated that CTGF is a potent inductor of gliogenesis during development. Our results showed that in vitro addition of recombinant CTGF protein to an embryonic mouse neural precursor cell culture increased the number of GFAP- and GFAP/Nestin-positive cells. Surprisingly, CTGF also increased the number of Sox2-positive cells. Moreover, this induction seemed not to involve cell proliferation. In addition, exogenous CTGF activated p44/42 but not p38 or JNK MAPK signaling, and increased the expression and deposition of the fibronectin extracellular matrix protein. Finally, CTGF was also able to induce GFAP as well as Nestin expression in a human malignant glioma stem cell line, suggesting a possible role in the differentiation process of gliomas. These results implicate ctgf as a key gene for astrogenesis during development, and suggest that its mechanism may involve activation of p44/42 MAPK signaling

  5. Chondrogenic Differentiation of Human Umbilical Cord Blood-Derived Unrestricted Somatic Stem Cells on A 3D Beta-Tricalcium Phosphate-Alginate-Gelatin Scaffold

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    Masoud Soleimani

    2014-03-01

    Full Text Available Objective: Finding cell sources for cartilage tissue engineering is a critical procedure. The purpose of the present experimental study was to test the in vitro efficacy of the beta-tricalcium phosphate-alginate-gelatin (BTAG scaffold to induce chondrogenic differentiation of human umbilical cord blood-derived unrestricted somatic stem cells (USSCs. Materials and Methods: In this experimental study, USSCs were encapsulated in BTAG scaffold and cultured for 3 weeks in chondrogenic medium as chondrogenic group and in Dulbecco’s Modified Eagle’s Medium (DMEM as control group. Chondrogenic differentiation was evaluated by histology, immunofluorescence and RNA analyses for the expression of cartilage extracellular matrix components. The obtain data were analyzed using SPSS version 15. Results: Histological and immunohistochemical staining revealed that collagen II was markedly expressed in the extracellular matrix of the seeded cells on scaffold in presence of chondrogenic media after 21 days. Reverse transcription-polymerase chain reaction (RT-PCR showed a significant increase in expression levels of genes encoded the cartilage-specific markers, aggrecan, type I and II collagen, and bone morphogenetic protein (BMP-6 in chondrogenic group. Conclusion: This study demonstrates that BTAG can be considered as a suitable scaffold for encapsulation and chondrogenesis of USSCs.

  6. Evaluation of Magnetic Nanoparticle-Labeled Chondrocytes Cultivated on a Type II Collagen–Chitosan/Poly(Lactic-co-Glycolic Acid Biphasic Scaffold

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    Juin-Yih Su

    2017-01-01

    Full Text Available Chondral or osteochondral defects are still controversial problems in orthopedics. Here, chondrocytes labeled with magnetic nanoparticles were cultivated on a biphasic, type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold in an attempt to develop cultures with trackable cells exhibiting growth, differentiation, and regeneration. Rabbit chondrocytes were labeled with magnetic nanoparticles and characterized by scanning electron microscopy (SEM, transmission electron (TEM microscopy, and gene and protein expression analyses. The experimental results showed that the magnetic nanoparticles did not affect the phenotype of chondrocytes after cell labeling, nor were protein and gene expression affected. The biphasic type II collagen–chitosan/poly(lactic-co-glycolic acid scaffold was characterized by SEM, and labeled chondrocytes showed a homogeneous distribution throughout the scaffold after cultivation onto the polymer. Cellular phenotype remained unaltered but with increased gene expression of type II collagen and aggrecan, as indicated by cell staining, indicating chondrogenesis. Decreased SRY-related high mobility group-box gene (Sox-9 levels of cultured chondrocytes indicated that differentiation was associated with osteogenesis. These results are encouraging for the development of techniques for trackable cartilage regeneration and osteochondral defect repair which may be applied in vivo and, eventually, in clinical trials.

  7. Lamin A deregulation in human mesenchymal stem cells promotes an impairment in their chondrogenic potential and imbalance in their response to oxidative stress.

    Science.gov (United States)

    Mateos, Jesús; De la Fuente, Alexandre; Lesende-Rodriguez, Iván; Fernández-Pernas, Pablo; Arufe, María C; Blanco, Francisco J

    2013-11-01

    In the present study, we examined the effect of the over-expression of LMNA, or its mutant form progerin (PG), on the mesoderm differentiation potential of mesenchymal stem cells (MSCs) from human umbilical cord (UC) stroma using a recently described differentiation model employing spheroid formation. Accumulation of lamin A (LMNA) was previously associated with the osteoarthritis (OA) chondrocyte phenotype. Mutations of this protein are linked to laminopathies and specifically to Hutchinson-Gilford Progeria Syndrome (HGPS), an accelerated aging disease. Some authors have proposed that a deregulation of LMNA affects the differentiation potential of stem cells. The chondrogenic potential is defective in PG-MSCs, although both PG and LMNA transduced MSCs, have an increase in hypertrophy markers during chondrogenic differentiation. Furthermore, both PG and LMNA-MSCs showed a decrease in manganese superoxide dismutase (MnSODM), an increase of mitochondrial MnSODM-dependent reactive oxygen species (ROS) and alterations in their migration capacity. Finally, defects in chondrogenesis are partially reversed by periodic incubation with ROS-scavenger agent that mimics MnSODM effect. Our results indicate that over-expression of LMNA or PG by lentiviral gene delivery leads to defects in chondrogenic differentiation potential partially due to an imbalance in oxidative stress.

  8. Influence of age on rat bone-marrow mesenchymal stem cells potential.

    Science.gov (United States)

    Fafián-Labora, J; Fernández-Pernas, P; Fuentes, I; De Toro, J; Oreiro, N; Sangiao-Alvarellos, S; Mateos, J; Arufe, M C

    2015-11-19

    Mesenchymal stem cells promising role in cell-based therapies and tissue engineering appears to be limited due to a decline of their regenerative potential with increasing donor age. Six age groups from bone marrow mesenchymal stem cells of Wistar rats were studied (newborn, infant, young, pre-pubertal, pubertal and adult). Quantitative proteomic assay was performance by iTRAQ using an 8-plex iTRAQ labeling and the proteins differentially expressed were grouped in pluripotency, proliferative and metabolism processes. Proliferation makers, CD117 and Ki67 were measure by flow cytometry assay. Real time polymerase chain reaction analysis of pluripotency markers Rex1, Oct4, Sox2 and Nanog were done. Biological differentiation was realized using specific mediums for 14 days to induce osteogenesis, adipogenesis or chondrogenesis and immunostain analysis of differentiated cell resulting were done. Enzimoimmunoassay analysis of several enzymes as L-lactate dehydrogenase and glucose-6-phosphate isomerase were also done to validate iTRAQ data. Taking together these results indicate for the first time that mesenchymal stem cells have significant differences in their proliferative, pluripotency and metabolism profiles and those differences are age depending.

  9. Craniosynostosis of coronal suture in Twist1+/- mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture

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    Bjorn eBehr

    2011-07-01

    Full Text Available Craniosynostosis, the premature closure of cranial suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during suture closure. We have previously demonstrated that physiological closure of the posterior frontal (PF suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt signaling in the sagittal suture leads to endochondral ossification of the suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1+/- mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1+/- mice. Our data indicate that coronal suture closure in Twist1+/- mice occurs between postnatal day 9 to 13 by endochondral ossification, as shown by histology, gene expression analysis and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1+/- mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.

  10. A Direct Sulfation Process of a Marine Polysaccharide in Ionic Liquid

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    Nathalie Chopin

    2015-01-01

    Full Text Available GY785 is an exopolysaccharide produced by a mesophilic bacterial strain Alteromonas infernus discovered in the deep-sea hydrothermal vents. GY785 highly sulfated derivative (GY785 DRS was previously demonstrated to be a promising molecule driving the efficient mesenchymal stem cell chondrogenesis for cartilage repair. This glycosaminoglycan- (GAG- like compound was modified in a classical solvent (N,N′-dimethylformamide. However, the use of classical solvents limits the polysaccharide solubility and causes the backbone degradation. In the present study, a one-step efficient sulfation process devoid of side effects (e.g., polysaccharide depolymerization and/or degradation was developed to produce GAG-like derivatives. The sulfation of GY785 derivative (GY785 DR was carried out using ionic liquid as a reaction medium. The successful sulfation of this anionic and highly branched heteropolysaccharide performed in ionic liquid would facilitate the production of new molecules of high specificity for biological targets such as tissue engineering or regenerative medicine.

  11. Limb development in the gekkonid lizard Gonatodes albogularis: A reconsideration of homology in the lizard carpus and tarsus.

    Science.gov (United States)

    Leal, Francisca; Tarazona, Oscar A; Ramírez-Pinilla, Martha Patricia

    2010-11-01

    Despite the attention squamate lizards have received in the study of digit and limb loss, little is known about limb morphogenesis in pentadactyl lizards. Recent developmental studies have provided a basis for understanding lizard autopodial element homology based on developmental and comparative anatomy. In addition, the composition and identity of some carpal and tarsal elements of lizard limbs, and reptiles in general, have been the theme of discussions about their homology compared to non-squamate Lepidosauromorpha and basal Amniota. The study of additional embryonic material from different lizard families may improve our understanding of squamate limb evolution. Here, we analyze limb morphogenesis in the gekkonid lizard Gonatodes albogularis describing patterns of chondrogenesis and ossification from early stages of embryonic development to hatchlings. Our results are in general agreement with previous developmental studies, but we also show that limb development in squamates probably involves more chondrogenic elements for carpal and tarsal morphogenesis, as previously recognized on the grounds of comparative anatomy. We provide evidence for the transitory presence of distal carpale 1 and intermedium in the carpus and tibiale, intermedium, distal centralia, and distal tarsale 2 in the tarsus. Hence, we demonstrate that some elements that were believed to be lost in squamate evolution are conserved as transitory elements during limb development. However, these elements do not represent just phylogenetic burden but may be important for the morphogenesis of the lizard autopodium.

  12. An Injectable Enzymatically Crosslinked Carboxymethylated Pullulan/Chondroitin Sulfate Hydrogel for Cartilage Tissue Engineering.

    Science.gov (United States)

    Chen, Feng; Yu, Songrui; Liu, Bing; Ni, Yunzhou; Yu, Chunyang; Su, Yue; Zhu, Xinyuan; Yu, Xiaowei; Zhou, Yongfeng; Yan, Deyue

    2016-01-28

    In this study, an enzymatically cross-linked injectable and biodegradable hydrogel system comprising carboxymethyl pullulan-tyramine (CMP-TA) and chondroitin sulfate-tyramine (CS-TA) conjugates was successfully developed under physiological conditions in the presence of both horseradish peroxidase (HRP) and hydrogen peroxide (H2O2) for cartilage tissue engineering (CTTE). The HRP crosslinking method makes this injectable system feasible, minimally invasive and easily translatable for regenerative medicine applications. The physicochemical properties of the mechanically stable hydrogel system can be modulated by varying the weight ratio and concentration of polymer as well as the concentrations of crosslinking reagents. Additionally, the cellular behaviour of porcine auricular chondrocytes encapsulated into CMP-TA/CS-TA hydrogels demonstrates that the hydrogel system has a good cyto-compatibility. Specifically, compared to the CMP-TA hydrogel, these CMP-TA/CS-TA composite hydrogels have enhanced cell proliferation and increased cartilaginous ECM deposition, which significantly facilitate chondrogenesis. Furthermore, histological analysis indicates that the hydrogel system exhibits acceptable tissue compatibility by using a mouse subcutaneous implantation model. Overall, the novel injectable pullulan/chondroitin sulfate composite hydrogels presented here are expected to be useful biomaterial scaffold for regenerating cartilage tissue.

  13. Mammalian limb loading and chondral modeling during ontogeny.

    Science.gov (United States)

    Hammond, Ashley S; Ning, Jie; Ward, Carol V; Ravosa, Matthew J

    2010-04-01

    The adaptive growth response of cartilage, or chondral modeling, can result in changes in joint and limb proportions during ontogeny and ultimately contribute to the adult form. Despite Hamrick's (1999) reevaluation of the mechanisms of chondral modeling, the process of chondral modeling remains poorly studied in animal models. Here, we characterize the macro- and microanatomical responses of the femoral growth plate, articular cartilage, and bone in 15 juvenile Sus scrofa domestica subjected to different locomotor activity patterns. The exercised animals exhibit thinner cartilage zones, greater cellularity and larger proliferative chondrocyte areas in the growth plate, as well as larger femoral dimensions and a more elongate femoral head compared with sedentary controls. In general, the growth plate demonstrates greater adaptive changes than articular cartilage. Moreover, chondrocyte hypertrophy and proliferation were found to be responsive to locomotor loading and thus more important factors in chondral modeling than the extracellular matrix variables that were examined herein. In sum, the underlying mechanisms of adaptive chondrogenesis and bone plasticity are key to informing evolutionary and translational studies regarding determinants of variation in joint form and function. Given the disparity between the predictions of chondral modeling theory and our experimental findings, this suggests a need for further evaluation of chondral modeling responses during ontogeny.

  14. Palovarotene Inhibits Heterotopic Ossification and Maintains Limb Mobility and Growth in Mice With the Human ACVR1(R206H) Fibrodysplasia Ossificans Progressiva (FOP) Mutation.

    Science.gov (United States)

    Chakkalakal, Salin A; Uchibe, Kenta; Convente, Michael R; Zhang, Deyu; Economides, Aris N; Kaplan, Frederick S; Pacifici, Maurizio; Iwamoto, Masahiro; Shore, Eileen M

    2016-09-01

    Fibrodysplasia ossificans progressiva (FOP), a rare and as yet untreatable genetic disorder of progressive extraskeletal ossification, is the most disabling form of heterotopic ossification (HO) in humans and causes skeletal deformities, movement impairment, and premature death. Most FOP patients carry an activating mutation in a bone morphogenetic protein (BMP) type I receptor gene, ACVR1(R206H) , that promotes ectopic chondrogenesis and osteogenesis and, in turn, HO. We showed previously that the retinoic acid receptor γ (RARγ) agonist palovarotene effectively inhibited HO in injury-induced and genetic mouse models of the disease. Here we report that the drug additionally prevents spontaneous HO, using a novel conditional-on knock-in mouse line carrying the human ACVR1(R206H) mutation for classic FOP. In addition, palovarotene restored long bone growth, maintained growth plate function, and protected growing mutant neonates when given to lactating mothers. Importantly, palovarotene maintained joint, limb, and body motion, providing clear evidence for its encompassing therapeutic potential as a treatment for FOP. © 2016 American Society for Bone and Mineral Research.

  15. Laminins and Nidogens in the Pericellular Matrix of Chondrocytes: Their Role in Osteoarthritis and Chondrogenic Differentiation.

    Science.gov (United States)

    Schminke, Boris; Frese, Jenny; Bode, Christa; Goldring, Mary B; Miosge, Nicolai

    2016-02-01

    The aim of this study was to investigate the role of laminins and nidogen-2 in osteoarthritis (OA) and their potential to support chondrogenic differentiation. We applied immunohistochemistry, electron microscopy, siRNA, quantitative RT-PCR, Western blot, and proteome analysis for the investigation of cartilage tissue and isolated chondrocytes in three-dimensional culture obtained from patients with late-stage knee OA and nidogen-2 knockout mice. We demonstrate that subunits of laminins appear in OA cartilage and that nidogen-2-null mice exhibit typical osteoarthritic features. Chondrogenic progenitor cells (CPCs) produced high levels of laminin-α1, laminin-α5, and nidogen-2 in their pericellular matrix, and laminin-α1 enhanced collagen type II and reduced collagen type I expression by cultured CPCs. Nidogen-2 increased SOX9 gene expression. Knockdown of nidogen-2 reduced SOX9 expression, whereas it up-regulated RUNX2 expression. This study reveals that the influence of the pericellular matrix on CPCs is important for the expression of the major regulator transcription factors, SOX9 and RUNX2. Our novel findings that laminins and nidogen-2 drive CPCs toward chondrogenesis may help in the elucidation of new treatment strategies for cartilage tissue regeneration.

  16. Genome-wide association scan suggests basis for microtia in Awassi sheep.

    Science.gov (United States)

    Jawasreh, K; Boettcher, P J; Stella, A

    2016-08-01

    Hereditary underdevelopment of the ear, a condition also known as microtia, has been observed in several sheep breeds as well as in humans and other species. Its genetic basis in sheep is unknown. The Awassi sheep, a breed native to southwest Asia, carries this phenotype and was targeted for molecular characterization via a genome-wide association study. DNA samples were collected from sheep in Jordan. Eight affected and 12 normal individuals were genotyped with the Illumina OvineSNP50(®) chip. Multilocus analyses failed to identify any genotypic association. In contrast, a single-locus analysis revealed a statistically significant association (P = 0.012, genome-wide) with a SNP at basepair 34 647 499 on OAR23. This marker is adjacent to the gene encoding transcription factor GATA-6, which has been shown to play a role in many developmental processes, including chondrogenesis. The lack of extended homozygosity in this region suggests a fairly ancient mutation, and the time of occurrence was estimated to be approximately 3000 years ago. Many of the earless sheep breeds may thus share the causative mutation, especially within the subgroup of fat-tailed, wool sheep.

  17. Regeneration of human-ear-shaped cartilage by co-culturing human microtia chondrocytes with BMSCs.

    Science.gov (United States)

    Zhang, Lu; He, Aijuan; Yin, Zongqi; Yu, Zheyuan; Luo, Xusong; Liu, Wei; Zhang, Wenjie; Cao, Yilin; Liu, Yu; Zhou, Guangdong

    2014-06-01

    Previously, we had addressed the issues of shape control/maintenance of in vitro engineered human-ear-shaped cartilage. Thus, lack of applicable cell source had become a major concern that blocks clinical translation of this technology. Autologous microtia chondrocytes (MCs) and bone marrow stromal cells (BMSCs) were both promising chondrogenic cells that did not involve obvious donor site morbidity. However, limited cell availability of MCs and ectopic ossification of chondrogenically induced BMSCs in subcutaneous environment greatly restricted their applications in external ear reconstruction. The current study demonstrated that MCs possessed strong proliferation ability but accompanied with rapid loss of chondrogenic ability during passage, indicating a poor feasibility to engineer the entire ear using expanded MCs. Fortunately, the co-transplantation results of MCs and BMSCs (25% MCs and 75% BMSCs) demonstrated a strong chondroinductive ability of MCs to promote stable ectopic chondrogenesis of BMSCs in subcutaneous environment. Moreover, cell labeling demonstrated that BMSCs could transform into chondrocyte-like cells under the chondrogenic niche provided by co-cultured MCs. Most importantly, a human-ear-shaped cartilaginous tissue with delicate structure and proper elasticity was successfully constructed by seeding the mixed cells (MCs and BMSCs) into the pre-shaped biodegradable ear-scaffold followed by 12 weeks of subcutaneous implantation in nude mouse. These results may provide a promising strategy to construct stable ectopic cartilage with MCs and stem cells (BMSCs) for autologous external ear reconstruction.

  18. Efficiency of Human Epiphyseal Chondrocytes with Differential Replication Numbers for Cellular Therapy Products

    Directory of Open Access Journals (Sweden)

    Michiyo Nasu

    2016-01-01

    Full Text Available The cell-based therapy for cartilage or bone requires a large number of cells; serial passages of chondrocytes are, therefore, needed. However, fates of expanded chondrocytes from extra fingers remain unclarified. The chondrocytes from human epiphyses morphologically changed from small polygonal cells to bipolar elongated spindle cells and to large polygonal cells with degeneration at early passages. Gene of type II collagen was expressed in the cells only at a primary culture (Passage 0 and Passage 1 (P1 cells. The nodules by implantation of P0 to P8 cells were composed of cartilage and perichondrium. The cartilage consisted of chondrocytes with round nuclei and type II collagen-positive matrix, and the perichondrium consisted of spindle cells with type I collage-positive matrix. The cartilage and perichondrium developed to bone with marrow cavity through enchondral ossification. Chondrogenesis and osteogenesis by epiphyseal chondrocytes depended on replication number in culture. It is noteworthy to take population doubling level in correlation with pharmaceutical efficacy into consideration when we use chondrocytes for cell-based therapies.

  19. Acquiring Chondrocyte Phenotype from Human Mesenchymal Stem Cells under Inflammatory Conditions

    Directory of Open Access Journals (Sweden)

    Masahiro Kondo

    2014-11-01

    Full Text Available An inflammatory milieu breaks down the cartilage matrix and induces chondrocyte apoptosis, resulting in cartilage destruction in patients with cartilage degenerative diseases, such as rheumatoid arthritis or osteoarthritis. Because of the limited regenerative ability of chondrocytes, defects in cartilage are irreversible and difficult to repair. Mesenchymal stem cells (MSCs are expected to be a new tool for cartilage repair because they are present in the cartilage and are able to differentiate into multiple lineages of cells, including chondrocytes. Although clinical trials using MSCs for patients with cartilage defects have already begun, its efficacy and repair mechanisms remain unknown. A PubMed search conducted in October 2014 using the following medical subject headings (MeSH terms: mesenchymal stromal cells, chondrogenesis, and cytokines resulted in 204 articles. The titles and abstracts were screened and nine articles relevant to “inflammatory” cytokines and “human” MSCs were identified. Herein, we review the cell biology and mechanisms of chondrocyte phenotype acquisition from human MSCs in an inflammatory milieu and discuss the clinical potential of MSCs for cartilage repair.

  20. Unique expression patterns of multiple key genes associated with the evolution of mammalian flight.

    Science.gov (United States)

    Wang, Zhe; Dai, Mengyao; Wang, Yao; Cooper, Kimberly L; Zhu, Tengteng; Dong, Dong; Zhang, Junpeng; Zhang, Shuyi

    2014-05-22

    Bats are the only mammals capable of true flight. Critical adaptations for flight include a pair of dramatically elongated hands with broad wing membranes. To study the molecular mechanisms of bat wing evolution, we perform genomewide mRNA sequencing and in situ hybridization for embryonic bat limbs. We identify seven key genes that display unique expression patterns in embryonic bat wings and feet, compared with mouse fore- and hindlimbs. The expression of all 5'HoxD genes (Hoxd9-13) and Tbx3, six known crucial transcription factors for limb and digit development, is extremely high and prolonged in the elongating wing area. The expression of Fam5c, a tumour suppressor, in bat limbs is bat-specific and significantly high in all short digit regions (the thumb and foot digits). These results suggest multiple genetic changes occurred independently during the evolution of bat wings to elongate the hand digits, promote membrane growth and keep other digits short. Our findings also indicate that the evolution of limb morphology depends on the complex integration of multiple gene regulatory networks and biological processes that control digit formation and identity, chondrogenesis, and interdigital regression or retention.

  1. Requirement of the mouse I-mfa gene for placental development and skeletal patterning.

    Science.gov (United States)

    Kraut, N; Snider, L; Chen, C M; Tapscott, S J; Groudine, M

    1998-11-02

    The bHLH-repressor protein I-mfa binds to MyoD family members, inhibits their activity, and blocks their nuclear import and binding to DNA. In situ hybridization analysis demonstrated that mouse I-mfa was highly expressed in extraembryonic lineages, in the sclerotome, and subsequently within mesenchymal precursors of the axial and appendicular skeleton, before chondrogenesis occurs. Targeted deletion of I-mfa in a C57Bl/6 background resulted in embryonic lethality around E10.5, associated with a placental defect and a markedly reduced number of trophoblast giant cells. Overexpression of I-mfa in rat trophoblast (Rcho-1) stem cells induced differentiation into trophoblast giant cells. I-mfa interacted with the bHLH protein Mash2, a negative regulator of trophoblast giant cell formation, and inhibited its transcriptional activity in cell culture. In contrast, I-mfa did not interfere with the activity of the bHLH protein Hand1, a positive regulator of giant cell differentiation. Interestingly, I-mfa-null embryos on a 129/Sv background had no placental defect, generally survived to adulthood, and exhibited delayed caudal neural tube closure and skeletal patterning defects that included fusions of ribs, vertebral bodies and abnormal formation of spinous processes. Our results indicate that I-mfa plays an important role in trophoblast and chondrogenic differentiation by negatively regulating a subset of lineage-restricted bHLH proteins.

  2. FGFR3 Deficiency Causes Multiple Chondroma-like Lesions by Upregulating Hedgehog Signaling.

    Directory of Open Access Journals (Sweden)

    Siru Zhou

    2015-06-01

    Full Text Available Most cartilaginous tumors are formed during skeletal development in locations adjacent to growth plates, suggesting that they arise from disordered endochondral bone growth. Fibroblast growth factor receptor (FGFR3 signaling plays essential roles in this process; however, the role of FGFR3 in cartilaginous tumorigenesis is not known. In this study, we found that postnatal chondrocyte-specific Fgfr3 deletion induced multiple chondroma-like lesions, including enchondromas and osteochondromas, adjacent to disordered growth plates. The lesions showed decreased extracellular signal-regulated kinase (ERK activity and increased Indian hedgehog (IHH expression. The same was observed in Fgfr3-deficient primary chondrocytes, in which treatment with a mitogen-activated protein kinase (MEK inhibitor increased Ihh expression. Importantly, treatment with an inhibitor of IHH signaling reduced the occurrence of chondroma-like lesions in Fgfr3-deficient mice. This is the first study reporting that the loss of Fgfr3 function leads to the formation of chondroma-like lesions via downregulation of MEK/ERK signaling and upregulation of IHH, suggesting that FGFR3 has a tumor suppressor-like function in chondrogenesis.

  3. Simulated microgravity alters the expression of key genes involved in fracture healing

    Science.gov (United States)

    McCabe, N. Patrick; Androjna, Caroline; Hill, Esther; Globus, Ruth K.; Midura, Ronald J.

    2013-11-01

    Fracture healing in animal models has been shown to be altered in both ground based analogs of spaceflight and in those exposed to actual spaceflight. The molecular mechanisms behind altered fracture healing as a result of chronic exposure to microgravity remain to be elucidated. This study investigates temporal gene expression of multiple factors involved in secondary fracture healing, specifically those integral to the development of a soft tissue callus and the transition to that of hard tissue. Skeletally mature female rats were subjected to a 4 week period of simulated microgravity and then underwent a closed femoral fracture procedure. Thereafter, they were reintroduced to the microgravity and allowed to heal for a 1 or 2 week period. A synchronous group of weight bearing rats was used as a normal fracture healing control. Utilizing Real-Time quantitative PCR on mRNA from fracture callus tissue, we found significant reductions in the levels of transcripts associated with angiogenesis, chondrogenesis, and osteogenesis. These data suggest an altered fracture healing process in a simulated microgravity environment, and these alterations begin early in the healing process. These findings may provide mechanistic insight towards developing countermeasure protocols to mitigate these adaptations.

  4. Nanoparticulate Mineralized Collagen Scaffolds and BMP-9 Induce a Long-Term Bone Cartilage Construct in Human Mesenchymal Stem Cells.

    Science.gov (United States)

    Ren, Xiaoyan; Weisgerber, Daniel W; Bischoff, David; Lewis, Michael S; Reid, Russell R; He, Tong-Chuan; Yamaguchi, Dean T; Miller, Timothy A; Harley, Brendan A C; Lee, Justine C

    2016-07-01

    Engineering the osteochondral junction requires fabrication of a microenvironment that supports both osteogenesis and chondrogenesis. Multiphasic scaffold strategies utilizing a combination of soluble factors and extracellular matrix components are ideally suited for such applications. In this work, the contribution of an osteogenic nanoparticulate mineralized glycosaminoglycan scaffold (MC-GAG) and a dually chondrogenic and osteogenic growth factor, BMP-9, in the differentiation of primary human mesenchymal stem cells (hMSCs) is evaluated. Although 2D cultures demonstrate alkaline phosphatase activity and mineralization of hMSCs induced by BMP-9, MC-GAG scaffolds do not demonstrate significant differences in the collagen I expression, osteopontin expression, or mineralization. Instead, BMP-9 increases expression of collagen II, Sox9, aggrecan (ACAN), and cartilage oligomeric protein. However, the hypertrophic chondrocyte marker, collagen X, is not elevated with BMP-9 treatment. In addition, histologic analyses demonstrate that while BMP-9 does not increase mineralization, BMP-9 treatment results in an increase of sulfated glycosaminoglycans. Thus, the combination of BMP-9 and MC-GAG stimulates chondrocytic and osteogenic differentiation of hMSCs.

  5. Matrigel supports neural, melanocytic and chondrogenic differentiation of trunk neural crest cells.

    Science.gov (United States)

    Ramos-Hryb, Ana B; Da-Costa, Meline C; Trentin, Andréa G; Calloni, Giordano W

    2013-01-01

    The neural crest (NC) is composed of highly multipotent precursor cells able to differentiate into both neural and mesenchymal phenotypes. Until now, most studies focusing on NC cell differentiation have been performed with traditional two-dimensional (2D) cell culture systems. However, such culture systems do not reflect the complex three-dimensional (3D) microenvironments of in vivo NC cells. To address this limitation, we have developed a method of Matrigel™ coating to create 2D and 3D microenvironments in the same culture well. When we performed cultures of trunk neural crest cells (TNCCs) on three different lots of basement membrane matrix (Matrigel™), we observed that all analyzed Matrigel™ lots were equally efficient in allowing the appearance of glial cells, neurons, melanocytes, smooth muscle cells and chondrocytes. We further observed that chondrocytes were found predominantly in the 3D microenvironment, whereas smooth muscle cells were almost exclusively located in the 2D microenvironment. Glial cells were present in both environments, but with broader quantities on the 2D surface. Melanocytes and neurons were equally distributed in both 2D and 3D microenvironments, but with distinct morphologies. It is worth noting the higher frequency of chondrocytes detected in this study using the 3D Matrigel™ microenvironment compared to previous reports of chondrogenesis obtained from TNCCs on traditional 2D cultures. In conclusion, Matrigel™ represents an attractive scaffold to study NC multipotentiality and differentiation, since it permits the appearance of the major NC phenotypes.

  6. Enhanced human bone marrow mesenchymal stem cell functions in novel 3D cartilage scaffolds with hydrogen treated multi-walled carbon nanotubes.

    Science.gov (United States)

    Holmes, Benjamin; Castro, Nathan J; Li, Jian; Keidar, Michael; Zhang, Lijie Grace

    2013-09-13

    Cartilage tissue is a nanostructured tissue which is notoriously hard to regenerate due to its extremely poor inherent regenerative capacity and complex stratified architecture. Current treatment methods are highly invasive and may have many complications. Thus, the goal of this work is to use nanomaterials and nano/microfabrication methods to create novel biologically inspired tissue engineered cartilage scaffolds to facilitate human bone marrow mesenchymal stem cell (MSC) chondrogenesis. To this end we utilized electrospinning to design and fabricate a series of novel 3D biomimetic nanostructured scaffolds based on hydrogen (H2) treated multi-walled carbon nanotubes (MWCNTs) and biocompatible poly(L-lactic acid) (PLLA) polymers. Specifically, a series of electrospun fibrous PLLA scaffolds with controlled fiber dimension were fabricated in this study. In vitro MSC studies showed that stem cells prefer to attach in the scaffolds with smaller fiber diameter. More importantly, the MWCNT embedded scaffolds showed a drastic increase in mechanical strength and a compressive Young's modulus matching to natural cartilage. Furthermore, our MSC differentiation results demonstrated that incorporation of the H2 treated carbon nanotubes and poly-L-lysine coating can induce more chondrogenic differentiations of MSCs than controls. After two weeks of culture, PLLA scaffolds with H2 treated MWCNTs and poly-L-lysine can achieve the highest glycosaminoglycan synthesis, making them promising for further exploration for cartilage regeneration.

  7. 3D printing of composite tissue with complex shape applied to ear regeneration.

    Science.gov (United States)

    Lee, Jung-Seob; Hong, Jung Min; Jung, Jin Woo; Shim, Jin-Hyung; Oh, Jeong-Hoon; Cho, Dong-Woo

    2014-06-01

    In the ear reconstruction field, tissue engineering enabling the regeneration of the ear's own tissue has been considered to be a promising technology. However, the ear is known to be difficult to regenerate using traditional methods due to its complex shape and composition. In this study, we used three-dimensional (3D) printing technology including a sacrificial layer process to regenerate both the auricular cartilage and fat tissue. The main part was printed with poly-caprolactone (PCL) and cell-laden hydrogel. At the same time, poly-ethylene-glycol (PEG) was also deposited as a sacrificial layer to support the main structure. After complete fabrication, PEG can be easily removed in aqueous solutions, and the procedure for removing PEG has no effect on the cell viability. For fabricating composite tissue, chondrocytes and adipocytes differentiated from adipose-derived stromal cells were encapsulated in hydrogel to dispense into the cartilage and fat regions, respectively, of ear-shaped structures. Finally, we fabricated the composite structure for feasibility testing, satisfying expectations for both the geometry and anatomy of the native ear. We also carried out in vitro assays for evaluating the chondrogenesis and adipogenesis of the cell-printed structure. As a result, the possibility of ear regeneration using 3D printing technology which allowed tissue formation from the separately printed chondrocytes and adipocytes was demonstrated.

  8. Pituitary Adenylate Cyclase Activating Polypeptide (PACAP Pathway Is Induced by Mechanical Load and Reduces the Activity of Hedgehog Signaling in Chondrogenic Micromass Cell Cultures

    Directory of Open Access Journals (Sweden)

    Tamás Juhász

    2015-07-01

    Full Text Available Pituitary adenylate cyclase activating polypeptide (PACAP is a neurohormone exerting protective function during various stress conditions either in mature or developing tissues. Previously we proved the presence of PACAP signaling elements in chicken limb bud-derived chondrogenic cells in micromass cell cultures. Since no data can be found if PACAP signaling is playing any role during mechanical stress in any tissues, we aimed to investigate its contribution in mechanotransduction during chondrogenesis. Expressions of the mRNAs of PACAP and its major receptor, PAC1 increased, while that of other receptors, VPAC1, VPAC2 decreased upon mechanical stimulus. Mechanical load enhanced the expression of collagen type X, a marker of hypertrophic differentiation of chondrocytes and PACAP addition attenuated this elevation. Moreover, exogenous PACAP also prevented the mechanical load evoked activation of hedgehog signaling: protein levels of Sonic and Indian Hedgehogs and Gli1 transcription factor were lowered while expressions of Gli2 and Gli3 were elevated by PACAP application during mechanical load. Our results suggest that mechanical load activates PACAP signaling and exogenous PACAP acts against the hypertrophy inducing effect of mechanical load.

  9. Versican expression in myoepithelial cells from carcinomas in canine mixed mammary tumors.

    Science.gov (United States)

    Damasceno, Karine A; Bertagnolli, Angélica C; Estrela-Lima, Alessandra; Rabelo, Bruna S; Campos, Liliane C; Ribeiro, Lorena G R; Cassali, Geovanni D

    2014-04-01

    The matrix of canine mixed mammary tumors (CMMTs) consists of proliferating spindle cells of possible myoepithelial origin, as well as myxomatous tissue, cartilage matrix and/or bone. Among the multiple components of this tumor extracellular matrix, versican probably plays a prominent role due to its importance in tumor progression, cell proliferation and differentiation. However, there are few data related to a possible association between versican expression and the state of myoepithelial cell differentiation in CMMTs. Using immunohistochemistry and histochemistry, the objective of this study was to evaluate the expression of versican, sulfated proteoglycans and mucopolysaccharides in myoepithelial cells at different stages of differentiation and to explore a potential relationship with p63 and α-smooth muscle actin (SMA) expression. A significant difference in versican expression was observed among the different stages of myoepithelial cell differentiation with an inverse correlation between versican and p63/SMA expression. These results suggest that at an early stage of proliferation, myoepithelial cells acquire a phenotype consistent with a role in chondrogenesis. Moreover, myoepithelial cells showed an affinity for safranin and periodic acid-Schiff staining at different stages of proliferation supporting the myoepithelial origin of spindle cells from CMMTs.

  10. Comparative evaluation of leukocyte- and platelet-rich plasma and pure platelet-rich plasma for cartilage regeneration

    Science.gov (United States)

    Xu, Zhengliang; Yin, Wenjing; Zhang, Yuelei; Qi, Xin; Chen, Yixuan; Xie, Xuetao; Zhang, Changqing

    2017-01-01

    Platelet-rich plasma (PRP) has gained growing popularity in the treatment of articular cartilage lesions in the last decade. However, the potential harmful effects of leukocytes in PRP on cartilage regeneration have seldom been studied in vitro, and not at all in vivo yet. The objective of the present study is to compare the effects of leukocyte- and platelet-rich plasma (L-PRP) and pure platelet-rich plasma (P-PRP) on cartilage repair and NF-κB pathway, in order to explore the mechanism underlying the function of leukocytes in PRP in cartilage regeneration. The constituent analysis showed that P-PRP had significantly lower concentrations of leukocytes and pro-inflammatory cytokines compared with L-PRP. In addition, cell proliferation and differentiation assays indicated P-PRP promoted growth and chondrogenesis of rabbit bone marrow mesenchymal stem cells (rBMSC) significantly compared with L-PRP. Despite similarity in macroscopic appearance, the implantation of P-PRP combining rBMSC in vivo yielded better cartilage repair results than the L-PRP group based on histological examination. Importantly, the therapeutic effects of PRP on cartilage regeneration could be enhanced by removing leukocytes to avoid the activation of the NF-κB pathway. Thus, PRP without concentrated leukocytes may be more suitable for the treatment of articular cartilage lesions. PMID:28265109

  11. Delivery of the Sox9 gene promotes chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells in an in vitro model

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    Wang, Z.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Li, X.L. [Department of Dermatology, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); He, X.J. [Department of Orthopedics, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Wu, B.J.; Xu, M. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Chang, H.M. [Department of Otolaryngology - Head and Neck Surgery, Affiliated Hospital of Xi' an Medical University, Xi' an (China); Zhang, X.H. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China); Xing, Z. [Department of Clinical Dentistry, Faculty of Dentistry, Center for Clinical Dental Research, University of Bergen, Bergen (Norway); Jing, X.H.; Kong, D.M.; Kou, X.H.; Yang, Y.Y. [Department of Otolaryngology - Head and Neck Surgery, The Second Hospital, Xi' an Jiaotong University, Xi' an (China)

    2014-03-18

    SRY-related high-mobility-group box 9 (Sox9) gene is a cartilage-specific transcription factor that plays essential roles in chondrocyte differentiation and cartilage formation. The aim of this study was to investigate the feasibility of genetic delivery of Sox9 to enhance chondrogenic differentiation of human umbilical cord blood-derived mesenchymal stem cells (hUC-MSCs). After they were isolated from human umbilical cord blood within 24 h after delivery of neonates, hUC-MSCs were untreated or transfected with a human Sox9-expressing plasmid or an empty vector. The cells were assessed for morphology and chondrogenic differentiation. The isolated cells with a fibroblast-like morphology in monolayer culture were positive for the MSC markers CD44, CD105, CD73, and CD90, but negative for the differentiation markers CD34, CD45, CD19, CD14, or major histocompatibility complex class II. Sox9 overexpression induced accumulation of sulfated proteoglycans, without altering the cellular morphology. Immunocytochemistry demonstrated that genetic delivery of Sox9 markedly enhanced the expression of aggrecan and type II collagen in hUC-MSCs compared with empty vector-transfected counterparts. Reverse transcription-polymerase chain reaction analysis further confirmed the elevation of aggrecan and type II collagen at the mRNA level in Sox9-transfected cells. Taken together, short-term Sox9 overexpression facilitates chondrogenesis of hUC-MSCs and may thus have potential implications in cartilage tissue engineering.

  12. Implants composed of carbon fiber mesh and bone-marrow-derived, chondrocyte-enriched cultures for joint surface reconstruction.

    Science.gov (United States)

    Robinson, D; Efrat, M; Mendes, D G; Halperin, N; Nevo, Z

    1993-01-01

    The current study integrates two distinct approaches in joint resurfacing into a combined type of implant, composed of carbon fiber mesh impregnated and coated with a hyaluronic-acid-based delivery substance containing cultured cells. Rabbit autogeneic chondrocyte-enriched cultures obtained from mesenchymal stem cells (chondroprogenitor cells) derived from adult rabbit bone marrow were grown in vitro under conditions favoring chondrogenesis. The improvement in quality of repair when a combined implant containing both cells and a carbon scaffold was used, in comparison to the utilization of carbon fiber mesh alone, was clearly demonstrated using clinical, histological, biochemical, and biomechanical examinations. Evaluations of the joints were performed at 6 weeks and 6 months after implantation. The repair tissue in the cell-implanted joints consisted of a typical hyaline cartilage, which was more cellular and thicker than the repair tissue in the hyaluronic-acid-impregnated carbon-fiber-implanted control joints. The hyaline cartilage in the experimental group formed a superficial layer above the carbon fibers, flush with the joint surface. In the controls, in which carbon fiber and the delivery substance alone were implanted, a histologically and biochemically fibrous tissue that was inferior biomechanically to the new cartilage was formed by the cells containing implants.

  13. Craniosynostosis of coronal suture in twist1 mice occurs through endochondral ossification recapitulating the physiological closure of posterior frontal suture.

    Science.gov (United States)

    Behr, Björn; Longaker, Michael T; Quarto, Natalina

    2011-01-01

    Craniosynostosis, the premature closure of cranial suture, is a pathologic condition that affects 1/2000 live births. Saethre-Chotzen syndrome is a genetic condition characterized by craniosynostosis. The Saethre-Chotzen syndrome, which is defined by loss-of-function mutations in the TWIST gene, is the second most prevalent craniosynostosis. Although much of the genetics and phenotypes in craniosynostosis syndromes is understood, less is known about the underlying ossification mechanism during suture closure. We have previously demonstrated that physiological closure of the posterior frontal suture occurs through endochondral ossification. Moreover, we revealed that antagonizing canonical Wnt-signaling in the sagittal suture leads to endochondral ossification of the suture mesenchyme and sagittal synostosis, presumably by inhibiting Twist1. Classic Saethre-Chotzen syndrome is characterized by coronal synostosis, and the haploinsufficient Twist1(+/-) mice represents a suitable model for studying this syndrome. Thus, we seeked to understand the underlying ossification process in coronal craniosynostosis in Twist1(+/-) mice. Our data indicate that coronal suture closure in Twist1(+/-) mice occurs between postnatal day 9 and 13 by endochondral ossification, as shown by histology, gene expression analysis, and immunohistochemistry. In conclusion, this study reveals that coronal craniosynostosis in Twist1(+/-) mice occurs through endochondral ossification. Moreover, it suggests that haploinsufficiency of Twist1 gene, a target of canonical Wnt-signaling, and inhibitor of chondrogenesis, mimics conditions of inactive canonical Wnt-signaling leading to craniosynostosis.

  14. PTH stimulated growth and decreased Col-X deposition are phosphotidylinositol-3,4,5 triphosphate kinase and mitogen activating protein kinase dependent in avian sterna.

    Science.gov (United States)

    Harrington, Erik Kern; Coon, David J; Kern, Matthew F; Svoboda, Kathy K H

    2010-02-01

    Type X collagen (Col-X) deposition is a marker of terminal differentiation during chondrogenesis, in addition to appositional growth and apoptosis. The parathyroid hormone/parathyroid hormone related peptide (PTH/PTHrP) receptor, or PPR, is a G-Protein coupled receptor (GPCR), which activates several downstream pathways, moderating chondrocyte differentiation, including suppression of Col-X deposition. An Avian sterna model was used to analyze the PPR GPCR downstream kinase role in growth rate and extracellular matrix (ECM) including Col-II, IX, and X. Phosphatidylinositol kinase (PI3K), mitogen activating protein kinase (MAPK) and protein kinase A (PKA) were inhibited with specific established inhibitors LY294002, PD98059, and H89, respectively to test the hypothesis that they could reverse/inhibit the PTH/PTHrP pathway. Excised E14 chick sterna were PTH treated with or without an inhibitor and compared to controls. Sternal length was measured every 24 hr. Cultured sterna were immuno-stained using specific antibodies for Col-II, IX, or X and examined via confocal microscopy. Increased growth in PTH-treated sterna was MAPK, PI3K, and PKA dose dependent, suggesting growth was regulated through multiple pathways. Col-X deposition was rescued in PTH-treated sterna in the presence of PI3K or MAPK inhibitors, but not with the PKA inhibitor. All three inhibitors moderately disrupted Col-II and Col-IX deposition. These results suggest that PTH can activate multiple pathways during chondrocyte differentiation.

  15. Engineered Microtissues Formed by Schiff Base Crosslinking Restore the Chondrogenic Potential of Aged Mesenchymal Stem Cells.

    Science.gov (United States)

    Millan, Christopher; Cavalli, Emma; Groth, Thomas; Maniura-Weber, Katharina; Zenobi-Wong, Marcy

    2015-06-24

    A universal method for reproducibly directing stem cell differentiation remains a major challenge for clinical applications involving cell-based therapies. The standard approach for chondrogenic induction by micromass pellet culture is highly susceptible to interdonor variability. A novel method for the fabrication of condensation-like engineered microtissues (EMTs) that utilizes hydrophilic polysaccharides to induce cell aggregation is reported here. Chondrogenesis of mesenchymal stem cells (MSCs) in EMTs is significantly enhanced compared to micromass pellets made by centrifugation measured by type II collagen gene expression, dimethylmethylene blue assay, and histology. MSCs from aged donors that fail to differentiate in pellet culture are successfully induced to synthesize cartilage-specific matrix in EMTs under identical media conditions. Furthermore, the EMT polysaccharides support the loading and release of the chondroinduction factor transforming growth factor β3 (TGF-β3). TGF-β-loaded EMTs (EMT(+TGF) ) facilitate cartilaginous tissue formation during culture in media not supplemented with the growth factor. The clinical potential of this approach is demonstrated in an explant defect model where EMT(+TGF) from aged MSCs synthesize de novo tissue containing sulfated glycosaminoglycans and type II collagen in situ.

  16. Exposure to Excess Phenobarbital Negatively Influences the Osteogenesis of Chick Embryos

    Science.gov (United States)

    Yan, Yu; Cheng, Xin; Yang, Ren-Hao; Li, He; Chen, Jian-Long; Ma, Zheng-Lai; Wang, Guang; Chuai, Manli; Yang, Xuesong

    2016-01-01

    Phenobarbital is an antiepileptic drug that is widely used to treat epilepsy in a clinical setting. However, a long term of phenobarbital administration in pregnant women may produce side effects on embryonic skeletogenesis. In this study, we aim to investigate the mechanism by which phenobarbital treatment induces developmental defects in long bones. We first determined that phenobarbital treatment decreased chondrogenesis and inhibited the proliferation of chondrocytes in chick embryos. Phenobarbital treatment also suppressed mineralization in both in vivo and in vitro long bone models. Next, we established that phenobarbital treatment delayed blood vessel invasion in a cartilage template, and this finding was supported by the down-regulation of vascular endothelial growth factor in the hypertrophic zone following phenobarbital treatment. Phenobarbital treatment inhibited tube formation and the migration of human umbilical vein endothelial cells. In addition, it impaired angiogenesis in chick yolk sac membrane model and chorioallantoic membrane model. In summary, phenobarbital exposure led to shortened lengths of long bones during embryogenesis, which might result from inhibiting mesenchyme differentiation, chondrocyte proliferation, and delaying mineralization by impairing vascular invasion. PMID:27746734

  17. Histogenesis and possible mechanism of chondroid changes in mixed tumour of the skin: immunohistochemical evaluation of bone morphogenetic protein, glycosaminoglycans, keratin, vimentin and neuronal markers.

    Science.gov (United States)

    Mori, M; Shrestha, P; Sakamoto, F; Yang, L J; Qin, C; Tsujimura, T

    1994-01-01

    The distribution of immunoreactivity of bone morphogenetic protein (BMP), the glycosaminoglycans chondroitin 4-sulphate (C4SPG), chondroitin 6-sulphate (C6SPG), dermatan sulphate (DSPG) and keratan sulphate proteoglycans (KSPG), cytokeratin (K8.12), vimentin, glial fibrillary acidic protein (GFAP), actin, desmin, S-100 protein and neuron-specific enolase (NSE) in mixed tumour of the skin was investigated using immunohistochemical methods using monoclonal (MoAb) and polyclonal antibodies (PoAb). A strong BMP immunoreactivity was found characteristically in outer tumour cells of tubuloductal structures and modified myoepithelial cells. Modified myoepithelial cells and chondroidally changed cells showed positive immunoreactivity for C4SPG, C6SPG and DSPG; and KSPG was more pronounced in the modified myoepithelial cells. Vimentin, S-100 protein, GFAP and NSE, but not actin and desmin, were distribute in the outer tumour cells and modified myoepithelial cells in chondroidally changed tissue. Two factors show that chondrogenesis in mixed tumour of the skin is associated with the modified myoepithelial cells through the activity of BMP and biosynthesis of glycosaminoglycans as matrix substance. First, outer or basal tumour cells in mixed tumour of the skin is characterized by the presence of positive immunoreactivity for BMP, KSPG, vimentin, cytokeratin K8.12, S-100 protein, GFAP and NSE, and second, there is a matrix of chondroidally changed tissue containing the reaction products of C4SPG, C6SPG, DSPF and KSPG.

  18. β-Catenin Does Not Confer Tumorigenicity When Introduced into Partially Transformed Human Mesenchymal Stem Cells

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    Sajida Piperdi

    2012-01-01

    Full Text Available Although osteosarcoma is the most common primary malignant bone tumor in children and adolescents, its cell of origin and the genetic alterations are unclear. Previous studies have shown that serially introducing hTERT, SV40 large TAg, and H-Ras transforms human mesenchymal stem cells into two distinct sarcomas cell populations, but they do not form osteoid. In this study, β-catenin was introduced into mesenchymal stem cells already containing hTERT and SV40 large TAg to analyze if this resulted in a model which more closely recapitulated osteosarcoma. Results. Regardless of the level of induced β-catenin expression in the stable transfectants, there were no marked differences induced in their phenotype or invasion and migration capacity. Perhaps more importantly, none of them formed tumors when injected into immunocompromised mice. Moreover, the resulting transformed cells could be induced to osteogenic and chondrogenic differentiation but not to adipogenic differentiation. Conclusions. β-catenin, although fostering osteogenic differentiation, does not induce the malignant features and tumorigenicity conveyed by oncogenic H-RAS when introduced into partly transformed mesenchymal stem cells. This may have implications for the role of β-catenin in osteosarcoma pathogenesis. It also may suggest that adipogenesis is an earlier branch point than osteogenesis and chondrogenesis in normal mesenchymal differentiation.

  19. Low-frequency, low-magnitude vibrations (LFLM enhances chondrogenic differentiation potential of human adipose derived mesenchymal stromal stem cells (hASCs

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    Krzysztof Marycz

    2016-02-01

    Full Text Available The aim of this study was to evaluate if low-frequency, low-magnitude vibrations (LFLM could enhance chondrogenic differentiation potential of human adipose derived mesenchymal stem cells (hASCs with simultaneous inhibition of their adipogenic properties for biomedical purposes. We developed a prototype device that induces low-magnitude (0.3 g low-frequency vibrations with the following frequencies: 25, 35 and 45 Hz. Afterwards, we used human adipose derived mesenchymal stem cell (hASCS, to investigate their cellular response to the mechanical signals. We have also evaluated hASCs morphological and proliferative activity changes in response to each frequency. Induction of chondrogenesis in hASCs, under the influence of a 35 Hz signal leads to most effective and stable cartilaginous tissue formation through highest secretion of Bone Morphogenetic Protein 2 (BMP-2, and Collagen type II, with low concentration of Collagen type I. These results correlated well with appropriate gene expression level. Simultaneously, we observed significant up-regulation of α3, α4, β1 and β3 integrins in chondroblast progenitor cells treated with 35 Hz vibrations, as well as Sox-9. Interestingly, we noticed that application of 35 Hz frequencies significantly inhibited adipogenesis of hASCs. The obtained results suggest that application of LFLM vibrations together with stem cell therapy might be a promising tool in cartilage regeneration.

  20. Stem cell application for osteoarthritis in the knee joint: A minireview

    Institute of Scientific and Technical Information of China (English)

    Kristin; Uth; Dimitar; Trifonov

    2014-01-01

    Knee osteoarthritis is a chronic, indolent disease that will affect an ever increasing number of patients, especially the elderly and the obese. It is characterized by degeneration of the cartilage substance inside the knee which leads to pain, stiffness and tenderness. By some estimations in 2030, only in the United States, this medical condition will burden 67 million people. While conventional treatments like physiotherapy or drugs offer temporary relief of clinical symptoms, restoration of normal cartilage function has been difficult to achieve. Moreover, in severe cases of knee osteoarthritis total knee replacement may be required. Total knee replacements come together with high effort and costs and are not always successful. The aim of this review is to outline the latest advances in stem cell therapy for knee osteoarthritis as well as highlight some of the advantages of stem cell therapy over traditional approaches aimed at restoration of cartilage function in the knee. In addition to the latest advances in the field, challenges associated with stem cell therapy regarding knee cartilage regeneration and chondrogenesis in vitro and in vivo are also outlined and analyzed. Furthermore, based on their critical assessment of the present academic literature the authors of this review share their vision about the future of stem cell applications in the treatment of knee osteoarthritis.

  1. Fibroblast-like synoviocytes induce calcium mineral formation and deposition.

    Science.gov (United States)

    Sun, Yubo; Mauerhan, David R; Franklin, Atiya M; Zinchenko, Natalia; Norton, Harry James; Hanley, Edward N; Gruber, Helen E

    2014-01-01

    Calcium crystals are present in the synovial fluid of 65%-100% patients with osteoarthritis (OA) and 20%-39% patients with rheumatoid arthritis (RA). This study sought to investigate the role of fibroblast-like synoviocytes (FLSs) in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla) protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s). The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  2. Fibroblast-Like Synoviocytes Induce Calcium Mineral Formation and Deposition

    Directory of Open Access Journals (Sweden)

    Yubo Sun

    2014-01-01

    Full Text Available Calcium crystals are present in the synovial fluid of 65%–100% patients with osteoarthritis (OA and 20%–39% patients with rheumatoid arthritis (RA. This study sought to investigate the role of fibroblast-like synoviocytes (FLSs in calcium mineral formation. We found that numerous genes classified in the biomineral formation process, including bone gamma-carboxyglutamate (gla protein/osteocalcin, runt-related transcription factor 2, ankylosis progressive homolog, and parathyroid hormone-like hormone, were differentially expressed in the OA and RA FLSs. Calcium deposits were detected in FLSs cultured in regular medium in the presence of ATP and FLSs cultured in chondrogenesis medium in the absence of ATP. More calcium minerals were deposited in the cultures of OA FLSs than in the cultures of RA FLSs. Examination of the micromass stained with nonaqueous alcoholic eosin indicated the presence of birefringent crystals. Phosphocitrate inhibited the OA FLSs-mediated calcium mineral deposition. These findings together suggest that OA FLSs are not passive bystanders but are active players in the pathological calcification process occurring in OA and that potential calcification stimuli for OA FLSs-mediated calcium deposition include ATP and certain unidentified differentiation-inducing factor(s. The OA FLSs-mediated pathological calcification process is a valid target for the development of disease-modifying drug for OA therapy.

  3. Insights from amphioxus into the evolution of vertebrate cartilage.

    Directory of Open Access Journals (Sweden)

    Daniel Meulemans

    Full Text Available Central to the story of vertebrate evolution is the origin of the vertebrate head, a problem difficult to approach using paleontology and comparative morphology due to a lack of unambiguous intermediate forms. Embryologically, much of the vertebrate head is derived from two ectodermal tissues, the neural crest and cranial placodes. Recent work in protochordates suggests the first chordates possessed migratory neural tube cells with some features of neural crest cells. However, it is unclear how and when these cells acquired the ability to form cellular cartilage, a cell type unique to vertebrates. It has been variously proposed that the neural crest acquired chondrogenic ability by recruiting proto-chondrogenic gene programs deployed in the neural tube, pharynx, and notochord. To test these hypotheses we examined the expression of 11 amphioxus orthologs of genes involved in neural crest chondrogenesis. Consistent with cellular cartilage as a vertebrate novelty, we find that no single amphioxus tissue co-expresses all or most of these genes. However, most are variously co-expressed in mesodermal derivatives. Our results suggest that neural crest-derived cartilage evolved by serial cooption of genes which functioned primitively in mesoderm.

  4. Potential of human fetal chorionic stem cells for the treatment of osteogenesis imperfecta.

    Science.gov (United States)

    Jones, Gemma N; Moschidou, Dafni; Abdulrazzak, Hassan; Kalirai, Bhalraj Singh; Vanleene, Maximilien; Osatis, Suchaya; Shefelbine, Sandra J; Horwood, Nicole J; Marenzana, Massimo; De Coppi, Paolo; Bassett, J H Duncan; Williams, Graham R; Fisk, Nicholas M; Guillot, Pascale V

    2014-02-01

    Osteogenesis imperfecta (OI) is a genetic bone pathology with prenatal onset, characterized by brittle bones in response to abnormal collagen composition. There is presently no cure for OI. We previously showed that human first trimester fetal blood mesenchymal stem cells (MSCs) transplanted into a murine OI model (oim mice) improved the phenotype. However, the clinical use of fetal MSC is constrained by their limited number and low availability. In contrast, human fetal early chorionic stem cells (e-CSC) can be used without ethical restrictions and isolated in high numbers from the placenta during ongoing pregnancy. Here, we show that intraperitoneal injection of e-CSC in oim neonates reduced fractures, increased bone ductility and bone volume (BV), increased the numbers of hypertrophic chondrocytes, and upregulated endogenous genes involved in endochondral and intramembranous ossification. Exogenous cells preferentially homed to long bone epiphyses, expressed osteoblast genes, and produced collagen COL1A2. Together, our data suggest that exogenous cells decrease bone brittleness and BV by directly differentiating to osteoblasts and indirectly stimulating host chondrogenesis and osteogenesis. In conclusion, the placenta is a practical source of stem cells for the treatment of OI.

  5. Chondrocytes, Mesenchymal Stem Cells, and Their Combination in Articular Cartilage Regenerative Medicine.

    Science.gov (United States)

    Nazempour, A; Van Wie, B J

    2016-05-01

    Articular cartilage (AC) is a highly organized connective tissue lining, covering the ends of bones within articulating joints. Its highly ordered structure is essential for stable motion and provides a frictionless surface easing load transfer. AC is vulnerable to lesions and, because it is aneural and avascular, it has limited self-repair potential which often leads to osteoarthritis. To date, no fully successful treatment for osteoarthritis has been reported. Thus, the development of innovative therapeutic approaches is desperately needed. Autologous chondrocyte implantation, the only cell-based surgical intervention approved in the United States for treating cartilage defects, has limitations because of de-differentiation of articular chondrocytes (AChs) upon in vitro expansion. De-differentiation can be abated if initial populations of AChs are co-cultured with mesenchymal stem cells (MSCs), which not only undergo chondrogenesis themselves but also support chondrocyte vitality. In this review we summarize studies utilizing AChs, non-AChs, and MSCs and compare associated outcomes. Moreover, a comprehensive set of recent human studies using chondrocytes to direct MSC differentiation, MSCs to support chondrocyte re-differentiation and proliferation in co-culture environments, and exploratory animal intra- and inter-species studies are systematically reviewed and discussed in an innovative manner allowing side-by-side comparisons of protocols and outcomes. Finally, a comprehensive set of recommendations are made for future studies.

  6. Chondrogenic potential of canine articular cartilage derived cells (cACCs

    Directory of Open Access Journals (Sweden)

    Nowak Urszula

    2016-01-01

    Full Text Available In the present paper, the potential of canine articular cartilage-derived cells (cACCs for chondrogenic differentiation was evaluated. The effectiveness of cACCs’ lineage commitment was analyzed after 14 days of culture in chondorgenic and non-chondrogenic conditions. Formation of proteoglycan-rich extracellular matrix was assessed using histochemical staining – Alcian Blue and Safranin-O, while elemental composition was determined by means of SEM-EDX. Additionally, ultrastructure of cACCs was evaluated using TEM. The expression of genes involved in chondrogenesis was monitored with quantitative Real Time PCR. Results obtained indicate that the potential of cACCs for cartilagous extracellular matrix formation may be maintained only in chondrogenic cultures. The formation of specific chondro-nodules was not observed in a non-chondrogenic culture environment. The analysis of cACCs’ ultrastructure, both in non-chondrogenic and chondrogenic cultures, revealed well-developed rough endoplasmatic reticulum and presence of mitochondria. The cACCs in chondrogenic medium shed an increased number of microvesicles. Furthermore, it was shown that the extracellular matrix of cACCs in chondrogenic cultures is rich in potassium and molybdenum. Additionally, it was determined that gene expression of collagen type II, aggrecan and SOX-9 was significantly increased during chondrogenic differentiation of cACCs. Results obtained indicate that the culture environment may significantly influence the cartilage phenotype of cACCs during long term culture.

  7. The use of fibrin and poly(lactic-co-glycolic acid hybrid scaffold for articular cartilage tissue engineering: an in vivo analysis

    Directory of Open Access Journals (Sweden)

    S Munirah

    2008-02-01

    Full Text Available Our preliminary results indicated that fibrin and poly(lactic-co-glycolic acid (PLGA hybrid scaffold promoted early chondrogenesis of articular cartilage constructs in vitro. The aim of this study was to evaluate in vivo cartilaginous tissue formation by chondrocyte-seeded fibrin/PLGA hybrid scaffolds. PLGA scaffolds were soaked carefully, in chondrocyte-fibrin suspension, and polymerized by dropping thrombin-calcium chloride (CaCl2 solution. PLGA-seeded chondrocytes were used as a control. Resulting constructs were implanted subcutaneously, at the dorsum of nude mice, for 4 weeks. Macroscopic observation, histological evaluation, gene expression and sulphated-glycosaminoglycan (sGAG analyses were performed at each time point of 1, 2 and 4 weeks post-implantation. Cartilaginous tissue formation in fibrin/PLGA hybrid construct was confirmed by the presence of lacunae and cartilage-isolated cells embedded within basophilic ground substance. Presence of proteoglycan and glycosaminoglycan (GAG in fibrin/PLGA hybrid constructs was confirmed by positive Safranin O and Alcian Blue staining. Collagen type II exhibited intense immunopositivity at the pericellular matrices. Chondrogenic properties were further demonstrated by the expression of gene encoded cartilage-specific markers, collagen type II and aggrecan core protein. The sGAG production in fibrin/PLGA hybrid constructs was higher than in the PLGA group. In conclusion, fibrin/PLGA hybrid scaffold promotes cartilaginous tissue formation in vivo and may serve as a potential cell delivery vehicle and a structural basis for articular cartilage tissue-engineering.

  8. In vitro investigation of a tissue-engineered cell-tendon complex mimicking the transitional architecture at the ligament-bone interface.

    Science.gov (United States)

    Wang, Zhibing; Zhang, Yuan; Zhu, Jie; Dong, Shiwu; Jiang, Tao; Zhou, Yue; Zhang, Xia

    2015-03-01

    Restoration of the transitional ligament-bone interface is critical for graft-bone integration. We postulated that an allogenic scaffold mimicking the fibrogenic, chondrogenic, and osteogenic transition gradients could physiologically promote ligament-bone incorporation. The aim of this study was to construct and characterize a composite tendon scaffold with a continuous and heterogeneous transition region mimicking a native ligament insertion site. Genetically modified heterogeneous cell populations were seeded within specific regions of decellularized rabbit Achilles tendons to fabricate a stratified scaffold containing three biofunctional regions supporting fibrogenesis, chondrogenesis, and osteogenesis. The observed morphology, architecture, cytocompatibility, and biomechanics of the scaffolds demonstrated their improved bio-physico-chemical properties. The formation of the transitional regions was augmented via enhanced delivery of two transcription factors, sex determining region Y-box 9 and runt-related transcription factor 2, which also triggered early up-regulated expression of cartilage- and bone-relevant markers, according to quantitative PCR and immunoblot analyses. Gradient tissue-specific matrix formation was also confirmed within the predesignated regions via histological staining and immunofluorescence assays. These results suggest that a transitional interface could be replicated on an engineered tendon through stratified tissue integration. The scaffold offers the advantages of a multitissue transition involving controlled cellular interactions and matrix heterogeneity, which can be applied for the regeneration of the ligament-bone interface.

  9. The Regulatory Role of Signaling Crosstalk in Hypertrophy of MSCs and Human Articular Chondrocytes.

    Science.gov (United States)

    Zhong, Leilei; Huang, Xiaobin; Karperien, Marcel; Post, Janine N

    2015-08-14

    Hypertrophic differentiation of chondrocytes is a main barrier in application of mesenchymal stem cells (MSCs) for cartilage repair. In addition, hypertrophy occurs occasionally in osteoarthritis (OA). Here we provide a comprehensive review on recent literature describing signal pathways in the hypertrophy of MSCs-derived in vitro differentiated chondrocytes and chondrocytes, with an emphasis on the crosstalk between these pathways. Insight into the exact regulation of hypertrophy by the signaling network is necessary for the efficient application of MSCs for articular cartilage repair and for developing novel strategies for curing OA. We focus on articles describing the role of the main signaling pathways in regulating chondrocyte hypertrophy-like changes. Most studies report hypertrophic differentiation in chondrogenesis of MSCs, in both human OA and experimental OA. Chondrocyte hypertrophy is not under the strict control of a single pathway but appears to be regulated by an intricately regulated network of multiple signaling pathways, such as WNT, Bone morphogenetic protein (BMP)/Transforming growth factor-β (TGFβ), Parathyroid hormone-related peptide (PTHrP), Indian hedgehog (IHH), Fibroblast growth factor (FGF), Insulin like growth factor (IGF) and Hypoxia-inducible factor (HIF). This comprehensive review describes how this intricate signaling network influences tissue-engineering applications of MSCs in articular cartilage (AC) repair, and improves understanding of the disease stages and cellular responses within an OA articular joint.

  10. Transient receptor potential vanilloid 4: The sixth sense of the musculoskeletal system?

    Science.gov (United States)

    Guilak, Farshid; Leddy, Holly A; Liedtke, Wolfgang

    2010-03-01

    The critical discovery in the past two decades of the transient receptor potential (TRP) superfamily of ion channels has revealed the potential mechanisms by which cells sense diverse stimuli beyond the prototypical "five senses," identifying ion channels that are gated by heat, cold, mechanical loading, osmolarity, and other physical and chemical stimuli. TRP vanilloid 4 (TRPV4) is a Ca(2+)-permeable nonselective cation channel that appears to play a mechanosensory or osmosensory role in several musculoskeletal tissues. In articular cartilage, TRPV4 exhibits osmotic sensitivity, controlling cellular volume recovery, and other physiologic responses to osmotic stress. TRPV4 is expressed in both osteoblasts and osteoclasts, and the absence of TRPV4 prevents disuse-induced bone loss. TRPV4 activation promotes chondrogenesis by inducing SOX9 transcription, whereas a TRPV4 gain-of-function mutation leads to a developmental skeletal dysplasia, suggesting a critical role for TRPV4 in skeletal development. These studies provide mounting evidence for a regulatory role for the sensory channel TRPV4 in control of musculoskeletal tissues.

  11. Direct influence of culture dimensionality on human mesenchymal stem cell differentiation at various matrix stiffnesses using a fibrous self-assembling peptide hydrogel.

    Science.gov (United States)

    Hogrebe, Nathaniel J; Gooch, Keith J

    2016-09-01

    Much is unknown about the effects of culture dimensionality on cell behavior due to the lack of biomimetic substrates that are suitable for directly comparing cells grown on two-dimensional (2D) and encapsulated within three-dimensional (3D) matrices of the same stiffness and biochemistry. To overcome this limitation, we used a self-assembling peptide hydrogel system that has tunable stiffness and cell-binding site density as well as a fibrous microarchitecture resembling the structure of collagen. We investigated the effect of culture dimensionality on human mesenchymal stem cell differentiation at different values of matrix stiffness (G' = 0.25, 1.25, 5, and 10 kPa) and a constant RGD (Arg-Gly-Asp) binding site concentration. In the presence of the same soluble induction factors, culture on top of stiff gels facilitated the most efficient osteogenesis, while encapsulation within the same stiff gels resulted in a switch to predominantly terminal chondrogenesis. Adipogenesis dominated at soft conditions, and 3D culture induced better adipogenic differentiation than 2D culture at a given stiffness. Interestingly, initial matrix-induced cell morphology was predictive of these end phenotypes. Furthermore, optimal culture conditions corresponded to each cell type's natural niche within the body, highlighting the importance of incorporating native matrix dimensionality and stiffness into tissue engineering strategies. © 2016 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 104A: 2356-2368, 2016.

  12. Tissue-engineering strategies to repair joint tissue in osteoarthritis: nonviral gene-transfer approaches.

    Science.gov (United States)

    Madry, Henning; Cucchiarini, Magali

    2014-10-01

    Loss of articular cartilage is a common clinical consequence of osteoarthritis (OA). In the past decade, substantial progress in tissue engineering, nonviral gene transfer, and cell transplantation have provided the scientific foundation for generating cartilaginous constructs from genetically modified cells. Combining tissue engineering with overexpression of therapeutic genes enables immediate filling of a cartilage defect with an engineered construct that actively supports chondrogenesis. Several pioneering studies have proved that spatially defined nonviral overexpression of growth-factor genes in constructs of solid biomaterials or hydrogels is advantageous compared with gene transfer or scaffold alone, both in vitro and in vivo. Notably, these investigations were performed in models of focal cartilage defects, because advanced cartilage-repair strategies based on the principles of tissue engineering have not advanced sufficiently to enable resurfacing of extensively degraded cartilage as therapy for OA. These studies serve as prototypes for future technological developments, because they raise the possibility that cartilage constructs engineered from genetically modified chondrocytes providing autocrine and paracrine stimuli could similarly compensate for the loss of articular cartilage in OA. Because cartilage-tissue-engineering strategies are already used in the clinic, combining tissue engineering and nonviral gene transfer could prove a powerful approach to treat OA.

  13. The effect of acute and chronic stress on growth.

    Science.gov (United States)

    Sävendahl, Lars

    2012-10-23

    Impaired bone growth is observed in many children exposed to stress, but whether the underlying cause is psychological or secondary to a variety of chronic disorders is unclear. The growth plate is specifically targeted by stress through many different mechanisms, including increased serum concentrations of proinflammatory cytokines and cortisol, as well as impaired actions of the growth hormone (GH)-insulin-like growth factor-1 (IGF-1) axis. Both glucocorticoids, such as cortisol, and proinflammatory cytokines adversely affect several aspects of chondrogenesis in the growth plate, and these effects can be ameliorated by raising local IGF-1 concentrations. However, this intervention does not completely normalize growth. In children with stress related to chronic inflammation, the cornerstone of improving stress-impaired growth remains the judicious use of glucocorticoids while ensuring effective control of the disease process. Specific immunomodulatory therapy that targets the actions of tumor necrosis factor-α (TNFα) is at least partially effective at rescuing linear growth in many children with juvenile idiopathic arthritis (JIA). Patients who do not respond to anti-TNF treatment may be candidates for therapeutic agents that target other proinflammatory cytokines and for GH intervention. Although GH treatment rescues linear growth in some patients with JIA, it is unknown whether GH can rescue growth in those patients who do not respond to anticytokine therapy. Further experimental and clinical studies are needed to explore these and other new potential treatment strategies that could improve bone growth in patients who do not respond to conventional therapy.

  14. The versatile functions of Sox9 in development, stem cells, and human diseases

    Science.gov (United States)

    Jo, Alice; Denduluri, Sahitya; Zhang, Bosi; Wang, Zhongliang; Yin, Liangjun; Yan, Zhengjian; Kang, Richard; Shi, Lewis L.; Mok, James; Lee, Michael J.; Haydon, Rex C.

    2014-01-01

    The transcription factor Sox9 was first discovered in patients with campomelic dysplasia, a haploinsufficiency disorder with skeletal deformities caused by dysregulation of Sox9 expression during chondrogenesis. Since then, its role as a cell fate determiner during embryonic development has been well characterized; Sox9 expression differentiates cells derived from all three germ layers into a large variety of specialized tissues and organs. However, recent data has shown that ectoderm- and endoderm-derived tissues continue to express Sox9 in mature organs and stem cell pools, suggesting its role in cell maintenance and specification during adult life. The versatility of Sox9 may be explained by a combination of post-transcriptional modifications, binding partners, and the tissue type in which it is expressed. Considering its importance during both development and adult life, it follows that dysregulation of Sox9 has been implicated in various congenital and acquired diseases, including fibrosis and cancer. This review provides a summary of the various roles of Sox9 in cell fate specification, stem cell biology, and related human diseases. Ultimately, understanding the mechanisms that regulate Sox9 will be crucial for developing effective therapies to treat disease caused by stem cell dysregulation or even reverse organ damage. PMID:25685828

  15. Induction of systemic bone changes by preconditioning total body irradiation for bone marrow transplantation

    Energy Technology Data Exchange (ETDEWEB)

    Miyazaki, Osamu; Okamoto, Reiko; Masaki, Hidekazu [National Centre for Child Health and Development, Department of Radiology, Tokyo (Japan); Nishimura, Gen [Tokyo Metropolitan Kiyose Children' s Hospital, Department of Radiology, Tokyo (Japan); Kumagai, Masaaki; Shioda, Yoko [National Centre for Child Health and Development, Department of Oncology, Tokyo (Japan); Nozawa, Kumiko [Saitama Children' s Medical Centre, Department of Radiology, Saitama (Japan); Kitoh, Hiroshi [Nagoya University Hospital, Department of Orthopaedic Surgery, Nagoya, Aichi (Japan)

    2009-01-15

    Preconditioning total body irradiation (TBI) prior to bone marrow transplantation (BMT) has been believed to be a safe procedure that does not cause late morbidity; yet, a recent report raises the suspicion that TBI-induced chondroosseous abnormalities do occur. To evaluate the radiological manifestations of TBI-induced skeletal alterations and their orthopaedic morbidity. Subjects included 11 children with TBI-induced skeletal changes, including 9 in our hospital and 2 in other hospitals. The former were selected from 53 children who had undergone TBI with BMT. Radiographic examinations (n=11), MRI (n=3), CT (n=2), and medical records in the 11 children were retrospectively reviewed. The skeletal alterations included abnormal epiphyseal ossification and metaphyseal fraying (8/11), longitudinal metaphyseal striations (8/11), irregular metaphyseal sclerosis (6/11), osteochondromas (4/11), slipped capital femoral epiphysis (2/10), genu valgum (3/10), and platyspondyly (2/3). MRI demonstrated immature primary spongiosa in the metaphysis. Of the 11 children, 9 had clinical symptoms. TBI can induce polyostotic and/or generalized bone changes, mainly affecting the epiphyseal/metaphyseal regions and occasionally the spine. The epi-/metaphyseal abnormalities represent impaired chondrogenesis in the epiphysis and growth plate and abnormal remodelling in the metaphysis. Generalized spine changes may lead to misdiagnosis of a skeletal dysplasia. (orig.)

  16. Effect of FGFR inhibitors on chicken limb development.

    Science.gov (United States)

    Horakova, Dana; Cela, Petra; Krejci, Pavel; Balek, Lukas; Moravcova Balkova, Simona; Matalova, Eva; Buchtova, Marcela

    2014-10-01

    Fibroblast growth factor (FGF) signalling appears essential for the regulation of limb development, but a full complexity of this regulation remains unclear. Here, we addressed the effect of three different chemical inhibitors of FGF receptor tyrosine kinases (FGFR) on growth and patterning of the chicken wings. The inhibitor PD173074 caused shorter and thinner wing when using lower concentration. Microinjection of higher PD173074 concentrations (25 and 50 mmol/L) into the wing bud at stage 20 resulted in the development of small wing rudiment or the total absence of the wing. Skeletal analysis revealed the absence of the radius but not ulna, deformation of metacarpal bones and/or a reduction of digits. Treatment with PD161570 resembled the effects of PD173074. NF449 induced shortening and deformation of the developing wing with reduced autopodium. These malformed embryos mostly died at the stage HH25-29. PD173074 reduced chondrogenesis also in the limb micromass cultures together with early inhibition of cartilaginous nodule formation, evidenced by lack of sulphated proteoglycan and peanut agglutinin expression. The effect of FGFR inhibition on limb development observed here was unlikely mediated by excessive cell death as none of the inhibitors caused massive apoptosis at low concentrations. More probably, FGFR inhibition decreased both the proliferation and adhesion of mesenchymal chondroprogenitors. We conclude that FGFR signalling contributes to the regulation of the anterior-posterior patterning of zeugopod during chicken limb development.

  17. The Application of Three-Dimensional Printing in Animal Model of Augmentation Rhinoplasty.

    Science.gov (United States)

    Kim, Yoo Suk; Shin, Yoo Seob; Park, Do Yang; Choi, Jae Won; Park, Joo Kyung; Kim, Dong Ho; Kim, Chul Ho; Park, Su A

    2015-09-01

    The role of three-dimensional (3D) printing has expanded in diverse areas in medicine. As plastic surgery needs to fulfill the different demands from diverse individuals, the applications of tailored 3D printing will become indispensable. In this study, we evaluated the feasibility of using 3D-printed polycaprolactone (PCL) scaffold seeded with fibrin/chondrocytes as a new dorsal augmentation material for rhinoplasty. The construct was surgically implanted on the nasal dorsum in the subperiosteal plane of six rabbits. The implants were harvested 4 and 12 weeks after implantation and evaluated by gross morphological assessment, radiographic imaging, and histologic examination. The initial shape of the implant was unchanged in all cases, and no definite post-operative complications were seen over the 3-month period. Radiologic evaluation confirmed that implants remained in the initial location without migration or extrusion. Histologic evaluations showed that the scaffold architectures were maintained with minimal inflammatory reactions; however, expected neo-chondrogenesis was not definite in the constructs. A new PCL scaffold designed by 3D printing method seeded with fibrin/chondrocytes can be a biocompatible augmentation material in rhinoplasty in the future.

  18. Selenoprotein O deficiencies suppress chondrogenic differentiation of ATDC5 cells.

    Science.gov (United States)

    Yan, Jidong; Fei, Yao; Han, Yan; Lu, Shemin

    2016-10-01

    Selenoprotein O (Sel O) is a selenium-containing protein, but its function is still unclear. In the present study, we observed that the mRNA and protein expression levels of Sel O increased during chondrogenic induction of ATDC5 cells. The effects of Sel O on chondrocyte differentiation were then examined through shRNA-mediated gene silencing technique. The expression of Sel O was significantly suppressed at both mRNA and protein levels in a stable cell line transfected with a Sel O-specific target shRNA construct. Thereafter, we demonstrated that Sel O deficiencies suppress chondrogenic differentiation of ATDC5 cells. Sel O deficiencies inhibited expression of chondrogenic gene Sox9, Col II, and aggrecan. Sel O-deficient cells also accumulated a few cartilage glycosaminoglycans (GAGs) and decreased the activity of alkaline phosphatase (ALP). In addition, Sel O deficiencies inhibited chondrocyte proliferation through delayed cell cycle progression by suppression of cyclin D1 expression. Moreover, Sel O deficiencies induced chondrocyte death through cell apoptosis. In summary, we describe the expression patterns and the essential roles of Sel O in chondrocyte viability, proliferation, and chondrogenic differentiation. Additionally, Sel O deficiency-mediated impaired chondrogenesis may illustrate the mechanisms of Se deficiency in the pathophysiological process of the endemic osteoarthropathy.

  19. A synthetic thermo-sensitive hydrogel for cartilage bioprinting and its biofunctionalization with polysaccharides

    Science.gov (United States)

    Blokzijl, Maarten M.; Gawlitta, Debby; Dhert, Wouter J. A.; Hennink, Wim E.; Malda, Jos; Vermonden, Tina

    2016-01-01

    Hydrogels based on triblock copolymers of polyethylene glycol and partially methacrylated poly(N-(2-hydroxypropyl) methacrylamide mono/dilactate) are an attractive class of biomaterials due to their biodegradability, cytocompatibility, and tunable thermo-responsive and mechanical properties. By fine-tuning these properties, the hydrogels can be 3D bioprinted, to generate e.g. constructs for cartilage repair. This study investigated whether hydrogels based on the above mentioned polymer with a 10% degree of methacrylation (M10P10), support cartilage formation by chondrocytes, and whether the incorporation of methacrylated chondroitin sulfate (CSMA) or methacrylated hyaluronic acid (HAMA) can improve the mechanical properties, long-term stability, and printability. Chondrocyte-laden M10P10 hydrogels were cultured for 42 days to evaluate chondrogenesis. M10P10 hydrogels with or without polysaccharides were evaluated for their mechanical properties (before and after UV photo-cross-linking), degradation kinetics, and printability. Extensive cartilage matrix production occurred in M10P10 hydrogels, highlighting their potential for cartilage repair strategies. The incorporation of polysaccharides increased the storage modulus of polymer mixtures and decreased the degradation kinetics in cross-linked hydrogels. Addition of HAMA to M10P10 hydrogels improved printability and resulted in 3D constructs with excellent cell viability. Hence, this novel combination of M10P10 with HAMA forms an interesting class of hydrogels for cartilage bioprinting. PMID:27171342

  20. Evaluation of a mPEG-polyester-based hydrogel as cell carrier for chondrocytes.

    Science.gov (United States)

    Peng, Sydney; Yang, Shu-Rui; Ko, Chao-Yin; Peng, Yu-Shiang; Chu, I-Ming

    2013-11-01

    Temperature-sensitive hydrogels are attractive alternatives to porous cell-seeded scaffolds and is minimally invasive through simple injection and in situ gelling. In this study, we compared the performance of two types of temperature-sensitive hydrogels on chondrocytes encapsulation for the use of tissue engineering of cartilage. The two hydrogels are composed of methoxy poly(ethylene glycol)- poly(lactic-co-valerolactone) (mPEG-PVLA), and methoxy poly(ethylene glycol)-poly(lactic- co-glycolide) (mPEG-PLGA). Osmolarity and pH were optimized through the manipulation of polymer concentration and dispersion medium. Chondrocytes proliferation in mPEG-PVLA hydrogels was observed as well as accumulation of GAGs and collagen. On the other hand, chondrocytes encapsulated in mPEG-PLGA hydrogels showed low viability and chondrogenesis. Also, mPEG-PVLA hydrogel, which is more hydrophobic, retained physical integrity after 14 days while mPEG-PLGA hydrogel underwent full degradation due to faster hydrolysis rate and more pronounced acidic self-catalyzed degradation. The mPEG-PVLA hydrogel can be furthered tuned by manipulation of molecular weights to obtain hydrogels with different swelling and degradation characteristics, which may be useful as producing a selection of hydrogels compatible with different cell types. Taken together, these results demonstrate that mPEG-PVLA hydrogels are promising to serve as three-dimensional cell carriers for chondrocytes and potentially applicable in cartilage tissue engineering.

  1. Mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-induced Smad1/5/8 phosphorylation

    Science.gov (United States)

    Liu, Jia; Saito, Kan; Maruya, Yuriko; Nakamura, Takashi; Yamada, Aya; Fukumoto, Emiko; Ishikawa, Momoko; Iwamoto, Tsutomu; Miyazaki, Kanako; Yoshizaki, Keigo; Ge, Lihong; Fukumoto, Satoshi

    2016-01-01

    Bone morphogenetic proteins (BMPs) regulate hard tissue formation, including bone and tooth. Growth differentiation factor 5 (GDF5), a known BMP, is expressed in cartilage and regulates chondrogenesis, and mutations have been shown to cause osteoarthritis. Notably, GDF5 is also expressed in periodontal ligament tissue; however, its role during tooth development is unclear. Here, we used cell culture and in vivo analyses to determine the role of GDF5 during tooth development. GDF5 and its associated BMP receptors are expressed at the protein and mRNA levels during postnatal tooth development, particularly at a stage associated with enamel formation. Furthermore, whereas BMP2 was observed to induce evidently the differentiation of enamel-forming ameloblasts, excess GDF5 induce mildly this differentiation. A mouse model harbouring a mutation in GDF5 (W408R) showed enhanced enamel formation in both the incisors and molars, but not in the tooth roots. Overexpression of the W408R GDF5 mutant protein was shown to induce BMP2-mediated mRNA expression of enamel matrix proteins and downstream phosphorylation of Smad1/5/8. These results suggest that mutant GDF5 enhances ameloblast differentiation via accelerated BMP2-signalling. PMID:27030100

  2. Thyroid development in zebrafish lacking Taz.

    Science.gov (United States)

    Pappalardo, Andrea; Porreca, Immacolata; Caputi, Luigi; De Felice, Elena; Schulte-Merker, Stephan; Zannini, Mariastella; Sordino, Paolo

    2015-11-01

    Taz is a signal-responsive transcriptional coregulator implicated in several biological functions, from chondrogenesis to regulation of organ size. Less well studied, however, is its role in thyroid formation. Here, we explored the in vivo effects on thyroid development of morpholino (MO)-mediated knockdown of wwtr1, the gene encoding zebrafish Taz. The wwtr1 gene is expressed in the thyroid primordium and pharyngeal tissue of developing zebrafish. Compared to mammalian cells, in which Taz promotes expression of thyroid transcription factors and thyroid differentiation genes, wwtr1 MO injection in zebrafish had little or no effect on the expression of thyroid transcription factors, and differentially altered the expression of thyroid differentiation genes. Analysis of wwtr1 morphants at later stages of development revealed that the number and the lumen of thyroid follicles, and the number of thyroid follicle cells, were significantly smaller. In addition, Taz-depleted larvae displayed patterning defects in ventral cranial vessels that correlate with lateral displacement of thyroid follicles. These findings indicate that the zebrafish Taz protein is needed for the normal differentiation of the thyroid and are the first to suggest that Taz confers growth advantage to the endocrine gland.

  3. Current strategies of tissue engineering in talus chondral defects.

    Science.gov (United States)

    Martinelli, Niccolo; Bonifacini, Carlo; Longo, Umile Giuseppe; Marinozzi, Andrea; Florio, Pino; Khan, Wasim S; Denaro, Vincenzo

    2013-05-01

    Due to the nature of articular cartilage of being poorly vascularized the capabilities of self repair are limited. Mesenchymal stem cells transplantation is a modern technique which has been developed after the high success rates obtained by microfracturing and drilling techniques which promote the release of growth factors and the infiltration of bone marrow derived cells in the lesion. In order to increase the concentration of bone marrow derived cells appropriate devices, the scaffolds, are necessary. These three dimensional constructs mimic the physiological ambient of chondrogenesis.The race for new scaffold materials, which will show high biocompatibility to prevent inflammatory response, high cellular adhesion properties with three dimensional architecture, high bioactivity to deliver growth factor appropriately and possibly high biodegrability has just begun. New studies will concentrate on the role, on the interaction and on the temporal sequence of growth factors to improve ostheocondral differentiation, but the necessity to increase the number of clinical studies with more patients and longer follow ups seems mandatory. The aim of this review is to update and summarise the evidence-based knowledge of treatment of talus chondral defect with new tissue engineering techniques.

  4. The Chondrogenic Induction Potential for Bone Marrow-Derived Stem Cells between Autologous Platelet-Rich Plasma and Common Chondrogenic Induction Agents: A Preliminary Comparative Study

    Directory of Open Access Journals (Sweden)

    Shan-zheng Wang

    2015-01-01

    Full Text Available The interests in platelet-rich plasma (PRP and their application in stem cell therapy have contributed to a better understanding of the basic biology of the prochondrogenesis effect on bone marrow-derived stem cells (BMSCs. We aimed at comparing the effect of autologous PRP with common chondrogenic induction agents (CCIAs on the chondrogenic differentiation of BMSCs. Rabbit BMSCs were isolated and characterized by flow cytometry and differentiated towards adipocytes and osteoblasts. The chondrogenic response of BMSCs to autologous PRP and CCIAs which included transforming growth factor-β1 (TGF-β1, dexamethasone (DEX, and vitamin C (Vc was examined by cell pellet culture. The isolated BMSCs after two passages highly expressed CD29 and CD44 but minimally expressed CD45. The osteogenic and adipogenic differentiation potentials of the isolated BMSCs were also confirmed. Compared with common CCIAs, autologous PRP significantly upregulated the chondrogenic related gene expression, including Col-2, AGC, and Sox-9. Osteogenic related gene expression, including Col-1 and OCN, was not of statistical significance between these two groups. Thus, our data shows that, compared with common chondrogenic induction agents, autologous PRP can be more effective in promoting the chondrogenesis of BMSCs.

  5. Sox9 potentiates BMP2-induced chondrogenic differentiation and inhibits BMP2-induced osteogenic differentiation.

    Science.gov (United States)

    Liao, Junyi; Hu, Ning; Zhou, Nian; Lin, Liangbo; Zhao, Chen; Yi, Shixiong; Fan, Tingxu; Bao, Wei; Liang, Xi; Chen, Hong; Xu, Wei; Chen, Cheng; Cheng, Qiang; Zeng, Yongming; Si, Weike; Yang, Zhong; Huang, Wei

    2014-01-01

    Bone morphogenetic protein 2 (BMP2) is one of the key chondrogenic growth factors involved in the cartilage regeneration. However, it also exhibits osteogenic abilities and triggers endochondral ossification. Effective chondrogenesis and inhibition of BMP2-induced osteogenesis and endochondral ossification can be achieved by directing the mesenchymal stem cells (MSCs) towards chondrocyte lineage with chodrogenic factors, such as Sox9. Here we investigated the effects of Sox9 on BMP2-induced chondrogenic and osteogenic differentiation of MSCs. We found exogenous overexpression of Sox9 enhanced the BMP2-induced chondrogenic differentiation of MSCs in vitro. Also, it inhibited early and late osteogenic differentiation of MSCs in vitro. Subcutaneous stem cell implantation demonstrated Sox9 potentiated BMP2-induced cartilage formation and inhibited endochondral ossification. Mouse limb cultures indicated that BMP2 and Sox9 acted synergistically to stimulate chondrocytes proliferation, and Sox9 inhibited BMP2-induced chondrocytes hypertrophy and ossification. This study strongly suggests that Sox9 potentiates BMP2-induced MSCs chondrogenic differentiation and cartilage formation, and inhibits BMP2-induced MSCs osteogenic differentiation and endochondral ossification. Thus, exogenous overexpression of Sox9 in BMP2-induced mesenchymal stem cells differentiation may be a new strategy for cartilage tissue engineering.

  6. Zebrafish Fins as a Model System for Skeletal Human Studies

    Directory of Open Access Journals (Sweden)

    Manuel Marí-Beffa

    2007-01-01

    Full Text Available Recent studies on the morphogenesis of the fins of Danio rerio (zebrafish during development and regeneration suggest that a number of inductive signals involved in the process are similar to some of those that affect bone and cartilage differentiation in mammals and humans. Akimenko et al. (2002 has shown that bone morphogenetic protein-2b (BMP2b is involved in the induction of dermal bone differentiation during fin regeneration. Many other groups have also shown that molecules from the transforming growth factor-beta superfamily (TGFβ, including BMP2, are effective in promoting chondrogenesis and osteogenesis in vivo in higher vertebrates, including humans. In the present study, we review the state of the art of this topic by a comparative analysis of skeletal tissue development, regeneration and renewal processes in tetrapods, and fin regeneration in fishes. A general conclusion of this study states that lepidotrichia is a special skeletal tissue different to cartilage, bone, enamel, or dentine in fishes, according to its extracellular matrix (ECM composition. However, the empirical analysis of inducing signals of skeletal tissues in fishes and tetrapods suggests that lepidotrichia is different to any responding features with main skeletal tissues. A number of new inductive molecules are arising from fin development and regeneration studies that might establish an empirical basis for further molecular approaches to mammal skeletal tissues differentiation. Despite the tissue dissimilarity, this empirical evidence might finally lead to clinical applications to skeletal disorders in humans.

  7. Stable, position-related responses to retinoic acid by chick limb-bud mesenchymal cells in serum-free cultures.

    Science.gov (United States)

    Paulsen, D F; Solursh, M; Langille, R M; Pang, L; Chen, W D

    1994-03-01

    Retinoic acid (RA) has dramatic effects on limb-skeletal patterning in vivo and may well play a pivotal role in normal limb morphogenesis. RA's effects on the expression of pattern-related genes in the developing limb are probably mediated by cytoplasmic RA-binding proteins and nuclear RA-receptors. Little is known, however, about how RA modifies specific cellular behaviors required for skeletal morphogenesis. Earlier studies supported a role for regional differences in RA concentration in generating the region-specific cell behaviors that lead to pattern formation. The present study explores the possibility that position-related, cell-autonomous differences in the way limb mesenchymal cells respond to RA might have a role in generating pattern-related cell behavior. Mesenchymal cells from different proximodistal regions of stage 21-22 and 23-24 chick wing-buds were grown in chemically defined medium and exposed to 5 or 50 ng/ml of RA for 4 days in high-density microtiter cultures. The effects of RA on chondrogenesis in these cultures clearly differed depending on the limb region from which the cells were isolated. Regional differences in RA's effects on growth over 4 days in these cultures were less striking. The region-dependent responses of these cells to RA proved relatively stable in culture despite ongoing cytodifferentiation. This serum-free culture model will be useful in exploring the mechanisms underlying the region-dependent responsiveness of these cells to RA.

  8. Expression of two nonallelic type II procollagen genes during Xenopus laevis embryogenesis is characterized by stage-specific production of alternatively spliced transcripts.

    Science.gov (United States)

    Su, M W; Suzuki, H R; Bieker, J J; Solursh, M; Ramirez, F

    1991-10-01

    The pattern of type II collagen expression during Xenopus laevis embryogenesis has been established after isolating specific cDNA and genomic clones. Evidence is presented suggesting that in X. laevis there are two transcriptionally active copies of the type II procollagen gene. Both genes are activated at the beginning of neurula stage and steady-state mRNA levels progressively increase thereafter. Initially, the transcripts are localized to notochord, somites, and the dorsal region of the lateral plate mesoderm. At later stages of development and parallel to increased mRNA accumulation, collagen expression becomes progressively more confined to chondrogenic regions of the tadpole. During the early period of mRNA accumulation, there is also a transient pattern of expression in localized sites that will later not undergo chondrogenesis, such as the floor plate in the ventral neural tube. At later times and coincident with the appearance of chondrogenic tissues in the developing embryo, expression of the procollagen genes is characterized by the production of an additional, alternatively spliced transcript. The alternatively spliced sequences encode the cysteine-rich globular domain in the NH2-propeptide of the type II procollagen chain. Immunohistochemical analyses with a type II collagen monoclonal antibody documented the deposition of the protein in the extracellular matrix of the developing embryo. Type II collagen expression is therefore temporally regulated by tissue-specific transcription and splicing factors directing the synthesis of distinct molecular forms of the precursor protein in the developing Xenopus embryo.

  9. Evaluation of magnesium alloys with alternative surface finishing for the proliferation and chondro-differentiation of human mesenchymal stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Trinidad, J; Arruebarrena, G; De Argandona, E Saenz; De Eguino, G Ruiz; Infante, A; RodrIguez, C I, E-mail: jtrinidad@eps.mondragon.edu

    2010-11-01

    Articular cartilage has little capacity for self-repair. As a result, continuous mechanical stress can lead to the degradation of articular cartilage, culminating in progressive damage and joint degeneration. Tissue engineering has arisen as a promising therapeutic approach to cartilage repair. Magnesium alloys are one of the most important metallic biomaterials emerging in this area due to their biocompatibility, bio-absorbability and especially to their mechanical properties. These properties make magnesium alloys a promising biomaterial in the regeneration of cartilage tissue. Objective. This study was undertaken to analyze the influence of surface characteristics of magnesium alloys in the adhesion, proliferation and differentiation of human mesenchymal stem cells (MSCs). Methods. Two commercial magnesium alloys (AZ31B and ZM21) were subjected to different treatments in order to obtain four different surfaces in each alloy. Human MSCs were seeded into the magnesium alloys and analyzed for their proliferation and chondrogenesis differentiation ability. Results. Human MSCs showed a greater proliferation and chondro-differentiation when cultured in the ZM21 magnesium alloy with a surface finishing of fine sanding, polishing, and etching.

  10. Stem cell application for osteoarthritis in the knee joint: A minireview.

    Science.gov (United States)

    Uth, Kristin; Trifonov, Dimitar

    2014-11-26

    Knee osteoarthritis is a chronic, indolent disease that will affect an ever increasing number of patients, especially the elderly and the obese. It is characterized by degeneration of the cartilage substance inside the knee which leads to pain, stiffness and tenderness. By some estimations in 2030, only in the United States, this medical condition will burden 67 million people. While conventional treatments like physiotherapy or drugs offer temporary relief of clinical symptoms, restoration of normal cartilage function has been difficult to achieve. Moreover, in severe cases of knee osteoarthritis total knee replacement may be required. Total knee replacements come together with high effort and costs and are not always successful. The aim of this review is to outline the latest advances in stem cell therapy for knee osteoarthritis as well as highlight some of the advantages of stem cell therapy over traditional approaches aimed at restoration of cartilage function in the knee. In addition to the latest advances in the field, challenges associated with stem cell therapy regarding knee cartilage regeneration and chondrogenesis in vitro and in vivo are also outlined and analyzed. Furthermore, based on their critical assessment of the present academic literature the authors of this review share their vision about the future of stem cell applications in the treatment of knee osteoarthritis.

  11. BMP-mediated induction of GATA4/5/6 blocks somitic responsiveness to SHH.

    Science.gov (United States)

    Daoud, Georges; Kempf, Hervé; Kumar, Deepak; Kozhemyakina, Elena; Holowacz, Tamara; Kim, Dae-Won; Ionescu, Andreia; Lassar, Andrew B

    2014-10-01

    The relative timing of SHH and BMP signals controls whether presomitic mesoderm (PSM) cells will adopt either a chondrogenic or lateral plate mesoderm fate. Here we document that SHH-mediated induction of Nkx3.2 maintains the competence of somitic cells to initiate chondrogenesis in response to subsequent BMP signals by repressing BMP-dependent induction of GATA genes. Conversely, administration of BMP signals to PSM or forced expression of GATA family members in chick PSM explants blocks induction of hedgehog-dependent gene expression. We demonstrate that GATA factors can interact with Gli factors and can recruit the transcriptional co-factor FOG1 (ZFPM1) to the regulatory region of the mouse Gli1 gene, repressing the induction of Gli1 by SHH by binding to both GATA and Gli binding sites. Knockdown of FOG1 reverses the ability of GATA factors to repress Gli1 expression. Our findings uncover a novel role for GATA transcription factors as repressors of hedgehog signaling, and document that NKX3.2 maintains the ability of sclerotomal cells to express SHH transcriptional targets in the presence of BMP signals by repressing the induction of Gata4/5/6.

  12. Enhancement of chondrogenic differentiation of rabbit mesenchymal stem cells by oriented nanofiber yarn-collagen type I/hyaluronate hybrid

    Energy Technology Data Exchange (ETDEWEB)

    Zheng, Xianyou; Wang, Wei; Liu, Shen [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Wu, Jinglei [Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biological Engineering, Donghua University, Shanghai 201620 (China); Li, Fengfeng [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Cao, Lei [Shanghai Key Laboratory of Orthopaedic Implants, Department of Orthopaedics, Shanghai Ninth People' s Hospital, Shanghai Jiao Tong University School of Medicine, 639 Zhizaoju Road, Shanghai 200011 (China); Liu, Xu-dong [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China); Mo, Xiumei, E-mail: xmm@dhu.edu.cn [Biomaterials and Tissue Engineering Laboratory, College of Chemistry and Chemical Engineering and Biological Engineering, Donghua University, Shanghai 201620 (China); Fan, Cunyi, E-mail: fancunyish@126.com [Department of Orthopaedics, Shanghai Jiao Tong University Affiliated Sixth People' s Hospital, 600 Yishan Road, Shanghai 200233 (China)

    2016-01-01

    Cartilage defects cause joint pain and loss of mobility. It is crucial to induce the chondrogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) by both biological and structural signals in cartilage tissue engineering. Sponge-like scaffolds fabricated using native cartilage extracellular matrix components can induce the BMSC differentiation by biological signals and limited structural signals. In this study, an oriented poly(L-lactic acid)-co-poly(ε-caprolactone) P(LLA-CL)/collagen type I (Col-I) nanofiber yarn mesh, fabricated by dynamic liquid electrospinning served as a skeleton for a freeze-dried Col-I/hyaluronate (HA) chondral phase (SPONGE) containing both structural and biological signals to guide BMSC chondrogenic differentiation. In vitro results show that the Yarn Col-I/HA hybrid scaffold (Yarn-CH) promotes orientation, adhesion and proliferation of BMSCs better than SPONGE. Furthermore, BMSCs seeded on the Yarn-CH scaffold demonstrated a large increase in the glycosaminoglycan content and expression of collagen type II following a 21-day culture. - Highlights: • An oriented yarn was used as the skeleton of the sponge-like scaffold. • Both structural and biological signals were given for BMSC chondrogenic differentiation. • Yarn-CH promotes orientation and chondrogenesis differentiation of BMSCs. • Yarn-CH reproduces the superficial zone of the cartilage.

  13. Cloning and study of adult-tissue-specific expression of Sox9 in Cyprinus carpio

    Indian Academy of Sciences (India)

    Du Qi-Yan; Wang Feng-Yu; Hua Hui-Ying; Chang Zhong-Jie

    2007-08-01

    The Sox9 gene is one of the important transcription factors in the development of many tissues and organs, particularly in sex determination and chondrogenesis. We amplified the genomic DNA of Cyprinus carpio using degenerate primers, and found that there were two versions of Sox9 in this species: Sox9a and Sox9b, that differ in having an intron of different length (704 bp and 616 bp, respectively) in the conserved HMG box region that codes for identical amino acid sequences. We used a two-phase rapid amplification of cDNA ends (RACE) for the isolation of full-length cDNA of Sox9b. Sequence analyses revealed a 2447-bp cDNA containing 233-bp 5′ untranslated region, a 927-bp 3′ untranslated region, including poly(A), and a 1287 bp open reading frame (ORF) encoding a protein of 428 amino acids. The HMG box of 79 amino acid motif was confirmed from positions 96–174. Sequence alignment showed that the identity of amino acids of Sox9 among ten animal species, including C. carpio, is 75%, indicating that the Sox9 gene is evolutionarily quite conserved. The expression level of Sox9b gene varied among several organs of adult C. carpio, with the level of expression being highest in the brain and testis.

  14. Expression of TGF-β in Fractures Fixed by Nitinol Swan-like Memory Compressive Connectors

    Science.gov (United States)

    Li, M.; Zhang, C. C.; Xu, S. G.; Fu, Q. G.

    2011-07-01

    In this article, the effect of internal fixation of a Nitinol swan-like memory compressive connector (SMC) on the temporal expression of transforming growth factor-β (TGF-β) at fracture sites is evaluated. Specimens were collected from 35 New Zealand rabbits modeled for bilateral humeral fracture fixed with either SMC or stainless dynamic compression plate (DCP). Five rabbits each were killed at day 1, 3, 7, 14, 21, 28, and 56. The local positive staining potency, positive area ratio, and positive index of TGF-β were measured using an immunohistochemistry approach (EnVision) in combination with a computerized image analysis system. TGF-β staining was seen in mesenchymal cells, osteoblasts, chondrocytes, and in the extracellular matrix of fractures fixed in both the SMC and the DCP samples without a significant difference in staining at both the early stages (days 1 and 3) and day 56. A higher TGF-β content was observed in the fractures fixed with SMC when compared to that of DCP from day 7 to 28. As a conclusion, TGF-β is highly expressed in fractures fixed with SMC during chondrogenesis stage and entochondrostosis stage. Finally, the mechanism of how SMC promoting synthesis and secretion of TGF-β in the process of fracture healing has been discussed.

  15. Tailoring chemical and physical properties of fibrous scaffolds from block copolyesters containing ether and thio-ether linkages for skeletal differentiation of human mesenchymal stromal cells.

    Science.gov (United States)

    Chen, Honglin; Gigli, Matteo; Gualandi, Chiara; Truckenmüller, Roman; van Blitterswijk, Clemens; Lotti, Nadia; Munari, Andrea; Focarete, Maria Letizia; Moroni, Lorenzo

    2016-01-01

    Bioactive scaffolds for tissue engineering call for demands on new materials which can enhance traditional biocompatibility requirements previously considered for clinical implantation. The current commercially available thermoplastic materials, such as poly(lactic acid) (PLA), poly(glycolic acid) (PGA), poly(ε-caprolactone) (PCL) and their copolymers, have been used to fabricate scaffolds for regenerative medicine. However, these polymers have limitations including lacking of broadly tuning mechanical and degradable properties, and activation of specific cell-scaffold interactions, which limit their further application in tissue engineering. In the present study, electrospun scaffolds were successfully fabricated from a new class of block poly(butylene succinate)-based (PBS-based) copolyesters containing either butylene thiodiglycolate (BTDG) or butylene diglycolate (BDG) sequences. The polyesters displayed tunable mechanical properties and hydrolysis rate depending on the molecular architecture and on the kind of heteroatom introduced along the polymer backbone. To investigate their potential for skeletal regeneration, human mesenchymal stromal cells (hMSCs) were cultured on the scaffolds in basic, osteogenic and chondrogenic media. Our results demonstrated that PBS-based copolyesters containing thio-ether linkages (i.e. BTDG segments) were more favorable for chondrogenesis of hMSCs than those containing ether linkages (i.e. BDG sequences). In contrast, PBS-based copolyesters containing ether linkages showed enhanced mineralization. Therefore, these new functional scaffolds might hold potential for osteochondral tissue engineering applications.

  16. Effects of Bone Morphogenic Proteins on Engineered Cartilage

    Science.gov (United States)

    Gooch, Keith, J.; Blunk, Torsten; Courter, Donald L.; Sieminski, Alisha; Vunjak-Novakovic, Gordana; Freed, Lisa E.

    2007-01-01

    A report describes experiments on the effects of bone morphogenic proteins (BMPs) on engineered cartilage grown in vitro. In the experiments, bovine calf articular chondrocytes were seeded onto biodegradable polyglycolic acid scaffolds and cultured in, variously, a control medium or a medium supplemented with BMP-2, BMP-12, or BMP-13 in various concentrations. Under all conditions investigated, cell-polymer constructs cultivated for 4 weeks macroscopically and histologically resembled native cartilage. At a concentration of 100 ng/mL, BMP-2, BMP-12, or BMP-13 caused (1) total masses of the constructs to exceed those of the controls by 121, 80, or 62 percent, respectively; (2) weight percentages of glycosaminoglycans in the constructs to increase by 27, 18, or 15, respectively; and (3) total collagen contents of the constructs to decrease to 63, 89, or 83 percent of the control values, respectively. BMP-2, but not BMP-12 or BMP-13, promoted chondrocyte hypertrophy. These observations were interpreted as suggesting that the three BMPs increase the growth rates and modulate the compositions of engineered cartilage. It was also concluded that in vitro engineered cartilage is a suitable system for studying effects of BMPs on chondrogenesis in a well-defined environment.

  17. Evolution of Autologous Chondrocyte Repair and Comparison to Other Cartilage Repair Techniques

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    Ashvin K. Dewan

    2014-01-01

    Full Text Available Articular cartilage defects have been addressed using microfracture, abrasion chondroplasty, or osteochondral grafting, but these strategies do not generate tissue that adequately recapitulates native cartilage. During the past 25 years, promising new strategies using assorted scaffolds and cell sources to induce chondrocyte expansion have emerged. We reviewed the evolution of autologous chondrocyte implantation and compared it to other cartilage repair techniques. Methods. We searched PubMed from 1949 to 2014 for the keywords “autologous chondrocyte implantation” (ACI and “cartilage repair” in clinical trials, meta-analyses, and review articles. We analyzed these articles, their bibliographies, our experience, and cartilage regeneration textbooks. Results. Microfracture, abrasion chondroplasty, osteochondral grafting, ACI, and autologous matrix-induced chondrogenesis are distinguishable by cell source (including chondrocytes and stem cells and associated scaffolds (natural or synthetic, hydrogels or membranes. ACI seems to be as good as, if not better than, microfracture for repairing large chondral defects in a young patient’s knee as evaluated by multiple clinical indices and the quality of regenerated tissue. Conclusion. Although there is not enough evidence to determine the best repair technique, ACI is the most established cell-based treatment for full-thickness chondral defects in young patients.

  18. 23(S),25(R)-1,25-dihydroxyvitamin D3-26,23-lactone stimulates murine bone formation in vivo

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    Shima, M.; Tanaka, H.; Norman, A.W.; Yamaoka, K.; Yoshikawa, H.; Takaoka, K.; Ishizuka, S.; Seino, Y. (Osaka Univ. School of Medicine (Japan))

    1990-02-01

    23(S),25(R)-1,25-Dihydroxyvitamin D3-26,23-lactone (1,25-lactone) has been shown to have unique actions different from those of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). In contrast to 1,25-(OH)2D3, 1,25-lactone causes a significant reduction in the serum Ca2+ level, stimulates collagen production in an osteoblastic cell line, and inhibits bone resorption induced by 1,25-(OH)2D3. A possible effect of 1,25-lactone on bone formation was examined in experiments on ectopic bone formation using a bone-inducing factor derived from Dunn osteosarcomas. 1,25-Lactone, a metabolite of 1,25-(OH)2D3, increased (3H)proline uptake at the stage of chondrogenesis and {sup 85}Sr uptake during bone formation. Significantly enlarged bone was also induced by this compound 3 weeks after implantation. These results suggest that the 1,25-lactone may be able to stimulate bone formation under in vivo conditions.

  19. Noninvasive Measurement of Ear Cartilage Elasticity on the Cellular Level: A New Method to Provide Biomechanical Information for Tissue Engineering

    Science.gov (United States)

    Bos, Ernst Jan; van der Laan, Koen; Helder, Marco N.; Mullender, Margriet G.; Iannuzzi, Davide

    2017-01-01

    Background: An important feature of auricular cartilage is its stiffness. To tissue engineer new cartilage, we need objective tools to provide us with the essential biomechanical information to mimic optimal conditions for chondrogenesis and extracellular matrix (ECM) development. In this study, we used an optomechanical sensor to investigate the elasticity of auricular cartilage ECM and tested whether sensitivity and measurement reproducibility of the sensor would be sufficient to accurately detect (subtle) differences in matrix compositions in healthy, diseased, or regenerated cartilage. Methods: As a surrogate model to different cartilage ECM compositions, goat ears (n = 9) were subjected to different degradation processes to remove the matrix components elastin and glycosaminoglycans. Individual ear samples were cut and divided into 3 groups. Group 1 served as control and was measured within 2 hours after animal death and at 24 and 48 hours, and groups 2 and 3 were measured after 24- and 48-h hyaluronidase or elastase digestion. Per sample, 9 consecutive measurements were taken ±300 μm apart. Results: Good reproducibility was seen between consecutive measurements with an overall interclass correlation coefficient average of 0.9 (0.81–0.98). Although degradation led to variable results, overall, a significant difference was seen between treatment groups after 48 hours (control, 4.2 MPa [±0.5] vs hyaluronidase, 2.0 MPa [±0.3], and elastase, 3.0 MPa [±0.4]; both P development of tissue-engineered auricular cartilage.

  20. Neocartilage formation from mesenchymal stem cells grown in type II collagen-hyaluronan composite scaffolds.

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    Yeh, Hsi-Yi; Lin, Ting-Yu; Lin, Chen-Huan; Yen, B Linju; Tsai, Ching-Lin; Hsu, Shan-Hui

    2013-01-01

    Three-dimensional (3D) collagen type II-hyaluronan (HA) composite scaffolds (CII-HA) which mimics the extracellular environment of natural cartilage were fabricated in this study. Rheological measurements demonstrated that the incorporation of HA increased the compression modulus of the scaffolds. An initial in vitro evaluation showed that scaffolds seeded with porcine chondrocytes formed cartilaginous-like tissue after 8 weeks, and HA functioned to promote the growth of chondrocytes into scaffolds. Placenta-derived multipotent cells (PDMC) and gingival fibroblasts (GF) were seeded on tissue culture polystyrene (TCPS), CII-HA films, and small intestinal submucosa (SIS) sheets for comparing their chondrogenesis differentiation potentials with those of adipose-derived adult stem cells (ADAS) and bone marrow-derived mesenchymal stem cells (BMSC). Among different cells, PDMC showed the greatest chondrogenic differentiation potential on both CII-HA films and SIS sheets upon TGF-β3 induction, followed by GF. This was evidenced by the up-regulation of chondrogenic genes (Sox9, aggrecan, and collagen type II), which was not observed for cells grown on TCPS. This finding suggested the essential role of substrate materials in the chondrogenic differentiation of PDMC and GF. Neocartilage formation was more obvious in both PDMC and GF cells plated on CII-HA composite scaffolds vs. 8-layer SIS at 28 days in vitro. Finally, implantation of PDMC/CII-HA constructs into NOD-SCID mice confirmed the formation of tissue-engineered cartilage in vivo.

  1. Versican Expression during Synovial Joint Morphogenesis

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    John B. Shepard, Heidi A. Krug, Brooklynn A. LaFoon, Stanley Hoffman, Anthony A. Capehart

    2007-01-01

    Full Text Available The extracellular matrix (ECM plays a critical role in governing cell behavior and phenotype during limb skeletogenesis. Chondroitin sulfate proteoglycans (Cspgs are highly expressed in the ECM of precartilage mesenchymal condensations and are important to limb chondrogenesis and cartilage structure, but little is known regarding their involvement in formation of synovial joints in the embryonic limb. Matrix versican Cspg expression has previously been reported in the epiphysis of developing long bones and presumptive joint; however, detailed analysis has not yet been conducted. In the present study we immunolocalized versican and aggrecan Cspgs during chick elbow joint morphogenesis between HH st25-41 of development. In this study we show that versican and aggrecan expression initially overlapped in the incipient cartilage model of long bones in the wing, but versican was also highly expressed in the perichondrium and presumptive joint interzone during early stages of morphogenesis (HH st25-34. By HH st36-41 versican localization was restricted to the future articular surfaces of the developing joint and surrounding joint capsule while aggrecan localized in an immediately adjacent and predominately non-overlapping region of chondrogenic cells at the epiphyses. These results suggest a potential role for versican proteoglycan in development and maintenance of the synovial joint interzone.

  2. Identification of a novel population of human cord blood cells with hematopoietic and chondrocytic potential

    Institute of Scientific and Technical Information of China (English)

    Karen E JAY; Anne ROULEAU; T Michael UNDERHILL; Mickie BHATIA

    2004-01-01

    With the exception of mature erythrocytes, cells within the human hematopoietic system are characterized by the cell surface expression of the pan-leukocyte receptor CD45. Here, we identify a novel subset among mononuclear cord blood cells depleted of lineage commitment markers (Lin-) that are devoid of CD45 expression. Surprisingly, functional examination of Lin-CD45- cells also lacking cell surface CD34 revealed they were capable of multipotential hematopoietic progenitor capacity. Co-culture with mouse embryonic limb bud cells demonstrated that Lin-CD45-CD34- cells were capable of contributing to cartilage nodules and differentiating into human chondrocytes. BMP-4, a mesodermal factor known to promote chondrogenesis, significantly augmented Lin-CD45-CD34- differentiation into chondrocytes.Moreover, unlike CD34+ human hematopoietic stem cells, Lin-CD45-CD34- cells were unable to proliferate or survive in liquid cultures, whereas single Lin-CD45-CD34- cells were able to chimerize the inner cell mass (ICM) of murine blastocysts and proliferate in this embryonic environment. Our study identifies a novel population of Lin-CD45-CD34-cells capable of commitment into both hematopoietic and chondrocytic lineages, suggesting that human cord blood may provide a more ubiquitous source of tissue with broader developmental potential than previously appreciated.

  3. Articular Cartilage Repair Using Marrow Stimulation Augmented with a Viable Chondral Allograft: 9-Month Postoperative Histological Evaluation

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    James K. Hoffman

    2015-01-01

    Full Text Available Marrow stimulation is frequently employed to treat focal chondral defects of the knee. However, marrow stimulation typically results in fibrocartilage repair tissue rather than healthy hyaline cartilage, which, over time, predisposes the repair to failure. Recently, a cryopreserved viable chondral allograft was developed to augment marrow stimulation. The chondral allograft is comprised of native viable chondrocytes, chondrogenic growth factors, and extracellular matrix proteins within the superficial, transitional, and radial zones of hyaline cartilage. Therefore, host mesenchymal stem cells that infiltrate the graft from the underlying bone marrow following marrow stimulation are provided with the optimal microenvironment to undergo chondrogenesis. The present report describes treatment of a trochlear defect with marrow stimulation augmented with this novel chondral allograft, along with nine month postoperative histological results. At nine months, the patient demonstrated complete resolution of pain and improvement in function, and the repair tissue consisted of 85% hyaline cartilage. For comparison, a biopsy obtained from a patient 8.2 months after treatment with marrow stimulation alone contained only 5% hyaline cartilage. These outcomes suggest that augmenting marrow stimulation with the viable chondral allograft can eliminate pain and improve outcomes, compared with marrow stimulation alone.

  4. Collagen hydrogel as an immunomodulatory scaffold in cartilage tissue engineering.

    Science.gov (United States)

    Yuan, Tun; Zhang, Li; Li, Kuifeng; Fan, Hongsong; Fan, Yujiang; Liang, Jie; Zhang, Xingdong

    2014-02-01

    A collagen type I hydrogel was constructed and used as the scaffold for cartilage tissue engineering. Neonatal rabbit chondrocytes were seeded into the hydrogel, and the constructs were cultured in vitro for 7, 14, and 28 days. The immunomodulatory effect of the hydrogel on seeded chondrocytes was carefully investigated. The expressions of major histocompatibility complex classes I and II of seeded chondrocytes increased with the time, which indicated that the immunogenicity also increased with the time. Meanwhile, the properly designed collagen type I hydrogel could prompt the chondrogenesis of engineered cartilage. The extracellular matrix (ECM) synthesis ability of seeded chondrocytes and the accumulated ECM in the constructs continuously increased with the culture time. Both the isolation and protection, which come from formed ECM and hydrogel scaffold, can effectively control the adverse immunogenicity of seeded chondrocytes and even help to lessen the immunogenicity of the whole engineered cartilage. As the result, the levels of mixed lymphocyte chondrocyte reactions of seed cells and the constructs decreased gradually. The stimulation on allogeneic lymphocytes of the whole constructs was obviously lower than that of the retrieved cells from the constructs. Therefore, properly designed collagen type I hydrogel can give certain immunogenicity-reducing effects on engineered cartilage based on chondrocytes, and it may be a potential immunomodulatory biomaterial in tissue engineering.

  5. Human Articular Cartilage Progenitor Cells Are Responsive to Mechanical Stimulation and Adenoviral-Mediated Overexpression of Bone-Morphogenetic Protein 2.

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    Alexander J Neumann

    Full Text Available Articular cartilage progenitor cells (ACPCs represent a new and potentially powerful alternative cell source to commonly used cell sources for cartilage repair, such as chondrocytes and bone-marrow derived mesenchymal stem cells (MSCs. This is particularly due to the apparent resistance of ACPCs to hypertrophy. The current study opted to investigate whether human ACPCs (hACPCs are responsive towards mechanical stimulation and/or adenoviral-mediated overexpression of bone morphogenetic protein 2 (BMP-2. hACPCs were cultured in fibrin-polyurethane composite scaffolds. Cells were cultured in a defined chondro-permissive medium, lacking exogenous growth factors. Constructs were cultured, for 7 or 28 days, under free-swelling conditions or with the application of complex mechanical stimulation, using a custom built bioreactor that is able to generate joint-like movements. Outcome parameters were quantification of BMP-2 and transforming growth factor beta 1 (TGF-β1 concentration within the cell culture medium, biochemical and gene expression analyses, histology and immunohistochemistry. The application of mechanical stimulation alone resulted in the initiation of chondrogenesis, demonstrating the cells are mechanoresponsive. This was evidenced by increased GAG production, lack of expression of hypertrophic markers and a promising gene expression profile (significant up-regulation of cartilaginous marker genes, specifically collagen type II, accompanied by no increase in the hypertrophic marker collagen type X or the osteogenic marker alkaline phosphatase. To further investigate the resistance of ACPCs to hypertrophy, overexpression of a factor associated with hypertrophic differentiation, BMP-2, was investigated. A novel, three-dimensional, transduction protocol was used to transduce cells with an adenovirus coding for BMP-2. Over-expression of BMP-2, independent of load, led to an increase in markers associated with hypertropy. Taken together ACPCs

  6. Vitamin a is a negative regulator of osteoblast mineralization.

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    Thomas Lind

    Full Text Available An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1 with the active metabolite of vitamin A; retinoic acid (RA, a retinoic acid receptor (RAR antagonist (AGN194310, and a Cyp26 inhibitor (R115866 which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  7. Chordoma-derived cell line U-CH1-N recapitulates the biological properties of notochordal nucleus pulposus cells.

    Science.gov (United States)

    Fujita, Nobuyuki; Suzuki, Satoshi; Watanabe, Kota; Ishii, Ken; Watanabe, Ryuichi; Shimoda, Masayuki; Takubo, Keiyo; Tsuji, Takashi; Toyama, Yoshiaki; Miyamoto, Takeshi; Horiuchi, Keisuke; Nakamura, Masaya; Matsumoto, Morio

    2016-08-01

    Intervertebral disc degeneration proceeds with age and is one of the major causes of lumbar pain and degenerative lumbar spine diseases. However, studies in the field of intervertebral disc biology have been hampered by the lack of reliable cell lines that can be used for in vitro assays. In this study, we show that a chordoma-derived cell line U-CH1-N cells highly express the nucleus pulposus (NP) marker genes, including T (encodes T brachyury transcription factor), KRT19, and CD24. These observations were further confirmed by immunocytochemistry and flow cytometry. Reporter analyses showed that transcriptional activity of T was enhanced in U-CH1-N cells. Chondrogenic capacity of U-CH1-N cells was verified by evaluating the expression of extracellular matrix (ECM) genes and Alcian blue staining. Of note, we found that proliferation and synthesis of chondrogenic ECM proteins were largely dependent on T in U-CH1-N cells. In accordance, knockdown of the T transcripts suppressed the expression of PCNA, a gene essential for DNA replication, and SOX5 and SOX6, the master regulators of chondrogenesis. On the other hand, the CD24-silenced cells showed no reduction in the mRNA expression level of the chondrogenic ECM genes. These results suggest that U-CH1-N shares important biological properties with notochordal NP cells and that T plays crucial roles in maintaining the notochordal NP cell-like phenotype in this cell line. Taken together, our data indicate that U-CH1-N may serve as a useful tool in studying the biology of intervertebral disc. © 2016 The Authors. Journal of Orthopaedic Research Published by Wiley Periodicals, Inc. on behalf of Orthopaedic Research Society. J Orthop Res 34:1341-1350, 2016.

  8. The effects of dynamic compression on the development of cartilage grafts engineered using bone marrow and infrapatellar fat pad derived stem cells.

    Science.gov (United States)

    Luo, Lu; Thorpe, Stephen D; Buckley, Conor T; Kelly, Daniel J

    2015-09-21

    Bioreactors that subject cell seeded scaffolds or hydrogels to biophysical stimulation have been used to improve the functionality of tissue engineered cartilage and to explore how such constructs might respond to the application of joint specific mechanical loading. Whether a particular cell type responds appropriately to physiological levels of biophysical stimulation could be considered a key determinant of its suitability for cartilage tissue engineering applications. The objective of this study was to determine the effects of dynamic compression on chondrogenesis of stem cells isolated from different tissue sources. Porcine bone marrow (BM) and infrapatellar fat pad (FP) derived stem cells were encapsulated in agarose hydrogels and cultured in a chondrogenic medium in free swelling (FS) conditions for 21 d, after which samples were subjected to dynamic compression (DC) of 10% strain (1 Hz, 1 h d(-1)) for a further 21 d. Both BM derived stem cells (BMSCs) and FP derived stem cells (FPSCs) were capable of generating cartilaginous tissues with near native levels of sulfated glycosaminoglycan (sGAG) content, although the spatial development of the engineered grafts strongly depended on the stem cell source. The mechanical properties of cartilage grafts generated from both stem cell sources also approached that observed in skeletally immature animals. Depending on the stem cell source and the donor, the application of DC either enhanced or had no significant effect on the functional development of cartilaginous grafts engineered using either BMSCs or FPSCs. BMSC seeded constructs subjected to DC stained less intensely for collagen type I. Furthermore, histological and micro-computed tomography analysis showed mineral deposition within BMSC seeded constructs was suppressed by the application of DC. Therefore, while the application of DC in vitro may only lead to modest improvements in the mechanical functionality of cartilaginous grafts, it may play an important

  9. Substrate stiffness and oxygen as regulators of stem cell differentiation during skeletal tissue regeneration: a mechanobiological model.

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    Darren Paul Burke

    Full Text Available Extrinsic mechanical signals have been implicated as key regulators of mesenchymal stem cell (MSC differentiation. It has been possible to test different hypotheses for mechano-regulated MSC differentiation by attempting to simulate regenerative events such as bone fracture repair, where repeatable spatial and temporal patterns of tissue differentiation occur. More recently, in vitro studies have identified other environmental cues such as substrate stiffness and oxygen tension as key regulators of MSC differentiation; however it remains unclear if and how such cues determine stem cell fate in vivo. As part of this study, a computational model was developed to test the hypothesis that substrate stiffness and oxygen tension regulate stem cell differentiation during fracture healing. Rather than assuming mechanical signals act directly on stem cells to determine their differentiation pathway, it is postulated that they act indirectly to regulate angiogenesis and hence partially determine the local oxygen environment within a regenerating tissue. Chondrogenesis of MSCs was hypothesized to occur in low oxygen regions, while in well vascularised regions of the regenerating tissue a soft local substrate was hypothesised to facilitate adipogenesis while a stiff substrate facilitated osteogenesis. Predictions from the model were compared to both experimental data and to predictions of a well established computational mechanobiological model where tissue differentiation is assumed to be regulated directly by the local mechanical environment. The model predicted all the major events of fracture repair, including cartilaginous bridging, endosteal and periosteal bony bridging and bone remodelling. It therefore provides support for the hypothesis that substrate stiffness and oxygen play a key role in regulating MSC fate during regenerative events such as fracture healing.

  10. Analysis of the chondrogenic potential and secretome of mesenchymal stem cells derived from human umbilical cord stroma.

    Science.gov (United States)

    Arufe, Maria C; De la Fuente, Alexandre; Mateos, Jesus; Fuentes, Isaac; De Toro, Francisco J; Blanco, Francisco J

    2011-07-01

    Mesenchymal stem cells (MSCs) from umbilical cord stroma were isolated by plastic adherence and characterized by flow cytometry, looking for cells positive for OCT3/4 and SSEA-4 as well as the classic MSC markers CD44, CD73, CD90, Ki67, CD105, and CD106 and negative for CD34 and CD45. Quantitative reverse transcriptase-polymerase chain reaction analysis of the genes ALP, MEF2C, MyoD, LPL, FAB4, and AMP, characteristic for the differentiated lineages, were used to evaluate early and late differentiation of 3 germ lines. Direct chondrogenic differentiation was achieved through spheroid formation by MSCs in a chondrogenic medium and the presence of chondrogenic markers at 4, 7, 14, 28, and 46 days of culture was tested. Immunohistochemistry and quantitative reverse transcriptase-polymerase chain reaction analyses were utilized to assess the expression of collagen type I, collagen type II, and collagen type X throughout the time studied. We found expression of all the markers as early as 4 days of chondrogenic differentiation culture, with their expression increasing with time, except for collagen type I, which decreased in expression in the formed spheroids after 4 days of differentiation. The signaling role of Wnt during chondrogenic differentiation was studied by western blot. We observed that β-catenin expression decreased during the chondrogenic process. Further, a secretome study to validate our model of differentiation in vitro was performed on spheroids formed during the chondrogenesis process. Our results indicate the multipotential capacity of this source of human cells; their chondrogenic capacity could be useful for future cell therapy in articular diseases.

  11. The Effects of Class-Specific Histone Deacetylase Inhibitors on the Development of Limbs During Organogenesis.

    Science.gov (United States)

    Paradis, France-Hélène; Hales, Barbara F

    2015-11-01

    Histone deacetylases (HDACs) play a major role in chromatin remodeling, gene regulation, and cellular signaling. While the role of each class of HDAC during normal development is unclear, several HDAC inhibitors are embryotoxic; the mechanisms leading to the teratogenicity of HDAC inhibitors are not known. Here, we investigated the effects of class-specific HDAC inhibitors on the development of organogenesis-stage murine limbs. Timed-pregnant COL2A1-ECFP, COL10A1-mCherry, and COL1A1-YFP CD1 reporter mice were euthanized on gestation day 12; embryonic forelimbs were excised and cultured in vitro for 1, 3, and 6 days in the presence or absence of MS275 (a class I HDAC inhibitor), MC1568 (a class III HDAC inhibitor), Sirtinol (a class II HDAC inhibitor), or valproic acid, our positive control. Fluorescently tagged COL2A1, COL10A1, and COL1A1 served as markers of the differentiation of proliferative chondrocytes, hypertrophic chondrocytes, and osteoblasts, respectively. MS275 and valproic acid caused a reduction in expression of all three markers, suggesting effects on both chondrogenesis and osteogenesis. MC1568 had no effect on chondrocyte markers and mildly inhibited COL1A1 expression at 6 days. Sirtinol had no effect on COL2A1 expression or chondrocyte differentiation 1 day following exposure; however, it caused a drastic regression in limb cartilage and reduced the expression of all three differentiation markers to nearly undetectable levels at 6 days. MS275 and Sirtinol caused a 2.2- and 2.7-fold increase, respectively, in cleaved-caspase 3, a marker of apoptosis, suggesting embryotoxicity. These data demonstrate that inhibition of class I or III HDACs causes severe developmental toxicity and is highly teratogenic.

  12. Imaging mesenchymal stem cells containing single wall nanotube nanoprobes in a 3D scaffold using photo-thermal optical coherence tomography

    Science.gov (United States)

    Connolly, Emma; Subhash, Hrebesh M.; Leahy, Martin; Rooney, Niall; Barry, Frank; Murphy, Mary; Barron, Valerie

    2014-02-01

    Despite the fact, that a range of clinically viable imaging modalities, such as magnetic resonance imaging (MRI), computed tomography (CT), photo emission tomography (PET), ultrasound and bioluminescence imaging are being optimised to track cells in vivo, many of these techniques are subject to limitations such as the levels of contrast agent required, toxic effects of radiotracers, photo attenuation of tissue and backscatter. With the advent of nanotechnology, nanoprobes are leading the charge to overcome these limitations. In particular, single wall nanotubes (SWNT) have been shown to be taken up by cells and as such are effective nanoprobes for cell imaging. Consequently, the main aim of this research is to employ mesenchymal stem cells (MSC) containing SWNT nanoprobes to image cell distribution in a 3D scaffold for cartilage repair. To this end, MSC were cultured in the presence of 32μg/ml SWNT in cell culture medium (αMEM, 10% FBS, 1% penicillin/streptomycin) for 24 hours. Upon confirmation of cell viability, the MSC containing SWNT were encapsulated in hyaluronic acid gels and loaded on polylactic acid polycaprolactone scaffolds. After 28 days in complete chondrogenic medium, with medium changes every 2 days, chondrogenesis was confirmed by the presence of glycosaminoglycan. Moreover, using photothermal optical coherence tomography (PT-OCT), the cells were seen to be distributed through the scaffold with high resolution. In summary, these data reveal that MSC containing SWNT nanoprobes in combination with PT-OCT offer an exciting opportunity for stem cell tracking in vitro for assessing seeding scaffolds and in vivo for determining biodistribution.

  13. Chondroinduction from Naturally Derived Cartilage Matrix: A Comparison Between Devitalized and Decellularized Cartilage Encapsulated in Hydrogel Pastes.

    Science.gov (United States)

    Beck, Emily C; Barragan, Marilyn; Libeer, Tony B; Kieweg, Sarah L; Converse, Gabriel L; Hopkins, Richard A; Berkland, Cory J; Detamore, Michael S

    2016-04-01

    Hydrogel precursors are liquid solutions that are prone to leaking after surgical placement. This problem was overcome by incorporating either decellularized cartilage (DCC) or devitalized cartilage (DVC) microparticles into traditional photocrosslinkable hydrogel precursors in an effort to achieve a paste-like hydrogel precursor. DCC and DVC were selected specifically for their potential to induce chondrogenesis of stem cells, given that materials that are chondroinductive on their own without growth factors are a revolutionary goal in orthopedic medicine. We hypothesized that DVC, lacking the additional chemical processing steps in DCC to remove cell content, would lead to a more chondroinductive hydrogel with rat bone marrow-derived mesenchymal stem cells. Hydrogels composed of methacrylated hyaluronic acid (MeHA) and either DCC or DVC microparticles were tested with and without exposure to transforming growth factor (TGF)-β3 over a 6 week culture period, where swelling, mechanical analysis, and gene expression were observed. For collagen II, Sox-9, and aggrecan expression, MeHA precursors containing DVC consistently outperformed the DCC-containing groups, even when the DCC groups were exposed to TGF-β3. DVC consistently outperformed all TGF-β3-exposed groups in aggrecan and collagen II gene expression as well. In addition, when the same concentrations of MeHA with DCC or DVC microparticles were evaluated for yield stress, the yield stress with the DVC microparticles was 2.7 times greater. Furthermore, the only MeHA-containing group that exhibited shape retention was the group containing DVC microparticles. DVC appeared to be superior to DCC in both chondroinductivity and rheological performance of hydrogel precursors, and therefore DVC microparticles may hold translational potential for cartilage regeneration.

  14. A Novel Regulatory Mechanism of Type II Collagen Expression via a SOX9-dependent Enhancer in Intron 6.

    Science.gov (United States)

    Yasuda, Hideyo; Oh, Chun-do; Chen, Di; de Crombrugghe, Benoit; Kim, Jin-Hoi

    2017-01-13

    Type II collagen α1 is specific for cartilaginous tissues, and mutations in its gene are associated with skeletal diseases. Its expression has been shown to be dependent on SOX9, a master transcription factor required for chondrogenesis that binds to an enhancer region in intron 1. However, ChIP sequencing revealed that SOX9 does not strongly bind to intron 1, but rather it binds to intron 6 and a site 30 kb upstream of the transcription start site. Here, we aimed to determine the role of the novel SOX9-binding site in intron 6. We prepared reporter constructs that contain a Col2a1 promoter, intron 1 with or without intron 6, and the luciferase gene. Although the reporter constructs were not activated by SOX9 alone, the construct that contained both introns 1 and 6 was activated 5-10-fold by the SOX9/SOX5 or the SOX9/SOX6 combination in transient-transfection assays in 293T cells. This enhancement was also observed in rat chondrosarcoma cells that stably expressed the construct. CRISPR/Cas9-induced deletion of intron 6 in RCS cells revealed that a 10-bp region of intron 6 is necessary both for Col2a1 expression and SOX9 binding. Furthermore, SOX9, but not SOX5, binds to this region as demonstrated in an electrophoretic mobility shift assay, although both SOX9 and SOX5 bind to a larger 325-bp fragment of intron 6 containing this small sequence. These findings suggest a novel mechanism of action of SOX5/6; namely, the SOX9/5/6 combination enhances Col2a1 transcription through a novel enhancer in intron 6 together with the enhancer in intron 1.

  15. Aislamiento y caracterización de células “stem” mesenquimales de médula ósea humana según criterios de la Sociedad Internacional de Terapia Celular

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    Jose Alejandro Aristizabal-Castellanos

    2010-12-01

    Full Text Available Isolation and characterization of mesenchymal stem cells from human bone marrow according to the criteria of the InternationalSociety for Cellular Therapy. Bone marrow (BM is an important source for isolating mesenchymal stem cells (MSC useful inimmunomodulation and tissue regeneration therapies. Objective. To isolate and characterize mesenchymal stem cells obtained from BMmeeting the requirements of the International Society for Cellular Therapy. Materials and methods. BM samples were collected fromvolunteer donors attending the Orthopedics Service of the San Ignacio University Hospital (Bogotá, Colombia. Morphological characteristicswere evaluated by inverted microscopy and the immunophenotype was determined by flow cytometry. Protocols were developed foradipogenic, osteogenic and chondrogenic differentiation using the Oil Red O, alkaline phosphatase and safranin stains, respectively.Results. We collected 24 samples of BM from patients with total hip replacement (volume of BM sample: 5-45 ml. Cells with afibroblastoid morphology were isolated from 21 BM samples (isolation efficiency: 87.5%. No statistical significant differences were foundbetween the hematopoyetic antigens (CD34 and CD45, p>0.05 in the immunophenotypic evaluation (of MSC from BM; on the contrary,there were differences (p=0.006 between the hematopoyetic antigen CD45 and the mesenchymal antigens (CD13, CD44, CD73, CD90,CD105, HLA-I, and HLA-DR. Oil Red O stain revealed the presence of multilocular adipocytes, in the osteogenic induction we observedlocalized mineralization nodules, and chondrogenesis was positive as revealed by the safranin stain. Conclusion. MSC were satisfactorilyisolated from BM and characterized according to the international standards.

  16. Increased adipogenesis in cultured embryonic chondrocytes and in adult bone marrow of dominant negative Erg transgenic mice.

    Directory of Open Access Journals (Sweden)

    Sébastien Flajollet

    Full Text Available In monolayer culture, primary articular chondrocytes have an intrinsic tendency to lose their phenotype during expansion. The molecular events underlying this chondrocyte dedifferentiation are still largely unknown. Several transcription factors are important for chondrocyte differentiation. The Ets transcription factor family may be involved in skeletal development. One family member, the Erg gene, is mainly expressed during cartilage formation. To further investigate the potential role of Erg in the maintenance of the chondrocyte phenotype, we isolated and cultured chondrocytes from the rib cartilage of embryos of transgenic mice that express a dominant negative form of Erg (DN-Erg during cartilage formation. DN-Erg expression in chondrocytes cultured for up to 20 days did not affect the early dedifferentiation usually observed in cultured chondrocytes. However, lipid droplets accumulated in DN-Erg chondrocytes, suggesting adipocyte emergence. Transcriptomic analysis using a DNA microarray, validated by quantitative RT-PCR, revealed strong differential gene expression, with a decrease in chondrogenesis-related markers and an increase in adipogenesis-related gene expression in cultured DN-Erg chondrocytes. These results indicate that Erg is involved in either maintaining the chondrogenic phenotype in vitro or in cell fate orientation. Along with the in vitro studies, we compared adipocyte presence in wild-type and transgenic mice skeletons. Histological investigations revealed an increase in the number of adipocytes in the bone marrow of adult DN-Erg mice even though no adipocytes were detected in embryonic cartilage or bone. These findings suggest that the Ets transcription factor family may contribute to the homeostatic balance in skeleton cell plasticity.

  17. Isolation and Characterization of Multipotential Mesenchymal Stromal Cells from Congenital Pseudoarthrosis of the Tibia: Case Report.

    Science.gov (United States)

    Diaz-Solano, Dylana; Wittig, Olga; Mota, Jose D; Cardier, Jose E

    2015-10-01

    Congenital pseudoarthrosis of the tibia (CPT) is an uncommon disease whose etiology and pathogenesis is unknown. Several evidences suggest that decreased osteogenic capacities, impaired local vascularization, and microenvironment alterations may play a role in the pathogenesis of CPT. Additionally, it is not clear if the pathogenesis of this disease is related to the absence of cells with osteogenic capacity of differentiation. In this work, a two-year-old patient diagnosed with CPT underwent an orthopedic surgery to promote bone union in a pseudoarthrosis lesion. Tissue from CPT lesion was excised, and histological evaluation and tissue culture were performed. Histologic analysis of the soft CPT lesion showed the presence of highly cellular fibrous tissue, vascularization, and abundant extracellular matrix. Fusiform cells of mesenchymal appearance were observed but osteoblasts, osteoclasts, chondrocytes, and adipose cells were not found. There was no evidence of osteogenesis. CPT tissue cultured as explants showed, after one month of culture, evidence of osteogenesis, chondrogenesis, and adipogenesis. Cells isolated from explants of CPT tissue showed a fibroblast-like morphology and expressed the mesenchymal stromal cell (MSC) markers: CD105, CD73, and CD90 (CPT-MSC). Functional analysis showed that CPT-MSC differentiate, in vitro, into osteogenic, chondrogenic, and adipocytic cells. CPT-MSC expressed osteocalcin and agrecan. CPT-MSC produced collagen in the presence of ascorbic acid. MSC from BM of normal individuals were used as control. In summary, our results indicate that CPT tissue contains MSC with osteogenic capacity of differentiation. It is possible that CPT microenvironment may contribute to impair the osteogenic capacity of differentiation of CPT-MSC.

  18. Analysis of the Fgfr2C342Y mouse model shows condensation defects due to misregulation of Sox9 expression in prechondrocytic mesenchyme

    Science.gov (United States)

    Peskett, Emma; Kumar, Samin; Baird, William; Jaiswal, Janhvi; Li, Ming; Patel, Priyanca; Britto, Jonathan A.

    2017-01-01

    ABSTRACT Syndromic craniosynostosis caused by mutations in FGFR2 is characterised by developmental pathology in both endochondral and membranous skeletogenesis. Detailed phenotypic characterisation of features in the membranous calvarium, the endochondral cranial base and other structures in the axial and appendicular skeleton has not been performed at embryonic stages. We investigated bone development in the Crouzon mouse model (Fgfr2C342Y) at pre- and post-ossification stages to improve understanding of the underlying pathogenesis. Phenotypic analysis was performed by whole-mount skeletal staining (Alcian Blue/Alizarin Red) and histological staining of sections of CD1 wild-type (WT), Fgfr2C342Y/+ heterozygous (HET) and Fgfr2C342Y/C342Y homozygous (HOM) mouse embryos from embryonic day (E)12.5-E17.5 stages. Gene expression (Sox9, Shh, Fgf10 and Runx2) was studied by in situ hybridisation and protein expression (COL2A1) by immunohistochemistry. Our analysis has identified severely decreased osteogenesis in parts of the craniofacial skeleton together with increased chondrogenesis in parts of the endochondral and cartilaginous skeleton in HOM embryos. The Sox9 expression domain in tracheal and basi-cranial chondrocytic precursors at E13.5 in HOM embryos is increased and expanded, correlating with the phenotypic observations which suggest FGFR2 signalling regulates Sox9 expression. Combined with abnormal staining of type II collagen in pre-chondrocytic mesenchyme, this is indicative of a mesenchymal condensation defect. An expanded spectrum of phenotypic features observed in the Fgfr2C342Y/C342Y mouse embryo paves the way towards better understanding the clinical attributes of human Crouzon–Pfeiffer syndrome. FGFR2 mutation results in impaired skeletogenesis; however, our findings suggest that many phenotypic aberrations stem from a primary failure of pre-chondrogenic/osteogenic mesenchymal condensation and link FGFR2 to SOX9, a principal regulator of skeletogenesis

  19. Epimorphic regeneration in mice is p53-independent.

    Science.gov (United States)

    Arthur, L Matthew; Demarest, Renee M; Clark, Lise; Gourevitch, Dmitri; Bedelbaeva, Kamila; Anderson, Rhonda; Snyder, Andrew; Capobianco, Anthony J; Lieberman, Paul; Feigenbaum, Lionel; Heber-Katz, E

    2010-09-15

    The process of regeneration is most readily studied in species of sponge, hydra, planarian and salamander (i.e., newt and axolotl). The closure of MRL mouse ear pinna through-and-through holes provides a mammalian model of unusual wound healing/regeneration in which a blastema-like structure closes the ear hole and cartilage and hair follicles are replaced. Recent studies, based on a broad level of DNA damage and a cell cycle pattern of G₂/M "arrest," showed that p21(Cip1/Waf1) was missing from the MRL mouse ear and that a p21-null mouse could close its ear holes. Given the p53/p21 axis of control of DNA damage, cell cycle arrest, apoptosis and senescence, we tested the role of p53 in the ear hole regenerative response. Using backcross mice, we found that loss of p53 in MRL mice did not show reduced healing. Furthermore, cross sections of MRL. p53(-/-) mouse ears at 6 weeks post-injury showed an increased level of adipocytes and chondrocytes in the region of healing whereas MRL or p21(-/-) mice showed chondrogenesis alone in this same region, though at later time points. In addition, we also investigated other cell cycle-related mutant mice to determine how p21 was being regulated. We demonstrate that p16 and Gadd45 null mice show little healing capacity. Interestingly, a partial healing phenotype in mice with a dual Tgfβ/Rag2 knockout mutation was seen. These data demonstrate an independence of p53 signaling for mouse appendage regeneration and suggest that the role of p21 in this process is possibly through the abrogation of the Tgfβ/Smad pathway.

  20. A novel fibrin gel derived from hyaluronic acid-grafted fibrinogen

    Energy Technology Data Exchange (ETDEWEB)

    Yang, Chiung L; Chen, Hui W; Wang, Tzu C; Wang, Yng J, E-mail: wang@ym.edu.tw [Institute of Biomedical Engineering, National Yang Ming University, No. 155, Sec. 2, Li-Nung St., Shih-Pai, Taipei, Taiwan 112 (China)

    2011-04-15

    Fibrinogen is a major plasma protein that forms a three-dimensional fibrin gel upon being activated by thrombin. In this study, we report the synthesis and potential applications of hybrid molecules composed of fibrinogen coupled to the reducing ends of short-chain hyaluronic acids (sHAs) by reductive amination. The grafting of sHAs to fibrinogen was verified by analyzing particle size, zeta potential and gel-electrophoretic mobility of the hybrid molecules. The sHA-fibrinogen hybrid molecules with graft ratios (sHA/fibrinogen) of up to 6.5 retained the ability to form gels in response to thrombin activation. The sHA-fibrin gels were transparent in appearance and exhibited high water content, which were characteristics distinct from those of gels formed by mixtures of sHAs and fibrinogen. The potential applications of the sHA-fibrin gels were evaluated. The sHA-fibrinogen gel with a graft ratio of 3.6 (S3.6F) was examined for its ability to encapsulate and support the differentiation of ATDC5 chondrocyte-like cells. Compared with the fibrinogen-formed gel, cells cultured in the S3.6F gel exhibited increased lacunae formation; moreover, the abundance of cartilaginous extracellular matrix molecules and the expression of chondrocyte marker genes, such as aggrecan, collagen II and Sox9, were also significantly increased. Our data suggest that the three-dimensional gel formed by the sHA-fibrinogen hybrid is a better support than the fibrin gel for chondrogenesis induction.

  1. Analysis of chick (Gallus gallus middle ear columella formation

    Directory of Open Access Journals (Sweden)

    Mercer Kathryn J

    2010-02-01

    Full Text Available Abstract Background The chick middle ear bone, the columella, provides an accessible model in which to study the tissue and molecular interactions necessary for induction and patterning of the columella, as well as associated multiple aspects of endochondral ossification. These include mesenchymal condensation, chondrogenesis, ossification of the medial footplate and shaft, and joint formation between the persistent cartilage of the extracolumella and ossified columella. Middle and external ear defects are responsible for approximately 10% of congenital hearing defects. Thus, understanding the morphogenesis and the molecular mechanisms of the formation of the middle ear is important to understanding normal and abnormal development of this essential component of the hearing apparatus. Results The columella, which arises from proximal ectomesenchyme of the second pharyngeal arch, is induced and patterned in a dynamic multi-step process. From the footplate, which inserts into the inner ear oval window, the shaft spans the pneumatic middle ear cavity, and the extracolumella inserts into the tympanic membrane. Through marker gene and immunolabeling analysis, we have determined the onset of each stage in the columella's development, from condensation to ossification. Significantly, a single condensation with the putative shaft and extracolumella arms already distinguishable is observed shortly before initiation of five separate chondrogenic centers within these structures. Ossification begins later, with periosteum formation in the shaft and, unexpectedly, a separate periosteum in the footplate. Conclusions The data presented in this study document the spatiotemporal events leading to morphogenesis of the columella and middle ear structures and provide the first gene expression data for this region. These data identify candidate genes and facilitate future functional studies and elucidation of the molecular mechanisms of columella formation.

  2. Structural determinants of heparin-transforming growth factor-β1 interactions and their effects on signaling.

    Science.gov (United States)

    Lee, Jonathan; Wee, Sheena; Gunaratne, Jayantha; Chua, R J E; Smith, Raymond A A; Ling, Ling; Fernig, David G; Swaminathan, Kunchithapadam; Nurcombe, Victor; Cool, Simon M

    2015-12-01

    Transforming growth factor-β1 (TGF-β1, Uniprot: P01137) is a heparin-binding protein that has been implicated in a number of physiological processes, including the initiation of chondrogenesis by human mesenchymal stem cells (hMSCs). Here, we identify the molecular features in the protein and in heparin required for binding and their effects on the potentiation of TGF-β1's activity on hMSCs. Using a proteomics "Protect and Label" approach, lysines K291, K304, K309, K315, K338, K373, K375 and K388 were identified as being directly involved in binding heparin (Data are available via ProteomeXchange with identifier PXD002772). Competition assays in an optical biosensor demonstrated that TGF-β1 does require N- and 6-O-sulfate groups for binding but that 2-O-sulfate groups are unlikely to underpin the interaction. Heparin-derived oligosaccharides as short as degree of polymerization (dp) 4 have a weak ability to compete for TGF-β1 binding to heparin, which increases with the length of the oligosaccharide to reach a maximum between dp18 and dp24. In cell-based assays, heparin, 2-O-, 6-O- and N-desulfated re-N-acetylated heparin and oligosaccharides 14-24 saccharides (dp14-24) in length all increased the phosphorylation of mothers against decapentaplegic homolog 2 (SMAD2) after 6 h of stimulation with TGF-β1. The results provide the structural basis for a model of heparin/heparan sulfate binding to TGF-β1 and demonstrate that the features in the polysaccharide required for binding are not identical to those required for sustaining the signaling by TGF-β1 in hMSCs.

  3. Chondroregulatory action of prolactin on proliferation and differentiation of mouse chondrogenic ATDC5 cells in 3-dimensional micromass cultures

    Energy Technology Data Exchange (ETDEWEB)

    Seriwatanachai, Dutmanee [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Krishnamra, Nateetip [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand); Charoenphandhu, Narattaphol, E-mail: naratt@narattsys.com [Center of Calcium and Bone Research (COCAB), Faculty of Science, Mahidol University, Bangkok (Thailand); Department of Physiology, Faculty of Science, Mahidol University, Bangkok (Thailand)

    2012-03-30

    Highlights: Black-Right-Pointing-Pointer Mouse chondrogenic ATDC5 cells expressed PRL receptor mRNAs and proteins. Black-Right-Pointing-Pointer Low PRL concentration (10 ng/mL) increased chondrocyte viability and differentiation. Black-Right-Pointing-Pointer Higher PRL concentrations ( Greater-Than-Or-Slanted-Equal-To 100 ng/mL) decreased viability and increased apoptosis. -- Abstract: A recent investigation in lactating rats has provided evidence that the lactogenic hormone prolactin (PRL) increases endochondral bone growth and bone elongation, presumably by accelerating apoptosis of hypertrophic chondrocytes in the growth plate and/or subsequent chondrogenic matrix mineralization. Herein, we demonstrated the direct chondroregulatory action of PRL on proliferation, differentiation and apoptosis of chondrocytes in 3-dimensional micromass culture of mouse chondrogenic ATDC5 cell line. The results showed that ATDC5 cells expressed PRL receptor (PRLR) transcripts, and responded typically to PRL by downregulating PRLR expression. Exposure to a low PRL concentration of 10 ng/mL, comparable to the normal levels in male and non-pregnant female rats, increased chondrocyte viability, differentiation, proteoglycan accumulation, and mRNA expression of several chondrogenic differentiation markers, such as Sox9, ALP and Hspg2. In contrast, high PRL concentrations of Greater-Than-Or-Slanted-Equal-To 100 ng/mL, comparable to the levels in pregnancy or lactation, decreased chondrocyte viability by inducing apoptosis, with no effect on chondrogenic marker expression. It could be concluded that chondrocytes directly but differentially responded to non-pregnant and pregnant/lactating levels of PRL, thus suggesting the stimulatory effect of PRL on chondrogenesis in young growing individuals, and supporting the hypothesis of hypertrophic chondrocyte apoptosis in the growth plate of lactating rats.

  4. Regenerative potential of the cartilaginous tissue in mesenchymal stem cells: update, limitations, and challenges.

    Science.gov (United States)

    Cruz, Ivana Beatrice Mânica da; Severo, Antônio Lourenço; Azzolin, Verônica Farina; Garcia, Luiz Filipe Machado; Kuhn, André; Lech, Osvandré

    2017-01-01

    Advances in the studies with adult mesenchymal stem cells (MSCs) have turned tissue regenerative therapy into a promising tool in many areas of medicine. In orthopedics, one of the main challenges has been the regeneration of cartilage tissue, mainly in diarthroses. In the induction of the MSCs, in addition to cytodifferentiation, the microenvironmental context of the tissue to be regenerated and an appropriate spatial arrangement are extremely important factors. Furthermore, it is known that MSC differentiation is fundamentally determined by mechanisms such as cell proliferation (mitosis), biochemical-molecular interactions, movement, cell adhesion, and apoptosis. Although the use of MSCs for cartilage regeneration remains at a research level, there are important questions to be resolved in order to make this therapy efficient and safe. It is known, for instance, that the expansion of chondrocytes in cultivation, needed to increase the number of cells, could end up producing fibrocartilage instead of hyaline cartilage. However, the latest results are promising. In 2014, the first stage I/II clinical trial to evaluate the efficacy and safety of the intra-articular injection of MSCs in femorotibial cartilage regeneration was published, indicating a decrease in injured areas. One issue to be explored is how many modifications in the articulate inflammatory environment could induce differentiation of MSCs already allocated in that region. Such issue arose from studies that suggested that the suppression of the inflammation may increase the efficiency of tissue regeneration. Considering the complexity of the events related to the chondrogenesis and cartilage repair, it can be concluded that the road ahead is still long, and that further studies are needed.

  5. Influence of extremely low frequency, low energy electromagnetic fields and combined mechanical stimulation on chondrocytes in 3-D constructs for cartilage tissue engineering.

    Science.gov (United States)

    Hilz, Florian M; Ahrens, Philipp; Grad, Sibylle; Stoddart, Martin J; Dahmani, Chiheb; Wilken, Frauke L; Sauerschnig, Martin; Niemeyer, Philipp; Zwingmann, Jörn; Burgkart, Rainer; von Eisenhart-Rothe, Rüdiger; Südkamp, Norbert P; Weyh, Thomas; Imhoff, Andreas B; Alini, Mauro; Salzmann, Gian M

    2014-02-01

    Articular cartilage, once damaged, has very low regenerative potential. Various experimental approaches have been conducted to enhance chondrogenesis and cartilage maturation. Among those, non-invasive electromagnetic fields have shown their beneficial influence for cartilage regeneration and are widely used for the treatment of non-unions, fractures, avascular necrosis and osteoarthritis. One very well accepted way to promote cartilage maturation is physical stimulation through bioreactors. The aim of this study was the investigation of combined mechanical and electromagnetic stress affecting cartilage cells in vitro. Primary articular chondrocytes from bovine fetlock joints were seeded into three-dimensional (3-D) polyurethane scaffolds and distributed into seven stimulated experimental groups. They either underwent mechanical or electromagnetic stimulation (sinusoidal electromagnetic field of 1 mT, 2 mT, or 3 mT; 60 Hz) or both within a joint-specific bioreactor and a coil system. The scaffold-cell constructs were analyzed for glycosaminoglycan (GAG) and DNA content, histology, and gene expression of collagen-1, collagen-2, aggrecan, cartilage oligomeric matrix protein (COMP), Sox9, proteoglycan-4 (PRG-4), and matrix metalloproteinases (MMP-3 and -13). There were statistically significant differences in GAG/DNA content between the stimulated versus the control group with highest levels in the combined stimulation group. Gene expression was significantly higher for combined stimulation groups versus static control for collagen 2/collagen 1 ratio and lower for MMP-13. Amongst other genes, a more chondrogenic phenotype was noticed in expression patterns for the stimulated groups. To conclude, there is an effect of electromagnetic and mechanical stimulation on chondrocytes seeded in a 3-D scaffold, resulting in improved extracellular matrix production.

  6. Carbon nanotube array inducing osteogenic differentiation of human mesenchymal stem cells.

    Science.gov (United States)

    Xu, Baiyao; Ju, Yang; Cui, Yanbin; Song, Guanbin

    2015-06-01

    Carbon nanotubes (CNTs) are a kind of nanomaterials which have been shown a promising application for biomedicine. There are a lot of studies to use CNTs to induce the differentiation of mesenchymal stem cells (MSCs). However, the cellular behavior of MSCs on the top layer of CNT array was still not well understood. In this study, we evaluated the morphology, the gene expressions of the osteogenic differentiation related markers, and the gene expressions of collagen type II (Col II, a marker of chondrogenesis), PPARγ (a marker of adipogenesis) and scleraxis (SCX, a marker of tenogenesis) in human mesenchymal stem cells (hMSCs) cultured on multi-walled carbon nanotube (MWCNT) array. The effect of MWCNT array on the mineralization of hMSCs which were cultured in osteogenic differentiation medium (ODM) was further assayed. Our results showed that the hMSCs cultured on MWCNT array spread well, formed numerous spiral shaped cell colons and showed perinuclear morphology. Compared to hMSCs cultured on dish, the gene expression of osteocalcin (OCN) was increased while the gene expressions of collagen type II (Col II), PPARγ and scleraxis (SCX) were decreased in hMSCs which were cultured on MWCNT array without any differentiation factors. Furthermore, compared with hMSCs on dish, the gene expressions of collagen type I (Col I), osteocalcin (OCN), osteopontin (OPN) and RUNX2, and the mineralization of hMSCs on MWCNT array were enhanced when they were cultured in osteogenic differentiation medium (ODM). Our results indicated that MWCNT array was able to promote the osteogenesis of hMSCs.

  7. Nell1-deficient mice have reduced expression of extracellular matrix proteins causing cranial and vertebral defects

    Energy Technology Data Exchange (ETDEWEB)

    Desai, Jayashree [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Shannon, Mark E. [Applied Biosystems; Johnson, Mahlon D. [University of Tennessee Graduate School of Medicine; Ruff, David W. [Applied Biosystems; Hughes, Lori A [ORNL; Kerley, Marilyn K [ORNL; Carpenter, D A [ORNL; Johnson, Dabney K [ORNL; Rinchik, Eugene M. [University of Tennessee, Knoxville (UTK) & Oak Ridge National Laboratory (ORNL); Culiat, Cymbeline T [ORNL

    2006-01-01

    The mammalian Nell1 gene encodes a protein kinase C-b1 (PKC-b1) binding protein that belongs to a new class of cell-signaling molecules controlling cell growth and differentiation. Over-expression of Nell1 in the developing cranial sutures in both human and mouse induces craniosynostosis, the premature fusion of the growing cranial bone fronts. Here, we report the generation, positional cloning and characterization of Nell16R, a recessive, neonatal-lethal point mutation in the mouse Nell1 gene, induced by N-ethyl-N-nitrosourea. Nell16R has a T!A base change that converts a codon for cysteine into a premature stop codon [Cys(502)Ter], resulting in severe truncation of the predicted protein product and marked reduction in steady-state levels of the transcript. In addition to the expected alteration of cranial morphology, Nell16R mutants manifest skeletal defects in the vertebral column and ribcage, revealing a hitherto undefined role for Nell1 in signal transduction in endochondral ossification. Real-time quantitative reverse transcription-PCR assays of 219 genes showed an association between the loss of Nell1 function and reduced expression of genes for extracellular matrix (ECM) proteins critical for chondrogenesis and osteogenesis. Several affected genes are involved in the human cartilage disorder Ehlers-Danlos Syndrome and other disorders associated with spinal curvature anomalies. Nell16R mutant mice are a new tool for elucidating basic mechanisms in osteoblast and chrondrocyte differentiation in the developing skull and vertebral column and understanding how perturbations in the production of ECM proteins can lead to anomalies in these structures.

  8. Effect of Reishi polysaccharides on human stem/progenitor cells.

    Science.gov (United States)

    Chen, Wan-Yu; Yang, Wen-Bin; Wong, Chi-Huey; Shih, Daniel Tzu-Bi

    2010-12-15

    The polysaccharide fraction of Ganoderma lucidum (F3) was found to benefit our health in many ways by influencing the activity of tissue stem/progenitor cells. In this study, F3 was found to promote the adipose tissue MSCs' aggregation and chondrosphere formation, with the increase of CAM (N-CAM, I-CAM) expressions and autokine (BMP-2, IL-11, and aggrecan) secretions, in an in vitro chondrogenesis assay. In a stem cell expansion culture, it possesses the thrombopoietin (TPO) and GM-CSF like functions to enhance the survival/renewal abilities of primitive hematopoietic stem/progenitor cells (HSCs). F3 was found to promote the dendrite growth of blood mononuclear cells (MNCs) and the expression of cell adhesion molecules in the formation of immature dendritic cells (DC). On the other hand, F3 exhibited inhibitory effects on blood endothelial progenitor (EPC) colony formation, with concomitant reduction of cell surface endoglin (CD105) and vascular endothelial growth factor receptor-3 (VEGFR-3) marker expressions, in the presence of angiogenic factors. A further cytokine array analysis revealed that F3 indeed inhibited the angiogenin synthesis and enhanced IL-1, MCP-1, MIP-1, RANTES, and GRO productions in the blood EPC derivation culture. Collectively, we have demonstrated that the polysaccharide fraction of G. lucidum F3 exhibits cytokine and chemokine like functions which are beneficial to human tissue stem/progenitor cells by modulating their CAM expressions and biological activities. These findings provide us a better the observation that F3 glycopolysaccharides indeed possesses anti-angiogenic and immune-modulating functions and promotes hematopoietic stem/progenitor cell homing for better human tissue protection, reducing disease progression and health.

  9. Effects of intermittent versus continuous parathyroid hormone administration on condylar chondrocyte proliferation and differentiation

    Energy Technology Data Exchange (ETDEWEB)

    Liu, Qi; Wan, Qilong; Yang, Rongtao; Zhou, Haihua [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Li, Zubing, E-mail: lizubing0827@163.com [The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) and Key Laboratory of Oral Biomedicine Ministry of Education, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China); Department of Oral and Maxillofacial Surgery, School and Hospital of Stomatology, Wuhan University, 237 Luoyu Road, Wuhan 430079 (China)

    2012-07-20

    Highlights: Black-Right-Pointing-Pointer Different PTH administration exerts different effects on condylar chondrocyte. Black-Right-Pointing-Pointer Intermittent PTH administration suppresses condylar chondrocyte proliferation. Black-Right-Pointing-Pointer Continuous PTH administration maintains condylar chondrocyte proliferating. Black-Right-Pointing-Pointer Intermittent PTH administration enhances condylar chondrocyte differentiation. -- Abstract: Endochondral ossification is a complex process involving chondrogenesis and osteogenesis regulated by many hormones and growth factors. Parathyroid hormone (PTH), one of the key hormones regulating bone metabolism, promotes osteoblast differentiation and osteogenesis by intermittent administration, whereas continuous PTH administration inhibits bone formation. However, the effects of PTH on chondrocyte proliferation and differentiation are still unclear. In this study, intermittent PTH administration presented enhanced effects on condylar chondrocyte differentiation and bone formation, as demonstrated by increased mineral nodule formation and alkaline phosphatase (ALP) activity, up-regulated runt-related transcription factor 2 (RUNX2), ALP, collagen type X (COL10a1), collagen type I (COL1a1), osteocalcin (OCN), bone sialoprotein (BSP), bone morphogenetic protein 2 (BMP2) and osterix (OSX) mRNA and/or protein expression. On the contrary, continuous PTH administration promoted condylar chondrocyte proliferation and suppressed its differentiation, as demonstrated by up-regulated collagen type II (COL2a1) mRNA expression, reduced mineral nodule formation and down-regulated expression of the mRNAs and/or proteins mentioned above. Our data suggest that PTH can regulate condylar chondrocyte proliferation and differentiation, depending on the type of PTH administration. These results provide new insight into the effects of PTH on condylar chondrocytes and new evidence for using local PTH administration to cure mandibular

  10. Comparison the repair potential for cartilage defect in knee joints with different sourced free periosteal autografts in rabbits model%家兔不同部位自体游离骨膜移植修复关节软骨缺损的实验比较

    Institute of Scientific and Technical Information of China (English)

    李亚伟; 王伟民; 霍建河; 沈建农

    2004-01-01

    AIM:To investigate the repair potential for cartilage defect with different sourced free periosteal autografts in rabbits model.METHODS:A total of 32 rabbits with 5 mm× 8 mm full thickness of cartilage defects on femoral joint surface were used in this study,free iliac and tibia periosteal autografts were performed.Four and 8 weeks later,specimens were taken out for histological observation. RESULTS:Hyaline and premature cartilage dominated in the newly formed tissue after 8 weeks.The iliac periosteal autografts showed a better regeneration quality than those from tibia.CONCLUSION:Iliac free periosteal autografts possessed a stronger chondrogenesis potential than those of tibia,so iliac may be an optimal donor for free periosteal autografts.%目的 :探讨不同部位自体游离骨膜修复关节软骨缺损的能力. 方法 : 用大耳兔 32只,在股骨关节面造成 5 mm× 8 mm全层软骨缺损,分别行自体髂骨骨膜和胫骨骨膜游离移植术,术后 4及 8周取材做组织学观察比较. 结果 : 术后 8周自体髂骨骨膜移植侧、胫骨骨膜移植侧的优势组织性质分别为类透明软骨、幼稚软骨,前者形成软骨的质量优于后者. 结论 : 自体髂骨骨膜游离移植的成软骨能力优于自体胫骨骨膜,是较理想的自体骨膜游离移植取材部位

  11. Clonal analysis of the differentiation potential of human adipose-derived adult stem cells.

    Science.gov (United States)

    Guilak, Farshid; Lott, Kristen E; Awad, Hani A; Cao, Qiongfang; Hicok, Kevin C; Fermor, Beverley; Gimble, Jeffrey M

    2006-01-01

    Pools of human adipose-derived adult stem (hADAS) cells can exhibit multiple differentiated phenotypes under appropriate in vitro culture conditions. Because adipose tissue is abundant and easily accessible, hADAS cells offer a promising source of cells for tissue engineering and other cell-based therapies. However, it is unclear whether individual hADAS cells can give rise to multiple differentiated phenotypes or whether each phenotype arises from a subset of committed progenitor cells that exists within a heterogeneous population. The goal of this study was to test the hypothesis that single hADAS are multipotent at a clonal level. hADAS cells were isolated from liposuction waste, and ring cloning was performed to select cells derived from a single progenitor cell. Forty-five clones were expanded through four passages and then induced for adipogenesis, osteogenesis, chondrogenesis, and neurogenesis using lineage-specific differentiation media. Quantitative differentiation criteria for each lineage were determined using histological and biochemical analyses. Eighty one percent of the hADAS cell clones differentiated into at least one of the lineages. In addition, 52% of the hADAS cell clones differentiated into two or more of the lineages. More clones expressed phenotypes of osteoblasts (48%), chondrocytes (43%), and neuron-like cells (52%) than of adipocytes (12%), possibly due to the loss of adipogenic ability after repeated subcultures. The findings are consistent with the hypothesis that hADAS cells are a type of multipotent adult stem cell and not solely a mixed population of unipotent progenitor cells. However, it is important to exercise caution in interpreting these results until they are validated using functional in vivo assays.

  12. Projection Stereolithographic Fabrication of Human Adipose Stem Cell-incorporated Biodegradable Scaffolds for Cartilage Tissue Engineering

    Directory of Open Access Journals (Sweden)

    Aaron X Sun

    2015-08-01

    Full Text Available Poor self-healing ability of cartilage necessitates the development of methods for cartilage regeneration. Scaffold construction with live stem cell incorporation and subsequent differentiation presents a promising route. Projection stereolithography (PSL offers high resolution and processing speed as well as the ability to fabricate scaffolds that precisely fit the anatomy of cartilage defects using medical imaging as the design template. We report here the use of a visible-light based PSL (VL-PSL system to encapsulate human adipose-derived stem cells (hASCs into a biodegradable polymer (poly-D,L-lactic acid/polyethylene glycol/ poly-D,L-lactic acid (PDLLA-PEG/hyaluronic acid (HA matrix to produce live cell constructs with customized architectures. After fabrication, hASCs showed high viability (84% and were uniformly distributed throughout the constructs, which possessed high mechanical property with a compressive modulus of 780 kPa. The hASC-seeded constructs were then cultured in Control or TGF-β3-containing chondrogenic medium for up to 28 days. In chondrogenic medium treated group (TGF-β3 group hASCs maintained 77% viability and expressed chondrogenic genes Sox9, collagen type II, and aggrecan at 11, 232, and 2.29 x 10(5 fold increases, respectively, compared to levels at day 0 in non-chondrogenic medium. The TGF-β3 group also produced a collagen type II and glycosaminoglycan (GAG-rich extracellular matrix, detected by immunohistochemistry, and Alcian blue and Safranin O staining suggesting robust chondrogenesis within the scaffold. Without chondroinductive addition (Control group, cell viability decreased with time (65% at 28 days and showed poor cartilage matrix deposition. After 28 days, mechanical strength of the TGF-β3 group remained high at 240 kPa. Thus, the PSL- and PLLA-PEG/HA based fabrication method using adult stem cells is a promising approach in producing mechanically competent engineered cartilage for joint cartilage

  13. Embryonic development of Python sebae - II: Craniofacial microscopic anatomy, cell proliferation and apoptosis.

    Science.gov (United States)

    Buchtová, Marcela; Boughner, Julia C; Fu, Katherine; Diewert, Virginia M; Richman, Joy M

    2007-01-01

    This study explores the microscopic craniofacial morphogenesis of the oviparous African rock python (Python sebae) spanning the first two-thirds of the post-oviposition period. At the time of laying, the python embryo consists of largely undifferentiated mesenchyme and epithelium with the exception of the cranial base and trabeculae cranii, which are undergoing chondrogenesis. The facial prominences are well defined and are at a late stage, close to the time when lip fusion begins. Later (11-12d), specializations in the epithelia begin to differentiate (vomeronasal and olfactory epithelia, teeth). Dental development in snakes is different from that of mammals in several aspects including an extended dental lamina with the capacity to form 4 sets of generational teeth. In addition, the ophidian olfactory system is very different from the mammalian. There is a large vomeronasal organ, a nasal cavity proper and an extraconchal space. All of these areas are lined with a greatly expanded olfactory epithelium. Intramembranous bone differentiation is taking place at stage 3 with some bones already ossifying whereas most are only represented as mesenchymal condensations. In addition to routine histological staining, PCNA immunohistochemistry reveals relatively higher levels of proliferation in the extending dental laminae, in osseous mesenchymal condensations and in the olfactory epithelia. Areas undergoing apoptosis were noted in the enamel organs of the teeth and osseous mesenchymal condensations. We propose that localized apoptosis helps to divide a single condensation into multiple ossification centres and this is a mechanism whereby novel morphology can be selected in response to evolutionary pressures. Several additional differences in head morphology between snakes and other amniotes were noted including a palatal groove separating the inner and outer row of teeth in the upper jaw, a tracheal opening within the tongue and a pharyngeal adhesion that closes off the

  14. Generation, Characterization, and Multilineage Potency of Mesenchymal-Like Progenitors Derived from Equine Induced Pluripotent Stem Cells.

    Science.gov (United States)

    Lepage, Sarah I; Nagy, Kristina; Sung, Hoon-Ki; Kandel, Rita A; Nagy, Andras; Koch, Thomas G

    2016-01-01

    Multipotent mesenchymal stromal cells (MSCs) are more and more frequently used to treat orthopedic injuries in horses. However, these cells are limited in their expandability and differentiation capacity. Recently, the first equine-induced pluripotent stem cell (iPSC) lines were reported by us [ 1 ]. In vitro differentiation of iPSCs into MSC-like cells is an attractive alternative to using MSCs derived from other sources, as a much larger quantity of patient-specific cells with broad differentiation potential could be generated. However, the differentiation capacity of iPSCs to MSCs and the potential for use in tissue engineering have yet to be explored. In this study, equine iPSCs were induced to differentiate into an MSC-like population. Upon induction, the iPSCs changed morphology toward spindle-shaped cells similar to MSCs. The ensuing iPSC-MSCs exhibited downregulation of pluripotency-associated genes and an upregulation of MSC-associated genes. In addition, the cells expressed the same surface markers as MSCs derived from equine umbilical cord blood. We then assessed the multilineage differentiation potential of iPSC-MSCs. Although chondrogenesis was not achieved after induction with transforming growth factor-beta 3 (TGFβ3) and/or bone morphogenic protein 4 (BMP-4) in 3D pellet culture, mineralization characteristic of osteogenesis and lipid droplet accumulation characteristic of adipogenesis were observed after chemical induction. We demonstrate a protocol for the derivation of MSC-like progenitor populations from equine iPS cells.

  15. Loss of VHL in mesenchymal progenitors of the limb bud alters multiple steps of endochondral bone development.

    Science.gov (United States)

    Mangiavini, Laura; Merceron, Christophe; Araldi, Elisa; Khatri, Richa; Gerard-O'Riley, Rita; Wilson, Tremika LeShan; Rankin, Erinn B; Giaccia, Amato J; Schipani, Ernestina

    2014-09-01

    Adaptation to low oxygen tension (hypoxia) is a critical event during development. The transcription factors Hypoxia Inducible Factor-1α (HIF-1α) and HIF-2α are essential mediators of the homeostatic responses that allow hypoxic cells to survive and differentiate. Von Hippel-Lindau protein (VHL) is the E3 ubiquitin ligase that targets HIFs to the proteasome for degradation in normoxia. We have previously demonstrated that the transcription factor HIF-1α is essential for survival and differentiation of growth plate chondrocytes, whereas HIF-2α is not necessary for fetal growth plate development. We have also shown that VHL is important for endochondral bone development, since loss of VHL in chondrocytes causes severe dwarfism. In this study, in order to expand our understanding of the role of VHL in chondrogenesis, we conditionally deleted VHL in mesenchymal progenitors of the limb bud, i.e. in cells not yet committed to the chondrocyte lineage. Deficiency of VHL in limb bud mesenchyme does not alter the timely differentiation of mesenchymal cells into chondrocytes. However, it causes structural collapse of the cartilaginous growth plate as a result of impaired proliferation, delayed terminal differentiation, and ectopic death of chondrocytes. This phenotype is associated to delayed replacement of cartilage by bone. Notably, loss of HIF-2α fully rescues the late formation of the bone marrow cavity in VHL mutant mice, though it does not affect any other detectable abnormality of the VHL mutant growth plates. Our findings demonstrate that VHL regulates bone morphogenesis as its loss considerably alters size, shape and overall development of the skeletal elements.

  16. A GDF5 point mutation strikes twice--causing BDA1 and SYNS2.

    Directory of Open Access Journals (Sweden)

    Elisa Degenkolbe

    Full Text Available Growth and Differentiation Factor 5 (GDF5 is a secreted growth factor that belongs to the Bone Morphogenetic Protein (BMP family and plays a pivotal role during limb development. GDF5 is a susceptibility gene for osteoarthritis (OA and mutations in GDF5 are associated with a wide variety of skeletal malformations ranging from complex syndromes such as acromesomelic chondrodysplasias to isolated forms of brachydactylies or multiple synostoses syndrome 2 (SYNS2. Here, we report on a family with an autosomal dominant inherited combination of SYNS2 and additional brachydactyly type A1 (BDA1 caused by a single point mutation in GDF5 (p.W414R. Functional studies, including chondrogenesis assays with primary mesenchymal cells, luciferase reporter gene assays and Surface Plasmon Resonance analysis, of the GDF5(W414R variant in comparison to other GDF5 mutations associated with isolated BDA1 (p.R399C or SYNS2 (p.E491K revealed a dual pathomechanism characterized by a gain- and loss-of-function at the same time. On the one hand insensitivity to the main GDF5 antagonist NOGGIN (NOG leads to a GDF5 gain of function and subsequent SYNS2 phenotype. Whereas on the other hand, a reduced signaling activity, specifically via the BMP receptor type IA (BMPR1A, is likely responsible for the BDA1 phenotype. These results demonstrate that one mutation in the overlapping interface of antagonist and receptor binding site in GDF5 can lead to a GDF5 variant with pathophysiological relevance for both, BDA1 and SYNS2 development. Consequently, our study assembles another part of the molecular puzzle of how loss and gain of function mutations in GDF5 affect bone development in hands and feet resulting in specific types of brachydactyly and SYNS2. These novel insights into the biology of GDF5 might also provide further clues on the pathophysiology of OA.

  17. An injectable hydrogel incorporating mesenchymal precursor cells and pentosan polysulphate for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Cameron, Andrew R; Menzies, Donna J; Ghosh, Peter; Whitehead, Darryl L; Gronthos, Stan; Zannettino, Andrew C W; Cooper-White, Justin J

    2013-12-01

    Intervertebral disc (IVD) degeneration is one of the leading causes of lower back pain and a major health problem worldwide. Current surgical treatments include excision or immobilisation, with neither approach resulting in the repair of the degenerative disc. As such, a tissue engineering-based approach in which stem cells, coupled with an advanced delivery system, could overcome this deficiency and lead to a therapy that encourages functional fibrocartilage generation in the IVD. In this study, we have developed an injectable hydrogel system based on enzymatically-crosslinked polyethylene glycol and hyaluronic acid. We examined the effects of adding pentosan polysulphate (PPS), a synthetic glycosaminoglycan-like factor that has previously been shown (in vitro and in vivo) to this gel system in order to induce chondrogenesis in mesenchymal precursor cells (MPCs) when added as a soluble factor, even in the absence of additional growth factors such as TGF-β. We show that both the gelation rate and mechanical strength of the resulting hydrogels can be tuned in order to optimise the conditions required to produce gels with the desired combination of properties for an IVD scaffold. Human immunoselected STRO-1+ MPCs were then incorporated into the hydrogels. They were shown to retain good viability after both the initial formation of the gel and for longer-term culture periods in vitro. Furthermore, MPC/hydrogel composites formed cartilage-like tissue which was significantly enhanced by the incorporation of PPS into the hydrogels, particularly with respect to the deposition of type-II-collagen. Finally, using a wild-type rat subcutaneous implantation model, we examined the extent of any immune reaction and confirmed that this matrix is well tolerated by the host. Together these data provide evidence that such a system has significant potential as both a delivery vehicle for MPCs and as a matrix for fibrocartilage tissue engineering applications.

  18. Ex vivo Expansion and Differentiation of Mesenchymal Stem Cells from Goat Bone Marrow

    Directory of Open Access Journals (Sweden)

    Mohamadreza Baghaban Eslaminejad

    2009-06-01

    Full Text Available Objective(sMesenchymal stem cells (MSCs from large animals as goat which is genetically more closely related tohuman have rarely been gained attentions. The present study tried to isolate and characterize MSCs fromgoat bone marrow.Materials and MethodsFibroblastic cells appeared in goat marrow cell culture were expanded through several subcultures.Passaged-3 cells were then differentiated among the osteogenic, adipogenic and chondrogenic cell lineagesto determine their MSC nature. Differentiations were determined by RT-PCR analysis of related geneexpression. To identify the best culture conditions for propagation, passage-3 cells were plated either atvarying cell densities or different fetal bovine serum (FBS concentrations for a week, at the end of whichthe cultures were statistically compared with respect to the cell proliferation. In this study, we alsodetermined goat MSC population doubling time (PDT as the index of their in vitro expansion rate.ResultsPassage-3 fibroblastic cells tended to differentiate into skeletal cell lineages. This was evident in bothspecific staining as well as the specific gene expression profile. Moreover, there appeared to be moreexpansion when the cultures were initiated at 100 cells/cm2 in a medium supplemented with 15% FBS. Arelatively short PDT (24.94±2.67 hr was a reflection of the goat MSC rapid rate of expansion.ConclusionTaken together, fibroblastic cells developed at goat marrow cell culture are able to differentiate into skeletalcell lineages. They undergo extensive proliferation when being plated at low cell density in 15% FBSconcentration.Keywords: Adipogenesis, Bovine serum, Cell seeding density, Chondrogenesis, Goat mesenchymal stemcells, Osteogenesis

  19. Mesenchymal stem cells in regenerative medicine: Focus on articular cartilage and intervertebral disc regeneration.

    Science.gov (United States)

    Richardson, Stephen M; Kalamegam, Gauthaman; Pushparaj, Peter N; Matta, Csaba; Memic, Adnan; Khademhosseini, Ali; Mobasheri, Reza; Poletti, Fabian L; Hoyland, Judith A; Mobasheri, Ali

    2016-04-15

    Musculoskeletal disorders represent a major cause of disability and morbidity globally and result in enormous costs for health and social care systems. Development of cell-based therapies is rapidly proliferating in a number of disease areas, including musculoskeletal disorders. Novel biological therapies that can effectively treat joint and spine degeneration are high priorities in regenerative medicine. Mesenchymal stem cells (MSCs) isolated from bone marrow (BM-MSCs), adipose tissue (AD-MSCs) and umbilical cord (UC-MSCs) show considerable promise for use in cartilage and intervertebral disc (IVD) repair. This review article focuses on stem cell-based therapeutics for cartilage and IVD repair in the context of the rising global burden of musculoskeletal disorders. We discuss the biology MSCs and chondroprogenitor cells and specifically focus on umbilical cord/Wharton's jelly derived MSCs and examine their potential for regenerative applications. We also summarize key components of the molecular machinery and signaling pathways responsible for the control of chondrogenesis and explore biomimetic scaffolds and biomaterials for articular cartilage and IVD regeneration. This review explores the exciting opportunities afforded by MSCs and discusses the challenges associated with cartilage and IVD repair and regeneration. There are still many technical challenges associated with isolating, expanding, differentiating, and pre-conditioning MSCs for subsequent implantation into degenerate joints and the spine. However, the prospect of combining biomaterials and cell-based therapies that incorporate chondrocytes, chondroprogenitors and MSCs leads to the optimistic view that interdisciplinary approaches will lead to significant breakthroughs in regenerating musculoskeletal tissues, such as the joint and the spine in the near future.

  20. Longitudinal analysis of osteogenic and angiogenic signaling factors in healing models mimicking atrophic and hypertrophic non-unions in rats.

    Directory of Open Access Journals (Sweden)

    Susann Minkwitz

    Full Text Available Impaired bone healing can have devastating consequences for the patient. Clinically relevant animal models are necessary to understand the pathology of impaired bone healing. In this study, two impaired healing models, a hypertrophic and an atrophic non-union, were compared to physiological bone healing in rats. The aim was to provide detailed information about differences in gene expression, vascularization and histology during the healing process. The change from a closed fracture (healing control group to an open osteotomy (hypertrophy group led to prolonged healing with reduced mineralized bridging after 42 days. RT-PCR data revealed higher gene expression of most tested osteogenic and angiogenic factors in the hypertrophy group at day 14. After 42 days a significant reduction of gene expression was seen for Bmp4 and Bambi in this group. The inhibition of angiogenesis by Fumagillin (atrophy group decreased the formation of new blood vessels and led to a non-healing situation with diminished chondrogenesis. RT-PCR results showed an attempt towards overcoming the early perturbance by significant up regulation of the angiogenic regulators Vegfa, Angiopoietin 2 and Fgf1 at day 7 and a further continuous increase of Fgf1, -2 and Angiopoietin 2 over time. However µCT angiograms showed incomplete recovery after 42 days. Furthermore, lower expression values were detected for the Bmps at day 14 and 21. The Bmp antagonists Dan and Twsg1 tended to be higher expressed in the atrophy group at day 42. In conclusion, the investigated animal models are suitable models to mimic human fracture healing complications and can be used for longitudinal studies. Analyzing osteogenic and angiogenic signaling patterns, clear changes in expression were identified between these three healing models, revealing the importance of a coordinated interplay of different factors to allow successful bone healing.

  1. Multi-lineage differentiation of hMSCs encapsulated in thermo-reversible hydrogel using a co-culture system with differentiated cells.

    Science.gov (United States)

    Park, Ji Sun; Yang, Han Na; Woo, Dae Gyun; Kim, Hyemin; Na, Kun; Park, Keun-Hong

    2010-10-01

    The micro-environment is an important factor in the differentiation of cultured stem cells for the purpose of site specific transplantation. In an attempt to optimize differentiation conditions, co-culture systems composed of both stem cells and primary cells or cell lines were used in hydrogel with in vitro and in vivo systems. Stem cells encapsulated in hydrogel, under certain conditions, can undergo increased differentiation both in vitro and in vivo; therefore, reconstruction of transplanted stem cells in a hydrogel co-culture system is important for tissue regeneration. In order to construct such a co-culture system, we attempted to create a hydrogel scaffold which could induce neo-tissue growth from the recipient bed into the material. This material would enable encapsulation of stem cells in vitro after which they could be transferred to an in vivo system utilizing nude mice. In this case, the hydrogel was implanted in the subfascial space of nude mice and excised 4 weeks later. Cross-sections of the excised samples were stained with von Kossa or safranin-O and tubular formations into the gel were observed with and tested by doppler imaging. The data showed that the hydrogel markedly induced growth of osteogenic, chondrogenic, and vascular-rich tissue into the hydrogel by 4 weeks, which surpassed that after transplantation in a co-culture system. Further, a co-culture system with differentiated cells and stem cells potentially enhanced chondrogenesis, osteogenesis, and vascularization. These findings suggest that a co-culture system with hydrogel as scaffold material for neo-tissue formation is a useful tools for multi-lineage stem cell differentiation.

  2. Cyclic compressive stress-induced scinderin regulates progress of developmental dysplasia of the hip.

    Science.gov (United States)

    Wang, Cheng-Long; Wang, Hui; Xiao, Fei; Wang, Chuan-Dong; Hu, Guo-Li; Zhu, Jun-Feng; Shen, Chao; Zuo, Bin; Cui, Yi-Min; Li, De; Yuan-Gao; Zhang, Xiao-Ling; Chen, Xiao-Dong

    2017-02-14

    Developmental dysplasia of the hip (DDH) is a common musculoskeletal disorder characterized by a mismatch between acetabulum and femoral head. Mechanical force plays an important role during the occurrence and development of abnormities in acetabulum and femoral head. In this study, we established a mechanical force model named cyclic compressive stress (Ccs). To analyze the effect of Ccs on DDH, we detected special genes in chondrocytes and osteoblasts. Results showed that Ccs downregulated chondrogenesis of ADTC5 in a concentration-dependent manner. Moreover, the mRNA level of Scinderin (Scin) considerably increased. We established lentivirus-SCIN(GV144-SCIN) to transfect hBMSCs, which were treated with different Ccs levels (0.25 Hz*5 cm, 0.5 Hz*5 cm, and 1 Hz*10 cm); the result showed that overexpression of Scin upregulated osteogenesis and osteoclastogenesis. By contrast, expression of chondrocyte-specific genes, including ACAN, COL-2A, and Sox9, decreased. Further molecular investigation demonstrated that Scin promoted osteogenesis and osteoclastogenesis through activation of the p-Smad1/5/8, NF-κB, and MAPK P38 signaling pathways, as well as stimulated the expression of key osteoclast transcriptional factors NFATc1 and c-Fos. Moreover, Scin-induced osteogenesis outweighed osteoclastogenesis in defective femur in vivo. The results of the analysis of Micro-CT confirmed these findings. Overall, Ccs influenced the development of DDH by promoting osteogenesis and cartilage degradation. In addition, Scin played a vital role in the development of DDH.

  3. A microRNA signature associated with chondrogenic lineage commitment

    Indian Academy of Sciences (India)

    Behnaz Bakhshandeh; Masoud Soleimani; Seyed Hassan Paylakhi; Nasser Ghaemi

    2012-08-01

    Generating appropriate cartilage for clinical applications to heal skeletal tissue loss is a major health concern. In this regard, cell-based approaches offer a potential therapeutic strategy for cartilage repair, although little is known about the precise mechanism of chondrogenesis. Unrestricted somatic stem cell (USSC) is considered as a suitable candidate because of its potential for differentiating into multiple cell types. Recent studies show that microRNAs (miRNAs) are involved in several biological processes including development and differentiation. To identify the chondro-specific miRNA signature, miRNA patterns of USSCs and differentiated chondrocytes were investigated using microarrays and validation by qPCR. Prior to these analyses, chondrogenic commitment of differentiated USSCs was verified by immunocytochemistry, specific staining and evaluation of some main chondrogenic marker genes. Various in silico explorations (for both putative targets and signalling pathways) and empirical analyses (miRNA transfections followed by qPCR of some chondrogenic indicators) were carried out to support our results. Transient modulation of multiple chondro-miRs (such as mir-630, mir-624 and mir-376) with chondrocyte targets (such as TGFbR, MAP3K, collagens, SMADs and cadherins) as mediators of chondrogenic signalling pathways including cell–cell interactions, TGF-beta, and MAPK signalling suggests a mechanism for genetic induction of chondrogenic differentiation. In conclusion, this research reveals more details about the allocation of USSCs into the chondrocytes through identification of miRNA signature which modulates targets and pathways required for chondrogenic lineage and could provide guidelines for future clinical treatments and anti-miRNA therapies.

  4. Additive manufactured polymeric 3D scaffolds with tailored surface topography influence mesenchymal stromal cells activity.

    Science.gov (United States)

    Neves, Sara C; Mota, Carlos; Longoni, Alessia; Barrias, Cristina C; Granja, Pedro L; Moroni, Lorenzo

    2016-05-24

    Additive manufactured three-dimensional (3D) scaffolds with tailored surface topography constitute a clear advantage in tissue regeneration strategies to steer cell behavior. 3D fibrous scaffolds of poly(ethylene oxide terephthalate)/poly(butylene terephthalate) block copolymer presenting different fiber surface features were successfully fabricated by additive manufacturing combined with wet-spinning, in a single step, without any post-processing. The optimization of the processing parameters, mainly driven by different solvent/non-solvent combinations, led to four distinct scaffold types, with average surface roughness values ranging from 0.071 ± 0.012 μm to 1.950 ± 0.553 μm, average pore sizes in the x- and y-axis between 351.1 ± 33.6 μm and 396.1 ± 32.3 μm, in the z-axis between 36.5 ± 5.3 μm and 70.7 ± 8.8 μm, average fiber diameters between 69.4 ± 6.1 μm and 99.0 ± 9.4 μm, and porosity values ranging from 60.2 ± 0.8% to 71.7 ± 2.6%. Human mesenchymal stromal cells (hMSCs) cultured on these scaffolds adhered, proliferated, and produced endogenous extracellular matrix. The effect of surface roughness and topography on hMSCs differentiation was more evident for cells seeded at lower density, where the percentage of cells in direct contact with the surface was higher compared to more densely seeded scaffolds. Under osteogenic conditions, lower surface roughness values (0.227 ± 0.035 μm) had a synergistic effect on hMSCs behavior, while chondrogenesis was favored on rougher surfaces (1.950 ± 0.553 μm).

  5. Resveratrol protects bone marrow mesenchymal stem cell derived chondrocytes cultured on chitosan-gelatin scaffolds from the inhibitory effect of interleukin-1β

    Institute of Scientific and Technical Information of China (English)

    Ming LEI; Shi-qing LIU; Yu-lan LIU

    2008-01-01

    Aim: To investigate the effects of resveratrol on interleukin-lbeta (IL-1β) induced catabolism in bone marrow mesenchymal stem cell (MSC) derived chon-drocytes cultured on chitosan-gelatin scaffolds (CGS). Methods: The chondro-genesis of alginate-encapsulated MSCs was evaluated by toluidine blue staining, RT-PCR, and immunostaing. MSC-derived chondrocyte morphology cultured on CGS was evaluated by a scanning electron microscope (SEM) and a laser confocal microscope (LCM). When these cells on CGS were pre-stimulated with IL-1β or co-treated with IL-1β and resveratrol in the absence and presence of the specific β1-integrin blocking antibody, collagen type Ⅱ, aggrecan, matrix metalloproteinase-13 (MMP-13) expression, and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blot analysis. Results: Transforming growth factor beta 3 (TGF-β3) combined with insulin-like growth factor Ⅰ (IGF-Ⅰ) induced the cartilage-specific collagen type Ⅱ, aggrecan expression and extracellular matrix (ECM) accumulation at the end of a 3-week culture. CGS supported those differentiated chondrocytes' attachment, proliferation, migration, and ECM formation. When those cells cultured on CGS were stimulated with IL-1β alone, collagen type Ⅱ and aggrecan expression was inhibited. However, MMP-13 expression increased. Resveratrol reversed the catabolic effects by reducing the translocation of NF-κB. A specific β1-integrin blocking antibody abrogated the effects of resveratrol on IL-1β stimulated MSC-derived chondrocytes. Conclusion: These results indicated that resveratrol acta as a NF-κB inhibitor to protect MSC-derived chondrocytes on the CGS from the IL-1β catabolism and these effects are mediated by β1-integrin.

  6. Mutations in CDC45, Encoding an Essential Component of the Pre-initiation Complex, Cause Meier-Gorlin Syndrome and Craniosynostosis.

    Science.gov (United States)

    Fenwick, Aimee L; Kliszczak, Maciej; Cooper, Fay; Murray, Jennie; Sanchez-Pulido, Luis; Twigg, Stephen R F; Goriely, Anne; McGowan, Simon J; Miller, Kerry A; Taylor, Indira B; Logan, Clare; Bozdogan, Sevcan; Danda, Sumita; Dixon, Joanne; Elsayed, Solaf M; Elsobky, Ezzat; Gardham, Alice; Hoffer, Mariette J V; Koopmans, Marije; McDonald-McGinn, Donna M; Santen, Gijs W E; Savarirayan, Ravi; de Silva, Deepthi; Vanakker, Olivier; Wall, Steven A; Wilson, Louise C; Yuregir, Ozge Ozalp; Zackai, Elaine H; Ponting, Chris P; Jackson, Andrew P; Wilkie, Andrew O M; Niedzwiedz, Wojciech; Bicknell, Louise S

    2016-07-07

    DNA replication precisely duplicates the genome to ensure stable inheritance of genetic information. Impaired licensing of origins of replication during the G1 phase of the cell cycle has been implicated in Meier-Gorlin syndrome (MGS), a disorder defined by the triad of short stature, microtia, and a/hypoplastic patellae. Biallelic partial loss-of-function mutations in multiple components of the pre-replication complex (preRC; ORC1, ORC4, ORC6, CDT1, or CDC6) as well as de novo stabilizing mutations in the licensing inhibitor, GMNN, cause MGS. Here we report the identification of mutations in CDC45 in 15 affected individuals from 12 families with MGS and/or craniosynostosis. CDC45 encodes a component of both the pre-initiation (preIC) and CMG helicase complexes, required for initiation of DNA replication origin firing and ongoing DNA synthesis during S-phase itself, respectively, and hence is functionally distinct from previously identified MGS-associated genes. The phenotypes of affected individuals range from syndromic coronal craniosynostosis to severe growth restriction, fulfilling diagnostic criteria for Meier-Gorlin syndrome. All mutations identified were biallelic and included synonymous mutations altering splicing of physiological CDC45 transcripts, as well as amino acid substitutions expected to result in partial loss of function. Functionally, mutations reduce levels of full-length transcripts and protein in subject cells, consistent with partial loss of CDC45 function and a predicted limited rate of DNA replication and cell proliferation. Our findings therefore implicate the preIC as an additional protein complex involved in the etiology of MGS and connect the core cellular machinery of genome replication with growth, chondrogenesis, and cranial suture homeostasis.

  7. Neocartilage formation from predifferentiated human adipose derived stem cells in vivo

    Institute of Scientific and Technical Information of China (English)

    Xiao-bing JIN; Yong-sheng SUN; Ke ZHANG; Jing WANG; Xiao-dong JU; Si-quan LOU

    2007-01-01

    Aim: To examine the chondrogenic potential of human adipose derived stem cells (hASC) induced by human transforming growth factor beta2 (hTGF beta2) in vitro, and to investigate if predifferentiated hASC can produce neocartilage in vivo. Methods: hASC were isolated from subcutaneous adipose tissue and cul-tured in pellets with the addition of hTGF beta2. Chondrogenic differentiation was assayed by RT-PCR, Western blotting, toluidine blue staining, and immuno-histochemistry staining for collagen type Ⅱ. For the in vivo study, intact induced cell pellets or the released cells embedded in alginate gel with different concentra-tions were implanted subcutaneously in nude mice. Specimens were harvested at different time points and carried with histological and immunohistochemistry ex-amination to evaluate the cartilage formation. Results: RT-PCR analysis revealed that hASC produced aggrecan and collagen type Ⅱ after 7 d of induction and continued throughout the culture period. This was also demonstrated by the Western blot analysis, positive staining of toluidine blue, and immunohistochem-istry for collagen type Ⅱ. After reseeding in the monolayer, the cells isolated from the pellets displayed a polygonal morphology compared with the primary spindle shape, hASC were released from the induced cell pellets when embedded in alginate gel (implanted cell concentration=5x106/mL or higher). They produced neocartilage after 12 weeks in vivo culture; however, intact induced cell pellets implanted subcutaneously rapidly lost their differentiated phenotype. Conclusion:Chondrogenesis of hASC in vitro can be induced by combining pellet culture and hTGF beta2 treatment. Predifferentiated hASC embedded in alginate gel have the ability of producing neocartilage in vivo.

  8. Characterization of the collagen component of cartilage repair tissue of the talus with quantitative MRI: comparison of T2 relaxation time measurements with a diffusion-weighted double-echo steady-state sequence (dwDESS)

    Energy Technology Data Exchange (ETDEWEB)

    Kretzschmar, M.; Hainc, N.; Studler, U. [University Hospital Basel, Department of Radiology, Basel (Switzerland); Bieri, O. [University Hospital Basel, Division of Radiological Physics, Basel (Switzerland); Miska, M. [University Hospital, Department of Orthopedics, Heidelberg (Germany); Wiewiorski, M.; Valderrabano, V. [University Hospital Basel, Department of Orthopedic Surgery, Basel (Switzerland)

    2015-04-01

    The purpose of this study was to characterize the collagen component of repair tissue (RT) of the talus after autologous matrix-induced chondrogenesis (AMIC) using quantitative T2 and diffusion-weighted imaging. Mean T2 values and diffusion coefficients of AMIC-RT and normal cartilage of the talus of 25 patients with posttraumatic osteochondral lesions and AMIC repair were compared in a cross-sectional design using partially spoiled steady-state free precession (pSSFP) for T2 quantification, and diffusion-weighted double-echo steady-state (dwDESS) for diffusion measurement. RT and cartilage were graded with modified Noyes and MOCART scores on morphological sequences. An association between follow-up interval and quantitative MRI measures was assessed using multivariate regression, after stratifying the cohort according to time interval between surgery and MRI. Mean T2 of the AMIC-RT and cartilage were 43.1 ms and 39.1 ms, respectively (p = 0.26). Mean diffusivity of the RT (1.76 μm{sup 2}/ms) was significantly higher compared to normal cartilage (1.46 μm{sup 2}/ms) (p = 0.0092). No correlation was found between morphological and quantitative parameters. RT diffusivity was lowest in the subgroup with follow-up >28 months (p = 0.027). Compared to T2-mapping, dwDESS demonstrated greater sensitivity in detecting differences in the collagen matrix between AMIC-RT and cartilage. Decreased diffusivity in patients with longer follow-up times may indicate an increased matrix organization of RT. (orig.)

  9. Japanese medaka Hox paralog group 2: insights into the evolution of Hox PG2 gene composition and expression in the Osteichthyes.

    Science.gov (United States)

    Davis, Adam; Scemama, Jean-Luc; Stellwag, Edmund J

    2008-12-15

    Hox paralog group 2 (PG2) genes function to specify the development of the hindbrain and pharyngeal arch-derived structures in the Osteichthyes. In this article, we describe the cDNA cloning and embryonic expression analysis of Japanese medaka (Oryzias latipes) Hox PG2 genes. We show that there are only two functional canonical Hox genes, hoxa2a and b2a, and that a previously identified hoxa2b gene is a transcribed pseudogene, psihoxa2b. The functional genes, hoxa2a and b2a, were expressed in developing rhombomeres and pharyngeal arches in a manner that was relatively well conserved compared with zebrafish (Danio rerio) but differed significantly from orthologous striped bass (Morone saxatilis) and Nile tilapia (Oreochromis niloticus) genes, which, we suggest, may be owing to effects of post-genome duplication loss of a Hox PG2 gene in the medaka and zebrafish lineages. psihoxa2b was expressed at readily detectable levels in several noncanonical Hox expression domains, including the ventral aspect of the neural tube, the pectoral fin buds and caudal-most region of the embryonic trunk, indicative that regulatory control elements needed for spatio-temporal expression have diverged from their ancestral counterparts. Comparative expression analyses showed medaka hoxa2a and b2a expression in the 2nd pharyngeal arch (PA2) beyond the onset of chondrogenesis, which, according to previous hypotheses, suggests these genes function redundantly as selector genes of PA2 identity. We conclude that Hox PG2 gene composition and expression have diverged significantly during osteichthyan evolution and that this divergence in teleosts may be related to lineage-dependent differential gene loss following an actinopterygian-specific whole genome duplication.

  10. Implication of NOD1 and NOD2 for the differentiation of multipotent mesenchymal stem cells derived from human umbilical cord blood.

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    Hyung-Sik Kim

    Full Text Available Toll-like receptors (TLRs and Nod-like receptors (NLRs are known to trigger an innate immune response against microbial infection. Although studies suggest that activation of TLRs modulate the function of mesenchymal stem cells (MSCs, little is known about the role of NLRs on the MSC function. In this study, we investigated whether NOD1 and NOD2 regulate the functions of human umbilical cord blood-derived MSCs (hUCB-MSCs. The genes of TLR2, TLR4, NOD1, and NOD2 were expressed in hUCB-MSCs. Stimulation with each agonist (Pam(3CSK(4 for TLR2, LPS for TLR4, Tri-DAP for NOD1, and MDP for NOD2 led to IL-8 production in hUCB-MSC, suggesting the expressed receptors are functional in hUCB-MSC. CCK-8 assay revealed that none of agonist influenced proliferation of hUCB-MSCs. We next examined whether TLR and NLR agonists affect osteogenic-, adipogenic-, and chondrogenic differentiation of hUCB-MSCs. Pam(3CSK(4 and Tri-DAP strongly enhanced osteogenic differentiation and ERK phosphorylation in hUCB-MSCs, and LPS and MDP also slightly did. Treatment of U0126 (MEK1/2 inhibitor restored osteogenic differentiation enhanced by Pam(3CSK(4. Tri-DAP and MDP inhibited adipogenic differentiation of hUCB-MSCs, but Pam(3CSK(4 and LPS did not. On chondrogenic differentiation, all TLR and NLR agonists could promote chondrogenesis of hUCB-MSCs with difference in the ability. Our findings suggest that NOD1 and NOD2 as well as TLRs are involved in regulating the differentiation of MSCs.

  11. Sox9基因的结构、功能及进化%Sox9 gene: structure, function and evolution

    Institute of Scientific and Technical Information of China (English)

    陈维; 王昌留; 王磊

    2012-01-01

    Sox9 is the first member of Sox gene family which has intron fragments after the discovery of SRY gene. About 671 Sox9 gene sequences are recorded in the Genebank, which are widely distributed in many species from mammals and bird to coelenterate. Sox9 is a multifunctional gene which takes part in sex determination, chondrogenesis, formation of nervous system and pancreas. Furthermore, this gene can also play roles in oncogenesis. This paper reviews the research progress of the structure, function and evolution of Sox9,to provide references for further study on Sox9 gene.%Sox9基因是继SRY基因之后人们发现的第一个具有内含子的Sox家族成员.在Genebank中可查的Sox9基因序列共671个,广泛分布于哺乳动物和鸟类直至水母等低等动物.近年来的研究发现,Sox9基因的功能呈现多样性,该基因在发育过程中参与性别的决定、软骨的形成以及神经系统和胰腺的形成,另外Sox9基因在肿瘤发生中也有一定的作用.本文从Sox9基因的结构、功能以及进化等方面进行了综述,为进一步研究Sox9基因提供参考资料.

  12. Time kinetics of bone defect healing in response to BMP-2 and GDF-5 characterised by in vivo biomechanics

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    D Wulsten

    2011-02-01

    Full Text Available This study reports that treatment of osseous defects with different growth factors initiates distinct rates of repair. We developed a new method for monitoring the progression of repair, based upon measuring the in vivo mechanical properties of healing bone. Two different members of the bone morphogenetic protein (BMP family were chosen to initiate defect healing: BMP-2 to induce osteogenesis, and growth-and-differentiation factor (GDF-5 to induce chondrogenesis. To evaluate bone healing, BMPs were implanted into stabilised 5 mm bone defects in rat femurs and compared to controls. During the first two weeks, in vivo biomechanical measurements showed similar values regardless of the treatment used. However, 2 weeks after surgery, the rhBMP-2 group had a substantial increase in stiffness, which was supported by the imaging modalities. Although the rhGDF-5 group showed comparable mechanical properties at 6 weeks as the rhBMP-2 group, the temporal development of regenerating tissues appeared different with rhGDF-5, resulting in a smaller callus and delayed tissue mineralisation. Moreover, histology showed the presence of cartilage in the rhGDF-5 group whereas the rhBMP-2 group had no cartilaginous tissue.Therefore, this study shows that rhBMP-2 and rhGDF-5 treated defects, under the same conditions, use distinct rates of bone healing as shown by the tissue mechanical properties. Furthermore, results showed that in vivo biomechanical method is capable of detecting differences in healing rate by means of change in callus stiffness due to tissue mineralisation.

  13. Turning Bone Morphogenetic Protein 2 (BMP2) on and off in Mesenchymal Cells.

    Science.gov (United States)

    Rogers, Melissa B; Shah, Tapan A; Shaikh, Nadia N

    2015-10-01

    The concentration, location, and timing of bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) gene expression must be precisely regulated. Abnormal BMP2 levels cause congenital anomalies and diseases involving the mesenchymal cells that differentiate into muscle, fat, cartilage, and bone. The molecules and conditions that influence BMP2 synthesis are diverse. Understandably, complex mechanisms control Bmp2 gene expression. This review includes a compilation of agents and conditions that can induce Bmp2. The currently known trans-regulatory factors and cis-regulatory elements that modulate Bmp2 expression are summarized and discussed. Bone morphogenetic protein 2 (BMP2, HGNC:1069, GeneID: 650) is a classical morphogen; a molecule that acts at a distance and whose concentration influences cell behavior. In mesenchymal cells, the concentration of BMP2 influences myogenesis, adipogenesis, chondrogenesis, and osteogenesis. Because the amount, timing, and location of BMP2 synthesis influence the allocation of cells to muscle, fat, cartilage, and bone, the mechanisms that regulate the Bmp2 gene are crucial. Key early mesodermal events that require precise Bmp2 regulation include heart specification and morphogenesis. Originally named for its osteoinductive properties, healing fractures requires BMP2. The human Bmp2 gene also has been linked to osteoporosis and osteoarthritis. In addition, all forms of pathological calcification in the vasculature and in cardiac valves involve the pro-osteogenic BMP2. The diverse tissues, mechanisms, and diseases influenced by BMP2 are too numerous to list here (see OMIM: 112261). However, in all BMP2-influenced pathologies, changes in the behavior and differentiation of pluripotent mesenchymal cells are a recurring theme. Consequently, much effort has been devoted to identifying the molecules and conditions that influence BMP2 synthesis and the complex mechanisms that control Bmp2 gene expression. This review begins with an

  14. BMP2 induces chondrogenic differentiation, osteogenic differentiation and endochondral ossification in stem cells.

    Science.gov (United States)

    Zhou, Nian; Li, Qi; Lin, Xin; Hu, Ning; Liao, Jun-Yi; Lin, Liang-Bo; Zhao, Chen; Hu, Zhen-Ming; Liang, Xi; Xu, Wei; Chen, Hong; Huang, Wei

    2016-10-01

    Bone morphogenetic protein 2 (BMP2), a member of the transforming growth factor-β (TGF-β) super-family, is one of the main chondrogenic growth factors involved in cartilage regeneration. BMP2 is known to induce chondrogenic differentiation in various types of stem cells in vitro. However, BMP2 also induces osteogenic differentiation and endochondral ossification in mesenchymal stem cells (MSCs). Although information regarding BMP2-induced chondrogenic and osteogenic differentiation within the same system might be essential for cartilage tissue engineering, few studies concerning these issues have been conducted. In this study, BMP2 was identified as a regulator of chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. BMP2 was used to regulate chondrogenic and osteogenic differentiation in stem cells within the same culture system in vitro and in vivo. Any changes in the differentiation markers were assessed. BMP2 was found to induce chondrogenesis and osteogenesis in vitro via the expression of Sox9, Runx2 and its downstream markers. According to the results of the subcutaneous stem cell implantation studies, BMP2 not only induced cartilage formation but also promoted endochondral ossification during ectopic bone/cartilage formation. In fetal limb cultures, BMP2 promoted chondrocyte hypertrophy and endochondral ossification. Our data reveal that BMP2 can spontaneously induce chondrogenic differentiation, osteogenic differentiation and endochondral bone formation within the same system. Thus, BMP2 can be used in cartilage tissue engineering to regulate cartilage formation but has to be properly regulated for cartilage tissue engineering in order to retain the cartilage phenotype.

  15. Combination of Collagen-Based Scaffold and Bioactive Factors Induces Adipose-Derived Mesenchymal Stem Cells Chondrogenic Differentiation In vitro

    Science.gov (United States)

    Calabrese, Giovanna; Forte, Stefano; Gulino, Rosario; Cefalì, Francesco; Figallo, Elisa; Salvatorelli, Lucia; Maniscalchi, Eugenia T.; Angelico, Giuseppe; Parenti, Rosalba; Gulisano, Massimo; Memeo, Lorenzo; Giuffrida, Raffaella

    2017-01-01

    Recently, multipotent mesenchymal stem cells (MSCs) have attracted much attention in the field of regenerative medicine due to their ability to give rise to different cell types, including chondrocytes. Damaged articular cartilage repair is one of the most challenging issues for regenerative medicine, due to the intrinsic limited capability of cartilage to heal because of its avascular nature. While surgical approaches like chondral autografts and allografts provide symptoms and function improvement only for a short period, MSC based stimulation therapies, like microfracture surgery or autologous matrix-induced chondrogenesis demonstrate to be more effective. The use of adult chondrocytes, which are the main cellular constituent of cartilage, in medical practice, is indeed limited due to their instability in monolayer culture and difficulty to collect donor tissue (articular and nasal cartilage). The most recent cartilage engineering approaches combine cells, biomaterial scaffold and bioactive factors to promote functional tissue replacements. Many recent evidences demonstrate that scaffolds providing specific microenvironmental conditions can promote MSCs differentiation toward a functional phenotype. In the present work, the chondrogenic potential of a new Collagen I based 3D scaffold has been assessed in vitro, in combination with human adipose-derived MSCs which possess a higher chondrogenic potential compared to MSCs isolated from other tissues. Our data indicate that the scaffold was able to promote the early stages of chondrogenic commitment and that supplementation of specific soluble factors was able to induce the complete differentiation of MSCs in chondrocytes as demonstrated by the appearance of cartilage distinctive markers (Sox 9, Aggrecan, Matrilin-1, and Collagen II), as well as by the cartilage-specific Alcian Blue staining and by the acquisition of typical cellular morphology. Such evidences suggest that the investigated scaffold formulation could

  16. The detection of a precartilage, blastema-specific marker.

    Science.gov (United States)

    Aulthouse, A L; Solursh, M

    1987-04-01

    Mesenchymal cell aggregates, termed blastema in vivo, precede cartilage differentiation in vivo and in high-density cell cultures. The galactose specific lectin, peanut agglutinin (PNA), has been shown to be blastema specific (B. Zimmermann and M. Thies, 1984, Histochemistry 81, 353-361). PNA appears to be a marker for precartilage cellular aggregates both in vivo and in vitro. Frozen sections of stage 24 chick wing buds were double stained with PNA-rhodamine and by indirect immunofluorescence with antibody directed against type II collagen. The PNA stained the humeral blastema intensely and extended distal to the level of type II collagen. High-density cultures of stage 24 chick wing buds were also evaluated for the distribution of PNA binding. Sixteen-hour cultures showed the earliest consistent appearance of PNA binding. The PNA-stained areas coincided with hematoxylin-stained cell aggregates. PNA staining was inhibited by 50 mM D(+)-galactose and was not sensitive to 1% testicular hyaluronidase pretreatment. No Alcian blue-staining nodules were present yet at 16 hr. The presence of a precartilage, blastema-specific marker in situ, as well as in precartilage aggregates in cultures, suggests the similarities in chondrogenesis between these two conditions. Stage 19 limb bud cultures did not form nodules but did form aggregates that were PNA positive. Furthermore, single cells that differentiated into chondrocytes on collagen gels or after cytochalasin D treatment lacked PNA-binding material. These results suggest that this material is specific to precartilage aggregates. The PNA-positive material was extracellular in distribution and was removed after brief extraction with 0.5 M guanidine hydrochloride.

  17. Vitamin a is a negative regulator of osteoblast mineralization.

    Science.gov (United States)

    Lind, Thomas; Sundqvist, Anders; Hu, Lijuan; Pejler, Gunnar; Andersson, Göran; Jacobson, Annica; Melhus, Håkan

    2013-01-01

    An excessive intake of vitamin A has been associated with an increased risk of fractures in humans. In animals, a high vitamin A intake leads to a reduction of long bone diameter and spontaneous fractures. Studies in rodents indicate that the bone thinning is due to increased periosteal bone resorption and reduced radial growth. Whether the latter is a consequence of direct effects on bone or indirect effects on appetite and general growth is unknown. In this study we therefore used pair-feeding and dynamic histomorphometry to investigate the direct effect of a high intake of vitamin A on bone formation in rats. Although there were no differences in body weight or femur length compared to controls, there was an approximately halved bone formation and mineral apposition rate at the femur diaphysis of rats fed vitamin A. To try to clarify the mechanism(s) behind this reduction, we treated primary human osteoblasts and a murine preosteoblastic cell line (MC3T3-E1) with the active metabolite of vitamin A; retinoic acid (RA), a retinoic acid receptor (RAR) antagonist (AGN194310), and a Cyp26 inhibitor (R115866) which blocks endogenous RA catabolism. We found that RA, via RARs, suppressed in vitro mineralization. This was independent of a negative effect on osteoblast proliferation. Alkaline phosphatase and bone gamma carboxyglutamate protein (Bglap, Osteocalcin) were drastically reduced in RA treated cells and RA also reduced the protein levels of Runx2 and Osterix, key transcription factors for progression to a mature osteoblast. Normal osteoblast differentiation involved up regulation of Cyp26b1, the major enzyme responsible for RA degradation, suggesting that a drop in RA signaling is required for osteogenesis analogous to what has been found for chondrogenesis. In addition, RA decreased Phex, an osteoblast/osteocyte protein necessary for mineralization. Taken together, our data indicate that vitamin A is a negative regulator of osteoblast mineralization.

  18. Electrospun scaffolds for multiple tissues regeneration in vivo through topography dependent induction of lineage specific differentiation.

    Science.gov (United States)

    Yin, Zi; Chen, Xiao; Song, Hai-Xin; Hu, Jia-Jie; Tang, Qiao-Mei; Zhu, Ting; Shen, Wei-Liang; Chen, Jia-Lin; Liu, Huanhuan; Heng, Boon Chin; Ouyang, Hong-Wei

    2015-03-01

    Physical topographic cues from various substrata have been shown to exert profound effects on the growth and differentiation of stem cells due to their niche-mimicking features. However, the biological function of different topographic materials utilized as bio-scaffolds in vivo have not been rigorously characterized. This study investigated the divergent differentiation pathways of mesenchymal stem cells (MSCs) and neo-tissue formation trigged by aligned and randomly-oriented fibrous scaffolds, both in vitro and in vivo. The aligned group was observed to form more mature tendon-like tissue in the Achilles tendon injury model, as evidenced by histological scoring and collagen I immunohistochemical staining data. In contrast, the randomly-oriented group exhibited much chondrogenesis and subsequent bone tissue formation through ossification. Additionally, X-ray imaging and osteocalcin immunohistochemical staining also demonstrated that osteogenesis in vivo is driven by randomly oriented topography. Furthermore, MSCs on the aligned substrate exhibited tenocyte-like morphology and enhanced tenogenic differentiation compared to cells grown on randomly-oriented scaffold. qRT-PCR analysis of osteogenic marker genes and alkaline phosphatase (ALP) staining demonstrated that MSCs cultured on randomly-oriented fiber scaffolds displayed enhanced osteogenic differentiation compared with cells cultured on aligned fiber scaffolds. Finally, it was demonstrated that cytoskeletal tension release abrogated the divergent differentiation pathways on different substrate topography. Collectively, these findings illustrate the relationship between topographic cues of the scaffold and their inductive role in tissue regeneration; thus providing an insight into future development of smart functionalized bio-scaffold design and its application in tissue engineering.

  19. Isolation of mesenchymal stem cells from equine umbilical cord blood

    Directory of Open Access Journals (Sweden)

    Thomsen Preben D

    2007-05-01

    Full Text Available Abstract Background There are no published studies on stem cells from equine cord blood although commercial storage of equine cord blood for future autologous stem cell transplantations is available. Mesenchymal stem cells (MSC have been isolated from fresh umbilical cord blood of humans collected non-invasively at the time of birth and from sheep cord blood collected invasively by a surgical intrauterine approach. Mesenchymal stem cells isolation percentage from frozen-thawed human cord blood is low and the future isolation percentage of MSCs from cryopreserved equine cord blood is therefore expectedly low. The hypothesis of this study was that equine MSCs could be isolated from fresh whole equine cord blood. Results Cord blood was collected from 7 foals immediately after foaling. The mononuclear cell fraction was isolated by Ficoll density centrifugation and cultured in a DMEM low glucose based media at 38.5°C in humidified atmosphere containing 5% CO2. In 4 out of 7 samples colonies with MSC morphology were observed. Cellular morphology varied between monolayers of elongated spindle-shaped cells to layered cell clusters of cuboidal cells with shorter cytoplasmic extensions. Positive Alizarin Red and von Kossa staining as well as significant calcium deposition and alkaline phosphatase activity confirmed osteogenesis. Histology and positive Safranin O staining of matrix glycosaminoglycans illustrated chondrogenesis. Oil Red O staining of lipid droplets confirmed adipogenesis. Conclusion We here report, for the first time, the isolation of mesenchymal-like stem cells from fresh equine cord blood and their differentiation into osteocytes, chondrocytes and adipocytes. This novel isolation of equine cord blood MSCs and their preliminary in vitro differentiation positions the horse as the ideal pre-clinical animal model for proof-of-principle studies of cord blood derived MSCs.

  20. Sec24D-dependent transport of extracellular matrix proteins is required for zebrafish skeletal morphogenesis.

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    Swapnalee Sarmah

    Full Text Available Protein transport from endoplasmic reticulum (ER to Golgi is primarily conducted by coated vesicular carriers such as COPII. Here, we describe zebrafish bulldog mutations that disrupt the function of the cargo adaptor Sec24D, an integral component of the COPII complex. We show that Sec24D is essential for secretion of cartilage matrix proteins, whereas the preceding development of craniofacial primordia and pre-chondrogenic condensations does not depend on this isoform. Bulldog chondrocytes fail to secrete type II collagen and matrilin to extracellular matrix (ECM, but membrane bound receptor beta1-Integrin and Cadherins appear to leave ER in Sec24D-independent fashion. Consequently, Sec24D-deficient cells accumulate proteins in the distended ER, although a subset of ER compartments and Golgi complexes as visualized by electron microscopy and NBD C(6-ceramide staining appear functional. Consistent with the backlog of proteins in the ER, chondrocytes activate the ER stress response machinery and significantly upregulate BiP transcription. Failure of ECM secretion hinders chondroblast intercalations thus resulting in small and malformed cartilages and severe craniofacial dysmorphology. This defect is specific to Sec24D mutants since knockdown of Sec24C, a close paralog of Sec24D, does not result in craniofacial cartilage dysmorphology. However, craniofacial development in double Sec24C/Sec24D-deficient animals is arrested earlier than in bulldog/sec24d, suggesting that Sec24C can compensate for loss of Sec24D at initial stages of chondrogenesis, but Sec24D is indispensable for chondrocyte maturation. Our study presents the first developmental perspective on Sec24D function and establishes Sec24D as a strong candidate for cartilage maintenance diseases and craniofacial birth defects.

  1. Isolation and characterization of multipotential mesenchymal cells from the mouse synovium.

    Directory of Open Access Journals (Sweden)

    Ippei Futami

    Full Text Available The human synovium contains mesenchymal stem cells (MSCs, which are multipotential non-hematopoietic progenitor cells that can differentiate into a variety of mesenchymal lineages and they may therefore be a candidate cell source for tissue repair. However, the molecular mechanisms by which this can occur are still largely unknown. Mouse primary cell culture enables us to investigate the molecular mechanisms underlying various phenomena because it allows for relatively easy gene manipulation, which is indispensable for the molecular analysis. However, mouse synovial mesenchymal cells (SMCs have not been established, although rabbit, cow, and rat SMCs are available, in addition to human MSCs. The aim of this study was to establish methods to harvest the synovium and to isolate and culture primary SMCs from mice. As the mouse SMCs were not able to be harvested and isolated using the same protocol for human, rat and rabbit SMCs, the protocol for humans was modified for SMCs from the Balb/c mouse knee joint. The mouse SMCs obtained showed superior proliferative potential, growth kinetics and colony formation compared to cells derived from muscle and bone marrow. They expressed PDGFRá and Sca-1 detected by flow cytometry, and showed an osteogenic, adipogenic and chondrogenic potential similar or superior to the cells derived from muscle and bone marrow by demonstrating in vitro osteogenesis, adipogenesis and chondrogenesis. In conclusion, we established a primary mouse synovial cell culture method. The cells derived from the mouse synovium demonstrated both the ability to proliferate and multipotentiality similar or superior to the cells derived from muscle and bone marrow.

  2. Donor-Matched Comparison of Chondrogenic Potential of Equine Bone Marrow- and Synovial Fluid-Derived Mesenchymal Stem Cells: Implications for Cartilage Tissue Regeneration

    Science.gov (United States)

    Zayed, Mohammed; Caniglia, Christopher; Misk, Nabil; Dhar, Madhu S.

    2017-01-01

    Mesenchymal stem cells (MSCs) have been demonstrated to be useful for cartilage tissue regeneration. Bone marrow (BM) and synovial fluid (SF) are promising sources for MSCs to be used in cartilage regeneration. In order to improve the clinical outcomes, it is recommended that prior to clinical use, the cellular properties and, specifically, their chondrogenic potential must be investigated. The purpose of this study is to compare and better understand the in vitro chondrogenic potential of equine bone marrow-derived mesenchymal stem cells (BMMSCs) and synovial fluid-derived mesenchymal stem cells (SFMSCs) populated from the same equine donor. BM- and SF-derived MSCs cultures were generated from five equine donors, and the MSCs were evaluated in vitro for their morphology, proliferation, trilineage differentiation, and immunophenotyping. Differences in their chondrogenic potentials were further evaluated quantitatively using glycosaminoglycan (GAG) content and via immunofluorescence of chondrogenic differentiation protein markers, SRY-type HMG box9, Aggrecan, and collagen II. The BMMSCs and SFMSCs were similar in cellular morphology, viability, and immunophenotype, but, varied in their chondrogenic potential, and expression of the key chondrogenic proteins. The SFMSCs exhibited a significant increase in GAG content compared to the BMMSCs (P < 0.0001) in three donors, suggesting increased levels of chondrogenesis. The expression of the key chondrogenic proteins correlated positively with the GAG content, suggesting that the differentiation process is dependent on the expression of the target proteins in these three donors. Our findings suggest that even though SFMSCs were hypothesized to be more chondrogenic relative to BMMSCs, there was considerable donor-to-donor variation in the primary cultures of MSCs which can significantly affect their downstream application.

  3. Therapeutic options in the treatment of cartilage defects: techniques and indications; Therapieoptionen zur Behandlung von Knorpelschaeden: Techniken und Indikationen

    Energy Technology Data Exchange (ETDEWEB)

    Resinger, C.; Vecsei, V.; Marlovits, S. [Universitaetsklinik fuer Unfallchirurgie, Medizinische Universitaet Wien (Austria)

    2004-08-01

    Cartilage is composed of chondrocytes embedded within an extracellular matrix of collagens, proteoglycans, and noncollagenous proteins. Together, these structures maintain the unique mechanical properties and manifest its striking inability to heal even the most minor injury. This review presents the principles of cartilage structure and the biological background of cartilage repair and gives information about the surgical techniques for treating cartilage defects. The response of cartilage to injuries differs from that of other tissues because of its avascularity, the immobility of chondrocytes and the limited ability of mature chondrocytes to proliferate and alter their synthetic patterns. Surgical therapeutic efforts in treating cartilage defects have focused on bringing new cells and tissues capable of chondrogenesis into the lesions and facilitating the access to the vascular system. The right indication and the treatment of joint instability and axis deformation are essential for the successful use of cartilage repair procedures. (orig.) [German] Das artikulaere Knorpelgewebe besteht aus einer einzelnen Zellpopulation, integriert in ein dreidimensionales Netzwerk hochorganisierter Matrixstrukturen. Dieser feingewebliche Aufbau bestimmt die einzigartigen mechanischen Eigenschaften, limitiert aber auch die physiologischen Reparationsmoeglichkeiten von Knorpeldefekten. Diese Uebersicht beschreibt die Grundlagen der Knorpelbiologie und die Mechanismen der Knorpelreparatur und behandelt die klinischen Ergebnisse chirurgischer Techniken zur Therapie umschriebener Knorpeldefekte. Die chirurgischen Techniken zur Therapie lokalisierter Defekte der Gelenkoberflaeche versuchen durch die Integration biologischer Mechanismen die mangelnde Regenerationsfaehigkeit artikulaeren Knorpels zu ueberwinden. Die Techniken der Transplantation chondrogener Gewebe wurden in juengster Zeit durch die Defektauffuellung mit autologen Chondrozyten erweitert. Eine klare Indikationsstellung

  4. Characterization of human adipose-derived stem cells and expression of chondrogenic genes during induction of cartilage differentiation

    Directory of Open Access Journals (Sweden)

    Adila A Hamid

    2012-01-01

    Full Text Available OBJECTIVES: Understanding the changes in chondrogenic gene expression that are involved in the differentiation of human adipose-derived stem cells to chondrogenic cells is important prior to using this approach for cartilage repair. The aims of the study were to characterize human adipose-derived stem cells and to examine chondrogenic gene expression after one, two, and three weeks of induction. MATERIALS AND METHODS: Human adipose-derived stem cells at passage 4 were evaluated by flow cytometry to examine the expression of surface markers. These adipose-derived stem cells were tested for adipogenic and osteogenic differentiation capacity. Ribonucleic acid was extracted from the cells for quantitative polymerase chain reaction analysis to determine the expression levels of chondrogenic genes after chondrogenic induction. RESULTS: Human adipose-derived stem cells were strongly positive for the mesenchymal markers CD90, CD73, CD44, CD9, and histocompatibility antigen and successfully differentiated into adipogenic and osteogenic lineages. The human adipose-derived stem cells aggregated and formed a dense matrix after chondrogenic induction. The expression of chondrogenic genes (collagen type II, aggrecan core protein, collagen type XI, COMP, and ELASTIN was significantly higher after the first week of induction. However, a significantly elevated expression of collagen type X was observed after three weeks of chondrogenic induction. CONCLUSION: Human adipose-derived stem cells retain stem cell characteristics after expansion in culture to passage 4 and serve as a feasible source of cells for cartilage regeneration. Chondrogenesis in human adiposederived stem cells was most prominent after one week of chondrogenic induction.

  5. Detecting new microRNAs in human osteoarthritic chondrocytes identifies miR-3085 as a human, chondrocyte-selective, microRNA

    Science.gov (United States)

    Crowe, N.; Swingler, T.E.; Le, L.T.T.; Barter, M.J.; Wheeler, G.; Pais, H.; Donell, S.T.; Young, D.A.; Dalmay, T.; Clark, I.M.

    2016-01-01

    Summary Objective To use deep sequencing to identify novel microRNAs (miRNAs) in human osteoarthritic cartilage which have a functional role in chondrocyte phenotype or function. Design A small RNA library was prepared from human osteoarthritic primary chondrocytes using in-house adaptors and analysed by Illumina sequencing. Novel candidate miRNAs were validated by northern blot and qRT-PCR. Expression was measured in cartilage models. Targets of novel candidates were identified by microarray and computational analysis, validated using 3′-UTR-luciferase reporter plasmids. Protein levels were assessed by western blot and functional analysis by cell adhesion. Results We identified 990 known miRNAs and 1621 potential novel miRNAs in human osteoarthritic chondrocytes, 60 of the latter were expressed in all samples assayed. MicroRNA-140-3p was the most highly expressed microRNA in osteoarthritic cartilage. Sixteen novel candidate miRNAs were analysed further, of which six remained after northern blot analysis. Three novel miRNAs were regulated across models of chondrogenesis, chondrocyte differentiation or cartilage injury. One sequence (novel #11), annotated in rodents as microRNA-3085-3p, was preferentially expressed in cartilage, dependent on chondrocyte differentiation and, in man, is located in an intron of the cartilage-expressed gene CRTAC-1. This microRNA was shown to target the ITGA5 gene directly (which encodes integrin alpha5) and inhibited adhesion to fibronectin (dependent on alpha5beta1 integrin). Conclusion Deep sequencing has uncovered many potential microRNA candidates expressed in human cartilage. At least three of these show potential functional interest in cartilage homeostasis and osteoarthritis (OA). Particularly, novel #11 (microRNA-3085-3p) which has been identified for the first time in man. PMID:26497608

  6. The Experimental Studies of the Tissue Engineering Cartilage by Co-Culturing Microtia Chondrocytes and Adipose Tissue-Derived Stem Cells in Vivo%残耳软骨细胞与脂肪干细胞共培养体内构建软骨的实验研究

    Institute of Scientific and Technical Information of China (English)

    张洁; 蒋海越; 何乐仁; 赵延勇; 杨庆华; 韩娟; 宋宇鹏

    2011-01-01

    Objective To explore the feasibility of the chondrogenesis by co-culturing microtia chondrocytes and human adipose tissue-derived stem cells in vivo. Methods hADSCs and microtia chondrocytes were isolated in vitro. 24 nude mice were randomly divided into 4 groups: ①Exp group, injected with microtia chondrocytes and hADSCs by a mixing ratio of 1:1 and the cell concentration was 5.0×l07 cells/mL; ②Ctrl 1 group, injected with only microtia chondrocytes and the cell concentration was 5.0×107 cellshnL; ③Ctrl 2 group, injected with only hADSCs and the cell concentration was 5.0×l07 cells/mL;④Ctrl 3 group, injected with only microtia chondrocytes and the cell concentration was 2.5×107 cells/mL. 6 nude mice were injected each group at a dose of 0.2 mL. All samples were harvested 10 weeks after culturingin vivo. Gross observation, average wet weights, glycosaminoglycan (GAG) quantification, histology and immunohistochemisty were used to evaluate the chondrogenesis of all groups. Results In Exp, Ctrl 1, and Ctrl 3 group, all the specimens formed homogeneous cartilagelike tissue with typical histological structure at different extent. In Ctrl 2 group, the specimens formed fiber-like tissue.Average wet weight and GAG content of specimens in Exp group were more than 88% of Ctrl 1 group while they were less than 40% in Ctrl 3 group. Cartilage lacuna was detected by HE staining in Exp, Ctrl 1 and Ctrl 3 group at different extent,but not in Ctrl 2 group. Collagen type Ⅱ was detected by immunohistochemistry in Exp, Ctrl 1 and Ctrl 3 group at different extent, but not in Ctrl 2 group. Conclusion Microtia chondrocytes could promote chondrogenesis of ADSCs in vivo under the co-culturing system. Tissue engineering cartilage by co-culturing microtia chondrocytes and ADSCs in vivo is feasible.%目的 验证残耳软骨细胞与脂肪来源的间充质干细胞(Adipose derived stem cells,ADSCs)共培养,体内构建软骨的可行性.方法 分离培养同一先天性

  7. Comparison of drug and cell-based delivery: engineered adult mesenchymal stem cells expressing soluble tumor necrosis factor receptor II prevent arthritis in mouse and rat animal models.

    Science.gov (United States)

    Liu, Linda N; Wang, Gang; Hendricks, Kyle; Lee, Keunmyoung; Bohnlein, Ernst; Junker, Uwe; Mosca, Joseph D

    2013-05-01

    Rheumatoid arthritis (RA) is a systemic autoimmune disease with unknown etiology where tumor necrosis factor-α (TNFα) plays a critical role. Etanercept, a recombinant fusion protein of human soluble tumor necrosis factor receptor II (hsTNFR) linked to the Fc portion of human IgG1, is used to treat RA based on the rationale that sTNFR binds TNFα and blocks TNFα-mediated inflammation. We compared hsTNFR protein delivery from genetically engineered human mesenchymal stem cells (hMSCs) with etanercept. Blocking TNFα-dependent intercellular adhesion molecule-1 expression on transduced hMSCs and inhibition of nitric oxide production from TNFα-treated bovine chondrocytes by conditioned culture media from transduced hMSCs demonstrated the functionality of the hsTNFR construction. Implanted hsTNFR-transduced mesenchymal stem cells (MSCs) reduced mouse serum circulating TNFα generated from either implanted TNFα-expressing cells or lipopolysaccharide induction more effectively than etanercept (TNFα, 100%; interleukin [IL]-1α, 90%; and IL-6, 60% within 6 hours), suggesting faster clearance of the soluble tumor necrosis factor receptor (sTNFR)-TNFα complex from the animals. In vivo efficacy of sTNFR-transduced MSCs was illustrated in two (immune-deficient and immune-competent) arthritic rodent models. In the antibody-induced arthritis BalbC/SCID mouse model, intramuscular injection of hsTNFR-transduced hMSCs reduced joint inflammation by 90% compared with untransduced hMSCs; in the collagen-induced arthritis Fischer rat model, both sTNFR-transduced rat MSCs and etanercept inhibited joint inflammation by 30%. In vitro chondrogenesis assays showed the ability of TNFα and IL1α, but not interferon γ, to inhibit hMSC differentiation to chondrocytes, illustrating an additional negative role for inflammatory cytokines in joint repair. The data support the utility of hMSCs as therapeutic gene delivery vehicles and their potential to be used in alleviating inflammation

  8. Optimization and translation of MSC-based hyaluronic acid hydrogels for cartilage repair

    Science.gov (United States)

    Erickson, Isaac E.

    2011-12-01

    Traumatic injury and disease disrupt the ability of cartilage to carry joint stresses and, without an innate regenerative response, often lead to degenerative changes towards the premature development of osteoarthritis. Surgical interventions have yet to restore long-term mechanical function. Towards this end, tissue engineering has been explored for the de novo formation of engineered cartilage as a biologic approach to cartilage repair. Research utilizing autologous chondrocytes has been promising, but clinical limitations in their yield have motivated research into the potential of mesenchymal stem cells (MSCs) as an alternative cell source. MSCs are multipotent cells that can differentiate towards a chondrocyte phenotype in a number of biomaterials, but no combination has successfully recapitulated the native mechanical function of healthy articular cartilage. The broad objective of this thesis was to establish an MSC-based tissue engineering approach worthy of clinical translation. Hydrogels are a common class of biomaterial used for cartilage tissue engineering and our initial work demonstrated the potential of a photo-polymerizable hyaluronic acid (HA) hydrogel to promote MSC chondrogenesis and improved construct maturation by optimizing macromer and MSC seeding density. The beneficial effects of dynamic compressive loading, high MSC density, and continuous mixing (orbital shaker) resulted in equilibrium modulus values over 1 MPa, well in range of native tissue. While compressive properties are crucial, clinical translation also demands that constructs stably integrate within a defect. We utilized a push-out testing modality to assess the in vitro integration of HA constructs within artificial cartilage defects. We established the necessity for in vitro pre-maturation of constructs before repair to achieve greater integration strength and compressive properties in situ. Combining high MSC density and gentle mixing resulted in integration strength over 500 k

  9. Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype.

    Science.gov (United States)

    Zhang, Qi; Lin, Shuxian; Liu, Ying; Yuan, Baozhi; Harris, Steph E; Feng, Jian Q

    2016-01-01

    Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis

  10. Hedgehog信号调控骨髓间充质干细胞成软骨细胞分化:调控方式及其串话机制尚待研究

    Institute of Scientific and Technical Information of China (English)

    刘宽; 吴兴

    2014-01-01

    背景:Hedgehog 信号通路在骨髓间充质干细胞成软骨细胞分化过程中发挥重要的调控作用,但具体的调控机制,以及与其他信号通路的串话机制仍需进一步的研究,是近来研究的热点。目的:介绍hedgehog信号在调控骨髓间充质干细胞成软骨分化过程中信号转导机制的研究现状与发展趋势,以及与其他信号通路的相互串话。方法:通过搜索CNKI,PubMed及Google Scholar等数据库,以“hedgehog,骨髓间充质干细胞,软骨形成,软骨细胞”和“hedgehog,IHH,SHH,bone marrow mesenchymal stem cel ,chondrogenesis, cartilage, chondrocyte”为检索词,查阅有关hedgehog信号与骨髓间充质干细胞分化相关的文献,最终共纳入36篇文献进行综述。结果与结论:骨髓间充质干细胞是目前公认的组织工程种子细胞来源,hedgehog信号通路是运动系统发育过程中重要的信号分子。Hedgehog信号蛋白IHH和SHH参与调控骨髓间充质干细胞的增殖与成软骨分化,以及软骨形成后的表型维持,且与其他信号通路发挥协同作用。然而,具体的调控方式以及与其他信号的串话机制,仍需进一步的研究,是这一领域未来研究的方向。

  11. Powerful bivariate genome-wide association analyses suggest the SOX6 gene influencing both obesity and osteoporosis phenotypes in males.

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    Yao-Zhong Liu

    Full Text Available BACKGROUND: Current genome-wide association studies (GWAS are normally implemented in a univariate framework and analyze different phenotypes in isolation. This univariate approach ignores the potential genetic correlation between important disease traits. Hence this approach is difficult to detect pleiotropic genes, which may exist for obesity and osteoporosis, two common diseases of major public health importance that are closely correlated genetically. PRINCIPAL FINDINGS: To identify such pleiotropic genes and the key mechanistic links between the two diseases, we here performed the first bivariate GWAS of obesity and osteoporosis. We searched for genes underlying co-variation of the obesity phenotype, body mass index (BMI, with the osteoporosis risk phenotype, hip bone mineral density (BMD, scanning approximately 380,000 SNPs in 1,000 unrelated homogeneous Caucasians, including 499 males and 501 females. We identified in the male subjects two SNPs in intron 1 of the SOX6 (SRY-box 6 gene, rs297325 and rs4756846, which were bivariately associated with both BMI and hip BMD, achieving p values of 6.82x10(-7 and 1.47x10(-6, respectively. The two SNPs ranked at the top in significance for bivariate association with BMI and hip BMD in the male subjects among all the approximately 380,000 SNPs examined genome-wide. The two SNPs were replicated in a Framingham Heart Study (FHS cohort containing 3,355 Caucasians (1,370 males and 1,985 females from 975 families. In the FHS male subjects, the two SNPs achieved p values of 0.03 and 0.02, respectively, for bivariate association with BMI and femoral neck BMD. Interestingly, SOX6 was previously found to be essential to both cartilage formation/chondrogenesis and obesity-related insulin resistance, suggesting the gene's dual role in both bone and fat. CONCLUSIONS: Our findings, together with the prior biological evidence, suggest the SOX6 gene's importance in co-regulation of obesity and osteoporosis.

  12. The effects of dynamic and three-dimensional environments on chondrogenic differentiation of bone marrow stromal cells

    Energy Technology Data Exchange (ETDEWEB)

    Jung, Youngmee; Kim, Sang-Heon; Kim, Soo Hyun [Biomaterials Research Center, Korea Institute of Science and Technology, PO Box 131, Cheonryang, Seoul, 130-650 (Korea, Republic of); Kim, Young Ha, E-mail: soohkim@kist.re.k [Department of Materials Science and Engineering, Gwangju Institute of Science and Technology, 261 Cheomdan-gwagiro, Buk-gu, Gwangju 500-712 (Korea, Republic of)

    2009-10-15

    Articular cartilage is subjected to complex loading, which plays a major role in its growth, development and maintenance. Previously, we found that mechanical stimuli enhanced the development and function of engineered cartilage tissues in elastic mechano-active poly(lactide-co-caprolactone) (PLCL) scaffolds. In addition, it is well known that the three-dimensional spatial organization of cells and extracellular matrices in hydrogels is crucial to chondrogenesis. This study was conducted to enhance the chondrogenic differentiation of bone marrow stromal cells (BMSCs) in the hybrid scaffolds of fibrin gels and PLCL scaffolds in dynamic environments by compression. A highly elastic scaffold was fabricated from very elastic PLCL with 85% porosity and a 300-500{mu}m pore size using a gel-pressing method. A mixture of rabbit BMSCs and fibrin gels was then seeded onto the PLCL scaffolds and subjected to continuous compressive deformation of 5% strain at 0.1 Hz for 10 days in a chondrogenic medium containing 10 ng ml{sup -1} TGF-beta{sub 1}. The BMSCs-seeded scaffold constructs were then implanted subcutaneously into nude mice. As a control, the cell-PLCL scaffold constructs were cultured under dynamic conditions or the cell-PLCL/fibrin hybrid scaffold constructs and the cell-PLCL scaffold constructs were cultured under static conditions for 10 days in vitro. The results revealed that cells adhered onto the hybrid scaffolds of fibrin gels and PLCL scaffolds cultured under dynamic conditions. In addition, the accumulation of the extracellular matrix of cell-scaffold constructs, which was increased through mechanical stimulation, showed that chondrogenic differentiation was sustained and enhanced significantly in the stimulated hybrid scaffold constructs. Overall, the results of this study indicate that the proper periodic application of dynamic compression and the three-dimensional environments of the hybrid scaffolds composed of fibrin gels and elastic PLCL can encourage

  13. Immortalisation with hTERT Impacts on Sulphated Glycosaminoglycan Secretion and Immunophenotype in a Variable and Cell Specific Manner.

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    Tina P Dale

    Full Text Available Limited options for the treatment of cartilage damage have driven the development of tissue engineered or cell therapy alternatives reliant on ex vivo cell expansion. The study of chondrogenesis in primary cells is difficult due to progressive cellular aging and senescence. Immortalisation via the reintroduction of the catalytic component of telomerase, hTERT, could allow repeated, longitudinal studies to be performed while bypassing senescent phenotypes.Three human cell types: bone marrow-derived stromal cells (BMA13, embryonic stem cell-derived (1C6 and chondrocytes (OK3 were transduced with hTERT (BMA13H, 1C6H and OK3H and proliferation, surface marker expression and tri-lineage differentiation capacity determined. The sulphated glycosaminoglycan (sGAG content of the monolayer and spent media was quantified in maintenance media (MM and pro-chondrogenic media (PChM and normalised to DNA.hTERT expression was confirmed in transduced cells with proliferation enhancement in 1C6H and OK3H cells but not BMA13H. All cells were negative for leukocyte markers (CD19, CD34, CD45 and CD73 positive. CD14 was expressed at low levels on OK3 and OK3H and HLA-DR on BMA13 (84.8%. CD90 was high for BMA13 (84.9% and OK3 (97.3% and moderate for 1C6 (56.7%, expression was reduced in BMA13H (33.7% and 1C6H (1.6%. CD105 levels varied (BMA13 87.7%, 1C6 8.2%, OK3 43.3% and underwent reduction in OK3H (25.1%. 1C6 and BMA13 demonstrated osteogenic and adipogenic differentiation but mineralised matrix and lipid accumulation appeared reduced post hTERT transduction. Chondrogenic differentiation resulted in increased monolayer-associated sGAG in all primary cells and 1C6H (p<0.001, and BMA13H (p<0.05. In contrast OK3H demonstrated reduced monolayer-associated sGAG in PChM (p<0.001. Media-associated sGAG accounted for ≥55% (PChM-1C6 and ≥74% (MM-1C6H.In conclusion, hTERT transduction could, but did not always, prevent senescence and cell phenotype, including

  14. A novel therapeutic approach with Caviunin-based isoflavonoid that en routes bone marrow cells to bone formation via BMP2/Wnt-β-catenin signaling.

    Science.gov (United States)

    Kushwaha, P; Khedgikar, V; Gautam, J; Dixit, P; Chillara, R; Verma, A; Thakur, R; Mishra, D P; Singh, D; Maurya, R; Chattopadhyay, N; Mishra, P R; Trivedi, R

    2014-09-18

    Recently, we reported that extract of Dalbergia sissoo made from leaves and pods have antiresorptive and bone-forming effects. The positive skeletal effect attributed because of active molecules present in the extract of Dalbergia sissoo. Caviunin 7-O-[β-D-apiofuranosyl-(1-6)-β-D-glucopyranoside] (CAFG), a novel isoflavonoid show higher percentage present in the extract. Here, we show the osteogenic potential of CAFG as an alternative for anabolic therapy for the treatment of osteoporosis by stimulating bone morphogenetic protein 2 (BMP2) and Wnt/β-catenin mechanism. CAFG supplementation improved trabecular micro-architecture of the long bones, increased biomechanical strength parameters of the vertebra and femur and decreased bone turnover markers better than genistein. Oral administration of CAFG to osteopenic ovariectomized mice increased osteoprogenitor cells in the bone marrow and increased the expression of osteogenic genes in femur and show new bone formation without uterine hyperplasia. CAFG increased mRNA expression of osteoprotegerin in bone and inhibited osteoclast activation by inhibiting the expression of skeletal osteoclastogenic genes. CAFG is also an effective accelerant for chondrogenesis and has stimulatory effect on the repair of cortical bone after drill-hole injury at the tissue, cell and gene level in mouse femur. At cellular levels, CAFG stimulated osteoblast proliferation, survival and differentiation. Signal transduction inhibitors in osteoblast demonstrated involvement of p-38 mitogen-activated protein kinase pathway stimulated by BMP2 to initiate Wnt/β-catenin signaling to reduce phosphorylation of GSK3-β and subsequent nuclear accumulation of β-catenin. Osteogenic effects were abrogated by Dkk1, Wnt-receptor blocker and FH535, inhibitor of TCF-complex by reduction in β-catenin levels. CAFG modulated MSC responsiveness to BMP2, which promoted osteoblast differentiation via Wnt/β-catenin mechanism. CAFG at 1 mg/kg(/)day dose in

  15. A balanced t(5;17 (p15;q22-23 in chondroblastoma: frequency of the re-arrangement and analysis of the candidate genes

    Directory of Open Access Journals (Sweden)

    Wijers-Koster Pauline

    2009-11-01

    originate from a distinct neoplastic clone, although the occurrence of two distinct clones is unlikely. Impairment of the CA10 gene might be pathogenetically relevant, as low expression was found in four cases. Diffuse expression of SRD5A1 and sex steroid signalling-related molecules confirms their role in neoplastic chondrogenesis.

  16. Effects of bound versus soluble pentosan polysulphate in PEG/HA-based hydrogels tailored for intervertebral disc regeneration.

    Science.gov (United States)

    Frith, Jessica E; Menzies, Donna J; Cameron, Andrew R; Ghosh, P; Whitehead, Darryl L; Gronthos, S; Zannettino, Andrew C W; Cooper-White, Justin J

    2014-01-01

    Previous reports in the literature investigating chondrogenesis in mesenchymal progenitor cell (MPC) cultures have confirmed the chondro-inductive potential of pentosan polysulphate (PPS), a highly sulphated semi-synthetic polysaccharide, when added as a soluble component to culture media under standard aggregate-assay conditions or to poly(ethylene glycol)/hyaluronic acid (PEG/HA)-based hydrogels, even in the absence of inductive factors (e.g. TGFβ). In this present study, we aimed to assess whether a 'bound' PPS would have greater activity and availability over a soluble PPS, as a media additive or when incorporated into PEG/HA-based hydrogels. We achieved this by covalently pre-binding the PPS to the HA component of the gel (forming a new molecule, HA-PPS). We firstly investigated the activity of HA-PPS compared to free PPS, when added as a soluble factor to culture media. Cell proliferation, as determined by CCK8 and EdU assay, was decreased in the presence of either bound or free PPS whilst chondrogenic differentiation, as determined by DMMB assay and histology, was enhanced. In all cases, the effect of the bound PPS (HA-PPS) was more potent than that of the unbound form. These results alone suggest wider applications for this new molecule, either as a culture supplement or as a coating for scaffolds targeted at chondrogenic differentiation or maturation. We then investigated the incorporation of HA-PPS into a PEG/HA-based hydrogel system, by simply substituting some of the HA for HA-PPS. Rheological testing confirmed that incorporation of either HA-PPS or PPS did not significantly affect gelation kinetics, final hydrogel modulus or degradation rate but had a small, but significant, effect on swelling. When encapsulated in the hydrogels, MPCs retained good viability and rapidly adopted a rounded morphology. Histological analysis of both GAG and collagen deposition after 21 days showed that the incorporation of the bound-PPS into the hydrogel resulted in

  17. Dmp1 Null Mice Develop a Unique Osteoarthritis-like Phenotype

    Science.gov (United States)

    Zhang, Qi; Lin, Shuxian; Liu, Ying; Yuan, Baozhi; Harris, Steph E; Feng, Jian Q.

    2016-01-01

    Patients with hypophosphatemia rickets (including DMP1 mutations) develop severe osteoarthritis (OA), although the mechanism is largely unknown. In this study, we first identified the expression of DMP1 in hypertrophic chondrocytes using immunohistochemistry (IHC) and X-gal analysis of Dmp1-knockout-lacZ-knockin heterozygous mice. Next, we characterized the OA-like phenotype in Dmp1 null mice from 7-week-old to one-year-old using multiple techniques, including X-ray, micro-CT, H&E staining, Goldner staining, scanning electronic microscopy, IHC assays, etc. We found a classical OA-like phenotype in Dmp1 null mice such as articular cartilage degradation, osteophyte formation, and subchondral osteosclerosis. These Dmp1 null mice also developed unique pathological changes, including a biphasic change in their articular cartilage from the initial expansion of hypertrophic chondrocytes at the age of 1-month to a quick diminished articular cartilage layer at the age of 3-months. Further, these null mice displayed severe enlarged knees and poorly formed bone with an expanded osteoid area. To address whether DMP1 plays a direct role in the articular cartilage, we deleted Dmp1 specifically in hypertrophic chondrocytes by crossing the Dmp1-loxP mice with Col X Cre mice. Interestingly, these conditional knockout mice didn't display notable defects in either the articular cartilage or the growth plate. Because of the hypophosphatemia remained in the entire life span of the Dmp1 null mice, we also investigated whether a high phosphate diet would improve the OA-like phenotype. A 8-week treatment of a high phosphate diet significantly rescued the OA-like defect in Dmp1 null mice, supporting the critical role of phosphate homeostasis in maintaining the healthy joint morphology and function. Taken together, this study demonstrates a unique OA-like phenotype in Dmp1 null mice, but a lack of the direct impact of DMP1 on chondrogenesis. Instead, the regulation of phosphate homeostasis

  18. Endochondral ossification for enhancing bone regeneration: converging native extracellular matrix biomaterials and developmental engineering in vivo.

    Science.gov (United States)

    Dennis, S Connor; Berkland, Cory J; Bonewald, Lynda F; Detamore, Michael S

    2015-06-01

    Autologous bone grafting (ABG) remains entrenched as the gold standard of treatment in bone regenerative surgery. Consequently, many marginally successful bone tissue engineering strategies have focused on mimicking portions of ABG's "ideal" osteoconductive, osteoinductive, and osteogenic composition resembling the late reparative stage extracellular matrix (ECM) in bone fracture repair, also known as the "hard" or "bony" callus. An alternative, less common approach that has emerged in the last decade harnesses endochondral (EC) ossification through developmental engineering principles, which acknowledges that the molecular and cellular mechanisms involved in developmental skeletogenesis, specifically EC ossification, are closely paralleled during native bone healing. EC ossification naturally occurs during the majority of bone fractures and, thus, can potentially be utilized to enhance bone regeneration for nearly any orthopedic indication, especially in avascular critical-sized defects where hypoxic conditions favor initial chondrogenesis instead of direct intramembranous ossification. The body's native EC ossification response, however, is not capable of regenerating critical-sized defects without intervention. We propose that an underexplored potential exists to regenerate bone through the native EC ossification response by utilizing strategies which mimic the initial inflammatory or fibrocartilaginous ECM (i.e., "pro-" or "soft" callus) observed in the early reparative stage of bone fracture repair. To date, the majority of strategies utilizing this approach rely on clinically burdensome in vitro cell expansion protocols. This review will focus on the confluence of two evolving areas, (1) native ECM biomaterials and (2) developmental engineering, which will attempt to overcome the technical, business, and regulatory challenges that persist in the area of bone regeneration. Significant attention will be given to native "raw" materials and ECM-based designs that

  19. BMP-2 functions independently of SHH signaling and triggers cell condensation and apoptosis in regenerating axolotl limbs

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    Finnson Kenneth

    2010-02-01

    Full Text Available Abstract Background Axolotls have the unique ability, among vertebrates, to perfectly regenerate complex body parts, such as limbs, after amputation. In addition, axolotls pattern developing and regenerating autopods from the anterior to posterior axis instead of posterior to anterior like all tetrapods studied to date. Sonic hedgehog is important in establishing this anterior-posterior axis of limbs in all tetrapods including axolotls. Interestingly, its expression is conserved (to the posterior side of limb buds and blastemas in axolotl limbs as in other tetrapods. It has been suggested that BMP-2 may be the secondary mediator of sonic hedgehog, although there is mounting evidence to the contrary in mice. Since BMP-2 expression is on the anterior portion of developing and regenerating limbs prior to digit patterning, opposite to the expression of sonic hedgehog, we examined whether BMP-2 expression was dependent on sonic hedgehog signaling and whether it affects patterning of the autopod during regeneration. Results The expression of BMP-2 and SOX-9 in developing and regenerating axolotl limbs corresponded to the first digits forming in the anterior portion of the autopods. The inhibition of sonic hedgehog signaling with cyclopamine caused hypomorphic limbs (during development and regeneration but did not affect the expression of BMP-2 and SOX-9. Overexpression of BMP-2 in regenerating limbs caused a loss of digits. Overexpression of Noggin (BMP inhibitor in regenerating limbs also resulted in a loss of digits. Histological analysis indicated that the loss due to BMP-2 overexpression was the result of increased cell condensation and apoptosis while the loss caused by Noggin was due to a decrease in cell division. Conclusion The expression of BMP-2 and its target SOX-9 was independent of sonic hedgehog signaling in developing and regenerating limbs. Their expression correlated with chondrogenesis and the appearance of skeletal elements has

  20. Expression of transmembrane carbonic anhydrases, CAIX and CAXII, in human development

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    Lerman Michael I

    2009-03-01

    Full Text Available Abstract Background Transmembrane CAIX and CAXII are members of the alpha carbonic anhydrase (CA family. They play a crucial role in differentiation, proliferation, and pH regulation. Expression of CAIX and CAXII proteins in tumor tissues is primarily induced by hypoxia and this is particularly true for CAIX, which is regulated by the transcription factor, hypoxia inducible factor-1 (HIF-1. Their distributions in normal adult human tissues are restricted to highly specialized cells that are not always hypoxic. The human fetus exists in a relatively hypoxic environment. We examined expression of CAIX, CAXII and HIF-1α in the developing human fetus and postnatal tissues to determine whether expression of CAIX and CAXII is exclusively regulated by HIF-1. Results The co-localization of CAIX and HIF-1α was limited to certain cell types in embryonic and early fetal tissues. Those cells comprised the primitive mesenchyma or involved chondrogenesis and skin development. Transient CAIX expression was limited to immature tissues of mesodermal origin and the skin and ependymal cells. The only tissues that persistently expressed CAIX protein were coelomic epithelium (mesothelium and its remnants, the epithelium of the stomach and biliary tree, glands and crypt cells of duodenum and small intestine, and the cells located at those sites previously identified as harboring adult stem cells in, for example, the skin and large intestine. In many instances co-localization of CAIX and HIF-1α was not evident. CAXII expression is restricted to cells involved in secretion and water absorption such as parietal cells of the stomach, acinar cells of the salivary glands and pancreas, epithelium of the large intestine, and renal tubules. Co-localization of CAXII with CAIX or HIF-1α was not observed. Conclusion The study has showed that: 1 HIF-1α and CAIX expression co- localized in many, but not all, of the embryonic and early fetal tissues; 2 There is no evidence of

  1. Direct lentiviral-cyclooxygenase 2 application to the tendon-bone interface promotes osteointegration and enhances return of the pull-out tensile strength of the tendon graft in a rat model of biceps tenodesis.

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    Charles H Rundle

    Full Text Available This study sought to determine if direct application of the lentiviral (LV-cyclooxygenase 2 (COX2 vector to the tendon-bone interface would promote osteointegration of the tendon graft in a rat model of biceps tenodesis. The LV-COX2 gene transfer strategy was chosen for investigation because a similar COX2 gene transfer strategy promoted bony bridging of the fracture gap during bone repair, which involves similar histologic transitions that occur in osteointegration. Briefly, a 1.14-mm diameter tunnel was drilled in the mid-groove of the humerus of adult Fischer 344 rats. The LV-COX2 or βgal control vector was applied directly into the bone tunnel and onto the end of the tendon graft, which was then pulled into the bone tunnel. A poly-L-lactide pin was press-fitted into the tunnel as interference fixation. Animals were sacrificed at 3, 5, or 8 weeks for histology analysis of osteointegration. The LV-COX2 gene transfer strategy enhanced neo-chondrogenesis at the tendon-bone interface but with only marginal effect on de novo bone formation. The tendon-bone interface of the LV-COX2-treated tenodesis showed the well-defined tendon-to-fibrocartilage-to-bone histologic transitions that are indicative of osteointegration of the tendon graft. The LV-COX2 in vivo gene transfer strategy also significantly enhanced angiogenesis at the tendon-bone interface. To determine if the increased osteointegration was translated into an improved pull-out mechanical strength property, the pull-out tensile strength of the LV-COX2-treated tendon grafts was determined with a pull-out mechanical testing assay. The LV-COX2 strategy yielded a significant improvement in the return of the pull-out strength of the tendon graft after 8 weeks. In conclusion, the COX2-based in vivo gene transfer strategy enhanced angiogenesis, osteointegration and improved return of the pull-out strength of the tendon graft. Thus, this strategy has great potential to be developed into an

  2. Macro- and micro-designed chitosan-alginate scaffold architecture by three-dimensional printing and directional freezing.

    Science.gov (United States)

    Reed, Stephanie; Lau, Grace; Delattre, Benjamin; Lopez, David Don; Tomsia, Antoni P; Wu, Benjamin M

    2016-01-07

    , while achieving porous zones that mimic articular cartilage zonal architecture. In future applications, precisely controlled micro- and macro-channels have the potential to assist immediate endogenous bone marrow uptake, stimulate chondrogenesis, and encourage vascularization of bone in an osteochondral scaffold.

  3. Cartilage Derived from Bone Marrow Mesenchymal Stem Cells Expresses Lubricin In Vitro and In Vivo.

    Directory of Open Access Journals (Sweden)

    Yusuke Nakagawa

    Full Text Available Lubricin expression in the superficial cartilage will be a crucial factor in the success of cartilage regeneration. Mesenchymal stem cells (MSCs are an attractive cell source and the use of aggregates of MSCs has some advantages in terms of chondrogenic potential and efficiency of cell adhesion. Lubricin expression in transplanted MSCs has not been fully elucidated so far. Our goals were to determine (1 whether cartilage pellets of human MSCs expressed lubricin in vitro chondrogenesis, (2 whether aggregates of human MSCs promoted lubricin expression, and (3 whether aggregates of MSCs expressed lubricin in the superficial cartilage after transplantation into osteochondral defects in rats.For in vitro analysis, human bone marrow (BM MSCs were differentiated into cartilage by pellet culture, and also aggregated using the hanging drop technique. For an animal study, aggregates of BM MSCs derived from GFP transgenic rats were transplanted to the osteochondral defect in the trochlear groove of wild type rat knee joints. Lubricin expression was mainly evaluated in differentiated and regenerated cartilages.In in vitro analysis, lubricin was detected in the superficial zone of the pellets and conditioned medium. mRNA expression of Proteoglycan4 (Prg4, which encodes lubricin, in pellets was significantly higher than that of undifferentiated MSCs. Aggregates showed different morphological features between the superficial and deep zone, and the Prg4 mRNA expression increased after aggregate formation. Lubricin was also found in the aggregate. In a rat study, articular cartilage regeneration was significantly better in the MSC group than in the control group as shown by macroscopical and histological analysis. The transmission electron microscope showed that morphology of the superficial cartilage in the MSC group was closer to that of the intact cartilage than in the control group. GFP positive cells remained in the repaired tissue and expressed lubricin in

  4. Gene expression pattern of functional neuronal cells derived from human bone marrow mesenchymal stromal cells

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    Bron Dominique

    2008-04-01

    Full Text Available Abstract Background Neuronal tissue has limited potential to self-renew or repair after neurological diseases. Cellular therapies using stem cells are promising approaches for the treatment of neurological diseases. However, the clinical use of embryonic stem cells or foetal tissues is limited by ethical considerations and other scientific problems. Thus, bone marrow mesenchymal stomal cells (BM-MSC could represent an alternative source of stem cells for cell replacement therapies. Indeed, many studies have demonstrated that MSC can give rise to neuronal cells as well as many tissue-specific cell phenotypes. Methods BM-MSC were differentiated in neuron-like cells under specific induction (NPBM + cAMP + IBMX + NGF + Insulin. By day ten, differentiated cells presented an expression profile of real neurons. Functionality of these differentiated cells was evaluated by calcium influx through glutamate receptor AMPA3. Results Using microarray analysis, we compared gene expression profile of these different samples, before and after neurogenic differentiation. Among the 1943 genes differentially expressed, genes down-regulated are involved in osteogenesis, chondrogenesis, adipogenesis, myogenesis and extracellular matrix component (tuftelin, AGC1, FADS3, tropomyosin, fibronectin, ECM2, HAPLN1, vimentin. Interestingly, genes implicated in neurogenesis are increased. Most of them are involved in the synaptic transmission and long term potentialisation as cortactin, CASK, SYNCRIP, SYNTL4 and STX1. Other genes are involved in neurite outgrowth, early neuronal cell development, neuropeptide signaling/synthesis and neuronal receptor (FK506, ARHGAP6, CDKRAP2, PMCH, GFPT2, GRIA3, MCT6, BDNF, PENK, amphiregulin, neurofilament 3, Epha4, synaptotagmin. Using real time RT-PCR, we confirmed the expression of selected neuronal genes: NEGR1, GRIA3 (AMPA3, NEF3, PENK and Epha4. Functionality of these neuron-like cells was demonstrated by Ca2+ influx through glutamate

  5. Periosteal BMP2 activity drives bone graft healing.

    Science.gov (United States)

    Chappuis, Vivianne; Gamer, Laura; Cox, Karen; Lowery, Jonathan W; Bosshardt, Dieter D; Rosen, Vicki

    2012-10-01

    Bone graft incorporation depends on the orchestrated activation of numerous growth factors and cytokines in both the host and the graft. Prominent in this signaling cascade is BMP2. Although BMP2 is dispensable for bone formation, it is required for the initiation of bone repair; thus understanding the cellular mechanisms underlying bone regeneration driven by BMP2 is essential for improving bone graft therapies. In the present study, we assessed the role of Bmp2 in bone graft incorporation using mice in which Bmp2 has been removed from the limb prior to skeletal formation (Bmp2(cKO)). When autograft transplantations were performed in Bmp2cKO mice, callus formation and bone healing were absent. Transplantation of either a vital wild type (WT) bone graft into a Bmp2(cKO) host or a vital Bmp2(cKO) graft into a WT host also resulted in the inhibition of bone graft incorporation. Histological analyses of these transplants show that in the absence of BMP2, periosteal progenitors remain quiescent and healing is not initiated. When we analyzed the expression of Sox9, a marker of chondrogenesis, on the graft surface, we found it significantly reduced when BMP2 was absent in either the graft itself or the host, suggesting that local BMP2 levels drive periosteal cell condensation and subsequent callus cell differentiation. The lack of integrated healing in the absence of BMP2 was not due to the inability of periosteal cells to respond to BMP2. Healing was achieved when grafts were pre-soaked in rhBMP2 protein, indicating that periosteal progenitors remain responsive in the absence of BMP2. In contrast to the requirement for BMP2 in periosteal progenitor activation in vital bone grafts, we found that bone matrix-derived BMP2 does not significantly enhance bone graft incorporation. Taken together, our data show that BMP2 signaling is not essential for the maintenance of periosteal progenitors, but is required for the activation of these progenitors and their subsequent

  6. Evidence for sequential appearance of cartilage matrix proteins in developing mouse limbs and in cultures of mouse mesenchymal cells.

    Science.gov (United States)

    Franzen, A; Heinegard, D; Solursh, M

    1987-01-01

    The initiation of synthesis and the accumulation of four cartilage matrix proteins (type II collagen and three noncollagenous proteins, one of Mr 148, one of Mr 59, and an oligometric protein of Mr above 500 with 100-kDa subunits, respectively) were studied in developing mouse limbs and in cultures of limb bud mesenchyme by means of immunolocalization. On day 13 of gestation, type II collagen was observed throughout the entire humerus, whereas the 148-kDa protein was localized only in the central portion. Neither the 100-kDa-subunit protein nor the 59-kDa protein could be demonstrated in the humerus at that stage. On day 14 1/2, type II collagen and the 148-kDa protein were codistributed throughout the humerus. The 100-kDa-subunit protein was detectable in the periphery of the humerus, whereas little 59-kDa protein could yet be demonstrated. On day 18, all four proteins being studied could be detected immunologically in the developing mouse humerus. They differed in immunolocalization. Type Ii collagen, the 148-kDa protein, and the 100-kDa-subunit protein were codistributed throughout the distal and proximal parts of the cartilage. However, the 148-kDa protein could no longer be detected immunochemically in the outermost part of the cartilage in the proximal shoulder joint. The 148-kDa protein codistributed with type II collagen and the 100-kDa-subunit protein in the distal cartilaginous region, where joint development was less advanced. On the other hand, the 59-kDa protein was not demonstrated directly within the hyaline cartilaginous structures, but surrounded the entire structure. This protein was also present in the same part of the proximal joint region as that in which the 148-kDa protein was not detected. To develop an in vitro model for studies of skeletogenesis, mesenchymal cells prepared from mouse limb buds were cultured as micromass cultures at high initial cell density to favor chondrogenesis. On day 3 of culture, type II collagen was the only protein

  7. SOX9 governs differentiation stage-specific gene expression in growth plate chondrocytes via direct concomitant transactivation and repression.

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    Victor Y L Leung

    2011-11-01

    Full Text Available Cartilage and endochondral bone development require SOX9 activity to regulate chondrogenesis, chondrocyte proliferation, and transition to a non-mitotic hypertrophic state. The restricted and reciprocal expression of the collagen X gene, Col10a1, in hypertrophic chondrocytes and Sox9 in immature chondrocytes epitomise the precise spatiotemporal control of gene expression as chondrocytes progress through phases of differentiation, but how this is achieved is not clear. Here, we have identified a regulatory element upstream of Col10a1 that enhances its expression in hypertrophic chondrocytes in vivo. In immature chondrocytes, where Col10a1 is not expressed, SOX9 interacts with a conserved sequence within this element that is analogous to that within the intronic enhancer of the collagen II gene Col2a1, the known transactivation target of SOX9. By analysing a series of Col10a1 reporter genes in transgenic mice, we show that the SOX9 binding consensus in this element is required to repress expression of the transgene in non-hypertrophic chondrocytes. Forced ectopic Sox9 expression in hypertrophic chondrocytes in vitro and in mice resulted in down-regulation of Col10a1. Mutation of a binding consensus motif for GLI transcription factors, which are the effectors of Indian hedgehog signaling, close to the SOX9 site in the Col10a1 regulatory element, also derepressed transgene expression in non-hypertrophic chondrocytes. GLI2 and GLI3 bound to the Col10a1 regulatory element but not to the enhancer of Col2a1. In addition to Col10a1, paired SOX9-GLI binding motifs are present in the conserved non-coding regions of several genes that are preferentially expressed in hypertrophic chondrocytes and the occurrence of pairing is unlikely to be by chance. We propose a regulatory paradigm whereby direct concomitant positive and negative transcriptional control by SOX9 ensures differentiation phase-specific gene expression in chondrocytes. Discrimination between

  8. MR imaging features of gadofluorine-labeled matrix-associated stem cell implants in cartilage defects.

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    Hossein Nejadnik

    Full Text Available OBJECTIVES: The purpose of our study was to assess the chondrogenic potential and the MR signal effects of GadofluorineM-Cy labeled matrix associated stem cell implants (MASI in pig knee specimen. MATERIALS AND METHODS: Human mesenchymal stem cells (hMSCs were labeled with the micelle-based contrast agent GadofluorineM-Cy. Ferucarbotran-labeled hMSCs, non-labeled hMSCs and scaffold only served as controls. Chondrogenic differentiation was induced and gene expression and histologic evaluation were performed. The proportions of spindle-shaped vs. round cells of chondrogenic pellets were compared between experimental groups using the Fisher's exact test. Labeled and unlabeled hMSCs and chondrocytes in scaffolds were implanted into cartilage defects of porcine femoral condyles and underwent MR imaging with T1- and T2-weighted SE and GE sequences. Contrast-to-noise ratios (CNR between implants and adjacent cartilage were determined and analyzed for significant differences between different experimental groups using the Kruskal-Wallis test. Significance was assigned for p0.017. However, hMSC differentiation into chondrocytes was superior for unlabeled and GadofluorineM-Cy-labeled cells compared with Ferucarbotran-labeled cells, as evidenced by a significantly higher proportion of spindle cells in chondrogenic pellets (p<0.05. GadofluorineM-Cy-labeled hMSCs and chondrocytes showed a positive signal effect on T1-weighted images and a negative signal effect on T2-weighted images while Ferucarbotran-labeled cells provided a negative signal effect on all sequences. CNR data for both GadofluorineM-Cy-labeled and Ferucarbotran-labeled hMSCs were significantly different compared to unlabeled control cells on T1-weighted SE and T2*-weighted MR images (p<0.017. CONCLUSION: hMSCs can be labeled by simple incubation with GadofluorineM-Cy. The labeled cells provide significant MR signal effects and less impaired chondrogenesis compared to Ferucarbotran-labeled h

  9. Differentiation of cartilaginous anlage in entire embryonic mouse limbs cultured in a rotating bioreactor.

    Science.gov (United States)

    Duke, P.; Oakley, C.; Montufar-Solis, D.

    The embryonic mammalian limb is sensitive both in vivo and in vitro to changes in gravitational force. Hypergravity of centrifugation and microgravity of space decreased size of elements due to precocious or delayed chondrogenesis respectively. In recapitulating spaceflight experiments, premetatarsals were cultured in suspension in a low stress, low sheer rotating bioreactor, and found to be shorter than those cultured in standard culture dishes, and cartilage development was delayed. This study only measured length of the metatarsals, and did not account for possible changes in width and/or in form of the skeletal elements. Shorter cartilage elements in limbbuds cultured in the bioreactor may be due to the ability of the system to reproduce a more in vivo 3D shape than traditional organ cultures. Tissues subjected to traditional organ cultures become flattened by their own weight, attachment to the filter, and restrictions imposed by nutrient diffusion. The purpose of the current experiment was to determine if entire limb buds could be successfully cultured in the bioreactor, and to compare the effects on 3D shape with that of culturing in a culture dish system. Fore and hind limbs from E11-E13 ICR mouse embryos were placed either in the bioreactor, in Trowell culture, or fixed as controls. Limbbuds were cultured for six days, fixed, and processed either as whole mounts or embedded for histology. Qualitative analysis revealed that the Trowell culture specimens were flattened, while bioreactor culture specimens had a more in vivo-like 3D limb shape. Sections of limbbuds from both types of cultures had excellent cartilage differentiation, with apparently more cell maturation, and hypertrophy in the specimens cultured in the bioreactor. Morphometric quantitation of the cartilaginous elements for comparisons of the two culture systems was complicated due to some limb buds fusing together during culture. This problem was especially noticeable in the younger limbs, and

  10. Differential properties of human ACL and MCL stem cells may be responsible for their differential healing capacity

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    Fu Freddie H

    2011-06-01

    Full Text Available Abstract Background The human anterior cruciate ligament (hACL and medial collateral ligament (hMCL of the knee joint are frequently injured, especially in athletic settings. It has been known that, while injuries to the MCL typically heal with conservative treatment, ACL injuries usually do not heal. As adult stem cells repair injured tissues through proliferation and differentiation, we hypothesized that the hACL and hMCL contain stem cells exhibiting unique properties that could be responsible for the differential healing capacity of the two ligaments. Methods To test the above hypothesis, we derived ligament stem cells from normal hACL and hMCL samples from the same adult donors using tissue culture techniques and characterized their properties using immunocytochemistry, RT-PCR, and flow cytometry. Results We found that both hACL stem cells (hACL-SCs and hMCL stem cells (hMCL-SCs formed colonies in culture and expressed stem cell markers nucleostemin and stage-specific embryonic antigen-4 (SSEA-4. Moreover, both hACL-SCs and hMCL-SCs expressed CD surface markers for mesenchymal stem cells, including CD44 and CD90, but not those markers for vascular cells, CD31, CD34, CD45, and CD146. However, hACL-SCs differed from hMCL-SCs in that the size and number of hACL-SC colonies in culture were much smaller and grew more slowly than hMCL-SC colonies. Moreover, fewer hACL-SCs in cell colonies expressed stem cell markers STRO-1 and octamer-binding transcription factor-4 (Oct-4 than hMCL-SCs. Finally, hACL-SCs had less multi-differentiation potential than hMCL-SCs, evidenced by differing extents of adipogenesis, chondrogenesis, and osteogenesis in the respective induction media. Conclusions This study shows for the first time that hACL-SCs are intrinsically different from hMCL-SCs. We suggest that the differences in their properties contribute to the known disparity in healing capabilities between the two ligaments.

  11. Chondrogenic potential of rat mesenchymal stem cells in vitro under the influence of electromagnetic fields%低频电磁场干预大鼠骨髓间充质干细胞体外成软骨分化的研究

    Institute of Scientific and Technical Information of China (English)

    虞冀哲; 杨勇; 刘朝旭; 宋明宇; 刘阳; 吴华

    2014-01-01

    目的 探讨在体外条件下低频电磁场诱导大鼠骨髓间充质干细胞向软骨细胞分化的可行性.方法 大鼠骨髓间充质于细胞传代至第5代后,采用微团培养法在含成纤维生长因子-2和转化生长因子-β3的培养基中生长,并给予正弦波电磁场(1.0 mT,50 Hz)干预.3周后进行阿利新蓝染色以检测软骨基质生成量,采用荧光定量聚合酶链反应(FQ-PCR)技术检测软骨特异性蛋白的基因表达水平,并使用二甲基-亚甲蓝(DMMB)染料结合法评估细胞微团中糖胺多糖水平.结果 在电磁场和生长因子的协同作用下,大鼠骨髓间充质干细胞(BMSCs)细胞微团向软骨分化,细胞微团中Ⅱ型、X型胶原及蛋白多糖基因表达水平显著增加,而性别决定区Y框蛋白9(SOX9)的表达没有明显变化.与非暴磁组比较,暴磁组细胞糖胺多糖(GAG)/DNA比例较高(3.108±0.341).结论 电磁场促进大鼠BMSCs成软骨分化可能与细胞表达Ⅱ、X型胶原及糖胺多糖增多有关.电磁场在生长因子的协同作用下可诱导及维持间充质干细胞成软骨分化,但单因素电磁场刺激不能诱导大鼠骨髓间充质干细胞成软骨分化.%Objective To determine the chondrogenic potential of mesenchymal stem cells (BMSCs) under exposure of extremely low frequency electromagnetic fields (ELF-EMF) and discuss the application prospect of EMF in cartilage tissue engineering.Methods Cell pellets of rat bone marrow-derived BMSCs were cultured in vitro under the addition of fibroblast growth factor 2 (FGF-2) and transforming growth factor-β3 (TGF-β3).Pellet cultures were exposed to sinusoidal ELF-EMF (1.0 mT,50 Hz).After 3 weeks of inductive differentiation culture,chondrogenesis was detected by alcian blue staining,and quantitative real-time polymerase chain reaction (real-time PCR) was used for detection of chondrogenesis related proteins.Glycosaminoglycan (GAG) contents of pellet cultures were determined using the

  12. Biodegradable chitosan scaffolds containing microspheres as carriers for controlled transforming growth factor-β1 delivery for cartilage tissue engineering

    Institute of Scientific and Technical Information of China (English)

    CAI Dao-zhang; ZENG Chun; QUAN Da-ping; BU Li-si; WANG Kun; LU Hua-ding; LI Xiao-feng

    2007-01-01

    Background Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-β1 (TGF-β1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-β1 delivery and the effect of released TGF-β1 on the chondrogenic potential of chondrocytes.Methods Chitosan scaffolds and chitosan microspheres loaded with TGF-β1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-β1 microspheres and incubated in vitro for 3 weeks. Histological examination and type Ⅱ collagen immunohistochemical staining was performed to evaluate the effects of released TGF-β1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.Results TGF-β1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-β1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-β1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-β1 was 37.4%, 50.7%,61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type Ⅱ collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type Ⅱ in control group (32.7%± 10.4%) was significantly lower than that in the controlled

  13. 脂肪组织来源干细胞的分化潜能和应用%DIFFERENTIATION POTENTIAL AND APPLICATION OF STEM CELLS FROM ADIPOSE TISSUE

    Institute of Scientific and Technical Information of China (English)

    史琳丽; 杨向群

    2012-01-01

    目的 介绍脂肪组织来源的干细胞种类、分化潜能及在再生医学中的应用和优势. 方法 广泛查阅近年关于BMSCs、脂肪来源干细胞(adipose-derived stem cells,ADSCs)和去分化脂肪(dedifferentiated fat,DFAT)细胞的实验研究及临床研究文献,并进行整理、综合与分析. 结果 从脂肪基质成分可以分离得到ADSCs,ADSCs具有多向分化潜能,可分化为脂肪、骨、软骨、内皮、肌以及神经细胞等,并已较成功地应用于再生医学各领域.成熟脂肪细胞经过天花板培养法可以去分化为成纤维样细胞,即DFAT细胞,获得了多向分化潜能,也可以像ADSCs一样分化为脂肪、骨、软骨、内皮、肌以及神经细胞等.相比较目前常用的成体干细胞BMSCs,ADSCs、DFAT细胞来源广泛、取材更容易;而相对于ADSCs,DFAT细胞均一性高,增殖能力强. 结论 脂肪组织作为人体干细胞的重要来源,可能会给临床组织缺损的修复与再生带来新希望.%Objective To introduce types and differentiation potentials of stem cells from adipose tissue, and its applications on regenerative medicine and advantages. Methods The literature of original experimental study and clinical research about bone marrow mesenchymal stem cells (BMSCs), adipose-derived stem cells (ADSCs), and dedifferentiated fat (DFAT) cells was extensively reviewed and analyzed. Results ADSCs can be isolated from stromal vascular fraction. As ADSCs have multi-lineage potentials, such as adipogenesis, osteogenesis, chondrogenesis, angiogenesis, myogenesis, and neurogenesis, they have already been successfully used in regenerative medicine areas. Dramatically, mature fat cells can be dedifferentiated and changed into fibroblast-like cells, named DFAT cells, via ceiling culture method. DFAT cells also had the same multi-lineage potentials as ADSCs, differentiating into adipocytes, osteocytes, chondrocytes, endothelial cells, muscle cells, and nerve cells. Compared

  14. Effect of self-assembled peptide–mesenchymal stem cell complex on the progression of osteoarthritis in a rat model

    Directory of Open Access Journals (Sweden)

    Kim JE

    2014-05-01

    metalloproteinases 1, and matrix metalloproteinase 9 were diffusely stained in controls, whereas localized or minimal staining was observed in other groups. Modified Mankin scores were significantly lower in the SAP and SAP-MSC groups than in controls (P=0.001 and 0.013. Although not statistically significant, synovial inflammation scores were lower in the SAP (1.3±0.3 and SAP-MSC (1.3±0.2 groups than in controls (2.6±0.2. However, neither the cytokine level nor the behavioral score was significantly different between groups.Conclusion: Injection of SAP-MSC hydrogels showed evidence of chondroprotection, as measured by the histologic grading and decreased expression of biochemical markers of inflammation and apoptosis. It also lowered subchondral bone mineral density, which can be increased by OA. This suggests that the SAP-MSC complex may have clinical potential to inhibit OA progression.Keywords: self-assembled peptide, mesenchymal stem cell, osteoarthritis, apoptosis, chondrogenesis

  15. Collagen complex gradient tricalcium phosphate composite scaffold with chondrocytes cultured in vitro%胶原复合梯度磷酸三钙负载软骨细胞的体外培养

    Institute of Scientific and Technical Information of China (English)

    查国庆; 廖威明; 黄保丁; 吴刚

    2009-01-01

    Objective To evaluate the feasibility of collagen complex gradient tricalcium phosphate composite scaffolds for cartilage tissue engineering by observing the compatibility and adhesion of chondrocytes. Methods High purified chondrocytes from knee joints of 8-week-old New Zealand rabbits were obtained by enzyme digestion, then the chondrocytes of the third generation were cultured combined with collagen complex gradient tricalcium phosphate composite scaffolds in vitro.Chondrocyte morphology, collagen Ⅱ expression, chondrogenesis, the compatibility of chondroeyte and scaffolds were evaluated with inverted microscope, HE staining, scanning electron microscope (SEM) and immunohistochemistry.Results By SEM, the scaffold had regular, porous architecture of communicated microholes.The average pore diameter was 100-150 μm.Observation revealed good hydrophilicity of the composite scaffold.The chondrocytes adhered to the surface, proliferated and migrated into the inside of the scaffold.Chondrocytes attached to wall of microholes of the scaffold had largely maintained morphology and could secrete the extracellular matrix on the porous scaffold.Conclusions Collagen complex gradient tricalcium phosphate composite scaffold possesses good cellular compatibility.%目的 观察新型三维支架材料胶原复合梯度磷酸三钙在体外与软骨细胞的相容性和黏附性,评价其作为软骨组织工程支架的可行性.方法 取8周龄新两兰大白兔膝关节软骨,以酶消化法获得高纯度软骨细胞,培养3代后与三维支架材料胶原复合梯度磷酸三钙在体外复合培养.用倒置相差显微镜、HE染色、免疫组织化学及扫描电镜观察软骨细胞形态、Ⅱ型胶原表达及成软骨能力,同时观察支架材料与软骨细胞的相容性.结果 扫描电镜观察显示支架材料具有疏松多孔结构,孔隙结构规则,孔径100~150 μm,材料内部孔与孔之间贯通良好.支架亲水性好.软骨细胞吸附于支

  16. Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair.

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    Pengzhen Wang

    Full Text Available Articular cartilage has poor capability for repair following trauma or degenerative pathology due to avascular property, low cell density and migratory ability. Discovery of novel therapeutic approaches for articular cartilage repair remains a significant clinical need. Hypoxia is a hallmark for cartilage development and pathology. Hypoxia inducible factor-1alpha (HIF-1α has been identified as a key mediator for chondrocytes to response to fluctuations of oxygen availability during cartilage development or repair. This suggests that HIF-1α may serve as a target for modulating chondrocyte functions. In this study, using phenotypic cellular screen assays, we identify that Icariin, an active flavonoid component from Herba Epimedii, activates HIF-1α expression in chondrocytes. We performed systemic in vitro and in vivo analysis to determine the roles of Icariin in regulation of chondrogenesis. Our results show that Icariin significantly increases hypoxia responsive element luciferase reporter activity, which is accompanied by increased accumulation and nuclear translocation of HIF-1α in murine chondrocytes. The phenotype is associated with inhibiting PHD activity through interaction between Icariin and iron ions. The upregulation of HIF-1α mRNA levels in chondrocytes persists during chondrogenic differentiation for 7 and 14 days. Icariin (10-6 M increases the proliferation of chondrocytes or chondroprogenitors examined by MTT, BrdU incorporation or colony formation assays. Icariin enhances chondrogenic marker expression in a micromass culture including Sox9, collagen type 2 (Col2α1 and aggrecan as determined by real-time PCR and promotes extracellular matrix (ECM synthesis indicated by Alcian blue staining. ELISA assays show dramatically increased production of aggrecan and hydroxyproline in Icariin-treated cultures at day 14 of chondrogenic differentiation as compared with the controls. Meanwhile, the expression of chondrocyte catabolic

  17. Effects of Ionizing Radiation on Murine Gene Expression in Skin and Bone

    Science.gov (United States)

    Terada, Masahiro; Schreurs, Ann-Sofie; Shirazi-Fard, Yasaman; Alwood, Joshua; Tahimic, Candice; Sowa, Marianne B.; Globus, Ruth K.

    2017-01-01

    Long duration spaceflight causes a negative calcium balance and reduces bone density in astronauts. The potential for exposure to space radiation to contribute to lasting decrements in bone mass is not yet understood. Sustained changes to bone mass have a relatively long latency for development, however skin is a radiation sensitive organ and changes in skin gene expression may serve as an early radiation biomarker of exposures and may correlate with adverse effects on skeletal tissue. Previous studies have shown that FGF18 gene expression levels of hair follicles collected from astronauts on the ISS rose over time. In the hair follicle, FGF18 signaling mediates radioresistance in the telogen by arresting the cell cycle, and FGF18 has the potential to function as a radioprotector. In bone, FGF18 appears to regulate cell proliferation and differentiation positively during osteogenesis and negatively during chondrogenesis. Cellular defense responses to radiation are shared by a variety of organs, hence in this study, we examined whether radiation induced gene expression changes in skin may be predictive of the responses of skeletal tissue to radiation exposure. We have examined oxidative stress and growth arrest pathways in mouse skin and long bones by measuring gene expression levels via quantitative polymerase chain reaction (qPCR) after exposure to total body irradiation (TBI). To investigate the effects of irradiation on gene expression, we used skin and femora (cortical shaft) from the following treatment groups: control (normally loaded, sham-irradiated), and TBI (0.5 Gy Fe-56 600 MeV/n and 0.5 Gy H-1 150 MeV/n). Animals were euthanized one and 11 days post-IR. Statistical analysis was performed via a Student's ttest. In skin samples one day after IR, skin expression of FGF18 was significantly greater (3.8X) than sham-irradiated controls (3.8X), but did not differ 11 days post TBI. Expression levels of other radiation related genes (Nfe2l2, Trp53, Cdkn1a, FoxO3

  18. Optimization of the protocols for in vitro culture and induction of hepatic differentiation of rat mesenchymal stem cells%骨髓间充质干细胞体外培养及肝向分化方案的优化

    Institute of Scientific and Technical Information of China (English)

    向俊西; 郑幸龙; 祝旭龙; 杨丽斐; 高睿; 李建辉; 刘学民; 吕毅

    2015-01-01

    目的 优化一套简便、可靠、稳定的大鼠骨髓间充质干细胞分离、培养、鉴定及肝向分化的方案.方法 采用全骨髓差速贴壁法分离提取大鼠骨髓间充质干细胞,通过优化的1.5 h差速贴壁结合12 h首次全量换液方法,及体外改良培养方案实现细胞高效纯化扩增.流式细胞术表面标志物检测结合成脂、成骨、成软骨诱导分化鉴定细胞群.采用添加bFGF、HGF、EGF等多种生长因子的三步诱导法促使骨髓间充质干细胞向肝系分化,并进行形态学、免疫学、基因水平评估.结果 分离纯化的细胞群阳性表达CD29、CD44、CD90,阴性表达CD34、CD45,经成脂、成骨、成软骨诱导液作用后,油红O、茜素红、甲苯胺蓝染色均呈阳性.经三步法肝向诱导处理后,细胞群呈肝样细胞形态改变,表达肝脏特异性标志物ALB、AFP,肝系相关基因表达水平呈时间依赖性逐渐上升.结论 优化后的方案能够简便、可靠、稳定地获得高纯度的大鼠骨髓间充质干细胞,并能在特定微环境作用下实现肝向分化.%Objective To optimize the protocols for isolation, in vitro culture, identification and induction of hepatic differentiation of rat bone marrow mesenchymal stem cells (BMSCs). Methods Rat BMSCs were separated and purified by differential adherent culture for 1.5 h with the first medium change at 12 h. The surface markers of BMSCs were detected by flow cytometry. The cells were induced to differentiate into adipogenic, osteogenic, and chondrogenesis lineages. A 3-step protocol including sequential addition of growth factors, cytokines and hormones was used to induce the BMSCs to differentiate into hepatocyte-like cells. Results The cells isolated using this protocol were positive for CD29, CD44, and CD90 and negative for CD29 and CD45. The adipogenic, osteogenic, and chondrogenic differentiation of the BMSCs were verified by Oil red, Alizarin red, and toluidine blue staining

  19. 大鼠骨髓间充质干细胞成软骨过程中微小RNA的表达%The expression of miRNAs of rat bone marrow mesenchymal stem cells during chondrogenic differentiation

    Institute of Scientific and Technical Information of China (English)

    陈松; 符培亮; 吴海山; 许震宇

    2014-01-01

    Objective To explore the expression of miRNAs of rat bone marrow mesenchymal stem cells (BMSCs) during chondrogenic differentiation induced by Transforming growth factor-β3 (TGF-β3),and study the effect of over-expression of miR-90a on the differentiation of BMSCs toward chondrocytes.Methods The rat BMSCs were isolated and cultured invitro and were divided into two groups:treated with and without TGF-β3.The express of miRNAs were analyzed by microarray gene chip at 7,14,21 d,and the results were verified by real-time reverse transcriptase-polymerase chain reaction (RT-qPCR).Meanwhile the BMSCs treated with TGF-β3 were also divided into two groups,transfected with miR-90a mimics or mimics-NC,to explore the effect of over-expression of miR-90a on the chondrogenesis of BMSCs.Results The BMSCs were isolated from the rat marrow successful.Microarray gene chip screening resulted in 16 miRNAs whose expression differences were more than 2 folds compared with the controls.Of them,9 were up-regulated and 7 down-regulated.At the same time,the miR-340,miR-140,miR-21 and miR-132 were determined by RT-qPCR,and the results showed that RT-qPCR and microarray gene chip results are consistent.The group transfected with miR-90a mimics express fewer glycosaminoglycans (GAG) than that of transfected with mimics-NC.Conclusion Many miRNAs were involved in the process of BMSCs differentiation into chondrocytes,and over-expression of miR-90a could inhibit the process.%目的 探讨转化生长因子-β3(TGF-β3)诱导骨髓间充质干细胞(BMSCs)向软骨分化过程中微小RNA(miRNAs)的表达,并观察过表达miR-90a对BMSCs成软骨分化的影响.方法 体外分离培养大鼠BMSCs,取第3代BMSCs,在全培养基基础上将其分成两组:实验组加入TGF-β3诱导成软骨分化,对照组不加TGF-β3.在诱导BMSCs向软骨分化第7、14、21天,采用微阵列基因芯片技术分析分化过程中miRNAs的表达,并采用实时定量逆转录聚合酶链反应(RT-qPCR

  20. 软骨形态发生蛋白1诱导的瘢痕成纤维细胞体内成软骨能力的实验研究%Chondrogenic Differentiation of Hypertrophic Scar -Derived Fibroblasts Initiated by Cartilage -derived Morphogenetic Protein 1 in Vivo

    Institute of Scientific and Technical Information of China (English)

    沈聪聪; 柴岗; 曲淼; 侯亦康; 许祐荣; 张艳

    2014-01-01

    目的:探讨软骨形态发生蛋白1(CDMP1)诱导的瘢痕成纤维细胞在体内环境下的软骨构建能力。方法取瘢痕切除术后丢弃的增生性瘢痕组织,提取瘢痕成纤维细胞。将瘢痕成纤维细胞与PGA/PLA支架复合,CDMP1软骨诱导液(CDMP1终浓度为100 ng/mL)进行诱导培养2周,设为诱导组(n=10);将常规培养液培养的瘢痕成纤维细胞-材料复合物植入裸鼠体内作为阴性对照,设为非诱导组(n=4);将软骨细胞-材料复合物植入裸鼠体内作为阳性对照,设为软骨组(n=4)。分别于4周和8周后取材,进行各组湿重、糖胺聚糖(GAG)含量测定,HE染色、Safranine-O染色和Ⅱ型胶原免疫组化染色。结果体内培养4周、8周后各组湿重、GAG含量测定显示,诱导组均高于非诱导组(P<0.05)。体内培养4周后,诱导组HE染色结果显示,瘢痕成纤维细胞诱导后出现软骨细胞陷窝结构;Safranine-O染色结果示,GAG均匀分布于基质;免疫组织化学染色示部分瘢痕成纤维细胞基质中COLⅡ阳性表达。8周时,诱导组的类软骨结果相对4周时更加成熟,更加符合软骨结构分布。结论在CDMP1诱导下,瘢痕成纤维细胞与PGA/PLA材料复合,在体内可以形成类软骨组织,具备一定的成软骨能力。%Objective To explore the chondrogenesis potential of hypertrophic scar fibroblasts (HSFBs) induced by cartilage-derived morphogenetic protein 1 (CDMP1) in vivo. Methods Hypertrophic scar fibroblasts (HSFBs) were isolated from discarded hypertrophic scar. HSFBs combining with PGA/PLA scaffold as HSFBs- PGA/PLA complex were induced by CDMP1 (100 ng/mL) for 2 weeks. Then the complex were transplanted into nude mice as induced group (n=10). The complex that not induced were treated as negative control, named as un-induced group ( n=4), while the chondrocytes-PGA/PLA complex were treated as positive control, named as

  1. The expression of IGF-2 in tetrachlorodibenzodioxin-induced congenital skeleton malformation%IGF一2在四氯二苯并二恶英诱导的大鼠骨骼畸形中的表达

    Institute of Scientific and Technical Information of China (English)

    郭磊; 赵玉岩; 张世亮; 朱世博; 刘魁

    2008-01-01

    analysis.respectively.Results TCDD(15 μg/kg)induced single or coalescent developmental skeleton defect in fetal rat(33.3%),such as cross-foot,cleft spine,cleft palate,rumplesand so on,The chondrogenesis center of tarsal bones and quantity of chondrocyte were decreased.There were expanding rough endoplasmic reticulum,degradation of nuclear matrix,and disorder of mitochondria cristae in cell nucleus of chondrocyte.IGF-2 protein level in TCDD-treated cartilaginous'tissue was decreased significantly.In chondrocyte treated with TCDD(1 × 10-8 mol/L)in vitro,the expression of IGF-2 mRNA and protein were decreased 80%and 60%,respectively(P<0.05).Conelusions The low expression of IGF-2 in chondrocyte may be related to the TCDD-induced developmentalskeleton defect.

  2. Successful isolation, in vitro expansion and characterization of stem cells from Human Dental Pulp

    Directory of Open Access Journals (Sweden)

    Preethy SP

    2010-01-01

    acids (5 .Cell counting was done by Trypan Blue dye exclusion method and the cells were seeded in 6 well culture plates. The plates with cells were incubated at 37˚C with 5% CO2 for varying periods from 14 days-28 days. The cells were observed daily and media change was done every three days. RESULTS: Viable Dental Pulp tissue-cells were obtained after transportation of up to 48 hrs and the in vitro growth of cells was initially slow but colonies were identified from the 10th day onwards. The cells were harvested at different intervals of 14-28 days for each sample based on their growth and subjected to H & E staining .The H & E staining of the cultured cells of all the samples showed positive resultsCONCLUSION: We are able to transport extracted teeth and derive viable dental pulp tissue cells after enzymatic digestion and multiply them in culture after a maximum of 48 hrs after transportation. The cells could be grown in culture with a morphology resembling dental pulp stem cells while in culture expansion and in H&E studies. Further characterization of the cells is necessary to confirm their Stemness. References1.Gronthos S, Mankani M, Brahim J, Robey PG, Shi S. Postnatal human dental pulp stem cells (DPSCs in vitro and in vivo. Proc Natl Acad Sci U S A. 20002.Nosrat IV, Widenfalk J, Olson L, Nosrat CA. Dental pulp cells produce neurotrophic factors, interact with trigeminal neurons in vitro, and rescue motoneurons after spinal cord injury. Dev Biol. 2001 Oct 3.Iohara K, Zheng L, Ito M, Tomokiyo A, Matsushita K, Nakashima M. Side population cells isolated from porcine dental pulp tissue with self-renewal and multipotency for dentinogenesis, chondrogenesis, adipogenesis, and neurogenesis. Stem Cells. 2006 Nov4.Gandia C, Armiñan A, García-Verdugo JM, Lledó E, Ruiz A, Miñana MD, Sanchez-Torrijos J, Payá R, Mirabet V, Carbonell-Uberos F, Llop M, Montero JA, Sepúlveda P. Human dental pulp stem cells improve left ventricular function, induce angiogenesis, and reduce

  3. In vitro fibrocartilage construction with bone marrow-derived mesenchymal stem cells-fully demineralized bone matrix%完全脱钙骨基质与骨髓间充质干细胞体外诱导构建组织工程纤维软骨

    Institute of Scientific and Technical Information of China (English)

    王鹏; 崔一民; 陈晓东

    2011-01-01

    背景:含有骨形态发生蛋白的完全脱钙骨基质可使骨髓间充质干细胞向软骨细胞分化并维持其软骨细胞的特性,在软骨发生过程中发挥重要作用.目的:将骨髓间充质干细胞与完全脱钙骨基质在体外诱导培养成纤维软骨组织以及观察培养体系与支架材料对细胞凋亡的影响.方法:采用全骨髓培养法分离培养猪骨髓间充质干细胞,取第1代骨髓间充质干细胞均匀接种到完全脱钙骨基质上,以含有转化生长因子β1、胰岛素样生长因子、地塞米松、抗坏血酸及体积分数10%FBS的高糖DMEM诱导培养液培养作为实验组,将单层诱导培养、单层基础培养的骨髓间充质干细胞、空白完全脱钙骨基质作为对照,于培养第1,2,3,4周进行大体形态观察以及Ⅰ、Ⅱ型胶原及聚集蛋白聚糖的RT-PCR定性检测;培养4周进行细胞凋亡检测.结果与结论:实验组可检测到Ⅰ型胶原的持续表达,Ⅱ型胶原及聚集蛋白聚糖表达逐渐增加.单层诱导培养组可检测到Ⅱ型胶原、聚集蛋白聚糖的表达及Ⅰ型胶原的持续表达,但表达量远低于实验组;实验组细胞凋亡率高于单层诱导培养组、单层基础培养组.说明转化生长因子β1、胰岛素样生长因子1体外诱导培养的骨髓间充质干细胞-完全脱钙骨基质可以表达特异性软骨基质,三维环境培养细胞外基质的表达量明显高于单层诱导培养;静态培养系统对三维培养的细胞凋亡有影响.%BACKGROUND:The fully demineralized bone matrix containing bone morphogenetic protein can induce bone marrow-derived mesenchymal stem cells (BMSCs) differentiate into cartilage cells, and keep it characteristics of cartilage cells. It plays an important role in the process of chondrogenesis.OBJECTIVE:To observe the complexes of BMSCs and fully demineralized bone matrix induced into the cartilage tissue in vitro and to detect the effects on cell apoptosis of

  4. Porcine Fetal Bone Marrow Mesenchymal Stem Cells in Cartilage Tissue Engineering%胎猪BMSC体外构建软骨的实验研究

    Institute of Scientific and Technical Information of China (English)

    刘李娜; 何爱娟; 周广东; 曹卫刚

    2013-01-01

    Objective To investigate the optimal seed cell for cartilage engineering by comparing the chondrogenesis capability of porcine fetal bone marrow mesenchymal stem cells (fBMSCs) and adult porcine BMSCs (aBMSCs). Methods Fetal pigs with gestational age of 70 days were obtained by the uterine-incision delivery, and primary fBMSCs were isolated from the bone marrow. Primary aBMSCs were isolated from the bone marrow which was aspirated from iliac. Cell morphology of the two kinds of cells at passage 3 were observed after in vitro proliferation. The two kinds of cells at passages 3 were characterized by their osteogenic, adipogenic and chondrogenic differentiation potential. Then the fBMSCs and aBMSCs were separately seeded onto a polyglycolic acid/polylactic acid (PGA/PLA) scaffold with the concentration of 1 ×108 cells/mL. All specimens were harvested after 8 weeks' culture in vitro. Gross observation, glycosaminoglycan (GAG) quantification, total collagen quantification and histology were used to compare related characteristic differences of engineered cartilage formed by the two kinds of cells. Results fBMSCs had better proliferation and multiple differentiation capacity than aBMSCs. The two kinds of cells both formed mature cartilage after 8 weeks of culture in vitro, and the engineered cartilage of aBMSCs group had better appearance. The GAG content and total collagen content of the cartilage formed by fBMSCs were both higher than the cartilage formed by aBMSCs (P<0.01). Histology and immunohistochemistry demonstrated that the cartilage formed by fBMSCs have more compact tissue structure. The cartilage matrix staining of cartilage formed by fBMSCs were stronger than that of cartilage formed by aBMSCs. Conclusion The fBMSCs seems to be the optimal seed cells for cartilage tissue engineering.%目的比较胎猪骨髓间充质干细胞(Bone Marrow Mesenchymal Stem Cells,BMSCs)和成年猪BMSCs构建软骨能力的差异,寻找合适的同种异体组织工

  5. The study of resveratrol protective effects and mechanism on tissue-engineered cartilage%白藜芦醇对组织工程化软骨的保护作用及其机制的实验研究

    Institute of Scientific and Technical Information of China (English)

    崔文岗; 雷鸣; 石岩; 肖德明

    2013-01-01

    Objective To investigate the mechanism of protective effects of resveratrol on tissueengineered cartilage.Methods The chondrogenesis of alginate-encapsulated bone marrow mesenchymal stem cells (BMSCs) were evaluated by toluidine blue staining and immunostain.The morphology of BMSCs-derived chondrocytes cultured on chitosan-gelatin scaffolds (CGS) was evaluated by scanning electron microscope and laser confocal microscope.When these cells on CGS were pre-stimulated with interleukin-1β (IL-1β) or cotreated with IL-1β and resveratrol in the absence and presence of specific β1-integrin blocking antibody,collagen type Ⅱ,aggrecan,matrix metalloproteinase-13 (MMP-13) expression,and the translocation of nuclear factor kappaB (NF-κB) were analyzed by Western blotting.ANOVA was used for statistical analysis.Results Alginate bead culture plus conditional medium together could induce the cartilage-specific collagen type Ⅱ,aggrecan expression and extracellular matrix accumulation in differentiated chondrocytes.CGS supported differentiated cell attachment,proliferation,and migration.When those cells cultured on CGS were stimulated with IL-1β alone,collagen type Ⅱ and aggrecan expression was inhibited.However,MMP-13 expression increased.By Western blotting semi-quantitative analysis,the expression level of cartilage-specific collagen type Ⅱ of the control group was 0.484±0.006; the expression level of resveratrol intervention group was 0.474±0.014.The difference between these two groups was not statistically significant (P>0.05).The expression level of the IL-1β intervention group reduced to 0.155±0.009,which was statistically significant different from the above two groups(P<0.05).Resveratrol could antagonist the negative effect of IL-1β,and increase collagen type Ⅱ to 0.468±0.014,the difference between these two was statistically significant (P<0.05),and no significant difference when compared to the control group (P>0.05).Specific β1-integrin