Expression Of Chondrocyte Markers Research Articles

Hydrogels derived from cartilage matrices promote induction of human mesenchymal stem cell chondrogenic differentiation.

Limited supplies of healthy autologous or allogeneic cartilage sources have inspired a growing interest in xenogeneic cartilage matrices as biological scaffolds for cartilage tissue engineering. The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible. Human MSCs cultured on hydrogels from shark skull cartilage, pig articular cartilage, and pig auricular cartilage ECM had increased expression of chondrocyte markers and decreased secretion of angiogenic factors VEGF-A and FGF2 in comparison to MSCs cultured on tissue culture polystyrene (TCPS) at one week. MSCs grown on shark ECM gels had decreased type-1 collagen mRNA as compared to all other groups. Degradation products of the cartilage ECM gels and soluble factors released by the matrices increased chondrogenic and decreased angiogenic mRNA levels, indicating that the processed ECM retains biochemically active proteins that can stimulate chondrogenic differentiation. In conclusion, this work supports the use of cartilage matrix-derived hydrogels for chondrogenic differentiation of MSCs and cartilage tissue engineering. Longer-term studies and positive controls will be needed to support these results to definitively demonstrate stimulation of chondrocyte differentiation, and particularly to verify that calcification without endochondral ossification does not occur as it does in shark cartilage. The objectives of this study were to determine if shark and pig cartilage extracellular matrix (ECM) hydrogels can stimulate chondrocytic differentiation of mesenchymal stem cells (MSCs) without exogenous growth factors and to determine if the soluble factors retained by these ECM hydrogels are responsible for this induction. Sharks are an especially interesting model for cartilage regeneration because their entire skeleton is composed of cartilage and they do not undergo endochondral ossification. Culturing human MSCs on porcine and shark cartilage ECM gels directly, with ECM gel conditioned media, or degradation products increased mRNA levels of chondrogenic factors while decreasing angiogenic factors. These studies indicate that xenogeneic cartilage ECMs have potential as biodegradable scaffolds capable of stimulating chondrogenesis while preventing angiogenesis for regenerative medicine applications and that ECM species selection can yield differential effects.

Hif-3α Fine-tunes Extracellular Matrix Production in Nucleus Pulposus Cell under Hypoxia

Introduction Nucleus pulposus (NP), the gelatinous tissue core in the intervertebral disc (IVD), is enriched with extracellular matrix type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1). Dysregulation of extracellular matrix proteins in the NP is tightly associated with disc degeneration, ultimately leading to a loss of mechanical function in motion segments.1 Expression of SOX9, the major chondrogenic transcription factor for COL2A1 and AGC1 gene activation, was evidenced in the normal and degenerated human NP, but weak in the annulus fibrosus (AF) region.2 Considering the virtually avascular characteristic of the NP, NP cells are regulated within a hypoxic microenvironment. Among the family of hypoxia inducible factor-α subunits (HIF-α), HIF-1α and HIF-2α (EPAS1) have been well illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the NP.3–5 However, the understanding of HIF-3α, the dominant negative regulator of HIF-1α,6 in IVD is limited. Here, we hypothesized that HIF-3α may regulate the SOX9-dependent transcription of the extracellular matrix genes in NP cells under hypoxia. We aimed at characterizing the sub-cellular expression patterns of HIF-1α and HIF-3α in mouse IVD tissues and human NP cells cultured under different oxygen tensions. Moreover, we tested the modulatory effects of HIF-1α/HIF-3α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods All animal and human works were approved by local ethical committee. IVD were harvested from wild-type C57BL/6N mice at 3 and 6 months-old. Lumbar IVD of scoliosis patients were collected under informed consent. Cells were harvested by digestion with 0.5 × of TrpLE Express ( Gibco) 37°C, 30 minute and 0.25mg/ml Collagenase II ( Gibco) 37°C, 90 minute in DMEM-HG ( Gibco). Human NP cells were expanded in DMEM-HG supplemented with 10% fetal bovine serum. Subsequently, human NP cells were subjected to ambient oxygen tension (21% O2) or intermediate hypoxia (5% O2) or extreme hypoxia (1% O2) for 72 hours prior to fixation or RNA collection. Immunohistochemistry staining of Hif-1α and Hif-3α was studied with mouse IVD paraffin sections and different sub-cellular locations of HIF-1α, HIF-3α and SOX9 expression were identified by immunofluorescence in human NP cells cultured under different oxygen tensions. To determine transcriptional activity of Col2a1 and Agc1, luciferase-based promoter assays on specific Col2a1 or Agc1 cis-acting elements with the overexpression for each of the HIF-α subunits and/or Sox9 in ATDC5 cells were performed. Results Hif-1α and Hif-3α expression pattern was identified in mouse NP, AF and endplate (EP) tissue. Expression of HIF-1α and SOX9 was detected in the nuclei of human NP cells independent of different oxygen tensions. Strikingly, induction of HIF-3α expression in nuclei was observed in human NP cells cultured under hypoxia (5% and 1% O2) versus normoxia (21% O2). Additionally, AGC1 gene expression was reduced in human NP cells cultured under hypoxia in comparison with normoxia. Furthermore, luciferase-based promoter assay showed that Hif-3α overexpression inhibited the Sox9–dependent transcriptional up-regulation of Col2a1 and Agc1. Conclusion Our findings illustrated that induction of HIF-3α in human NP cell nuclei was evidenced in hypoxia versus normoxia. We further demonstrated that Hif-3α has a function in inhibiting Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of HIF-3α in fine-tuning the extracellular matrix protein production in NP cells under hypoxia. Detailed molecular mechanism of HIF-3α in regulating human NP cell and matrix homeostasis under hypoxia warrants further investigations. References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol 2010;222(1):23–32 Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55(2):91–97 Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res 2002;308(3):401–407 Risbud MV, Guttapalli A, Stokes DG, et al. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 2006;98(1):152–159 Agrawal A, Gajghate S, Smith H, et al. Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 2008;58(12):3798–3808 Dengler VL, Galbraith MD, Espinosa JM. Transcriptional regulation by hypoxia inducible factors. Crit Rev Biochem Mol Biol 2014;49(1):1–15

Epigenetically mediated spontaneous reduction of NFAT1 expression causes imbalanced metabolic activities of articular chondrocytes in aged mice

Abnormal metabolic activities of chondrocytes may cause articular cartilage (AC) degradation, but key transcription factors regulating metabolic activities in AC of aging individuals remain unknown. This study aimed to investigate the role of transcription factor NFAT1 in regulating the expression of anabolic and catabolic molecules in AC of aged mice. The hip, knee, and shoulder joints of BALB/c mice were harvested at 6, 12, 15, 18, and 24 months of age for histopathological and immunohistochemical (IHC) analyses. Total RNA was isolated from AC for gene expression. Genomic DNA and chromatin were prepared from AC for methylated DNA immunoprecipitation (MeDIP) and chromatin immunoprecipitation (ChIP) assays. NFAT1 expression in AC of mice was significantly decreased after 12 months of age, which was associated with reduced proteoglycan staining, decreased expression of chondrocyte markers, and increased expression of interleukin-1β. Forced Nfat1 expression in chondrocytes from aged mice significantly reversed the abnormal metabolic activities. ChIP assays confirmed that NFAT1 bound to the promoter of the Acan, Col2a1, Col9a1, Col11a1, Il1b, Mmp13 and Tnfa genes in articular chondrocytes of aged mice. ChIP and MeDIP assays revealed that reduced NFAT1 expression in AC of aged mice was regulated by epigenetic histone methylation at the promoter region and was correlated with increased DNA methylation at introns 1 and 10 of the Nfat1 gene. NFAT1 is a transcriptional regulator of multiple anabolic and catabolic genes in AC of aged mice. Epigenetically mediated reduction of NFAT1 expression causes imbalanced metabolic activities of articular chondrocytes in aged mice.

MicroRNA-200a Regulates the Development of Mandibular Condylar Cartilage

Mandibular condylar cartilage (MCC) is classified as secondary cartilage, the histologic structure of which is unique from that of primary cartilage. MicroRNA (miRNA) is a small noncoding RNA that binds to the messenger RNA (mRNA) target to repress its translation and plays an important role in cell differentiation, proliferation, and death. Microarray analysis revealed that miR-200a was characteristically expressed during embryonic development. We hypothesized that miR-200a may be involved in regulating the formation of cartilage during MCC growth. We investigated the function of miR-200a by transfecting an inhibitor or mimic into MCC organ and cell cultures. A histologic examination revealed the localized inhibitory effects of the miR-200a mimic and widespread enhancing effects of the inhibitor on chondrocytic differentiation in the MCC organ culture system. An immunohistochemical examination and gene expression analysis demonstrated that the miR-200a inhibitor enhanced chondrogenesis, while the mimic had the opposite effect by enhancing cell proliferation. Quantitative reverse transcription polymerase chain reaction analysis revealed that miR-200a downregulated the gene expression of chondrocyte markers. Moreover, transfection of the miR-200a mimic into ATDC5 cells repressed the formation of the cartilaginous matrix. These results indicate that miR-200a contributed to chondrogenesis in developing MCC by controlling proliferation and differentiation in MCC cells.

Osthole Inhibits Proliferation and Induces Catabolism in Rat Chondrocytes and Cartilage Tissue

Background/Aims: Cartilage destruction is thought to be the major mediator of osteoarthritis. Recent studies suggest that inhibition of subchrondral bone loss by anti-osteoporosis (OP) drug can protect cartilige erosion. Osthole, as a promising agent for treating osteoporosis, may show potential in treating osteoarthritis. The purpose of this study was to investigate whether Osthole affects the proliferation and catabolism of rat chondrocytes, and the degeneration of cartilage explants. Methods: Rat chondrocytes were treated with Osthole (0 μM, 6.25 μM, 12.5 μM, and 25 μM) with or without IL1-β (10ng/ml) for 24 hours. The expression levels of type II collagen and MMP13 were detected by western Blot. Marker genes for chondrocytes (A-can and Sox9), matrix metalloproteinases (MMPs), aggrecanases (ADAMTS5) and genes implicated in extracellular matrix catabolism were evaluated by qPCR. Cell proliferation was assessed by measuring proliferating cell nuclear antigen (PCNA) expression and fluorescence activated cell sorter. Wnt7b/β-catenin signaling was also investigated. Cartilage explants from two-week old SD rats were cultured with IL-1β, Osthole and Osthole plus IL-1β for four days and glycosaminoglycan (GAG) synthesis was assessed with toluidine blue staining and Safranine O/Fast Green FCF staining, collagen type II expression was detected by immunofuorescence. Results: Osthole reduced expression of chondrocyte markers and increased expression of MMP13, ADAMTS5 and MMP9 in a dose-dependent manner. Catabolic gene expression levels were further improved by Osthole plus IL-1β. Osthole inhibited chondrocyte proliferation. GAG synthesis and type II collagen were decreased in both the IL-1β groups and the Osthole groups, and significantly reduced by Osthole plus IL-1β. Conclusions: Our data suggested that Osthole increases the catabolism of rat chondrocytes and cartilage explants, this effect might be mediated through inhibiting Wnt7b/β-catenin pathway.

Controlled chondrogenesis from adipose-derived stem cells by recombinant transforming growth factor-β3 fusion protein in peptide scaffolds

Adipose-derived stem cells (ADSCs) are promising for cartilage repair due to their easy accessibility and chondrogenic potential. Although chondrogenesis of transforming growth factor-β (TGF-β) mediated mesenchymal stem cells (MSCs) is well established in vitro, clinical tissue engineering requires effective and controlled delivery of TGF-β in vivo. In this work, a self-assembled peptide scaffold was employed to construct cartilages in vivo through the chondrogenesis from ADSCs controlled by recombinant fusion protein LAP-MMP-mTGF-β3 that was transfected by lentiviral vectors. During this course, the addition of matrix metalloproteinases (MMPs) can trigger the release of mTGF-β3 from the recombinant fusion protein of LAP-MMP-mTGF-β3 in the combined scaffolds, thus stimulating the differentiation of ADSCs into chondrogenesis. The specific expression of cartilage genes was analyzed by real-time polymerase chain reaction and Western blot. The expression of chondrocytic markers was obviously upregulated to a higher level compared to the one by commonly used TGF-β3 alone. After 3weeks of in vitro culturing, the hybrids with differentiated chondrogenesis were then injected subcutaneously into nude mice and retrieved after 4weeks of culturing in vivo. Histological analysis also confirmed that the recombinant fusion protein was more effective for the formation of cartilage matrix than the cases either with TGF-β3 alone or without LAP-MMP-mTGF-β3 (P<0.05). This study demonstrates that controlled local delivery of the LAP-MMP-mTGF-β3 constructs can accelerate differentiation of ADSCs into the cartilage in vivo, which indicates the great potential of this hybrid in rapid therapy of osteoarthritis.

Hif-αs Modulation of Sox9-Dependent Extracellular Matrix Production: Insight into Intervertebral Disc Cell Activity under Hypoxia

Introduction Intervertebral disc (IVD) degeneration leads to loss of mechanical function of motion segment and is attributed to deregulated extracellular matrix components in the nucleus pulposus (NP). The NP is rich in type II collagen (encoded by COL2A1) and aggrecan (encoded by AGC1), which are known to be regulated by the master chondrogenic transcription factor, Sox9.1 In situ studies of human degenerated NP illustrated a reduction of COL2A1 and absence of AGC1 expression, whereas SOX9 expression level remained high.2 This indicates that there may be other factors in the IVD that can regulate the Sox9-dependent matrix expression. Recent studies in aged rabbit IVD suggest that the abnormal NP cells may be contributed by a migration of the Sox9-expressing prechondrocytic cells from the annulus fibrosus (AF) adjacent to the endplate (EP).3, 4 Among the hypoxia inducible factor-α subunits (Hif-α), Hif-1α and Hif-2α (EPAS1) have been illustrated as important transcription factors in maintaining disc cell and matrix homeostasis, particularly in the hypoxic NP region.5-7 We hypothesized that Hif-αs may regulate the Sox9-dependent transcription of the extracellular matrix genes in response to IVD degeneration. First, we aimed at identification of Sox9 and Hif-αs co-expression in cells of non-degenerated and degenerated IVD. Second, we tested the modulatory effects of Hif-1α/Hif-2α on the expression of Col2a1 and Agc1 using Sox9-expressing mouse prechondrocytic cells as a model. Materials and Methods IVD were harvested from wild-type C57BL/6N mice at 3 and 6 month old. Disc degeneration was induced by an insertion of 25G needle into contralateral AF of 4-month old Lewis rat and discs were harvested post 2 to 4 weeks of operation. Lumbar IVD of scoliosis patients and degenerative disc disease patient were collected. All animal and human works were approved by local ethical committee. Comparative immunohistochemistry staining of Hif-1α, Hif-2α and Sox9 were studied in parrafilm sections of IVD. Luciferase-based promoter assays based on specific Col2a1 or Agc1 cis-acting elements via the overexpression for each of the HIF-α subunits and/or for Sox9 in ATDC5 cells were performed. Results Relative co-expression patterns of Hif-1α, Hif-2α and Sox9 were detected in AF, NP and EP of the mouse, rat and human IVD. Intracellular expression of Hif-1α, Hif-2α, and Sox9 were confirmed by immunofluorescence, indicating a possible role for Hif-αs-Sox9 tonic activity in the IVD. By luciferase-based promoter assay, Sox9 is demonstrated as a dominant transcription factor in the activation of transcription of Col2a1 and Agc1 in mouse chondroprogenitor cells. Overt additive transcriptional upregulation of Col2a1 and Agc1 was observed with a co-expression of HIF-1α and Sox9. Strikingly, Hif-2α inhibited the Sox9-dependent transcriptional upregulation of Col2a1 and Agc1. Conclusion Our findings indicate the presence of Hif-αs/Sox9 expressing cells in IVD. In addition, our data suggest that Hif-αs can modulate Sox9-mediated transcriptional regulation of Col2a1 and Agc1 in prechondrocytic cells, implicating a possible role of dynamic Hif-αs-Sox9 interaction in the IVD degeneration process. Further investigations of the orchestration mechanisms of Hif-αs/Sox9 in the IVD may provide insights in potential strategies to alleviate disc degeneration. Disclosure of Interest None declared References Richardson SM, Hoyland JA, Mobasheri R, Csaki C, Shakibaei M, Mobasheri A. Mesenchymal stem cells in regenerative medicine: opportunities and challenges for articular cartilage and intervertebral disc tissue engineering. J Cell Physiol 2010;222(1):23–32 Sive JI, Baird P, Jeziorsk M, Watkins A, Hoyland JA, Freemont AJ. Expression of chondrocyte markers by cells of normal and degenerate intervertebral discs. Mol Pathol 2002;55(2):91–97 Henriksson H, Thornemo M, Karlsson C, et al. Identification of cell proliferation zones, progenitor cells and a potential stem cell niche in the intervertebral disc region: a study in four species. Spine 2009;34(21):2278–2287 Henriksson HB, Svala E, Skioldebrand E, Lindahl A, Brisby H. Support of concept that migrating progenitor cells from stem cell niches contribute to normal regeneration of the adult mammal intervertebral disc: a descriptive study in the New Zealand white rabbit. Spine 2012;37(9):722–732 Rajpurohit R, Risbud MV, Ducheyne P, Vresilovic EJ, Shapiro IM. Phenotypic characteristics of the nucleus pulposus: expression of hypoxia inducing factor-1, glucose transporter-1 and MMP-2. Cell Tissue Res 2002;308(3):401–407 Risbud MV, Guttapalli A, Stokes DG, et al. Nucleus pulposus cells express HIF-1 alpha under normoxic culture conditions: a metabolic adaptation to the intervertebral disc microenvironment. J Cell Biochem 2006;98(1):152–159 Agrawal A, Gajghate S, Smith H, et al. Cited2 modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus pulposus cells of the rat intervertebral disc. Arthritis Rheum 2008;58(12):3798–3808

Generation of Col2a1-EGFP iPS Cells for Monitoring Chondrogenic Differentiation

Induced pluripotent stem cells (iPSC) are a promising cell source for cartilage regenerative medicine; however, the methods for chondrocyte induction from iPSC are currently developing and not yet sufficient for clinical application. Here, we report the establishment of a fluorescent indicator system for monitoring chondrogenic differentiation from iPSC to simplify screening for effective factors that induce chondrocytes from iPSC. We generated iPSC from embryonic fibroblasts of Col2a1-EGFP transgenic mice by retroviral transduction of Oct4, Sox2, Klf4, and c-Myc. Among the 30 clones of Col2a1-EGFP iPSC we established, two clones showed high expression levels of embryonic stem cell (ESC) marker genes, similar to control ESC. A teratoma formation assay showed that the two clones were pluripotent and differentiated into cell types from all three germ layers. The fluorescent signal was observed during chondrogenic differentiation of the two clones concomitant with the increase in chondrocyte marker expression. In conclusion, Col2a1-EGFP iPSC are useful for monitoring chondrogenic differentiation and will contribute to research in cartilage regenerative medicine.

Wnt signaling is involved in human articular chondrocyte de-differentiation in vitro

Osteoarthritis is the most prevalent form of arthritis in the world. Certain signaling pathways, such as the wnt pathway, are involved in cartilage pathology. Osteoarthritic chondrocytes undergo morphological and biochemical changes that lead to chondrocyte de-differentiation. We investigated whether the Wnt pathway is involved in de-differentiation of human articular chondrocytes in vitro. Human articular chondrocytes were cultured for four passages in the presence or absence of IL-1 in monolayer or micromass culture. Changes in cell morphology were monitored by light microscopy. Protein and gene expression of chondrocyte markers and Wnt pathway components were determined by Western blotting and qPCR after culture. After culturing for four passages, chondrocytes exhibited a fibroblast-like morphology. Collagen type II and aggrecan protein and gene expression decreased, while collagen type I, matrix metalloproteinase 13, and nitric oxide synthase expressions increased. Wnt molecule expression profiles changed; Wnt5a protein expression, the Wnt target gene, c-jun, and in Wnt pathway regulator, sFRP4 increased. Treatment with IL-1 caused chondrocyte morphology to become more filament-like. This change in morphology was accompanied by extinction of col II expression and increased col I, MMP13 and eNOS expression. Changes in expression of the Wnt pathway components also were observed. Wnt7a decreased significantly, while Wnt5a, LRP5, β-catenin and c-jun expressions increased. Culture of human articular chondrocytes with or without IL-1 not only induced chondrocyte de-differentiation, but also changed the expression profiles of Wnt components, which suggests that the Wnt pathway is involved in chondrocyte de-differentiation in vitro.

The Oncogene LRF Stimulates Proliferation of Mesenchymal Stem Cells and Inhibits Their Chondrogenic Differentiation.

Objective. The oncogene leukemia/lymphoma-related factor (LRF) enhances chondrosarcoma proliferation and malignancy. This study aimed to investigate the roles of LRF in chondrogenic differentiation of primary human bone marrow–derived mesenchymal stem cells (BMSCs). Design. LRF was overexpressed in BMSC by lentiviral transduction. Chondrogenic differentiation of BMSC was induced by high-density pellet culture. Western blotting and real-time polymerase chain reaction were used to investigate changes in protein and mRNA levels, respectively, during chondrogenesis. Safranin-O staining, immunohistochemistry, and glycoaminoglycan contents were used to assess cartilage matrix deposition. BMSC proliferation was determined by mitochondrial dehydrogenase activity and cell counting. Cell cycle profiling was performed by flow cytometry. Results. LRF overexpression effectively inhibited protein and mRNA expression of chondrocyte markers and cartilage matrix deposition during chondrogenesis of BMSC. Endogenous LRF expression was constitutively high in undifferentiated BMSC but remained low in primary articular chondrocytes. Endogenous LRF protein was downregulated in a time-dependent manner during chondrogenesis. BMSCs overexpressing LRF had higher proliferation rates and cell population in the S phase. LRF suppressed p53 expression during chondrogenesis and this might prevent differentiating chondrocytes from entering a quiescent state. Conclusion. Our data showed that LRF is important for stimulating stem cell proliferation and cell cycle progression. It is known that LRF is highly expressed in the mouse limb buds prior to overt chondrogenesis; thus, LRF might function to prevent premature chondrogenic differentiation of stem cells.

Influence of cryopreservation, cultivation time and patient’s age on gene expression in Hyalograft® C cartilage transplants

Our aim was to evaluate the impact of cryopreservation, cultivation time and patient's age on the expression of specific chondrogenic markers in Hyalograft® C transplants. Gene expression of chondrocyte markers [collagen type I (COL1A1), COL2A1, aggrecan, versican, melanoma inhibitory activity (MIA) and interleukin (IL)-1β] was analysed in cartilage biopsies (n = 17) and Hyalograft® C transplant samples (non-cryopreserved = 78, cryopreserved = 13) by quantitative real-time polymerase chain reaction (PCR). Correlation analyses were performed to evaluate the influence of the above-named parameters on the level of gene expression. Cryopreservation of cells was found to decrease COL2A1 and MIA significantly (4.6-fold, p < 0.01 and 2-fold, p < 0.045, respectively). The duration of cryopreservation had no further influence on the expression of these factors. No correlation was detected between cultivation time (75 ± 31 days) and the expression level of any gene. Cartilage transplants from older patients (>35 years) exhibited a significantly higher IL-1β expression (3.7-fold, p < 0.039) than transplants from younger patients (≤ 35 years). Our data demonstrate that cryopreservation has a profound impact on chondrocyte metabolic activity by decreasing the expression of COL2A1 and MIA in Hyalograft® C transplants, independent of the duration of cryopreservation.

Changes in chondrogenesis differentiation potential of bone marrow mesenchymal stem cells according to donor age

Purpose: Mesenchymal stem cells (MSC) are good candidates to be used in regenerative medicine due to their differentiation potential. Different studies propose these cells for the development of osteoarticular tissues. However, while MSC from bone marrow are well characterized, recent studies have evocated a decrease in proliferation and differentiation capability of these cells when donor's age increase. The aim of this work was to study the influence of donor's age on the chondrogenic differentiation of bone marrow mesenchymal stem cells. Methods: Two groups of donors were analyzed (<20 years and >60 years). Proliferation and clonogenicity of cells were tested for a culture period of 10 weeks. Chondrogenic differentiation of cells was induced in pellet culture using a chondrogenic differentiation medium and TGF-β3. After that, proteoglycan production was assayed using alcian blue staining and gene expression of chondrocyte markers quantified by qRT-PCR. Results: Cells from the young donor's group an increased proliferation capacity and clonogenicity as compared with cells from old donors. In parallel, young donor group's cells were characterized by high level expression of type II collagen, aggrecan and SOX-9 genes while cells from the old donors group showed a poor chondrogenic differentiation. Conclusions: This study shows that differences on proliferation and chondrogenic differentiation exist between mesenchymal stem cells isolated from bone marrow obtained from young or old donors. This could be considered in the choise of MSC for cartilage tissue engineering.

Human cartilage fragments in a composite scaffold for single-stage cartilage repair: an in vitro study of the chondrocyte migration and the influence of TGF-β1 and G-CSF

Minced chondral fragments are becoming popular as a source of cells for cartilage repair, as a growing interest is developing towards one-stage procedures to treat cartilage lesions. The purpose of this study is to (A) compare cell outgrowth from cartilage fragments of adult and young donors using two different types of scaffolds and (B) evaluate the influence of transforming-growth-factor-β1 (TGF-β1) and granulocyte colony-stimulating factor (G-CSF) on chondrocyte behaviour. In part (A) cartilage fragments from adult and young donors were either loaded onto an HA-derivative injectable paste scaffold or onto an HA-derivative membrane scaffold. Construct sections were then examined for cell counting after 1, 2 and 3months. In part (B) only membrane scaffolds were prepared using cartilage fragments from young donors. Constructs were cultured either in standard growth medium or in the presence of specific growth factors, such as TGF-β1 or G-CSF or TGF-β1+G-CSF. After 1month, construct sections were examined for cell counting. Expression of chondrocyte markers (SOX9, CD151, CD49c) and proliferative markers (β-catenin, PCNA) was assessed using immunofluorescence techniques, both in unstimulated construct sections and in cells from unstimulated and stimulated construct cultures. Part (A): histological analysis showed age-dependent and time-dependent chondrocyte migration. A significant difference (p<0.05) was observed between young and older donors at the same time point. No difference was detected between the two types of scaffolds within the same group at the same time point. Part (B): after 1month, the number of migrating cells/area significantly increased due to exposure to TGF-β1 and/or G-CSF (p<0.05). Immunofluorescence revealed that outgrowing cells from unstimulated scaffold sections were positive for SOX9, CD151, CD49c and G-CSF receptor. Immunofluorescence of cells from construct cultures showed an increase in β-catenin in all stimulated groups and an increased PCNA expression in G-CSF-exposed cultures (p<0.05). Outgrowing cells may represent a subset of chondrocytes undergoing a phenotypic shift towards a proliferative state. TGF-β1, and to a greater extent G-CSF, may accelerate this outgrowth. The clinical relevance of this study may involve a potential future clinical application of scaffolds preloaded with growth factors as an additional coating for chondral fragments. Indeed, a controlled delivery of G-CSF, widely employed in various clinical settings, might improve the repair process driven by minced human cartilage fragments during one-stage cartilage repair.

Role and epigenetic regulation of p16Ink4a-MMP-1 axis in OA-associated senescence-like phenotype and hypertrophy of chondrocytes

Backgroundand objectivesOsteoarthritic (OA) chondrocyte is characterised by altogether DNA damage accumulation, eroded telomeres, expression of senescence marker such as p16Ink4a and establishment of one specific secretome including IL-1b, IL-8 and...

Inhibitory effect of miR-29a on the chondrogenic differentiation of mesenchymal stem cells

Backgroundand objectivesMesenchymal stem or stromal cells (MSC) are multipotent cells that can differentiate into different lineages, particularly osteoblasts and chondrocytes. The differentiation process of MSC is regulated by various molecules...

Antifibrotic effect of adipose stromalcells on chondrocytes from osteoarthritic patients

PurposesOsteoarthritis (OA) characterised by degeneration of articular cartilage is the most frequent rheumatic disease. Mesenchymal stem cells (MSC) isolated from bone marrow (MSC) or adipose tissues (ASC) secrete a large...

SOX9 Protein Induces a Chondrogenic Phenotype of Mesangial Cells and Contributes to Advanced Diabetic Nephropathy

Diabetic nephropathy (DN) is the most important chronic kidney disease. We previously reported that Smad1 transcriptionally regulates the expression of extracellular matrix in DN. Phenotypic change in mesangial cells (MCs) is a key pathologic event in the progression of DN. The aim of this study is to investigate a novel mechanism underlying chondrogenic phenotypic change in MCs that results in the development of DN. MCs showed chondrogenic potential in a micromass culture, and BMP4 induced the expression of chondrocyte markers (SRY-related HMG Box 9 (SOX9) and type II collagen (COL2)). Advanced glycation end products induced the expression of chondrocyte marker proteins downstream from the BMP4-Smad1 signaling pathway in MCs. In addition, hypoxia also induced the expression of BMP4, hypoxia-inducible factor-1α (HIF-1α), and chondrocyte markers. Overexpression of SOX9 caused ectopic expression of proteoglycans and COL2 in MCs. Furthermore, forced expression of Smad1 induced chondrocyte markers as well. Dorsomorphin inhibited these inductions. Glomerular expressions of HIF-1α, BMP4, and chondrocyte markers were observed in diabetic nephropathy mice. These positive stainings were observed in mesangial sclerotic lesions. SOX9 was partially colocalized with HIF-1α and BMP4 in diabetic glomeruli. BMP4 knock-in transgenic mice showed not only similar pathological lesions to DN, but also the induction of chondrocyte markers in the sclerotic lesions. Here we demonstrate that HIF-1α and BMP4 induce SOX9 expression and subsequent chondrogenic phenotype change in DN. The results suggested that the transdifferentiation of MCs into chondrocyte-like cells in chronic hypoxic stress may result in irreversible structural change in DN.

P2Y2 receptors and GRK2 are involved in oscillatory fluid flow induced ERK1/2 responses in chondrocytes

Mechanical loading is an important factor regulating cartilage metabolism maintained by chondrocytes. However, some of its underlying mechanisms remain poorly understood. In this study, we employed a chondrogenic cell line ATDC5 to investigate roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction. We first confirmed the expression of chondrocyte markers in differentiated ATDC5 cells. We then exposed both differentiated and undifferentiated ATDC5 cells to oscillatory fluid flow, and found that differentiated ATDC5 cells responded to oscillatory fluid flow by increasing COX-2 and aggrecan expressions. More importantly, fluid flow induced ERK1/2 response in differentiated cells was increased more than 10 times compared to those in undifferentiated cells. Furthermore, we found that P2Y(2) mRNA and protein levels in differentiated ATDC5 cells were significantly higher than those in undifferentiated cells. In contrast, GRK2 protein levels in differentiated cells were significantly lower than those in undifferentiated cells. Finally, overexpressions of P2Y(2) and GRK2 in differentiated ATDC5 cells result in a 34% increase and a 21% decrease of the ERK1/2 phosphorylation, respectively, in response to oscillatory fluid flow, suggesting important roles of P2Y(2) and GRK2 in chondrocyte mechanotransduction.

The Chondrogenic Potential of Mesenchymal Cells and Chondrocytes from Osteoarthritic Subjects: A Comparative Analysis.

Objective:The multipotential nature of stem or progenitor cells apparently makes them the ideal choice for any cell therapy, but this as yet remains to be proven. This study (30 subjects) was designed to compare the potential to repair articular cartilage of chondrocytes taken from different regions in osteoarthritic cartilage with that of mesenchymal stem cells prepared from bone marrow of the same subject.Design:Cartilage biopsies, bone marrow, and blood samples were taken from each of 30 individuals with chronic osteoarthritis (aged 62-85 years) undergoing total knee replacement. The chondrogenic potential of chondrocytes isolated from cartilage biopsies taken from different regions of osteoarthritic cartilage was compared with that of mesenchymal cells by quantitative analysis of several chondrocyte specific markers and an ex vivo cartilage differentiation assay.Results:Cartilage-derived articular chondrocytes are superior to bone marrow–derived cells when compared for their ex vivo chondrogenic potential. Interestingly, there was marked and significant difference in the expression of chondrocytic markers between chondrocytes derived from adjacent, visually distinct regions of the diseased cartilage. When cultured in the presence of a fibroblast growth factor 2 variant, all cell samples from both tissues showed a high degree of chondrogenic potential.Conclusions:Although bone marrow–derived mesenchymal cells, when supplemented with the appropriate chondrogenic factors, are a suitable source for autologous cartilage implantation, adult chondroprogenitor cells, particularly those from moderately affected regions of the osteoarthritic joints, demonstrate superior chondrogenic potential.

Electrospun cross‐linked gelatin fibers with controlled diameter: The effect of matrix stiffness on proliferative and biosynthetic activity of chondrocytes cultured in vitro

Nanofibrous scaffolds were prepared from gelatin solutions and were further cross-linked with glutaraldehyde (GA). The fiber diameter was varied from 100 to 1000 nm by controlling the applied voltage (4-15 kV) and the concentration of the gelatin solution (4-15%). The tensile moduli and the tensile strength of the noncross-linked scaffolds varied from 20 to 120 MPa and 0.5 to 3.5 MPa, respectively. Cross-linking with GA led to an increase in both the tensile modulus and strength and correlated with cross-linker concentration. Gelatin-based matrices were characterized by Fourier transform infrared spectroscopy and differential scanning calorimetry. High cellular viabilities and rounded morphology of chondrocytes was observed at the end of 7 days in culture with added matrix deposition and flattening of cells at 15 days. Matrix stiffness was noted to impact cell densities and the expression of chondrocytic markers, especially aggrecan. The ratios of collagen-II (C-II) to collagen-I (C-I) of 0.62 and 1.33 were noted on gelatin nanofibrous scaffolds cross-linked with 0.1% GA at the end of 7 and 15 days in culture, respectively. C-II/C-I ratios of 1.30 and 2.58 were noted on scaffolds cross-linked with 1.0% GA at the end of 7 and 15 days in culture, respectively.