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At least 19 recordsLinked to original sources
PubMed2003

Functional analysis of fibronectin isoforms in chondrogenesis: Full-length recombinant mesenchymal fibronectin reduces spreading and promotes condensation and chondrogenesis of limb mesenchymal cells.

Fibronectin (FN), a large dimeric glycoprotein, functions primarily as a connecting molecule in the extracellular matrices of tissues by mediating both cell-matrix and matrix-matrix interactions. All members of the FN family are products of a single FN gene; heterogeneity arises from the alternative splicing of at least three regions (IIIB, IIIA, and V) during processing of a common primary transcript. During chick embryonic limb chondrogenesis, FN structure changes from B+A+ in precartilage mesenchyme to B+A- in differentiated cartilage, and exon IIIA has been shown to be necessary for the process of mesenchymal cellular condensation, a requisite event that precedes overt expression of chondrocyte phenotype. This study aims to investigate the mechanistic action of the FN isoforms in mesenchymal chondrogenesis and, in particular, to identify the specific cellular function in mesenchymal condensation mediated by the mesenchymal (B+A+) FN isoform. Full-length cDNAs corresponding to four splice variants (B+A+, B+A-, B-A+, B-A-) of FN were constructed, and expressed the corresponding proteins using a baculovirus expression vector system. Cell adhesion assays with purified proteins showed that, although the relative levels of cell attachment were approximately the same, chick limb-bud mesenchymal cells spread up to 40 % less on mesenchymal (B+A+) FN than on cartilage (B+A-) FN, (B-A+) FN, or plasma (B-A-) FN. Cellular condensation and chondrogenic differentiation were also promoted in high-density micromass cultures of limb mesenchymal cells plated onto B+A+ FN. These observations suggest that the process of mesenchymal condensation is mediated at least in part by the enhanced ability of chondrogenic mesenchymal cells to migrate and aggregate as a consequence of residing in and interacting with mesenchymal FN. Our findings are consistent with and provide a mechanistic basis for previous observations that rounding of limb mesenchymal cells precedes the onset of chondrogenesis.

Animals↗
PubMed2004

Histochemical evidence of the initial chondrogenesis and osteogenesis in the periosteum of a rib fractured model: implications of osteocyte involvement in periosteal chondrogenesis.

We have examined cellular events at the early stages of periosteal chondrogenesis and osteogenesis induced by bone fracture, using a well-standardized rib fracture model of the mouse. The initial cellular event was recognized as considerable proliferation in the deeper layer referred to as the "cambium layer" of the periosteum, as evidenced by numerous proliferating cell nuclear antigen-positive cells. The periosteal cartilage and bone were then regenerated directly from the region of the most-differentiated cell, i.e., mature osteoblasts of the cambium layer both close to and distant from the fracture site. Therefore, periosteal osteoblasts appeared to have the potential to differentiate into chondrogenic and osteoblastic lineages. CD31-positive blood vessels were uniformly localized along the periosteum that was regenerating cartilage and bone, being therefore indicative of less influence on the initiation of osteochondrogenesis. In contrast, however, the regenerated periosteal cartilage or bone extended from the cortical bones included dead or living osteocytes, respectively. Empty lacunae and lacunae embedded with amorphous materials were found close to the regenerated cartilage, while intact osteocytes persisted adjacent to the regenerated bone. The embedded lacunae with amorphous materials would render the tissue fluid, nutrients, oxygen, and several secretory factors such as dentin matrix protein-1 impossible to be delivered to the periosteal osteoblasts that interconnect osteocytes via gap junctions. Our study thus provides two major clues on initial cellular events in response to bone fracture: the potentiality of periosteal osteoblastic differentiation into a chondrogenic lineage, and a putative involvement of osteocytes in periosteal cartilage and bone regeneration.

Animals↗
PubMed2000

Opposing role of mitogen-activated protein kinase subtypes, erk-1/2 and p38, in the regulation of chondrogenesis of mesenchymes.

The present studies were performed to determine subtype-specific roles of mitogen-activated protein kinase in chondrogenesis. Erk-1/2 activities, downstream of protein kinase C, decreased as chondrogenesis proceeded, whereas p38 activities, independent of protein kinase C, continuously increased during chondrogenesis. Inhibition of Erk-1/2 with PD98059 enhanced chondrogenesis up to 1. 7-fold, whereas inhibition of p38 with SB203580 reduced it to about 30% of the control level. Inhibition of Erk-1/2 or p38 did not affect precartilage condensation. However, cartilage nodule formation was significantly blocked by the inhibition of p38, whereas Erk-1/2 inhibition did not affect it. Modulation of chondrogenesis by the inhibition of Erk-1/2 and p38 was accompanied by altered expression of adhesion molecules in an opposite way. Expression of N-cadherin was reduced as chondrogenesis proceeded. Inhibition of p38 caused sustained expression of N-cadherin, whereas Erk-1/2 inhibition accelerated the reduction of N-cadherin expression. Expression of integrin alpha5beta1 and fibronectin were found to transiently increase during chondrogenesis. Inhibition of p38 caused continuous increase of expression of these molecules, whereas Erk-1/2 inhibition accelerated the decrease of expression of these molecules at a later period of chondrogenesis. Because temporal expression of these adhesion molecules regulates chondrogenesis, the above results indicate that Erk-1/2 and p38 conversely regulate chondrogenesis at post-precartilage condensation stages by modulating expression of adhesion molecules.

Animals↗
PubMed2000

Protein kinase A regulates chondrogenesis of mesenchymal cells at the post-precartilage condensation stage via protein kinase C-alpha signaling.

Chondrogenesis of mesenchymal cells during in vitro micromass culture requires the generation of cyclic adenosine monophosphate (cAMP) and subsequent activation of cAMP-dependent protein kinase A (PKA). In this study, we investigated the regulatory activity of PKA during chondrogenesis of chick limb bud mesenchymal cells. PKA activity was high in 1-day and 2-day cultures, which was followed by a slight decrease in 4-day and 5-day old cultures. Inhibition of PKA blocked chondrogenesis. It did not affect precartilage condensation, but it blocked the progression from the precartilage condensation stage to cartilage nodule formation. The PKA inhibition-induced blockage of chondrogenesis was accompanied by an altered expression of N-cadherin. Although expression of N-cadherin was detected during the early period of chondrogenesis, it became reduced as chondrogenesis proceeded. Still, inhibition of PKA maintained expression of N-cadherin throughout the micromass culture period. The inhibition of PKA did not affect expression of protein kinase C-alpha (PKCalpha), PKCepsilon, PKCdelta, and PKClambda/iota, which are the isoforms expressed in differentiating mesenchymal cells. However, PKA inhibition completely blocked activation of PKCalpha. Because PKC activity regulates N-cadherin expression and chondrogenesis, the PKA-mediated regulation of PKCalpha appears to be responsible for the PKA regulation of N-cadherin expression and chondrogenesis. Taken together, our results suggest that PKA regulates chondrogenesis by activating PKCalpha at the stage of post-precartilage condensation.

Animals↗
PubMed1997

Cyr61, product of a growth factor-inducible immediate-early gene, regulates chondrogenesis in mouse limb bud mesenchymal cells.

Chondrogenesis during embryonic skeletal development involves the condensation of mesenchymal cells followed by their differentiation into chondrocytes. We describe herein a previously unrecognized regulator of mammalian chondrogenesis encoded by a murine growth factor-inducible immediate-early gene, cyr61. The Cyr61 protein is a secreted, heparin-binding protein (379 amino acids with 38 conserved cysteines) that promotes cell adhesion, migration, and proliferation. The expression pattern of the cyr61 gene during embryogenesis is tissue specific and temporally regulated. Most notably, cyr61 is transiently expressed in mesenchymal cells of both mesodermal and neuroectodermal origins undergoing chondrogenesis, suggesting that Cyr61 may play a role in the development of the embryonic skeleton. In this communication, we demonstrate that the Cyr61 protein promotes chondrogenesis in micromass cultures of limb bud mesenchymal cells in vitro and is likely to play a similar role in vivo based on the following observations: (1) Cyr61 is present in the embryonic limb mesenchyme during chondrogenesis in vivo and in vitro; (2) purified recombinant Cyr61 protein added exogenously to micromass cultures promotes chondrogenesis as judged by precocious expression of type II collagen, increased [35S]sulfate incorporation, and larger Alcian blue-staining cartilage nodules; (3) Cyr61 enhances cell-cell aggregation, an initial step in chondrogenesis, and promotes chondrogenic differentiation in cultures plated at subthreshold cell densities that are otherwise unable to support differentiation; and (4) neutralization of the endogenous Cyr61 with specific antibodies inhibits chondrogenesis. Taken together, these results identify Cyr61 as a novel player in chondrogenesis that contributes to the development of the mammalian embryonic skeleton.

Animals↗
PubMed1998

Protein kinase C regulates chondrogenesis of mesenchymes via mitogen-activated protein kinase signaling.

A possible regulatory mechanism of protein kinase C (PKC) in the chondrogenesis of chick limb bud mesenchymes has been investigated. Inhibition or down-regulation of PKC resulted in the activation of a mitogen-activated protein kinase subtype Erk-1 and the inhibition of chondrogenesis. On the other hand, inhibition of Erk-1 with PD98059 enhanced chondrogenesis and relieved PKC-induced blockage of chondrogenesis. Erk-1 inhibition, however, did not affect expression and subcellular distribution of PKC isoforms expressed in mesenchymes nor cell proliferation. The results suggest that PKC regulates chondrogenesis by modulating Erk-1 activity. Inhibition or depletion of PKC inhibited proliferation of chondrogenic competent cells, and Erk-1 inhibition did not affect PKC modulation of cell proliferation. However, PKC-induced modulation of expression of cell adhesion molecules involved in precartilage condensation was reversed by the inhibition of Erk-1. Expression of N-cadherin was detected at the early period of chondrogenesis. Inhibition or depletion of PKC induced sustained expression of N-cadherin, and Erk-1 inhibition blocked the effects of PKC modulation. The expression of integrin alpha5 beta1 and fibronectin was found to be increased transiently during chondrogenesis. Depletion or inhibition of PKC caused a continuous increase of the expression of these molecules throughout the culture period, and Erk-1 inhibition abolished the modulating effects of PKC. Because reduction of the examined cell adhesion molecule expression is a prerequisite for the progression of chondrogenesis after cell condensation, our results indicate that PKC regulates chondrogenesis by modulating expression of these molecules via Erk-1 signaling.

Animals↗
PubMed1999

Effects of transforming growth factor-beta signaling on chondrogenesis in mouse chondrogenic EC cells, ATDC5.

Cellular condensation of chondroprogenitors is a distinct cellular event in chondrogenesis. During this process, N-cadherin mediates cell-cell interactions responsible for the initial stage of cellular condensation and subsequently fibronectin contributes to cell-matrix interactions mediating a progression of chondrogenesis. We previously showed that chondrogenesis in mouse chondrogenic EC cells, ATDC5, was induced, at a high incidence in the presence of insulin, through formation of cellular condensation. In this study, we took advantage of the sequential progression of chondrogenesis in ATDC5 cells and evaluated, in vitro in these cells, the role of endogenous transforming growth factor (TGF)-beta in chondrogenesis. ATDC5 cells expressed TGF-beta2 mRNA at a cellular condensation stage. The treatment of undifferentiated ATDC5 cells with anti-TGF-beta32 neutralizing antibody inhibited the accumulation of Alcian blue stainable proteoglycan in a dose-dependent manner. Transfection of a dominant-negative mutant of mouse TGF-beta type II receptor to undifferentiated ATDC5 cells completely inhibited cellular condensation. Moreover, exogenously administered TGF-beta2 upregulated the expression of fibronectin and type II collagen (a phenotypic marker gene of chondrogenesis) mRNAs and downregulated that of N-cadherin mRNA in time- and dose-dependent manners. These results indicate that TGF-beta stimulates chondrogenesis via initiation of cellular condensation by transition from an initial N-cadherin-contributing stage to a fibronectin-contributing stage during processes of chondrogenesis in ATDC5 cells.

Animals↗
PubMed2005

Transcriptional coactivator PGC-1alpha regulates chondrogenesis via association with Sox9.

Chondrogenesis is a multistep pathway in which multipotential mesenchymal stem cells (MSC) differentiate into chondrocytes. The transcription factor Sox9 (SRY-related high mobility group-Box gene 9) regulates chondrocyte differentiation and cartilage-specific expression of genes, such as Col2a1 (collagen type II alpha1). However, Sox9 expression is detected not only in chondrogenic tissue but also in nonchondrogenic tissues, suggesting the existence of a molecular partner(s) required for Sox9 to control chondrogenesis and chondrogenic gene expression. Here, we report identification of peroxisome proliferator-activated receptor gamma co-activator 1alpha (PGC-1alpha) as a coactivator for Sox9 during chondrogenesis. Expression of PGC-1alpha is induced at chondrogenesis sites during mouse embryonic limb development and during chondrogenesis in human MSC cultures. PGC-1alpha directly interacts with Sox9 and promotes Sox9-dependent transcriptional activity, suggesting that PGC-1alpha acts as a transcriptional coactivator for Sox9. Consistent with this finding, PGC-1alpha disruption in MSC by small interfering RNA inhibits Col2a1 expression during chondrogenesis. Furthermore, overexpression of both PGC-1alpha and Sox9 induced expression of chondrogenic genes, including Col2a1, followed by chondrogenesis in the MSC and developing chick limb. Together, our results suggest a transcriptional mechanism for chondrogenesis that is coordinated by PGC-1alpha.

Animals↗
PubMed1990

Characterization of bone-derived chondrogenesis-stimulating activity on embryonic limb mesenchymal cells in vitro.

Demineralized bone matrix contains factors which stimulate chondrogenesis and osteogenesis in vivo. A water-soluble extract of bone has been shown to stimulate chondrogenesis in vitro in embryonic limb mesenchymal cells (Syftestad, Lucas & Caplan, 1985). The aim of this study was to analyse the cellular mechanism of the bone-derived chondrogenesis-stimulating activity, with particular attention on how normal requirements for chondrogenesis may be altered. The effects of bovine bone extract (BBE) on chondrogenesis in vitro were studied using micromass cultures of chick limb bud mesenchyme isolated from embryos at Hamburger-Hamilton (HH) stage 23/24, an experimental system which is capable of undergoing chondrogenic differentiation. Bovine diaphyseal long bones were demineralized and extracted with guanidine-HCl to prepare BBE (Syftestad & Caplan, 1984). High-density mesenchyme cultures (30 x 10(6) cells/ml) were exposed to different doses of BBE (0.01-1.0 mg ml-1) and chondrogenesis was quantified based on cartilage nodule number and [35S]sulphate incorporation. BBE was tested on micromass cultures of varying plating densities (2-30 x 10(6) cells/ml), on cultures of 'young' limb bud cells (HH stage 17/18), and on cultures enriched with chondroprogenitor cells obtained from subridge mesoderm. Since poly-L-lysine (PL) has recently been shown (San Antonio & Tuan, 1986) to promote chondrogensis, PL and BBE were introduced together in different doses, in the culture medium, to determine if their actions were synergistic. Our results show that BBE stimulates chondrogenesis in a dose-dependent manner and by a specific, direct action on the chondroprogenitor cells but not in normally non-chondrogenic, low density or 'young' limb bud cell cultures. The effects of PL and BBE are additive and these agents appear to act by separate mechanisms to stimulate chondrogenesis; PL primarily enhances nodule formation, and BBE appears to promote nodule growth.

Animals↗
PubMed1995

Differential expression of neural cell adhesion molecule (NCAM) during osteogenesis and secondary chondrogenesis in the embryonic chick.

Progenitor cells in the periosteum-perichondrium of the posterior hook of the quadratojugal (QJ, a membrane bone) in the embryonic chick are bipotential for osteogenesis and chondrogenesis. These cells switch from osteogenesis to chondrogenesis between 10 to 11 days in normal (mobile) embryos but not in paralyzed (immobile) embryos. Expression of neural cell adhesion molecule (NCAM) was studied using a monoclonal antibody in QJ hooks from normal and paralyzed chick embryos between 10 and 21 days of incubation. NCAM is expressed in osteoprogenitor cells and osteoblasts but not in chondroprogenitor cells, chondroblasts, or chondrocytes. The switch of progenitor cell differentiation from an osteogenic to a chondrogenic pathway between 10 and 11 days of incubation coincides with down-regulation of NCAM expression. Both initiation of secondary chondrogenesis and down-regulation of NCAM depend on biomechanical stimulation. In embryos paralyzed at 9 days, secondary cartilage fails to form and progenitor cells remain positive for NCAM. Furthermore, paralysis influences NCAM expression in progenitor cells before secondary chondrogenesis morphologically begins, indicating that NCAM may play a role in the initiation of secondary chondrogenesis. In 15-day normal embryos, NCAM-positive cells accumulate between the perichondrium and secondary cartilage in a position that prevents further cartilage formation in the hook. In 19-day embryos, these cells lose their NCAM expression and restart chondrogenesis in a second phase of differentiation, forming an articular cartilage. Loss of NCAM expression in this cell layer and re-commencement of chondrogenesis do not occur in embryos paralyzed at 13 days, and therefore also require biomechanical stimulation. Hence, down-regulation of NCAM expression correlates with two phases of secondary chondrogenesis in embryonic life, both of which are dependent upon embryonic movement.

Animals↗
PubMed2000

Positionally-dependent chondrogenesis induced by BMP4 is co-regulated by Sox9 and Msx2.

Cranial neural crest cells emigrate from the posterior midbrain and anterior hindbrain to populate the first branchial arch and eventually differentiate into multiple cell lineages in the maxilla and mandible during craniofacial morphogenesis. In the developing mouse mandibular process, the expression profiles of BMP4, Msx2, Sox9, and type II collagen demonstrate temporally and spatially restrictive localization patterns suggestive of their functions in the patterning and differentiation of cartilage. Under serumless culture conditions, beads soaked in BMP4 and implanted into embryonic day 10 (E10) mouse mandibular explants induced ectopic cartilage formation in the proximal position of the explant. However, BMP4-soaked beads implanted at the rostral position did not have an inductive effect. Ectopic chondrogenesis was associated with the up-regulation of Sox9 and Msx2 expression in the immediate vicinity of the BMP4 beads 24 hours after implantation. Control beads had no effect on cartilage induction or Msx2 and Sox9 expression. Sox9 was induced at all sites of BMP4 bead implantation. In contrast, Msx2 expression was induced more intensely at the rostral position when compared with the proximal position, and suggested that Msx2 expression was inhibitory to chondrogenesis. To test the hypothesis that over-expression of Msx2 inhibits chondrogenesis, we ectopically expressed Msx2 in the mandibular process organ culture system using adenovirus gene delivery strategy. Microinjection of the Msx2-adenovirus to the proximal position inhibited BMP4-induced chondrogenesis. Over-expression of Msx2 also resulted in the abrogation of endogenous cartilage and the down-regulation of type II collagen expression. Taken together, these results suggest that BMP4 induces chondrogenesis, the pattern of which is positively regulated by Sox9 and negatively by Msx2. Chondrogenesis only occurs at sites where Sox9 expression is high relative to that of Msx2. The combinatorial action of these transcription factors appear to establish a threshold for Sox9 function and thereby restricts the position of chondrogenesis.

Animals↗
PubMed1999

N-Cadherin expression and signaling in limb mesenchymal chondrogenesis: stimulation by poly-L-lysine.

Cellular condensation is a requisite step in the initiation of mesenchymal chondrogenesis in the embryonic limb bud. We have previously shown that cellular condensation of limb chondroprogenitor mesenchymal cells is accompanied by elevated expression of N-cadherin during chondrogenesis both in vivo and in vitro. N-Cadherin-mediated cell-cell interaction is also functionally required for proper mesenchymal chondrogenesis both in vivo and in vitro. In this report, we have further analyzed the functional importance of N-cadherin in the cellular condensation-chondrogenesis pathway by examining N-cadherin expression and related activities in high density micromass cultures of chick limb mesenchymal cells in which chondrogenesis is being stimulated with the cationic polymer, poly-L-lysine (PL). The chondrogenesis-promoting action of PL is thought to involve the clustering of cells via ionic cross-linking, perhaps mimicking the action of an endogenous matrix component. Immunohistochemistry, immunoblotting, and Northern blot analysis all show that PL treatment results in a time-dependent increase in N-cadherin expression at both the protein and mRNA levels. In addition, inhibition of N-cadherin function with a neutralizing monoclonal antibody directed to its extracellular domain inhibits the chondrogenesis-stimulating effect of PL. PL treatment also alters the tyrosine-phosphorylation state of the N-cadherin associated signaling protein, beta-catenin. These results suggest that N-cadherin-mediated cell adhesion is a requisite regulatory component of the limb mesenchymal chondrogenic differentiation program, involving at least in part beta-catenin tyrosine phosphorylation as a signaling step.

Animals↗
PubMed2001

The enhancement of periosteal chondrogenesis in organ culture by dynamic fluid pressure.

Cartilage repair by autologous periosteal arthroplasty is enhanced by continuous passive motion (CPM) of the joint after transplantation of the periosteal graft. However, the mechanisms by which CPM stimulate chondrogenesis are unknown. Based on the observation that an oscillating intra-synovial pressure fluctuation has been reported to occur during CPM (0.6-10 kPa), it was hypothesized that the oscillating pressure experienced by the periosteal graft as a result of CPM has a beneficial effect on the chondrogenic response of the graft. We have developed an in vitro model with which dynamic fluid pressures (DFP) that mimic those during CPM can be applied to periosteal explants while they are cultured in agarose gel suspension. In this study periosteal explants were treated with or without DFP during suspension culture in agarose, which is conducive to chondrogenesis. Different DFP application times (30 min, 4 h, 24 h/day) and pressure magnitudes (13, 103 kPa or stepwise 13 to 54 to 103 kPa) were compared for their effects on periosteal chondrogenesis. Low levels of DFP (13 kPa at 0.3 Hz) significantly enhanced chondrogenesis over controls (34 +/- 7% vs 14 +/- 5%; P < 0.05), while higher pressures (103 kPa at 0.3 Hz) completely inhibited chondrogenesis, as determined from the percentage of tissue that was determined to be cartilage by histomorphometry. Application of low levels of DFP to periosteal explants also resulted in significantly increased concentrations of Collagen Type II protein (43 +/- 8% vs 10 +/- 5%; P < 0.05). New proteoglycan synthesis, as measured by 35S-sulphate uptake was increased by 30% in periosteal explants stimulated with DFP (350 +/- 50 DPM vs 250 +/- 75 DPM of 35S-sulphate uptake/microg total protein), when compared to controls though this difference was not statistically significant. The DFP effect at low levels was dose-dependant for time of application as well, with 4 h/day stimulation causing significantly higher chondrogenesis than just 30 min/day (34 +/- 7 vs 12 +/- 4% cartilage; P < 0.05) and not significantly less than that obtained with 24 h/day of DFP (48 +/- 9% cartilage, P > 0.05). These observations may partially explain the beneficial effect on cartilage repair by CPM. They also validate an in vitro model permitting studies aimed at elucidating the mechanisms of action of mechanical factors regulating chondrogenesis. The fact that these tissues were successfully cultured in a mechanical environment for six weeks makes it possible to study the actions of mechanical factors on the entire chondrogenic pathway, from induction to maturation. Finally, these data support the theoretical predictions regarding the role of hydrostatic compression in fracture healing.

Animals↗
PubMed1998

The G3 domain of versican inhibits mesenchymal chondrogenesis via the epidermal growth factor-like motifs.

Versican is a highly expressed proteoglycan in zones of developing tissues. To investigate whether versican plays a role in cell differentiation, we studied its role in mesenchymal condensation and chondrogenesis. Here we report that a mini-versican gene product inhibits mesenchymal chondrogenesis but not condensation. The mini-versican-treated mesenchymal cultures form fewer, smaller cartilaginous nodules and produced lower levels of link protein and type II collagen. The versican G3 domain alone, but not G1, was sufficient to inhibit mesenchymal chondrogenesis. Deletion of two epidermal growth factor (EGF)-like motifs in the G3 domain abolished the effect of versican. The G3 domain of aggrecan, which does not contain an EGF-like motif, did not inhibit mesenchymal chondrogenesis. We also generated a chimera construct containing the two EGF-like motifs of versican and the G3 domain of aggrecan, and we observed that this chimera construct inhibited chondrogenesis to a lesser extent than did the full-length versican G3 construct. Direct transfection of mesenchymal cells with different constructs produced similar results. Furthermore, treatment with versican antisense oligonucleotides and transfection with a versican antisense construct promoted chondrogenesis. Taken together, our results strongly suggest that versican inhibits mesenchymal chondrogenesis via its EGF-like motifs.

3T3 Cells↗
PubMed2006

In vitro chondrogenesis of human synovium-derived mesenchymal stem cells: optimal condition and comparison with bone marrow-derived cells.

There are increasing reports that mesenchymal stem cells (MSCs) are present in various tissues other than bone marrow, including synovium. Here we investigated the optimal conditions for in vitro chondrogenesis of human synovium-derived MSCs and compared these cells with bone marrow-derived MSCs, especially in terms of their chondrogenesis potential. Synovium and bone marrow were harvested from six donors during knee operations for ligament injuries. Digested synovium cells or nucleated cells from bone marrow were expanded clonally. A pellet culture system was used for chondrogenesis, and the best combination of up to three cytokines of the seven assessed. Synovium-derived MSCs plated at a lower density expanded more rapidly. Contrary to previous reports, a combination of TGFbeta and dexamethasone was not sufficient to induce chondrogenesis. However, addition of BMP2 to TGFbeta and dexamethasone dramatically increased cartilage pellet size and the synthesis of cartilage matrix. The cartilage pellets were also analyzed by electron microscopy and immunohistology. DNA content per pellet decreased during chondrogenesis, indicating the pellet increased its size through the accumulation of newly synthesized extracellular matrix. Sequential chondrogenic gene expression was demonstrated by RT-PCR. Synovium-derived MSCs looked similar to the bone marrow-derived MSCs in their surface epitopes and proliferation potential; however, cartilage pellets from synovium were significantly larger than those from bone marrow in patient-matched comparisons. We demonstrated that the combination of TGFbeta, dexamethasone, and BMP2 was optimal for in vitro chondrogenesis of synovium-derived MSCs and that the synovium-derived MSCs have a greater chondrogenesis potential than bone marrow-derived MSCs.

Adolescent↗
PubMed1995

Expression of protein kinase C isozymes that are required for chondrogenesis of chick limb bud mesenchymal cells.

Protein kinase C (PKC) has been suggested to be involved in the chondrogenesis of chick limb bud mesenchymal cells. This study examined the expression and the role of PKC isozymes in chondrogenesis. Multiple PKC isozymes such as conventional PKC (cPKC alpha and gamma), new PKC (nPKC epsilon), and atypical PKC (aPKC zeta, lambda, and tau) were expressed in chondroblasts but cPKC beta and nPKC delta were not detected. The amounts of expressed cPKC and nPKC isozymes, namely cPKC alpha and gamma and nPKC epsilon, were increased as chondrogenesis proceeds while the level of aPKC isozymes was not changed. Treatment of cells with specific PKC inhibitors blocked chondrogenesis. Prolonged exposure of cells to phorbol ester which down regulates both cPKC and nPKC also blocked chondrogenic differentiation. The inhibition of chondrogenesis was the most effective when PKC activity was blocked at the early stage of chondrogenesis (i.e., for the first 24 hours of micromass culture). Down regulation of PKC blocked both proliferation of cells and synthesis of sulfated proteoglycans, indicating that expression of cPKC and nPKC is required at early stage of chondrogenesis.

Animals↗
PubMed1994

Chondrogenesis of neural crest cells: effect of poly-L-lysine and bone extract.

The mechanisms of chondrogenic differentiation are generally studied in vitro by analyzing the action of agents that promote or affect chondrogenesis in embryonic mesenchyme, such as cells of the embryonic limb bud. However, it is not known whether progenitor cells of the craniofacial skeleton, which are of a different embryonic origin and derived from the neural crest, are similarly responsive to such agents. To gain insight into the regulation of chondrogenic differentiation in cells derived from neural crest, we have treated chick embryonic neural crest explants in vitro with poly-L-lysine (PL, M(r) 380 kDa) or bovine bone extract (BBE), two agents known to enhance chondrogenesis of limb mesenchymal cells. Both cephalic (normally chondrogenic) and trunk (normally nonchondrogenic) neural crest cells were analyzed. Chondrogenic differentiation was determined by histological, immunohistochemical and autoradiographic methods. Our results indicate that both PL (380 kDa) and BBE significantly enhance chondrogenesis of cephalic neural crest cells, suggesting that the mechanism of chondrogenesis of these ectodermally derived cells is similar to that of mesodermally derived limb mesenchymal cells. However, trunk neural crest cells did not undergo chondrogenesis in response to PL or BBE. These data show that chondrogenesis can be enhanced in cranial ectodermal neural crest cells in a manner similar to that in the limb mesenchyme. However, since nonchondrogenic trunk neural crest cells are not responsive, an inherent potential for cartilaginous differentiation is necessary for exogenous stimulation of chondrogenesis.

Animals↗