Chondrogenic differentiation of murine C3H10T1/2 multipotential mesenchymal cells: I. Stimulation by bone morphogenetic protein-2 in high-density micromass cultures

Chondrogenic differentiation of mesenchymal cells is generally thought to be initiated by the inductive action of specific growth factors and depends on intimate cell-cell interactions. In this study, we have used multipotential murine C3H10T1/2 cells to analyze the effect and mechanism of action of bone morphogenetic protein 2 (BMP-2) on chondrogenesis. C3H10T1/2 cells have been previously shown to undergo multiple differentiation pathways. While chondrogenesis, osteogenesis, myogenesis and adipogenesis have been observed, chondrocytes appear significantly less frequently than the other cell types, and the appearance of chondrocytes exclusive of the other cell types has not been observed. We report here that the appearance of chondrocytes in C3H10T1/2 cells is markedly enhanced as a result of culture under conditions favorable for chondrogenesis, i.e. plating as high-density micromass and treatment with BMP-2. Such cultures contain chondrocyte-like cells, elaborate an Alcian blue stained cartilage-like matrix, express link protein and type II collagen, both cartilage matrix markers, and show increased [35S]sulfate incorporation. The appearance of Alcian blue positive material and increased sulfate incorporation are dependent on the dose of BMP-2, culture time, and cell plating density of the micromass cultures. Differentiation of cells within the micromass was specific to the chondrogenic lineage, as alkaline phosphatase staining revealed only faint staining in the micromass at the highest BMP-2 concentration. The importance of enhanced cell-cell interaction in the chondroinductive effects of BMP-2 on high-density C3H10T1/2 cultures was further implicated by the additional promotion of chondrogenesis in the presence of the polycationic compound, poly-L-lysine, which has been previously reported to enhance cellular interactions and chondrogenesis in embryonic limb mesenchymal cells. Taken together, these findings suggest that chondrogenesis in C3H10T1/2 cells is inducible by BMP-2 and requires cell-cell interaction.     

Efficient chondrogenic differentiation of mesenchymal cells in micromass culture by retroviral gene transfer of BMP-2

The multipotential murine embryonic C3H10T1/2 mesenchymal cell line is able to undergo chondrogenesis in vitro, in a high density micromass environment, following treatment with soluble human bone morphogenetic protein-2 (BMP-2). To enhance this process, the human BMP-2 cDNA was cloned into a retroviral expression vector and a high titer, infectious retrovirus (replication defective) was generated. Infection of C3HIOT1/2 cells with this retroviral construct resulted in an infection efficiency of 90-95% and was highly effective in converting cells in micromass culture to a chondrocyte phenotype, as assessed by positive Alcian blue staining for extracellular matrix proteoglycans, increased sulfate incorporation, increased expression of the cartilage marker genes collagen type II and aggrecan, and decreased expression of collagen type I. Interestingly, BMP-2 expression in the micromass cultures also induced the expression of the cell cycle inhibitory protein/differentiation factor p21/WAF1, suggesting its functional involvement in chondrogenesis. The chondrogenic effect of retrovirally expressed BMP-2 in these high-density cultures was limited to the infected cells, since uninfected cells did not chondrify when co-cultured as a nonoverlapping micromass adjacent to BMP-2 expressing cells. These data indicate that retrovirally expressed BMP-2 is highly effective at inducing a chondrocyte phenotype in a multipotential mesenchymal cell line in vitro, and its action is restricted to the infected cell population. These findings should provide a framework for the optimization of chondrogenesis in culture using mesenchymal stem cells and retroviral gene transfer.     

Regulation of adipose-derived adult stem cells differentiating into chondrocytes with the use of rhBMP-2

BACKGROUND: Adipose tissue has been demonstrated to contain a population of progenitor cells that can differentiate into bone and cartilage. Studies have suggested that adipose-derived adult stem (ADAS) cells can be induced to differentiate into chondrocytes by transforming growth factor-beta (TGF-beta). In this study, we examined whether bone morphogenetic protein-2 (BMP-2), as a member of the TGF-beta superfamily, could regulate ADAS cells to differentiate into a chondrolineage. METHODS: ADAS cells were isolated and induced by rhBMP-2. These cells were cultured in pellets for 2 weeks, and the chondrogenic phenotype was observed in vitro and in vivo. ADAS cells cultured without BMP-2 were used as controls. RESULTS: After 2 weeks of culture, the differentiated ADAS cells reacted positively to Alcian blue and collagen II, and the content of collagen II protein was obviously up-regulated at day 14. Glycosaminoglycan (GAG) content gradually increased from day 2 to day 14 (P < 0.05). However, H&E staining and collagen II expression were weak, and there was a little collagen II protein and GAG detected in the control group. Additionally, the pellets of ADAS cells induced by rhBMP-2 were transplanted into BALB/C nude mice and formed cartilage lacuna at week 8 in vivo. DISCUSSION: These data demonstrate that rhBMP-2 induce ADAS cells to differentiate into chondrocytes in vitro and in vivo. This is useful for basic and clinical studies aimed at repairing cartilage damage. But in a control group, ADAS cells tended towards differentiation into chondrocytes, which was affected by ITS. We will be exploring the mechanism further.     

Efficient differentiation of human iPSC‐derived mesenchymal stem cells to chondroprogenitor cells

Induced pluripotent stem cells (iPSC) hold tremendous potential for personalized cell‐based repair strategies to treat musculoskeletal disorders. To establish human iPSCs as a potential source of viable chondroprogenitors for articular cartilage repair, we assessed the in vitro chondrogenic potential of the pluripotent population versus an iPSC‐derived mesenchymal‐like progenitor population. We found the direct plating of undifferentiated iPSCs into high‐density micromass cultures in the presence of BMP‐2 promoted chondrogenic differentiation, however these conditions resulted in a mixed population of cells resembling the phenotype of articular cartilage, transient cartilage, and fibrocartilage. The progenitor cells derived from human iPSCs exhibited immunophenotypic features of mesenchymal stem cells (MSCs) and developed along multiple mesenchymal lineages, including osteoblasts, adipocytes, and chondrocytes in vitro. The data indicate the derivation of a mesenchymal stem cell population from human iPSCs is necessary to limit culture heterogeneity as well as chondrocyte maturation in the differentiated progeny. Moreover, as compared to pellet culture differentiation, BMP‐2 treatment of iPSC‐derived MSC‐like (iPSC–MSC) micromass cultures resulted in a phenotype more typical of articular chondrocytes, characterized by the enrichment of cartilage‐specific type II collagen (Col2a1), decreased expression of type I collagen (Col1a1) as well as lack of chondrocyte hypertrophy. These studies represent a first step toward identifying the most suitable iPSC progeny for developing cell‐based approaches to repair joint cartilage damage. J. Cell. Biochem. 114: 480–490, 2013. © 2012 Wiley Periodicals, Inc.     

Bone morphogenetic protein (BMP) localization in developing human and rat growth plate, metaphysis, epiphysis, and articular cartilage.

We assessed the distribution and relative staining intensity of bone morphogenetic protein (BMP)-1-7 by immunohistochemistry in tibial growth plates, epiphyses, metaphyses, and articular cartilage in one 21-week and one 22-week human fetus and in five 10-week-old Sprague-Dawley rats. In the rats, articular cartilage was also examined. BMP proteins were mostly cytoplasmic, with negligible matrix staining. Highest BMP levels were seen in (a) hypertrophic and calcifying zone chondrocytes of growth plate (BMP-1-7), (b) osteoblasts and/or osteoprogenitor fibroblasts and vascular cells of the metaphyseal cortex and medulla (BMP-1-6), (c) osteoclasts of the metaphysis and epiphysis (BMP-1,-4,-5, and -6), and (d) mid to deep zone articular chondrocytes of weanling rats (BMP-1-7). BMP staining in osteoclasts, an unexpected finding, was consistently strong with BMP-4, -5, and -6 but was variable and dependent on osteoclast location with BMP-2,-3, and -7. BMP-1-7 were moderately to intensely stained in vascular canals of human fetal epiphyseal cartilage by endothelial cells and pericytes. BMP-1,-3,-5,-6, and -7 were localized in hypertrophic chondrocytes adjacent to cartilage canals. We conclude that BMP expression is associated with maturing chondrocytes of growth plate and articular cartilage, and may play a role in chondrocyte differentiation and/or apoptosis. BMP appears to be expressed by osteoclasts and might be involved in the intercellular "cross-talk" between osteoclasts and neighboring osteoprogenitor cells at sites of bone remodeling.     

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

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-β1 and BMP-7. Real-time PCR analysis revealed the SZP expression was up-regulated by TGF-β1 and BMP-7. Chondrogenically differentiated IFP progenitor cells were stimulated by TGF-β1 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-β 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     

Chondroitin sulfate sulfation motifs as putative biomarkers for isolation of articular cartilage progenitor cells.

Osteoarthritis is a chronic, debilitating joint disease characterized by progressive destruction of articular cartilage. Recently, a number of studies have identified a chondroprogenitor cell population within articular cartilage with significant potential for repair/regeneration. As yet, there are few robust biomarkers of these cells. In this study, we show that monoclonal antibodies recognizing novel chondroitin sulfate sulfation motif epitopes in glycosaminoglycans on proteoglycans can be used to identify metabolically distinct subpopulations of cells specifically within the superficial zone of the tissue and that flow cytometric analysis can recognize these cell subpopulations. Fluorochrome co-localization analysis suggests that the chondroitin sulfate sulphation motifs are associated with a range of cell and extracellular matrix proteoglycans within the stem cell niche that include perlecan and aggrecan but not versican. The unique distributions of these sulphation motifs within the microenvironment of superficial zone chondrocytes, seems to designate early stages of stem/progenitor cell differentiation and is consistent with these molecules playing a functional role in regulating aspects of chondrogenesis. The isolation and further characterization of these cells will lead to an improved understanding of the role novel chondroitin sulfate sulfation plays in articular cartilage development and may contribute significantly to the field of articular cartilage repair.     

Protein kinase C delta null mice exhibit structural alterations in articular surface,intra-articular and subchondral compartments

IntroductionStructural alterations in intra-articular and subchondral compartments are hallmarks of osteoarthritis, a degenerative disease that causes pain and disability in the aging population. Protein kinase C delta (PKC-δ) plays versatile functions in cell growth and differentiation, but its role in the articular cartilage and subchondral bone is not known.MethodsHistological analysis including alcian blue, safranin O staining and fluorochrome labeling were used to reveal structural alterations at the articular cartilage surface and bone–cartilage interface in PKC-δ knockout (KO) mice. The morphology and organization of chondrocytes were studied using confocal microscopy. Glycosaminoglycan content was studied by micromass culture of chondrocytes of PKC-δ KO mice.ResultsWe uncovered atypical structural demarcation between articular cartilage and subchondral bone of PKC-δ KO mice. Histology analyses revealed a thickening of the articular cartilage and calcified bone–cartilage interface, and decreased safranin O staining accompanied by an increase in the number of hypertrophic chondrocytes in the articular cartilage of PKC-δ KO mice. Interestingly, loss of demarcation between articular cartilage and bone was concomitant with irregular chondrocyte morphology and arrangement. Consistently, in vivo calcein labeling assay showed an increased intensity of calcein labeling in the interface of the growth plate and metaphysis in PKC-δ KO mice. Furthermore, in vitro culture of chondrocyte micromass showed a decreased alcian blue staining of chondrocyte micromass in the PKC-δ KO mice, indicative of a reduced level of glycosaminoglycan production.ConclusionsOur data imply a role for PKC-δ in the osteochondral plasticity of the interface between articular cartilage and the osteochondral junction.

Electronic supplementary material

The online version of this article (doi:10.1186/s13075-015-0720-4) contains supplementary material, which is available to authorized users.     

Rat extramedullary adipose tissue as a source of osteochondrogenic progenitor cells

Human liposuction aspirates contain pluripotent adipose-derived mesodermal stem cells that have previously been shown to differentiate into various mesodermal cell types, including osteoblasts and chondrocytes. To develop an autologous research model of bone and cartilage tissue engineering, the authors sought to determine whether rat inguinal fat pads contain a similar population of osteochondrogenic precursor cells. It was hypothesized that the rat inguinal fat pad contains adipose-derived multipotential cells that resemble human adipose-derived mesodermal stem cells in their osteochondrogenic capacity. To test this, the authors assessed the ability of cells isolated from the rat inguinal fat pad to differentiate into osteoblasts and chondrocytes by a variety of lineage-specific histologic stains.Rat inguinal fat pads were isolated and processed from Sprague-Dawley rats into a fibroblast-like cell population. Cell cultures were placed in pro-osteogenic media containing dexamethasone, ascorbic acid, and beta-glycerol phosphate. Osteogenic differentiation was assessed at 2, 4, and 6 weeks. Alkaline phosphatase activity and von Kossa staining were performed to assess osteoblastic differentiation and the production of a calcified extracellular matrix. Cell cultures were also placed in prochondrogenic conditions and media supplemented with transforming growth factor-beta1, insulin, transferrin, and ascorbic acid. Chondrogenic differentiation was assessed at 2, 7, and 14 days by the presence of positive Alcian blue staining and type II collagen immunohistochemistry. Cells placed in osteogenic conditions changed in structure to a more cuboidal shape, formed bone nodules, stained positively for alkaline phosphatase activity, and secreted calcified extracellular matrix by 2 weeks. Cells placed in chondrogenic conditions formed cartilaginous nodules within 48 hours that stained positively for Alcian blue and type II collagen. The authors identified the rat inguinal fat pad as a source of osteochondrogenic precursors and developed a straightforward technique to isolate osteochondrogenic precursors from a small animal source. This relatively easily obtained source of osteochondrogenic cells from the rat may be useful for study of tissue engineering strategies and the basic science of stem cell biology.     

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

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 represent a potential alterative cell source for cartilage tissue engineering applications.     

Two subpopulations of differentiated chondrocytes identified with a monoclonal antibody to keratan sulfate

We have prepared a monoclonal antibody, named MZ15, that specifically binds keratan sulfate. Immunofluorescence studies showed that the distribution of keratan sulfate in articular cartilage was not uniform: the amount of keratan sulfate increased with distance from the articular surface. Two subpopulations of chondrocytes could be distinguished after isolation from cartilage by the presence or absence of cell surface keratan sulfate. Keratan sulfate-negative chondrocytes were shown to come from the upper cartilage layers. There was therefore a direct correlation between biochemical heterogeneity of cartilage matrix and heterogeneity within the chondrocyte population. During growth in monolayer culture, superficial chondrocytes began to synthesize keratan sulfate, but the cells could still be distinguished from cultures of deep or unfractionated chondrocytes by their reduced substrate adhesiveness and tendency to remain rounded.     

Biomechanics of single zonal chondrocytes

Articular cartilage has a distinct zonal architecture, and previous work has shown that chondrocytes from different zones exhibit variations in gene expression and biosynthesis. In this study, the material properties of single chondrocytes from the superficial and middle/deep zones of bovine distal metatarsal articular cartilage were determined using unconfined compression and digital videocapture. To determine the viscoelastic properties of zonal chondrocytes, unconfined creep compression experiments were performed and the resulting creep curves of individual cells were fit using a standard linear viscoelastic solid model. In the model, a fixed value of the Poisson's ratio was used, determined optically from direct compression of middle/deep chondrocytes. The two approaches used in this study yielded the following average material properties of single chondrocytes: Poisson's ratio of 0.26+/-0.08, instantaneous modulus of 1.06+/-0.82 kPa, relaxed modulus of 0.78+/-0.58 kPa, and apparent viscosity of 4.08+/-7.20 kPa s. Superficial zone chondrocytes were found to be significantly stiffer than middle/deep zone chondrocytes. Attachment time did not affect the stiffness of the cells. The zonal variation in viscoelastic properties may result from the distinct mechanical environments experienced by the cells in vivo. Identifying intrinsic differences in the biomechanics of superficial and middle/deep zone chondrocytes is an important component in understanding how biomechanics influence articular cartilage health and disease.     

Toward regeneration of articular cartilage

Articular cartilage is classified as permanent hyaline cartilage and has significant differences in structure, extracelluar matrix components, gene expression profile, and mechanical property from transient hyaline cartilage found in the epiphyseal growth plate. In the process of synovial joint development, articular cartilage originates from the interzone, developing at the edge of the cartilaginous anlagen, and establishes zonal structure over time and supports smooth movement of the synovial joint through life. The cascade actions of key regulators, such as Wnts, GDF5, Erg, and PTHLH, coordinate sequential steps of articular cartilage formation. Articular chondrocytes are restrictedly controlled not to differentiate into a hypertrophic stage by autocrine and paracrine factors and extracellular matrix microenvironment, but retain potential to undergo hypertrophy. The basal calcified zone of articular cartilage is connected with subchondral bone, but not invaded by blood vessels nor replaced by bone, which is highly contrasted with the growth plate. Articular cartilage has limited regenerative capacity, but likely possesses and potentially uses intrinsic stem cell source in the superficial layer, Ranvier's groove, the intra‐articular tissues such as synovium and fat pad, and marrow below the subchondral bone. Considering the biological views on articular cartilage, several important points are raised for regeneration of articular cartilage. We should evaluate the nature of regenerated cartilage as permanent hyaline cartilage and not just hyaline cartilage. We should study how a hypertrophic phenotype of transplanted cells can be lastingly suppressed in regenerating tissue. Furthermore, we should develop the methods and reagents to activate recruitment of intrinsic stem/progenitor cells into the damaged site. Birth Defects Research (Part C) 99:192–202, 2013 . © 2013 Wiley Periodicals, Inc .     

Flavonoid Compound Icariin Activates Hypoxia Inducible Factor-1α in Chondrocytes and Promotes Articular Cartilage Repair

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 marker genes including Mmp2, Mmp9, Mmp13, Adamts4 and Adamts5 was downregulated following Icariin treatment for 14 days. In a differentiation assay using bone marrow mesenchymal stem cells (MSCs) carrying HIF-1α floxed allele, the promotive effect of Icariin on chondrogenic differentiation is largely decreased following Cre recombinase-mediated deletion of HIF-1α in MSCs as indicated by Alcian blue staining for proteoglycan synthesis. In an alginate hydrogel 3D culture system, Icariin increases Safranin O positive (SO+) cartilage area. This phenotype is accompanied by upregulation of HIF-1α, increased proliferating cell nuclear antigen positive (PCNA+) cell numbers, SOX9+ chondrogenic cell numbers, and Col2 expression in the newly formed cartilage. Coincide with the micromass culture, Icariin treatment upregulates mRNA levels of Sox9, Col2α1, aggrecan and Col10α1 in the 3D cultures. We then generated alginate hydrogel 3D complexes incorporated with Icariin. The 3D complexes were transplanted in a mouse osteochondral defect model. ICRS II histological scoring at 6 and 12 weeks post-transplantation shows that 3D complexes incorporated with Icariin significantly enhance articular cartilage repair with higher scores particularly in selected parameters including SO+ cartilage area, subchondral bone and overall assessment than that of the controls. The results suggest that Icariin may inhibit PHD activity likely through competition for cellular iron ions and therefore it may serve as an HIF-1α activator to promote articular cartilage repair through regulating chondrocyte proliferation, differentiation and integration with subchondral bone formation.     

Induction of superficial zone protein (SZP)/lubricin/PRG 4 in muscle-derived mesenchymal stem/progenitor cells by transforming growth factor-β1 and bone morphogenetic protein-7

Introduction

Articular cartilage (AC) is an avascular tissue with precise polarity and organization. The three distinct zones are: surface, middle and deep. The production and accumulation of the superficial zone protein (SZP), also known as lubricin, by the surface zone is a characteristic feature of AC. To date, there is a wealth of evidence showing differentiation of AC from mesenchymal stem cells. Most studies that described chondrogenic differentiation did not focus on AC with characteristic surface marker SZP/lubricin. The present investigation was initiated to determine the induction of SZP/lubricin in skeletal muscle-derived mesenchymal stem/progenitor cells (MDMSCs) by transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-7 (BMP-7).

Methods

MDMSCs were cultured as a monolayer at a density of 1 × 10⁵ cells/well in 12-well tissue culture plates. Cell cultures were treated for 3, 7 and 10 days with TGF-β1 and BMP-7. The medium was analyzed for SZP. The cells were used to isolate RNA for RT-PCR assays for SZP expression.

Results

The SZP/lubricin increased in a time-dependent manner on Days 3, 7 and 10 in the medium. As early as Day 3, there was a three-fold increase in response to 3 ng/ml of TGF-β1 and 300 ng/ml of BMP-7. This was confirmed by immunochemical localization of SZP as early as Day 3 after treatment with TGF-β1. The expression of SZP mRNA was enhanced by TGF-β1.

Conclusions

The present investigation demonstrated the efficient and reproducible induction of SZP/lubricin accumulation by TGF-β1 and BMP-7 in skeletal MDMSCs. Optimization of the experimental conditions may permit the utility of MDMSCs in generating surface zone-like cells with phenotypic markers of AC and, therefore, constitute a promising cell source for tissue engineering approaches of superficial zone cartilage.     

Differentiation and mineralization of murine mesenchymal C3H10T1/2 cells in micromass culture

The murine mesenchymal cell line, C3H10T1/2 in micromass culture undergoes chondrogenic differentiation with the addition of BMP-2. This study compares the use of BMP-2 vs. insulin, transferrin, and sodium selenite (ITS) to create a chondrogenic micromass cell culture system that models cartilage calcification in the presence of 4 mM inorganic phosphate. BMP-2 treated cultures showed more intense alcian blue staining for proteoglycans than ITS treated cultures at early time points. Both ITS and BMP-2 treated cultures showed similar mineral deposition in cultures treated with 4 mM phosphate via von Kossa staining, however FTIR spectroscopy of cultures showed different matrix properties. ITS treated cultures produced matrix that more closely resembled mouse calcified cartilage by FTIR analysis. ⁴⁵Ca uptake curves showed delayed onset of mineralization in cultures treated with BMP-2, however they had an increased rate of mineralization (initial slope of ⁴⁵Ca uptake curve) when compared to the cultures treated with ITS. Immunohistochemistry showed the presence of both collagens type I and type II in BMP-2 and ITS treated control (1 mM inorganic phosphate) and mineralizing cultures. BMP-2 treated mineralizing cultures displayed more intense staining for collagen type II than all other cultures. Collagen type X staining was detected at Day 9 only in mineralizing cultures treated with ITS. Western blotting of Day 9 cultures confirmed the presence of collagen type X in the mineralizing ITS cultures, and also showed very small amounts of collagen type X in BMP-2 treated cultures and control ITS cultures. By Day 16 all cultures stained positive for collagen type X. These data suggest that BMP-2 induces a more chondrogenic phenotype, while ITS treatment favors maturation and hypertrophy of the chondrocytes in the murine micromass cultures.     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice     

Immunohistochemical localization of fibroblast growth factor-2 in normal and brachymorphic mouse tibial growth plate and articular cartilage

Epiphyses of the proximal tibiae of 7-week-old normal and homozygous recessive brachymorphic mice (bm/bm) were immunostained using a monoclonal antibody to basic fibroblast growth factor to determine its expression in growth plate cartilage, osteoblasts on the surfaces of the primary spongiosa and articular cartilage. In the normal growth plate, the immunoreactive factor was present in chondrocytes of the proliferating and upper hypertrophic zones but absent from lower hypertrophic chondrocytes. Immunostaining was present only in the territorial extracellular matrix immediately adjacent to the chondrocytes of the proliferating and upper hypertrophic zones. Osteoblasts of the primary spongiosa stained heavily in normal mice. Strong staining was observed in intermediate zone articular chondrocytes. Cells in the superficial layer of articular cartilage were unstained. The extracellular matrix of the articular cartilage was completely free of immunostaining. In contrast, the reduced size of bm/bm growth plates was accompanied by significantly reduced staining intensity in proliferating and upper hypertrophic chondrocytes, and staining was absent from the territorial extracellular matrix of all zones of the bm/bm growth plate. Osteoblasts of the primary spongiosa of bm/bm mice stained less than those of normal mice. Articular cartilage chondrocytes in the intermediate zone stained with less intensity in bm/bm mice, and the cells of the superficial layer were unstained. The extracellular matrix of bm/bm articular cartilage was completely free of staining. Brachymorphic epiphyseal growth plate and articular chondrocytes, and osteoblasts in the primary spongiosa, express reduced amounts of immunoreactive fibroblast growth factor-2. This phenotypical characteristic may be associated with abnormal endochondral ossification and development of bone in brachymorphic mice