Skeletal dysplasia and defective chondrocyte differentiation by targeted overexpression of fibroblast growth factor 9 in transgenic mice.

Mutations in fibroblast growth factor receptor 3 (FGFR3) cause several human chondrodysplasias, including achondroplasia, the most common form of dwarfism in humans. From in vitro studies, the skeletal defects observed in these disorders have been attributed to constitutive activation of FGFR3. Here we show that FGF9 and FGFR3, a high-affinity receptor for this ligand, have similar developmental expression patterns, particularly in areas of active chondrogenesis. Targeted overexpression of FGF9 to cartilage of transgenic mice disturbs postnatal skeletal development and linear bone growth. The growth plate of these mice exhibits reduced proliferation and terminal differentiation of chondrocytes similar to that observed in the human disorders. The observations provide evidence that targeted, in vivo activation of endogenous FGFR3 inhibits bone growth and demonstrate that signals derived from FGF9-FGFR3 interactions can physiologically block endochondral ossification to produce a phenotype characteristic of the achondroplasia group of human chondrodysplasias.     

Systems genetics analysis of mouse chondrocyte differentiation

One of the goals of systems genetics is the reconstruction of gene networks that underlie key processes in development and disease. To identify cartilage gene networks that play an important role in bone development, we used a systems genetics approach that integrated microarray gene expression profiles from cartilage and bone phenotypic data from two sets of recombinant inbred strains. Microarray profiles generated from isolated chondrocytes were used to generate weighted gene coexpression networks. This analysis resulted in the identification of subnetworks (modules) of coexpressed genes that then were examined for relationships with bone geometry and density. One module exhibited significant correlation with femur length (r = 0.416), anteroposterior diameter (r = 0.418), mediolateral diameter (r = 0.576), and bone mineral density (r = 0.475). Highly connected genes (n = 28) from this and other modules were tested in vitro using prechondrocyte ATDC5 cells and RNA interference. Five of the 28 genes were found to play a role in chondrocyte differentiation. Two of these, Hspd1 and Cdkn1a, were known previously to function in chondrocyte development, whereas the other three, Bhlhb9, Cugbp1, and Spcs3, are novel genes. Our integrative analysis provided a systems‐level view of cartilage development and identified genes that may be involved in bone development. © 2011 American Society for Bone and Mineral Research.     

Bovine articular chondrocyte function in vitro depends upon oxygen tension

Articular cartilage is a physiologically hypoxic tissue with a proposed gradient of oxygen tension ranging from about 10% oxygen at the cartilage surface to less than 1% in the deepest layers. The position of the chondrocyte within this gradient may modulate the cell's behavior and phenotype. Moreover, the oxygen gradient is likely to be disturbed during joint diseases in which the pO(2)of the synovial fluid declines which may cause changes in chondrocyte behavior and gene expression. Thus, there is a need to understand the chondrocyte's response to different oxygen tensions. We compared the behavior of bovine articular chondrocytes cultured in alginate beads for 7 days in medium maintained at <0.1, 5, 10 or 20% oxygen. The chondrocytes' survival, differentiation, cell division, viability and matrix production were assessed at each oxygen tension and rRNA and mRNA abundance was measured.Chondrocytes were able to survive under all oxygen tensions for at least 7 days but cells cultured under anoxic conditions were metabolically less active than cells maintained in higher oxygen tensions; this was associated with a decrease in matrix production. In <0.1% oxygen there was a marked decrease in rRNA and mRNA abundance in the cells. There were no differences in cell division or differentiation between any oxygen tensions. These findings indicate that articular chondrocytes can be cultured successfully in the pO(2)range in which they are thought to exist in vivo (5-10% pO(2)) and are fully active under these conditions. Under anoxic conditions (<0.1% pO(2)) function is severely compromised.     

Bioassay of chondrocyte differentiation by bone morphogenetic protein

For sensitive bioassay of cartilage development in response to bone morphogenetic protein (BMP), the supernatant of interstitial fluid containing crud BMP was obtained from an implanted diffusion chamber, applied to a cellulose acetate film, and stained with Alcian blue. The optical density of the Alcian blue-stained spot was measured by a densitometer for quantitative analysis. Density of the Alcian blue-stained spot was markedly increased when cartilage developed outside the chamber. Occurrence of cartilage-specific proteoglycan-H (PG-H) and Type II collagen was seen in this intensely stained spot by dot-blot analysis. Electron microscopic examination revealed that the residue of interstitial fluid in the implanted diffusion chamber contained many collagen fibrils and proteoglycan granules.     

The use of debrided human articular cartilage for autologous chondrocyte implantation: maintenance of chondrocyte differentiation and proliferation in type I collagen gels.

Autologous chondrocyte implantation (ACI) is the most promising surgical treatment for large full thickness knee joint articular cartilage (AC) defects where cells from healthy non-weight bearing area AC are multiplied in vitro and implanted into such defects. In the routine surgical procedure for symptomatic knee full thickness AC defects, damaged AC surrounding the edge and the base of such defects is usually debrided and discarded. The purpose of this study was to examine if chondrocytes from this 'debrided' AC can proliferate, synthesize a cartilage specific matrix and thus can be used for ACI. METHODS: Biopsies were retrieved from 12 patients (debrided articular cartilage: DAC, aged 35-61) and from two autopsies (normal articular cartilage: NAC, aged 21 and 25). Chondrocytes were isolated, seeded at low density in type I collagen gels and as monolayer cultures for 4 weeks without passage. RESULTS: After 4 weeks cultures in type I collagen gels, cell proliferation from DAC (18.34 +/- 1.95 fold) was similar to cells from NAC (11.24 +/- 1.02 fold). Syntheses of proteoglycan and collagen in DAC were also similar to NAC. Newly synthesized matrices in gel cultures consisted predominantly of type II collagen as shown by immuno-labelling and SDS-PAGE followed by fluorography. Chondrocytes from 'debrided human AC' cultured at low density in type I collagen gels may be used for the ACI procedure as they provide sufficient viable cell numbers for ACI and maintain their chondrocyte phenotype as they synthesize a cartilage-like matrix.     

Wnt gene expression in the post-natal growth plate: regulation with chondrocyte differentiation

Longitudinal growth of long bones occurs at the growth plate by endochondral ossification. In the embryonic mouse, this process is regulated by Wnt signaling. Little is known about which members of the Wnt family of secreted signaling proteins might be involved in the regulation of the postnatal growth plate. We used microdissection and real-time PCR to study mRNA expression of Wnt genes in the mouse growth plate. Of the 19 known members of the Wnt family, only six were expressed in postnatal growth plate. Of these, Wnts -2b, -4, and -10b signal through the canonical beta-catenin pathway and Wnts -5a, -5b, and -11 signal through the noncanonical calcium pathway. The spatial expression for these six Wnts was remarkably similar, showing low mRNA expression in the resting zone, increasing expression as the chondrocytes differentiated into the proliferative and prehypertrophic state and then (except Wnt-2b) decreasing expression as the chondrocytes underwent hypertrophic differentiation. This overall pattern is broadly consistent with previous studies of embryonic mouse growth cartilage suggesting that Wnt signaling modulates chondrocyte proliferation and hypertrophic differentiation. We also found that mRNA expression of these Wnt genes persisted at similar levels at 4 weeks, when longitudinal bone growth is waning. In conclusion, we have identified for the first time the specific Wnt genes that are expressed in the postnatal mammalian growth plate. The six identified Wnt genes showed a similar pattern of expression during chondrocyte differentiation, suggesting overlapping or interacting roles in postnatal endochondral bone formation.     

BMP-6 is an autocrine stimulator of chondrocyte differentiation.

While parathyroid hormone-related protein (PTHrP) has been characterized as an important negative regulator of chondrocyte maturation in the growth plate, the autocrine or paracrine factors that stimulate chondrocyte maturation are not well characterized. Cephalic sternal chondrocytes were isolated from 13-day embryos, and the role of bone morphogenetic protein-6 (BMP-6) as a positive regulator of chondrocyte maturation was examined in monolayer cultures. Progressive maturation, which was accelerated in the presence of ascorbate, occurred in the cultures. During maturation, the cultures expressed high levels of BMP-6 mRNA which preceded the induction of type X collagen mRNA. Treatment of the cultures with PTHrP (10(-7) M) at the time of plating completely abolished BMP-6 and type X collagen mRNA expression. Removal of PTHrP after 6 days was followed by the rapid (within 24 h) expression of BMP-6 and type X collagen mRNA, with BMP-6 again preceding type X collagen expression. The addition of exogenous BMP-6 (100 ng/ml) to the cultures accelerated the maturation process both in the presence and absence of ascorbate and resulted in the highest levels of type X collagen. When exogenous BMP-6 was added to PTHrP containing cultures, maturation occurred with the expression of high levels of type X collagen, despite the presence of PTHrP in the cultures. Furthermore, BMP-6 did not stimulate expression of its own mRNA in the PTHrP treated cultures, but it did stimulate the expression of Indian hedgehog (Ihh) mRNA. These latter findings suggest that while PTHrP directly inhibits BMP-6, it indirectly regulates Ihh expression through BMP-6. Other phenotypic changes associated with chondrocyte differentiation were also stimulated by BMP-6, including increased alkaline phosphatase activity and decreased proliferation. The results suggest that BMP-6 is an autocrine factor that initiates chondrocyte maturation and that PTHrP may prevent maturation by inhibiting the expression of BMP-6.     

Leptin regulates chondrocyte differentiation and matrix maturation during endochondral ossification

Leptin has been suggested to mediate a variety of actions, including bone development, via its ubiquitously expressed receptor (Ob-Rb). In this study, we investigated the role of leptin in endochondral ossification at the growth plate. The growth plates of wild-type and ob/ob mice were analyzed. Effects of leptin on chondrocyte gene expression, cell cycle, apoptosis and matrix mineralization were assessed using primary chondrocyte culture and the ATDC5 cell differentiation culture system. Immunohistochemistry and in situ hybridization showed that leptin was localized in prehypertrophic chondrocytes in normal mice and that Ob-Rb was localized in hypertrophic chondrocytes in normal and ob/ob mice. Growth plates of ob/ob mice were more fragile than those of wild-type mice in a mechanical test and were broken easily at the chondro-osseous junction. The growth plates of ob/ob mice showed disturbed columnar structure, decreased type X collagen expression, less organized collagen fibril arrangement, increased apoptosis and premature mineralization. Leptin administration in ob/ob mice led to an increase in femoral and humeral lengths and decrease in the proportional length of the calcified hypertrophic zone to the whole hypertrophic zone. In primary chondrocyte culture, the matrix mineralization in ob/ob chondrocytes was stronger than that of wild-type mice; this mineralization in both types of mice was abolished by the addition of exogenous leptin (10 ng/ml). During ATDC5 cell differentiation culture, exogenous leptin at a concentration of 1-10 ng/ml (equivalent to the normal serum concentration of leptin) altered type X collagen mRNA expression and suppressed apoptosis, cell growth and matrix calcification. In conclusion, we demonstrated that leptin modulates several events associated with terminal differentiation of chondrocytes. Our finding that the growth plates of ob/ob mice were fragile implies a disturbance in the differentiation/maturation process of growth plates due to depletion of leptin signaling in ob/ob mice. These findings suggest that peripheral leptin signaling plays an essential role in endochondral ossification at the growth plate.     

In vitro chondrocyte differentiation using costochondral chondrocytes as a source of primary rat chondrocyte cultures: an improved isolation and cryopreservation method

INTRODUCTION: Isolating and culturing primary chondrocytes such that they retain their cell type and differentiate to a hypertrophic state is central to many investigations of skeletal growth and its regulation. The ability to store frozen chondrocytes has additional scientific and tissue engineering interest. Previous work has produced approaches of varying yield and complexity but does not permit frozen storage of cells for subsequent differentiation in culture. Investigations of growth plate dysplasias secondary to defective osteoclastogenesis in rodent models of osteopetrosis led us to adapt and modify a culture method and to cryopreserve neonatal rat costochondral chondrocytes. METHODS: Chondrocytes were isolated from dissected ribs of 3-day-old rat pups by collagenase, hyaluronidase, and trypsin serial digestions. This was done either immediately or after the isolation was interrupted following an initial protease treatment to allow the chondrocytes, still in partially digested rib rudiments, to be frozen and later thawed for culture. Cells were plated in flat-bottom wells and allowed to adhere and grow under different conditions. Choice of media permitted cells to be maintained or induced to differentiate. Cell growth was monitored, as was expression of several relevant genes: collagen types II and X; osteocalcin, Sox9, adipocyte FABP, MyoD, aggrecan, and others. Mineralization was measured by alizarin red binding, and cultures were examined by light, fluorescence, and electron microscopy. RESULTS: Cells retained their chondrocyte phenotype and ability to differentiate and mineralize the collagen-rich extracellular matrix even after freezing-thawing. RT-PCR showed retention of chondrocyte-specific gene expression, including aggrecan and collagen II. The cells had a flattened, "proliferating zone" appearance initially, and by 2 weeks post-confluence, exhibited swelling and other salient features of hypertrophic cells seen in vivo. Collagen fibrils were abundant in the extracellular matrix, along with matrix vesicles. The switch to collagen type X as marker for hypertrophy was not rigidly temporally regulated as happens in vivo, but its expression increased during hypertrophic differentiation. CONCLUSIONS: This method should prove valuable as a means of studying chondrocyte regulation and has the advantages of simpler initial dissection, yields of a purer chondrocyte population, and the ability to stockpile frozen raw material for subsequent studies.     

Gene expression and cell differentiation in matrix-associated chondrocyte transplantation grafts: a comparative study

Objective

Although scaffold composition and architecture are considered to be important parameters for tissue engineering, their influence on gene expression and cell differentiation is rarely investigated in scaffolds used for matrix-associated autologous chondrocyte transplantation (MACT). In this study we have therefore comparatively analyzed the gene expression of important chondrogenic markers in four clinical applied cell-graft systems with very different scaffold characteristics.

Methods

Residuals (n = 165) of four different transplant types (MACI^®, Hyalograft^®C, CaReS^® and Novocart^®3D) were collected during surgery and analyzed for Col1, Col2, aggrecan, versican, melanoma inhibitory activity (MIA) and IL-1β by real-time PCR. Scaffold and cell morphology were evaluated by histology and electron microscopy.

Results

Despite the cultivation on 3D scaffolds, the cell differentiation on all transplant types didn’t reach the levels of native cartilage. Gene expression highly differed between the transplant types. The highest differentiation of cells (Col2/Col1 ratio) was found in CaReS^®, followed by Novocart^®3D, Hyalograft^®C and MACI^®. IL-1β expression also exhibited high differences between the scaffolds showing low expression levels in Novocart^®3D and CaReS^® and higher expression levels in MACI^® and Hyalograft^®C.

Conclusions

Our data indicate that scaffold characteristics as well as culture conditions highly influence gene expression in cartilage transplants and that these parameters may have profound impact on the tissue regeneration after MACT.     

EXT1 regulates chondrocyte proliferation and differentiation during endochondral bone development

Multiple Hereditary Exostoses (MHE) is an autosomal dominant skeletal disorder most frequently caused by mutations in the EXT1 gene. MHE affects proper development of endochondral bones, such that all affected individuals present with exostoses adjacent to the growth plate of long bones, while some individuals exhibit additional bone deformities. EXT1 functions as a heparan sulfate (HS) co-polymerase, and when defective causes improper elongation of glycosaminoglycan side chains on core proteins of HS proteoglycans. Although analysis of heterozygous EXT1-deficient mice has failed to reveal any significant gross morphological variations in skeletal development, significant alterations in molecular signaling occur in the developing long bones. Our results indicate that defects in EXT1 and the resulting reduction in HS lead to enhanced Indian Hedgehog diffusion causing an increase in chondrocyte proliferation and delayed hypertrophic differentiation.     

EXT 1 gene mutation induces chondrocyte cytoskeletal abnormalities and defective collagen expression in the exostoses.

Hereditary multiple exostoses (HME), an autosomal skeletal disorder characterized by cartilage-capped excrescences, has been ascribed to mutations in EXT 1 and EXT 2, two tumor suppressor-related genes encoding glycosyltransferases involved in the heparan sulfate proteoglycan (HSPG) biosynthesis. Taking advantage of the availability of three different exostoses from a patient with HME harboring a premature termination codon in the EXT 1 gene, morphological, immunologic, and biochemical analyses of the samples were carried out. The cartilaginous exostosis, when compared with control cartilage, exhibited alterations in the distribution and morphology of chondrocytes with abundant bundles of actin filaments indicative of cytoskeletal defects. Chondrocytes in the exostosis were surrounded by an extracellular matrix containing abnormally high amounts of collagen type X. The unexpected presence of collagen type I unevenly distributed in the cartilage matrix further suggested that some of the hypertrophic chondrocytes detected in the cartilaginous caps of the exostoses underwent accelerated differentiation. The two mineralized exostoses presented lamellar bone arrangement undergoing intense remodeling as evidenced by the presence of numerous reversal lines. The increased electrophoretic mobility of chondroitin sulfate and dermatan sulfate proteoglycans (PGs) extracted from the two bony exostoses was ascribed to an absence of the decorin core protein. Altogether, these data indicate that EXT mutations might induce a defective endochondral ossification process in exostoses by altering actin distribution and chondrocyte differentiation and by promoting primary calcification through decorin removal.     

Low frequency EMF regulates chondrocyte differentiation and expression of matrix proteins.

This study describes the enhancement of chondrogenic differentiation in endochondral ossification by extremely low frequency pulsed electric/magnetic fields (EMFs). The demineralized bone matrix (DBM)-induced endochondral ossification model was used to examine the effects of EMF stimulation. [35S]-Sulfate and [3H]-thymidine incorporation and glycosaminoglycan (GAG) content were determined by standard methods. Proteoglycan (PG) and GAG molecular size and composition were determined by gel chromatography and sequential enzyme digestion. Immunohistochemical and Western blot analysis of PGs were done with antibodies 2B6, 3B3, 2D3 and 5D4. Northern analysis of total RNA extracts was performed for aggrecan, and type II collagen. All data was compared for significance by Student's t- or analysis of variance (ANOVA)-tests. The EMF field accelerated chondrogenesis as evidenced by an increase in: (1) 35SO4 incorporation and GAG content, (2) the number of chondrocytes at day 8 of development, (3) the volumetric density of cartilage and (4) the extent of immunostaining for 3B3 and 5D4. No differences in DNA content or [3H]-thymidine incorporation were observed between control and stimulated ossicles, suggesting the absence of enhanced cell proliferation or recruitment as a mechanism for the acceleration. PG and GAG molecular sizes and GAG chemical composition were similar in stimulated and control ossicles, indicating that stimulation resulted in an accelerated synthesis of normal cartilage molecules. The increased expression of PG and type II collagen mRNA as well as a greater immunoreactivity of 3B3 and 5D4 suggest an increase in the rate of differentiation of chondrocytes and enhanced phenotypic maturation.     

Nitric oxide-nitric oxide synthase regulates key maturational events during chondrocyte terminal differentiation

The goal of this investigation was to explore the mechanism by which NOS and NO serve to regulate events linked to chondrocyte terminal differentiation. NOS isoform expression and NO adducts in chick growth cartilage were detected by immunohistochemistry and Western blot analysis. All NOS isoforms were expressed in chick growth plate chondrocytes with the highest levels present in the hypertrophic region. The enzymes were active since nitrosocysteine and nitrotyrosine residues were detected in regions of the epiphysis with the highest levels of NOS expression. Maturing chick sternal chondrocytes evidenced an increase in NO release and a rise in NOS protein levels. When treated with NOS inhibitors, there was a decrease in the alkaline phosphatase activity of the hypertrophic cells. On the other hand, NO donors caused a small but significant elevation in alkaline phosphatase activity. Transient transfections of chondrocytes with an endothelial NOS isoform caused an increase in collagen type X promoter activity. Induction of both collagen type X expression and alkaline phosphatase activity was blocked by inhibitors of the cGMP pathway. These findings indicate that NO is generated by three NOS isoforms in terminally differentiated chondrocytes. The expression of NOS and the generation of NO enhanced maturation by upregulating alkaline phosphatase and collagen type X expression. Since expression of these two determinants was blocked by inhibitors of the cGMP pathway, it is concluded that NO metabolism is required for development of the mature chondrocyte phenotype.