Chondrogenesis and hypertrophy in response to aggregate behaviors of human mesenchymal stem cells on a dendrimer-immobilized surface.

OBJECTIVES: To investigate the behaviors of aggregates of human mesenchymal stem cells (hMSCs) on chondrogenesis and chondrocyte hypertrophy using spatiotemporal expression patterns of chondrogenic (type II collagen) and hypertrophic (type X collagen) markers during chondrogenesis. RESULTS: hMSCs were cultured on either a polystyrene surface or polyamidoamine dendrimer surface with a fifth generation (G5) dendron structure in chondrogenic medium and growth medium. At day 7, cell aggregates without stress fibers formed on the G5 surface and triggered differentiation of hMSCs toward the chondrogenic fate, as indicated by type II collagen being observed while type X collagen was undetectable. In contrast, immunostaining of hMSCs cultured on polystyrene, which exhibited abundant stress fibers and did not form aggregates, revealed no evidence of either type II and or type X collagen. At day 21, the morphological changes of the cell aggregates formed on the G5 surface were suppressed as a result of stress fiber formation. Type II collagen was observed throughout the aggregates whereas type X collagen was detected only at the basal side of the aggregates. Change of cell aggregate behaviors derived from G5 surface alone regulated chondrogenesis and hypotrophy, and this was enhanced by chondrogenic medium. CONCLUSIONS: Incubation of hMSCs affects the expression of type II and X collagens via effects on cell aggregate behavior and stress fiber formation.

TLR signalling can modify the mineralization of tooth germ.

OBJECTIVE: The aim of this work is to investigate the possible role of Toll-like receptor 4 (TLR4) during the development of mouse tooth germ. TLR4 is well known to inhibit mineralization and cause inflammation in mature odontoblasts and dental pulp cells. However, unlike these pathological functions of TLR4, little is known about the developmental function(s) of TLR4 during tooth development. MATERIALS AND METHODS: TLR4 expression was studied via Western blot in developing lower mouse incisors from E13.5 to E18.5. To generate functional data about the effects of TLR4, a specific agonist (LPS) was applied to the medium of in vitro tooth germ cultures, followed by Western blot, histochemical staining, ELISA assay, in situ hybridization and RT-qPCR. RESULTS: Increased accumulation of biotin-labelled LPS was detected in the enamel organ and in preodontoblasts. LPS treatment induced degradation of the inhibitor molecule (IκB) of the NF-κB signalling pathway. However, no morphological alterations were detected in cultured tissue after LPS addition at the applied dosage. Activation of TLR4 inhibited the mineralization of enamel and dentin, as demonstrated by alizarin red staining and as decreased levels of collagen type X. mRNA expression of ameloblastin was elevated after LPS administration. CONCLUSION: These results demonstrate that TLR4 may decrease the mineralization of hard tissues of the tooth germ and may trigger the maturation of ameloblasts; it can give valuable information to understand better congenital tooth abnormalities.

Mastication markedly affects mandibular condylar cartilage growth, gene expression, and morphology.

INTRODUCTION: Mandibular growth is believed to be strongly related to mastication. Furthermore, mandibular condylar cartilage is known to be derived from neural crest cells. We examined whether the degree of chewing affects condylar cartilage growth of the mandible. METHODS: Mice were fed diets with varying hardness. Genes specific to neural crest-derived cells were measured by real-time polymerase chain reaction to compare the expression changes between the mandibular and tibia cartilages. The mandibular condylar cartilage was then evaluated histologically, and proliferation was evaluated using proliferating cell nuclear antigen. Immunostaining was conducted for osteopontin, type X collagen, and Musashi1, and real-time polymerase chain reaction was used to assess the expression levels of osteopontin and type X collagen. RESULTS: Markers including P75, Wnt-1, Musashi1, and Nestin were upregulated in the mandibular condylar cartilage as compared with the tibial cartilage. Histologic assessment of the mandibular cartilage showed that the hypertrophic chondrocyte zone was statistically significantly thicker in mice fed a hard diet. Chondrocyte proliferation and Musashi1 expression were lower in mice fed a hard diet. After 4 weeks, numerous osteopontin and type X collagen-positive cells were observed in mice fed a mixed diet. CONCLUSIONS: Mastication affects the balance between differentiation and proliferation in the mandibular condylar cartilage. This phenomenon might be attributed to the presence of neural crest-derived cells.

Investigation of chondrocyte hypertrophy and cartilage calcification in a full-depth articular cartilage explants model.

Articular cartilage deterioration, which includes cartilage degradation and chondrocyte hypertrophy, is a hallmark of degenerative joint diseases (DJD). Chondrocyte hypertrophy is initiated in the deep layer of the cartilage; thus, a robust explants model for investigation of hypertrophy should include this zone. The aim of this study was to characterize and investigate the hypertrophy-promoting potential of different endogenous factors on an ex vivo articular cartilage model. The full-depth cartilage explants were harvested from bovine femoral condyle and cultured for 13 days in different conditions: 10 ng/ml oncostatin M + 20 ng/ml TNF-α; 100 ng/ml IGF1; 10-100 ng/ml bFGF; 10-100 ng/ml BMP2; 50 µg/ml ascorbic acid in combination with 10 mM ß-glycerophosphate; and 20-100 ng/ml triiodothyronine. The cellular activity and morphology, degradation, formation and calcification, and expression level of hypertrophic markers were investigated. The hypertrophic factors tested all induced cellular activity and marked morphological changes starting at day 4, however, not in a synchronized manner. Both cartilage degradation and formation were induced by T3 (P < 0.05). Only T3 had a full hypertrophic gene expression profile (P < 0.05). We developed and characterized a novel model for investigation of chondrocyte hypertrophy. We speculated that this can become an important investigatory tool for investigation of matrix turnover, chondrocyte hypertrophy and cartilage calcification that are associated with DJD pathogenesis.

Investigating the role of culture conditions on hypertrophic differentiation in human cartilage endplate cells.

Cartilage endplate degeneration/calcification has been linked to the onset and progression of intervertebral disc degeneration and there is a critical need to understand mechanisms, such as hypertrophic differentiation, of cartilage endplate degeneration/calcification to inform treatment strategies for discogenic back pain. In vitro cell culture conditions capable of inducing hypertrophic differentiation are used to study pathophysiological mechanisms in articular chondrocytes, but culture conditions capable of inducing a hypertrophic cartilage endplate cell phenotype have yet to be explored. The goal of this study was to investigate the role of culture conditions capable of inducing hypertrophic differentiation in articular chondrocytes on hypertrophic differentiation in human cartilage endplate cells. Isolated human cartilage endplate cells were cultured as pellets for 21 days at either 5% O2 (physiologic for cartilage) or 20.7% O2 (hyperoxic) and treated with 10% fetal bovine serum or Wnt agonist, two stimuli used to induce hypertrophic differentiation in articular chondrocytes. Cartilage endplate cells did not exhibit a hypertrophic cell morphology in response to fetal bovine serum or Wnt agonist but did display other hallmarks of chondrocyte hypertrophy and degeneration such as hypertrophic gene and protein expression, and a decrease in healthy proteoglycans and an increase in fibrous collagen accumulation. These findings demonstrate that cartilage endplate cells take on a degenerative phenotype in response to hypertrophic stimuli in vitro, but do not undergo classical changes in morphology associated with hypertrophic differentiation regardless of oxygen levels, highlighting potential differences in the response of cartilage endplate cells versus articular chondrocytes to the same stimuli.

Analysis of Collagen type X alpha 1 (COL10A1) expression and prognostic significance in gastric cancer based on bioinformatics.

Collagen type X alpha 1 (COL10A1) is a member of the collagen family and the main matrix component. However, COL10A1 expression and prognosis relationship remains unclear in gastric cancer (GC). Through the analysis of database of Oncomine, the Cancer Genome Atlas (TCGA) as well as the Gene Expression Omnibus (GEO), in contrast to the tissue of normal gastric, COL10A1 in gastric cancer, had been upregulated. The high expression of COL10A1 was obviously related to T stage (P = 0.025) and lymph node metastasis (P = 0.025). It has been illustrated by the analysis of logistic regression that COL10A1's heightened expression in gastric cancer had been essentially linked with pathological stage, tumor differentiation, and T classification. The Kaplan-Meier curve in the Kaplan-Meier plotter database (P = 0.0371) and GSE84437 (P = 0.002) indicate that patients with high COL10A1 expression possess poor prognosis, specifically GC patients with lymph node metastasis have it. TCGA's Multivariate analysis (P = 0.025) and GSE84437 dataset (P = 0.034) show that high expression COL10A1 is a key independent predictor of poor overall survival. Searching KEGG pathway enrichment by GSEA, the results suggested that 29 pathways were enriched. qRT-PCR technique was used for verification of the COL10A1's high expression in gastric cancer in contrast to the normal gastric tissues. In conclusion, COL10A1 is of great importance in predicting the survival rate of GC patients.

Norms for Clinical Use of CXM, a Real-Time Marker of Height Velocity.

CONTEXT: Height velocity (HV) is difficult to assess because growth is very slow. The current practice of calculating it from measurements taken at several-month intervals is insufficient for managing children with growth disorders. We identified a bone growth by-product (collagen X biomarker, CXM) in blood that in preliminary analysis in healthy children correlated strongly with conventionally determined HV and displayed a pattern resembling published norms for HV vs age. OBJECTIVE: The goal was to confirm our initial observations supporting the utility of CXM as an HV biomarker in a larger number of individuals and establish working reference ranges for future studies. DESIGN, SETTINGS, AND PARTICIPANTS: CXM was assessed in archived blood samples from 302 healthy children and 10 healthy adults yielding 961 CXM measurements. A total of 432 measurements were plotted by age, and sex-specific reference ranges were calculated. Serial values from 116 participants were plotted against observed HV. Matched plasma, serum, and dried blood spot readings were compared. RESULTS: A correlation of blood CXM with conventional HV was confirmed. Scatter plots of CXM vs age showed a similar pattern to current HV norms, and CXM levels demarcated the pubertal growth spurt both in girls and boys. CXM levels differed little in matched serum, plasma, and dried blood spot samples. CONCLUSIONS: Blood CXM offers a potential means to estimate HV in real time. Our results establish sex-specific, working reference ranges for assessing skeletal growth, especially over time. CXM stability in stored samples makes it well suited for retrospective studies.

Hypertrophic differentiation and calcification during intervertebral disc degeneration.

BACKGROUND: In degenerative intervertebral discs (IVDs) collagen type X expression and calcifications have been demonstrated, resembling advanced osteoarthritis (OA), which is associated with hypertrophic differentiation, characterized by the production of collagen type X, Runt-related transcription factor 2 (Runx2), osteoprotegerin (OPG), alkaline phosphatase (ALP) and calcifications. OBJECTIVE: The aim of this study was to determine if hypertrophic differentiation occurs during IVD degeneration. METHODS: IVDs from all Thompson degeneration grades were prepared for histology, extraction of nucleus pulposus (NP) and annulus fibrosis (AF) tissue (N=50) and micro-CT (N=27). The presence of collagen type X, OPG and Runx2 was determined by immunohistochemistry, with OPG levels also determined by Enzyme-linked immunosorbent assay (ELISA). The presence of calcification was determined by micro-CT, von Kossa and Alizarin Red staining. RESULTS: Immunohistochemical staining for collagen type X, OPG, Runx2 appeared more intense in the NP of degenerative compared to healthy IVD samples. OPG levels correlated significantly with degeneration grade (NP: P<0.000; AF: P=0.002) and the number of microscopic calcifications (NP: P=0.002; AF: P=0.008). The extent of calcifications on micro-CT also correlated with degeneration grade (NP: P<0.001, AF: P=0.001) as did von Kossa staining (NP: P=0.015, AF: P=0.016). ALP staining was only incidentally seen in the transition zone of grades IV and V degenerated IVDs. CONCLUSION: This study for the first time demonstrates that hypertrophic differentiation occurs during IVD degeneration, as shown by an increase in OPG levels, the presence of ALP activity, increased immunopositivity of Runx2 and collagen type X.

Applying an excessive mechanical stress alters the effect of subchondral osteoblasts on chondrocytes in a co-culture system.

Osteoarthritis (OA) sometimes occurs as a consequence of repeated microtrauma involved in parafunction, which may lead to microfracture in the subchondral bone. The aim of this in vitro study was to evaluate the effects of subchondral osteoblasts in loading with repeated excessive mechanical stress on the metabolism of overlying chondrocytes. A high-magnitude cyclic tensile stress of 15 kPa (30 cycles min(-1)) was applied to the cultured osteoblasts obtained from porcine mandibular condyles. The chondrocytes in alginate beads were then co-cultured with mechanically stressed or unstressed osteoblasts. Chondrocytes co-cultured with unstressed osteoblasts showed a phenotypic shift to hypertrophic chondrocytes, characterized by decreased expression of type II collagen, aggrecan, Sry-related HMG box (SOX-9), and cartilage oligomeric matrix protein (COMP) genes and increased expression of type X collagen and bone sialoprotein (BSP) genes, suggesting that the co-culture may change the chondrocyte differentiation to some extent. These changes were more distinct in chondrocytes co-cultured with excessively mechanically stressed osteoblasts. After co-culture with stressed osteoblasts, the expressions of matrix metalloproteinase (MMP)1, MMP3 and MMP13 genes were also enhanced and the synthesis of DNA, proteoglycan and collagen were significantly decreased in chondrocytes. These results demonstrate that alterations in cartilage metabolism can be induced by stressed osteoblasts, indicating a possible explanation for the onset and progression of OA.

Differential gene expression in the perichondrium and cartilage of the neonatal mouse temporomandibular joint.

Our goal was to discover genes differentially expressed in the perichondrium (PC) of the mandibular condylar cartilage (MCC) that might enhance regenerative medicine or orthopaedic therapies directed at the tissues of the temporomandibular joint. We used targeted gene arrays (osteogenesis, stem cell) to identify genes preferentially expressed in the PC and the cartilaginous (C) portions of the MCC in 2-day-old mice. Genes with higher expression in the PC sample related to growth factor ligand-receptor interactions [FGF-13 (6.4x), FGF-18 (4x), NCAM (2x); PGDF receptors, transforming growth factor (TGF)-beta and IGF-1], the Notch isoforms (especially Notch 3 and 4) and their ligands or structural proteins/proteoglycans [collagen XIV (21x), collagen XVIII (4x), decorin (2.5x)]. Genes with higher expression in the C sample consisted mostly of known cartilage-specific genes [aggrecan (11x), procollagens X (33x), XI (14x), IX (4.5x), Sox 9 (4.4x) and Indian hedgehog (6.7x)]. However, the functional or structural roles of several genes that were expressed at higher levels in the PC sample are unclear [myogenic factor (Myf) 9 (9x), tooth-related genes such as tuftelin (2.5x) and dentin sialophosphoprotein (1.6x), VEGF-B (2x) and its receptors (3-4x) and sclerostin (1.7x)]. FGF, Notch and TGF-beta signalling may be important regulators of MCC proliferation and differentiation; the relatively high expression of genes such as Myf6 and VEGF-B and its receptors suggests a degree of unsuspected plasticity in PC cells.

[Developmental changes of acetabular cartilage complex: an experimental study of a straight-leg swaddle model of newborn rats].

OBJECTIVE: By establishing a model of straight-leg swaddle of newborn rats and observing the experimental animals'hips morphologically and pathologically, this study explored the changes of gross appearance of the acetabulum and the maturity of cartilage cells in the different regions of acetabular cartilage complex. METHODS: The legs and hips were fixed by adhesive tape for 10 days in the position of hip extension and adduction in 31 newborn Wistar rats (experimental group). The other 31 newborn rats without legs and hips treatment were used as the control group. After 10 days raising in the same condition, all the rats were sacrificed. The gross appearance, histological observations and VEGF and type X collagen immunohistochemistry were used for examining the acetabulum changes. RESULTS: A straight leg swaddle model of newborn rats was established successfully. In the experimental group the acetabulum became shallow and small and surrounded by more soft tissues. There were 49 dislocated hips (49/54) in the experimental group and 2 hips dislocated (2/60) in the control group (p<0.01). Fake acetabulum appeared in the experimental group. In the control group, the shape of the acetabulum was normol, and no fake acetabulum was found. The safranin O-fast green staining showed that the orange-red cartilage in the experimental group was wider than the control group. Immunohistochemistry observations showed VEGF and type X collagen immunoreactivities in the hypertrophic layer of the acetabular cartilage complex in the experimental group were lower than those in the control group. The percentages of VEGF positive and type X collagen positive cells in the iliac hypertrophic layer of the acetabular articular cartilage were significantly higher than those in the ischiadic ramus and the pubic branch in the experimental group. CONCLUSIONS: VEGF and type X collagen immunoreactivities in acetabular cartilage cells decrease in a straight-leg swaddle model of newborn rats. This suggests that this position might lead to dysmaturity of the acetabular cartilage cells and affect the development of the acetabulum.

Use of Allogeneic Hypoxic Mesenchymal Stem Cells For Treating Disc Degeneration in Rabbits.

Intervertebral discs (IVDs) are important biomechanical components of the spine. Once degenerated, mesenchymal stem cell (MSC)-based therapies may aid in the repair of these discs. Although hypoxic preconditioning enhances the chondrogenic potential of MSCs, it is unknown whether bone marrow MSCs expanded under hypoxic conditions (1% O2 , here referred to as hypoxic MSCs) are better than bone marrow MSCs expanded under normoxic conditions (air, here referred to as normoxic MSCs) with regards to disc regeneration capacity. The purpose of this study was to compare the therapeutic effects of hypoxic and normoxic MSCs in a rabbit needle puncture degenerated disc model after intra-disc injection. Six weeks after needle puncture, MSCs were injected into the IVD. A vehicle-treated group and an un-punctured sham-control group were included as controls. The tissues were analyzed by histological and immunohistochemical methods 6 and 12 weeks post-injection. At 6 and 12 weeks, less disc space narrowing was evident in the hypoxic MSC-treated group compared to the normoxic MSC-treated group. Significantly better histological scores were observed in the hypoxic MSC group. Discs treated with hypoxic MSCs also demonstrated significantly better extracellular matrix deposition in type II and XI collagen. Increased CD105 and BMP-7 expression were also observed upon injection of hypoxic MSCs. In conclusion, hypoxic MSC injection was more effective than normoxic MSC injection for reducing IVD degeneration progression in vivo. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 37:1440-1450, 2019.

Wnt/beta-catenin signaling regulates cranial base development and growth.

Wnt proteins and beta-catenin signaling regulate major processes during embryonic development, and we hypothesized that they regulate cranial base synchondrosis development and growth. To address this issue, we analyzed cartilage-specific beta-catenin-deficient mice. Mutant synchondroses lacked typical growth plate zones, and endochondral ossification was delayed. In reciprocal transgenic experiments, cartilage overexpression of a constitutive active Lef1, a transcriptional mediator of Wnt/beta-catenin signaling, caused precocious chondrocyte hypertrophy and intermingling of immature and mature chondrocytes. The developmental changes seen in beta-catenin-deficient synchondroses were accompanied by marked reductions in Ihh and PTHrP as well as sFRP-1, an endogenous Wnt signaling antagonist and a potential Ihh signaling target. Thus, Wnt/beta-catenin signaling is essential for cranial base development and synchondrosis growth plate function. This pathway promotes chondrocyte maturation and ossification events, and may exert this important role by dampening the effects of Ihh-PTHrP together with sFRP-1.

An immunohistochemical and ultrastructural study of the pericellular matrix of uneroded hypertrophic chondrocytes in the mandibular condyle of aged c-src-deficient mice.

OBJECTIVE: Previous studies indicate that hypertrophic chondrocytes can transdifferentiate or dedifferentiate and redifferentiate into bone cells during the endochondral bone formation. Mandibular condyle in aged c-src-deficient mice has incremental line-like striations consisting of cartilaginous and non-cartilaginous layers, and the former contains intact hypertrophic chondrocytes in uneroded lacunae. The purpose of this study is to determine the phenotype changes of uneroded hypertrophic chondrocytes. DESIGN: Immunohistochemical and ultrastructural examinations of the pericellular matrix of hypertrophic chondrocytes in the upper, middle, and lower regions of the mandibular condyle were conducted in aged c-src-deficient mice, using several antibodies of cartilage/bone marker proteins. RESULTS: Co-localisation of aggrecan, type I collagen, and dentin matrix protein-1 (DMP-1) or matrix extracellular phosphoprotein (MEPE) was detected in the pericellular matrix of the middle region. Ultrastructurally, granular substances in the pericellular matrix of the middle region were the remains of upper region chondrocytes, which were mixed with thick collagen fibrils. In the lower region, the width of the pericellular matrix and the amount of collagen fibrils were increased. Versican, type I collagen, DMP-1, and MEPE were detected in the osteocyte lacunae. Additionally, DMP-1 and MEPE were detected in the pericellular matrix of uneroded hypertrophic chondrocytes located in the lower, peripheral region of the mandibular condyle in younger c-src-deficient mice, but not in the aged wild-type mice. CONCLUSIONS: These results indicate that long-term survived, uneroded hypertrophic chondrocytes, at least in a part, acquire osteocytic characteristics.

[Indirect coculture of human infrapatellar fat pad-derived stem cells with osteoarthritic chondrocytes induces their chondrogenesis].

Objective: To promote phenotype recovery of osteoarthritic articular chondrocytes( OACs) and induce chondrogenic differentiation of infrapatellar fat pad-derived stem cells( IPFPSCs) by indirectly coculturing these two types of cells. Methods: The OACs and IPFPSCs were isolated and cultured in vitro. This experiment included single IPFPSCs group,single OACs group,and coculture group. After cells were cultured in vitro with chondrogenic medium for 21 days,the chondrocyte phenotypes were determined by HE staining( cell morphology),Alcian blue staining( glycosaminoglycan content) and immunofluorescence cytochemistry( collagen 1,collagen 2,collagen 3,aggrecan,SOX9). Results: In coculture group,the OACs aggregated into microspheres,and the IPFPSCs were oval in shape. In single culture groups,the OACs were less aggregated and the spheres were smaller; and the IPFPSCs were spindle in shape. HE staining showed that,in the coculture group,the nuclei of OACs spheres were dark,and the IPFPSCs were rich in cytoplasm; while in single culture groups,the nuclei of OAC spheres were less dark,and the IPFPSCs were less stained compared with the coculture group. Alcian blue staining indicated that glycosaminoglycan content was higher in the coculture group than in single culture groups. Immunofluorescent staining showed that the intensity of chondrogenic markers( collagen 2,aggrecan,and SOX9)was stronger,while the intensity of collagen 1 and collagen 10 was weaker in the coculture group as compared with single culture groups. Conclusion: The indirect coculture of IPFPSCs with OACs can contribute to the phenotype recovery of OACs and induce the chondrogenic differentiation of IPFPSCs.

Thyroid hormone-stimulated differentiation of primary rib chondrocytes in vitro requires thyroid hormone receptor beta.

The active thyroid hormone, triiodothyronine (T(3)), binds to thyroid hormone receptors (TR) and plays an essential role in the control of chondrocyte proliferation and differentiation. Hypo- and hyperthyroidism alter the structure of growth plate cartilage and modify chondrocyte gene expression in vivo, whilst TR mutations or deletions in mice result in altered growth plate architecture. Nevertheless, the particular roles of individual TR isoforms in mediating T(3) action in chondrocytes have not been studied and are difficult to determine in vivo because of complex cellular and molecular interactions that regulate growth plate maturation. Therefore, we studied the effects of TRalpha and TRbeta on chondrocyte growth and differentiation in primary cultures of neonatal rib chondrocytes isolated from TRalpha- and TRbeta-deficient mice. T(3) decreased proliferation but accelerated differentiation of rib chondrocytes from wild-type mice. T(3) treatment resulted in similar effects in TRalpha-deficient chondrocytes, but in TRbeta-deficient chondrocytes, all T(3) responses were abrogated. Furthermore, T(3) increased TRbeta1 expression in wild-type and TRalpha-deficient chondrocytes. These data indicate that T(3)-stimulated differentiation of primary rib chondrocytes in vitro requires TRbeta and suggest that the TRbeta1 isoform mediates important T(3) actions in mouse rib chondrocytes.

Ceramide inhibition of chondrocyte proliferation and bone growth is IGF-I independent.

Proinflammatory cytokines inhibit growth plate development. However, their underlying mechanisms of action are unclear. These effects may be mediated by ceramide, a sphingosine-based lipid second messenger, which is elevated in a number of chronic inflammatory diseases. To test this hypothesis, we determined the effects of C2-ceramide, a cell permeable ceramide analogue, on the growth of the ATDC5 chondrogenic cell line and on cultured fetal mice metatarsals. In ATDC5 cells, C2-ceramide significantly induced apoptosis at both 40 (82%; P < 0.05) and 25 microM (53%; P < 0.05). At 40 microM, C2-ceramide significantly reduced proliferation ([3H]-thymidine uptake/mg protein) (62%; P < 0.05). C2-ceramide did not markedly alter the differentiation state of the cells as judged by the expression of markers of chondrogenesis and differentiation (sox 9, collagen II and collagen X). The IGF-I signalling pathway is the major autocrine/paracrine regulator of bone growth. Both in the presence and absence of IGF-I, C2-ceramide (25 microM) induced an equivalent reduction in proliferation (60%; P < 0.001). Similarly, C2-ceramide (40 microM) induced a 31% reduction in fetal metatarsal growth both in the presence and absence of IGF-I (both P < 0.001). Furthermore, C2-ceramide reduced ADCT5 proliferation in the presence of AG1024, an IGF-I and insulin receptor blocker. Therefore, C2-ceramide-dependent inhibition appears to be independent of IGF-mediated stimulation of bone growth. Indeed, biochemical studies demonstrated that C2-ceramide (25 microM) pretreatment did not alter IGF-I-stimulated phosphorylation of insulin receptor substrate-1, Akt or P44/42 MAP kinase. In conclusion, C2-ceramide inhibits proliferation and induces apoptosis in growth plate chondrocytes through an IGF-I independent mechanism.

Forward deviation of the mandibular condyle enhances endochondral ossification of condylar cartilage indicated by increased expression of type X collagen.

OBJECTIVE: Using type X collagen as a marker, this research was designed to examine the alteration of condylar growth in response to mandibular condylar forward positioning. METHODS: One hundred female Sprague-Dawley rats with 5 weeks of age were randomly divided into five experimental and five control groups. In the experimental groups, bite jumping appliances created forward positioning of the condyle. The experimental rats, together with the age-matched controls, were sacrificed on days 3, 7, 14, 21 and 30, respectively. Tissue sections were cut in the sagittal plane through the mandibular condyle and were processed for in situ hybridization and immunostaining of type X collagen and then for quantitative imaging analyses. RESULTS: (1) Both type X collagen mRNA in situ hybridization signals and type X collagen immunostaining were localized within the hypertrophic zone of the condylar cartilage. (2) With condylar forward positioning, the level of type X collagen mRNA signals (8,541 +/- 74 microm(2) at peak) was 300% higher than that in the controls (2,117 +/- 78 microm(2) at peak); type X collagen immunostaining in condylar advancing groups (54,864 +/- 134 microm(2) at peak) was 254% more than that in the controls (15,470 +/- 121 microm(2) at peak). (3) The amount of type X collagen mRNA signals and immunostaining in experimental and control groups reached the highest levels at day 14 and day 21, respectively, indicating that an increase in endochondral ossification occurred 21 days after condylar forward deviation. CONCLUSION: Condylar forward repositioning provokes an enhanced maturation of condylar chondrocytes resulting in increased synthesis of type X collagen, a extracellular protein that attributes to endochondral ossification.

In vitro analysis of differential expression of collagens, integrins, and growth factors in cultured human chondrocytes.

OBJECTIVES: Tissue engineering represents a promising method for the construction of autologous chondrogenic grafts for reconstructive surgery. In cultured chondrocytes, the dedifferentiation and proliferation of the cells are critical factors that influence the generation of transplants. The aim of our study was to find and characterize markers for cell proliferation and dedifferentiation in cultured chondrocytes. STUDY DESIGN AND SETTING: Human chondrocytes were isolated from septal cartilage and held in primary cell culture. Cells were harvested after 1, 6, and 21 days. The differentiation of the cells was investigated with bright-field microscopy, the expression patterns of various proteins using immunohistochemistry, and the expression of distinct genes with the microarray technique. RESULTS: The chondrocytes showed a strong proliferation. After 6 and 21 days, collagen 9 and 10 were downregulated; collagen 11 was activated. Collagen 1 and 2 were downregulated after 6 days but were reactivated after 21 days. Tumor growth factor beta (TGF-beta)1 was strongly expressed on days 1, 6, and 21, TGF-beta2 was never expressed, and TGF-beta3 and -beta4 were upregulated from day 1 to day 21. The TGF-beta receptor III was expressed on days 1, 6, and 21. Integrin beta1, beta5, and alpha5 were upregulated from day 1 to day 21; integrin beta3 was downregulated. CONCLUSION AND SIGNIFICANCE: Collagens 3, 4, 8, 9, and 11 might be new markers for the dedifferentiation of chondrocytes. Collagen 2 might be a marker for the synthetic activity of the cells rather than the dedifferentiation. TGF-beta3 and -beta4 might influence the dedifferentiation, which is fortified by the expression of TGF-beta receptor III. Integrin beta1, beta5, and alpha5 might be involved in signal transmission for the dedifferentiation.

An in situ hybridization and histochemical study of development and postnatal changes of mouse mandibular angular cartilage compared with condylar cartilage.

To investigate the origin and postnatal changes of mouse mandibular angular cartilage, in situ hybridization for cartilaginous marker proteins, histochemistry for alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP), and bromodeoxyuridine (BrDU) analyses were performed. Chondrocytes of the mandibular angular cartilage were derived from ALP-positive progenitor cells and first detected at embryonic day (E) 15.5. Newly formed chondrocytes rapidly differentiated into hypertrophic chondrocytes and hypertrophic cell zone rapidly extended in subsequent a few days. During this period, bone sialoprotein mRNA was more widely expressed than osteopontin mRNA in cartilage. Endochondral bone formation started at E 17.5 with the resorption of the bone collar by osteoclasts. These characteristics were consistent with those of the condylar cartilage, although developmental process was 0.5-1.5 day delayed relative to the condylar cartilage. During the postnatal period, contrast to the condylar cartilage, the angular cartilage constantly decreased in volume with advancing age. Reduction of proliferating activity estimated by BrDU incorporation accounts for this phenomenon. We demonstrate new structural features of the mandibular angular cartilage that may contribute to a coming research for the secondary cartilage.