Immunohistochemical examination of epiphyseal growth plates of Japanese Brown cattle with chondrodysplasia.

A new type of inherited chondrodysplasia is described in Japanese Brown cattle, but the basic defects of the epiphyseal growth plate (EGP) in the limb long bones, and proliferation and differentiation of the chondrocytes in the EGP, are not yet understood. In the present study, the EGPs of the limb long bones in eight cases of chondrodysplasia and four normal (control) cattle were examined histologically and immunohistochemically. In the control cattle, proliferative chondrocytes (PCs) and hypertrophic chondrocytes (HCs) were arranged in columns parallel to the long axis of the bone, and HCs were situated on the metaphyseal side of the EGP. In all the affected cattle, many chondrocytes with a hypertrophic appearance were detected in the inner areas of the central portion of the EGP. The PC columns were short and arranged irregularly. Bone tissue and small blood vessels were found frequently in these areas. Six affected cattle showed complete EGP-closure. Backscattered electron (BSE) imaging showed that the calcified cartilage matrix was restricted to the lower region of the hypertrophic zone (HZ) of the EGP in the control cattle, while the calcified cartilage matrix and bone tissue were scattered in the inner areas of the EGP in all the chondrodysplastic cattle. Immunohistochemistry revealed type X collagen in the HCs and cartilage matrix of the HZ in the control cattle. In all the affected cattle, type X collagen was detected in apparently hypertrophic chondrocytes in the inner areas of the EGP. Type II collagen was detected in the entire EGP in all the affected cattle, as in the controls. BrdU (5-bromo-2'-deoxyuridine), injected intravenously 1h before euthanasia was detected in many PCs in the EGP in the control cattle; none, however, was detected in the central portion of the EGP in any affected animal. These observations indicate that differentiation into HCs and calcification of cartilage matrix occur in the inner areas of the central portion of the EGP in chondrodysplasia of Japanese Brown cattle. Differentiation into the HCs at this abnormal site may be caused by the inadequate proliferation and disorganization of the PCs. Premature EGP-closure, observed commonly in chondrodysplasia of Japanese Brown cattle, was thought to be caused by replacement of the calcified cartilage in the inner areas of the EGP by bone tissue.

A static, closed and scaffold-free bioreactor system that permits chondrogenesis in vitro.

OBJECTIVE: To characterise in vitro engineered cartilaginous constructs made employing a novel static, scaffold-free and closed chamber system. DESIGN: Chondrocytes derived from slaughter age pigs (3-6 months) were seeded at high density (20 x 10(6)) into cylindrical chambers (1.0 x 0.5cm) and were maintained between an upper and a lower membrane (100 kDa) for 21 days and subsequently cultured in open culture for 7 additional days. RESULTS: Viable constructs produced were approximately 10 mmx2mm in size and were stable enough to be handled by surgical pincers. Histology and electron microscopy evaluations revealed a neo-cartilage structure of high cell density with a comprehensive extracellular matrix. Predominately collagen type II and negligible amounts of collagen types I and X were detected using RT-PCR and SDS-PAGE analyses. CONCLUSIONS: In this study, we provide evidence of a scaffold-free system that can produce immature hyaline-like cartilaginous constructs suitable for in vivo implantation, or that may be useful for in vitro studies of events related to the process of chondrogenesis.

Impact of mutations of cartilage matrix genes on matrix structure, gene activity and chondrogenesis.

OBJECTIVE: Chondrocytes in the growth plate at different stages of differentiation synthesize characteristic extracellular matrix (ECM) components. Mutations in some ECM genes result in chondrodysplasia in humans and mice. We aimed to evaluate the impact of loss- and gain-of-function mutations of ECM genes on matrix structure, gene expression and formation of the growth plate. DESIGN: We review information on the impact of deficiencies in proteoglycans, and types X and II collagens on skeletal development. Additionally, we compare the impact of a glycine904 to cysteine (G904C) mutation in the triple helical coding domain of mouse Col2a1 with two previously reported Col2a1 mutations (exon7 deletion (Del1) and G85C). The G904C Col2a1 gene was introduced as a transgene into mice. Transgenic newborn mice were examined for skeletal development. The histology of the epiphyseal cartilage and the growth plate, and the ultrastructure of chondrocytes and collagen fibrillar morphology in the ECM were studied in 18.5-day transgenic and wild-type fetuses. The distribution of the mRNAs for Col2a1, Col11a1, Col9a1, Matn1, Agc and Ihh in the growth plate of 18.5-day G904C/G904C and wild type fetuses were compared by in situ hybridization. RESULTS: Heterozygous transgenic mice harbouring five copies of the G904C Col2a1 transgene developed skeletal abnormalities and dwarfism. Homozygous G904C/G904C mice died at birth, showing cleft palate, disrupted zonation of chondrocytes and reduction of the zone of hypertrophic chondrocytes. Fewer collagen fibrils were found in ECM of the cartilage. Rough endoplasmic reticulum of the chondrocytes of G904C/+ and G904C/G904C mice was distended. In G904C/G904C mutant mice, Agc gene activity was extended to the hypertrophic zone. Expression of the other genes studied was unchanged. Calcified materials that were not found normally in the maturing and only at low abundance in the hypertrophic zones of the wild type growth plate, were present in these zones in G904C/G904C mice. Despite phenotypic similarities for the G904C and Del1 mice, reduced expression of types I, II, IX, X collagens and aggrecan were reported for the latter mutation. Changes in gene activity and matrix organization in the growth plate also accompanied deficiencies in aggrecan, perlecan and collagen II. CONCLUSIONS: The data suggest that a single amino acid alteration in collagen II could lead to skeletal abnormalities through multiple secondary effects on the synthesis and assembly of ECM components. The functional impact of mutations of ECM genes reveals that chondrodysplasia is caused not just by the formation of abnormal matrix molecules, but that the alteration of one ECM component may lead to a cascade of disruption of other gene activities in chondrocytes which collectively contribute to the pathological changes in the architecture of the growth plate.