Quantitative analysis of collagen network structure and fibril dimensions in cartilage repair with autologous chondrocyte transplantation.

OBJECTIVE: The aim of this study was to undertake a stereological analysis to quantify the dimensions of the collagen network in the repair tissue of porcine joints after they had been subjected to autologous chondrocyte transplantation (ACT). METHOD: ACT was used to repair cartilage lesions in knee joints of pigs. Electron-microscopic stereology, immunostaining for type II collagen, and quantitative polarized-light microscopy were utilized to study the collagen fibrils in the repair tissue 3 and 12 months after the operation. RESULTS: The collagen volume density (V(V)) was lower in the repair tissue than in normal cartilage at 3 months (20.4 vs. 23.7%) after the operation. The collagen surface density (S(V), 1.5·10(-2) vs. 3.1·10(-2) nm(2)/nm(3)) and V(V) increased with time in the repair tissue (20.4 vs. 44.7%). Quantitative polarized-light microscopy detected a higher degree of collagen parallelism in the repair tissue at 3 months after the operation (55.7 vs. 49.7%). In contrast, 1 year after the operation, fibril parallelism was lower in the repair tissue than in the control cartilage (47.5 vs. 69.8%). CONCLUSION: Following ACT, V(V) and S(V) increased in the repair tissue with time, reflecting maturation of the tissue. One year after the operation, there was a lower level of fibril organization in the repair tissue than in the control cartilage. Thus, the newly synthesized collagen fibrils in the repair tissue appeared to form a denser network than in the control cartilage, but the fibrils remained more randomly oriented.

Cartilage collagen fibril network in newborn transgenic mice analyzed by electron microscopic stereology.

The objective was to investigate by electron microscopic stereology the properties of the cartilage collagen fibril network in newborn transgenic mice. The mice harbored transgenes targeted to affect the structure or assembly of the collagen fibrils. The mouse lines investigated here harbored either (i) one or (ii) two human COL2A1 genes with Arg519Cys mutation in addition to one or (iii) no active allele(s) of the murine COL2A1 gene, (iv) two inactive alleles of the procollagen N-proteinase genes, or (v) a human COL2A1 gene with deleted exons 16-27. In all newborn mice carrying the COL2A1 transgene with Arg519Cys mutation, the growth plate collagen fibrils were thinner than in the wild-type (wt) mice and showed clearly reduced volume fraction of the fibril network. In mice with the inactive procollagen N-proteinase genes, the fibril thickness and the volume fraction of collagen did not differ from the wt mice. In mice harboring the transgene of human COL2A1 gene with internally deleted exons 16-27, the collagen fibril diameter remained the same, but the volume density of collagen network was reduced. Using the indirect stereology, the differences in the collagen fibril stereological estimates could be reliably detected in newborn mice harboring mutations that affect the structure and assembly of collagen fibrils. The EM stereology permitted early detection of altered phenotype of the collagen fibril network in newborn transgenic mice. It is recommended that the indirect model-based stereological technique is utilized instead of the direct design-based technique for the estimation of collagen volume, surface, and length densities.

Collagenase-induced changes in articular cartilage as detected by electron-microscopic stereology, quantitative polarized light microscopy and biochemical assays.

The purpose of this study was to compare the ability of electron-microscopic (EM) stereology with quantitative polarized light microscopy (PLM) and biochemical collagen (hydroxyproline) and crosslink (pyridinoline) analyses to detect changes in the superficial collagen network of bovine articular cartilage after digestion in vitro with purified bacterial (Clostridium histolyticum) collagenase (30 U/ml) for 24 and 48 h. Collagen volume (V(V)) and surface (S(V)) densities of the uppermost third of the superficial zone were estimated indirectly from zonal isotropic uniform random sections using collagen length density (L(V)) and average collagen fibril diameter, or its average second power. Collagenase digestion caused a significant decrease in fibril diameter (64 to 62%), V(V) (89 to 95%) and S(V) (64 to 86%) after incubation for 24 and 48 h. Collagen L(V) remained unchanged after 24 h incubation but decreased 63% after 48 h. Collagen concentration per dry weight, assayed biochemically from the whole superficial zone, decreased also significantly (29 to 60%) after 24 and 48 h digestions, respectively. The pyridinoline concentration per dry weight of the superficial zone decreased (31 to 57%) whereas the pyridinoline concentration per collagen remained unchanged. PLM revealed that the birefringence of the uppermost third of the superficial zone was decreased by 36% after digestion for 24 h though the total birefringence of the whole zone was not reduced. However, after 48 h, the birefringence of the whole superficial zone was significantly reduced (76%). All of the techniques compared in this study could detect collagen network degradation in bovine articular cartilage but the EM stereological technique was more sensitive at detecting the changes than PLM or biochemical assays.

A human COL2A1 gene with an Arg519Cys mutation causes osteochondrodysplasia in transgenic mice.

OBJECTIVE: An arginine-to-cysteine substitution at position 519 of the COL2A1 gene causes early generalized osteoarthritis with mild chondrodysplasia in humans. In this study, a human COL2A1 gene with the same mutation was introduced into a murine genome having 1 or no alleles of the murine Col2a1 gene, and the skeletal phenotypes of the transgenic mice were compared with those of control mice. METHODS: Mice with 1 allele of the normal murine Col2a1 gene and 1 allele of the mutated human COL2A1 gene (n = 10), those with no murine Col2a1 gene and 2 alleles of the mutated human COL2A1 gene (n = 13), those with no murine Col2a1 gene and only 1 allele of the mutated COL2A1 gene (n = 9), and normal control mice (n = 11) were studied for skeletal abnormalities, using radiographic imaging and light microscopic analyses of histologic sections. The collagen network of cartilage was also investigated with transmission electron microscopy. RESULTS: At 2 months of age, all transgenic mice had dysplastic changes in their long bones, flattened vertebral bodies, and osteoarthritic changes in their joints. The intervertebral discs of the transgenic animals were degenerated, and their histologic structure was disturbed. The changes were more severe in mice with no murine Col2a1 allele. CONCLUSION: The human COL2A1 gene with the Arg519Cys mutation causes osteochondrodysplasia in mice, as it does in humans.