Cathepsin K (EC 3.4.22.38) is a recently described enzyme that has been shown to cleave type I collagen in its triple helix. The aim of this study was to determine if it also cleaves type II collagen in the triple helix and to identify the helical cleavage site(s) in types I and II collagens. Soluble human and bovine type II collagen, and rat type I collagen, were incubated with cathepsin K before the reaction was stopped with trans-epoxysuccinyl-l-leucylamido-(4-guanidino)butane (E-64). Analysis by SDS/PAGE of the collagen digests showed that optimal activity of cathepsin K against native type II collagen was between pH 5.0 and 5.5 and against denatured collagen between pH 4.0 and 7.0. The enzyme cleaved telopeptides as well as the alpha1(II) chains, generating multiple fragments in the range 90-120 kDa. The collagenolytic activity was not due to a contaminating metalloenzyme or serine proteinase as it was not inhibited by 1,10-phenanthroline, EDTA or 3,4-dichloroisocoumarin. Western blotting with anti-peptide antibodies to different regions of the alpha1(II) chain suggested that cathepsin K cleaved native alpha1(II) chains in the N-terminal region of the helical domain rather than at the well-defined collagenase cleavage site. This was confirmed by N-terminal sequencing of one of the fragments, revealing cleavage at a Gly-Lys bond, 58 residues from the N-terminus of the helical domain. By using a similar approach, cathepsin K was found to cleave native type I collagen close to the N-terminus of its triple helix. These results indicate that cathepsin K could have a role in the turnover of type II collagen, as well as type I collagen.

Adult chondrocytes are less chondrogenic than immature cells, yet it is likely that autologous cells from adult patients will be used clinically for cartilage engineering. The aim of this study was to compare the postexpansion chondrogenic potential of adult nasal and articular chondrocytes. Bovine or human chondrocytes were expanded in monolayer culture, seeded onto polyglycolic acid (PGA) scaffolds, and cultured for 40 days. Engineered cartilage constructs were processed for histological and quantitative analysis of the extracellular matrix and mRNA. Some engineered constructs were implanted in athymic mice for up to six additional weeks before analysis. Using adult bovine tissues as a cell source, nasal chondrocytes generated a matrix with significantly higher fractions of collagen type II and glycosaminoglycans as compared with articular chondrocytes. Human adult nasal chondrocytes proliferated approximately four times faster than human articular chondrocytes in monolayer culture, and had a markedly higher chondrogenic capacity, as assessed by the mRNA and protein analysis of in vitro-engineered constructs. Cartilage engineered from human nasal cells survived and grew during 6 weeks of implantation in vivo whereas articular cartilage constructs failed to survive. In conclusion, for adult patients nasal septum chondrocytes are a better cell source than articular chondrocytes for the in vitro engineering of autologous cartilage grafts. It remains to be established whether cartilage engineered from nasal cells can function effectively when implanted at an articular site.

Three-dimensional hyaline cartilage can be engineered using BMSCs from patients with OA. This method could thus be used for the repair of cartilage lesions.

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When bone marrow stem cells were differentiated into chondrocytes, adipocytes, or osteocytes, nucleostemin expression was 70%-90% lower than in the undifferentiated cells retained in monolayer culture. We conclude that nucleostemin is a marker of undifferentiated human adult bone marrow stem cells and that it is involved in the regulation of proliferation of these cells. STEM CELLS