Integrative Cartilage Repair Research Articles

Integrative Cartilage Repair: Inhibition by β-Aminopropionitrile

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Integrative cartilage repair: inhibition by beta-aminopropionitrile.

The effects of beta-aminopropionitrile, a known inhibitor of lysyl oxidase, on the extractability of newly synthesized collagen and integrative cartilage repair were determined in explant cultures of adult bovine articular cartilage. Dose-escalation studies indicated that treatment of cartilage explants for 6 days with beta-aminopropionitrile caused a dose-dependent inhibition of proteoglycan synthesis ([35S]sulfate incorporation) with a 50% inhibition at 2.2 mM. However, 0.25 mM beta-aminopropionitrile had no detectable effect on proteoglycan synthesis and was thus used for subsequent experiments. Treatment of cartilage with beta-aminopropionitrile for 14 days increased the extractability of newly synthesized collagen with 4 M guanidine-HCl while having little effect on proteoglycan synthesis, proteoglycan deposition, collagen synthesis (formation of [3H]hydroxyproline after labeling with [3H]proline), collagen deposition, or cartilage cellularity (DNA content). In untreated cultures, the percentage of radiolabeled collagen ([3H]hydroxyproline) that was extractable after 1 day of radiolabeling, 6 days of radiolabeling, or 6 days of label and 6 days of chase decreased from 81 to 25 and 9%, respectively. In beta-aminopropionitrile-treated cultures, the extractability was relatively higher (96, 62, and 47%, respectively). Treatment with beta-aminopropionitrile after radiolabeling with [14C]lysine also significantly inhibited the formation of the reducible crosslink [14C]dihydroxylysinonorleucine without affecting the overall deposition in cartilage of [14C]lysine and [14C]hydroxylysine. In functional repair studies, treatment with beta-aminopropionitrile caused an almost complete inhibition of integration between pairs of cartilage explants maintained in apposition for 2 weeks. These results indicate that beta-aminopropionitrile blocks the formation of collagen crosslinks in cartilage explants and suggest that such crosslinks are critical to integrative cartilage repair.

Biomechanics of integrative cartilage repair

Cartilage repair is required in a number of orthopaedic conditions and rheumatic diseases. From a macroscopic viewpoint, the complete repair of an articular cartilage defect requires integration of opposing cartilage surfaces or the integration of repair tissue with the surrounding host cartilage. However, integrative cartilage repair does not occur readily or predictablyin vivo. Consideration of the ‘integrative cartilage repair process’, at least in the relatively early stages, as the formation of a adhesive suggests several biomechanical approaches for characterizing the properties of the repair tissue. Both strength of materials and fracture mechanics approaches for characterizing adhesives have recently been applied to the study of integrative cartilage repair. Experimental configurations, such as the single-lap adhesive test, have been adapted to determine the strength of the biological repair that occurs between sections of bovine cartilage during explant culture, as well as the strength of adhesive materials that are applied to opposing cartilage surfaces. A variety of fracture mechanics test procedures, such as the (modified) single edge notch, ‘T’ peel, dynamic shear, and trouser tear tests, have been used to assess Mode I, II, and III fracture toughness values of normal articular cartilage and, in some cases, cartilaginous tissue undergoing integrative repair. The relationships between adhesive biomechanical properties and underlying cellular and molecular processes during integrative cartilage repair remain to be elucidated. The determination of such relationships may allow the design of tissue engineering procedures to stimulate integrative cartilage repair.

Effect of static compression on proteoglycan biosynthesis by chondrocytes transplanted to articular cartilage in vitro.

Transplantation of chondrocytes by injection or within carrier matrices has shown promise for augmenting the repair of articular cartilage defects. In vivo, transplanted chondrocytes are exposed to mechanical forces. This in vitro study examined the effect of a step application of compressive load to chondrocytes after the cells had been seeded onto a cartilage surface. Bovine chondrocytes were transplanted onto bovine cartilage disks, allowed to attach for 1 hour or 4 days, and subjected to compression through overlying cartilage disks in a confined compression configuration. Before use, the disks were lyophilized to lyse the endogenous chondrocytes and thereby allow assessment of the metabolic activity of the transplanted cells. During a 16-hour application of compressive stress of 0.24-0.72 MPa, proteoglycan synthesis, assessed as [35S]sulfate incorporation into macromolecules, was inhibited by approximately 68% after the 1-hour attachment and by approximately 45% after the 4-day attachment. Cell retention after the application of load was assessed by use of [3H]thymidine-tagged chondrocytes and quantitation of the displacement of radioactivity. After the 1-hour seeding period, loading induced a dose-dependent dislodgment of [3H]radioactivity (as much as 35%) from the tissue bilayer. In contrast, after the 4-day seeding period, there was no detectable effect of loading on chondrocyte dislodgment with an 8-12% release of radioactivity. The inhibitory effect of a 16-hour compression of 0.48 MPa applied after the 4-day seeding period was studied further. This protocol did not appear to have an irreversible effect on chondrocyte metabolism; at 2 days after the release of load, proteoglycan synthesis by the loaded cells was stimulated by 41% compared with transplanted cells that were not subjected to loading. These results suggest that the application of static compressive stress to chondrocytes at a cartilage surface may affect biosynthesis by these cells and thus subsequent integrative cartilage repair. Such an effect may have implications for optimization of the tightness of the press fit of a cell-laden cartilaginous construct into an articular defect.