Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage.

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15 U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015 U/mL) over a longer duration (one week) was investigated. Treatment with 0.004 U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65 % greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

Shearing of synovial fluid activates latent TGF-ß.

OBJECTIVE: TGF-ß is synthesized in an inactive latent complex that is unable to bind to membrane receptors, thus unable to induce a cellular biological response until it has been activated. In addition to activation by chemical mediators, recent studies have demonstrated that mechanical forces may activate latent TGF-ßvia integrin-mediated cellular contractions, or mechanical shearing of blood serum. Since TGF-ß is present in synovial fluid in latent form, and since normal diarthrodial joint function produces fluid shear, this study tested the hypothesis that the native latent TGF-ß1 of synovial fluid can be activated by shearing. DESIGN: Synovial fluid from 26 bovine joints and three adult human joints was sheared at mean shear rates up to 4000 s(-1) for up to 15 h. RESULTS: Unsheared synovial fluid was found to contain high levels of latent TGF-ß1 (4.35 ± 2.02 ng/mL bovine, 1.84 ± 0.89 ng/mL human; mean ± radius of 95% confidence interval) and low amounts (<0.05 ng/mL) of the active peptide. Synovial fluid concentrations of active TGF-ß1 increased monotonically with shear rate and shearing duration, reaching levels of 2.64 ± 1.22 ng/mL for bovine and 0.60 ± 0.39 ng/mL for human synovial fluid. Following termination of shearing, there was no statistical change in these active levels over the next 8 h for either species, demonstrating long-term stability of the activated peptide. The unsheared control group continued to exhibit negligible levels of active TGF-ß1 at all times. CONCLUSIONS: Results confirmed the hypothesis of this study and suggest that shearing of synovial fluid might contribute an additional biosynthetic effect of mechanical loading of diarthrodial joints.