ABSTRACT
Articular cartilage possesses a remarkable, mechanically-robust extracellular matrix (ECM) that is organized and distributed throughout the tissue to resist physiologic strains and provide low friction during articulation. The ability to characterize the make-up and distribution of the cartilage ECM is critical to both understand the process by which articular cartilage undergoes disease-related degeneration and to develop novel tissue repair strategies to restore tissue functionality. However, the ability to quantitatively measure the spatial distribution of cartilage ECM constituents throughout the tissue has remained a major challenge. In this experimental investigation, we assessed the analytical ability of Raman micro-spectroscopic imaging to semi-quantitatively measure the distribution of the major ECM constituents in cartilage tissues. Raman spectroscopic images were acquired of two distinct cartilage tissue types that possess large spatial ECM gradients throughout their depth: native articular cartilage explants and large engineered cartilage tissue constructs. Spectral acquisitions were processed via multivariate curve resolution to decompose the "fingerprint" range spectra (800-1800 cm-1) to the component spectra of GAG, collagen, and water, giving rise to the depth dependent concentration profile of each constituent throughout the tissues. These Raman spectroscopic acquired-profiles exhibited strong agreement with profiles independently acquired via direct biochemical assaying of spatial tissue sections. Further, we harness this spectroscopic technique to evaluate local heterogeneities through the depth of cartilage. This work represents a powerful analytical validation of the accuracy of Raman spectroscopic imaging measurements of the spatial distribution of biochemical components in a biological tissue and shows that it can be used as a valuable tool for quantitatively measuring the distribution and organization of ECM constituents in native and engineered cartilage tissue specimens.
ABSTRACT
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.
