Assay Techniques

Relatively little is known regarding the specific characteristics of subpopulations of ligament and tendon fibroblasts. Particularly in ligaments, it is not clear whether there are distinct subpopulations. Differences have been found between fibroblasts derived from the medial collateral ligament (MCL), an extra-articular ligament, as compared to those derived from the anterior cruciate ligament (ACL), an intra-articular ligament [15]. Morphologically, ACL fibroblasts appear more hexagonal, whereas MCL cells seem more spindle shaped and elongated. Phalloidin staining revealed a larger number of microfilaments in ACL cells. Proliferation and migration appeared to be significantly lower in ACL cultures. A medium-dependent, differential response to fluid-induced shear stress has also been found [8]. Similarly, different responses to several growth factors have been found in cultured ligament cells [9]. However, these previously mentioned studies have used animal cells, and it is not known whether similar differences exist in human cell types. The only study involving human cells showed a difference in signal pathways upon binding to fibronectin [10]. Using a micropipette-micromanipulation system, the individual cell adhesiveness to a fibronectin-coated glass surface was determined. By using inhibitors of signaling pathways, it was found that MCL fibroblasts play a crucial role in cAMP and Ca2+/phospholipid signaling during integrin-mediated cell adhesion. In ACL fibroblasts, this signal pathway appears to play only a minor role. Whether this truly represents a phenotypic difference, and is therefore usable as an assay technique, is yet to be determined.

Cultured tendon cells, following separation into TSC and IF, can be evaluated [4]. Microscopy has revealed that TSC are relatively large cells with lipid-containing cytoplasmic vesicles. Conversely, IF are smaller, fusiform cells that are more sensitive to trypsin, as compared to TSC. When comparing proliferation rates, TSC have a shorter generation time. Immunohistologic staining for fibronectin, as well as ELISA and mRNA quantitation, have been used to distinguish TSC and IF in culture [16]. Fibronectin levels are markedly increased in TSC culture, as compared to IF culture. TSC and IF have different gene responses to growth factors and other agonists, particularly in response to mechanical loading [17, 18]. Studies on tendon cell subpopulations also involve the use of animal specimens. Although not published, we have been able to confirm similar findings in our laboratory using human specimens. However, because of the difficulties in obtaining fresh, normal human specimens, only a limited number of investigations have been done using human cells.

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