Tenascin C, undulin, collagen XIV and fibronectin are extracellular matrix glycoproteins with a partial DNA sequence homology. During embryogenesis, tenascin C is abundant in mesenchymal tissues but its distribution in human adult tissue is severely restricted. The levels of tenascin C expression are enhanced with skin inflammation, wound healing and hyperproliferative skin diseases and return to normal in normal scar tissue after wound contraction is completed. Undulin/collagen XIV is associated with collagen fibrils and fibronectin is present throughout the dermis in adult skin but it is produced by keloidal fibroblasts in an increased amount. In this study we investigated by immunohistochemistry the expression of the three extracellular matrix proteins in keloids and normal skin as well as in keloidal and normal fibroblasts in vitro. In keloids, increased tenascin C expression was observed especially in the reticular dermis associated with collagen fibrils sharply demarcating the limit of the lesion. In normal tissue, tenascin C was only expressed beneath the basal lamina and dermal-epidermal junction. Corresponding to the in vivo findings, tenascin C expression was increased in keloidal fibroblasts compared with normal fibroblasts in vitro (P < 0.003), whereas undulin/collagen XIV and fibronectin expression in keloids and keloidal fibroblasts was similar to that in normal tissue and normal fibroblasts, respectively. Therefore, tenascin C is a marker associated with keloids and we suggest that keloidal fibroblasts, once stimulated, continue to produce tenascin C independently from circulating factors.

In order to obtain a persuasive explanation for the beneficial clinical effect of cryotherapy on keloids, we developed a reproducible model to apply freezing temperatures on cell cultures, and investigated their influence on proliferation, viability, synthetic activity and differentiation of dermal fibroblasts in vitro. Cell cultures were established from 13 untreated keloids and 10 healthy skin specimens matched for age and skin localization to the donors. No significant influence of cell freezing on the proliferation rates of both keloidal and normal fibroblasts was documented, but mechanical cell destruction with a wide variation in lethality rates (29% average lethal effect on keloidal fibroblasts and 41% on normal ones) was observed. When comparing specimens of keloidal and normal tissue derived from the same four donors, the keloidal fibroblasts were similar regarding their synthetic activity but presented enhanced tenascin-C expression compared with the normal fibroblasts. After cryotherapy, delayed collagen III increase was detected in both cell types (P = 0.03). The collagen II/collagen I ratio increased from 1.6 to 2.8 in the keloidal and only from 1.9 to 2.2 in the normal fibroblasts after subcultivation. Normal fibroblasts exhibited a significantly lasting increase in fibronectin synthesis after freezing (P = 0.03). The intensity of staining against tenascin-C was decreased in five of nine keloidal fibroblast cultures after cryotherapy (P < 0.05) but increased in four of five normal fibroblast cultures (P = 0.016), so that the intensity of tenascin-C staining after freezing became identical in both cell types. Immunoblot studies in four patients and two controls confirmed a temporary decrease of tenascin-C in keloidal but not in normal fibroblasts immediately after freezing. Significantly decreased staining with two markers of myogenic differentiation, myosin in keloidal fibroblasts (P = 0.002) and desmin (P = 0.007) in normal fibroblasts, could also be detected after treatment. In summary, with the help of a model for controlled cell freezing in vitro, cryotherapy was found to modify collagen synthesis and differentiation of keloidal fibroblasts.

Cryosurgery is currently regarded as the treatment of choice for keloids and hypertrophic scars. Four techniques are established or currently under evaluation in this area. The first is cryosurgery as monotherapy. Using this regimen, 241 of 356 patients (68%) with keloids and 72 of 89 patients with hypertrophic scars (81%), showed a greater than 50% improvement or complete regression (five studies). Using the second technique, cryosurgery with intralesional corticosteroids, significant regression of keloids was found in 119 of 159 patients (75%; four studies). Cryosurgery induces tissue edema and facilitates intralesional injections; however, the combined therapy was not superior (90% greater than 50% reduction of lesional volume) to monotherapy (83.3%) in a study of 40 patients with keloids. The third technique is surgical debulkment prior to cryosurgery without corticosteroids. This regimen is unavoidable in large keloids; however, it can result in recurrences despite its shortterm promising effects. Intralesional cryosurgery, the fourth technique, was developed by modifying the technique by Weshahy and is under evaluation in our department. In addition, histological and immunohistological studies of keloids and keloidal fibroblasts in vitro treated by cryosurgery have detected changes indicating potential rejuvenation of the scars.