Advantages of the Presence of Living Dermal Fibroblasts Within Restylane for Soft Tissue Augmentation

Abstract

For the elimination of facial wrinkles and skin contour defects, injectable filler substances composed of commercially prepared nonanimal stabilized hyaluronic acid (Restylane) are now widely used. Although this method of suspension has been shown to be relatively safe and convenient, varying degrees of resorption have required repeated percutaneous injections. This study was undertaken to evaluate the feasibility of Restylane, which is a modified hyaluronic acid, combined with cultured human dermal fibroblasts, to enhance the longevity of injected implants. The histologic changes of the injected implants were also evaluated. For the test group, fibroblasts from the dermis of healthy adults were isolated and cultivated. The cultured fibroblasts were measured with a hemocytometer. Five × 105 fibroblasts suspended in 200 μl of Dulbecco phosphate-buffered saline (DPBS) were then dispersed in 200 μl of Restylane to form a 400-μl human fibroblast–Restylane mix. For the control group, 200 μl of DPBS without fibroblasts were mixed with 200 μl of Restylane. These implants were subcutaneously injected into the back of an athymic nude mouse at 6 sites, the 3 left sites composing the control group and the 3 right sites composing the test group. Twelve nude mice were injected for a total of 36 injections per group. The nodular swellings that resulted from the injections were excised to include skin beyond the swelling points down to the panniculus carnosus layer using 5-mm punches, and the weights were measured at 1, 2, 4, 8, 12, and 16 weeks after the injections. Histologic comparisons were also performed to confirm the presence of human collagen in the fibroblast-mixed Restylane group using immunohistochemical study with antihuman collagen type I polyclonal antibody. The mean weight of the control group nodules decreased throughout the examination period. The mean weight at the 16th week was 60% of the weight at the first week. On the other hand, the mean weight of the test-group nodules decreased only over the first 2 weeks. Beyond 2 weeks, there was no further significant weight change. The mean weight at the 16th week was 91% of the weight measured at the first week. Histologic examinations of the control group exhibited negative immunohistochemical staining for human collagen at each examination period. The test group exhibited positive staining after 2 weeks, indicating the presence of human collagen. These results indicate that Restylane mixed with cultured human dermal fibroblasts may be successfully injected as living grafts for long-term retention of implants.