Graft of autologous fibroblasts in gingival tissue in vivo after culture in vitro preliminary study on rats

Abstract

Several grafting techniques and guided tissue regeneration techniques (GTR) have been well-developed in periodontal surgery. However, these techniques could induce pain and side effects, such as a gingival recession during the healing period following the therapy. The graft of a small autologous connective tissue, using non-invasive surgical techniques could yield several benefits for the patients. Our preliminary study explores the feasibility of collecting healthy gingival tissues, culturing them in vitro to amplify rat gingival fibroblasts (RGF) and inoculating the obtained cells into autologous rat gingival tissues in vivo. Gingival tissues samples were cultured as explants as described by Freshney et al. and Adolphe. Confluent cells surrounding explants were detached after 7 d of culture from Petri dishes using 0.05% trypsin and designated "first transferred cells" (T1). At the third passage (T3), cells cultured as monolayer were either examined under microscopy--phase contrast, scanning, or transmission electron--or numerated after trypan blue exclusion test. Autologous RGF labelled with fluorochrome were inoculated at the vestibular and palatine site of gingival tissue close to the superior incisors. In this preliminary study, 12 Wistar rats were used; for each, 2 biopsies were dissected and fixed for phase contrast or fluorescence microscopy. On d 1, 3 and 7 after injection in rat gingival tissues, fluorochrome-labelled cells could be detected in all these.

A simple and established method of tissue culture of human gingival fibroblasts for gingival augmentation.

“…The cells were only isolated mechanically and cultured in DMEM supplemented with 10% FCS and penicillin (100 IU/mI), streptomycin (100 g/ml), and amphotericin B (100 μg/ml). Similarly to our methods, Simalin-Safo et al [11], in an in vitro preliminary study of rat gingival fibroblasts, used samples of epithelial connective tissue (2 × 2 × 2 mm) in DMEM with 20% FBS and penicillin (100 IU/ml), and streptomycin (100 μg/ml), but without amphotericin B. It is very important to use amphotericin B because it protects the cells against mycological infection.…”

“…In other methods, fibroblasts were isolated by dispase and collagenase. The cells were cultivated in a recombinant collagen sponge [12]. Earlier methods of gingival fibroblast isolation and culture involved very specific enzymes for cell separation.…”

“…Studies by Simain-Sato et al on animals in 1999 and by Pini Prato et al on humans in 2000 [10,11] described the possibility of culturing connective tissue from a small fragment of autologous host tissue. This method allows achieving gingival augmentation 1) with reducing the size of the donor site to a minimum (ca.…”

An Estimation of the Biological Properties of Fish Collagen in an Experimental In Vitro Study

“…Triple-helical conformation of collagen and its peptides were extracted from various animals including bovine skin, porcine skin, bird feet, frog skin, shark skin, rat tail tendons [8]. There have been many attempts to find an alternative source of equally efficient and in some opinion safer collagen and another form [3,8,9]. One of the prospective sources comes from marine organisms (sea urchins, fish scale and skin, jellyfish, shark skin).…”