Orientation of gingival fibroblasts and newly-synthesized collagen fibers in vitro. Resemblance to transseptal and dento-gingival fibers.

Abstract

One hundred and fourteen pairs of transversally cut root sections ∼150 μm thick were obtained from porcine premolars and were attached to the floor of 35 mm culture dishes leaving an interdental space (IDS) of ∼500 μm between their cemental surfaces. Following partial demineralization of half of the samples, human gingival fibroblasts (HGF) were seeded in each dish and supplemented daily with 50μg/ml of vitamin C. The cultures were observed daily using phase microscopy. Demineralized and non‐demineralized specimens were obtained at each of 12 h, 24 h, 5, 10, 20, and 30 days, stained with van Gieson's picro‐fuchsin for identification of collagen or processed for examination by scanning electron microscopy (SEM). Two of the 30‐day‐old cultures were processed for examination by transmission electron microscopy.Sheets of oriented cells anchored to the cemental surfaces of the root sections and the floor of the dish were evident at 5 days. After 10 days of culture, a multilayered bridge‐like structure of oriented cells and fibrils (50–100 nm thick) was seen to connect the opposing cemental surfaces bordering the IDS at one or more sites. Oriented fine fibrillar structures were observed at this time using van Gieson's staining. The IDS was completely filled with oriented cells and fibers after 20 days of culture. Similar structures extending between the cemental surfaces and the floor of the dish developed around the rest of the circumference of the root sections. The appearance of this material at 30 days as observed by van Gieson's staining was reminiscent of transseptal and dento‐gingival fibers in vivo. The staining by van Gieson's method, the size of the fibrils, and the banded fibrils observed by TEM in cultures 30 days old suggest that the fibrillar material was predominantly collagen.The system was significantly more reproducible when demineralized rather than mineralized sections of root were used. It is suggested that attachment, migration, and contraction of the HGF are crucial for the orientation of the cellular and endogeneously produced fibrillar components in vitro and that these processes may operate in the development and healing of oriented fiber systems in vivo. Further, partial demineralization may facilitate these processes in wound healing.