Inhibition of bone formation by gingival connective tissue cells

Objectives: Empirical observation suggests that the potential for bone regeneration may be limited by the presence of the gingival connective tissues. The aims of this study were to test the hypothesis that gingival connective cells can directly inhibit osteoblast differentiation. Methods: Primary rat osteoblast (ROB) cultures were obtained by enzymatic digestion of rat calvariae and in initial experiments from rat bone marrow stromal cells (BMSC); rat gingival (RGF) and periodontal ligament (PDL) fibroblast cultures were prepared from tissue explants. In initial experiments RGF cells were tested for inhibitory activity in co-cultures of cells in 3-D collagen gels. In further experiments 48 hours conditioned medium (CM) from RGF, PDL and ROB cultures were used to stimulate ROB cultures for 48 hours. Osteoblast differentiation was assessed by alkaline phosphatase (ALP) expression expressed in arbitrary OD units, and proliferation assessed by MTT assay. Results: In co-culture experiments the inclusion of BMSC cells in collagen gels with osteoblasts resulted in a 45% reduction in ALP expression (ALP values : BMSC 0.93 ± 0.18, BMSC + 3T3 0.52 ± 0.069, P = 0.05). CM from RGF inhibited ALP expression by 55% compared to control media; however PDL CM had no effects on ALP expression ( control media 3.9±0.21; PDL CM 4.06 ± 0.85; RGF CM 1.75 ± 0.11* * P < 0.0001 by ANOVA and Bonferoni tests). Neither CM caused significant changes in proliferation. In contrast ROB CM inhibited both ROB cell proliferation and ALP expression. Conclusions: The results suggest that gingival connective tissue may inhibit bone formation by the production of soluble factors that block osteoblast differentiation. Interestingly, PDL cells did not inhibit osteoblast differentiation. These data support the idea that superficial and deep connective tissues may have distinct roles in regulating the potential for bone regeneration. Supported by ITI Research Foundation