OSTEOGENESIS ENHANCED BY CHITOSAN (POLY-N-ACETYL GLUCOSAMINOGLYCAN) IN-VITRO

CHITOSAN, WITH A CHEMICAL STRUCTURE SIMILAR to hyaluronic acid, has be en implicated as a wound healing agent. The purpose of this research w as to evaluate the effect of chitosan on osteoblast differentiation an d bone formation in vitro. Mesenchymal stem cells were harvested from fetal Swiss Webster mice calvariae prior to osteoblast differentiation and calcification (12 to 13 days in utero). Stem cells were seeded in to 6-well culture plates at a density of 350,000 cells per well. Using this model, it was possible to quantify the influence of chitosan on osteoprogenitor differentiation and osteogenesis. Experimental wells w ere pretreated with 200 mu l chitosan (2 mg/ml in 0.2% acetic acid veh icle). Control wells were pretreated with 200 mu l vehicle (0.2% aceti c acid) or remained untreated. Cells were allowed to grow under optima l conditions for 14 days. Cell cultures were fixed with glutaraldehyde and stained with Von Kossa stain to identify bone forming colonies. P ositive staining colonies were identified and counted under light micr oscopy. Histologic cross-sections of representative positively stained colonies identified osteoblasts and confirmed bone formation. Examina tion of control wells revealed 3.6 +/- 0.6 colonies per well while exp erimental wells revealed a significantly greater average of 6.2 +/- 1. 2 colonies per well (P less than or equal to 0.01). Computer-assisted image analysis of the average area of bone formed by control colonies was 0.34 +/- 0.09 (relative units) while that of experimental colonies was 0.39 +/- 0.06 (relative units) per average bone forming colony. T he difference in mean size (control versus chitosan bone forming colon y) was not statistically significant (P = 0.4691). The results of this in vitro experiment suggest that chitosan potentiates the differentia tion of osteoprogenitor cells and may facilitate the formation of bone .