[Cytological study on osteoblasts and in-situ setting calcium phosphate cements].

Objective: To evaluate biocompatibility of three kinds of self-developed injectable calcium phosphate cements (CPCs): chitosan microspheres/CPC, β-tricalcium phosphate (β-TCP)/CPC, and K(+)/CPC and the viability of the osteogenic cells cultured with CPC pastes and discs for 10 days.

Methods: The rabbit marrow stromal cells (rMSCs), isolated from rabbit bone marrow with density gradient centrifugation and flow cytometer, were cultured, expended and induced into osteoblasts. Alizarin red staining was used to determine the function of ossification. Then, rMSCs were incubated randomly on both the pastes and solidified discs of the 3 kinds of CPCs. The cells cultured on a 24-well plate were as blank control. Each group had 4 samples. The proliferation and differentiation of each group were observed using acid phosphatase assay (APA) and by testing the activity of alkaline phosphatase (ALP) at day 1, 4, 7, and 10. After stained by acridine orange(AO), the cells were observed, counted and analyzed with an epifluorescence microscopy. The morphology of the cells on CPCs was observed with scanning electron microscope(SEM).The data was subjected to two-way ANOVA followed by LSD test to compare between groups.

Results: The process of solidification of the three kinds of CPC pastes has the toxic effect on cells, which is irreversible. The proliferation( the average absortion of pastes are 0.049,0.050,0.049; the discs are 0.898,0.867,0.909;P<0.001), function(the average ALP activity of pastes after ten days are 0.775,0.782,0.798 U/g protein; the discs are 49.288,49.631, 49.744 U/g protein;P<0.001) and number of cells(the average number of cells of pastes after ten days are 3.7,3.7,3.7; the discs are 91.1,89.7,93.7;P<0.001) directly exposed to CPC paste significantly decreased compared with those contacting with the discs. By contrast, cells on the three kinds of discs showed better viability, proliferation, and ossification and cell numbers increased obviously with culture days.

Conclusion: The process of solidification of the three kinds of CPC pastes has toxic effect on cells. Further study needs to explore a method to protect osteoblasts when seeded into the CPC paste.