Effect of heat treatment of poly(l-lactide) on the response of osteoblast-like MC3T3-E1 cells

Poly(L-lactide) (PLLA) products are molded by heat extrusion. These treatments may change chemical properties and biological response of the PLLA to cells. In this study, the effect of heat treatment of PLLA on osteoblast proliferation and differentiation was examined in vitro. Osteoblast-like MC3T3-E1 cells were cultured for 2 weeks on the PLLA subjected to various heating temperature and time combinations. The protein, DNA, and hydroxyproline (HYP) contents and alkaline phosphatase (ALP) activity of cells cultured on the untreated (non-heated) PLLA with a weight average molecular weight (Mw) of 1,000,000 (high Mw PLLA) were not significantly different from those of cells cultured on glass. The activation of osteoblast differentiation by the high Mw PLLA was weak. In contrast, increases in ALP activity and HYP content were found for cells cultured on the PLLA heated at a high temperature of 200 or 250 degrees C. Heat treatment of high Mw PLLA increased differentiation of MC3T3-E1 cells cultured upon it. Significant degradation of PLLA (decrease in molecular weight and increase in molecular weight distribution) were observed following heat treatment. The Mw of PLLA decreased from 1,000,000 to below 20,000, and 14.4 microg of L-lactic acid was released from 10 mg of PLLA by heating at 250 degrees C. Therefore, the effect of low Mw chemicals, which were expected to be the degradation products of high Mw PLLA after heat treatment, on MC3T3-E1 cell activities was examined. Increases in the protein, DNA and HYP amounts and ALP activity for cells cultured with L-lactide or L-lactic acid were observed at 100 microg/ml, but not at 10 microg/ml. When the cells were cultured on the low Mw PLLA (Mw 20,000), their biological parameters also increased. Twelve micrograms of L-lactic acid released from 10 mg of the low Mw PLLA during 2 weeks incubation. The concentration of L-lactic acid in the incubation solution of low Mw PLLA or heat-treated PLLA was too small to cause cell activation. These results suggested that increases in osteoblast differentiation on the heat-treated PLLA was not to due to soluble degradation chemicals, such as L-lactic acid, rather than the remaining low Mw PLLA.