Osteoblast Compatibility Of Polylactic Acid And Modified Polylactic Acid

Polyactic acid materials are synthetic and biodegradable materials. Their surfaces were modified, and acidity matters produced by degradation were neutralized, which will facilitate adhesion between materials and cells, then improve cellular compatibility of materials. Based on synthesizing this kind of modified polyactic acid materials, cellular compatibility of ethylenediamine modified poly(DL–lactic acid)(DMPLA), RGD modified poly(DL–lactic acid)(RGDPLA) and poly(DL–lactic acid)(PLA) were studied employing the way of in vitro primary osteoblast culture, and directly incolulating cells on the surfaces of materials. Cellular compatibility was compared in the aspects of cell morphology, cell attachment and spread, cell proliferation and cell cycle, cell physiological function. According to the experimental results, the biocompatibility of RGDPLA, DMPLA and PLA have been evaluated respectively. Themry gists were provided for clinical application. The tissue engineering materials which have good biocompatibility are expected to get. The main works and conclusions are included as follows: (1) Cell culture:osteoblasts were obtained and cultured from crania of neonatal rats by tissue blocks method. Cultured cell were identified by improved Von Kossa method. Results showed that cultured primary osteoblasts had characteristics of osteoblasts, which could be used for experimental research on cellular compatibility of materials. (2) Cell morphology analysis on the materials: growth conditions were observed on materials by phase contrast microscope, and recorded by taking pictures. The results indicated that the cell density on the RGDPLA was higher than those on the DMPLA and PLA. Furthermore, the cell form on the RGDPLA showed triangle or shuttle, when it was cultured 5 days, a majority of cells were connected with each other and became reticulation. Relatively speaking, the cell density were lower on the DMPLA and PLA. The experiment result suggested that: as morphology was regarded, RGDPLA had better cellular compatibility than other materials including not modified and modified. (3) Cells were cultured for 4,8,12,16 and 20h respectively on the different materials, then the cell numbers were counted, and the cell attachment for earlier period was analyzed and compared quantitatively. HE staining technique was employed to observe spreading morphology that the materials had effects on the osteoblasts. The experimental results showed that in the earlier period a lot of osteoblasts had attached on the RGDPLA, DMPLA and PLA dealt with osteoblasts inferior. Normal spreading osteoblasts are square or fusiform shape, and they have polydactyly centrifugal spreading pseudopodium. In the early days osteoblasts showed slice appearance, under low time microscope osteoblasts showed round shape, the edge was abnormity. The cell on RGDPLA spreaded a lot in the quantity and quality. The numbers of spreading cells on DMPLA and PLA were a little while RGDPLA wax beneficial for the attachment and spread of osteoblasts. (4) Cell proliferation was determined by MTT assay at 2,4,6,8d, and a growth curve was drawn. Through flow cytometer, cell cycle of the cells cultured for 12,24 and 48h respectively on the different materials were analysed. The results showed that proliferation power and growth curve of osteoblasts were different along with different materials. Osteoblasts on the three materials all possessed proliferation power. In those, the cell proliferation power on RGDPLA was notablely stronger than other two groups materials and glass control group. That of DMPLA was bigger than that of PLA. RGDPLA made cell enter prophase of cell division(G2/M)and segmentation(S)the cells during the two stages were more than that of DMPLA and PLA. This suggested that RGDPLA improved cells to divide and proliferate, and it had better cellular compatibility in the aspects of division and proliferation. (5)Coomassie brilliant blue was adopted to test the total protein content of cells on the different materials after cultured 4,7 and 10d, the effects that three materials had on osteoblast activity were compared. Alkaline phosphatase (ALP) activity of osteoblasts was determined by enzyme dynamical method at 4,7 and 10d employing chromometry, the content of Ca was estimated after cell-material compound culture for 4,7 and 10d. The results showed that RGDPLA had better compatibility with osteoblasts than DMPLA and PLA in cell physiological function including protein synthesis, ALP activity and inorganic calcium excretion. All the results summarized about cell morphology, cell attachmentand spread, cell proliferation and cell cycle, cell physiological function, and indicated that much better biocompatibility of RGDPLA with osteoblasts than DMPLA and PLA, suggesting the potentially wide applications of RGDPLA in the biomedical area, especially in the tissue engineering.