Cytocompatibility Of Polylactic Acid And Modified Polylactic Acid With MC3T3-E1

Polylactic acid is a synthetic biodegradable material. 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)(DPLA), maleic anhydride modified poly(DL–lactic acid)(MPLA)and poly(DL–lactic acid)(DLPLA)were studied employing the way of in vitro primary MC3T3-E1 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 DPLA,MPLA and DLPLA 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) Materials synthesis and performance evaluation;poly(D,L-lactic acid) was modified in the bulk with maleic anhydride (MA), aliphatic diamine (DA). The advantage of using MA is due to its highly reactive anhydride groups. Butanediamine (BDA) is expected to neutralize the acidic degrading products of DL-PLA. . The results of rhodamine-carboxyl interaction method showed the dosage of MA Ninhydrin method was used to detect the BDA content in DPLA material The properties of various poly(D,L-lactic acid) based substrate films were investigated, including surface wettability.The surface wettability evaluation was based on static water contact angle (sessile drop contact angle) and water adsorption ratio.(2)Cells culture:MC3T3-E1 cells used in bone tissue engineering scaffolds biocompatibility evaluation, is now widely used to study the ability of bone cells one cell can be used for material compatibility comparison studied.(3) Cells in the material films of the adhesion and spreading: phase-contrast microscopy observed the growth of cells in the different material films for 4h, 8h, 24h, and photographed record. HE staining observed using MC3T3-E1 cells and basal materials after 3d of culture growth, and photo documentation. The experimental results show that: DPLA group cell density than DLPLA group and the MPLA, and DPLA materials group basement cell morphology was on the triangle or short spindle, relatively speaking, and DLPLA MPLA cell density on the growth of smaller, showed that cells in the DPLA The superior performance in the morphology and DLPLA the MPLA. Performance on cell morphology, DPLA was better than the MPLA and DLPLA cells compatibility. And,the higher BDA grafting ratio in DPLA was better.Micro suction aspiration technique measured basal cell and composite materials training for 0.5h and 4h after the adhesion of size. The experimental results show that: DPLA more conducive to cell adhesion. The higher BDA grafting ratio in DPLA was better cells compatibility.(4)Cell proliferation was determined by MTT assay at 2,4,6,8d.Through flow cytometer,cell cycle of the cells cultured for 72h on the different materials were analysed.The results showed that proliferation power and growth curve of MC3T3-E1 were different along with different materials.MC3T3-E1 on the three materials all possessed proliferation power.In those,the cell proliferation power on DPLA was notablely stronger than other two groups materials. That of DPLA was bigger than that of DLPLA and MPLA..DPLA made cell enter prophase of cell division(G2/M)and segmentation(S)the cells during the two stages were more than that of MPLA and DLPLA.This suggested that DPLA improved cells to divide and proliferate,and it had better cellular compatibility in the aspects of division and proliferation.And higher grafting ratio in DPLA was better in cells proliferation.(5) BCA was adopted to test the total protein content of cells on the different materials after cultured 5,10 and 15d,the effects that three materials had on MC3T3-E1 activity were compared.Alkaline phosphatase(ALP)activity of MC3T3-E1 was determined by enzyme dynamical method at 7,14 and 21d employing chromometry,the content of Ca was estimated after cell-material compound culture for 5,10 and 15d.The results showed that DPLA had better compatibility with MC3T3-E1 than MPLA and DLPLA in cell physiological function including protein synthesis,ALP activity and inorganic calcium excretion(6) By RT-PCR detection MC3T3-E1 cellsⅠcollagen mRNA expression. Detected by Western blot analysis MC3T3-E1 cells collagen typeⅠand osteopontin (OPN) in the situation. The results showed that different substrate materials on cell-related gene expression could have different effects.All the results summarized about cell morphology,cell attachmentand spread,cell proliferation and cell cycle,cell physiological function,and indicated that much betterbiocompatibility of DPLA with MC3T3-E1than MPLA and DLPLA,suggesting the potentially wide applications of DPLA in the biomedical area,especially in the tissue engineering.