| Though absorbable plates have been widely used in craniofacial fractures and osteotomy operations, some complications such as infections, foreign body reactions, malocclusions and bone joint dislocations appeared in some cases, which maybe related with the biocompatibility, degradation speed, the changes of mechanical strength during degrading and the influence on the degradation products to the environment of the absorbable plates. The aim of this research was to evaluate the biocompatibility of the materials by study the influences of the PDLLA/nano-Ha plates on tissues and osteoblasts in vitro and in vivo, and to investigate the effects of PRP and CGP on the local microenvironment. Furthermore, animal experiments were also used to evaluate the feasibility of the PDLLA/nano-Ha plates in rigid fixation of the craniofacial fractures.To establish a SD fetal rat primary osteoblastic cells culture method to lay the foundation of Research on biological properties of osteoblasts and function of various biological materials to osteoblast in vitro, cells obtained by two kinds of mixed enzymatic digestion for three stpes from day21of gestation fetal rat calvarial, and purified through differential attachment purification method. We used inverted microscope to observe the Cells. The cells were identified by real-time fluorescence quantitative and modified Von Kossa calcified nodule staining. The result sowed the osteoblasts were larger and scale-like with abundant cytoplasm and multiple pseudopodia under the inverted microscope. BGP, collagen and ?-actin gene amplification curve were smoothing, had an obvious exponential amplification stage and amplification efficiency. Modified Von Kossa's method showed a large number of black nodules formed. Conclusion:Through the method of two kinds of mixed enzyme digestion and differential velocity adherent purification, osteoblasts could be obtained with the advantage of large quantity, high purity and vigorous growth.To investigate the effects of the PDLLA plates on the growth and proliferation of fetal rat calvaria osteoblasts, the forth passage fetal rat calvaria osteoblasts were seeded on the PDLLA plates or co-cultured with medium being soaked with PDLLA/nano-Ha plates for24hours that were cut into10mm*7mm*3mm dimensions. The results showed that primary calvarial osteoblasts represent large cell volume. scale-like with abundant cytoplasm and pseudopodia under phase contrast microscope. RT-PCR showed the genes of osteocalcin, type I collagen and internal control ?-actin amplified with smooth curves, and obvious exponential amplification stage. Modified Von Kossa calcified nodules staining represented volumes of black nodules developed on the bottom of the culture plates. The results indicated that Repeated digestion and anchorage could obtain massive purified vigorous osteoblasts.The structure and proliferation of osteoblasts that were co-cultured on the surface of PDLLA plates were observed with scanning electronic microscope and CCK-8method respectively. Otherwise, the cell cycles of the osteoblasts were also investigated. The results were analyzed with SPSS10.0software. The results showed that being co-cultured with PDLLA/nano-Ha after Id,4d,7d. the osteoblasts represents intact cell morphology with processes connected with each other. The surfaces of the cells were rough with plenty of needle-like processes. Furthermore, the cells became flat and tightly fit with the material in the wake of increasing number of cells.The results of cck-8showed that the cells proliferate at the peak value after being incubated14days. There was no significant difference between the experiment and control groups. The flow cytometer results showed that at the4th day, PDLLA/nano-Ha could obviously induce the osteoblasts retaining in the G2/M stage (P<0.05). At4th and7th day. the cells incubated with the PDLLA/nano-Ha in the S stage were significantly lower compared with those of control group. In PDLLA/nano-Ha group, there were obviously increased cells being detained in G0/G1stage at the21st day than the other three time points (P?0.05). Otherwise, in the experiment group, the osteoblasts in the G2/M stage were significantly reduced in the21st day with the4th,7th and14th day. The PI value increased at the4th and7th day and started to reduce at the14th day.The results showed that the osteoblasts could not only adhere and proliferate properly on the surface of PDLLA/nano-Ha plates, but also display developing proficiency.The influence on the local microenvironment of absorbable plates may be one of the factors that induce postoperative complications. In this study, the mRNA expression level of Cbf?1?ALP?Col-I and BGP in osteoblasts and the changes of calcium and phosphorus concentration in the medium were both studied to investigate the mechanism of PDLLA/nano-Ha on the mRNA of osteoblasts and the local microenvironment. According to the mRNA sequence of P-actin?ALP?Col-I?Cbf?1and BGP from the GenBank, the primers were designed by Clone Manager software. The data were analyzed by SPSS10.0software.The results showed that the PDLLA/nano-Ha could promote the proliferation and differentiation of osteoblasts incessantly during the proliferative stage, while reduce the differentiation process in the maturation and mineralization stages. Otherwise, the changes of the local calcium and phosphorus concentration could accelerate the mineralization outside the cells, which was beneficial to sclerotized.-(bone forming)In the animal study, PRP was added into the fractured locations to investigate the variety of related index during the agglutination. Thirty-two adult male New Zealand rabbits weighted between2.5-3.0kilograms were randomly divided into four groups as2weeks,4weeks,8weeks and12weeks after operation. The animals belong in every time point were also randomly divided into two groups as being deal with PDLLA/nano-Ha or PDLLA/nano-Ha plus PRP. The animals were operated according to the mandible fracture model and executed at the four time points after operation. The methods including gross structure investigation, X-ray examination, HE staining and TEM were applied to observe the healing process. The results showed that the PDLLA/nano-Ha not only stimulate osteogenic action obviously and promote the growth and reconstruction of the callus, but also have no interference on the fracture healing.In the clinical experiment, a comparative study of the effectiveness osteosynthesis, process of fracture healing and biocompatibility was carried out between the absorbable and titanium plates. CGP were used in one of the experiment groups. The results showed that the absorbable plates not only provide rigid fixation but earlier exercises.The conclusions were obtained that PDLLA/nano-Ha plates both have good biocompatibility, moderate degradation rate and provide stable rigid fixation. Furthermore, the material accelerated the processing of fracture healing by improve the local microenvironment. |
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