Studies On Preparation And Biological Compatibility Experiments Of Porous PLLA/n-HA/PES Composite Material

ObjectiveIn:this study we have prepared the high porosity of composite porous PLLA/n-HA/PES materials in our lab, based on analysis of the PLLA composite, porous material production, material characterization, in vitro degradation, cell experiments, and animal in vivo experiments, in order to explore the use of this new composite porous materials for bone defect repairing, and provide theoretical basis for clinical application.Methods(1)Based on the solvent casting-particle leaching method, the PLLA/n-HA/PES composite porous materials were fabricated via blending by a certain viscosity average molecular weight of PLLA (poly-L-lactide), which has been prepared with Ring-opening polymerization in our lab before, with n-HA(nano-hydroxyapatite) powders and PES (Polyethylene succinate).(2)Different proportion of porous PLLA/n-HA/PES composite materials included the combination of the different ratio of PLLA/n-HA and the constant content of PES. The different proportions of porous composite materials have been degraded in PBS buffer solution. Degradation of materials was detected under different time point in the weightlessness rate, bibulous rate, PH value, the change of molecular weight and the visual change under the scanning electron microscopy (SEM) for verifying the mechanism of its degradation, and the best ratio of proportion for animal in vivo experiment.(3)The porous composite materials were developed together with the osteogenesis cells. Detection of the indicators were used for assessment of biological compatibility. The adhesion and proliferation of the cells on the surface of the material were determined by MTT method. Cell count with acridine orange staining, and alkaline phosphatase (ALP) level detection was performed, too. Spray gold to the surface of the material after drying and observe the cells morphology on it with SEM. Skin sensitization test results evaluated according to the contact patch sensitization reaction scoring standard; Acute systemic toxicity test evaluated according to materials toxicity evaluation grading; Subacute toxicity test analyzed according to the organ index and statistical data; Muscular implant test evaluated according to the histological evaluation standard.(4) Thirty rabbits of half male and half female were divided into three groups of ten ones each, called the blank group, autologous bone implant group and porous composite implant group respectively. Killed the groups of animals at2w,4w,6w,8w,12w, and16w respectively. Observed with X-ray and analyzed by the Lane-Sandhu X-ray score analysis. Scanned the boundary between bone and material with SEM and observed the porous material growth situation in bone tissue and morphology changes on its surface according to energy spectrum analysis. Bone mineral density(BMD) and biomechanical experiment was detected for evaluating the situation of bone defection. All of the results were analyzed by statistical analysis software.Results(1) Have prepared the high porosity of composite porous PLLA/n-HA/PES materials successfully, and the purity and structure were consistent with theory according to results of the experiments. The aperture size is between150to300μm by SEM and holes in the materials connected to each other and its bracket structure was in good condition, therefore it had a certain toughness and strength against the stress.(2) Adjusted the different ratio of PLLA/n-HA of100/10;100/20;100/30;100/40;100/50with the constant content of PES. The different proportions of porous composite material degraded in PBS buffer solution, according to the results of the weightlessness rate, bibulous rate, and PH, we concluded in that the most appropriate mass fraction of PES porous composite material was PLLA/HA=100/40.(3) Osteoblast cells grew well in the leaching solution of porous PLLA/n-HA/PES composite that indicated it has no cytotoxicity. The results of MTT text and the changes of alkaline phosphatase indicated that materials can be induced osteogenesis. The material culture together with osteogenesis cells and the cells could adhere to the surface and holes of the composite materials and then proliferated. The rats showed no toxicity by skin sensitization test and toxicity tests. Implanted materials did not cause organ canker, muscle atrophy or inflammation, and the wound was healing well.(4) Implanted materials were healing well in New Zealand white rabbit radial bone defect model. The results of X-ray, biopsy and SEM showed that implanted porous composite materials were tightly bound with the bone tissues and completely degraded and replaced by bone tissue after16weeks. BMD text showed that there was no significant difference between material implant group and autologous bone implant group (p>0.05). Mechanical testing also indicated there was no significant difference between them (p>0.05). All the results showed that bone formation capacity between material implant group and autologous bone implant group had no significant difference.Conclusion(1) The high porosity of PLLA/n-HA/PES porous materials with a good bracket structure had enough space for cell growth.(2) The most appropriate degradation of Porous composite material wasPLLA/HA=100/40.(3)Porous composite material of PLLA/n-HA/PES had good biological compatibility that could induce bone growth and promote bone defect repair.