The Experimental Study Of Repairing Large Bone Defect With Biomimetic Poly (L-latic Acid)/Chitosan/Hydroxylapatite Composite Scaffolds

Objective:To evaluate the biocompatibility of the biomimetic poly (L-latic acid)/chitosan nano-fibers/nano-hydroxylapatite (PLLA/CSNF/nHA) composite scaffolds, to explore the feasibility of repairing the large bone defect in radius of New Zealand rabbits with it, which combined with bone marrow mesenchymal stem cells (BMSCs),and a new way of repairing the large bone defects with bone tissue engineering.Methods:1. The BMSCs were isolated from the New Zealand rabbits by the density-gradient centrifugation and cultured and purified.2. The BMSCs were identified by morphology observations with the optical microscope and the exterior antigen through the flow cytometry method.3. PLLA/CSNF biomimetic scaffolds were prepared through the dual phase separation technique, then mineralized into biomimetic PLLA/CSNF/nHA composite scaffolds. The poriness and the biomimetic structure were evaluated.4. The scaffolds were co-cultured with BMSCs. The cellular compatibility was evaluated through the adherent rate and proliferation activity of BMSCs.5. The PLLA/CSNF/nHA composite scaffolds were implanted into the subcutaneous tissues of the rabbit. The tissue compatibility was evaluated by the general observation and HE staining at 4th,8th and 12th week.6. The large defects (1.5cm) in radius of rabbits were prepared and experimented in three groups. PLLA/CSNF/nHA/BMSCs compound was planted into the defected position in the experimental group; PLLA/CSNF/nHA scaffolds in the control; and nothing in the blank. The curative effect was evaluated by general observation, radiology examination and HE staining at the 4th,10th and 16th week. Result:The BMSCs of New Zealand rabbits could be obtained through density-gradient centrifugation and cultured in vitro.1. CD29 and CD44 of BMSCs were expressed at 92.01% and 90.52% respectively while CD34 and CD45 at 0.36% and 0.78%. The shape of BMSCs were spindle and tufted.2. The pore diameters of PLLA/CSNF/nHA biomimetic scaffolds are 300μm to 450μm with good connectivity,and 200nm to 500nm of chitosan nanofibers in them.All the scaffolds showed a high porosity of 90% and good mechanical property3. The BMSCs had well adhesion in the scaffolds with the adherent rate (12h) of 78.3%, cytoactivity (14d) of 96.27%, G0-G1 period of 92.33%.4. The PLLA/CSNF/nHA biomimetic scaffolds had been degraded at 12th week after implanted in the subcutaneous tissues,witout hydroncus, necrosis or inflammation, reject reaction in surrounding tissues.5. The defects were repaired perfectly with the scaffolds degraded in the experimental group; partially repaired and degraded in the control; and full of fibrillar connective tissues in the blank,by the last evaluation.Conclusion:1. The BMSCs of New Zealand rabbit, that can be obtained through the density-gradient centrifugation and cultured stably for long term in vitro, are the ideal choice for tissue engineering.2. The structure of PLLA/CSNF/nHA biomimetic scaffolds,that prepared through the dual phase separation technique,with good biocompatibility and degradation,is analogous to that of extracellular matrix.3. PLLA/CSNF/nHA biomimetic scaffolds composited with BMSCs of rabbits have a good ability to accelerate the formation of new bone and repair the large bone defect.