Preparation And Performance Of Nano-structured Bone Repair Materials Based On Carboxylated PLGA

Bone defect or bone nonunion treatment has been a clinical problem in the field of orthopeadic which have not been solved. The rapid development of bone tissue engineering and regenerative medicine provided a new method for for bone defects and nonunion treatment. As one of the most important elements of bone tissue engineering, the scaffolds are receiving extensive attention of researchers. At present, many bone regenreration composite materials based on inorganic particles and poly lactic-co-glycollic acid polymer have been developed, and the composite can combine the advantages of both materials. Conventional method of preparing inorganic particles/poly lactic-co-glycollic acid polymer composite is put the inorganic particles into the poly lactic-co-glycollic acid polymer and then mixed, which led to the inorganic ions dispersed unevenly. After implantation the component can not effectively contact with host cell and play its biological effects. Therefore, the preparation of composite scaffold with a porous and uniform structure based on inorganic particles/poly lactic-co-glycollic acid has an important clinical significance.ObjectiveTo investigate a method of preparing carboxylated poly lactic-co-glycollic acid polymer (PLGA-COOH); To investigate a method of preparing two new nano bone repair composite materials based on carboxylated PLGA, carboxylated poly lactic-co-glycollic acid polymer/nano-hydroxyapatite (PLGA-COOH/HAP) and carboxylated poly lactic-co-glycollic acid polymer/nano-tricalcium phosphate (PLGA-COOH/TCP); Study the characterization, mechanical property and degradation property of two composites. To explore the biocompatibility of nano-structured bone repair materials in vitro and its ectopic osteogenesis property and bone repair property in vivo.Materials and Methods1. Carboxylated PLGA was prepared, and further mixed with nano HAP and nano TCP, blended, stirring, washing salt and freeze-dried. Studying its characterization, mechanical property and degradation property.2. The scaffolds and MC3T3-E1 cells were cultured together to test the biocompatibility and bioactivity of scaffolds. MC3T3-E1 cells were cultured in medium with different concentrations of extract liquid, and observe the effect on cell proliferation and cell cycle by the CCK-8 test and flow cytometry. Detection of alkaline phosphatase levels (ALP) to clarify the effect of the scaffolds on MC3T3-E1 differentiation. The MC3T3-E1 was cultured on material surface to observe the cell adhesion property under scanning electron microscopy and CLSM.3. The scaffolds were loaded with rhBMP-2 in vitro and test the release property. Scaffolds loaded with or without rhBMP-2 were implanted into SD mice in vivo to observe its osteogenic properties.4. Evaluation the bone regeneration ability of scaffolds in a rabbit cranial critical bone defect model.5. Evaluation the bone regeneration ability of scaffolds loaded with or without rhBMP-2 in a rabbit ulna critical segmental bone defect model.Results1.1H NMR and molecular weight analysis showed that the molecular weight of PLGA polymer was about 120000 and carboxylated PLGA was successfully fabricated. SEM and Micro CT scan showed that the new composite scaffolds have a porous structure and pore diameter was uniform. Mechanical test shows that there was no significant different between two scaffolds. In vitro degradation experiments showed that the weight of scaffolds reduced with time. XRD results showed that the crystal structure of inorganic components of the composite scaffold unchanged. PLGA polymer molecular weight reduced over time. EDX analysis confirmed the two scaffolds characteristic elements are calcium, phosphorus and carbon.2. CCK-8 and flow cytometry showed that the scaffolds have good biocompatibility and can promote cell proliferation. SEM and CLSM result of cell adhesion showed the composite scaffolds and carboxylated PLGA are conducive to cell adhesion and cell morphology is better. It confirmed the above results that the composite scaffolds and carboxylated PLGA have good biocompatibility. Alkaline phosphatase levels (ALP) indicated that the scaffolds can promote the process of MC3T3-E1 differentiation.3. The results of rhBMP-2 in vitro release test showed that the two scaffolds can be used as carrier system of rhBMP-2, the effective release time can be up to 360 hours. The scaffolds loaded with or without rhBMP-2 were implanted into mouse gluteus muscle. After 2 weeks and 4weeks, heterotopic ossification and new bone can be observed clearly. Heterotopic ossification results show that scaffold loaded with rhBMP-2 has good biological activity and it is conducive to new bone formation. Scaffolds alone did not result in significant inflammatory response, which indicates that the scaffolds have a good biological safety and can meet with national standards of biomedical materials.4. The composite scaffolds were implanted in rabbit cranial critical defect and the results show that the composite scaffolds have a good capacity of bone repair. Compared with pure PLGA copolymers, the composites are more conducive to new bone formation and bone remodeling.5. The composite scaffolds loaded with or without rhBMP-2 were implanted into rabbit ulna segmental critical defect and the results show that the composite scaffolds loaded with rhBMP-2 can repair long bone segmental defect.Conclusion1. Carboxylated PLGA polymer was successfully prepared. Based on carboxylated PLGA polymer, two novel nanocomposite scaffolds, PLGA-COOH/HAP and PLGA-COOH/TCP were fabricated. Its mechanical property, porosity and pore size, degradation property can basically meet the clinical standards.2. In vitro experiments show that the scaffolds have a good biocompatibility and can promote cell proliferation, differentiation and adhension without a significant cytotoxicity.3. The two scaffolds can be used as a carrier system of rhBMP-2. Scaffolds loaded with rhBMP-2 has good biological activity and it is conducive to new bone formation in vivo in heterotopic ossification experiment.4. PLGA-COOH/HAP and PLGA-COOH/TCP composite scaffolds loaded with rhBMP-2 have a strong bone repair capability of lone bone critical defect. PLGA-COOH/HAP and PLGA-COOH/TCP composite scaffolds have a strong bone repair capability of cranial critical bone defect, which indicates that the scaffolds a have potential value in clinical applications.