Experimental Study Of Loading BMP-9 NHA/Col?/MWCNT Composite Scaffold For Bone Defect Repairing In Rats

Objective:As the special part of the human skeleton system,craniofacial bones not only need to perform the important functions of pronunciation,speech and mastication,but also plays an indispensable role in the appearance of the human.In clinical practice,patients with different degrees of maxillofacial bone defects caused by trauma,tumor or congenital malformations are not uncommon to oral and maxillofacial surgeons.This disease has a serious of impact on language,pronunciation,swallowing and appearance.At the same time,it brings great impact on the patients'daily life and mental.In order to meet the need for bone defect treatment,a large number of researchers are devoting to the methods of promoting bone repairing.Bone tissue engineering is one of the effective methods,especially for a large size of bony defect.Scaffolds,cells and cytokines and chemokines promoting osteogenesis are regarded as three important factors in bone tissue engineering,and scaffold is usually the key factor of them.As an ideal material for bone tissue engineering,it should possess good biocompatibility,good surface activity,bone conductivity,osteoinduction,proper pore size and porosity,suitable mechanical strength,plasticity and strong effect on bone regeneration.Hydroxyapatite?HA?is considered as the most studied biomaterials for its proven biocompatibility and for being the main inorganic constituent of the bone.Nano-hydroxyapatite?nHA?,as biomaterial,is similar to the inorganic component of human bone,so it is considered to be more suitable for bone scaffolds.It is also an important source of calcium and phosphate,very important for the bone regeneration and bone remodeling.Collagen I?ColI?is a major extracellular matrix?ECM?component that has been widely used for constructive remodelling to facilitate cell growth and differentiation.It is therefore a suitable base material to fabricate a porous collagen scaffold.Carbon nanotubes?CNTs?,known as"Super Fiber",is a new nano-materials developed by Japanese scholar.It was applied in bone tissue engineering to increase the physical and chemical properties of scaffold for the first time.Bone morphogenetic protein 9?Bone Morphogenetic 9 Protein,BMP-9?more and more attention by scholars,also known as GDF-2?Growth Differentiation Factor 2?,in all known BMPs proteins,the strongest effect on bone formation.At present,more and more researches on the application of BMP-9 in bone tissue engineering have been carried out,and scholars have used different methods to construct the composite scaffold loaded with BMP-9 protein.Methods:1.Synthesis of nHA/Col I/MWCNT scaffold.2.The mechanical strength of the scaffold was measured by the universal mechanical testing instrument.3.The water absorption and swelling ratio were calculated by immersion in ddH₂O.4.The degradation rate of scaffold material and the analysis of PH value in the process of degradation;5.SD rats BMSCs were isolated,cultured,flow cytometry,osteogenic induced and alizarin Red S staining.6.CCK-8 kit was used to detect the cytotoxicity of the scaffolds.7.SEM was used to observe the cell adhesion.8.The effect of cytokine BMP-9 on the osteogenic differentiation of BMSCs and the expression of OCN,Akt and p-Akt by the Western Blot.9.Construction of BMP-9-nHA/ColI/MWCNT scaffold and the application of ELISA kit to detect the release of BMP-9.10.The adhesion rate was detected after BMSCs was co-cultured with BMP-9-nHA/ColI/MWCNT scaffolds.11.BMSCs were induced osteogenic differentiation and related protein expression was detected.12.The preparation of SD rats cranial bone defect and the implantation of different scaffolds.13.CT and histological examination after operation.14.Statistical analysis of experimental datas.Result:1.nHA/Col I/MWCNT scaffold with the concentration of 1%wt MWCNT is best suitable.The compressive strength is 5.91±0.58MPa,the compression modulus is0.66±0.72MPa,the absorption rate is 253±6%,the swelling rate is 34.5±1.3%,the porosity is 89±3.3%and the pore size is 129±23?m,with the concentration of MWCNT increasing,the compression strength and the compression modulus increasing,while the water absorption rate,the swelling rate,the porosity and the pore size were decreasing.2.SEM image showed that single round cells became flat,polygonal or triangular.Cell microfilaments and pseudopodia were tightly connected to the scaffold.CCK-8 assay kit demonstrated the scaffold is without cytotoxicity.3.The ALP activity and OCN expression were significantly increased in the process of BMSCs induced by BMP-9,and the phosphorylation level of Akt was increased.4.After BMP-9 was loaded with nHA/Col I/MWCNT scaffold,ELISA assay kit was used to verify that the BMP-9 release until the 17th day.Compared with the control group,the early adhesion rate of the new composite scaffold was significantly higher.And the activity of ALP and the expression of OCN were increased in the BMP-9-nHA/Col I/MWCNT scaffold compared with the blank group,and the phosphorylation of Akt was increased.5.Twelve weeks after implantation and analyzed by radiology and histology,BMP-9 loaded nHA/Col I/MWCNT composite scaffold shows good biocompatibility and promoting bone regeneration in vivo.Conclusion:1.nHA/Col I/MWCNT scaffold was fabricated by blending freeze-drying.The most suitable concentration of MWCNT is 1%wt.2.nHA/Col I/MWCNT scaffold has good mechanical strength,good biocompatibility,can be used as a scaffold for bone defect repair.3.BMP-9-nHA/Col I/MWCNT composite scaffold can promote the osteogenic differentiation of BMSCs in vitro,and the effect of the scaffold material on the phosphorylation of Akt was increased.4.The application of BMP-9 loaded nHA/Col I/MWCNT scaffolds to repair the CSD of rats,confirmed that the composite scaffold had significant osteogenic effect,can be used as a selection method of repairing bone defect for bone tissue engineering.