Biomimetic Synthesis Of Silk Fibroin/nano-hydroxyapatite Composite Biomaterial And Experimental Study Of Spinal Fusion In Rabbit

Spinal fusion is one of the most common spinal surgery primarily for treatment of spinal instability caused by trauma,degeneration,deformity,cancer and other spine diseases.internal fixation is used to provide immediate stability of the spine, providing the necessary biomechanical fusion environment,however,The long-term stability and biological healing of the spine must rely on bony fusion. In the current clinical use of spinal fusion materials, autogenous bone grafting is generally considered the "gold standard", the major disadvantages of autograft are the limited amount of graft material available as the potential morbidity of the harvest complications including prolonged pain at the donor site,nerve damage,fracture and infection.bone allograft has also been used,its disadvantages include immune response and potential risk of infectious transmission.therefore,we are looking for the ideal bone grafting biomaterial to promote spinal fusion.the bone substitutes currently available have specific disadvantages and none of them is entirely suitable for clinical application including metals,polymer and ceramics.so,current researth is focusing on how to combine the advantages of various material,overcome their shortcoming,to preparation of composite biomaterials with excellent performance.Silk fibroin has excellent biocompatibility, biodegradability and unique mechanical properties,and can induce hydroxyapatite crystal nucleation and growth as the template of inorganic mineralization.silk fibroin/nano-hydroxyapatite composite biomaterial simulates the composition and structure of nature bone tissue,improve nano-hydroxyapatites insufficient mechanical properties,and so is expected to be an ideal bone graft biomaterial.In this experiment, biomimetic synthesis of silk fibroin/nano-hydroxyapatite like-bone composite biomaterial is accomplished by coprecipitation method using silk fibroin,CaC12 and (NH4)2HP04 as raw materials,mimicing the organic and inorganic constituents of natural tissue.after that,the biological safety and biocompatibility with osteoblasts are evaluated, and the degradation performance and ectopic bone formation capacity of silk fibroin/nano-hydroxyapatite composite biomaterial is studied by putting it into bags of rabbit muscle.further,we evaluate the effect of silk fibroin/nano-hydroxyapatite material compound autogenous bone marrow stromal cells on the enhangcement of rabbit spinal fusion,in order to explore an ideal bone repair material to meet clinical need.Part 1:Biomimetic synthesis and characterization of silk fibroin/nano-hydroxyapatite composite biomaterialObjective:Simulating the composition and structure of nature bone tissue,biomimetic synthesis of silk fibroin/nano-hydroxyapatite composite.Method:silk fibroin/nano-hydroxyapetite composite biomaterial is synthesized by the coprecipitation method using silk fibroin,CaC12 and (NH4)2HP04 as raw materials.as organic/inorganic components ratio in nature bone is 30/70,composites biomaterial with 70% nano-hydroxyapatite are selected for further study.the microstructure,phase composition,chemical structure and Thermal stability are determined by scanning electron microscopy(SEM),transmission electron microscopy(TEM),X-ray diffraction(XRD), Fourier Fourier transform infrared spectrometer (FTIR), thermal gravimetric analysis (TGA) and so on.Results:The as-prepared SF/nHA composites contain silk fibroin 32%,simulating the proportion of organic ingradients in natural bone tissue,with calcium phosphorus ratio of 1.66,similar to the standard calcium phosphate ratio in hydroxyapatite. Characterization results show that the inorganic component in the composite is poorly crystalline HA containing carbonate ions. The typical HA crystallites possess 200-500 nm in length and around 20-30 nm in width. The SF is incorporated into the composite by strong chemical interactions between HA and SF, probably take place via the chemical bonding between calcium ions and the carbonyl groups of SF. the silk fibroin will induce self-organized orientation of HA nanocrystal as the template, the growth of HA crystal will progress spontaneously with preferred orientation (along its c-axis),which.is similar to the apatite crystals deposited on the surface of collagen fibers in natural bone tissue.Conclusion:Like-bone silk fibroin/nano-hydroxyapatites composite biomaterial is synthesized by the coprecipitation method,the composition and structure is similar to the nature bone tissue.Part 2:A study on the biocompatibility of SF/nHA composite biomaterial cocultureed with rat osteoblasts.Objective:to evaluate the biocompatibility of SF/nHA composite biomaterial with bone tissue functional cells-osteoblasts.Methods:MC3T3-E1 osteoblasts were seeded on the SF/nHA composite and co-cultured.the adherent cell number and morphological changes of oateoblasts were observed by scanning electron microscope.the adhesion and proliferation were determined by inverted microscope and scanning electron microscope,the cells were also stained for matrix minerlization.Results:the adherent number of MC3T3-E1 osteoblasts on the sueface of SF/nHA composites are higher than that of control,and the osteoblasts are more easily to adhere and spread on SF/nHA,the proliferation mineralization of steoblasts was improved also.Conlusion:biomimetic synthesized SF/nHA composite biomaterial has excellent biocompatibility for osteoblasts.Part 3:the study on the enhancement of rabbit Posterolateral spinal fusion with silk fibroin/nano-hydroxyapatite composite biomaterial compound autogenous bone marrow stromal cells. Objective:To evaluate the effect of the SF/nHA composite biomaterial on the enhancement of posterolateral spinal fusion in rabbit. Methods:Obtain bone marrow from Rabbit beneath the greater femur trochanter, Isolate and purify BMSCs in vitro and then induce BMSCs in osteogenic differentiation.observe cell's morphology,identify cell surface antigen,evaluate cell's ossification by alkali phosphatase stain and Von Kossa stain. SF/nHA composite biomaterial compound BMSCs were applied to New-Zealand rabbit Posterolateral lumbar spine fusion model. Gross and imaging observation,histological examination and manual palpation of the fusion masses were performed to evaluate the ability of enhancement of spinal fusion. Results:1.BMSCs cultured in the culture dish has good performance of adhesion,growth,proliferation,and showed good significant osteogenic activity after osteogenic induction..the results of Von Kossa stain and alkali phosphatase stain indicated BMSCs has osteogenesis potential.2. The rabbits were sacrificed at 12 weeks after the fusion surgery, and the samples were obtained.gross observation showed that there is obsvious new bone regeneration on the silk fibroin/nano-hydroxyapatite composite biomaterial sides similar to the autografted sides.X-ray,and palpation also evidenced the results.hietological examination demonstrated that SF/nHA composite biomaterial transformed into bone tissue gradually. Conclusions:1.purified rabbit BMSCs in vitro can been induced and differentiated into osteoblasts with high osteogenic activity,can satisfy the requirement of seeding cell for bone tissue engineering research and biocompatibility study.2.slik fibroin/nano-hydroxyapatite composite biomaterial used for rabbit posterolateral lumbar spinal fusion can effectively enhance bony fusion, similar to autografts.