Preparation Of Degradable Porus Silk Fibroin/Hydroxyapatite (SF/HA) Composite And Experimental Study On Repairing Segmental Bone Defect By The Composite

Partâ… : Preparation of Degradable Porus Silk Fibroin/Hydroxyapatite (SF/HA) Composite and Degradation of the Composite in SD rats in Vivo【Objective】To prepare a novel bone substitute of silk fibroin/hydroxyapatite (SF/HA) composite for bone tissue engineering, and to explore the composite degradation rate and tissue reaction on it in SD rats in vivo.【Methods】Silk fibroin (SF) and Hydroxyapatite (HA) composite powders were synthesized. 4 types of porous silk fibroin/hydroxyapatite (SF/HA) composite could be prepared when adding silk short fibers and using NaCl as porogen by isostatic pressing. Using HA as a control group, water absorption rate of 4 composites and HA were measured at 0.5, 2, 6, 12 and 24 h after these materials soaked in deionized water. These materials were implanted into SD rats'back subcutaneously to observe their degradation rate and tissue reaction on them. Specimens in experiment group were observed in general and from the aspect of histology at post-operative 2, 6, 12, 16, 20 and 24 weeks respectively, while control group at post-operative 2, 12 and 24 weeks.【Results】Pore size, porosity and strength of these 4 type of SF /HA were different when proportion of SF, HA, affix and porogen was different. Absorption rate was as follows: SF/HA₃ and SF/HA4>SF/HA₂ and SF/HA₁>HA. HA, SF/HA₁ and SF/HA₂ had little degradation. SF/HA₃ had complete degradation during post-operative 20～24 weeks, while SF/HA4 had complete degradation during post-operative 12～16 weeks. Histology showed that tissue surrounding SF/HA had no apomorphosis and necrosis.【Conclusions】SF/HA₁ is unsuitable for bone tissue engineering scaffold because its average pore size was 6.5μm, and its maximum pore size was only 15μm. SF/HA4 is also unsuitable for bone tissue engineering scaffold because its strength is only 1.59MPa and it quickly spread in water. In the future experiment, we will exclude these two composites, and only carry out study on SF/HA₃ and SF/HA₂. Partâ…¡: Experimental Studies on Cultivation of Bone Marrow Stromal Cells (BMSCs) from Rabbits and BMSCs Osteogenic Differentiation【Objective】To differentiate and proliferate bone marrow stromal cells (BMSCs) from rabbit's marrow into osteoblasts in vitro to prepare seed cells for bone tissue engineering and compatibility test.【Methods】Primarily cultured BMSCs isolated from a rabbit's bone marrow by density gradient centrifugation were subcultured in mineralization medium to induce their differentiation into osteoblasts, whose morphological characteristics and proliferation status were observed by phase-contrast microscope. Von Kossa staining and alkaline phosphatase (ALP) activity test were employed to assess BMSCs'osteoblastic differentiation and the generation of calcified extracellular matrix. MTT assay detected the osteoblast proliferation.【Results】BMSCs cultured in vitro showed obvious osteogenic capacity in mineralization DMEM. Von Kossa staining of the mineralized nodules and alkaline phosphatase detection of the passaged cells both yielded positive results. MTT assay showed the osteoblast proliferation was normal.【Conclusions】BMSCs could be cultured, differentiated and proliferated with active osteogenic function by differentiation culture medium in vitro. So it could be suitable for seed cell for bone tissue engineering.Partâ…¢: Study on Cellular Compatibility of the degradable porus composite of SF/HA【Objective】To evaluate SF/HA cellular compatibility as well as the feasibility of the composite to serve as a scaffold in tissue engineering by using co-culturing of osteogenic BMSCs and SF/HA.【Methods】The third passage BMSCs were transplanted into SF/HA₂ and SF/HA₃ after being induced to differentiate into osteoblasts and then seeded into the materials for 3 to 5 days. BMSCs alone were cultured at the same condition to act as controls. The cellular morphology and function (attachment, proliferation and differentiation) were assessed separately by means of phase contrast microscope, HE, SEM and MTT assay, ALP activity. The material leaching liquor were used to test cell toxicity. 【Results】BMSCs could adhere to SF/HA₂ and SF/HA₃, and proliferate and grow on the surface of the composites normally. The cellular activity and function were not affected by the materials, and no statistical difference was found between the two groups and the control group (P>0.05). Cell toxicity test discovered these materials had no toxic effect on BMSCs.【Conclusion】SF/HA₂ and SF/HA₃ have a good biocompatibility and can be used as a tissue engineering scaffold.Partâ…£: Studies on ectopic bone formation of SF/HA with rabbit BMSCs and the composite tissue compatibility【Objective】To explore the ectopic osteogenesised ability of the tissue engineered bone fabricated by osteoblasts which derived from rabbit BMSCs co-cultured with SF/HA, and to observe the material tissue compatibility by placing it into rabbits'muscle.【Methods】The differentiated osteoblasts which were derived from BMSCs, with 5×107/ml density, were seeded onto SF/HA (SF/HA₂+BMSCs as Group1; SF/HA₃+BMSCs as Group2). Then, co-cultured for 5 days, the compounds with cells and SF/HA were implanted into muscular pouch of back of rabbits and SF/HA alone was implanted as a control group. The effectiveness of bone formation was assessed separately by means of gross observation and histology after implantation for 4, 8, 12 weeks.【Results】New osteogenesis was detected at the end of the 4th, 8th and 12th week after implantation respectively in experimental group. The quantity of new osteogenesis in experimental group 1 was better than in experimental group 2 by histology morphometry (P<0.05). There was no new bone formation in the control group. Muscle surrounding all materials had no apomorphosis and necrosis from the aspect of histology.【Conclusion】Muscle implantation test, Subcutanea implantation test in Partâ… and cell compatibility in Partâ…¢show SF/HA₂ and SF/HA₃ have a good biocompatibility. The tissue engineered bone fabricated by osteoblasts which derived from BMSCs cultured onto SF/HA have a good ability of ectopia osteogenesis in vivo. It is supposed to be a good way to repair clinical bone defect. However, SF/HA₃ is more suitable than SF/HA₂ because SF/HA₃ has a better bio-degradation rate and ectopia osteogenesis effect. Partâ…¤: Experimental study on rabbit segmental radial defects repaired by SF/HA co-cultured with rabbit BMSCs【Objective】To discuss the bone formation of SF/HA₃ tissue engineered bone co-cultured with rabbit's BMSCs and the feasibility of SF/HA₃ as a bone substitute.【Methods】SF/HA₃ is named SF/HA in the current study. The purified, culture-expanded, and osteogenic BMSCs were combined with SF/HA in vitro according to condition of cell culture. A segmental bone defect (15 mm in length) was created at left radial in each rabbit. The composite grafts were implanted into the bone defects in rabbits through open operation. The curative effect was evaluated by radiographic examination, histology analysis and eyes observation in experimental groups, experimental control group and blank control group at post-operative 4, 8, 12, 16 week, respectively.【Results】The bone defects that had been treated with grafts exhibited new bone formation increased with time by radiography, histology and eye observation. The rate and quality of new bone formation were significantly different in the experimental groups, experimental control groups and blank control groups (P<0.05). The segmental bone defects were complete union in the experimental groups, while the defects were partial union in the experimental control groups. In the defects in blank groups, there were no formation of new bone after operation and bone defects were finally repaired only by fibrous tissue.【Conclusion】SF/HA has a suitable degradation rate and can be replaced by normal bone tissue appropriately. The bone formation ability of SF/HA combined with BMSCs is equivalent to the ability of autogenous bone. It is supposed to be a good way to repair clinical bone defect. However, the composite has no bone induction, and using the material alone has little bone formation in the segmental bone defect.