| After progressive atrophy of the jaw,bone trauma and/or tumor,the bone quantity and quality in the implant area usually insufficient.Bone graft material considered to be an important substitute to restore any bone defects.Currently,clinical bone graft materials are utilized to repair bone defects including autogenous,allogenic and synthetic bone grafts.Autogenous transplantation has always been considered to be the "gold standard" of bone transplantation substitutes.Whereas,the limited quantity of autologous graft is unable to meet the clinical needs which makes patients suffering a second surgery.Furthermore,disadvantages such as limited sources,high immunogenicity,and osteo-inductivity,pushed us to think about the bone tissue engineering method to repair bone defects.Imitating the natural structure of extracellular matrix is the main content of bone tissue engineering scaffold design.The injectable hydrogel is more suitable for the repair of the irregular bone defect due to the characteristics of in-situ forming,biocompatibility and beneficial to the delivery of the active carrier.However,most of the in situ synthesized hydrogels need to be completed under the function of cross-linking agents,which are toxic.Whereas in situ synthesized hydrogels with higher biocompatibility could be obtained through Schiff base reaction.Hydroxypropyl chitosan(HPCS)is a derivative of chitosan with water-soluble function.Amino groups in HPCS could form Schiff base bonds with aldehyde glucan,then new hydrogels will be obtained.Nevertheless,new hydrogel has inadequate mechanical strength and lack of osteo-inductivity in clinical application.In order to overcome these problems,nano hydroxyapatite particles with excellent biocompatibility and microspheres with slow release of active cytokines were proposed in this experiment.BMP-2 bioactive peptide-P24 was synthesized according to the core domain of BMP-2.As a short-chain polypeptide containing only 24 amino acids,which is easy to bind to cell surface receptors and has good osteogenic induction ability for the stable structure and fully exposed bioactive site.Nevertheless,P24 is similarly easy to be hydrolyzed or inactivated.So we need to construct appropriate drug carrier to achieve local controlled release.Poly(lactic acid hydroxyacetic acid)(PLGA)microspheres are widely used as carriers of bioactive factor because of their good biological safety,mechanical properties and controllable biodegradation.In this study,a composite hydrogel system loaded with active peptide microspheres was designed to control the release of P24 polypeptide.The effects and mechanisms of the biomimetic system in promoting osteogenesis in vivo and in vitro were verified experimentally.In the second chapter,a water-in-oil-in-water(W/O/W)double emulsion solvent evaporation method was used to prepare the loaded P24 polypeptide PLGA microspheres.The P24 microspheres and nano-hydroxyapatite were co-doped into the polysaccharide-based hydrogel to obtain the composite hydrogel with P24-loaded microspheres.Fourier transform infrared spectroscopy,field emission scanning electron microscopy and rheometer were used to characterize and observe the structure,morphology and gelation of the composite hydrogel,and the release kinetics of the composite hydrogel was measured in vitro.A three-dimensional reticulated composite hydrogel with P24-loaded microspheres with pores of about 300-600 mwas successfully prepared.The incorporation of P24-loaded microspheres and nano-hydroxyapatite did not affect the pore size of the composite hydrogel.However,it increased the mechanical strength of the hydrogel and prolonged the release period of peptides and alleviated the acid problem caused by the degradation of PLGA microspheres.In the third chapter,the effects of composite hydrogel with the composite hydrogel with P24-loaded micropheres on the cell biology of MC3T3-E1 cells were evaluated.The cells stretched well on the composite hydrogel with P24-loaded microspheres and many filamentous pseudopods were observed under the scanning electron microscope.The results of the detection of CCK-8,ALP activity and the Alizarin red S staining showed that the composite hydrogel with P24-loaded microspheres could promote the proliferation,differentiation and mineralization ofMC3T3-E1 cells.At the molecular level,the composite hydrogel with P24-loaded microspheres could promote the expression of osteoblast-related genes and proteins,demonstrating that it had the biological activity of promoting the osteogenesis of MC3T3-E1 cells in vitro.In the fourth chapter,the osteoinductive ability of hydrogel with P24-loaded microspheres was tested by establishing an ectopic osteogenic model of the rat dorsal muscle pocket.Through HE and Masson’s trichrome staining,there were more collagen and neovascularization in the hydrogels when the composite hydrogel with P24-loaded microspheres were implanted into the rat dorsal spinal muscle for 8 weeks.The results of immunohistochemical staining showed that the positive expression of OCN and CD31 was observed in the experimental group at 8 weeks and the expression area was significantly higher than the 4-week results.However,there was no expression of OCN found in the control group.The results showed that the composite hydrogel had positive osteoinductivity effects in vivo.In the fifth chapter,the rat calvaria defect model was established,and the bone defect was implanted with the composite hydrogel with P24-loaded microspheres to observe the ability of this composite system to repair the bone defect in vivo.Micro CT showed that bone defect can be repaired significantly in the experimental group at8 weeks.The results of H & E and Masson’s trichrome staining showed that there were more woven bones formed in the defect.From the results of immunohistochemical staining,we knew that the positive expression area of OCN and CD31 in the experimental group at 8 weeks was significantly higher than that at 4weeks and the control group.The results of this chapter showed that the composite hydrogel with P24-loaded microspheres had a positive ability of promoting bone regeneration in vivo.This study suggests that this new hydrogel system is a potential material for bone tissue engineering,and provides a theoretical basis for the application of P24-loaded microspheres composited hydrogel in the bone defects around the implant. |
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