| Objective: Maxillofacial bone defects not only directly cause facial deformities and affect the appearance,but also lead to some degree of dysfunction in the pronunciation,language,chewing and breathing.At present,although autologous or allogeneic tissue transplantation used in clinical treatment has good efficacy,it still has drawbacks that cannot be ignored.Therefore,the proposal of bone tissue engineering provides a new idea for solving this clinical problem.Bone tissue engineering is the combination of tissue repair materials and biologically active substances,and biological factors play a role in promoting bone tissue repair.At present,bone tissue engineering has gradually evolved to promote the repair and regeneration of bone tissue through the use of a combination of cell scaffolding materials and bioactive factors,so that the defective bone tissue can be repaired or reconstructed.The advantage of this method is that it is not limited to the donor's source and does not have the problem of immunological rejection.The reconstructed tissue can replace the original tissue and has ideal functions.The nano-hydroxyapatite(n HA)crystals with a particle size in the range of 1-100 nm,have a good surface energy and quantum efficiency,and are also excellent in degradability and biological activity,and are more easily integrated with bone tissues and strengthen bones.Implantation of n HA crystal into the body will not cause pathological damage to the body,and can promote the microcirculation of tissue fluid through the interconnected pores,thereby promoting the binding of new bone and the surrounding tissue.Gelatin originates from the organism itself and is the product of denaturation produced after partial hydrolysis of collagen.It has a very wide range of applications in the medical field.Gelatin comes from a wide variety of sources and is readily available;it has good biocompatibility and biodegradability.Compared with collagen,gelatin has less antigenicity,and the abundant amino acids contained in its molecular chain can increase the recognition site of cells and promote seed cells to adhere thereto.Traditionally,growth factors are usually added directly to scaffold materials.This type of preparation often leads to a large and rapid release of growth factors in a short period of time,so that the growth factors have a short duration of action and the concentration of local factors is too high.The role is often more harm than good.Gelatin microspheres(GMS)belong to a slow-release drug delivery system,which not only delays the release of drugs from the carrier,but also protects the growth factors contained therein.A single artificial bone substitute is usually only able to exert bone conduction and lacks osteoinduction.In view of this deficiency,biological scaffolds with good biocompatibility,osteoconductivity,and degradability can be combined with growth factors with good osteoinductive activity,so that dual properties of bone conduction and osteoinduction can be achieved.The repair and reconstruction of bone defects is a complex and precise process in which various types of cytokines participate in and regulate.Any type of growth factor does not play an independent role in the process of bone formation,but interacts and coordinates with other growth factors to form an organic regulatory network together to participate in the regulation of the bone repair process.Therefore,more and more scholars have chosen to combine the use of two growth factors in order to achieve better bone tissue regeneration.As a member of BMP-7 subtype in BMPs,bone morphogenetic protein-6(BMP-6)can induce osteogenesis-directed differentiation of bone marrow mesenchymal stem cells(BMMSCs).Platelet-derived growth factor(PDGF)acts as a mitogenic stimulator for various cells,and it can exert chemotaxis on mesenchymal stem cells and osteoblasts,and is also one of the most powerful bone growth factors.PDGF can promote the recruitment and migration of bone-forming cells in damaged areas,promote cell division,proliferation and differentiation,and play an important role in the reconstruction of local blood circulation.In this experiment,we constructed a nano-hydroxyapatite/gelatin-gelatin microsphere scaffold capable of sustained release of PDGF and BMP-6,and explored its physicochemical and biological properties.Composite scaffolds with rat BMMSCs were used for the critical bone of skull.The repair of the defect was used to evaluate the application effect of the composite stent material for bone tissue engineering.Methods: 1.Preparation of gelatin microspheres and nano-hydroxyapatite/gelatingelatin microsphere scaffolds by modified emulsification cross-linking method,direct dispersion + vacuum freeze-drying method.2.The universal tester tests the mechanical strength of the bracket.3.The water absorption and porosity of the scaffold materials were measured by weighing method and medium immersion method respectively.The structure of the scaffold was observed by scanning electron microscope and the pore size of the scaffold was measured.4.In vitro extraction,isolation and culture of bone marrow mesenchymal stem cells from rat bone marrow adherent method.5.Identification of surface markers of rat bone marrow mesenchymal stem cells.6.Bone marrow mesenchymal stem cells osteogenic and adipogenic differentiation and identification.7.The biosafety of the scaffold material was tested by in vitro cytotoxicity test,hemolysis test,intradermal reaction test,pyrogenic test,acute systemic toxicity test,local implantation response test and subchronic systemic toxicity test.8.The cells were seeded on the scaffolds and the scaffolds were judged for cell compatibility by early adherence rate,cell proliferation rate,scanning electron microscopy and confocal laser scanning microscopy.9.Construct a dual-factor-loaded scaffold and determine the in vitro release of the factor.10.Bone marrow-derived mesenchymal stem cells seeded on scaffolds were cultured for osteogenic induction.The ALP activity of cells after osteoblast induction was detected and the expression of related proteins was detected by western blot.11.Preparation of critical bone defect model of skull in SD rats and implantation of scaffold materials in different groups.12.Postoperative imaging and histological examination.13.Statistical analysis of experimental data.Result: 1.The optimal proportion of nano-hydroxyapatite for nano-hydroxyapatite/gelatin-gelatin microsphere scaffolds is 20 wt%.Scanning electron microscopy observation of the sidewall roughness of the scaffold material facilitates cell adhesion.2.The rat bone marrow mesenchymal stem cells extracted from the experiment were found to have stable phenotypes and high purity,with good activity and multi-directional differentiation potential.3.In vitro biocompatibility performance test showed that the composite scaffold material constructed in this experiment did not have cytotoxicity,pyrogenicity,sensitization,and did not cause acute and chronic body injury in experimental animals.It has good adhesion and proliferation ability on the scaffold and the cell morphology is good.4.The sustained-release two-factor composite scaffold constructed in the experiment was detected by the Elisa kit.The results showed that after14 days of in vitro release,the cumulative release of PDGF was 95.213±2.461%;the cumulative release of BMP-6 on the 18 th day was 97.037±1.863%,and the release curve showed a relatively gentle and gradual upward trend,suggesting that the factors encapsulated in the gelatin microspheres can achieve a certain degree of sustained release.5.After cell inoculation and osteogenic induction,related tests showed that the dual sustained-release PDGF and BMP-6 scaffolds can promote cell ALP activity and osteocalcin expression,and the phosphorylation of the osteoblast-related pathway protein Akt is increased.When the PI3 K inhibitor was added to the culture system,the expression of OCN decreased but was not completely inhibited.6.After 8 weeks of critical skull defect repair test in SD rats,postoperative radiation and histological examination,the dual-release PDGF,BMP-6 nano-hydroxyapatite/gelatin-gelatin microsphere scaffold showed good osteogenesis and biocompatibility,this result is consistent with in vitro experimental results.Conclusion: 1.In this experiment,we successfully constructed nano-hydroxyapatite/gelatin-gelatin microsphere scaffolds for dual-release PDGF and BMP-6.When the amount of nano-hydroxyapatite is 20% by weight,it has the best performance as a bone tissue engineering material.2.The nano-hydroxyapatite/gelatin-gelatin microsphere scaffold has good biological performance and mechanical strength,and meets the requirements of bone tissue engineering for scaffold materials.There was no obvious stimuli response in the animal's body and it could be used as a cell scaffold for the repair of bone defects.3.The sustained release of PDGF and BMP-6 nano-hydroxyapatite/gelatin-gelatin microsphere scaffolds can promote the osteogenic differentiation of BMMSCs in vitro.The osteogenic effect of the scaffold is related to the increased phosphorylation of Akt,the protein that is induced by PDGF,and there is a synergistic effect of PDGF and BMP-6 in promoting bone formation.4.In the experiment,we used sustained-release PDGF and BMP-6 nano-hydroxyapatite/gelatin-gelatin microsphere scaffolds to repair rat skull defects.The results confirmed that the composite scaffold material has a significant osteogenic effect and can be used as a tissue engineering method to repair bone defects. |
PDF Full Text Request