Construction Of Injectable Chitosan Sustained-Release Gene Vector And Experimental Study On Bone Defect Repairs

Bone defects caused by diseases such as trauma,tumors and infections are increasingly common among clinicians,seriously affecting the physical and mental health and quality of life of patients.At present,the clinically preferred treatment plan is still autologous bone or allogeneic bone transplantation,but there are problems such as limited sources,donor damage,immune rejection,etc.Therefore,in recent years,the hotspots of osteogenic research at home and abroad have gradually turned into researches on gene therapy and tissue engineering bone.One of the main reasons for insufficient bone volume in bone defect repair in physiological state is the lack of osteogenic cytokines induced over time.The application of exogenous protein growth factors in bone defects is limited by the defects of short half-life,easy dilution by body fluid,difficult control of dosage and high cost.Gene therapy provides a new and effective solution to this problem.How to make the appropriate,timely and stable expression and release of various intervening growth factors in gene therapy for bone defects is the difficulty in gene therapy research,which involves the selection of appropriate gene carriers.Currently,the main advantage of using virus or liposome as gene therapy vector is high transfection rate.However,viral vectors also have some defects such as immunogenicity and potential pathogenicity.However,liposomes,which are commonly used in non-viral gene vectors,are cytotoxic and transfected by serum,and therefore not suitable for in vivo therapy,although they can achieve high transfection rates.In fact,physiological osteogenesis takes 2-3 months,which is a complex process involving multiple cytokines,mainly bone morphogenetic proteins.At present,most gene vectors reported in experimental studies(except some viral vectors that can be integrated into host DNA)can only maintain the expression aging of transfected cells for about 1-2weeks,which cannot meet the requirements of physiological osteogenesis aging(2-3months).Therefore,how to develop a gene carrier with good biocompatibility and sustained release of genes is a technical problem that genetic engineering needs to overcome in the clinical work of bone defects in the future.In recent years,the application of injectable hydrogels in bone defects has become a hotspot in tissue regeneration medicine.The main advantages are that it can be used by minimally invasive injection,with less damage,simple operation and more accurate filling of irregular tissues.The currently reported hydrogels are mainly used as scaffold materials for the repair of bone defects,while hydrogels as gene carriers are rarely reported.If the gene carrier is also prepared in the form of a hydrogel,it can be used alone or in combination with a hydrogel-type scaffold material to exert the advantages of injectable therapy,and more in line with the principles of minimally invasive surgery in orthopedics.It will have broad application prospects in the future clinical treatment.In view of the above problems,we chose non-viral vector chitosan as the research object,hoping to construct injectable,slow-release gene vector to meet the requirements of bone defect repair.Chitosan,as cationic polysaccharides in non-viral vectors,has natural bioactivity that can be degraded,and its therapeutic safety is effectively guaranteed.As a gene carrier,the main disadvantage is that the transfection efficiency is not high,but because it contains the active group amino,using the molecular chain functional group modification,ligand modification method,can synthesize a variety of chitosan derivatives with different properties,fully expand the application range of chitosan.Previous experiments of the research group have confirmed that thiolatedN-alkylated chitosan(TACS)can be synthesized by modification of chitosan by alkylation and mercaptosylation.The transfection efficiency of TACS is obviously higher than that of ordinary chitosan.On this basis,in order to meet the needs of bone defect treatment and enhance the effect of sustained-release gene expression,we constructed TACS@EG-HBC/pDNA composite particles as a new gene vector through wrapping polyethylene glycol(PEG)grafted hydroxybutyl chitosan(HBC)on the outer layer of TACS/pDNA composite particles.Hydroxybutyl chitosan is constructed by hydroxyl butyl bonding to hydroxyl and amino groups of chitosan.Its unique thermos-sensitive characteristic enables TACS@EG-HBC/pDNA composite particles to change to gel state at 37 degrees Celsius,which lays a theoretical foundation for the use of composite particles in an injectable dosage form.The purpose of grafting PEG is to increase the water solubility and stability of the whole material and further improve the efficiency of transfection.Since the outer structure is wrapped with EG-HBC,the degradation process,gene expression and maintenance of TACS@EG-HBC/pDNA composite particles in the transfected cells are theoretically longer,which is more consistent with the sustained release requirement of gene therapy for bone defect repair.To confirm the hypothesis above,we selected bone morphogenetic protein BMP4plasmid(pBMP4-EGFP)which fused with green fluorescent protein(EGFP)as marker gene to construct TACS/pBMP4-EGFP and TACS@EG-HBC/pBMP4-EGFP composite particles,respectively.We examined the effects of the two kinds of composite particles' transfection on bone marrow mesenchymal stem cells(BMSCs)and osteogenesis in vitro,as well as their effect of gene therapy as injectable hydrogel on radius defect in rabbits,providing a new idea for clinical treatment of bone defect.Experiment 1 Construction,characterization and temperature sensitivity identification of injectable chitosan sustained release gene vectorObjective: To complete the modification of chitosan,synthesize thiol alkylated chitosan(TACS)and hydroxybutyl chitosan(EG-HBC)of polyethylene glycol,and construct a new injectable gene loading sustained release system by using TACS loaded gene plasmid and wrapping EG-HBC.The characterization and properties of the thermo-sensitive gel were evaluated.Methods:(1)To identify the synthesis results of TACS and EG-HBC by modified chitosan chemical synthesis,and to determine the response of EG-HBC to PH value and temperature,and the change of viscosity with the change of temperature,and to identify the characteristics of temperature sensitive gel.(2)The plasmids pBMP4-EGFP and TACS fused with EGFP marker gene were combined by recoagulation to form TACS/pBMP4-EGFP,and then TACS@ EG-HBC/pBMP4-EGFP was synthesized by physical deposition of EG-HBC on its outer layer.Two kinds of nanocomposite particles were characterized by dynamic light scattering instrument and transmission electron microscope,and their carrying and protective effects on gene plasmids were detected by gel electrophoresis.Results:(1)Fourier transform infrared spectroscopy(FTIR)showed that compared with CS,TACS had(-CH3)group,HBC had(-OH)group,EG-HBC had(-CH2)group and amide bond,which confirmed that the synthesis of TACS,HBC and EG-HBC was successful.(2)When the pH value of EG-HBC increased from 6 to 6.5 and 7,the solution changed from clear to muddy gel.When the temperature increased from 25?to 37?,the solubility of the solution decreased,and the state of the gel was turbid.The solution viscosity increases from125.6 mPa s(20?)to 157.5 mPa s(50?)with the change of temperature.(3)TACS@EG-HBC/pEGFP-BMP4 nanospheres prepared with N/P ratio of 20 were mostly spherical with a diameter of 192.6±16.11 nm.The zeta potential of TACS/pBMP4-EGFP composite particles is 24.79+1.3 mv,while that ofTACS@EG-HBC/pBMP4-EGFP composite particles is-21.65+1.1 mv.Gel electrophoresis experiments showed that compound particles TACS/pBMP4-EGFP and TACS@EG-HBC/pBMP4-EGFP could both protect plasmid from degradation by DNA enzyme.The heparin dissociation experiment proved that the plasmid released by TACS/pBMP4-EGFP maintained its original stability.In vitro sustained-release experiments showed that the release rate of TACS/pBMP4-EGFP composite particles was 80% in 5 days,while that of TACS@EG-HBC/pBMP4-EGFP composite particles was 37.8% in 30 days.It was confirmed that the sustained-release effect of TACS@EG-HBC/pBMP4-EGFP composite particles was better.In the presence of lysozyme,both composite particles released 80% of the gene plasmids within 3 days,which proved that the chitosan could still be degraded by lysozyme.Conclusion: The modification of chitosan in this chapter is successful.TACS and EG-HBC are prepared successfully.EG-HBC showed good temperature sensitivity and PH response,and gelatinization could occur at 37?.Both TACS@EG-HBC/BMP4-EGFP and TACS/ BMP4-EGFP showed good protection and stability to the gene vector.However,the TACS@ EG-HBC/BMP4-EGFP composite particle has a more obvious sustained release effect in vitro.Experiment 2 Effects of chitosan mediated BMP4-EGFP gene transfection on sustained release,osteogenesis of BMSC gene in rabbits and in vivo sustained release Objective: To investigate the effects of TACS/ pBMP4-EGFP and TACS@EG-HBC/pBMP4-EGFP as sustained-release gene vectors on the transfection efficiency,sustained-release gene expression and osteogenic differentiation of BMSC in rabbits.The in vivo transfection was carried out by intramuscular injection in mice toinvestigate the in vivo sustained release expression of the two kinds of gene vectors.Methods:(1)In vitro culture of rabbit BMSC,flow cytometry detection and osteogenesis,lipid induction,identification of its stem cell characteristics.(2)Two kinds of gene vector composite particles,TACS/ pBMP4-EGFP and TACS@EG-HBC/pBMP4-EGFP,were used to transfect rabbit BMSC.Liposome 2000 was used as the positive control,and the EGFP gene transfection efficiency and sustained release expression were tested by flow cytometry and fluorescence microscope.The sustained release expression of BMP4 in TACS group and TACS@ EG-HBC group was compared by Weston Blot and ELISA.(3)In the non-transfected group,alkaline phosphatase staining and activity determination,alisarin red staining were used to compare the effect of in vitro osteogenic induction and osteogenic properties of BMSC transfected with two kinds of compound particles.(4)The effect of tacs-@EG-HBC/pBMP4-EGFP injected into the skeletal muscle of mice was observed by laser confocal microscope.Results:(1)Rabbit BMSC was extracted and cultured by gradient centrifugation method,with long fusiform and vortex growth,and was passaged every 5-7 days.P3 generation of rabbits showed positive expression of CD44(96.6%),CD90(98.9%),negative expression of CD45(0.21%),CD34(0.06%).Red lipid droplets were visible in lipid-induced oil red staining,and Vocanssa stained dark brown nodules were visible after osteogenesis induction.(2)In vitro cell transfection experiments,the transfection rates of TACS/ pBMP4-EGFP for BMSC were 24.8±5.1%,31.1±3.3% and 34.5±4.6%at 3,7 and 14 days after transfection,respectively.The transfection rates of TACS@EG-HBC/pBMP4-EGFP at the corresponding time were 5.9±2.4%,17.5±3.9% and35.3±4.2%,respectively.The results obtained under fluorescence microscope were basically consistent with those obtained under fluorescence microscope.Both thetransfected BMSC expressed BMP4 by Weston Blot and ELISA,while the tacs-@EG-HBC group showed stronger sustained release expression.(3)The effect on osteogenic induction was compared with the non-transfected group.After osteogenic induction,the ALP phosphatase activity in the TACS@ EG-HBC group at 14 days was stronger than that in the TACS group and the non-transfected group,and the calcium nodules stained with alizarin red at 21 days were more obvious.(4)When TACS@EG-HBC/pBMP4-EGFP was injected into the skeletal muscle of mice,Laser confocal microscope observed that the sustained expression of the gene could maintain for 12 weeks.Conclusion: Both kinds of gene vector composite particles can be transfected into BMSC for sustained release expression of EGFP and BMP4.Compared with TACS/pBMP4-EGFP composite particles,TACS@EG-HBC/pBMP4-EGFP showed better sustained release expression of the gene,and the transfected BMSC had better osteogenic induction ability.Intramuscular injection of mice confirmed the sustained expression of TACS@EG-HBC/pBMP4-EGFP in vivo for 12 weeks.Experiment 3 Experimental study on bone defect repair by injectable sustained-release chitosan composite particles loaded with BMP4-EGFP Objective: To investigate the in vivo osteogenic effect of pBMP4-EGFP-loaded chitosan composite particles and the sustained release expression of plasmid in bone defect in rabbits.Methods: 24 New Zealand white rabbits were randomly divided into three groups to construct the 18 mm complete bone defect model of both forelimbs.Seven days after the establishment of the model,TACS @EG-HBC/pBMP4-EGFP composite particlesuspension or TACS/ pBMP4-EGFP composite particle suspension containing 80 ug plasmidome pBMP4-EGFP were injected into the bone defect respectively.The experimental animals were sacrificed at 2,4,8 and 12 weeks after the operation,and the gross specimens of the defect were taken for X ray,Lane-SandhuX score and HE-staining of the new bone tissue to understand the repair of the bone defect.Biomechanical tests were performed at 12 weeks to evaluate the load-bearing capacity of healed bone tissue.Immunohistochemistry of BMP4 in new bone tissue to understand the transfection and sustained release expression of gene in vivo.Results: All the experimental animals survived well,and the gross histological examination showed that there was a small amount of new bone growth in the blank group from week 4 to week 12.Irregular callus was observed at the end of the bone defect in the TACS group and the TACS@EG-HBC group at 2 weeks.As time went on,new bone was formed faster in the TASC@EG-HBC group than in the TACS group,and the bone defect was basically repaired in the TACS@EG-HBC group at 12 weeks.The biomechanical test showed that the strength of radius was weaker than that of normal radius,and the difference was statistically significant(n=4 P<0.05).The restoration effect of X-ray photography was consistent with that of general specimens.According to Lane-SandhuX score,there were statistically significant differences between different groups at week 4,8 and 12.The TACS@ EG-HBC group had higher scores at two time points at week 8,12 than the other two groups,and the difference was statistically significant(P<0.05,n=4).When HE-staining the new bone formation site for 12 weeks,trabecular structure was found in the blank control group,but the cortical structure was not obvious.Lamellar bone in TACS group was immature.In the TACS@ EG-HBC group,trabeculae were connected into slices,and a large number of lamellar bones were formed,with dense bone and clear boundary between skin and medulla.Immunohistochemistry suggested that a small number of BMP4 positive cells could stillbe observed in the new bone tissues of the TACS@EG-HBC group at 12 weeks,so the sustained release expression was stronger than that of the TACS group.Conclusion: TACS@EG-HBC/pBMP4-EGFP constructed in the early stage plays a good role in the sustained release expression of the gene and enhances the effect of new bone formation.When the observation node was 12 weeks old,local transfection of TACS@EG-HBC/pBMP4-EGFP composite particles containing 80 ug plasmid could realize bone at both ends of the defect to grow completely toward the middle junction?...