| Objective: At present,it is difficult to treat the osteomyelitis and osteomyelitis induced bone defects,easy to relapse,drug side effects,secondary or multiple operations,etc.How to design degradable composite biomaterials to effectively improve the local precise anti infection of antibiotics and complete the repair of bone defects has become a research hotspot.In this study,we constructed the drug loaded microspheres containing poly(lactic co glycolic acid)(PLGA)-chitosan(CS)-hyaluronic acid(HA)(PLGA-CS-HA)by means of power,and PLGA-CSHA was used to load vancomycin(VA).The drug loaded microspheres were respectively injected with nano hydroxyapatite(HAP),hydroxypropyl carboxymethyl cellulose and aminated hyaluronic acid injectable hydrogel(OHPMC-HA)composite microspheres and bone microspheres(VA@PLGA-CSHA/HAP)and antibacterial microspheres(VA@PLGA-CS-HA/OHPMC-HA)were prepared with biodegradable,good sustained release antibacterial effect and osteogenic potential.The physicochemical and biological properties of the microspheres and two kinds of composites were evaluated,and the osteogenic and osteoinductive antibacterial properties were evaluated.The repair of defects and the treatment of osteomyelitis provide scientific basis.Methods:(1)preparation and in vitro evaluation of VA@PLGA-CS-HA composite sustained-release microspheres: PLGA,CS and HA were used as materials to prepare porous microspheres with multilayer structure by emulsion polymerization,and VA@PLGA-CS-HA loaded with antibacterial microspheres was prepared.The physicochemical properties(scanning electron microscopy,drug loading,encapsulation efficiency and in vitro drug release)of the microspheres were prepared.At the same time,the best ratio of VA @ PLGA-CSHA was selected based on the above results;(2)the preparation and in vitro performance evaluation of VA@PLGA-CS-HA / HAp composite antibacterial sustained-release microspheres bone repair scaffold: nano HAP was used as the base material through hydrothermal method and VA@PLGA-CS-HA antibacterial slow-release method.The VA@PLGA-CS-HA / HAP sustained-release microsphere scaffold with biodegradable and antibacterial effect was prepared by compounding the microspheres,and its physical and chemical properties(scanning electron microscopy,mechanical properties,in vitro degradation and other experiments),biological properties(CCK-8 method to detect cytotoxicity,hemolysis experiment)and osteogenesis,bone induction(alkaline phosphatase activity experiment,Western test)were carried out The expression of the osteogenic genes Runx2,BMP2,OCN and COL-1 at m RNA and protein levels were detected by blotting and PCR,and the in vitro antibacterial performance(bacteriostatic circle test,flask shaking method)was evaluated.At the same time,the best ratio of VA@PLGA-CS-HA / HAP was selected based on the above results.(3)preparation and performance evaluation of VA@PLGA-CSHA/HPMC-HA injectable antimicrobial microspheres for bone repair hydrogel.Injectable hydrogel was prepared from hydroxypropyl carboxymethyl cellulose and aminated hyaluronic acid as matrix,and microspheres VA@PLGA-CS-HA was added to hydrogels to prepare injectable hydrogels with biodegradability and antibacterial properties,and their physicochemical properties(gelation time)were also studied.Determination,rheological analysis,swelling performance),biological performance(CCK-8 method to detect cytotoxicity,hemolysis test),osteogenesis,osteoinduction(alkaline phosphatase activity test,western The expression of osteogenic genes Runx2,BMP2,OCN and COL-1 at m RNA and protein levels were detected by blotting and PCR,and the antibacterial properties(bacteriostatic circle test and flask shaking method)were evaluated.At the same time,the best ratio of VA@PLGA-CS-HA / HPMC-HA was selected based on the above results.(4)animal osteomyelitis model experiment: the best match was selected by establishing the rabbit tibial osteomyelitis defect model Compared with VA@PLGA-CS-HA/HAP,the antibacterial microsphere bone repair scaffolds and VA@PLGA-CS-HA/HPMC-HA injectable antimicrobial microspheres were used to repair the hydrogel.The above-mentioned two materials were used in the animal model of chronic osteomyelitis,and then the anti-bacterial condition and bone repair ability of the two materials to osteomyelitis and bone defect were evaluated by X-ray,pathological section and microbiological examinationResults:(1)the optimal ratio of PLGA / CS / HA microspheres was: PLGA: CS:HA mass concentration ratio: 10% wt: 1.5% wt: 1% wt,the average diameter of PLGA / CS / HA microspheres was 58.77 ± 32.23 um.(2)The concentration of vancomycin solution 50 mg / ml in VA@PLGA-CS-HA microspheres was the best concentration(VA@PLGA-CS-HA-50),the encapsulation efficiency and drug loading were(8.64 ± 0.39)% and(86.41 ± 3.91)% respectively,and the microspheres had no obvious cytotoxicity.The release experiment in vitro for 56 days showed that the concentration of vancomycin was above the minimum antibacterial concentration.(3)The best preparation ratio of VA@PLGA-CS-HA/ HAP-50: VA@PLGA-CS-HA-50: HAP mass concentration ratio is 0.2g/ml:0.3g/ml,the range of mechanical pressure that can bear is 7.43 ± 0.81 MPa,the degradation rate of the scaffold at 56 days is(22.69 ± 1.97)%;there is no obvious cytotoxicity and hemolysis;VA@PLGA-CS-HA / HAP-50 has certain osteogenic and osteogenic effects,The diameter of bacteriostatic circle of Staphylococcus aureus was 21.3 ± 0.7mm at 24 hours,and the relative CFU was less than 1% at56 days.(4)The best preparation ratio of VA@PLGA-CS-HA/OHPMC-HA: the mass concentration ratio of OHPMC: HA-ADA is 2%: 2%,after equal volume mixing,VA@PLGA-CS-HA/OHPMC-HA is prepared by adding VA@PLGACS-HA-50 microspheres according to 0.2g/ml.The gelation time is 243±12.5S,and the rheological test meets the requirement of injectable hydrogel.VA@PLGA-CS-HA/OHPMC-HA has no obvious cytotoxicity and cell hemolysis.It has certain osteogenic and promoting bone differentiation effects;the inhibition zone diameter of Staphylococcus aureus is 22.3 ±0.5 mm for 24 hours,and the Relative CFU is less than 1% at fifty-sixth days,which has a significant inhibitory effect on the growth of Staphylococcus aureus.(5)Animal osteomyelitis model experiment,8 weeks after tibial osteomyelitis: evaluation of the infection degree:the bone infection degree of group A was significantly heavier than that of group B(group VA@PLGA-CS-HA/OHPMC-HA),C(VA@PLGA-CS-HA/HAP),D(hydrogel stent combination group)(`X=6.25 `X=6.25,P < 0.05),B and C two groups had no difference in bone infection rate(P=0.594 > 0.05),and the bone infection rate of the group was the lightest.P<0.05)? Evaluation of bone repair degree: the bone regeneration degree of group A was significantly worse than that of group B,C and D(`x = 2.67,P < 0.05).There was no difference in bone regeneration between group B and C(P = 0.242 > 0.05).The bone regeneration degree of group D was the best among the four groups(`x = 10.17 P < 0.05).Pathological score: compared with group B,C and D,group A had less new bone regeneration and more inflammatory cells and intramedullary tissue hyperplasia(`x = 10.17 P < 0.05),group B and C had no difference(P = 0.133 > 0.05),group D had better new bone regeneration and less inflammatory cells and fibrous tissue hyperplasia(`x = 2.67 P < 0.05).Microbiological examination: the number of bacteria per gram of bone specimen in group A was significantly higher than that in group B,C and D(P < 0.05),but there was no statistical significance between groups B,C and D(P > 0.05).Conclusion: VA@PLGA-CS-HA/HAP antibacterial microsphere bone repair scaffolds and VA@PLGA-CS-HA/OHPMC-HA injectable microsphere hydrogel polymer composites have good sustained release,antibacterial,osteogenic and osteoinductive abilities,and have good biocompatibility and biodegradability.They are potential composite drug loading materials for osteomyelitis complicated with bone defects. |
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