| BackgroundAs the people's health requirements increase,the number of people participating in physical exercise has increased significantly,and the resulting in more and more sports-related injuries such as tendon-bone junction injury.As one of the common tendon-bone junction injuries,acute rotator cuff injuries will change into chronic injuries in this area and affect the physiological function of the corresponding parts without timely and correct treatment.Strong tendon-bone healing,which is in line with physiological function,is a necessary condition for restoring the physiological function after injury of various tendon-bone junctions.Therefore,a correct understanding of the characteristics,the pathological characteristics and healing process of tendon-bone junction after acute injury is a prerequisite for successful treatment,and will provide a strong guarantee for the rational and correct choice of treatment methods.As one of the important research contents in orthopaedics,surgical repair of the damaged tendon-bone junction and functional recovery of the damaged area after surgery has become a clinical problem to be solved urgently.In recent years,in tissue engineering research,the choice of appropriate biomaterials and the use of reasonable materials and drugs to make drug delivery system for the repair of injured sites has become a research hotspot,and may become the preferred treatment for tendon-bone junction injury in the future.In more and more drug delivery systems,polymer microspheres with the advantages of low cost,easy fabrication and strong plasticity have gradually become a popular drug delivery system in research.Polymer microspheres are usually made of natural or artificial polymers with good biocompatibility and biodegradability.They have the functions of drug protection and sustained release after drug loading.They can form drug delivery systems together with tissue engineering scaffolds and participate in tissue repair at the site of injury.Simvastatin,as an inhibitor of HMG-CoA reductase,is widely used in clinic to reduce low density lipoprotein,triglyceride and total cholesterol in blood.Simvastatin is a kind of white powder at room temperature,insoluble in water,but soluble in organic solvents such as trichloromethane and ethanol.With the in-depth study of simvastatin,it was found that simvastatin could promote osteoblast differentiation and inhibit osteolysis and osteoclast activity by inhibiting BMP-2 signal pathway inhibitor and matrix metalloproteinase-9 pathway.Simvastatin can also enhance the activity of macrophages and other immunocompetent cells to stimulate local bone formation and promote bone repair.However,due to its dose-related drug functions,hepatotoxicity and low oral utilization,serious toxicity and side effects may occur after high-dose application.Since local application of simvastatin can effectively avoid its toxic and side effects,and can directly affect tissues and organs,we need to send it to the injured site accurately by means of drug delivery system.The preparation and assembly of drug delivery system is the guarantee of stable drug release in dogs.In this study,two kinds of natural polymers and hydroxyapatite were combined to form a core-shell microsphere system,and simvastatin was loaded into the core layer to achieve the goal of slowly releasing simvastatin from the whole system as the polymer degraded,thus bringing a new way for the treatment of tendon-bone junction sites.Our experimental study is divided into four parts:(I)Preparation of core-shell microspheres with simvastatin-loaded function;(II)Isolation,culture and identification of bone marrow mesenchymal stem cells in SD rats;(III)Biocompatibility and osteogenic differentiation of drug-loaded microspheres in vitro;(IV)Preparation of biological soft scaffolds containing drug-loaded microspheres and evaluate its promoting effect on tendon-bone healing in vivo.Part I Preparation of core-shell microspheres containing simvastatin ObjectiveHydroxyapatite and natural polymer chitosan and zein were used as raw materials to obtain novel sustained-release microspheres containing core-shell structure by electrospray technology.MethodChitosan solution and nano hydroxyapatite were used to prepare the shell solution,zein and simvastatin were used to prepare the core layer solution.The core-shell structure of sustained-release microspheres containing simvastatin were prepared by coaxial electrospray technology.The morphology of the microspheres were observed by environment scanning electron microscopy(ESEM),and the size and distribution of the microspheres were calculated at the same time.The core-shell structure of the sustained-release microspheres loaded with simvastatin was verified by laser scanning confocal microscopy(LSCM)and transmission electron microscopy(TEM).The composition of the microspheres was analyzed by energy dispersive spectrometer(EDS)and X-ray diffraction(XRD).The drug loading capacity,encapsulation efficiency rate and percentage yield of simvastatin-loaded sustained-release microspheres prepared with different concentration of chitosan in shell solution and the drug release rule in vitro were calculated.ResultA new loaded simvastatin sustained-release microspheres with core-shell structure can be obtained by electrospray technology.The average diameter of the microspheres is mainly between 1 and 2 ?m,the encapsulation efficiency is 66.5-69.9%,the drug loading capacity is about 0.2%,and the percentage yield is about 73.02-75.65%,which can be changed with the different concentration of chitosan.Simvastatin-loaded sustained-release microspheres with core-shell structure can reduce initial drug burst and release slowly for at least a week.Conclusion1.A new core-shell microsphere loaded with simvastatin was prepared by coaxial electrospray technology.The effects of the concentration of chitosan on the morphology,particle size,drug loading capacity,encapsulation efficiency and percentage yield of the microspheres were explored.The results showed that the size distribution of drug-loaded microspheres prepared by chitosan with 2%(w/v)concentration was uniform,and the yield and the encapsulation efficiency of SIM were ideal.2.It was proved that hydroxyapatite was successfully contained in the prepared microspheres,and simvastatin was uniformly dispersed in the microspheres and existed in amorphous state.3.The new CS-nHA/zein-SIM core-shell microspheres can significantly reduce the drug burst release at the initial stage and sustain the release of simvastatin within a week.Part II Isolation,culture and identification of bone marrow mesenchymal stem cells from SD rats ObjectiveTo establish a method for isolation and culture of bone marrow mesenchymal stem cells from SD rats,and to observe their morphology,cell viability,growth curve and cell identification.MethodBone marrow mesenchymal stem cells(BMSCs)were isolated and cultured from femur and tibia of SD rats by whole bone marrow adherence method.Their morphology was observed and recorded under inverted microscope.Cell viability was detected by CCK-8 along with trypan blue staining,and growth curve was drawn after detected.The surface antigen markers CD11 b,CD29,CD45 and CD90 of the third generation BMSCs were detected by flow cytometry.Alkaline phosphatase,alizarin X red S staining and oil red O staining were used to detect the differentiation potential of osteogenesis and adipogenesis.ResultAfter 24 hours of isolation from bone marrow,some cells adhered to the bone marrow.After 7-10 days of culture,the adherent cells distributed in a whirlpool-like colony.The cell morphology was uniform and the cell viability reached(94.93 ± 2.07)%.There was a significant logarithmic growth period within 3-5 days of culture.The positive rates of CD11 b and CD45 were 2.36% and 0.15% respectively.The positive rates of CD29 and CD90 were 99.61% and 99.84%,respectively.After 14 days of osteogenic differentiation,alkaline phosphatase staining showed dark purple or dark brown outline in the cytoplasm.After 28 days of induction,a number of calcium nodules appeared and could be stained by alizarin red S staining solution.After 21 days of fat induction,oil red O dyes were used to dye the fat droplets,which showed bright red color when combined with dyes.Conclusion1.Bone marrow mesenchymal stem cells were successfully extracted from femur and tibia of SD rats by whole bone marrow adherence method,with high cell viability and recovery rate.2.Bone marrow mesenchymal stem cells have high proliferation ability and purity.The growth curve is "S" shape,and the logarithmic growth period can be observed with vigorous growth.3.The surface antigens CD29 and CD90 of bone marrow mesenchymal stem cells were positive,while CD11 b and CD45 were negative,which accorded with the surface antigen characteristics of mesenchymal stem cells.Osteogenic differentiation of bone marrow mesenchymal stem cells showed a large amount of calcium deposition in extracellular matrix.Alkaline phosphatase and alizarin red staining were positive,which possessed the characteristics of osteoblasts.After induction of adipogenic differentiation,a large number of fat droplets could be seen in the cells.These results demonstrate that cultured bone marrow mesenchymal stem cells have multidirectional differentiation potential.Part III Biocompatibility and osteogenic differentiation of simvastatinloaded sustained-release microspheres in vitro ObjectiveTo study the biocompatibility of simvastatin sustained-release CS-nHA/zein-SIM core-shell microspheres in vitro and to identify the osteogenic differentiation ability of SD rat BMSCs in vitro.MethodSimvastatin-loaded sustained-release microspheres were co-cultured with BMSCs of the third generation of SD rats,and CCK-8 was used to detect cytotoxicity and cell proliferation.The cell cycle of co-cultured cells was detected,and the apoptosis and mineralization of co-cultured cells were also observed.The activity of alkaline phosphatase was measured quantitatively.The morphology of cells cultured with drug-loaded microspheres for 1,4 and 7 days was observed.The expression of osteogenic differentiation-related genes was detected by flow cytometry.ResultAfter co-culture of BMSCs and CS-nHA/zein-SIM microspheres with core-shell structure,the proliferation rate was significantly higher than that of blank control group(p < 0.05).The proportion of BMSCs in phase S and G2 was higher than other groups.The percentage of living cells was higher after 1,4 and 7 days of incubation with drug-loaded microspheres with core-shell structure.Quantitative analysis of alkaline phosphatase activity and alizarin red S staining showed that the absorption value was higher than that of other groups(p < 0.05).The cell morphology was normal and the cell extension was adequate.The expression of osteogenic differentiation related genes such as Runx2,BSP,OPN,OCN,OSX,ALP in each group was higher than that in other groups(p < 0.05).Conclusion 1.CS-nHA/zein-SIM core-shell microspheres showed good biocompatibility and low cytotoxicity in vitro,and could improve the proliferation and differentiation of BMSCs in SD rats.2.CS-nHA/zein-SIM core-shell microspheres can effectively promote the osteogenic differentiation of BMSCs in SD rats,and can induce the high expression of osteogenic differentiation-related genes in cells.Part IV Preparation of gelatin soft scaffolds loaded with simvastatin sustained-release microspheres and in vivo study on promoting tendon-bone healing ObjectiveTo study the biocompatibility of simvastatin-loaded sustained-release microspheres soft scaffolds in vitro and evaluate the effect of simvastatin-loaded microspheres on tendon-bone healing in vivo.MethodThe sustained-release microspheres loaded with simvastatin were evenly mixed with gelatin and glycerol to prepare drug release soft scaffolds.The drug release,cytotoxicity and proliferation of the system in vitro were determined.The model of tendon-bone junction injury in SD rats was established and treated with the soft scaffold.Histological observation of the treatment results and detection of related gene expression in tissue cells were carried out.ResultThe soft scaffolds loaded with simvastatin sustained-release microspheres were successfully prepared.The drug release rate was about(14.83 ± 2.35)% in the first 24 hours and could be sustained for about a week.After co-culture with SD rat BMSCs for 1 day,4 days and 7 days in vitro,CCK-8 assay showed that the system had no cytotoxicity and could significantly stimulate cell proliferation.The injury model of the tendon-bone junction of the supraspinatus muscle of SD rats was successfully established.After the treatment of the soft scaffold in the bone tunnel,the tissue around the bone tunnel was stained by HE and Masson staining method at 6 and 12 weeks after surgery.The tissue repair in the experimental group was faster,and more calcified fibrocartilage could be seen in the tunnel at the tendon-bone interface.The expressions of BSP,OPN,OCN,SCX,TNMD,TENC,COL-1?1 and vascular endothelial growth factor(VEGF)in the experimental group were higher than those in the control group,and part of the difference was statistically significant(p < 0.05).Conclusion1.The in vitro cell experiments of simvastatin-loaded microsphere soft scaffold showed that it had good biocompatibility and could improve the proliferation of BMSCs in SD rats.2.Simvastatin-loaded microsphere soft scaffolds can effectively promote cell differentiation and tissue repair and healing in the injured area of supraspinatus tendon and bone in SD rats,and increase the expression of related genes in the cells around the injured area. |
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