| Objective:(1)In this study,rifapentine polylactic acid sustained-release microspheres(RPSMs)were prepared.The in vitro drug release characteristics,antibacterial activity and histocompatibility of RPSMs were evaluated to provide basis for subsequent experiments.(2)In this study,hydroxyapatite/?-tricalcium phosphate(HA/?-TCP)composite was used as scaffold,loaded with RPSMs to construct complex.The effects of the RPSMs complex on cell proliferation,osteogenic differentiation and drug release characteristics were evaluated by in vitro experiments.(3)A rabbit model of spinal tuberculosis defect was established,and the RPSMs complex was used to treat spinal tuberculosis model,which was comprehensively evaluated from the two aspects of therapeutic effect and tissue repair effect of bone defect in spinal tuberculosis.In conclusion,the complex constructed of RPSMs and HA/?-TCP represents a potential new strategy for TB infections and bone defects.It will provide a potential new strategy for treatment of spinal tuberculosis.Methods:(1)With rifapentine as the target drug and polylactic acid as the drug carrier,RPSMs were prepared through the double emulsion-solvent evaporation method.The particle size,encapsulation rate and drug loading rate of RPSMs were measured,and the release ability and antibacterial activity of RPSMs was investigated in vitro;The effects of RPSMs on the morphology,adhesion,proliferation,osteogenesis and physicochemical properties of BMSCs were detected by microscope,CCK-8 method,alkaline phosphatase(ALP)staining and measurement,alizarin red staining.The biocompatibility was evaluated by cytotoxic test and hemolytic test.(2)The isolated rabbit BMSCs were identified by flow cytometry,and the ability of multilineage differentiation potential of BMSCs was investigated.In this study,hydroxyapatite/?-tricalcium phosphate(HA/?-TCP)composite was used as scaffold,loaded with RPSMs to construct complex.The drug release ability of the complex was evaluated by in vitro release test.BMSCs were divided into four groups: Control group(BMSCs),Induced group(induced BMSCs,IBMSCs),RPSMs-induced group(IBMSCs+RPSMs),complex-induced group(IBMSCs+RPSMs+HA/?-TCP).The effects of the complex on the osteogenic differentiation of rabbit BMSCs were evaluated by alkaline phosphatase and cecropin staining,real-time fluorescence quantitative PCR and Western blot;The effects of each group on the osteogenic differentiation of rabbit BMSCs were evaluated by alizarin red and ALP staining,quantitative real-time PCR(qRT-PCR)and Western blot.(3)To establish a rabbit model of spinal tuberculosis defect and evaluate the model with X-ray,MRI,HE staining and bacteriology.RPSMs complex was used to treat spinal tuberculosis.The therapeutic effect of the RPSMs complex on the spinal tuberculosis model was evaluated by inflammatory indexes such as erythrocyte sedimentation rate(ESR),C-reactive protein(CRP)and tumor necrosis factor-?(TNF-?)at 6 and 12 weeks of postoperation.The macroscopic observations,histological observations and Nilsson histological score confirmed the tissue repair effect of bone defect in spinal tuberculosis at 6 and 12 weeks of postoperation.The HPLC method was used to detect the concentration of drugs in tissues and observe the sustained release characteristics of RPSMs complex and the accumulation of drug in other tissues.Results:(1)The appearance of RPSMs is brick red and powdery.Under light and electron microscopes,they appeared spherical and uniformly dispersed;The mean particle size of the RPSMs was 27.67±2.05?m.The encapsulation and drug loading efficiency of the RPSMs were 78.11±1.16% and 35.57±0.85% respectively.The sustained release of RPSMs reached 48 days.The antibacterial activity of RPSMs results showed that RPSMs had a long-term inhibitory effect on the growth of mycobacterium tuberculosis.BMSCs cultured with RPSMs were positive for alizarin red and ALP staining,and compared with other groups,there was no significant difference in ALP activity and migration ability(P>0.05).The cytotoxicity of RPSMs was evaluated as grade 1,and the hemolysis rate was 1.6%.(2)The results of flow cytometry accorded with the standard of stem cell identification and BMSCs had a good ability for osteogeneic,adipogenic and chondrogenic differentiation.The in vitro release test showed that the drug release of RPSMs complex constructed was more stable with a release time of 58 days.The complex-induced group was positive for alizarin red and ALP staining.The results of qRT-PCR showed that the levels of collagen I of complex-induced group was significantly higher than that of the other three groups(P<0.01),and the levels of osteocalcin of complex-induced group was significantly higher than that of the RPSMs-induced group(P<0.01).The results of Western blot indicated that the expression levels of collagen I and osteocalcin protein in the complex-induced group was significantly higher than that of the other three groups(P<0.01).(3)In the spinal tuberculosis model group,The results of X-ray and MRI showed spinal fusion,deformities and tuberculous abscess formation in the infected segment.HE staining revealed characteristic lesions such as tuberculous nodules,and mycobacterium tuberculosis was found in bacteriological culture.RPSMs complex was used to treat spinal tuberculosis.The results demonstrated that ESR,CRP and TNF-? in the complex group decreased at 12 weeks,and there was no statistically significant difference compared with the level before tuberculosis infection(P>0.05).The results of gross observation and immunohistochemical staining showed that the defect area of the complex group was replaced by new bone tissue without obvious boundary with the surrounding bone tissue.The results of Nilsson histological score confirmed that the score of the complex group was significantly higher than that of the other two groups at 6 and 12 weeks of postoperation(P<0.01).The drug concentration test results showed that the complex released stably for 60 days which was consistent with the results of in vitro release,with no apparent accumulation of the drug in other tissues.Conclusion:(1)The prepared RPSMs have high encapsulation and drug loading efficiency,with good bacteriostatic ability;The results of in vitro tests confirmed that RPSMs had no significant effect on the proliferation,osteogenic differentiation,and had good histocompatibility.(2)In this study,the RPSMs complex constructed was more stable and longer duration of sustained release and retention,which could promote the osteogenic differentiation of BMSCs.(3)A rabbit model of spinal tuberculosis defect was successfully established;The spinal tuberculosis model was treated with RPSMs complex showed good therapeutic effect and repair effect;The drug release of the complex in vivo was stable similar to that of in vitro release. |
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