In Vitro And In Vive Characterizations Of Bone-like Hydroxyapatite/poly Amino Acid Loaded With Rifapentine Microspheres On Osteoarticular Tuberculosis

PART 1 EFFECT OF BONE-LIKE HYDROXYAPATITE/POLY AMINO ACID LOADED WITH RIFAPENTINE MICROSPHERES ON OSTEOARTICULAR TUBERCULOSIS IN VITROObjectiveTo investigate the effect of rifapentine microspheres loaded bone-like hydroxypatite/poly amino acid(BHA/PAA)composites on the bacterial growth of Mycobacterium tuberculosis(MTB),cell proliferation and differentiation in MTB H37Rv-infected MG63 cells.Furthermore,whether Wnt/?-catenin signaling pathway was activated by rifapentine microspheresloaded BHA/PAA composites were explored.MethodsThe in vitro antibacterial activity was determined by bacterial growth index.The alkaline phosphatase(ALP)activity,expression of runt-related transcription factor 2(Runx2)and osteocalcin(OCN)was examined by commercial kit,expression ofWnt1,LDL receptor related protein 6(Lrp6)and ?-catenin were determined by western blot analysis,to determine the effect of RPM-loaded BHA/PAA on MTB H37Rv-infected MG63 cell differentiation.ResultsThebacterial growth index of H37 Rv was significantly inhibited by rifapentine microspheresloaded BHA/PAA composites(p<0.05).The MTT assay showed that the optical density values of rifapentine microspheresloaded BHA/PAA groups were higher than that of the control groups(p<0.05).The ALPactivity and the expression of Runx2 andOCN were also higher than that of the control groups(p<0.05).Furthermore,the expression of Wnt1,LDL receptor related protein 6(Lrp6)and ?-catenin was significantly increased in H37Rv-infected MG63 cells following treatment with rifapentine microspheresloaded BHA/PAA composites(p<0.05).ConclusionRifapentine microspheresloaded BHA/PAA composites had an effective activity against MTB in vitro.The proliferation and differentiation of H37Rv-infected MG63 cells were promoted by rifapentine microspheresloaded BHA/PAA composites,by the activation of Wnt/?-catenin signaling.PART 2 IN VIVO RELEASE CHARACTERISTICS AND BIOCOMPABILITY OF BONE-LIKE HYDROXYAPATITE/POLY AMINO ACID LOADED WITH RIFAPENTINE MICROSPHERESObjectiveTo investigate the in vivo drug release characteristics and biocompability of rifapentine microspheresloaded BHA/PAA composites on a bone defect model in rabbits,hoping to get experiment data for the following study and clinical application.MethodsThe rifapentine microspheresloaded BHA/PAA compositeswere implanted into the bone defect made in New Zealand White rabbits.High-performance liquid chromatography(HPLC)method was adopted to determine the concentrations of rifapentine in the plasma as well as in the local muscle tissues.Hematoxylin-eosin(HE)staining,biochemical tests(alanine amiotransferase,aspertate aminotransferase,blood urea nitrogen,creatinine)were performed to elucidate the side effects of rifapentine microspheresloaded BHA/PAA composites on heart,liver and kidney.ResultsThe in vivo release tests showed that rifapentine microspheresloaded BHA/PAA composites exhibited sustained release profiles of rifapentine,and the drug concentration in the muscle tissues kept higher than the minimum inhibitory concentration of rifapentine against Mycobacterium tuberculosis for as long as 12 weeks.The biocompatibility and osteogenic ability of rifapentine microspheresloaded BHA/PAA composites were evaluated by HE staining.We found the material was completely degraded and absorbed at 12 weeks after implanting,and new trabecular bone and cartilage had formed.In addition,there were no significant differences of alanine amiotransferase,aspertate aminotransferase,blood urea nitrogen and creatinine between the rifapentine microspheresloaded BHA/PAA compositesand the control groups(p>0.05).ConclusionThisstudy firstly demonstrated that rifapentine microspheresloaded BHA/PAA composites slowly and continuously released rifapentine in vivo,and it has no side effects on heart,liver and kidney.In addition,rifapentine microspheresloaded BHA/PAA composites promoted new bone formation while the material was gradually degraded and absorbed.This study provided the theoretical basis for the advancement in treating osteoarticular tuberculosis.