Study On The Effect Of Polylactic Acid Three-dimensional Scaffold On The Sustained Release Of Active Ingredients Of Traditional Chinese Medicine For Tonifying Kidney And Activating Blood Circulation To Promote Bone Repai

Background:Polylactic acid,a biodegradable biomaterial,is made by corn,wheat,potatoes,waste paper.Its degradation products are carbon dioxide and water.It has the advantages of renewable materials and non-toxic degradation.Polylactic acid is a recognized safe material approved by the US Food and Drug Administration.Polylactic acid,which has high practical value in orthopedic field,is good in mechanical properties,antibacterial activity,biocompatibility,and biodegradability.That biodegradable polylactic acid loaded with bone repair drugs promotes bone injury repairing is currently a research focus and hotspot in the field of bone repair nowadays.Kidney-tonifying and blood-activating herbal medicine has the advantages of simplicity,convenience,low cost,curable,and has good clinical efficacy in repairing bone injury.Osteoporactic total flavone(OTF)combined with panax notoginseng saponin(PNS),the active ingredient of kidney-tonifying and blood-activating herbal medicine,are used as bone repair drugs,which are then loaded by poly-L-lactic acid(PLLA)material to fabricate sustainably released drug-loaded PLLA scaffold.Implanting it into local injury site allows drugs to reach the bone injury site directly and promote bone injury repair.It is an innovation in clinical administration of traditional Chinese medicine(TCM),and could improve the clinical efficacy of TCM active ingredients for treating bone injury in a certain extent.Objective:1 This research aims to fabricate the sustainably released PLLA scaffold which is loaded with OTF and PNS,the active ingredient of kidney-tonifying and blood-activating herbal medicine,and to assess its characterization and biological safety.2 This research aims to explore therapeutic effects and potential mechanisms of the sustainably released PLLA scaffold loaded with OTF and PNS on repairing different species(rabbit,rat)and different bone injuries(bone necrosis,bone defect)by promoting bone regeneration.Methods:1 The sustainably releasing drug loaded 3D scaffolds,including OTF/PLLA scaffold(100 mg OTF),OTF-PNS/PLLA scaffold(50 mg OTF+50 mg PNS),PNS/PLLA scaffold(100 mg PNS),were fabricated by using freeze-drying/phase separation technique.The characterizations of those scaffolds were assessed by scanning electron microscope,pycnometer method,strength testing machine,vanillin-perchloric acid chromogenic method and ultraviolet spectrophotometry.The in vitro cytotoxicity of those scaffolds was assessed by using CCK-8 method.The in vivo biological safety of those scaffolds was assessed by hematoxylin-eosin(HE)staining of local implanted bone tissue and main organs,and by testing hepatic and renal function(glutamic-pyruvic transaminase,glutamic oxaloacetic transaminase,creatinine,blood urea nitrogen).2 The sustainably releasing drug loaded scaffolds were implanted into hormone induced rabbit early femoral head necrosis.The bone regeneration effect of those scaffolds on repairing hormone induced rabbit early femoral head necrosis was assessed by Micro-CT analysis,HE staining,Masson staining.The expression and distribution of osteogenic(OPN,ALP)and angiogenic(VEGF,CD31)related factors at the lesion site were detected by immunostaining,fluorescence quantitative PCR and Western Blot techniques to evaluate the bone regeneration mechanism of those scaffolds on repairing hormone induced rabbit early femoral head necrosis.3 The sustainably releasing drug loaded scaffolds were implanted into ovariectomized osteoporosis rat bone defect.The bone regeneration effect of those scaffolds on repairing ovariectomized osteoporosis rat bone defect was assessed by Micro-CT analysis,HE staining,Masson staining.The expression and distribution of.osteogenic(OPN,ALP,Runx2)and angiogenic(VEGF,CD31,VWF)related factors at the lesion site were detected by immunohistochemistry staining,fluorescence quantitative PCR and Western Blot techniques to evaluate the regenerative repair mechanism of those scaffolds on repairing ovariectomized osteoporosis rat bone defect.Results:1 We fabricated drug loaded scaffolds including OTF/PLLA scaffold(100 mg OTF),OTF-PNS/PLLA scaffold(50 mg OTF+50 mg PNS),PNS/PLLA scaffold(100 mg PNS).These scaffolds were porous structures,aperture sizes varied,approximately 10-30 μm.The maximum compressive strength and porosity of these scaffolds met the strength requirements for bone repair and the porosity requirements for vascular growth.The OTF and PNS loaded by scaffolds showed a slowly sustained release.Those scaffolds had little cytotoxicity on MC3T3-E1 cells.Those scaffolds had no toxic and side effects,and showed good biosafety for local implanted bone tissue,main organs,hepatic and renal function.2 The sustainably releasing drug loaded scaffolds,especially OTF-PNS/PLLA scaffold,significantly promoted bone regeneration at implanted site of hormone induced rabbit early femoral head necrosis.These scaffolds significantly upregulated the expression and distribution of proteins and mRNA of osteogenic related factors(OPN,ALP)and angiogenic related factors(VEGF,CD31),activated the production and activation of osteogenic and angiogenic related factors in bone lesion sites.3 The sustainably releasing drug loaded scaffolds significantly promoted bone regeneration at implanted site of ovariectomized osteoporosis rat bone defect,the repair effect of OTF-PNS scaffold was better than others.These scaffolds significantly upregulated the expression and distribution of proteins and mRNA of osteogenic related factors(OPN,ALP,Runx2)and angiogenic related factors(VEGF,CD31,VWF),activated the production and activation of osteogenic and angiogenic related factors in bone lesion sites.Conclusions:1 The sustainably releasing drug loaded scaffolds we fabricated could meet the strength requirements for bone repair and the porosity requirements for vascular growth,show a slowly sustained release of OTF and PNS.Those scaffolds exhibit good biological safety.2 The sustainably releasing drug loaded scaffolds could significantly improve bone regenerative repair of hormone induced rabbit early femoral head necrosis.The effect mechanism might be inducing the expression of proteins and mRNA of osteogenic related factors(OPN,ALP)and angiogenic related factors(VEGF,CD31),increasing its distribution at bone lesion sites.3 The sustainably releasing drug loaded scaffolds could promote bone regenerative repair of ovariectomized osteoporosis rat bone defect.The effect mechanism might be inducing the expression of proteins and mRNA of osteogenic related factors(OPN,ALP,Runx2)and angiogenic related factors(VEGF,CD31,VWF),increasing its distribution at bone lesion sites.4 Compared with OTF/PLLA scaffold(100 mg OTF)and PNS/PLLA scaffold(100 mg PNS),OTF-PNS/PLLA scaffold(50 mg OTF+50 mg PNS)shows the best ability of promoting bone regeneration for repairing bone necrosis and bone defect of model animal.This reflects the bone regenerative advantage of combining osteogenesis and angiogenesis guided by kidney-tonifying and blood-activating theory,and confirms that drug loaded scaffolds implantation could promote bone regenerative repair of different species(rabbit,rat)and different bone injuries(bone necrosis,bone defect).