The Preparation Of Electrospun Lovastatin Loaded Antiosteoporosis Composite Scaffold “eLTPS” And Investigation Of Its Performance In Vitro And In Vivo

Osteoporosis(OP)is regarded as one of the most challenging comorbidity that often encountered in spine surgery cases.Implant failure,such as interbody cage subsidence,vertebral body split or fracture,pull out of pedicle screw,and broken screw caused by pseudoarthrosis or bony nonunion,are not uncommon for OP patients who had spinal fusion surgery due to impaired bone density and trabeculae structure.Also,with the impaired proliferation and osteogenesis function of osteoblast,OP patients tend to have prolonged bony healing period as well as decreased healing ability.Therefore,ideally extended pharmacological intervention is needed for the healing of bony defect under osteoporosis.However,the therapeutic effect of traditional systematic drug treatment is limited due to relatively poor utilization for local cite of bony defect.Also,the risk of hepatic and renal dysfunction,as well as calcium related heart disease are elevated for long-term systematic drug usage.Moreover,in clinical practice,extra bony tissue is often needed for the filling of bony defect and to achieve spinal fusion.Consequently,a composite bone scaffold that have long-term anti-osteoporosis drug delivery ability with favorable biomechanical property is potentially needed for the healing of osteoporotic bony defect as well as improvement of local bone quality.Stains,which are 3-hydroxy 3-methyl glutaril coenzyme A(HMG-Co A)reductase inhibitors,are recently widely considered to have the potential to promote bony healing and to improve bone formation through osteoblast apoptosis inhibition,improved osteoblast differentiation,and osteoclast function inhibition.However,stains have relatively low bone tissue affinity when systematically used while the osteogenesis effect promoted by locally delivered statins rely on the local drug dose,hence a proper local drug delivery system is needed.Electrospinning is a popular option for drug delivery in recent years.The advantage of electrospinning include convenience,low fabricate price,high compacity,favorable biocompatibility,and can achieve long-term controlled release.However,although electrospun material can itself be a tissue scaffold,it is reasonable to be combined with traditional bone tissue scaffold in order to obtain proper biomechanical solidity.The objective of the present study is to fabricate “eLTPS”(electrospun e-LOVPCL/?-TCP/PCL),a composite tissue scaffold that is able to sustained local release antiosteoporosis drug(Lovastatin,LOV),using electrospun LOV-loaded nano-yarn(e LOV/PCL)which then combined and with bone tissue scaffold(?-TCP/PCL)in a “sandwich-like” fashion.Also,the local cite drug releasing pattern is then accurately modified by changing the number of layers of e LOV/PCL and investigate the biocompatibility and osteogenesis effect of eLTPS in vitro.Moreover,the established and modified eLTPS is then implanted into the osteoporotic bone defect rabbit model,so as to investigate the antiosteoporosis and bony repairing ability offered by eLTPS in vivo.The present study may provide a novel effective option of the local strategy for the healing of osteoporotic bony defect.Part I : The preparation and investigate of surface characteristics of a composite bone tissue scaffold that make sustained release of antiosteoporosis drugObjective: To fabricate a composite bone tissue scaffold(“eLTPS”)that is able to make sustained local release antiosteoporosis drug(LOV),and to investigate its physical and chemical property as well as surface micro-structure characteristics.Methods: The LOV-loaded nano-yarn,“e LOV/PCL”,was fabricated using electrospinning technique with mixed LOV and PCL.The e PCL/LOV was then compressed and combined with physical property modified ?-TCP/PCL scaffold in a “sandwich-like” fashion.The compressive mechanical property of eLTPS was tested using universal material experiment machine,the surface micro-structure was investigated using scanning electron microscope(SEM),and energy dispersive spectrometer(EDS)was used to investigate the surface chemical composition.Results: We've successfully fabricated the antiosteoporosis drug-loaded composite bone tissue scaffold,eLTPS.All prepared materials have favorable mechanical property with strength over compressive strength over 70 MPa,among which the 1:1 ratio ?-TCP/PCL have the highest compressive strength(P?0.05);The chemical composition of prepared materials was further identified with EDS;SEM then revealed the roughness of surface on fabricated eLTPS,which distributed with enormous pores that allows cell adhesion and growth,while the size of pores are relatively even with diameter between about 250 ?m and 350?m.Conclusion:We have managed to accurately load antiosteoporosis drug,LOV,using electrospinning technique.The porous 3-dimensional composite bone scaffold was then successfully fabricated after combined e LOV/PCL with ?-TCP/PCL.The eLTPS can be loaded with desired layer(s)of e LOV/PCL which also have proper biomechanical strength,thus is theoretically suitable to be utilized in the local repair of osteoporotic bone defect.Part II: In vitro sustained release characteristic,investigate of biocompatibility ofeLTPS and its effect on the osteoblast proliferation and differentiationObjective: The objective of part II to is to investigate the eLTPS local LOV releasing feature,bio-safety,and its bioactive effect on osteoblastMethods: eLTPS was placed into phosphate buffered saline(PBS),the LOV dose was tested using ultraviolet(UV)spectrophotometer once every 2 days for 49 days.The LOV release profiles were drafted and compared between eLTPS loaded with different nano-yarn layers of e LOV/PCL.After co-cultured with MC3T3 cells,the cell proliferation of eLTPS and control groups were tested and compared using CCK-8 assay.The ALP staining and quantitative analysis were then applied to investigate the effect of eLTPS on osteoblast osteogenesis and mineralization.Moreover,the SEM was used to dynamically observe the osteoblast adhesion on eLTPS.Results: All groups of eLTPS' cumulative LOV release is around 20% after 3 days,and then the releasing speed gradually slowed down.eLTPS with 3 layers of LOV nanoyarn has smooth and steady releasing profile,whose cumulative releasing rate is only 62.5% after 49 days,with 37.5% of LOV unreleased.After co-culturing,the MC3 CT generally grew well in all eLTPS and control groups.On the first day,as a result of the initial burst release of LOV,the cell survival rate(c SR)of eLTPS group is relatively lower than that of the control groups,however the difference is not statistically significant.The proliferation of MC3T3 cells in eLTPS group improved gradually with prolonged culture time.The cytotoxicity of eLTPS is level one,indicating it has favorable biocompatibility.The ALP staining and quantitative analysis results demonstrated that the eLTPS group has the best ALP activity(P?0.05)compared with un-loaded TPS group and blank control group,with cells stained into dark blue.The SEM results shows that osteoblast adhesion started since the first day of co-culturing with eLTPS,as the cells were in good spreading shape.The adhered osteoblast increased with prolonged culture time,and some of the cells grew into the pores of eLTPS,while secreted matrix can be found around the cells.Conclusion: eLTPS is demonstrated to have favorable ability to make long-term controlled release of LOV;Cytotoxic experiment proved eLTPS has good biocompatibility;ALP staining indicated that eLTPS could promote osteogenic differentiation and mineralization;and SEM confirmed the adhesion and growth of osteoblast in eLTPS.Results of in vitro experiments initially demonstrated that eLTPS has good potential to promote osteoporotic bone healing.Part III: The in vivo osteogenesis effect of eLTPS in an osteoporosis animal modelExperiment I: The establishment and validation of an osteoporosis animal modelObjective: The objective of this part is to establish an irreversible osteoporosis rabbit model and to investigate its validation.Methods: New Zealand rabbits were used in animal experiment.The irreversible osteoporosis rabbit model was established with combined bilateral ovariectomy(OVX)and low dose methylprednisolone injection(MPi).The validation of osteoporosis model was reconfirmed with investigation of bone parameters on the region of interest(ROI)of lower section of femur using micro-CT.Results: All experiment animals survived without significant complication.Results of micro-CT demonstrated that the OVX group have thinner cortical bone,while the trabecula was sparser and slimmer with larger separation.The quantitative bone parameters of OVX+MPi group are detailed as follow: Tn =0.67±0.06 1/mm;Tb.Th =0.14±0.01 mm;BV/TV% =13.89%±1.07%;BMD =161.74±11.58 g/cc.All the above key bone parameters of OVX+MPi group are significantly lower than that of sham surgery group,with P?0.001,P?0.01,P?0.05,P?0.001,respectively.According to previous literature,the establishment of osteoporosis rabbit model can be considered as successful.Conclusion: We have prepared the irreversible osteoporosis animal model in rabbits by combined OVX and MPi.Micro-CT and quantitative analysis further validated the successful establishment of the osteoporosis rabbit model.Experiment II: The in vivo osteogenesis effect of eLTPS in an osteoporosis animalmodelObjective: To investigate the osteogenesis ability of eLTPS in the osteoporosis rabbit modelMethods: The established osteoporosis model was used for further in vivo experiment.The bone defect model was made on the femoral condyle according to the size and configuration of previous prepared eLTPS.The bone defect was then implanted with eLTPS and TPS(control).After 4 and 12 weeks,the animals were euthanatized to and specimens were harvested from the inferior femoral segment.The macroscopic observation of specimens was made,and micro-CT were then used for further investigate on the microstructure and bone parameters.The histological characteristics were then observed with hard tissue slicing and methyl aniline blue staining as well as HE staining.Results: All the implanted animals survived postoperatively,no significant complications was encountered.After 12 weeks,bony healing was achieved in all groups per macroscopic observation.Micro-CT results shows that eLTPS group have more and better structured trabecular bone in the ROI.The key bone parameters of eLTPS was better improved compared with the control group(P?0.05).Histological results show that eLTPS grew well in vivo with no significant surrounding inflammation.Compared with the control group,more surrounding new bone and well-structured trabecular was found in eLTPS group,while more ingrowth new bone and vascular-like tissue was also found in eLTPS group.Conclusion: Different healing patterns was found in eLTPS and control group in the osteoporotic rabbit femoral defect model.The microstructure of osteoporotic bone defect site was improved in eLTPS group with better bone parameters.Histological results further validated that eLTPS group has better local new bone and vascular ingrowth.To sum up,eLTPS can be considered to have promoted the healing process of osteoporotic bone defects in vivo.