The Osteogenic Effects Of PNIPAAM Modified Mesoporous Hydroxyapatite Controlling Simvastatin Release In Osteoporosis Model

Osteoporosis is a chronic bone disorder characterized by low bone mass and bone micro-architecture deterioration,which usually noticed in women whose estrogen quickly decreased after menopause[6-8].The pharmacological treatments of osteoporosis are mainly divided into anabolic agents,such as bisphosphonates,calcitonin,ipriflavone,vitamin D,estrogen,and estrogen receptor modulators;and bone formation-stimulating regimens,such as parathyroid hormone(PTH),fluoride,and strontium renelate[9].These drugs used in systematic definitely reduced bone loss in osteoporosis as well as accompany with side effects.Therefore,bone substitute local treatment tended to be an effective solution to accelerate bone healing and regeneration in osteoporosis[1].Mesoporous hydroxyapatite(MHA)possesses good biocompatibility,ordered mesopore structure,similar chemical composition,and hydroxyl group which can be chemically modified as an ideal drug carrier[2-4].Poly(N-isopropylacrylamide)(PNIPAAM),a thermal-responsive polymer used as an injectable hydrogel.Below the lower critical solution temperature(LCST,32?),PNIPAAM chains become hydrophilic and show extended conformations in water so that cells will detach mildly from the PNIPAAM modified surface.Above the LCST,the PNIPAAM chains become hydrophobic and collapse on the material surface.PNIPAAM modified MHA would release drug slowly and realize long-term release in local bone defect area[5].Thus,we set up three parts of the experiments to solve these problems:(1)We firstly synthetize mesoporous hydroxyapatite(MHA),modify it chemically using PNIPAAM,and carry simvastatin(SIM)as bone formation-stimulating regimens to be MHA-SIM-PNIPAAM nanoparticle.Then,surface morphology,interior structure,chemical composition,lattice parameter,and drug release were observed.(2)Bone mesenchymal stem cells(BMSCs)from female wistar rats was derived directly by flushing bone marrow out of femur and tibia.Then,BMSCs was identified by osteogenic,adipogenic,and chondrogenic differentiation,and the protein expression of CD90 and CD45 on the cell membrane was detected.In addition,we investigate cell compatibility and osteogenic RNA expression of BMSCs culturing in MHA-SIM-PNIPAAM extracts.(3)We establish rat ovariectomized model.We compare the osteogenesis of MHA-SIM-PNIPAAM and MHA in osteoporotic or sham femur defect model,including cell morphology and attachment,bone formation,mineralization,and osteoclastic activity.Part I Synthesis,characterizations of MHA-SIM-PNIPAAMObjective:The aim of this study is to present the synthesis process of a sustained release system of simvastatin(SIM)based on the mesoporous hydroxyapatite(MHA)capped with poly(N-isopropylacrylamide)(PNIPAAM)and to detect its physic characterizations.Materials and methods:The MHA nanoparticles were prepared by using cetyltrimethylammonium bromide(CTAB)as a template and the modified PNIPAAM layer on the surface of MHA was fabricated through surface-initiated atom transfer radical polymerization(SI-ATRP).Its characterizations were detected by Scanning electron microscopy(SEM),Tcanning electron microscopy(TEM),Spectra from Fourier transform infrared spectroscopy(FT-IR),Brunauer-Emmett-Teller(BET),X-ray photoelectron spectroscopy(XPS),and X-ray diffraction(XRD)analysis.The release profile of simvastatin(SIM)was tested by UV-vis spectrophotometer at 238 nm wavelength.Results:The MHA-PNIPAAM nanoparticles were fabricated as rod-like shapes at length of 40-50nm and width of 15-20nm with well-distributed 5nm diameter pores inside nanoparticles and 5 nm polymer layer modified around the surface of nanoparticles.XRD and BET analysis verified the mesoporous structures and successful modification of PNIPAAM?FT-IR and XPS analysis detected the chemical composition of MHA-SIM-PNIPAAM nanoparticles.The release profile showed that the release of SIM from MHA-SIM-PNIPAAM lasted 16 days and the cumulative amount of released SIM was almost seven-fold than MHA-SIM.Conclusion:The MHA-SIM-PNIPAAM nanoparticles were successfully synthesized and SIM drug can be controlled released for 16 days.Part ? The isolation and identification of rat BMSCs as well as the biological features of MHA-SIM-PNIPAAM nanoparticlesOjective:The aim of the study is to derive and then identify BMSCs from female wistar rats.The biocompatibility of MHA-SIM-PNIPAAM nanoparticles was also dectected.Materials and methods:The bone marrow mesenchymal stem cells(BMSCs)from wistar rats was derived directly by flushing bone marrow out of femur and tibia.To identify BMSCs,Alizarine Red S staining,Oil Red staining,and Alcian blue staining was taken to detect osteogenic,adipogenic,and chondrogenic differentiation.Flow cytometry was used to measure the protein expression of CD90 and CD45 on the cell membrane.BMSCs proliferation was assessed by Cell Counting Kit-8(CCK-8)assay.Cell morphology and attachment was observed by cell fluorescence staining.The RNA expression of RUNX2 and OPN of BMSCs induced by MHA or MHA-SIM-PNIPAAM nanoparticle extraction was detected by fluorescent quantitative PCR analysis.Results:BMSCs of wistar rats was successfully derived and the cells possessed the capacity of osteogenic,adipogenic,and chrondrogenic differentiation.The positive expression of CD90 cell is more 99%and negative expression of CD45 cells is less than 10%on the cell membrane.SIM loaded MHA-PNIPAAM exhibited better performance on cell proliferation and attachment.The protein expression of RUNX2 in MHA and MHA-SIM-PNIPAAM groups was enhanced at day 7,but at day 14 only MHA-SIM-PNIPAAM group promoted the RNA expression of RUNX2.Both two groups enhanced the RNA expression of OPN at two time points.Conclusion:BMSCs of wistar rats was successfully derived.MHA-SIM-PNIPAAM nanoparticles showed a good biological performance.Part III Study on the osteogenesis of MHA-SIM-PNIPAAM nanoparticles in osteoporotic bone defect:in vitro and in vivo studyOjective:The aim of the study was to evaluate the osteogenic potential of the sustained release system of simvastatin(SIM)based on the mesoporous hydroxyapatite(MHA)capped with poly(N-isopropylacrylamide)(PNIPAAM)in ovariectomized(OVX)model.Materials and methods:Ovariectomy was conducted to build rat osteoporosis model.Cell morphology and attachment was observed by cell fluorescence staining.The osteogenic differentiation was evaluated by alkaline phosphatase(ALP)activity and Alizarin Red staining.The therapeutic efficiency of MHA-SIM-PNIPAAM nanoparticle was evaluated by ?CT analysis,hematoxylin and eosin staining,immunohistochemical staining,and tartrate-resistant acid phosphatase(TRAP)in rat femur defect.Results:Rat osteoporosis model was successfully built after ovariectomy for 2 month confirmed by ?CT.The cell morphology pretended to be more short and flat in OVX BMSCs than in sham BMSCs,and the osteogenic differentiation weakened.MHA-SIM-PNIPAAM group showed better osteogenic performance than other groups in both cells.In vivo study,?CT and HE staining elucidated big discrepancy of new bone formation was observed at 3 weeks than 6 weeks in all groups and MHA-SIM-PNIPAAM group showed best bone repair especially at 6 weeks.Immunohistochemical staining showed OPN marker expressed stronger than BSP protein and both were strongest expressed in MHA-SIM-PNIPAAM group.The positive OPN staining was specific localizad around new forming bone lacuna and the nanoparticles remnant.TRAP staining showed the osteoclasts was the lest observed in MHA-SIM-PNIPAAM group.Conclusion:MHA-SIM-PNIPAAM nanoparticles is a potential bone substitute for bone defect in osteoporosis.