Studies On Bioactive Bone Regeneration Materials Based On Biodegradable Multifunctional Polyester Microspheres

The repair strategies for bone defects resulted from traumas,infections,tumor ablation and bone disorders meet rising demands in practical clinics.Autograft is deemed as the gold standard for bone regeneration,but it is restricted by its limited resource and donor morbility.Allograft is not affinitive to the native tissues,turns out to be inflammatory around the healthy tissues,and cannot shun the risk of virus propagation.The principles of tissue engineering provide an alternative strategy to face the challenges,in which,a key issue is to develop bioactive scaffolds with well-defined structures and multi-function to induce osteogenesis.With injectability and flexibility in preparation,biodegradable microsphere type scaffolds have attracted keen interests targeting tissue regeneration.Additionally,they can be filled into irregular defects via minimally invasive treatment to relieve the burden of the patients.Aliphatic polyesters(e.g.PLA,PGA,PCL and PLGA etc.)are among the most popular degradable biomaterials that have been investigated for decades.They have been approved available for implants by FDA of many countries,and now are widely applied in biomedical areas benefiting from their tunable degradation rates,good biocompatibility and excellent processability.For tissue regeneration,however,the microspheres made of aliphatic polyesters are showing deficiencies in aspects of hydrophobicity,acidic degradation products and lacking bioactivity,etc..To develop the polyester-based microspheres into a kind of promising bone defect filling materials,in this thesis,amphiphilic PLLA-PEG-PLLA copolymers were synthesized and made into rough-surfaced porous microspheres via the double emulsion-solvent evaporation method.Then modifications were performed on the microspheres for the purpose to endow them with activities as anti-bacteria,osteogensis and angiogensis.The feasibility of these functional modifications in inducing bone regeneration were verified by in vivo evaluations using different animal models.Briefly.the researches having been done in this thesis can be summarized as:(1)PLLA-PEG-PLLA triblock copolymers were synthesized via ring-open polymerization of L-lactide being initiated by the two end hydroxyls of PEG at different ratios Microspheres with different morphologies were prepared and modified with dopamine,gelatin and biomineralization.(2)Alendronate-loaded biomineralized microspheres were subcutaneously injected into the back of New Zealand rabbit to evaluate their biocompatibility and their capacity in inducing ectopic osteogenesis.(3)Dual purpose microspheres with both nanosilver and hydroxyapatite being loaded were prepared.Their antibacterial and osteoinductive abilities were evaluated by both the subcutaneous model in rabbit back and S.aureus-infected cranical defects in rats.(4)Vancomycin-and strontium-loaded biomineralized microspheres were prepared with multiactivities as antibacteria,osteogensis and angiogensis,which were verified via using both subcutaneous model and S.aureus-treated femur defect model in rabbits.More details are introduced as follows(1)Amphiphilic PLLA-PEG-PLLA triblock copolymers were synthesized using the hydrophilic and biocompatible PEG as the initiator by controlling its contents(10%,30%).The synthesized block copolymers were prepared into microspheres by double emulsion-solvent evaporation technology,followed by polydopamine or gelatin coating,and further apatite deposition in simulated body fluid(SBF).The coating of polydopamine or gelatin endowed the microspheres excellent cell affinity,provided reactive groups for further modification and nuclear sites for biomineralization.The introduced hydroxyapatite would not only be able to promote osteogenesis,but also able to partially neutralize the acidic degradation products of the copolymers.The microspheres made of PLLA-PEG-PLLA containing 10%PEG component were determined a proper choice for the following studies in view of their morphology and cell affinity.(2)Alendronate,a medicine for treating metabolic bone disorder,can improve the osteoblasts' activities to enhance bone regeneration via the up-regulation of biomineralization and the down-regulation of bone resorption.Therefore,alendronate was introduced into the aforementioned PLLA-PEG-PLLA(10%PEG)microspheres during the biomineralization in SBF.Gelatin-treated microspheres were used and different amounts of alendronate were introduced by dissolving gradient concentrations of alendronate in the SBF.Continuous release of alendronate was detected,showing apparent dependence on its loading amounts.These microspheres demonstrated non-cytotoxicity to BMSCs when the loaded alendronate was controlled below a critical value.It was identified that BMSCs could attach well and proliferate vigorously on the selected alendronate-loaded microspheres.At the meantime,the osteogenic differentiation of BMSCs were enhanced significantly in comparison with those cells cultured on biomineralized microspheres without alendronate.In vivo evaluation was carried out by injecting the alendronate-loaded microspheres into rabbit's back subcutaneously.The results revealed the microspheres possessed good,and more promisingly,they could significantly promote ectopic osteogenesis in comparison with biomineralized microspheres without alendronate,indicating the feasibility of using alendronate in bone tissue engineering.(3)Clinically,the traumatic bone tissues suffered with infectious syndromes,and the mostly severe infection invited ischemia to the dysfunction of bone repair,so biomaterials having both the antibacterial and osteogenic activity attracted wide attentions in great need for bone regeneration.This study was based on the modification of polydopamine coating,AgNPs loading and biomineralization onto the porous microspheres to fabricate the dual-functionalized microspheres for the regeneration of infected bone defects.The sustained release of silver ion,which was corresponding to its loading amount,represented the conspicuous inhibition for E.coli and S.aureus in vitro.The BMSCs cultured on the microspheres with proper AgNPs loading demonstrated comparable biocompatibility,celladhersion,spread,proliferation and osteogenic differentiation activities to those cells cultured on microspheres with no AgNPs.The subcutaneous implantation in rabbits indicated the capacity of the AgNPs-loaded microspheres in inducing ectopic osteogenesis while displaying no hint of adverse effect.Then the dual-functional microspheres were filled into the S.aureus-infected rat calvarial defects to evaluate their bacteria-resisted and bone-promoting performances.The consequences demonstrated the excellent antibaterial and osteogenic activity for the AgNPs-and HA-loaded microspheres compared with biomineralized microspheres containing no AgNPs and the blank control groups.(4)Although the microspheres loaded with AgNPs and HA had efficient effects on assays both in vitro and in vivo,the security of AgNPs still attracted much concern.In considering to a safer antibacterial biomaterial with increasing inhibited effect on killing S.aureus,we embeddedone of the most common antibiotics used clinically,vancomycin,and introduced an active element strontium into microsphere during biomineralization.Thus we have prepared a kind of multi-functionalized microspheres and characterized its performances(antibacterial,angiogenic,osteogenic)both in vitro and in vivo.To avoid the significant loss of vancomycin during microsphere preparation,MCF-26 mesoporous silica was used as a carrier to absorb the antibiotics and then embedded into the microspheres.The vancomycin and strontium were steadily released at levels in accordance with their loading amounts.The S.aureus cloning ability was significantly inhibited by the vancomycin-contained microspheres,but these microspheres demonstrated non-cytotoxicity,good affinity to cell adhersion,spread and proliferation at optimized vancomycin loading amount.With the increase amount of incorporated strontium,the markers and genes relative to the extents of angiogensis and osteogensis were extensively up-regulated.The subcutaneous implantation in rabbits demonstrated the biocompatibility of the microspheres with vancomycin,strontium and HA,while the introduction of strontium improved the impact on vessel-forming and ectopic bone formation.We introduced the multi-functional microspheres into the rabbit femurs infected with S.aureus in advance,it turned that the loadings of the two extra components led tos efficient bone regeneration,which was much superior to the vancomycin-only and blank groups.Taken together,the biodegradable aliphatic polyesters applied as bone regenerative materials have bright future in clinic therapy in the long run.The multi-functional microspheres developed in this thesis displayed advantages in several aspects over other scaffolding materials for bone tissue engineering.On one hand,they can be readily injected into sites and fill irregularly shaped defect with theminimally invasive treatments.On the other hand,the microspheres have strong flexibility in functional modification to incorporate activities as antibacteria,angiogensis and osteogensis.Thus,they are envisioned promising candidates for clinic use and provide alternatives to meet the complex situations of bone repairing.