Application Of Polylactic Acid And Polyethylene Glycol Copolymers In Skin And Bone Defects Repair

OBJECTIVE: Accidents-related severe skin,soft tissue injuries complicated with bone fractures are very common nowadays.The use of copolymer-based biomaterials to realize sustained release of growth factors or drugs provides new strategy for their treatment.In this study,PDLLA and PEG were used as raw materials to prepare a novel hydrogel and nanoparticle system,respectively.They were used as carriers for PRP and DFO.After characterizing the temperature-sensitive properties,drug-loading parameters,PRP sustained-release profile as well as cell toxicity of PLEL,we explored the effects of the released grow factors on migration and tube formation in HUVECs.We also investigated the impact of iron overload and DFO on the secretion of VEGF and BMP-2 in BMSCs.We assessd the effect of DFO on the osteogenic differentiation of BMSCs and the underlying molecular mechanism.Finally,we attempted to accelerate the pathophysiological processes such as epithelial creeping,angiogenesis and bone tissue regeneration in the injured sites by implanting PRP/PLEL dressing and tissue-engineered bone containing DFO/PLGA-PEG-PLGA nanoparticles,with the purpose of achieving the repair of full-thickness skin defects and critical-sized calvaria defects.METHODS: PDLLA-PEG-PDLLA triblock copolymers was synthesized by ringopening polymerization,and its physical and chemical properties were systematically characterized.PDLLA-PEG-PDLLA was dissolved in physiological saline to obtain different concentrations of copolymer solution(PLEL hydrogel),and then the temperature-sensitive properties were studied comprehensively.The PLEL hydrogel and its leaching solution were prepared with different concentrations and their toxic effects on HUVECs and fibroblasts were studied by CCK-8.PRP/PLEL was prepared by dissolving PRP into PLEL.Firstly,we depicted the release profile of PRP GFs,and then its effect on migration and tube formation capabilities of HUVECs was tested.PRP/PLEL was used as wound dressing to cure the full-thickness skin defect in rats.Through the general photos,Microfil perfusion+Micro-CT scan,HE,Masson's staining,immunohistochemistry and immunofluorescence,the effect of the new PRP/PLEL dressing on regeneration of blood vessels and on the promotion of skin wound healing was comprehensively evaluated.PLGA-PEG-PLGA copolymers was synthesized by ring-opening polymerization after introducing GA.Afterwards,the DFO-encapsulated PLGA-PEG-PLGA nanoparticles(DFO/PLGA-PEG-PLGA nanoparticles)were prepared by solvent extraction/evaporation method.The physical and chemical properties and drug loading properties of the nanoparticles were characterized by SEM,DLS,laser particle size analyzer and HPLC.The sustained release curve of DFO was obtained by a modified dialysis method.The effects of iron overload,DFO,DFO/PLGA-PEG-PLGA nanoparticles on proliferation,differentiation and secretion for relevant growth factors in BMSCs or HUVECs were studied by CCK-8,ALP activity assay,ARS staining,RTPCR,Western Blot and ELISA.And at the same time,the underlying molecular mechanism was explored.DFO/PLGA-PEG-PLGA nanoparticles and ?-TCP were used to construct the tissue engineering bone and it was implanted into the critical-sized calvaria defect in rats.Its osteogenic effect was evaluated by Micro-CT,Van Gieson staining and immunohistochemistry.RESULTS: The prepared PLEL hydrogel can undergo sol-gel phase transition at about 37?.PRP GFs in PLEL could be released for up to 14 days in a sustained manner.In vitro,PLEL hydrogel and its leachate showed good biocompatibility,with no significant toxic effects on HUVECs and fibroblasts.The PRP/PLEL could significantly improve the migration and tube formation capabilities of HUVECs.Animal experiments indicated that the PRP/PLEL significantly promoted wound closure,epithelial regeneration,angiogenesis and collagen deposition,and finally accelerated the healing of the full-thickness skin defect.The PLGA-PEG-PLGA nanoparticles were highly efficient in DFO encapsulation and could achieve a sustained release of DFO.DFO solution showed a significant toxicity on BMSCs and HUVECs,but promoted their secretion of VEGF and BMP-2.The cytotoxic effect of DFO encapsulated in PLGA-PEG-PLGA nanoparticles was significantly reduced,and at the same time the pharmacological effects of DFO were retained.Iron overload inhibited osteogenic differentiation,while DFO promoted osteogenesis through Wnt5 a,via the PI3K/AKT and NFATc1 pathways.In vivo,?-TCP+DFO/PLGA-PEG-PLGA group showed better bone regeneration in the defect area,comparing to the blank ?-TCP group and the ?-TCP + PLGA-PEG-PLGA group.CONCLUTIONS: The PDLLA-PEG-PDLLA and PLGA-PEG-PLGA block copolymers were synthesized by ring-opening polymerization,and were processed to prepare the PLEL hydrogel and the PLGA-PEG-PLGA nanoparticles.They can be used as PRP and DFO carriers,and at the same time maintain their activities.PRP/PLEL can promote migration and tube formation of HUVECs in vitro,while DFO/PLGA-PEGPLGA nanoparticles stimulate the secretion of osteogenesis-related growth factors in BMSCs and HUVECs,and facilitate osteogenic differentiation of BMSCs.In vivo results show that PRP/PLEL can promote the angiogenesis,epithelial neoplasms and collagen deposition,and finally accelerate skin wound healing;and the ?-TCP+DFO/PLGA-PEG-PLGA tissue-engineered bone can activate the endogenous bone repair function.In short,the PRP/PLEL composite hydrogel system and DFO/PLGA-PEG-PLGA nanoparticles are expected to exert huge clinical and economic benefits in the treatment of severe full-thickness skin defects and criticalsized bone fractures.