Microenvironment Responsive Hypoxia-mimetic DFO Composite Hydrogel For On-demand Neovascularization To Promote Tendon-to-bone Healing

BackgroundThe incidence of tendon-bone interface injuries is increasing year by year and has become a serious health problem,bringing huge economic pressure and medical burden to society,and greatly reducing the quality of life of millions of people around the world.Tendon-bone interfacial tissue engineering and regenerative medicine provide new strategies for tendon-bone interface injury.As an FDA-approved safe drug,deferoxamine mesylate(DFO)can effectively chelate iron ions,upregulate HIF-1α,promote angiogenesis,and effectively anti-ROS and regulate macrophage polarization.For the microenvironment of hypoxia,metaacidity and ROS enrichment after tendon-bone interface injury,multiple-response sustained-release gel(CAHPSDB)can effectively respond to changes in the microenvironment,release DFO on demand according to the needs of the microenvironment after loading DFO,so as to achieve on-demand vascularization,promote osteogenesis,reduce microenvironment ROS,and effectively reduce inflammatory response.Combined with the pathophysiological mechanism of the tendon-bone interface,we suspect that the multiple-response sustained-release gel(CAHPSDB-DFO)scaffold loaded with DFO has great scientific significance for the improvement of the tendon-bone interface microenvironment and the repair and regeneration of interface damage.ObjectiveTo design and construct a sustained-release gel scaffold with excellent mechanical properties and biocompatibility that can release DFO in response to changes in the microenvironment at the tendon-bone interface.To investigate the effect of interfacial tissue engineering on the repair and regeneration of tendon-bone interfacial injury and discuss the related mechanism.Method1.Different slow-release gel scaffolds were prepared and loaded with DFO for microscopic morphology observation,infrared spectroscopy,mechanical properties characterization and responsive slow-release performance measurement.2.In vitro experiments were conducted to evaluate the cytocompatibility of different sustained-release gel scaffolds,the expression levels of HIF-1α,VEGF,BMP-2 and RUNX2 in cells,the effect of angiogenesis and osteogenesis,antioxidant stress,regulation of macrophage polarization and other cell biological behaviors.3.A rat model of anterior cruciate ligament reconstruction was constructed,and CAHPSDB-DFO gel scaffold was wrapped in tendon graft.Imaging,biomechanical and histological analysis were performed at the tendine-bone interface to evaluate the imaging and histological repair and regeneration of the tendine-bone interface aweek 4,8 and 12,respectively.Results1.CAHPSDB-DFO gel scaffold showed a 3D three-dimensional structure of interpenetrating network,and the surface microstructure was rough and uneven,which was suitable for two-cell climbing,growth and migration.At the same time,it has good mechanical properties and can respond to the stimuli of the external microenvironment in multiple ways to achieve the role of releasing DFO on demand.2.CAHPSDB-DFO has excellent biocompatibility,and cells can gradually show laminated growth on the gel scaffold.Effectively up-regulate the expression levels of HIF-1α,VEGF,BMP-2 and RUNX2,and promote angiogenesis and osteogenesis.While,it showed good resistance to oxidative stress and regulation of macrophage differentiation towards M2 direction.3.CAHPSDB-DFO showed excellent repair effect of tendon-bone interface injury in imaging,biomechanics and histology at 4,8 and 12 weeks after surgery,and showed less inflammatory reaction at the tendon-bone interface,which could effectively realize early and rapid healing of the tendon-bone interface.ConclusionIn our study,we prepared a novel multi-responsive Interpenetrating network hydrogel scaffolds,which is adaptive to the tendon-bone interfacial microenvironment under a hypoxic,acidic,ROS,and related enzyme-enriched state for controllable release of the hypoxia mimicking agent DFO.We showed that the multifunctional CAHPSDB-DFO gel could achieve effective controlled release of DFO responsive to the hypoxic,acidic,ROS,and related enzyme-enriched microenvironment.The controllable release of DFO not only significantly reduced its toxicity,but also maintaining effective biological activity by up-regulating HIF-la and promoting the expression of VEGV,BMP-2 and Runx-2 on demand.At the same time,CAHPSDB-DFO gel scaffolds could effectively promote angiogenesis and osteogenesis,regulate immune response,resist oxidative stress,and exhibit anti-inflammatory effects.Therefore,after ACL reconstruction,CAHPSDB-DFO gel scaffolds can effectively promote the early and rapid healing of the tendon graft-bone interface,providing new treatment strategies and direction for tendon-bone interface injury.