Study On Promoting The Repair Of Atrophic Nonunion By Activating In Situ NUSC With Integrin-specific Hydrogel Constructed Based On Osteogenic Microenvironment

Atrophic nonunion is a serious complication of fracture,which is mainly characterized by the stagnation of bone repair,no callus formation,and the atrophy and absorption of the bone broken end showing a rat tail shape.At present,ensuring local mechanical stability while enhancing osteogenic activity is the main clinical treatment strategy.The mechanically stable microenvironment can be effectively obtained by replacing implantation or fixing method.However,how to improve the local osteogenic ability is still a problem.In recent years,the development of a variety of biomaterials with appropriate osteogenic bioactivities have brought new directions.However,most of the current research on these biomaterials focused on preventing the occurrence of nonunion,and the therapeutic effect of existing bone nonunion was not confirmed.Moreover,the design of these biomaterials took the properties of the materials as the starting point and ignored the needs of clinical diseases,which contributed to their unstable effects.In view of these problems,our study proposes to design and fabricate biomaterials that can specifically improve the local osteogenic microenvironment and initiate the local bone repair process based on the molecular mechanism in atrophic nonunion.Impaired blood supply in the fracture site has always been considered to be closely related to the occurrence of atrophic nonunion.In clinical practice,the incidence of atrophic nonunion is indeed significantly increased in patients with severely damaged blood supply at the fracture end,which makes a long-standing general view that after the formation of atrophic nonunion,the fracture end is characterized by severe lack of blood supply.However,with the deepening of research in recent years,more and more studies have shown that the blood supply of the broken ends in atrophic nonunion has been well regenerated and can be restored to normal levels during the process of nonunion,but the osteogenic activity is still stagnant.Besides blood supply,accurate coordination and interaction between local cells and their microenvironment components are also essential for bone healing.Recent studies have confirmed that there are enough stem cells,namely nonunion stem cells(NUSC),in the local site of atrophic nonunion,which have good osteogenic differentiation ability in the normal osteogenic microenvironment,but they are in a dormant state of being unable to osteogenic differentiation in the microenvironment of atrophic nonunion.Therefore,it is possible to greatly improve the therapeutic effect and promote bone repair by clarifying the molecular mechanism characteristics of abnormal osteogenic microenvironment at the fractured end after the formation of atrophic nonunion,and designing a treatment strategy based on the molecular mechanism characteristics to accurately regulate the microenvironment and specifically activate the osteogenic differentiation process of in-situ NUSC.According to this,this study performed the following four sections of experiment:(1)Microenvironment characteristics and molecular mechanisms of osteogenesis in atrophic nonunionIn the first section,an animal model of atrophic nonunion was constructed via a method simulating the clinical scenario.The histological,immunofluorescence and immunohistochemical staining for the microenvironment of atrophic nonunion showed that the atrophic nonunion site had similar blood supply as the normal bone repair,but the expression of osteogenic proteins was significantly reduced.Subsequently,the further in-depth studies of m RNA sequencing and bioinformatics analysis on the local microenvironment were conducted.The results showed that the low expression of collagen I(Col-Ι)/ integrin α2β1/focal adhesion kinase(FAK)/extracellular regulated protein kinases(ERK)pathway caused by the reduction of Col-Ι content in atrophic nonunion may be closely related to the decreased osteogenic ability.Col-Ι is the main organic component of bone extracellular matrix,which plays an important role in the construction of normal osteogenic microenvironment.Subsequently,we verified this conclusion by immunofluorescence and immunohistochemical staining.The results confirmed that the expression of Col-Ι/integrin α2β1/FAK/ERK pathway protein and its downstream osteogenic protein was significantly decreased.Moreover,NUSC from atroic nonunion was successfully extracted and the content of integrin α2β1 on its surface was confirmed similar to that of normal bone marrow mesenchymal stem cells.Furthermore,NUSC could recover good osteogenic ability in the normal microenvironment.However,the FAK/ERK pathway in NUSC could be inhibited and the recovery of osteogenic ability could be significantly reversed after specifically inhibiting the binding of Col-Ι to integrin receptor α2β1.Therefore,we initially confirmed that the low expression of Col-Ι/integrin α2β1/FAK/ERK pathway caused by the reduction of Col-Ι content is closely related to the decreased osteogenic ability of atrophic nonunion.(2)Construction and characterization of integrin-specific hydrogel based on the osteogenic microenvironment of atrophic nonunionGuided by the study on the microenvironment of atrophic nonunion,we first synthesized peptide containing GFOGER which are the domain in Col-Ι that can recognize and bind to the integrin α2β1 receptor by solid-phase synthesis method.The expected structural characteristics were confirmed by mass spectrometry and circular binary chromatography.Then,Calcein-AM/PI staining,CCK-8 cell proliferation assay,ALP staining and alizarin red staining were performed and the results determined the appropriate peptide concentration that had good biocompatibility and could promote the proliferation and osteogenic differentiation of NUSC.Subsequently,in order to enable GFOGER peptides to exert optimal therapeutic effect,the integrin-specific hydrogel with drug delivery function was prepared by simple mixing of GFOGER peptides,aminated modified polyethylene glycol and oxidized dextran.After that,scanning electron microscopy test,energy dispersive spectroscopy analysis,infrared spectrum analysis,rheological experiment,in vivo degradation experiment and in vitro release test were conducted and it was confirmed that the hydrogel had good microstructure and chemical structure characteristics,showed satisfactory injectable,self-healing,adhesion,biodegradability and GFOGER peptide sustained release.These properties make it suitable for the clinical application of atrophic nonunion treatment.(3)Effects of integrin specific hydrogel on NUSCIn the third section,we confirmed that the constructed integrin specific hydrogel had good cell compatibility and promoted the proliferation of NUSC by Calcein-AM/PI staining and CCK-8 cell proliferation assay.Subsequently,through fluorescence labeling,immunofluorescence and phalloidin staining,we confirmed that the integrin specific hydrogel could specifically bind to the integrin α2β1 receptor on the surface of NUSC and this interaction facilitated the formation of focal adhesion and the polymerization of cytoskeletal proteins,leading to a functional state conducive to osteogenic differentiation.Furthermore,ALP assay,alizarin red staining,RT-q PCR,western blot and other in vitro studies confirmed that the integrin specific hydrogel could promote the osteogenic differentiation of NUSC by specifically activating the integrin α2β1/FAK/ERK signaling pathway.(4)The efficacy of integrin specific hydrogel in promoting the repair of atrophic bone nonunion in animal modelIn the final section,the integrin-specific hydrogel was locally injected into radial atrophic nonunion in a rabbit model.The results of imaging,histological,immunohistochemical and immunofluorescence staining confirmed that the hydrogel could specifically improve the local osteogenic microenvironment,activate the osteogenic differentiation of NUSC and restart the bone repair process by activating the integrin α2β1/FAK/ERK signaling pathway and promoting the expression of downstream osteogenic related proteins.In conclusion,this study preliminarily confirmed that the low expression of Col-Ι/integrin α2β1/FAK/ERK pathway caused by the significant reduction of Col-Ι content was closely related to the loss of osteogenic activity in atrophic nonunion.The integrin specific hydrogel designed on this basis can activate integrin α2β1/FAK/ERK signaling pathway,improve the osteogenic microenvironment and promote bone repair.The precise biomaterials designed based on the osteogenic microenvironment in this study will bring new strategies for the treatment of atrophic nonunion and the development of orthopedic biomaterials.