Anti-foreign Body Response Of MSC-derived Extracellular Matrix Functionalized Electrospinning Fibrous Scaffold

Current state-of-the-art biomedical implants and tissue engineering methods promise technologies to improve or even restore the function of defective or diseased organs.However,one of the biggest challenges to clinical success is the lack of material-host functional integration.Foreign-body response(FBR)caused by biomaterial implantation which leads to the fibrosis and formation of collagen fibrous capsule on the surface of the material,accompanied by serious clinical problems such as persistent chronic inflammation,tissue fibrosis,tissue adhesion and scar,seriously impedes the long-term functional maintenance and stability of the implants.With a deep understanding of a series of cellular and molecular events following biomaterial implantation,it is recognized that the high functional plasticity of macrophage plays a key role in regulating the FBR and functional integration of implants.Therefore,researchers have developed a series of design strategies based on the regulation of macrophage behavior to modulate the immune response of the implant and thereby reduce FBR.Although these modification strategies(including physical modification,chemical modification and delivery of bioactive molecules)can alleviate the FBR and improve the functional integration of implants to a certain extent,the “immune regenerative biomaterial” that could improve local immune microenvironment are still lacking.Therefore,exploiting the novel "immunomodulatory" biomaterials resistant to FBR remains an important challenge in the biomedical fields.From the perspective of "learning from nature",materials modified with endogenous "immunomodulators" are likely to be more stable in directing immune responses in vivo and in vitro.The powerful immunomodulatory properties of mesenchymal stem cells(MSC),especially when stimulated by inflammatory mediators,provide insights for the design of novel "immunomodulatory" biomaterials.With the in-depth study on the immunomodulatory mechanism of MSC,it is widely believed that MSC exerts their immunomodulatory function mainly through the paracrine mechanism.MSC-derived extracellular matrix(ECM),as the main products of MSC paracrine,is expected to be used in tissue engineering and regenerative medicine.Therefore,we hypothesized that inflammatory mediators priming MSC-derived ECM(MSC-i ECM)could act as the endogenous "immunomodulators" to reduce FBR by regulating macrophage-mediated inflammatory response.Based on the hypothesis,we constructed a novel "immunomodulatory" biomaterial through the functionalization of MSC-i ECM that could regulate macrophage-mediated inflammatory response.First and foremost,we investigated the regulatory effects of i ECM functionalized scaffolds on macrophage polarization and immune function in vitro.Additionally,the in vivo FBR of i ECM functionalized scaffolds and their role in the prevention of Achilles tendon adhesion were investigated.Further,we analyzed two important functional components in MSC-derived ECM: bioactive factors and exosomes,which play a key role in regulating the inflammatory response of macrophage.The main contents and results of this study are listed as follows:(1)Construction of MSC-ECM functionalized fibrous scaffolds and their immunomodulatory effectMSC-ECM functionalized electrospun fibrous scaffolds were constructed by decellularization tissue engineering technology.Firstly,one-step emulsion electrospinning was used to fabricate polycaprolactone(PCL)/silk fibroin(SF)composite scaffolds as the substrate material for ECM deposition.Through the characterization of the physical and chemical properties of PCL/SF fibrous scaffolds,we found that PCL/SF composite fiber scaffolds showed a micro/nanoscale bimodal fiber structure.Cell experiments further demonstrated that the larger fiber diameter and pore size of the PCL/SF bimodal fiber scaffolds promoted the cell infiltration and ECM deposition into the scaffolds.Secondly,PCL/SF-ECM scaffolds were constructed by decellularization method.To enhance the immunoregulatory potential of MSC,we performed inflammatory licensing with IFN-γ to obtain immunomodulatory extracellular matrix(i ECM).Through the detection of decellularization effectiveness,we found that the decellularization method of physical freezing combined with NH4 OH treatment used in our experiment could effectively remove the immunogenic components of MSC,while retaining a large amount of extracellular matrix attached to the PCL/SF scaffolds.Meanwhile,the results of mechanical properties of ECM modified scaffolds indicated that the ECM functionalization improved the mechanical strength of PCL/SF scaffolds.In addition,the immunomodulatory effects of ECM functionalized fibrous scaffolds on macrophages were investigated.The results of immunofluorescence staining and RT-q PCR showed that i ECM functionalized fibrous scaffolds regulated macrophage phenotype from M1 to M2.Meanwhile,the expression levels of various inflammatory factors were significantly reduced.In general,the PCL/SF bimodal fiber scaffolds that possess high cell infiltration provide an ideal culture platform for obtaining more abundant extracellular matrix of stem cells.Additionally,ECM functionalized fibrous scaffolds can significantly reduce macrophage inflammatory response in vitro.(2)Study on in vivo FBR and prevention of Achilles tendon adhesion of MSC-ECM functionalized fibrous scaffoldTo investigate the in vivo FBR of i ECM functionalized scaffolds and their role in the prevention of Achilles tendon adhesion,we first constructed a rat subcutaneous implantation model in vivo.The results of H&E and Masson staining of tissue sections indicated that the i ECM functionalized scaffold significantly improved host cell infiltration in the early stage,and inhibited the formation of collagen fibrous capsule in the late stage.Meanwhile,the results of immunofluorescence staining showed that i ECM functionalized scaffolds significantly reduced FBR by regulating macrophage polarization to M2 type.Moreover,the i ECM functionalized fiber scaffold was used as a physical barrier to evaluate its effectiveness in preventing Achilles tendon adhesion.We established a tendon injury model in rat and treated the injured tendon with i ECM functionalized fibrous scaffolds as the anti-adhesive physical barrier.The peritendinous adhesions were further evaluated by gross observation and histological analysis.The results demonstrated that i ECM functionalized fibrous scaffolds showed obvious anti-adhesion effect due to their strong immunomodulatory function.In addition,the higher degree of vascularization in PCL/SF-i ECM scaffold group suggested that the scaffold not only possess excellent anti-adhesion effect,but also could potentially facilitate tendon regeneration.(3)The functional components analysis of MSC-ECMTo further identify the functional components of MSC-ECM for immunomodulation,we first conducted a large-scale proteomic study of extracellular matrix using quantitative proteomics techniques.Based on the data independent acquisition(DIA)quantitative method,a total of 2402 proteins were quantified in each type of protein sample.The majority of proteins in the 2402 detected proteins could be attributed to ECM proteins,signaling proteins and immune responsive proteins,which contributed to up to 69% of the detected proteins.Importantly,we detected a variety of bioactive factors and receptors in ECM samples,such as TGFBI,IGF2,HGF,PDGFA,VGFR3,TGFb R2,IL13RA1,etc;these bioactive factors play an essential role in regulating the inflammatory response of macrophage.In addition,the enrichment analysis of differentially expressed proteins under different conditions(2D,PCL/SF,PCL/SF+IFN-γ)showed that compared with 2D culture,PCL/SF fibrous scaffolds significantly affected multiple metabolic pathways of MSC,including amino acid metabolism,glucose metabolism and energy metabolism.In contrast,IFN-γ stimulation activated multiple signaling pathways involved in immune regulation of MSCs,including chemokine signaling pathway,NF-κB signaling pathway,B cell /T cell/Toll-like receptor signaling pathway,TGF-β signaling pathway,and complement cascade.Further,we identified the exosomes(Exo)embedded in ECM functionalized scaffolds by means of SEM,fluorescence labeling and Western blot,and verified the immunomodulatory function of MSC-derived exosomes.The results showed that micro/nano-sized extracellular vesicles(EVs)/exosomes were embedded in the ECM scaffolds in the form of aggregation.Additionally,MSC-derived exosomes significantly facilitated human/murine macrophage polarization to anti-inflammatory M2-type and inhibited the expression of various inflammatory factors in vitro.Importantly,the exosomes derived from IFN-γ pretreated-MSC showed stronger immunosuppressive effects.Taken together,the multiple bioactive factors and exosomes in MSC-derived ECM,as functional components of ECM,play a key role in regulating the inflammatory response of macrophage.The identification of these functional components provides a mechanism insight into the immunomodulatory effect of ECM functionalized biomaterials.In summary,a novel "immunoregulatory" scaffold was developed through functionalization of MSC-i ECM in this study.The i ECM functionalized fibrous scaffold successfully reduced the FBR and effectively prevented tissue adhesion by regulating macrophage phenotype from M1 to M2.Tissue engineering scaffolds based on MSC-ECM,as an acellular regenerative medicine strategy,have important research prospects and clinical application value.