Physical Characteristics Of ECM-derived Biomaterials Regulate Macrophage Responses And Influence Tissue Regeneration

The vigorous development of regenerative dentistry has brought new hope for dental therapeutics.As a basic element in regenerative medicine,biomaterials provide substrates for cell adhesion and templates for tissue remodeling,playing a key role in both exogenous and endogenous strategies for regenerative medicine.When biomaterials are implanted into the body,their regulation of immune cells(such as macrophages,CD4~+T cells,etc.)and tissue-forming cells(such as stem cells,progenitor cells,etc.)directly influence the outcome of tissue regeneration.In recent years,the development of immunomodulatory biomaterials(i.e.,biomaterials with the ability to modulate immune responses)has drawn wide attention of researchers in this field.Different types of biomaterials including inorganic materials,synthetic polymers,natural materials,can elicit unfavorable immune responses in various degree when they are used in vivo.Therefore,it is significant to explore how to optimize the immunomodulatory capacities of biomaterials by modifying their physical/chemical/biological characteristics.It has been shown that,besides chemical components and cytokine delivery capacity of biomaterials,which have already received extensive attention,physical characteristics of biomaterials also play a critical role in modulating immune responses.A previous study by our group has discovered that macrophages that were encapsulated in hydrogels of different stiffness had completely different destiny.In order to further reveal how the physical properties of biomaterials influence their immunomodulatory properties,the present study focused on topography and physical state of biomaterials,investigating the effect of physical characteristics on macrophages and tissue regeneration.Extracellular matrix(ECM)derived materials are produced by decellularizing native tissues or by using components purified from ECM.Considering their chemical similarity to natural ECM and great biological properties,ECM derived materials can not only be used as bioscaffolds in regenerative therapies of diverse tissues,but also be used as a research tool in developing biomaterial platform,which makes it feasible to further explore some complex cellular activities.In this study,we explored how the topography and the physical state of ECM derived materials(gelatin or decellularized bone matrix)affected macrophages and stem cells,avoiding the effect of material chemistry,which will pave way for designing immunomodulatory biomaterials and developing novel immunotherapies.1.The effect of scaffold topography on macrophage polarizationObjective:Topography is an important physical characteristic of biomaterials.Whatever the macro forms of the bio-scaffolds,cells sense the scaffold topography from the very beginning of their interaction and then initiate a series of biological responses.In this part we aimed to clarify the effect of the topography of ECM-derived materials on macrophage polarization.Methods:Gelatin scaffolds with either nanofibrous or flat topography were manufactured through electrospinning and casting respectively.The murine cell line RAW264.7 was chosen as the macrophage model.The effect of different topographies on macrophage polarization were evaluated by scanning electronic microscopy,real-time PCR,immunofluorescence staining,multi-plex cytokine assay,RNA sequence.Results:Compared with macrophages on the flat gelatin scaffolds,those on the nanofibrous gelatin scaffolds spread less,maintained higher ratio of round shape,but adhered more in number.Besides,macrophages on nanofibrous gelatin scaffolds expressed a lower level of M1polarization marker(iNOS)with or without the presence of MSC;they also expressed a lower level of M2 marker(MMR),but the presence of MSC eliminated this difference.Macrophages on flat and nanofibrous gelatin scaffolds secreted similar levels of inflammatory cytokines after 24-h culturing.According to the RNA sequence analysis,NF-?B,NOD,and microorganism infection related pathways displayed downward trends in macrophages cultured on the gelatin scaffolds with nanofibrous topography.Conclusions:The nanofibrous topography of ECM-derived materials can weaken M1polarization and inhibit pro-inflammatory responses,but cannot promote M2 polarization.It is suggested to add M2-inducive factors in relevant scaffold designing.2.The regulation of cell behaviors by gelatin nanofibersObjective:The ECM of native tissues often presents as a nanofibrous network mostly made up of collagens.Nanofibrous gelatin scaffolds greatly mimic the natural ECM in both physical and chemical properties,and thus possess a promising future in regenerative medicine.In order to exclude the possible influence of the genetic modification in immortalized cell lines,we used cells isolated from viable tissues to further clarify how the gelatin nanofibers regulate the behaviors of two important cell participants(i.e.,primary macrophages and stem cells)in regeneration.Methods:Rat bone marrow derived macrophages(BMDM)were used as the primary macrophage model.The effect of gelatin nanofibers on BMDM polarization was evaluated by SEM,real-time PCR,and immunofluorescent staining.Human dental pulp stem cells(DPSC)were used as the stem cell model,which were mainly analyzed from the perspectives of morphology and transcriptome.Results:When cultured on gelatin nanofibers,the M1 marker(iNOS)of BMDM was down-regulated but the M2 marker(MMR)was not affected.This trend was not affected by the presence of MSC modulation.According to the transcriptome analysis,nanofibers tended to inhibit DPSC mitosis and prevent cell senescence,and DPSC on nanofibers displayed the up-regulation of TGF-?pathway and signaling pathways regulating pluripotency of stem cell.Conclusions:Despite slightly different responses to the gelatin scaffolds between RAW264.7 and BMDM,the nanofibrous topography still significantly inhibits M1 polarization,which further confirms the previous conclusions.Considering the tendency to decrease cell proliferation of gelatin nanofibers,they may not be suitable for expanding cells in vitro,but can be used as artificial ECM to temporarily preserve stem cells to delay cell senescence and maintain pluripotency.3.The effect of scaffold physical state on macrophage responses and tissue regenerationObjective:ECM produced by decellularizing native tissues retain the natural structural and functional proteins,and possess the advantages of promoting tissue regeneration.However,ECM derived from different tissues vary from each other in the capacity to provoking immune responses and thus affect the outcomes of regeneration.Bone ECM(B-ECM)-based transplants have been reported to evoke adverse immune responses in many cases.In this part,we aimed to optimize the B-ECM in terms of their immunomodulatory properties to ensure downstream regenerative outcomes by changing the physical states of B-ECM.Methods:B-ECM particles were prepared by a series of procedures including decalcification,decellularization,lyophilization and freezing-milling.The commonly used B-ECM particles were further transformed into B-ECM gels using a simple digestion-neutralization protocol.The topography,surface composition and Young's modulus of B-ECM particles and gels were compared using SEM,ATR-FITR,and AFM.BMDM polarization states on B-ECM particles and gels were evaluated by SEM,mRNA expression,cytokine secretion,and NO production.Bone marrow mesenchymal stem cells(BMMSC)cultured on the two B-ECM scaffolds were analyzed by CCK-8 proliferation assay,ALP staining,extracellular ALP activity and osteogenic mRNA expression.The effects of B-ECM particles and gels on the regeneration of periodontal bone were evaluated in rat periodontal defect models.Results:B-ECM gels displayed a nanofibrous network morphology,and had a lower Young's modulus than B-ECM particles,and both types of B-ECM had similar surface composition.The states of B-ECM did not affect BMMSC in proliferation and osteogenesis.Instead of inducing macrophages toward M1 polarization,as reported in the literature and confirmed in the present study for B-ECM particles,the B-ECM gels were found to be more likely to polarize macrophages toward M2 phenotypes,leading to slightly enhanced bone regeneration in the periodontal defects.Conclusions:The particle-gel transformation is a simple,practical and economical strategy to modify the immunomodulatory properties of B-ECM before in vivo transplantation and hence has important implications that may facilitate the use of ECM-based bioscaffolds derived from diverse source tissues for regenerative purposes.