In Situ Tissue Engineering Scaffolds Sequentially-Released BFGF And BMP-2 Facilitate Periodontal Tissue Regeneration

Background and Objective:Chronic periodontitis is one of the most common bacterial infectious diseases,which can progressively destroy periodontal attachment and alveolar bone.The existing periodontal therapies can eliminate local irritants and control the development of the disease,but cannot achieve complete repair and regeneration of damaged periodontal tissues.Traditional tissue engineering relies on a combination of seed cells,scaffolds,and growth factors(GFs)to restore defect tissues.However,the acquisition and culture of seed cells are time-consuming and laborintensive.In addition,stem cell transplant may have a certain risk of tumorigenicity.It can be seen that the application of traditional tissue engineering is acceptable in some incurable diseases,but it’s hard to apply in the periodontitis treatment.In recent years,in situ tissue engineering has emerged as a novel strategy for periodontal tissue regeneration.It does not need to transplant stem cells,but relies on scaffolds and GFs.In situ tissue engineering can mobilize endogenous stem cells to defect areas and induce their directed differentiation to mimic the endogenous wound healing process.Meanwhile,periodontal tissue repair and regeneration is composed of a series of continuous and dynamic processes,therefore,it is difficult for a single GF to regulate the entire process,and multiple GFs are often required to coordinate with each other and play a role in a specific time period.Basic fibroblast growth factor(bFGF)acts a pivotal part in the homing and proliferation of mesenchymal stem cells(MSCs).And it has a strong role in promoting angiogenesis and immune regulation functions.Therefore,bFGF plays an important role in creating a good repair microenvironment for the wound area in the early stage of repair.Bone morphogenetic protein2(BMP-2)can significantly promote the osteogenic/cementogenic differentiation of MSCs and the formation of Sharpey fibers.Our group previously demonstrated that the sequential application of bFGF and BMP-2 in vitro was more effective than the application of a single GF or the simultaneous application of two GFs to promote osteogenic differentiation of periodontal ligament stem cells(PDLSCs).However,GFs are unstable and easily decomposed by proteases in vivo.At present,the excessive use of GFs has been wildly applied in clinical treatment to preserve efficient concentration,which can bring a wide variety of complications,like tissue edema,ectopic osteogenesis,and skin rash.Therefore,how to ensure the long-term activity of GFs and make their sustained release at different stages is the key to in situ periodontal tissue engineering.And the electrospinning technology has been used in tissue engineering and drugcontrolled release.Therefore,this study intended to construct an in situ tissue engineering scaffold(iTEscaffold)that can sequentially release bFGF and BMP-2 by electrospinning technology,to characterize the physical and chemical properties of the scaffolds,and to explore the biological effects of the iTE-scaffold on PDLSCs in vitro,the regulatory effects of the iTE-scaffold on immune cells in vitro,and the in vivo effects of the iTE-scaffold on the recruitment of endogenous stem cells,the regulation of immune microenvironment,bone remodeling,and functional periodontal tissue regeneration,so as to provide a scientific basis for the clinical translation of in situ tissue engineering technique.Materials and Methods:1.Preparation and characterizations of iTE-scaffolds sequentially-released bFGF and BMP-2Firstly,poly(lactide-co-glycolide)(PLGA)and poly(L-lactic acid)(PLLA)were dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol(HFIP),as a shell or core spinning solution.Secondly,bFGF or BMP-2 was dissolved in an aqueous solution under ultrasound,and then was added to the spinning solution of the shell layer or the core layer in a ratio of 1:10 to prepare a uniform emulsion.Afterwards,electrospinning was used to fabricated the iTE-scaffold.Then,the surface morphology of the scaffolds was visualized under an scanning electron microscopy(SEM).The core/shell structure of fibrous scaffold was evaluated by fluorescence staining.The chemical composition of the scaffolds was characterized by fourier-transform infrared spectroscopy(FTIR).What’s more,water contact angle(WCA)tests were performed on the scaffolds to evaluate the wettability of the scaffolds.The mechanical properties of the scaffolds were detected by mechanical testing.The release profiles of the scaffolds were detected by enzyme-linked immunosorbent assay(ELISA).PDLSCs were seeded on the scaffolds,live/dead cell staining and cell counting kit-8(CCK-8)were conducted to characterize the cell viability of the scaffolds.Then,confocal laser scanning microscope(CLSM)and SEM were used to observe the cell morphology on the scaffolds.2.The regulatory effects of iTE-scaffolds sequentially-released bFGF and BMP-2 on modulation of early immuno-inflammation in periodontal injuryRAW264.7 macrophages were seeded on the scaffolds,and the effect of the scaffolds on macrophages in vitro was investigated by immunofluorescence staining,flow cytometry,and quantitative real-time polymerase chain reaction(qRT-PCR).The effect of the scaffolds on the secretion of inflammatory-related factors and anti-inflammatory-related factors by macrophages was studied by ELISA;Then,the scaffolds were implanted into the Wistar rat periodontal defect model,and the regulation of the scaffolds on the polarization of macrophages in the defect area was evaluated by immunofluorescence double staining.The expression of inflammatory factors in the defect area was studied by immunohistochemical staining.Finally,flow cytometry was used to analyze the regulation of Treg/Th17 balance in the blood,spleen,and submandibular lymph nodes.3.The regulatory effects of iTE-scaffolds sequentially-released bFGF and BMP-2 on periodontal tissue repair and regenerationPDLSCs were seeded on the scaffolds,and the effects of the scaffolds on the proliferation and migration of cells were studied by CCK-8 and Transwell assay.Alkaline phosphatase(ALP)activity and qRT-PCR were used to assay the osteogenic capacity of the scaffolds.Then,the tube formation assay,qRT-PCR,and immunofluorescence staining were performed to study the angiogenesis ability of the scaffolds.Afterwards,the scaffolds were implanted into the periodontal defect model,and immunofluorescence double staining was used to evaluate the role of the scaffolds in promoting angiogenesis and recruiting MSCs.Afterwards,relevant histological staining methods were conducted to study the regenerative effects of the scaffolds on alveolar bone,periodontal ligament,and alveolar bone in the defect area.Results:1.The iTE-scaffolds sequentially-released bFGF and BMP-2 were successfully constructed and characterizedThe iTE-scaffolds sequentially-released bFGF and BMP-2 were successfully constructed.SEM results showed that the fibers interlaced each other,exhibiting a three-dimensional network structure.The morphology of the scaffolds was smooth and uniform.FTIR results showed that the scaffolds retained the characteristic groups of the raw materials PLGA and PLLA,and the chemical properties of the raw materials were unchanged.WCA experiment results showed that the iTE-scaffold exhibited a smaller contact angle.The mechanical properties test showed that iTE-scaffolds can provide sufficient mechanical support;ELISA results showed that bFGF and BMP-2 were sequentially released from the scaffolds.After PDLSCs were seeded on the scaffolds,the results of live/dead cell staining and CCK-8 assay showed that there was no obvious cytotoxicity on the cells;SEM and CLSM results revealed that the fibrous scaffolds were very conducive to the attachment and growth of cells.2.The iTE-scaffolds sequentially-released bFGF and BMP-2 effectively regulated the immune microenvironment in the early stage of periodontal injuryAfter RAW264.7 macrophages were seeded on the scaffolds,the results of immunofluorescence staining and flow cytometry showed that the iTE-scaffolds significantly reduced the proportion of lipopolysaccharide(LPS)-induced iNOS+M1 macrophages and increased the proportion of CD206+M2 macrophages.qRT-PCR revealed that the iTE-scaffolds could inhibit the expression level of M1 macrophage-related genes TNF-α,iNOS,and IL-1β,and while up-regulated the expression level of M2 macrophage-related genes Arg-1,CD206,and TGF-β.ELISA result showed that the iTE-scaffolds reduced the release of inflammatory factors IL-1β and TNF-α,while increased the secretion of pro-healing factors IL-10 and TGFβ.After the iTE-scaffolds were implanted into the periodontal defect,immunofluorescence staining results exhibited that in the early stage of wound healing(1,2 w),the number of M1 macrophages(iNOS+CD68+)in the defect area was remarkably reduced,while the number of M2 macrophages(CD206+CD68+)was significantly increased.Flow cytometry analysis showed that the iTE-scaffolds could maintain the Treg/Th17 balance in blood,spleen,and submandibular lymph nodes.Immunohistochemical staining exhibited that the iTE-scaffold group expressed lower TNF-α and IL-1β than NC and P-scaffold groups,while the expression of IL-10 was remarkably higher in iTE-scaffold group than the other two groups.3.The iTE scaffolds sequentially-released bFGF and BMP-2 promoted periodontal tissue repair and regenerationAfter PDLSCs were seeded on the scaffolds,the results of CCK-8 and Transwell assay showed that the iTE-scaffolds could promote the proliferation and migration of PDLSCs in an early stage.Moreover,the iTE-scaffolds significantly promoted ALP activity and the expression of osteogenesis-related genes of PDLSCs.After angiogenic induction,the results of the tube formation assay showed that the iTE-scaffolds significantly enhanced the ability of PDLSCs to form networks.qRT-PCR showed that the expression of angiogenesis-related genes of PDLSCs was significantly up-regulated.Immunofluorescence staining results also revealed that the iTEscaffolds could remarkably elevate the expression of CD31 in PDLSCs.After iTE-scaffolds were into the periodontal defect,the result of double immunofluorescence staining showed that the iTE-scaffolds not only significantly promoted the recruitment of MSCs to the periodontal defect areas,but also increased blood supply in the defect areas.TRAP staining showed that the iTE-scaffolds significantly increased the number of osteoclasts in the defect areas.What’s more,micro-CT analysis,H&E staining,and immunohistochemical staining showed that the Itescaffolds significantly enhanced bone repair in the defect area.At the same time,the regeneration of cementum and functionally arranged periodontal ligament could also be observed on the surface of the damaged root in Ite-scaffold group.Conclusions:1.The iTE-scaffolds sequentially-released bFGF and BMP-2 were successfully constructed which exhibited fibrous 3D structures and the frequency of the diameters was uniform.The iTE-scaffolds showed distinguished biocompatibility and could sequentially release bFGF and BMP-2.2.The iTE scaffolds sequentially-released bFGF and BMP-2 could induce macrophage polarization into pro-healing M2 phenotype and regulate Treg/Th17 balance to modulate inflammation.3.The iTE-scaffolds sequentially-released bFGF and BMP-2 could remarkably promote the proliferation,chemotaxis and angiogenic differentiation of PDLSCs in the early stage,and induce their osteogenic differentiation in the later stage.The iTE-scaffolds could enhance stem cell homing,angiogenesis,bone remodeling,and ultimately achieve functional regeneration of damaged periodontal tissue in vivo.