| BackgroundThe essence of inflammation is the body’s defense response to various traumatic stimuli,and inflammation is also the physiological basis for restoring tissue homeostasis and promoting self-repair.In nature,certain lower vertebrates such as zebrafish display superior regeneration capacity than that of mammals,an acute and transient sequential inflammatory response is essential and vital to efficient tissue regeneration.Meanwhile,compared to cutaneous wounds,the accelerated healing without scars in oral mucosa is also closely linked to a heightened inflammatory response primed at baseline and a rapid controllable inflammation,which serves as the main effective factor to promote tissue repair and physiological structure reconstruction.Recent studies have suggested that in the animal models of bone loss caused by severe trauma and cutaneous wounds induced by diabetes,a relatively insufficient,prolonged and dysregulated inflammation will reduce the regenerative efficiency of the body resulting in refractory lesions.Therefore,eliciting a transient acute inflammatory response would promote tissue construction and regeneration.Whether strategies of precise regulation of the inflammatory response mode will be potential for tissue regeneration（eliciting a heightened inflammatory response and timely promote inflammation resolution for shortening the inflammatory process）becomes a research hotspot.Inflammation is a tightly regulated physiological process.Neutrophils and macrophages,as the key cells of inflammation,play a coordinated role in clearing infections and damaged cells promoting tissue repair and regeneration.The recruitment of neutrophils represents the initiation of inflammatory response.Neutrophils typically serve as the first leukocytes being recruited to the sites of infection or injury,which play an important role in preventing the invasion of infections at the early stage of inflammation by secreting cytokines and chemokines and recruiting other immune cells.However,overexuberant infiltration or persistence of neutrophils results in chronic inflammation and even collateral tissue damage.Therefore,neutrophils must be removed in time after completing their missions of recruitment,activation and phagocytosis.Timely apoptosis of neutrophils is essential for reducing inflammatory mediators and resolving inflammation.The apoptotic neutrophils are subsequently engulfed by macrophages,which leads to macrophages reprogramming to anti-inflammatory phenotype,contributing to recruitment,proliferation and differentiation ofMSCs to restore homeostasis and promote tissue regeneration.According to previous studies,our group proposed the hypothesis:sequentially regulate the recruitment and apoptosis of neutrophils which then promote the transformation of macrophages into an anti-inflammatory phenotype,so as to reprogram the inflammatory response for tissue regeneration of refractory lesions.The migration of neutrophils is guided by a diverse groups of molecular guidance cues.N-formylmethionyl-leucyl-phenyl-alanine（fMLP）,generated by bacteria and damaged mitochondria,plays crucial roles in the early recruitment of neutrophils.While,the apoptosis of neutrophils is mainly induced by two kinds of cascade signals:apoptosis caused by the endogenous spontaneous apoptosis and apoptosis caused by the interaction between exogenous death receptors and ligands.FasL/Fas system represents a typical exogenous apoptotic signal of neutrophil apoptosis.Studies have shown that the novel controlled release biomaterial-hydrogel（Gel,etc.）and nanoparticle（SiO2,etc.）hybrid complex take the advantages of structural diversity and precise drug delivery,which can tempo-spatially regulate cell biological behavior by loading specific biomolecules.Therefore,this study intends to design and prepare a hybrid biomaterial Gel@fMLP/SiO2-FasL.Its principles are as follows:fMLP can be explosively released after application and neutrophils can be recruited rapidly.Then,the p H responsive hydrogel was degraded by acid generated after neutrophils activation,and FasL on the surface of SiO2 was exposed,thus subsequently inducing apoptosis of activated neutrophils.Finally,apoptotic neutrophils can be endocytosed by macrophages to stimulate their transformation into an anti-inflammatory phenotype,forming a microenvironment conducive to tissue regeneration.With the controlled release function of the novel hybrid biomaterial,the transiently heightened and sequential inflammatory response induced by neutrophils is regulated,so as to lay a research foundation for the treatment of refractory lesions.AimFirstly,the physical and chemical properties of Gel@fMLP/SiO2-FasL were tested to validate the ability of two stages of accurately controlled release with burst release of fMLP and p H-responsive release of SiO2-FasL.Secondly,a co-culture system was established to detect the sequential role of the hybrid biomaterial in promoting neutrophil recruitment and inducing activated neutrophil apoptosis,and to evaluate the phenotypic transformation of macrophages after endocytosis of the pretreated activated neutrophil,so as to verify the regulatory effect of Gel@fMLP/SiO2-FasL on the biological behavior of inflammatory cells.Then,a mice critical-sized bone defect model was constructed to observe the bone repair and the degree of local tissue inflammation after Gel@fMLP/SiO2-FasL application,and to clarify its function of regulating the sequential inflammatory response guided by neutrophils to promote the regeneration of critical-sized bone defect.Finally,a diabetic cutaneous wound model was constructed to observe the wound healing and local tissue inflammatory response pattern after the application of Gel@fMLP/SiO2-FasL,and to verify that it can promote diabetic wound healing by reprogramming the inflammatory response process.This study aims to construct a new technical system for sequentially regulation of neutrophil recruitment and apoptosis,reprogramming the inflammatory response process to promote tissue regeneration,and provide new ideas and strategies for the treatment of refractory lesions clinically.Methods1.Preparation and characterization of the hybrid biomaterial（Gel@fMLP/SiO2-FasL）.Firstly,the gel matrix was prepared using 4-formylbenzenboric acid modified chitosan（CS-FPBA）and its modification was evaluated by 1H NMR and UV-vis spectrophotometer.Secondly,the FasL-conjugated SiO2（SiO2-FasL）was synthesized and the morphology,size and zeta-potential of SiO2 and SiO2-FasL were measured by transmission electron microscope（TEM）and laser particle size analyzer.Then,the hybrid hydrogel complex was prepared with physically loaded fMLP and SiO2-FasL,and the morphology,contents and mechanical properties of the gel complex were observed by scanning electron microscopy（SEM）,fourier transform infrared spectroscopy（FT-IR）and rheometer.Finally,the released percentage of fMLP and SiO2-FasL was determined by monitoring the payload fluorescence using fluorescence spectrophotometer.2.The regulation of the inflammatory cell biological behavior by hybrid biomaterials in vitro.Primary neutrophils and macrophages from mice bone marrow were firstly isolated and characterized by flow cytometry.Then,neutrophils were co-cultured with hybrid biomaterials in transwell co-culture system,and the number of migrated cells was counted by flow cytometry and immunofluorescence.After being activated,neutrophils were co-cultured with nanoparticles,the percentage of apoptotic cells was determined by flow cytometry.The expression level of apoptosis-related proteins of neutrophils was detected by Western blot.Finally,neutrophils induced by nanoparticles（SiO2-FasL）were co-cultured with macrophages.Phenotypic change of macrophages was observed by immunofluorescence staining,phenotypic related protein expression of macrophages was detected by Western blot,and the expression levels of cytokines in the co-culture supernatants were evaluated by ELISA.3.The effect of Gel@fMLP/SiO2-FasL on repair of mice critical-sized calvarial bone defect.Firstly,the critical-sized calvarial bone defect was constructed and then Gel@fMLP/SiO2-FasL was applied locally.Micro-CT was used to detect the repair of bone defects 8 weeks post operation.H&E and Masson staining were used to observe the formation of bone tissues.Afterwards,immunofluorescence staining was used to observe the infiltration of neutrophils and macrophages,the proportion of neutrophil apoptosis and the expression of cytokines in the defects short-term after Gel@fMLP/SiO2-FasL application.4.Evaluation of Gel@fMLP/SiO2-FasL on wound healing of diabetic mice.Type I diabetic mice model was constructed and a full-thickness cutaneous defect centered on the back was generated.The wound healing speed in normal mice and diabetic mice was compared.The infiltration of neutrophils and macrophages in the cutaneous defects was detected by flow cytometry and immunofluorescence staining.After that,Gel@fMLP/SiO2-FasL was applied to wounds of diabetic mice,the effects of hybrid biomaterials were evaluated and compared.Tissue regeneration was observed by H&E and Masson staining.At last,flow cytometry and immunofluorescence staining were used to detect the infiltration of neutrophils and macrophages and the apoptosis of neutrophils was evaluated by immunofluorescence staining in the cutaneous wounds.Moreover,ELISA was used to detect the expression levels of inflammatory cytokines in the defects.Results1.Gel@fMLP/SiO2-FasL was constructed with the capability of fMLP burst release and p H-triggered on-demand delivery of SiO2-FasL.Firstly,the PBA-based polymeric hydrogel matrix（CS-FPBA）were formed using Chitosan（CS）modified by 4-Formylphenylboronic Acid.Then,FasL was immobilized onto the surface of SiO2 to get SiO2-FasL.Subsequently,fMLP and SiO2-FasL were successfully loaded into the p H-responsive hydrogel to form the hybrid biomaterial Gel@fMLP/SiO2-FasL,which could bear large deformation force and maintain a complete three-dimensional network structure with superior stability.Finally,the on-demand release profile showed that fMLP can be rapidly released and SiO2-FasL can be p H-responsive controlled released from the hydrogel.2.The hybrid biomaterial induces neutrophil migration and apoptosis,and macrophage phenotypic transformation in vitro.Firstly,the co-culture system verified that the hybrid biomaterials containing fMLP could promote neutrophils migration.Secondly,SiO2-FasL could induce activated neutrophil apoptosis.After co-cultured with the SiO2-FasL-induced neutrophils,macrophages could transform from pro-inflammatory phenotype to anti-inflammatory phenotype.The expression of pro-inflammatory cytokines TNF-αand IL-6 were downregulated,and the expression of anti-inflammatory cytokines TGF-βand IL-10 were upregulated in the supernatants.3.Gel@fMLP/SiO2-FasL improves repairing of critical-sized calvarial bone defects.It was observed that the hybrid biomaterial Gel@fMLP/SiO2-FasL promoted the repair of critical-sized calvarial bone defects in mice.Moreover,rapid recruitment and timely apoptosis of neutrophils were observed at the defect site after Gel@fMLP/SiO2-FasL application.Finally,the infiltration of pro-inflammatory macrophages and their phenotype transformation to anti-inflammatory macrophages were also promoted.The expression level of pro-inflammatory cytokine TNF-αwas downregulated,and the expression of anti-inflammatory cytokine TGF-βwas in turn upregulated.4.Gel@fMLP/SiO2-FasL accelerates wound healing of diabetic cutaneous defects.Type I diabetic mice model was successfully established.The cutaneous wound healing speed of diabetic mice was significantly lower than that of the healthy mice.In addition,the inflammatory response after injury was delayed and lasted longer than that of the healthy mice.However,wound healing was significantly accelerated in diabetic mice after Gel@fMLP/SiO2-FasL administration.For inflammatory cells observation,neutrophil recruitment and infiltration of pro-inflammatory macrophages were promoted after Gel@fMLP/SiO2-FasL treatment.Moreover,Gel@fMLP/SiO2-FasL could induce the apoptosis of neutrophils and macrophages transformation to anti-inflammatory phenotype.Meanwhile,the expression of pro-inflammatory cytokines was downregulated and the levels of anti-inflammatory cytokines were upregulated.Conclusion1.Gel@fMLP/SiO2-FasL was successfully constructed and it possessed the biological function to sequentially release fMLP and FasL.2.Hybrid biomaterials could promote neutrophil migration,induce activated neutrophils apoptosis,faciliate macrophage transformation into anti-inflammatory phenotype,which can be used to regulate inflammatory response progress.3.Gel@fMLP/SiO2-FasL could induce a transiently heightened inflammatory response stimulating endogenous regeneration potential and promoting bone defect repair.4.Gel@fMLP/SiO2-FasL could reprogram the chronic inflammatory response of diabetic cutaneous wound into a transiently heightened process to accelerate wound healing.Based on the innovative concept that inducing moderately acute inflammatory response could promote tissue repair and reconstruction,this work established the technical system for the construction of new hybrid biomaterial Gel@fMLP/SiO2-FasL for the first time.After that,its biological role in inducing sequential inflammatory response via precisely regulating neutrophil recruitment and apoptosis was identified.This work further verified the biological function of remodeling inflammatory response in injury site to promote repairing of critical-sized calvarial bone defects and diabetic cutaneous wound healing,laying a research foundation and providing a new strategy for treatment of complex cranial and maxillofacial injuries. |
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