| Skin wound has become a serious healthcare issue worldwide due to the increasing incidence of burns,tears,and chronic disease-caused skin ulcers.Wound healing is a dynamic biological process that can be divided into four different stages: hemostasis,inflammation,proliferation,and remodeling.Inflammatory response and immune microenvironment play important roles in wound healing.Wound dressings are commonly used to facilitate wound healing in clinics.The clinical inflammatory response of wounds is mainly contributed by three reasons: injury-induced stress,bacterial infection,and foreign body rejection(FBR)against medical dressings.Antibiotics can be used to reduce inflammation caused by bacterial infection.However,there remains a grand need for effective measures to control stress-induced inflammation and FBR-inflammation associated with medical dressings.Spermidine(SPD)is a biogenic polyamine originally identified in semen and later found to modulate immune responses to protect sperms,which are regarded as “foreign bodies” by the female immune system,to facilitate fertilization and embryo implantation.Inspired by the immunomodulatory function of SPD,this dissertation proposes to inhibit the inflammatory response triggered by wound dressing with SPD.Meanwhile,based on the regulatory effect of SPD on macrophage polarization,a wound dressing containing SPD will be developed to alleviate overall inflammatory response,improve immune microenvironment,and thus accelerate wound healing.Polypropylene carbonate(PPC),an aliphatic polycarbonate synthesized from carbon dioxide and propylene oxide,has been regarded as an environment-friendly material.However,due to the hydrophobicity,low cell and tissue adhesion of PPC,pure PPC cannot be used as a wound dressing directly,and as a synthetic polymer,PPC will also trigger FBR.Inspired by the composition of extracellular matrix(ECM),this dissertation plans to modify PPC with chitosan(CTS)and gelatin(GEL)to improve its biocompatibility.However,CTS obtained from shrimp shells and GEL obtained from pig skin are still exogenous to rats used in this work and humans,so they will still trigger FBR.Therefore,this dissertation intends to develop bio-inspired antiinflammatory dressing by introducing SPD into the biomodification of PPC,and expand the application of environment-friendly materials in medical fields.First,after the addition of glycerol(Gly)to the mixture of CTS and GEL a series of CTS-GEL-Gly(CGG)films have been prepared by casting,which is guided by the theory of moist wound healing.According to the results of swelling test and tensile test,the optimal Gly content is determined as 2.5%(v/v).The CGG2.5 film shows good mechanical property,which overcomes the defects of poor ductility and fragility of CTS-GEL film.Second,a crosslinker based on the Schiff base reaction between the amino groups of SPD and the aldehyde groups of terephthalaldehyde(TA)has been prepared with SPD concentrations of 0.0125,0.0250,0.0375,0.0500,0.0625,0.0750,0.0875 and0.1000 M,which is denoted as SPD crosslinker.The molar ratio of TA to SPD is 1.2:1.0,which ensures that the remaining aldehyde groups of the crosslinker can undergo another Schiff base reaction with amino groups in the CGG film.Thus,the SPD-loaded CGG film,i.e.,SCGG film,has been successfully prepared.Next,SCGG-PPC-SCGG(SPS)composite film has been fabricated with PPC as the mid-layer and SCGG films as the two outside layers by plasma treatment and spincoating.More importantly,diethylenetriamine(DETA),a sham molecule resembling SPD in chemical structure,has been chosen as a control for SPD,and DETA-loaded DCGG-PPC-DCGG(DPD)composite film has been prepared in parallel.The physicochemical characterizations show that both SPS and DPD composite films have obvious "sandwich" structures,good swelling properties,suitable water vapor permeability,excellent mechanical strength and thermal stability.Moreover,in vitro biological tests indicate that SPS and DPD composite films possess good cytocompatibility,scratch healing function,and broad-spectrum antibacterial properties within the crosslinker concentration of 0.0375-0.0750 M.However,the in vitro results of inflammatory assays exhibit that SPS composite film can reduce the levels of proinflammatory markers such as interleukin 6(IL-6),tumor necrosis factor alpha α(TNF-α),and nitric oxide(NO)by about 66.19%,52.37%,and 71.86%,respectively,and increase the concentration of anti-inflammatory marker interleukin 10(IL-10)by about48.21%.Such effects of SPS composite film also depend on the concentration of SPD.In contrast,DPD film shows a little favorable effect that fluctuates independent of the concentration of DETA.Besides,the double immunofluorescent staining against cluster of differentiation 206/inducible nitric oxide synthase(CD206/i NOS)displays that SPS composite film can promote the expression of Mφ2 macrophage marker CD206,and inhibit the expression of Mφ1 macrophage marker i NOS,while DPD film cannot achieve the same outcome.Taken together,SPS film containing SPD has demonstrated in vitro anti-inflammatory and immunomodulatory functions,while promoting the polarization of Mφ1 macrophage to Mφ2 macrophage.Then,full-thickness skin wound model has been established in SD rats and the wounds were treated with pure PPC films(PPC group),SPS films(SPS-6 group)and DPD films(DPD-6 group)to examine the wound healing and immunomodulatory properties in vivo.Compared to the untreated group(Blank),PPC group and DPD-6group,the SPS-6 group shows faster healing process and better healing outcome with no excessive deposition of type Ⅰ collagen,which is closer to the normal skin.The results of immunofluorescent staining against cluster of differentiation 11b(CD11b),cluster of differentiation 163/cluster of differentiation 68(CD163/CD68),and i NOS/CD68 display that SPS film can not only inhibit inflammation and immune cell infiltration in wound bed,but also accelerate the macrophage polarization from proinflammatory Mφ1 to repairing Mφ2.The results are also corroborated by Western blots,which exhibit that SPS film can significantly down-regulate the expression of pro-inflammatory cytokines including nuclear factor kappa-B(NF-κB),monocyte chemoattractant protein-1(MCP-1),IL-6,interleukin 2(IL-2),and TNF-α,and enhance the secretion of anti-inflammatory factors,such as transforming growth factor β(TGF-β)and IL-10.Meanwhile,SPS film can also up-regulate the expression of grow factors involved in epidermal and dermal regeneration,i.e.epidermal growth factor(EGF),fibroblast growth factor(b-FGF)and vascular endothelial growth factor(VEGF).Finally,in order to explore the mechanism for SPS film to promote Mφ2macrophage polarization and wound healing,proteomic analysis has been conducted on the tissues of Blank,SPS and DPD groups,which were collected on postsurgical Day 7.The mechanism for the effect of SPS film on macrophage polarization has been investigated by mutual comparison among the three groups,i.e.SPS vs.DPD,SPS vs.Blank,and DPD vs.Blank.The results suggest that,on one hand,SPD-loaded SPS film can down-regulate the expression of acetyl Co A carboxylase 1(Acaca)and thus inhibit the AMPK/NF-κB signaling pathway to suppress the expression of pro-inflammatory cytokines;and on the other hand,SPS film can enhance tricarboxylic acid cycle(TCA)and fatty acid oxidation(FAO),thereby accelerating the transition from Mφ1macrophage-dominated phase to Mφ2 macrophage-dominated phase during wound healing.Hence,SPD-loaded SPS film can reduce inflammatory response and modulate immune microenvironment through the above two aspects to promote wound healing and yield regenerated skin closer to normal skin. |
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