Preparation And Performance Investigation Of Bioinspired Anti-rejection Hydrogel For Artificial Skin

Owing to the rising demand and the development of innovative technologies,artificial skin has been more widely used in clinics over recent decades,which plays an indispensable role in saving lives and improving the quality of life.Hydrogels have been considered as an excellent and promising biomaterial to make artificial skins,due to their high water content and 3D porous network similar to native skins.However,implanted materials generally induce foreign body rejection(FBR)of hosts.Such a reaction against implants may cause implant failure and uncomfortableness in patients.Currently in clinics,the use of anti-inflammatory drugs,such as dexamethasone and rapamycin,is the major approach to regulate immune system and FBR.However,these immunosuppressants can cause undesirable side effects including liver toxicity,kidney toxicity,neurotoxicity and carcinogenesis.Hence,it remains a grand challenge to endow artificial skin with a close-to-native function that can induce a favorable and reparative immune response without detrimental side effects.In recent years,biomimetic design has become an important development trend in the field of biological manufacturing.Nature can help us find solutions to overcome immune rejection.Spermidine(SPD)is a natural polyamine widely exists in human body with known antiinflammatory,antioxidant and anti-aging functions,which holds a good potential to alleviate FBR.Therefore,in this dissertation,a hydrogel for artificial skin has been prepared based on SPD,which is expected to have capabilities of both immune regulation and promoting regeneration.First,in order to simulate the extracellular matrix and reduce immune rejection,gelatin and chitosan were selected to prepare hydrogels and modified with methacryloyl(MA)group.The chemical modification of MA not only improves mechanical properties of gelatin and solubility of chitosan in neutral solution,but also endows biopolymers with the characteristics of UV crosslinking.The obtained products are respectively named as gelatin methacryloyl(Gel MA)and chitosan methacryloyl(CTSMA).Gel MA hydrogel was prepared by Gel MA and a photo-crosslinking initiator(I2959).Meanwhile,Gel MA/CTS-MA hydrogel was prepared from the mixture of Gel MA and CTS-MA,which is named as single-network(SN)hydrogel hereafter.SN hydrogel shows better performance in swelling and mechanical properties than Gel MA hydrogel.Together with the results of cytocompatibility assay,it is determined that the optimal formulation of SN hydrogel is 10 wt% Gel MA,1.0 wt% CTS-MA,and 0.5 wt%I2959.Subsequently,the SPD crosslinker was prepared by the reversible Schiff base reaction between SPD and terephthalaldehyde(TA).Such an approach can ensure the slow release of SPD from the hydrogels,leveraging the reversibility of Schiff base reaction and avoiding undesirable burst release of SPD.Then,SPD double-network(SPD-DN)hydrogels were prepared in two-steps,i.e.Schiff base reaction with SPD crosslinker and addition polymerization under UV light.The results demonstrate that0.1 m M SPD-DN hydrogel has 3D networks with suitable pore size,good swelling properties,sufficient mechanical strength,great biodegradability,and excellent biocompatibility.In order to evaluate the immunoregulatory effects of our SPD-crosslinked hydrogels,murine macrophages,RAW 264.7,were used as an in vitro cell model to investigate the secretion and expression of pro-inflammatory cytokines as well as the transformation of macrophage phenotypes.Lipopolysaccharide(LPS),a major component of the outer membrane of bacteria,was used to induce the activation of macrophages,and the levels of pro-inflammatory cytokines secreted in the media of RAW 264.7 culture with SPD or SPD-DN hydrogels were measured by enzyme-linked immunosorbent assay(ELISA).It is important to mention that diethylenetriamine(DETA),which has a chemical structure close to SPD,is used as a control for free SPD,and DETA crosslinker,resembling SPD crosslinker,has also been prepared to crosslink control hydrogels for comparison.The results of enzyme linked immunosorbent assay(ELISA)show that SPD and SPD-DN hydrogels reduced the secretion of inflammatory mediators by macrophages induced by LPS in a dose-dependent manner.Especially when the free SPD concentration is 5 ?M,the productions of interleukin 6(IL-6),tumor necrosis factor(TNF-?)and nitric oxide(NO)will be reduced by 95%,47% and 95%respectively.In contrast,the secretions of IL-6,TNF-? and NO in presence of free DETA show fluctuating trends around the levels induced by LPS without significant decreases.As for the macrophages cultured on the hydrogels,the secretions of IL-6,TNF-? and NO in presence of SPD-DN hydrogels demonstrate decreases of 91%,68%and 52% respectively compared with those of SN hydrogels.In comparison,DETADN hydrogels cannot achieve the same effect.Macrophages are mainly classified into two groups: pro-inflammatory M1 phenotype and anti-inflammatory M2 phenotype.SPD and SPD-DN hydrogels can down-regulate the m RNA levels of marker genes of M1 macrophage and up-regulate the m RNA levels of marker genes of M2 macrophage.In addition,immunofluorescent staining exhibits that SPD and SPD-DN hydrogels can inhibit the expression of inducible nitric oxide synthase(i NOs),a marker of M1 macrophages,and promote the expression of CD206,a cell surface marker of M2 macrophages.Together,these results indicate that SPD and SPD-DN hydrogels can not only inhibit the polarization of M1 macrophages,but also promote the transformation of M2 macrophages.Next,a series of SPD-DN and DETA-DN hydrogels with different concentrations of crosslinker,as well as SN hydrogels,were embedded in rat skins to observe the overall immune response against a foreign object and to decide an optimal concentration of crosslinker for subsequent experiments.It is believed that SPD-DN hydrogels can alleviate the inflammatory response against a foreign body in a dosedependent manner and SPD-DN hydrogels with 500 ?M crosslinker have been selected for further in vivo characterizations.Then a full-thickness skin defect model in rats was established to evaluate the wound healing rate,immune response,vascularization and collagen deposition caused by SN,SPD-DN and DETA-DN hydrogels.Compared with SN hydrogel,DETA-DN hydrogel and the blank control group,fewer inflammatory cells are found to infiltrate the wound sites treated with SPD-DN hydrogel with lower levels of pro-inflammatory cytokines(IL-6 and TNF-?).Besides,the proportion of M2 macrophages in pan-macrophages is as high as 80%,indicating that SPD-DN hydrogel can effectively reduce inflammation and promote the transformation of M2 macrophages in vivo.Moreover,compared with SN hydrogel,DETA-DN hydrogel and the blank control group,the wounds treated with SPD-DN hydrogel have demonstrated faster wound closure rate based on external observation.Masson staining and Sirius scarlet staining results show that SPD-DN hydrogel treatment can cause more deposition of collagen in the early phase of wound healing and remodel the deposited collagen into fibers of higher strength.The results of immunofluorescent staining demonstrate that the SPD-DN hydrogel-treated group has a high expression of CD31 protein,an angiogenesis marker,in the early stage of wound healing,and a lower expression of CD31 in the later stage of wound healing.Furthermore,the thicknesses of new epidermis and granulation tissue at wound beds treated with SPD-DN hydrogel are closer to those of native skin.More importantly,skin appendix,such as hair follicles and sweat glands,are identified at the wound beds treated with SPD-DN hydrogel.In summary,it has been found that SPD-DN hydrogel can accelerate wound healing by promoting re-epithelialization,collagen deposition and maturation,granulation tissue regeneration,and angiogenesis.