In Situ Formed Hydrogels With Anti-Inflammatory Effect To Promote Full-Thickness Wound Healing

The human skin is the largest,most sensitive and most vulnerable tissue.When it is damaged,the wound becomes infected,heals slowly,and then becomes a chronic wound.It shows a long healing time and a state of inability to repair itself.The most fundamental reason is that it cannot heal in a timely and orderly stage,but stays in the second stage of wound healing:the self-perpetuating inflammatory phase.The persistence of inflammation damages the tissues,contributes to the non-healing of the wound,as well as triggers the development of other diseases.Therefore,the elimination of excessive inflammation is necessary for wound healing.The natural active ingredients of plants have attracted extensive attention due to their abundant sources,significant activity,safety and reliability.Traditional Chinese medicine uses the fruit and root of Xanthium as an anti-inflammatory agent for the treatment of associated inflammatory diseases.The biological function of xanthatin,an active ingredient extracted from Xanthium,needs further research.And the anti-inflammatory mechanism of xanthatin has not yet been clarified.Therefore,it is very necessary to explore the anti-inflammatory effect and mechanism of xanthatin in detail,and lay the foundation for the development of new antiinflammatory drugs.Wound dressings are also essential in the wound healing process.Some of the previous dry dressings such as gauze and bandages cannot provide a moist healing environment,tend to stick to the wound and can cause secondary damage when the dressing is removed.Hydrogels are widely used for tissue repair due to its high water content and similarity to extracellular matrix(ECM).It both absorbs tissue exudate,provides a moist environment to prevent wound dehydration and allows oxygen transport.The advantages of injectable hydrogels are the ability to fill irregularly shaped wounds,provide adhesion at the wound site and improve the interaction between the hydrogel dressing and the wound.Among them,injectable hydrogels have attracted much attention due to their advantages of filling irregularly shaped wounds and having adhesive properties at the wound site and improving the interaction between hydrogel dressings and wounds.The continuous in-depth research on wound healing does not only stop at the choice of wound dressings,but also requires functional active ingredients to make wound healing faster and better.Therefore,the development of suitable and functional wound dressings is urgent.The use of hyaluronic acid and hydroxyethyl starch as hydrogel matrices for wound dressings has not been reported in the literature,and the combination of their excellent properties will certainly provide a good dressing.Therefore,to explore the anti-inflammatory mechanism of xanthatin and combine it with injectable hydrogel,use Xanthium to achieve a sustained release effect from the hydrogel to have an anti-inflammatory effect.The combination of the excellent properties of hydrogels and xanthatin could provide a promising wound healing treatment strategy.In this study,we investigated the anti-inflammatory effect and mechanism of xanthatin.Meanwhile,we also prepared an in situ formed hydrogel using oxidized hydroxyethyl starch(OHES)and hydrazide hyaluronic acid(HA-ADH).In addition,we combined HA-ADH/OHES hydrogel with xanthatin to prepare a bioactive hydrogel that could provide a promising therapeutic strategy for wound healing.The main research contents and results are as follows:In this research,the efficacy and mechanism of xanthatin for the inflammation model established by LPS were studied.Xanthatin exhibits potent anti-inflammatory effects.LPSstimulated RAW 264.7 cells,xanthatin can inhibit the production of NO,the expression of IL-1β,IL-6 and TNF-α,and the transcription of IL-1β,IL-6,TNF-α,i NOS and COX-2.At the same time,the upstream signaling proteins were evaluated.Xanthatin reduced the expression levels of i NOS and COX-2 proteins,and also inhibited the expression levels of IκBα,p65,STAT3,ERK1/2,and SAPK/JNK phosphorylated proteins.In addition,xanthatin can lower ROS content to reduce cell damage and pathway activation.These changes could suggest that xanthatin inhibits the occurrence of inflammation through the interaction of NF-κB,MAPK and STATs pathways.As a whole,xanthatin improves the inflammatory response induced in RAW264.7 macrophages with being LPS-activated,which offers us ideas for understanding and developing Xanthium L.plants as a treatment for inflammation.In situ formed hydrogels were prepared by using a single Schiff base of HA-ADH and OHES.The physicochemical properties of HA-ADH/OHES hydrogels with different ratios were investigated.The HA-ADH/OHES hydrogel has suitable gel time,significant water retention capacity,suitable biodegradability and good biocompatibility.It can be suitable for wound healing applications.The HA-ADH/OHES hydrogel was combined with Xanthium to prepare a hydrogel with anti-inflammatory activity.The sustained release effect of the drug-loaded hydrogel was explored,and the release balance was reached after 48 h.in addition,the drug-loaded hydrogel could promote the proliferation of skin fibroblasts by about 20%,which could also lay a foundation for promoting wound healing.The most suitable ratio(HA-ADH:OHES=1:1)of hydrogel was chosen as the carrier for xanthatin.A creative,inflammation-responsive,injectable and self-healing hydrogel(XT+HAADH/OHES1)was designed and validated for wound healing.In vivo experiments showed that the XT+HA-ADH/OHES1 hydrogel accelerated wound healing mainly through antiinflammatory biological effects and the regulation of various wound-healing-related factors.It could accelerate the order and compaction of tissues,promote the formation of hair follicles and the production of collagen fibers,reduce the expression of pro-inflammatory factors(TNF-α)and up-regulate the expression of vascular-related proteins and factors(CD31 and VEGF).The results suggested that xanthatin and the hydrogel play a synergistic role in promoting wound healing.Injectable xanthatin-loaded hydrogels appear to be promising candidates for wound healing applications.