Application Of T-DAT-PRP Interpenetrating Network Hydrogel In Wound Repair And Fat Regeneration

Background: The skin is the largest surface organ in the human body,with multiple important physiological functions.During trauma,surgery,battle wounds and military training,skin tissues are highly susceptible to serious injury,and poor healing of injured skin can lead to prolonged trauma and possible formation of scarring contractures that can cause functional impairment.In addition,along with the skin damage,the incidence of soft tissue defects such as subcutaneous fat is very high,which in turn will affect the functional reconstruction of the injured.Severe soft tissue defects,especially craniofacial defects,can cause psychological trauma.Existing treatments have limitations and there is a lack of novel highly bioactive materials that can be used to promote both wound healing and adipose soft tissue regeneration.The lack of novel materials critical to the salvage of skin and adipose soft tissue defects is not conducive to solving the dilemma faced by clinical treatment and poses a serious threat to the health of patients.At the same time,it is also a pain point problem faced by the troops,which seriously affects the combat effectiveness of our army.Objective: In response to the current lack of novel clinical materials for both wound and soft tissue defect repair,we started from the improvement of the existing clinically used bioactive material,platelet-rich plasma(PRP),and explored the principle of crosslinking to prepare a novel t-PRP hydrogel.The feasibility of using the principle of crosslinking between fibronectin in t-PRP(temperature-controlled platelet-rich plasma,PRP)and collagen enriched in decellularized adipose tissue(DAT)hydrogel to prepare a novel t-DAT-PRP hydrogel was explored,and the mechanical properties,stability and bioactivity of the novel hydrogel were further evaluated.The in vivo and in vitro experiments were conducted to investigate the promotional effect of t-DAT-PRP hydrogel on wound healing and whether it could regenerate adipose tissue as a bioactive scaffold for soft tissue defect repair.Methods and results: We prepared t-DAT-PRP hydrogel by "one-step mutual mixing".By adjusting the different ratios of t-PRP and DAT pre gel,we prepared t-DAT-PRP hydrogel containing 30%,50% and 70% t-PRP(v/v).Furthermore,the biocompatibility and cytotoxicity of t-DAT-PRP hydrogels with different proportions were tested by hemolysis test and live/dead cell staining;The system proportion of t-DAT-PRP hydrogel was obtained by screening the function regulation of vascular endothelial cells.We further evaluate the temperature control characteristics,stability,and microstructure of t-DAT-PRP hydrogel;The release of growth factors from t-DAT-PRP hydrogel was measured by ELISA,and it was verified by immunofluorescence and q RT PCR experiments that t-DAT-PRP hydrogel could regulate macrophages to polarize into M2 type.The above results suggest that t-DAT-PRP hydrogel has good biocompatibility,high safety and no toxic effect on cells.And it has better mechanical properties and stability,can achieve sustained release of growth factors,promote macrophage polarization towards M2 type that promotes repair,and has enhanced regenerative repair function.In order to evaluate the effect of t-DAT-PRP hydrogel on promoting wound healing,we established the fullthickness skin defect models of nude mice and Bama pig,respectively.The above two animal models showed that they had good healing promoting effects.In vivo and in vitro studies showed that compared with t-PRP,the t-DAT-PRP hydrogel prepared by adding DAT could promote the M2 type polarization of macrophages and enhance the ability of promoting angiogenesis.To elucidate the possible mechanisms of action,we detected the active components in DAT through mass spectrometry analysis,and classified,summarized,and enriched the components.The results showed that DAT is rich in various growth factors and immune regulatory proteins,and has good ability to promote angiogenesis and polarization of macrophages towards M2 type.To evaluate the function of t-DAT-PRP hydrogel in promoting fat regeneration,we used the mouse back filling model to collect tissue samples of t-DAT-PRP hydrogel and DAT hydrogel at different time points after transplantation,and then recorded the volume and weight of all grafts for statistical analysis.Then HE,Masson,and immunofluorescence staining were used to evaluate the fat regeneration effect and neovascularisation.The results showed that tDAT-PRP hydrogel volume retention was increased,and both fat regeneration and neovascularisation were significantly enhanced.In order to further enhance the effect of t-DAT-PRP hydrogel on promoting fat regeneration,adipose derived stem cells(ADSCs)were loaded.Taking advantage of the enhanced function of platelets in PRP on ADSCs and the scaffold function of DAT,we chose to use the mouse back filling model again,and the evaluation method was the same as above.The results showed that ADSCs loaded on t-DAT-PRP hydrogel could further enhance the fat regeneration function.Conclusion: In this study,t-DAT-PRP hydrogels were prepared by a ""one-step mutual mixing" method,which effectively solved the limitations of traditional PRP,such as poor mechanical properties and stability,the use of exogenous additives,and the inability to be used for soft tissue regeneration.hrough the study of wound model and fat regeneration model,it was found that t-DAT-PRP hydrogel could better promote angiogenesis through the persistent release of growth factors and the combined use of DAT.And it exhibits immune regulatory function,which can induce macrophages to polarize towards M2 type that promotes repair,promoting skin and soft tissue defect repair.In order to further enhance the soft tissue regeneration function of t-DAT-PRP hydrogel,we use it to load ADSCs,and make full use of the enhanced function of platelets in PRP on ADSCs and the scaffold function of DAT.In conclusion,the t-DAT-PRP hydrogel constructed in this study has the excellent characteristics of simple preparation method,more ingenious design,promising clinical application,better mechanical properties,better stability performance and higher bioactivity.It can be used for regenerative repair of skin and adipose soft tissues,which solves the dilemma of lack of new materials with high bioactivity for both wound and soft tissue defect repair.Looking forward to the approval of DAT products in China,t-DAT-PRP hydrogel can be rapidly applied to clinical practice and equipment battlefield,so that patients and soldiers can benefit.