Efficacy Of Recombinant Human Collagen Hydrogel Loading With Adipose-Derived Stem Cells In Diabetic Wound Healing

Background:Diabetic ulcer,a common complication of diabetes,is characterized by a high incidence rate,tendency for recurrence,and severe cases may lead to amputation,imposing a heavy economic burden on patients,families,and nations.However,the conventional treatment approaches for clinical diabetic ulcers,such as glycemic control,wound debridement,infection control,and vascular reperfusion,still yield unsatisfactory outcomes.Factors like hyperglycemia,hypoxia,sustained inflammation,and dysregulated expression of cytokines and growth factors in diabetic wounds disrupt the consecutive and overlapping phases of hemostasis,inflammation,proliferation,and remodeling,leading to delayed wound healing.Therefore,there is an urgent need to explore effective therapeutic methods to regulate the healing process of diabetic wounds.In recent years,stem cells have shown remarkable potential in promoting the healing of diabetic wounds,owing to their multidirectional differentiation,cell recruitment,immune regulation,angiogenesis,and epithelialization functions.Among them,human adiposederived stem cells(hADSCs)have advantages such as wide availability and ease of acquisition compared to other stem cells,making them a preferred choice for clinical therapy.However,the low survival rate of cells due to inadequate delivery methods and adverse microenvironments during stem cell transplantation into diabetic wounds has been a challenge.Therefore,the use of scaffold materials such as hydrogels to deliver and protect stem cells has emerged as a highly promising research direction.Collagen,a major component of the extracellular matrix,exhibits high biocompatibility and good biodegradability.When prepared as a hydrogel,it can provide a three-dimensional environment conducive to the growth,proliferation,and functionality of stem cells.However,currently available collagen is mainly of animal origin,posing risks of disease transmission.In recent years,the rapid development of recombinant human collagen materials in China holds promise to overcome this dilemma.Recombinant human collagen,produced using genetic engineering technology through yeast and other carriers,shares high homology with human collagen,offering advantages such as high biocompatibility,superior quality control,ease of customization,and functionalization.Therefore,this study aims to prepare a photocurable recombinant human collagen type Ⅲ Methacryloyl(rHCIII-MA)hydrogel material,load and effectively deliver hADSCs,and investigate the role of this stem cellloaded hydrogel system in promoting the repair of diabetic wounds.Objectives:Part Ⅰ:Preparation of rHCⅢ-MA hydrogel loaded with hADSCs(termed as hADSCs@rHCⅢ-MA hydrogel).Part Ⅱ:Investigation of the material characterization and biocompatibility of the hADSCs@rHCⅢ-MA hydrogel.Part Ⅲ:Exploration of the effect of the hADSCs@rHCⅢ-MA hydrogel on the repair of full-thickness defect wounds in diabetic mice and preliminary exploration of its mechanism.Methods:Part Ⅰ:(1)Primary hADSCs were extracted,cultured,and passaged.Flow cytometry was employed to detect cell surface markers,and osteogenic,adipogenic,and chondrogenic induction were used to identify the multidirectional differentiation potential of the cells.(2)Recombinant human type Ⅲ collagen(rHCⅢ)was modified with Methacrylic anhydride(MA)groups through grafting reaction to synthesize rHCⅢ-MA.The characterization of compounds rHCⅢ and rHCⅢ-MA was conducted using proton nuclear magnetic resonance(1H NMR)and Fourier transform infrared spectroscopy(FTIR),and the degree of substitution in the grafting reaction was calculated.Gelation experiments and rheological measurements were performed to preliminarily determine the gelation concentration and required light exposure time for rHCⅢ-MA hydrogel.(3)hADSCs@rHCⅢ-MA hydrogel was constructed,and gelation experiments were conducted using different cell concentrations to further confirm the concentration of rHCⅢMA in the hydrogel.Part Ⅱ:(1)Material characterization of rHCⅢ-MA hydrogel and hADSCs@rHCⅢ-MA hydrogel was compared.Gelation experiments,rheological measurements,compression modulus tests,swelling and water retention experiments,enzymatic degradation experiments,scanning electron microscopy(SEM),and pathological section staining were performed to study the material properties of the hydrogel and observe the effect of stem cell introduction on the hydrogel system.(2)In vitro biocompatibility testing of hADSCs@rHCⅢ-MA hydrogel was conducted.The hydrogel was cultured in vitro,and TUNEL assay was used to detect whether the photocuring process and related reagents induced hADSCs apoptosis.Cell proliferation and metabolism were evaluated using CCK-8 and AlamarBlue assays,cell viability was assessed using live/dead fluorescence staining,and cell adhesion and spreading were observed using phalloidin staining.Part Ⅲ:(1)Small animal live imaging experiments were conducted to compare the survival time of hADSCs in vivo between hADSCs@rHCⅢ-MA hydrogel and pure hADSCs groups,verifying whether the hydrogel plays a protective role in stem cells.(2)A full-thickness skin defect model was established in diabetic mice,and blank control group,pure rHCⅢ-MA hydrogel group,pure hADSCs group,and hADSCs@rHCⅢ-MA hydrogel group were set up.The healing speed was evaluated through gross observation and statistical analysis of wound closure rate.The healing quality was assessed through pathological staining and statistical analysis:neutrophil immunohistochemical staining(MPO)and macrophage immunofluorescence staining(F4/80)were performed on postoperative day 3 tissues to evaluate the inflammatory phase,CD31 and Ki67 immunohistochemical staining were performed on postoperative day 9 tissues to evaluate the proliferation phase,Masson staining was performed on postoperative day 14 tissues,and Sirius red staining was performed on postoperative day 21 tissues to evaluate the effect of hADSCs@rHCⅢ-MA hydrogel on multiple stages of diabetic wound healing.(3)RNA sequencing(RNA-seq)was performed to compare the genomic differences between hADSCs cultured in two-dimensional culture dishes under normal conditions and hADSCs cultured in three-dimensional rHCⅢ-MA hydrogel,followed by validation through qRT-PCR to explore the effects of rHCⅢ-MA hydrogel on hADSCs and the mechanisms underlying the promotion of diabetic wound repair by stem cell-loaded hydrogel.Results:Part Ⅰ:(1)Primary hADSCs were successfully isolated and passaged in culture.Flow cytometry and trilineage induction differentiation results indicated that the extracted cells were hADSCs with multidirectional differentiation potential.(2)rHCⅢ-MA was successfully synthesized.1H NMR and FTIR analyses confirmed the grafting of MA onto rHCⅢ,with a calculated degree of substitution of 22.1%.Gelation experiments preliminarily selected 12.5%rHCⅢ-MA hydrogel for photocrosslinking under 405 nm wavelength light for 60 s.Further confirmation of 12.5%rHCⅢ-MA hydrogel suitability for wound repair was achieved through storage modulus(G’)testing.(3)hADSCs@rHCⅢ-MA hydrogel was successfully constructed.Gelation experiments indicated that 12.5%rHCⅢ-MA hydrogel could load and gel 1×106 cells/mL and 1×107 cells/mL concentrations of hADSCs.Part Ⅱ:(1)Material characterization of rHCⅢ-MA hydrogel and hADSCs@rHCⅢ-MA hydrogel was compared.Rheological testing results showed that both could fully gel within 60 s.Compression testing revealed that the introduction of hADSCs reduced the compressive modulus of the hydrogel.hADSCs@rHCⅢ-MA hydrogel exhibited superior swelling and water retention properties compared to rHCⅢ-MA hydrogel,with faster in vitro enzymatic degradation rate.SEM revealed the three-dimensional network structure of the hydrogel and cell adhesion morphology,which was further confirmed by HE staining of the hydrogel.(2)In vitro,rHCⅢ-MA hydrogel demonstrated good biocompatibility for encapsulating hADSCs:TUNEL assay showed minimal hADSCs apoptosis after 48 h of gelation;CCK-8 and AlamarBlue assays demonstrated significant increases in cell metabolism and proliferation from day 7 to 10;live/dead staining and phalloidin staining indicated that hADSCs could survive and gradually spread in rHCⅢ-MA hydrogel for up to 21 days of in vitro culture.Part Ⅲ:(1)In vivo small animal imaging experiments showed that hADSCs in the hADSCs@rHCⅢ-MA hydrogel group survived longer than those in the pure hADSCs group,demonstrating the hydrogel’s ability to protect stem cells and prolong their survival in vivo.(2)Diabetic mouse full-thickness skin defect healing experiments demonstrated that the hADSCs@rHCⅢ-MA hydrogel group had the fastest healing rate and the best healing quality.HE staining of wound tissues showed gradual degradation of the hydrogel as a temporary scaffold,replaced by newly formed tissue.Pathological staining of wound tissues indicated that rHCⅢ-MA hydrogel and hADSCs in the hADSCs@rHCⅢ-MA hydrogel group synergistically improved wound healing in multiple stages:the inflammatory phase showed more uniform distribution of neutrophils,with the highest and most evenly distributed macrophage count;the proliferation phase exhibited significantly increased neovascularization and cell proliferation,with better granulation tissue formation;Masson’s trichrome and Sirius red staining in the remodeling phase suggested a regular network of collagen deposition similar to normal skin tissue,with significantly increased collagen deposition and optimal matrix remodeling.(3)RNA-seq and qRT-PCR results indicated that the three-dimensional culture system of rHCⅢ-MA hydrogel might regulate the function of hADSCs by affecting the expression of genes related to cellular behaviors,inflammatory factors,matrix remodeling,growth factors,and their pathways,enhancing their ability to promote wound repair.Conclusions:In this study,we developed a convenient and biologically safe photocurable rHCⅢ-MA hydrogel for rapid and uniform encapsulation as well as efficient delivery of hADSCs.The hADSCs@rHCⅢ-MA hydrogel,when formed in situ on diabetic wounds,promoted diabetic mouse wound repair and tissue regeneration through immunomodulation,angiogenesis,cell proliferation,and enhanced matrix remodeling.In this hydrogel system,the rHCⅢ-MA hydrogel provided a suitable three-dimensional environment for hADSCs growth and functionality,while protecting stem cells and prolonging their survival time in the harsh microenvironment of diabetic wounds.The hADSCs acted as recruiters,attracting immune cells and wound repair-related cells into the hydrogel,gradually replacing the temporary scaffold and generating new tissue and matrix,thus offering a novel approach for achieving true in situ regeneration of wounds.