| Purpose:Proliferative scarring,a fibrous proliferative disease caused by abnormal wound healing,has been a great challenge in the treatment of skin defects.Based on nano-loading technology and 3D printing technology,this study developed a dermal extracellular matrix（DECM）hydrogel scaffold loaded with ginsenoside Rg3（GS/Rg3）for repairing the wound defect on rats and inhibiting the scar formation,which would provide some experimental basis for scar-free healing of skin defects.Methods:（1）Preparation and characterization of dermal extracellular matrix hydrogel（DECM）: porcine full-thickness skin was decellularized by mechanical stratification,freeze-thaw cycle and chemical-enzyme solution agitation;then the decellularized powder was dissolved in pepsin solution at 4°C to obtain DECM pre-gel solution,and the temperature was increased to crosslink the solution to form hydrogel;subsequently the DNA kit was used to detect the decellularization efficiency,and H＆E staining,Masson’s trichrome staining and DAPI staining were used to analyze the composition of DECM hydrogel.（2）Preparation and characterization of mesoporous silica（MSNs）and MSNs/Rg3 complexes: MSNs were synthesized by sol-gel method and characterized by transmission electron microscopy;MSNs/Rg3 solid dispersions were prepared by centrifugation and the encapsulation and loading rates of drugs were detected by high performance liquid chromatography（HPLC）.（3）Characterization and 3D printed DECM-MSNs/Rg3 bioinks: the cytotoxicity of hydrogels was detected by MTT method;the L929 cell proliferation was detected by the EDU-488 kit;the swelling and degradation properties of hydrogels were detected by weighing method;the rheological properties of bioinks were detected by stress-controlled rheometer;finally,the desired scaffold models were constructed by3 DMAXs software and printed by 3D extrusion printer.（4）Evaluation of the efficacy of each group of hydrogel scaffolds（Control,DECM,DECM-2MSNs,DECM-2MSNs/Rg3）on the full skin defects in rats: full skin defects models were constructed on the back of rats,and hydrogel scaffolds of the above groups were implanted into the defect area to observe the wound healing and scar formation at days 4,7 and 14,and the specific healing mechanism was analyzed by histological and immunohistochemical methods.Results:（1）DECM hydrogel is temperature-sensitive and can be crosslinked to form hydrogel when left at 37℃ for 40 min;DNA quantification results showed that98.567% of ds DNA was removed from the tissue after decellularization,which greatly reduced or even eliminated the immunogenicity of DECM;histological and immunohistochemical staining results also showed that there were few cellular remnants in DECM and collagen was still retained in DECM after decellularization,which made it similar in composition to the natural ECM and could provide a microenvironment conducive to cell growth and migration,thus facilitating wound healing.（2）The synthesized MSNs were in the form of white powder,and TEM showed that they formed uniform pore channels from the center to the periphery with a particle diameter of about 278 nm and an average pore size of 2.47 nm,which could be used for loading drugs;the detection by HPLC showed that the encapsulation rate of Rg3 was about 49.03%,and the drug loading rate was 14.05%,and the cumulative release time in vitro was up to 60 h.（3）MTT assay showed that all groups of hydrogels exhibited good cytocompatibility and EDU-488 assay showed that the introduction of Rg3 could inhibit the proliferation of fibroblasts;the swelling and degradation performance analyses showed that all groups of hydrogels had good water absorption and natural degradation ability;the rheological performance analysis proved that DECM hydrogel bioink had good printability,and the addition of MSNs increased the viscosity of DECM,which was conducive to improving the stability of the printed scaffold;the scaffold with good support and stability could be printed under the condition of 0.4bar,18 mm/s,37℃,and could be folded into different shapes without obvious deformation or fracture after crosslinking,which was similar to the properties of natural skin.（4）The results of in vivo experiments in rats showed that the wound surface of DECM-2MSN/Rg3 group on day 14 was smooth and the healing rate reached 99.04%.In contrast,73.71%,95.70% and 94.12% in the blank control group,DECM group and DECM-2MSNs group,respectively,were significantly lower than the experimental group and had different degrees of crusting on the wound surface.The results of histological and immunohistochemical analyses showed that the vascularization level and collagen content in DECM-2MSNs/Rg3 group were less than those in the other three control groups,indicating that Rg3 was able to reduce the inflammatory response and inhibit collagen proliferation,thereby reducing scar formation during the wound healing process.Conclusion:The experimental results showed that the 3D printed DECM scaffolds have good support,stability and uniform pore structure,which facilitate the exchange of nutrients and metabolic wastes,and MSNs encapsulated Rg3 can prolong its action time.The synergistic effect of the two can accelerate wound healing and inhibit scar formation,which has potential application value for scar-free wound healing. |
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