| BackgroundThe oral and maxillofacial region is located in a protruding and exposed position of the human body,which is extremely vulnerable to trauma and soft tissue defects.Different degrees of scars may be easily left after the maxillofacial trauma,which has a huge impact on the patient’s quality of life,mental health,and social adaptability.It has aroused the public urgent pursuit for more advanced and efficient medical methods in the field of wound healing.Wound healing is a dynamic and interactive process.Various cells,extracellular matrix components,and soluble media regulate coordinately regulate the various phases of the wound healing process,including coagulation,angiogenesis,inflammation,epithelialization,and remodeling.To promote the healing and reduce scars of a wound,multifunctional composite wound dressings are often used to improve this process.The ideal wound dressing should absorb excess exudates while maintaining the moist environment required for wound healing.It has a loose structure to facilitate gas exchange.Additionally,it is characterized by suitable mechanical properties,sufficient biocompatibility,biodegradability,and even cell-inducing function.ObjectiveRana chensinensis skin peptides(RCSPs)are a mixture of peptides and proteins extracted from the discarded skin of Rana chensinensis.RCSPs have been proven to have good biocompatibility,antioxidant and anti-apoptotic activities,and can regulate fibroblast adhesion and proliferation.In this study,we aim to fabricate a composite multifunctional dressing with the function of both sustained drug release and wound healing promotion,which is comprised of polyvinyl alcohol-gelatin hydrogel embedded calcium alginate core-shell microspheres loaded with RCSPs.The effect of the composite dressing on the compatibility of NIH-3T3 fibroblasts in vitro and the wound healing of rat full-thickness skin defect model wound be explored,to provide a new research direction for wound repair and skin tissue engineering.Methods1.With RCSPs solution as the inner solution and sodium alginate solution as the outer solution,the calcium alginate-RCSPs core-shell microspheres were obtained by using the coaxial electrostatic spraying method.The core-shell microspheres were added to a homogeneous solution of polyvinyl alcohol/gelatin,and the freeze-thaw cycle is repeated three times for cross-linking.Finally,the RCSPs-loaded core-shell microsphere/hydrogel composite dressing was obtained through a freeze-drying method.2.The morphological characteristics of core-shell microspheres and hydrogel were observed by inverted microscope and scanning electron microscope.The encapsulation efficiency of core-shell microspheres for RCSPs was determined by the coomassie brilliant blue staining method G-250.A general material testing machine was used to analyze the mechanical properties of the composite dressing.The water absorption performance and drug release performance of the composite dressing were measured by the weighing method and ultraviolet spectrophotometer,respectively.3.NIH-3T3 fibroblasts were inoculated into the prepared hydrogel/core-shell microsphere composite dressing.The cytocompatibility was tested by CCK-8 kit,and the cell proliferation was observed by Calcein-AM/PI staining.4.A full-thickness wound defect model was prepared on the back of Wistar rats,and the prepared hydrogel/core-shell microsphere composite dressing was transplanted.In vivo experiments were divided into three groups:the hydrogel with core-shell microspheres group(H-CSM),the hydrogel with solid microspheres group(H-SM),and the hydrogel without microspheres group(HO).We observed and recorded the wound healing state of the defects on the 4th,8th,and 12th days.At the same time,the skin tissues of the wound margin were stained with hematoxylin-eosin(H&E)for histological analysis.Results1.Calcium alginate-RCSPs microspheres with a significant core-shell structure were successfully prepared.As the ratio of internal and external axial flow rates increases,the thickness of the shell layer of the microspheres gradually decreases.After the core-shell microspheres were embedded in the polyvinyl alcohol/gelatin hydrogel,the composite dressing was observed under a scanning microscope to show a highly loose three-dimensional network structure and had a high porosity.2.Core-shell microspheres have higher encapsulation efficiency for RCSPs than solid microspheres.With the addition of core-shell microspheres,the water absorption of H-CSM hydrogel increased to 1105%.The composite dressing exhibited good mechanical properties suitable for the skin and could sustainedly release RCSPs within 9 days to ensure the continuous treatment of wounds.3.The results of the CCK-8 experiment showed that the cell survival rate of the H-CSM group,H-SM group,and H0 group was greater than 80%within 7 days,and the H-CSM group showed the highest cell survival rate(92.37%),indicating that the composite dressing has good biocompatibility to NIH-3T3.The results of Calcein-AM/PI staining also proved that NIH-3T3 cells could proliferate normally on the surface of the composite dressing used in this experiment.4.In vivo experiments,we successfully prepared a full-thickness wound defect model on the back of the rat.The wound healing rate of H-CSM group>H-SM group>H0 group>control group(P<0.05)within 4,8,and 12 days after dressing treatment,and the early wound healing rate of H-CSM group>50%.H&E staining analysis showed a uniform re-epithelialization layer in the wound edge tissue of the H-CSM group,the number of new blood vessels and fibroblasts increased greatly,and new hair follicles appeared.Compared with other groups,H-CSM significantly promoted wound tissue regeneration and shortened wound healing time.ConclusionsA hydrogel/core-shell microsphere composite scaffold with a drug sustained-release system was constructed,which was a combination of calcium alginate microspheres with a core-shell structure loaded with RCSPs and PVA/gelatin hydrogel.The composite scaffold could maintain the proliferation of NIH-3T3 fibroblasts,show good biocompatibility,and promote the wound healing and tissue regeneration of full-thickness skin defect in rats. |
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