A Highly Concentrated Collagen-chondroitin Sulfate Scaffold Composite With Platelet Rich Plasma Promotes Wound Healing In Porcine Exostosis

Background:Bone exposed wounds are often difficult to heal,requiring almost multiple surgeries,which increases the clinical burden.Currently,various biological materials are widely used in clinical wound repair,and most of these materials are collagen scaffolds.During the treatment of wounds with collagen scaffolds,The exudate locally in the wound would cause collapse of the collagen scaffold material at common concentrations unless the scaffolds were cross-linked by chemical methods or by other methods such as de-hydrothermal treatment.Moreover,insufficient vascular regeneration remains a major cause of poor wound healing.Platelet rich plasma contains a variety of growth factors that have been shown to promote vascular regeneration.Current animal choices regarding wound models are mainly murine.However,wound healing in mice is fundamentally different from that in humans.Porcine skin has significant similarities to human skin.At present,the animals used in skin wound models are mainly nude mice.However,the way wounds heal in nude mice is not the same as in humans.There are structural similarities between pig skin and human skin,so using pig as experimental animal to build wound model has advantages that other experimental animals do not have.Most importantly,both porcine and human wounds heal primarily by re epithelialization rather than contraction.Research purpose:To improve the collagen concentration,mechanical properties and water absorption characteristics of current common collagen scaffolds used in skin repair.As well as addressing the difficulty of revascularization during the healing of bone exposed wounds,this experiment utilized a centrifugation based method to obtain a high concentration collagen chondroitin sulfate scaffold by combining this material with platelet rich plasma,using the bone exposed wound of a large mammalian pig as a model,to investigate the role and possible mechanisms of high concentration collagen chondroitin sulfate scaffold composite platelet rich plasma in promoting wound healing and vascular regeneration.To provide data for further clinical applications.Research methods:(1)Synthesize high concentration collagen chondroitin sulfate scaffolds and examine their material characterization.(2)Prepare platelet rich plasma by gradient centrifugation,and analyze the platelet content and the concentrations of transforming growth factor β(TGF-β)、plateletderived growth factor AB(PDGF-AB),vascular endothelial cell growth factor(VEGF)were measured.(3)To construct a porcine exostosis wound model,a high concentration of collagen chondroitin sulfate scaffold combined with platelet rich plasma was applied to porcine exostosis wounds,and its pro wound healing effects and vascular regeneration effects were detected by gross observation,histological staining,immunohistochemical staining,and possible mechanisms.(4)High concentration collagen chondroitin sulfate scaffolds were combined with platelet rich plasma in vitro to explore the possible mechanism of high concentration collagen chondroitin sulfate combined with platelet rich plasma in promoting wound healing by CO culturing with human vascular endothelial cells and mouse fibroblasts,respectively,cell proliferation assay by MTT,gene expression by PCR,and expression of related proteins by Western blot.Experimental results:(1)A highly concentrated collagen chondroitin sulfate scaffold was successfully synthesized,which was significantly enhanced in mechanical properties and water absorption characteristics.(2)The concentration of platelets in the resulting platelet rich plasma,obtained by gradient centrifugation,is more than five times that of whole blood and contains TGF-β、PDGF-AB、VEGF and other growth factors.(3)A high concentration of collagen chondroitin sulfate scaffolds combined with platelet rich plasma significantly promoted the healing of porcine exostosis wounds.And histological staining showed that the amount of epithelial collagen deposition in the platelet-rich plasma group was significantly increased compared with the control group while immunohistochemical staining showed that vascular regeneration was increased in the group with the addition of platelet rich plasma.(4)In vitro cell experimental results demonstrated that platelet rich plasma could promote the proliferation of human vascular endothelial cells and mouse fibroblasts.Initial exploration at the molecular and protein levels showed that platelet rich plasma compounded with a high concentration of collagen chondroitin sulfate scaffold promoted the expression of genes and proteins related to the Notch1 Signaling Pathway in human vascular endothelial cells.Conclusion:High concentration collagen chondroitin sulfate scaffolds showed high collagen concentration,mechanical properties and water absorption characteristics.Its combination with platelet rich plasma significantly accelerates wound healing and promotes revascularization of porcine bone exteriorized.Furthermore,initial exploration at the molecular and protein levels found that platelet rich plasma compounded with a high concentration of collagen chondroitin sulfate scaffold promoted the expression of genes and proteins related to the Notch1 Signaling Pathway in human vascular endothelial cells.