Collagen/hyaluronan based hydrogels releasing sulfated hyaluronan improve dermal wound healing in diabetic mice via reducing inflammatory macrophage activity

Sustained inflammation associated with dysregulated macrophage activation prevents tissue formation and healing of chronic wounds.Control of inflammation and immune cell functions thus represents a promising approach in the development of advanced therapeutic strategies.Here we describe immunomodulatory hyaluronan/collagen(HA-AC/coll)-based hydrogels containing high-sulfated hyaluronan(sHA)as immunoregulatory component for the modulation of inflammatory macrophage activities in disturbed wound healing.Solute sHA downregulates inflammatory activities of bone marrow-derived and tissue-resident macrophages in vitro.This further affects macrophage-mediated pro-inflammatory activation of skin cells as shown in skin ex-vivo cultures.In a mouse model of acute skin inflammation,intradermal injection of sHA downregulates the inflammatory processes in the skin.This is associated with the promotion of an anti-inflammatory gene signature in skin macrophages indicating a shift of their activation profile.For in vivo translation,we designed HA-AC/coll hydrogels allowing delivery of sHA into wounds over a period of at least one week.Their immunoregulatory capacity was analyzed in a translational experimental approach in skin wounds of diabetic db/db mice,an established model for disturbed wound healing.The sHA-releasing hydrogels improved defective tissue repair with reduced inflammation,augmented pro-regenerative macrophage activation,increased vascularization,and accelerated new tissue formation and wound closure.

Covalent linkage of sulfated hyaluronan to the collagen scaffold Mucograft® enhances scaffold stability and reduces proinflammatory macrophage activation in vivo

Sulfated glycosaminoglycans(sGAG)show interaction with biological mediator proteins.Although collagen-based biomaterials are widely used in clinics,their combination with high-sulfated hyaluronan(sHA3)is unexplored.This study aims to functionalize a collagen-based scaffold(Mucograft®)with sHA3 via electrostatic(sHA3/PBS)or covalent binding to collagen fibrils(sHA3+EDC/NHS).Crosslinking without sHA3 was used as a control(EDC/NHS Ctrl).The properties of the sHA3-functionalized materials were characterized.In vitro growth factor and cytokine release after culturing with liquid platelet-rich fibrin was performed by means of ELISA.The cellular reaction to the biomaterials was analyzed in a subcutaneous rat model.The study revealed that covalent linking of sHA3 to collagen allowed only a marginal release of sHA3 over 28 days in contrast to electrostatically bound sHA3.sHA3+EDC/NHS scaffolds showed reduced vascular endothelial growth factor(VEGF),transforming growth factor beta 1(TGF-β1)and enhanced interleukin-8(IL-8)and epithelial growth factor(EGF)release in vitro compared to the other scaffolds.Both sHA3/PBS and EDC/NHS Ctrl scaffolds showed a high proinflammatory reaction(M1:CD-68+/CCR7+)and induced multinucleated giant cell(MNGC)formation in vivo.Only sHA3+EDC/NHS scaffolds reduced the proinflammatory macrophage M1 response and did not induce MNGC formation during the 30 days.SHA3+EDC/NHS scaffolds had a stable structure in vivo and showed sufficient integration into the implantation region after 30 days,whereas EDC/NHS Ctrl scaffolds underwent marked disintegration and lost their initial structure.In summary,functionalized collagen(sHA3+EDC/NHS)modulates the inflammatory response and is a promising biomaterial as a stable scaffold for full-thickness skin regeneration in the future.