Topical α-gal nanoparticles accelerate diabetic wound healing.

An inadequate response from macrophages, key orchestrators of the wound healing process, has been implicated in the pathophysiology of impaired healing in diabetes. This study explored the utility of nanoparticles presenting the α-gal (Galα1-3Galß1-4GlcNAc-R) epitope to induce anti-Gal antibody-mediated local transient recruitment of macrophages to accelerate wound closure and healing in a diabetic murine model. α1,3galactosyltrasferase knockout mice were stimulated to produce anti-Gal antibodies and subsequently diabetes was induced by streptozotocin-induced ß-cell destruction. Six mm full-thickness skin wounds were made and α-gal nanoparticles (AGN) were topically applied on postwounding days 0 and 1. Wounds were analysed histologically for macrophage invasion and markers of wound healing, including epithelialization, vascularization and granulation tissue deposition through postoperative day 12. We found that application of AGN transiently but significantly increased macrophage recruitment into the wounds of diabetic mice. Treated wounds demonstrated more rapid closure and enhanced wound healing as demonstrated by significantly accelerated rates of epithelialization, vascularization and granulation tissue deposition. Thus, topical treatment of full-thickness wounds in diabetic mice with α-gal nanoparticles induced a transient but significant increase in macrophage recruitment resulting in an accelerated rate of wound healing. Using α-gal nanoparticles as a topical wound healing adjunct is a simple, safe and effective means of augmenting dysregulated macrophage recruitment present in the diabetic state.

Antigen-Mediated, Macrophage-Stimulated, Accelerated Wound Healing Using α-Gal Nanoparticles.

BACKGROUND: Macrophages are known to be crucial to timely and efficacious wound healing. They have been shown to modulate inflammation and the migration and proliferation of regenerative cells, promoting tissue deposition and wound closure. This study explored the use of the natural antigen Galα1-3Galß1-4GlcNAc-R (α-gal), present in lower mammals yet absent in Old World primates and humans, to induce a transiently enhanced macrophage response and thereby direct accelerated wound closure and healing in a standard murine model. METHODS: α1,3galactosyltransferase knockout mice were stimulated to produce anti-Gal antibodies at levels comparable with humans. α-Gal-containing micelle nanoparticles were generated and applied to full-thickness splinted wounds on the mice. At 1, 2, 3, 6, and 9 days postoperatively, mice were killed, and wounds were analyzed histologically for macrophage invasion, epithelialization, vascularization, and granulation tissue deposition. Flow cytometry of wound tissue was performed to quantify relative levels of proinflammatory M1 to anti-inflammatory M2 macrophage subtypes. RESULTS: Treatment of splinted full-thickness murine wounds with α-gal-containing nanoparticles led to accelerated wound healing and closure as demonstrated by accelerated rates of keratinization, vascular growth, and wound tissue deposition. Furthermore, treated wounds demonstrated early and enhanced macrophage invasion, as well as a lower M1-M2 ratio. CONCLUSION: Application of α-gal-containing nanoparticles to wounds stimulated a transiently increased inflammatory response, accelerating the rate of wound healing. Use of α-gal may be a simple and effective way to stimulate the wound healing response in both normal and pathologic wound beds.

Facilitated self-assembly of a prevascularized dermal/epidermal collagen scaffold.

Introduction: Resurfacing complex full thickness wounds requires free tissue transfer which creates donor site morbidity. We describe a method to fabricate a skin flap equivalent with a hierarchical microvascular network. Materials & methods: We fabricated a flap of skin-like tissue containing a hierarchical vascular network by sacrificing Pluronic® F127 macrofibers and interwoven microfibers within collagen encapsulating human pericytes and fibroblasts. Channels were seeded with smooth muscle and endothelial cells. Constructs were topically seeded with keratinocytes. Results: After 28 days in culture, multiphoton microscopy revealed a hierarchical interconnected network of macro- and micro-vessels; larger vessels (>100 µm) were lined with a monolayer endothelial neointima and a subendothelial smooth muscle neomedia. Neoangiogenic sprouts formed in the collagen protodermis and pericytes self-assembled around both fabricated vessels and neoangiogenic sprouts. Conclusion: We fabricated a prevascularized scaffold containing a hierarchical 3D network of interconnected macro- and microchannels within a collagen protodermis subjacent to an overlying protoepidermis with the potential for recipient microvascular anastomosis.

Microstructured hydrogel scaffolds containing differential density interfaces promote rapid cellular invasion and vascularization.

INTRODUCTION: Insufficient vascularization of currently available clinical biomaterials has limited their application to optimal wound beds. We designed a hydrogel scaffold with a unique internal microstructure of differential collagen densities to induce cellular invasion and neovascularization. METHODS: Microsphere scaffolds (MSS) were fabricated by encasing 1% (w/v) type 1 collagen microspheres 50-150 µm in diameter in 0.3% collagen bulk. 1% and 0.3% monophase collagen scaffolds and Integra® disks served as controls. Mechanical characterization as well as in vitro and in vivo invasion assays were performed. Cell number and depth of invasion were analyzed using Imaris™. Cell identity was assessed immunohistochemically. RESULTS: In vitro, MSS exhibited significantly greater average depth of cellular invasion than Integra® and monophase collagen controls. MSS also demonstrated significantly higher cell counts than controls. In vivo, MSS revealed significantly more cellular invasion spanning the entire scaffold depth at 14 days than Integra®. CD31+ expressing luminal structures suggestive of neovasculature were seen within MSS at 7 days and were more prevalent after 14 days. Multiphoton microscopy of MSS demonstrated erythrocytes within luminal structures after 14 days. CONCLUSION: By harnessing simple architectural cues to induce cellular migration, MSS holds great potential for clinical translation as the next generation dermal replacement product. STATEMENT OF SIGNIFICANCE: Large skin wounds require tissue engineered dermal substitutes in order to promote healing. Currently available dermal replacement products do not always adequately incorporate into the body, especially in complex wounds, due to poor neovascularization. In this paper, we present a hydrogel with an innovative microarchitecture that is composed of dense type I collagen microspheres suspended in a less-dense collagen bulk. We show that cell invasion into the scaffold is driven solely by mechanical cues inherent within this differential density interface, and that this induces robust vascular cell invasion both in vitro and in a rodent model. Our hydrogel performs favorably compared to the current clinical gold standard, Integra®. We believe this hydrogel scaffold may be the first of the next generation of dermal replacement products.

Transient phase behavior of an elastomeric biomaterial applied to abdominal laparotomy closure.

Secure closure of the fascial layers after entry into the peritoneal cavity is crucial to prevent incisional hernia, yet appropriate purchase of the tissue can be challenging due to the proximity of the underlying protuberant bowel which may become punctured by the surgical needle or strangulated by the suture itself. Devices currently employed to provide visceral protection during abdominal closure, such as the metal malleable retractor and Glassman Visceral Retainer, are unable to provide complete protection as they must be removed prior to complete closure. A puncture resistant, biocompatible, and degradable matrix that can be left in place without need for removal would facilitate rapid and safe abdominal closure. We describe a novel elastomer (CC-DHA) that undergoes a rapid but controlled solid-to-liquid phase transition through the application of a destabilized carbonate cross-linked network. The elastomer is comprised of a polycarbonate cross-linked network of dihydroxyacetone, glycerol ethoxylate, and tri(ethylene glycol). The ketone functionality of the dihydroxyacetone facilitates hydrolytic cleavage of the carbonate linkages resulting in a rapidly degrading barrier that can be left in situ to facilitate abdominal fascial closure. Using a murine laparotomy model we demonstrated rapid dissolution and metabolism of the elastomer without evidence of toxicity or intraabdominal scarring. Furthermore, needle puncture and mechanical properties demonstrated the material to be both compliant and sufficiently puncture resistant. These unique characteristics make the biomaterial extraordinarily useful as a physical barrier to prevent inadvertent bowel injury during fascial closure, with the potential for wider application across a variety of medical and surgical applications. STATEMENT OF SIGNIFICANCE: Fascial closure after abdominal surgery requires delicate maneuvers to prevent incisional hernia while minimizing risk for inadvertent bowel injury. We describe a novel biocompatible and biodegradable polycarbonate elastomer (CC-DHA) comprised of dihydroxyacetone, glycerol ethoxylate, and tri(ethylene glycol), for use as a rapidly degrading protective visceral barrier to aid in abdominal closure. Rapid polymer dissolution and metabolism was demonstrated using a murine laparotomy model without evidence of toxicity or intraabdominal scarring. Furthermore, mechanical studies showed the material to be sufficiently puncture resistant and compliant. Overall, this new biomaterial is extraordinary useful as a physical barrier to prevent inadvertent bowel injury during fascial closure, with the potential for wider application across a variety of medical and surgical applications.