Dorsal Full-thickness Wounds Research Articles

619 IL-10 producing CD4+ T-cells mitigate dermal fibrosis

Background: CD4+ T-cells are essential in regulating dermal fibrosis and are a main source of IL-10. We have shown that IL-10 reduces dermal fibrosis by upregulating high molecular weight hyaluronan (HMW-HA). However, little of known the role of these cells in scarring. We hypothesize that IL-10 producing CD4+ cells attenuate dermal wound fibrosis via mediating inflammation and altering fibroblast extracellular matrix (ECM) production. Methods: C57BL/6J murine(6-10wk) splenocytes were enriched and sort into CD4+ IL-10 producing cells (Treg&Tr1). In vitro, fibroblasts were co-cultured with Treg or Tr1; fibrotic (Col1a1, aSMA) and ECM remodeling (HA synthases (HAS)1-3) markers were analyzed (qRT-PCR). In vivo, we performed bilateral 6mm dorsal full-thickness wounds on SCID mice (female,8-10wk), and adoptively transferred 106 CD4+, Treg, or Tr1 cells. Wounds were harvested at days 7/28 and analyzed for closure (H&E), fibrosis (trichrome), cell-cell spatial relationships (imaging mass cytometry) and inflammation panel (Luminex). Results: We confirmed the population (flow cytometry) and functionality (IL-10 ELISA) of Treg and Tr1. In the co-culture, Col1a1 in Tr1/Treg treated fibroblasts was reduced by 52.4% or 43.5%; and aSMA was reduced by 25.2% or 44.7%, respectively, compared to untreated fibroblasts. HAS2, a hyaluronan synthase which primarily produces HMW-HA, increased 3.11 or 2.95-fold with Tr1/Treg respectively. In vivo, Tr1 treated mice wound showed expedited wound closure at d7 and significantly greater aSMA (IHC), though both CD4+ and Tr1 treatments resulted in significantly reduced collagen content at d28 (trichrome). Reduced F4/80 were observed in all wounds treated with T cells (IHC). Luminex assay revealed wounds treated with Tr1 had decreased proinflammatory cytokines IL-6, MCP-1, and GM-CSF. Conclusions: IL-10 producing CD4+ cells regulate fibroblast ECM deposition and inflammatory cytokine balance to attenuate fibrosis. Promoting Tr1 recruitment, IL-10 production, and HMW-HA synthesis in wounds may a therapeutic target to improve wound healing.

PC4 serves as a negative regulator of skin wound healing in mice

BackgroundHuman positive cofactor 4 (PC4) was initially characterized as a multifunctional transcriptional cofactor, but its role in skin wound healing is still unclear. The purpose of this study was to explore the role of PC4 in skin wound healing through PC4 knock-in mouse model.MethodsA PC4 knock-in mouse model (PC4+/+) with a dorsal full-thickness wound was used to investigate the biological functions of PC4 in skin wound healing. Quantitative PCR, Western blot analysis and immunohistochemistry were performed to evaluate the expression of PC4; Sirius red staining and immunofluorescence were performed to explore the change of collagen deposition and angiogenesis. Proliferation and apoptosis were detected using Ki67 staining and TUNEL assay. Primary dermal fibroblasts were isolated from mouse skin to perform cell scratch experiments, cck-8 assay and colony formation assay.ResultsThe PC4+/+ mice were fertile and did not display overt abnormalities but showed an obvious delay in cutaneous healing of dorsal skin. Histological staining showed insufficient re-epithelialization, decreased angiogenesis and collagen deposition, increased apoptosis and decreased cell proliferation in PC4+/+ skin. Our data also showed decreased migration rate and proliferation ability in cultured primary fibroblasts from PC4+/+ mice in vitro.ConclusionsThis study suggests that PC4 might serve as a negative regulator of skin wound healing in mice.

Cationized Bombyx mori silk fibroin as a delivery carrier of the VEGF165-Ang-1 coexpression plasmid for dermal tissue regeneration.

The angiogenesis of an implanted construct is among the most important issues in tissue engineering. In this study, spermine was used to modify Bombyx mori silk fibroin (BSF) to synthesize cationized BSF (CBSF). BSF and CBSF were coated in sequence on the surface of polyethyleneimine (PEI)/vascular endothelial growth factor 165/angiopoietin-1 coexpression plasmid DNA (pDNA) complexes to form CBSF/BSF/PEI/pDNA quaternary complexes. BSF scaffolds loaded with carrier/pDNA complexes were prepared as dermal regeneration scaffolds by freeze-drying. In one set of experiments, scaffolds were used to cover a chick embryo chorioallantoic membrane (CAM) to investigate the influence of carrier/pDNA complexes on angiogenesis; in another set of experiments, scaffolds were implanted into dorsal full-thickness wounds in Sprague-Dawley rats to evaluate the effect of carrier/pDNA complex-loaded BSF scaffolds on neovascularization and dermal tissue regeneration. After modification with spermine, the surface zeta potential value of BSF rose to +11 mV from an initial value of -9 mV, and the isoelectric point of BSF increased from 4.20 to 9.04. The in vitro transfection of human umbilical vein endothelial cells (EA.hy926) with quaternary complexes revealed that the CBSF/BSF/PEI/pDNA complexes clearly exhibited lower cytotoxicity and higher transfection efficiency than the PEI/pDNA complexes. The CAM assay showed a more abundant branching pattern of blood vessels in BSF scaffolds loaded with CBSF/BSF/PEI/pDNA complexes than in BSF scaffolds without complexes or loaded with PEI/pDNA complexes. The in vivo experimental results demonstrated that the incorporation of CBSF/BSF/PEI/pDNA complexes could effectively enhance angiogenesis in the implanted BSF scaffolds, thereby promoting the regeneration of dermal tissue, providing a new scaffold for the regeneration of dermal tissue and other tissues containing blood vessels.

Fibrin improves skin wound perfusion in a diabetic rat model

The fibrin matrix of the thrombus that is formed directly after wounding, is an important determinant of the success of the early phase of wound healing. This phase is often impaired in patients with diabetes. A promising approach to improve skin wound healing is the application of a pro-angiogenic fibrin matrix onto the wound. We studied this in 59 female WAG/RijCrl diabetic rats, in which we created two dorsal full-thickness wounds of which one was treated with a human physiological fibrin matrix (2mg/ml) and one with PBS as control. Wound healing parameters were determined at different time points. The wound closure was significantly improved in fibrin-treated wounds on day 3 and 7. Also, fibrin-treated wounds showed a significantly higher perfusion on day 28 and 35 compared to control wounds (p<0.05). CD68 staining revealed that human fibrin did not induce an immune response. In conclusion: the application of a fibrin matrix on a diabetic wound showed improved perfusion and an increased early closure rate of the wound area.

A Novel Skin Splint for Accurately Mapping Dermal Remodeling and Epithelialization During Wound Healing.

The mouse excisional dorsal full-thickness wound model with a silicon splint fixed on the skin has been widely used to mimic human wound healing. However, the method cannot accurately quantify dermal remodeling, since the initial point of epithelialization on the wound surface is unclear. To overcome this limitation, we have developed a novel mouse excisional wound model to assess the degree of epithelial extension and regeneration, using a plastic ring-shaped splint fixed beneath the surrounding epidermal tissue. At the end of the experiment, tissue samples were fixed in formalin, the splint was excised, and paraffin sections were prepared. Splint holes, corresponding to the prior location of the splint, were evident on the tissue cross-sections, and the epidermis above the holes was considered the initial excision site. The epidermal contraction and epithelial regeneration, as independent essential tissue alterations in wound healing, could be distinguishable and quantified. Compared with previous splint models, this method provides an accurate evaluation of epidermal processes in wound healing, and can be a platform to assess the effects of various wound healing factors. J. Cell. Physiol. 232: 1225-1232, 2017. © 2016 Wiley Periodicals, Inc.

Functionalized silk fibroin dressing with topical bioactive insulin release for accelerated chronic wound healing

The healing of chronic wounds remains a key challenge in regenerative medicine. To promote wound healing, a bioactive wound dressing is required. In this study, a functionalized silk fibroin dressing with topical bioactive insulin release was prepared for the treatment of chronic wounds. For this purpose, insulin-encapsulated silk fibroin (SF) microparticles were prepared by coaxial electrospraying of aqueous SF solution under mild processing conditions. Insulin was successfully encapsulated in the inner layer of SF microparticles, providing a sustained insulin release for up to 28days. It was found that the insulin released from the microparticles could maintain original molecular conformation. Moreover, the cell migration assay based on human keratinocyte and endothelial cells confirmed that the insulin released from SF microparticles retained its native bioactivity. Furthermore, the insulin-encapsulated microparticles were loaded into a SF sponge, functioned as a bioactive wound dressing, and the in vivo therapeutic effect of the sponge dressing was evaluated on dorsal full thickness wounds of diabetic Sprague-Dawley rats. The results showed that an insulin-functionalized SF dressing accelerated wound closure, collagen deposition and vascularization, thus, significantly promoting wound healing. The insulin-functionalized SF dressing provides new treatment options for chronic wounds.

PDGF-BB does not accelerate healing in diabetic mice with splinted skin wounds.

Topical application of platelet-derived growth factor-BB (PDGF-BB) is considered to accelerate tissue repair of impaired chronic wounds. However, the vast literature is plagued with conflicting reports of its efficacy in animal models and this is often influenced by a wide array of experimental variables making it difficult to compare the results across the studies. To mitigate the confounding variables that influence the efficacy of topically applied PDGF-BB, we used a controlled full thickness splinted excisional wound model in db/db mice (type 2 diabetic mouse model) for our investigations. A carefully-defined silicone-splinted wound model, with reduced wound contraction, controlled splint and bandage maintenance, allowing for healing primarily by reepithelialization was employed. Two splinted 8 mm dorsal full thickness wounds were made in db/db mice. Wounds were topically treated once daily with either 3 µg PDGF-BB in 30 µl of 5% PEG-PBS vehicle or an equal volume of vehicle for 10 days. Body weights, wound contraction, wound closure, reepithelialization, collagen content, and wound bed inflammation were evaluated clinically and histopathologically. The bioactivity of PDGF-BB was confirmed by in vitro proliferation assay. PDGF-BB, although bioactive in vitro, failed to accelerate wound healing in vivo in the db/db mice using the splinted wound model. Considering that the predominant mechanism of wound healing in humans is by re-epeithelialization, the most appropriate model for evaluating therapeutics is one that uses splints to prevent excessive wound contraction. Here, we report that PDGF-BB does not promote wound closure by re-epithelialization in a murine splinted wound model. Our results highlight that the effects of cytoactive factors reported in vivo ought to be carefully interpreted with critical consideration of the wound model used.

Performance of an in situ formed bioactive hydrogel dressing from a PEG-based hyperbranched multifunctional copolymer

Hydrogel dressings have been widely used for wound management due to their ability to maintain a hydrated wound environment, restore the skin’s physical barrier and facilitate regular dressing replacement. However, the therapeutic functions of standard hydrogel dressings are restricted. In this study, an injectable hybrid hydrogel dressing system was prepared from a polyethylene glycol (PEG)-based thermoresponsive hyperbranched multiacrylate functional copolymer and thiol-modified hyaluronic acid in combination with adipose-derived stem cells (ADSCs). The cell viability, proliferation and metabolic activity of the encapsulated ADSCs were studied in vitro, and a rat dorsal full-thickness wound model was used to evaluate this bioactive hydrogel dressing in vivo. It was found that long-term cell viability could be achieved for both in vitro (21days) and in vivo (14days) studies. With ADSCs, this hydrogel system prevented wound contraction and enhanced angiogenesis, showing the potential of this system as a bioactive hydrogel dressing for wound healing.

Silk fibroin/chondroitin sulfate/hyaluronic acid ternary scaffolds for dermal tissue reconstruction

The fabrication of new dermal substitutes providing mechanical support and cellular cues is urgently needed in dermal reconstruction. Silk fibroin (SF)/chondroitin sulfate (CS)/hyaluronic acid (HA) ternary scaffolds (95–248μm in pore diameter, 88–93% in porosity) were prepared by freeze-drying. By the incorporation of CS and HA with the SF solution, the chemical potential and quantity of free water around ice crystals could be controlled to form smaller pores in the SF/CS/HA ternary scaffold main pores and improve scaffold equilibrium swelling. This feature offers benefits for cell adhesion, survival and proliferation. In vivo SF, SF/HA and SF/CS/HA (80/5/15) scaffolds as dermal equivalents were implanted onto dorsal full-thickness wounds of Sprague–Dawley rats to evaluate wound healing. Compared to SF and SF/HA scaffolds, the SF/CS/HA (80/5/15) scaffolds promoted dermis regeneration, related to improved angiogenesis and collagen deposition. Further, vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) expression in the SF/CS/HA (80/5/15) groups were investigated by immunohistochemistry to assess the mechanisms involved in the stimulation of secretion of VEGF, PDGF and bFGF and accumulation of these growth factors related to accelerated wound process. These new three-dimensional ternary scaffolds offer potential for dermal tissue regeneration.

Combination of stromal cell-derived factor-1 and collagen-glycosaminoglycan scaffold delays contraction and accelerates reepithelialization of dermal wounds in wild-type mice

While dermal substitutes can mitigate scarring and wound contraction, a significant drawback of current dermal replacement technologies is the apparent delay in vascular ingrowth compared with conventional skin grafts. Herein, we examined the effect of the chemokine stromal cell-derived factor-1 (SDF-1) on the performance of a porous collagen-glycosaminoglycan dermal analog in excisional wounds in mice. C57BL/6 mice with 1 cm × 1 cm dorsal full-thickness wounds were covered with a collagen-glycosaminoglycan scaffold, followed by four daily topical applications of 1 μg SDF-1 or phosphate-buffered saline vehicle. Some animals were also pretreated with five daily doses of 300 mg/kg granulocyte colony-stimulating factor. Animals treated with SDF-1 and no granulocyte colony-stimulating factor reepithelialized 36% faster than vehicle controls (16 vs. 25 days), and exhibited less wound contraction on postwounding day 18 (∼ 35% greater wound area) plus three-fold longer neoepidermis formed than controls. Conversely, granulocyte colony-stimulating factor promoted contraction and no epidermal regeneration. Early (postwounding Day 3) inflammatory cell infiltration in the SDF-1-treated group was 86% less, while the fraction of proliferating cells (positive Ki67 staining) was 32% more, when compared with controls. These results suggest that SDF-1 simultaneously delays contraction and promotes reepithelialization and may improve the wound-healing performance of skin substitutes.

065 Improving Integra™“Take” with Negatively Charged Methacrlylic Acid Beads

Introduction: Integra™ has gained acceptance as a dermal replacement for large burns with limited donor sites. Integra™ vascularization takes between 14 and 21 days. Accelerating the vascularization of Integra™ could improve “take” rates and potentially decrease the obligatory waiting period between application and autografting. Hypothesis: Application of negatively charged methacrylic acid (MAA) beads will enhance vascularization of Integra™ Methods Male Wistar rats (n = 11) had two 2 × 3 cm contra lateral dorsal full thickness wounds excised and grafted using Integra™. The MAA beads were applied under the Integra™ topically on the wound bed. Three experimental treatments were compared: Group 1‐ high‐dose MAA beads, Group 2‐ low‐dose MAA beads, Group 3‐ Integra only. Laser Doppler Imaging (LDI) to assess perfusion, as well as digital photography was done on days 7,10 and 14. Tissue samples for H + E and Factor VIII histological staining were collected on day 14. Results: The average “take” was 99%(p &lt; 0.05) for high‐dose MAA beads, 98%(p &lt; 0.05) for low‐dose MAA beads and 82% in the Integra™ only group at day 7. At day 7 (p &lt; 0.01) and day 10 (p &lt; 0.05), the low‐dose MAA group had significantly greater perfusion than the Integra™ only group. There were no statistically significant differences at day 14. Microvessel density (MVD) counts revealed a &gt;40% increase in the number of vessels in both the low‐dose MAA (p &lt; 0.05) and high‐dose MAA (p &lt; 0.05) groups when compared to the Integra™ only group. There was no difference in LDI perfusion or MVD counts between low and high‐dose MAA groups. Conclusion: Factor VIII staining revealed enhanced angiogenesis in Integra™ treated with low and high‐dose negatively charged MAA beads. Low‐dose negatively charged MAA beads improved and accelerated the vascularization of Integra™ in this rodent model.