Wound Healing In Murine Model Research Articles

Histone demethylase JARID1C/KDM5C regulates Th17 cells by increasing IL-6 expression in diabetic plasmacytoid dendritic cells.

Plasmacytoid dendritic cells (pDCs) are first responders to tissue injury, where they prime naive T cells. The role of pDCs in physiologic wound repair has been examined, but little is known about pDCs in diabetic wound tissue and their interactions with naive CD4+ T cells. Diabetic wounds are characterized by increased levels of inflammatory IL-17A cytokine, partly due to increased Th17 CD4+ cells. This increased IL-17A cytokine, in excess, impairs tissue repair. Here, using human tissue and murine wound healing models, we found that diabetic wound pDCs produced excess IL-6 and TGF-β and that these cytokines skewed naive CD4+ T cells toward a Th17 inflammatory phenotype following cutaneous injury. Further, we identified that increased IL-6 cytokine production by diabetic wound pDCs is regulated by a histone demethylase, Jumonji AT-rich interactive domain 1C histone demethylase (JARID1C). Decreased JARID1C increased IL-6 transcription in diabetic pDCs, and this process was regulated upstream by an IFN-I/TYK2/JAK1,3 signaling pathway. When inhibited in nondiabetic wound pDCs, JARID1C skewed naive CD4+ T cells toward a Th17 phenotype and increased IL-17A production. Together, this suggests that diabetic wound pDCs are epigenetically altered to increase IL-6 expression that then affects T cell phenotype. These findings identify a therapeutically manipulable pathway in diabetic wounds.

Performance of collagen-based matrices from Nile tilapia skin: A pilot proteomic study in a murine model of wound healing.

Full-thickness cutaneous trauma, due to the lack of dermis, leads to difficulty in epithelialization by keratinocytes, developing a fibrotic scar, with less elasticity than the original skin, which may have disorders in predisposed individuals, resulting in hypertrophic scar and keloids. Biomedical materials have excellent characteristics, such as good biocompatibility and low immunogenicity, which can temporarily replace traditional materials used as primary dressings. In this work, we developed two dermal matrices based on Nile tilapia collagen, with (M_GAG) and without (M) glycosaminoglycans, using a sugarcane polymer membrane as a matrix support. To assess the molecular mechanisms driving wound healing, we performed qualitative proteomic analysis on the wound bed in an in vivo study involving immunocompetent murine models at 14 and 21 days post-full-thickness skin injury. Gene Ontology and Pathway analysis revealed that both skins were markedly represented by modulation of the immune system, emphasizing controlling the acute inflammation response at 14 and 21 days post-injury. Furthermore, both groups showed significant enrichment of pathways related to RNA and protein metabolism, suggesting an increase in protein synthesis required for tissue repair and proper wound closure. Other pathways, such as keratinization and vitamin D3 metabolism, were also enriched in the groups treated with M matrix. Finally, both matrices improved wound healing in a full post-thick skin lesion. However, our preliminary molecular data reveals that the collagen-mediated healing matrix lacking glycosaminoglycan (M) exhibited a phenotype more favorable to tissue repair, making it more suitable for use before skin grafts.

Rational design and synthesis of lumican stapled peptides for promoting corneal wound healing

PurposeLumican is a major extracellular matrix (ECM) component in the cornea that is upregulated after injury and promotes corneal wound healing. We have recently shown that peptides designed based on the 13 C-terminal amino acids of lumican (LumC13 and LumC13C-A) are able to recapitulate the effects of lumican on promoting corneal wound healing. Herein we used computational chemistry to develop peptide mimetics derived from LumC13C-A with increased stability and half-life that are biologically active and non-toxic, thereby promoting corneal wound healing with increased pharmacological potential. MethodsDifferent peptides staples were rationalized using LumC13C-A sequence by computational chemistry, docked to TGFβRI and the interface binding energies compared. Lowest scoring peptides were synthesized, and the toxicity of peptides tested using CCK8-based cell viability assay. The efficacy of the stapled peptides at promoting corneal wound healing was tested using a proliferation assay, an in vitro scratch assay using human corneal epithelial cells and an in vivo murine corneal debridement wound healing model. ResultsBinding free energies were calculated using MMGBSA algorithm, and peptides LumC13C and LumC13S5 displayed superior binding to ALK5 compared to the non-stapled peptide LumC13C-A. The presence of the hydrocarbon staple in LumC13C enhances the stability of the α-helical conformation, thereby facilitating more optimal interactions with the ALK5 receptor. The stapled peptides do not present cytotoxic effects on human corneal epithelial cells at a 300 nM concentration. Similar to lumican and LumC13C-A, both C13C and LumC13S5 significantly promote corneal wound healing both in vitro and in vivo. ConclusionsHighly stable and non-toxic stapled peptides designed based on LumC13, significantly promote corneal wound healing. As a proof of principle, our data shows that more stable and pharmacologically relevant peptides can be designed based on endogenous peptide sequences for treating various corneal pathologies.

KY19382 Accelerates Cutaneous Wound Healing via Activation of the Wnt/β-Catenin Signaling Pathway.

The Wnt/β-catenin signaling pathway plays important roles in the multi-phases of wound healing: homeostasis, inflammation, proliferative, and remodeling phases. However, there are no clinically available therapeutic agents targeting the Wnt/β-catenin pathway. In this study, we tested the effect of 5, 6-dichloroindirubin-3'-methoxime (KY19382), a small molecule that activates the Wnt/β-catenin pathway via interference with the function of the negative feedback regulator CXXC5, on cutaneous wound healing. KY19382 significantly enhanced cell migration of human keratinocytes and dermal fibroblasts with increased levels of β-catenin, phalloidin, Keratin 14, proliferating cell nuclear antigen (PCNA), Collagen I, and alpha-smooth muscle actin (α-SMA) by activating the Wnt/β-catenin signaling pathway without causing significant cytotoxicity. In addition, levels of Collagen I, Keratin 14, PCNA, and stem cell markers were significantly increased by KY19382 in a cutaneous murine wound healing model. Moreover, KY19382 treatment accelerated re-epithelialization and neo-epidermis formation with collagen deposition and stem cell activation at an early stage of cutaneous wound healing. Overall, KY19382 accelerates wound healing via activating the Wnt/β-catenin pathway, and may have the potential to be used for the development of a new wound healing agent.

Topical Vanadate Improves Excisional Wound Healing in Murine Model

Lintel, Hendrik BS; Abbas, Darren MD; Mackay, Duncan JD MD, MBA; Griffin, Michelle; Spielman, Amanda Faith; Guardino, Nicholas BS; Churukian, Andrew BS; Guo, Jason L; Longaker, Michael T MD, MBA, FACS; Wan, Derrick C MD, FACS Author Information

Modulation of Lymphangiogenesis in Incisional Murine Diabetic Wound Healing Using Negative Pressure Wound Therapy.

Objective: Despite the significant function of lymphatics in wound healing, and frequent clinical use of Negative Pressure Wound Therapy (NPWT), the effect of mechanical force application on lymphangiogenesis remains to be elucidated. We utilize a murine incisional wound healing model to assess the mechanisms of lymphangiogenesis following NPWT. Approach: Dorsal incisional skin wounds were created on diabetic mice (genetically obese leptin receptor-deficient mice [db/db]; n = 30) and covered with an occlusive dressing (Control, n = 15) or NPWT (-125 mmHg, continuous, 24 h for 7 days; NPWT, n = 15). The wounds were macroscopically assessed for 28 days. Tissue was harvested on day 10 for analysis. Qualitative functional analysis of lymphatic drainage was performed on day 28 using Evans Blue staining (n = 2). Results: NPWT increased lymphatic vessel density (40 ± 20 vs. 12 ± 6 podoplanin [PDPN]+ and 25 ± 9 vs. 14 ± 8 lymphatic vessel endothelial receptor 1 [LYVE-1]+) and vessel diameter (28 ± 9 vs. 12 ± 2 μm). Western blotting verified the upregulation of LYVE-1 with NPWT. Leukocyte presence was higher with NPWT (22% ± 3.7% vs. 9.1% ± 4.1% lymphocyte common antigen [CD45]+) and the leukocytes were predominately B cells clustered within vessels (8.8% ± 2.5% vs. 18% ± 3.6% B-lymphocyte antigen CD20 [CD20]+). Macrophage presence was lower in the NPWT group. Lymphatic drainage was increased in the NPWT group, which exhibited greater Evans Blue positivity. Innovation: The lymphangiogenic effects take place independent of macrophage infiltration, appearing to correlate with B cell presence. Conclusion: NPWT promotes lymphangiogenesis in incisional wounds, significantly increasing the lymph vessel density and diameter. This study highlights the potential of NPWT to stimulate lymphatic drainage and wound healing of surgical incisions.

Endogenous Interleukin-10 Contributes to Wound Healing and Regulates Tissue Repair

IntroductionInterleukin-10 (IL-10) is essential in fetal regenerative wound healing and likewise promotes a regenerative phenotype in adult dermal wounds. However, the role of endogenous IL-10 in postnatal dermal wound healing is not well-established. We sought to determine the function of endogenous IL-10 in murine full thickness excisional wounds that are splinted to prevent contracture and mimic human patterns of wound closure. MethodsFull-thickness excisional wounds were made in wildtype (WT) and IL-10−/− mice on a C57BL/6J background (F/M, 8 wk old). In a subset of wounds, contraction was prevented by splinting with silicone stents (stenting) and maintaining a moist wound microenvironment using a semiocclusive dressing. Wounds were examined for re-epithelialization, granulation tissue deposition, and inflammatory cell infiltrate at day 7 and fibrosis and scarring at day 30 postwounding. ResultsWe observed no difference in wound healing rate between WT and IL-10−/− mice in either the stented or unstented group. At day 7, unstented IL-10−/− wounds had a larger granulation tissue area and more inflammatory infiltrate than their WT counterparts. However, we did observe more F4/80+ cell infiltrate in stented IL-10−/− wounds at day 7. At day 30, stented wounds had increased scar area and epithelial thickness compared to unstented wounds. ConclusionsThese data suggest that endogenous IL-10 expression does not alter closure of full thickness excisional wounds when wound hydration and excessive contraction of murine skin are controlled. However, the loss of IL-10 leads to increased inflammatory cell infiltration and scarring. These new findings suggest that IL-10 contributes to the regulation of inflammation without compromising the healing response. These data combined with previous reports of increased rates of healing in IL-10−/− mice wounds not controlled for hydration and contraction suggest an important role for murine wound healing models used in research studies of molecular mechanisms that regulate healing.

Sustained release of alginate hydrogel containing antimicrobial peptide Chol-37(F34-R) in vitro and its effect on wound healing in murine model of Pseudomonas aeruginosa infection.

Antibiotic resistance is a significant public health concern around the globe. Antimicrobial peptides exhibit broad-spectrum and efficient antibacterial activity with an added advantage of low drug resistance. The higher water content and 3D network structure of the hydrogels are beneficial for maintaining antimicrobial peptide activity and help to prevent degradation. The antimicrobial peptide released from hydrogels also hasten the local wound healing by promoting epithelial tissue regeneration and granulation tissue formation. This study aimed at developing sodium alginate based hydrogel loaded with a novel antimicrobial peptide Chol-37(F34-R) and to investigate the characteristics in vitro and in vivo as an alternative antibacterial wound dressing to treat infectious wounds. Hydrogels were developed and optimized by varying the concentrations of crosslinkers and subjected to various characterization tests like cross-sectional morphology, swelling index, percent water contents, water retention ratio, drug release and antibacterial activity in vitro, and Pseudomonas aeruginosa infected wound mice model in vivo. The results indicated that the hydrogel C proved superior in terms of cross-sectional morphology having uniformly sized interconnected pores, a good swelling index, with the capacity to retain a higher quantity of water. Furthermore, the optimized hydrogel has been found to exert a significant antimicrobial activity against bacteria and was also found to prevent bacterial infiltration into the wound site due to forming an impermeable barrier between the wound bed and external environment. The optimized hydrogel was found to significantly hasten skin regeneration in animal models when compared to other treatments in addition to strong inhibitory effect on the release of pro-inflammatory cytokines (interleukin-1β and tumor necrosis factor-α). Our results suggest that sodium alginate -based hydrogels loaded with Chol-37(F34-R) hold the potential to be used as an alternative to conventional antibiotics in treating infectious skin wounds.

Evaluating neem gum-polyvinyl alcohol (NGP-PVA) blend nanofiber mat as a novel platform for wound healing in murine model

Modern-day treatment demands scarless wound healing utilizing scaffolds in the form of nanofiber mats which are tissue and environment-friendly. Neem gum polysaccharide (NGP) in conjugation with Polyvinyl alcohol (PVA) in the form of nanofibers exhibits antimicrobial properties mimicking extracellular matrix for tissue growth. Different grades of nanofibers mats (NFM) were prepared by combining different ratios of NGP and PVA which were later crosslinked using glutaraldehyde vapors (25 % w/v in 0.5 M HCl), and optimized grade G14 exhibited maximum tensile strength with smooth surface morphology, hemocompatible properties, in-vitro biodegradability and antimicrobial action against S. aureus &E. coli. G14 was analytically characterized using different analytical techniques viz. Fourier-transform infrared spectroscopy (FTIR), Thermogravimetric analysis (TGA), which indicated polymer-polymer compatibility. The surface hydrophobicity as detected using Optical contact angle (OCA) confirmed the hydrophobicity of NFM with increased glutaraldehyde vapor for crosslinking when compared to non-crosslinked NFM. Histopathology slides indicated G14 CL-NFM accelerated the wound healing in mice with dense collagen and fibroblasts when compared to control mice suggesting the tissue engineering potential of the prepared device.

Abstract 10357: Pyruvate Dehydrogenase Kinase 4 is a Novel Regulator of Endothelial Cell Mitochondrial Respiration in Diabetes-Impaired Angiogenesis: Implications for Preventing Diabetic Vascular Complications

Introduction: Diabetic patients have a greater risk of experiencing chronic wounds and amputation than non-diabetic patients. Ischaemia-driven angiogenesis is essential for tissue repair but is impaired in diabetes due to aberrant endothelial cell (EC) responses to ischaemia. Pyruvate dehydrogenase kinase 4 (PDK4) normally suppresses mitochondrial respiration in ischaemia by phosphorylating the pyruvate dehydrogenase complex (pPDC) to preserve EC function. However, the role of the PDK4/PDC axis in angiogenesis and the effects of diabetes are unexplored. Methods and Results: In human coronary artery endothelial cells (HCAECs), siRNA-mediated PDK4 knockdown reduced tubulogenesis (49%, P <0.01) and migration (51%, P <0.01), revealing an essential role for PDK4 in angiogenesis. HCAECs were exposed to 5 or 25mM glucose for 72h and hypoxia for 6h. High glucose attenuated the induction of PDK4 and pPDC in response to hypoxia. This was associated with an aberrant increase in mitochondrial respiration (20%, P <0.01), measured by the Seahorse Bioanalyser system, and impaired tubulogenesis (52%, P <0.001) and migration (79%, P <0.0001). Lentiviral overexpression of PDK4 rescued this impairment in high glucose and hypoxia. Similarly, treatment with reconstituted high-density lipoprotein (rHDL), a known proangiogenic agent, restored the induction of PDK4 and pPDC in hypoxia and high glucose, and rescued tubulogenesis and migration. Mechanistically, rHDL increased binding of FOXO1 to the PDK4 promoter (78%, P <0.05), assessed by chromatin immunoprecipitation, compared to PBS controls. In a murine model of diabetic wound healing, diabetes impaired the early induction of PDK4 and pPDC in response to wound ischaemia, and reduced wound neovessels. Topical rHDL enhanced wound PDK4 and pPDC levels over 72h post-wounding (68-165%, P <0.05), and increased wound neovascularisation (62%, P <0.05) and wound closure (45%, P <0.001), compared to PBS controls. Conclusion: PDK4 is essential for hypoxia-driven angiogenesis. This response is impaired in diabetes and can be corrected by PDK4 overexpression and rHDL. We have identified a novel regulatory pathway for diabetes-impaired angiogenesis that can be targeted to prevent diabetic vascular complications.

Regenerative Potential of A Bovine ECM-Derived Hydrogel for Biomedical Applications.

Recent advancements in regenerative medicine have enhanced the development of biomaterials as multi-functional dressings, capable of accelerating wound healing and addressing the challenge of chronic wounds. Hydrogels obtained from decellularized tissues have a complex composition, comparable to the native extracellular environment, showing highly interesting characteristics for wound healing applications. In this study, a bovine pericardium decellularized extracellular matrix (dECM) hydrogel was characterized in terms of macromolecules content, and its immunomodulatory, angiogenic and wound healing potential has been evaluated. The polarization profile of human monocytes-derived macrophages seeded on dECM hydrogel was assessed by RT-qPCR. Angiogenic markers expression has been evaluated by Western blot and antibody array on cell lysates derived from endothelial cells cultured on dECM hydrogel, and a murine in vivo model of hindlimb ischemia was used to evaluate the angiogenic potential. Fibroblast migration was assessed by a transwell migration assay, and an in vivo murine wound healing model treated with dECM hydrogels was also used. The results showed a complex composition, of which the major component is collagen type I. The dECM hydrogel is biocompatible, able to drive M2 phenotype polarization, stimulate the expression of angiogenic markers in vitro, and prevent loss of functionality in hindlimb ischemia model. Furthermore, it drives fibroblast migration and shows ability to facilitate wound closure in vivo, demonstrating its great potential for regenerative applications.

81. The Effect of Masculinizing Hormone Therapy On Wound Healing in Sterilized Mice

Purpose: Wound healing is a topic of great interest in plastic surgery. Prior work has demonstrated sex-dependent differences in wound healing, but mechanisms underpinning these differences are poorly understood. There is general agreement among experts that transgender men on masculinizing hormone therapy heal wounds worse than those who aren’t. The morbidity of this poor wound healing is high and includes problems such as unsightly hypertrophic scars and urethral stenosis. It is known that androgens are associated with an increased local inflammatory response, decreased collagen matrix deposition, and delayed cutaneous wound healing. To further understand this phenomenon, we designed a novel mouse model and studied the effects of trans-sex hormones on wound healing in castrated mice. Methods: CB57BL/6J mice were randomized to 6 groups based on gender, hormone regimen and surgical castration (total n = 75). Ovariectomies, gonadectomies or sham occurred on day 0 and mice were assigned to receive either no-, mono-, or bi-weekly exogenous testosterone for 3 weeks. Bilateral dorsal excisional wounding (6mm punch biopsy) occurred on day 14, and wound were stented open with plastic 6mm rings (secured with suture) to ensure healing via reepithelization as opposed to contraction. Wounds were harvested on day 21 and assessed for planimetry, histology (H&E, trichrome), immunofluorescence, and qPCR (TNFα). Serum testosterone levels were obtained with mass spectrometry. All protocols were approved by Johns Hopkins ACUC. Difference in means was tested with one-way ANOVA with significance set at 0.05. Results: There was a statistically significant difference in mean normalized TNFα concentrations between the six groups (F = 2.50, F critic = 2.35, P = 0.039). Wound sample TNFα concentration was highest in female mice that were on exogenous testosterone. Wound histologic analysis showed poorer wound healing (decreased re-epithelization, larger wound size, increased granulation tissue) in female mice that received exogenous testosterone versus female groups that did not. Conclusion: Preliminary results in our novel murine transgender wound healing model suggest that testosterone increases inflammation and impairs wound healing with significantly more detrimental effects in XX as compared to XY mice, even with identical hormone levels. These effects may be in part mediated by TNFα activity. Ongoing work is focused on characterizing the hormone-mediated effects of TNFα in wound healing in transgender models and developing topical therapeutic interventions.

Evaluation of a Gel Containing a Propionibacterium Extract in an In Vivo Model of Wound Healing

Inappropriate wound healing (WH) management can cause significant comorbidities, especially in patients affected by chronic and metabolic diseases, such as diabetes. WH involves several different, partially overlapping processes, including hemostasis, inflammation, cell proliferation, and remodeling. Oxidative stress in WH contributes to WH impairment because of the overexpression of radical oxygen species (ROS) and nitrogen species (RNS). This study aimed to evaluate the in vitro antioxidative action of a gel containing a Propionibacterium extract (Emorsan® Gel) and assess its skin re-epithelialization properties in a mouse model of WH. The scavenging effects of the bacterial extract were assessed in vitro through the ABTS and DPPH assays and in L-929 murine fibroblasts. The effects of the Emorsan® Gel were studied in vivo in a murine model of WH. After WH induction, mice were treated daily with vehicle or Emorsan® Gel for 6 or 12 days. According to the in vitro tests, the Propionibacterium extract exerted an inhibitory effect on ROS and RNS, consequently leading to the reduction in malondialdehyde (MDA) and nitrite levels. Before proceeding with the in vivo study, the Emorsan® Gel was verified to be unabsorbed. Therefore, the observed effects could be ascribed to a local action. The results obtained in vivo showed that through local reduction of oxidative stress and inflammation (IL-1β, TNF-α), the Emorsan® Gel significantly reduced the infiltration of mast cells into the injured wound, leading to the amelioration of symptoms such as itch and skin irritation. Therefore, the Emorsan® Gel improved the speed and percentage of wound area closure by improving the tissue remodeling process, prompting vascular–endothelial growth factor (VEGF) and transforming growth factor (TGF)- β production and reducing the expression of adhesion molecules. Emorsan® Gel, by its ability to inhibit free radicals, could reduce local inflammation and oxidative stress, thus enhancing the speed of wound healing.

Mesenchymal Stem Cells From Mouse Hair Follicles Reduce Hypertrophic Scarring in a Murine Wound Healing Model

Wound healing of acute full-thickness injuries and chronic non-healing ulcers leads to delayed wound closure, prolonged recovery period and hypertrophic scarring, generating a demand for an autologous cell therapy and a relevant pre-clinical research models for wound healing. In this study, an immunocompetent model for wound healing was employed using a syngeneic murine cell line of mesenchymal stem cells cultured from the mouse whisker hair follicle outer root sheath (named moMSCORS). moMSCORS were isolated using an air-liquid interface method, expanded in vitro and characterized according to the MSC definition criteria - cell viability, in vitro proliferation, MSC phenotype and multi-lineage differentiations. Moreover, upon applying moMSCORS in an in vivo full-thickness wound model in the syngeneic C57BL/6 mice, the treated wounds displayed different morphology to that of the untreated wound beds. Quantitative evaluation of angiogenesis, granulation and wound closure involving clinical scoring and software-based quantification indicated a lower degree of inflammation in the treated wounds. Histological staining of treated wounds by the means of H&E, Alcian Blue, PicroSirius Red and αSMA immune labelling showed lower cellularity, less collagen filaments as well as thinner dermal and epidermal layers compared with the untreated wounds, indicating a general reduction of hypertrophic scars. The decreased inflammation, accelerated wound closure and non-hypertrophic scarring, which were facilitated by moMSCORS, hereby address a common problem of hypertrophic scars and non-physiological tissue properties upon wound closure, and additionally offer an in vivo model for the autologous cell-based wound healing.Graphic

Pathogenic Role of Diabetes-Induced Overexpression of Kallistatin in Corneal Wound Healing Deficiency Through Inhibition of Canonical Wnt Signaling.

It was reported previously that circulation levels of kallistatin, an endogenous Wnt signaling inhibitor, are increased in patients with diabetes. The current study was conducted to determine the role of kallistatin in delayed wound healing in diabetic corneas. Immunostaining and Western blot analysis showed kallistatin levels were upregulated in corneas from humans and rodents with diabetes. In murine corneal wound healing models, the canonical Wnt signaling was activated in nondiabetic corneas and suppressed in diabetic corneas, correlating with delayed wound healing. Transgenic expression of kallistatin suppressed the activation of Wnt signaling in the cornea and delayed wound healing. Local inhibition of Wnt signaling in the cornea by kallistatin, an LRP6-blocking antibody, or the soluble VLDL receptor ectodomain (an endogenous Wnt signaling inhibitor) delayed wound healing. In contrast, ablation of the VLDL receptor resulted in overactivation of Wnt/β-catenin signaling and accelerated corneal wound healing. Activation of Wnt signaling in the cornea accelerated wound healing. Activation of Wnt signaling promoted human corneal epithelial cell migration and proliferation, which was attenuated by kallistatin. Our findings suggested that diabetes-induced overexpression of kallistatin contributes to delayed corneal wound healing by inhibiting the canonical Wnt signaling. Thus, kallistatin and Wnt/β-catenin signaling in the cornea could be potential therapeutic targets for diabetic corneal complications.

Abdominoplasty Skin-Based Dressing for Deep Wound Treatment-Evaluation of Different Methods of Preparation on Therapeutic Potential.

The management of hard-to-heal wounds is a significant clinical challenge. Acellular dermal matrices (ADMs) have been successfully introduced to enhance the healing process. Here, we aimed to develop protocol for the preparation of novel ADMs from abdominoplasty skin. We used three different decellularization protocols for skin processing, namely, 1M NaCl and sodium dodecyl sulfate (SDS, in ADM1); 2M NaCl and sodium dodecyl sulfate (SDS, in ADM1); and a combination of recombinant trypsin and Triton X-100 (in hADM 3). We assessed the effectiveness of decellularization and ADM’s structure by using histochemical and immunochemical staining. In addition, we evaluated the therapeutic potential of novel ADMs in a murine model of wound healing. Furthermore, targeted transcriptomic profiling of genes associated with wound healing was performed. First, we found that all three proposed methods of decellularization effectively removed cellular components from abdominoplasty skin. We showed, however, significant differences in the presence of class I human leukocyte antigen (HLA class I ABC), Talin 1/2, and chondroitin sulfate proteoglycan (NG2). In addition, we found that protocols, when utilized differentially, influenced the preservation of types I, III, IV, and VII collagens. Finally, we showed that abdominoplasty skin-derived ADMs might serve as an effective and safe option for deep wound treatment. More importantly, our novel dressing (ADM1) improves the kinetics of wound closure and scar maturation in the proliferative and remodeling phases of wound healing. In conclusion, we developed a protocol for abdominoplasty skin decellularization suitable for the preparation of biological dressings. We showed that different decellularization methods affect the purity, structure, and therapeutic properties of ADMs.

A Novel Murine Model of Chronic Ischemic Cutaneous Wound Healing

A Novel Murine Model of Chronic Ischemic Cutaneous Wound Healing

Collagen-Containing Fish Sidestream-Derived Protein Hydrolysates Support Skin Repair via Chemokine Induction.

Restoring homeostasis following tissue damage requires a dynamic and tightly orchestrated sequence of molecular and cellular events that ensure repair and healing. It is well established that nutrition directly affects skin homeostasis, while malnutrition causes impaired tissue healing. In this study, we utilized fish sidestream-derived protein hydrolysates including fish collagen as dietary supplements, and investigated their effect on the skin repair process using a murine model of cutaneous wound healing. We explored potential differences in wound closure and histological morphology between diet groups, and analyzed the expression and production of factors that participate in different stages of the repair process. Dietary supplementation with fish sidestream-derived collagen alone (Collagen), or in combination with a protein hydrolysate derived from salmon heads (HSH), resulted in accelerated healing. Chemical analysis of the tested extracts revealed that Collagen had the highest protein content and that HSH contained the great amount of zinc, known to support immune responses. Indeed, tissues from mice fed with collagen-containing supplements exhibited an increase in the expression levels of chemokines, important for the recruitment of immune cells into the damaged wound region. Moreover, expression of a potent angiogenic factor, vascular endothelial growth factor-A (VEGF-A), was elevated followed by enhanced collagen deposition. Our findings suggest that a 5%-supplemented diet with marine collagen-enriched supplements promotes tissue repair in the model of cutaneous wound healing, proposing a novel health-promoting use of fish sidestreams.

Synthesis and evaluation of an amphiphilic deferoxamine:gallium-conjugated cationic random copolymer against a murine wound healing infection model of Pseudomonas aeruginosa

Multidrug resistant (MDR) Gram-negative bacteria are an urgent global health threat. We report on the design and evaluation of a xenosiderophore-conjugated cationic random copolymer (pGQ-DG) which exhibits selective antibacterial activity against Pseudomonas aeruginosa (P. aeruginosa) by targeting select outer membrane (OM) receptors for scavenging xenosiderophores such as deferoxamine (DFO), while possessing favorable cytocompatibility and exhibiting low hemolysis, to enhance and safely damage the bacterial OM. pGQ-DG demonstrated synergistic properties in combination with vancomycin (VAN) when evaluated in vitro against P. aeruginosa. In addition, pGQ-DG plus VAN cleared the P. aeruginosa infection and efficiently accelerated healing in a murine wound healing model as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against MDR bacteria. Statement of significanceP. aeruginosa exhibits intrinsic antibiotic resistance due to limited permeability of its outer membrane (OM). A triple combination antipseudomonal approach was investigated by 1) selectively targeting P. aeruginosa through the complex DFO:gallium, 2) disrupting the OM through a cationic random copolymer, and 3) enhancing bacteria sensitivity to VAN as a result of the OM disruption. Synthesis and characterization of the lead polymer pGQ-DG, mechanism of action, antimicrobial activity, and biocompatibility were investigated in vitro and in vivo. Overall pGQ-DG plus VAN cleared the P. aeruginosa infection and accelerated wound healing in mice as effectively as colistin, suggesting that this strategy could serve as an alternative to colistin against multidrug resistant P. aeruginosa.

Ex vivo conditioning of peripheral blood mononuclear cells of diabetic patients promotes vasculogenic wound healing.

The quality and quantity of endothelial progenitor cells (EPCs) are impaired in patients with diabetes mellitus patients, leading to reduced tissue repair during autologous EPC therapy. This study aimed to address the limitations of the previously described serum‐free Quantity and Quality Control Culture System (QQc) using CD34+ cells by investigating the therapeutic potential of a novel mononuclear cell (MNC)‐QQ. MNCs were isolated from 50 mL of peripheral blood of patients with diabetes mellitus and healthy volunteers (n = 13 each) and subjected to QQc for 7 days in serum‐free expansion media with VEGF, Flt‐3 ligand, TPO, IL‐6, and SCF. The vascular regeneration capability of MNC‐QQ cells pre‐ or post‐QQc was evaluated with an EPC colony‐forming assay, FACS, EPC culture, tube formation assay, and quantitative real time PCR. For in vivo assessment, 1 × 104 pre‐ and post‐MNC‐QQc cells from diabetic donors were injected into a murine wound‐healing model using Balb/c nude mice. The percentage of wound closure and angio‐vasculogenesis was then assessed. This study revealed vasculogenic, anti‐inflammatory, and wound‐healing effects of MNC‐QQ therapy in both in vitro and in vivo models. This system addresses the low efficiency and efficacy of the current naïve MNC therapy for wound‐healing in diabetic patients. As this technique requires a simple blood draw, isolation, and peripheral blood MNC suspension culture for only a week, it can be used as a simple and effective outpatient‐based vascular and regenerative therapy for patients with diabetes mellitus.