Temporal and differential proteomic profile of molecular mediators associated with chronic and acute wound healing.

The underlying pathophysiology of nonhealing chronic wounds is poorly understood due to the changes occurring at the gene level and the complexity arising in their proteomic profile. Here, we elucidated the temporal and differential profile of the normal and diabetic wound-healing mediators along with their interactions and associated pathways. Skin tissues corresponding to normal and diabetic wounds were isolated at Days 0, 3, 6, and 9 representing different healing phases. Temporal gene expression was analyzed by quantitative real-time PCR. Concurrently, differential protein patterns in the wound tissues were identified by Nano LC-ESI-TOF mass spectrometry and later confirmed by Western blot analysis. Gene ontology annotation, protein-protein interaction, and protein pathway analysis were performed using DAVID, PANTHER, and STRING bioinformatics resources. Uniquely identified proteins (complement C3, amyloid beta precursor protein, and cytoplasmic linker associated protein 2) in the diabetic wound tissue implied that these proteins are involved in the pathogenesis of diabetic wound. They exhibit enhanced catalytic activity, trigger pathways linked with inflammation, and negatively regulate wound healing. However, in the normal wound tissue, axin 1, chondroitin sulfate proteoglycan 4, and sphingosine-1-phosphate receptor were identified, which are involved in proliferation, angiogenesis, and remodeling. Our findings demonstrate the correlation between elevated gene expression of tumor necrosis factor-α, interleukin (IL)-1ß, and identified mediators: aryl hydrocarbon receptor nuclear translocator, 5'-aminolevulinate synthase 2, and CXC-family, that inflicted an inflammatory response by activating downstream MAPK, JAK-STAT, and NF-κB pathways. Similarly, in normal wound tissue, the upregulated IL-4 and hepatocyte growth factor levels in conjunction with the identified proteins, serine/threonine-protein kinase mTOR and peroxisome proliferator-activated receptor gamma, played a significant role in the cellular response to platelet-derived growth factor stimulus, dermal epithelialization, and cell proliferation, processes associated with the repair mechanism. Furthermore, Western blot analysis indicated elevated levels of inflammatory markers and reduced levels of proliferative and angiogenic factors in the diabetic wound.

Single dose human perinatal stem cells accelerate healing of cold-induced rat burn wound.

Temporal phases of wound healing and their corresponding healing factors are essential in wound regeneration. Mesenchymal stem cells (MSCs) accelerate wound healing via their paracrine secretions by enhancing cell migration, angiogenesis, and reducing inflammation. This study evaluated the local therapeutic effect of human umbilical cord MSCs (hUCMSCs) in the healing of cold-induced burn wounds. An in vitro wound (scratch) was developed in rat skin fibroblasts. The culture was maintained in the conditioned medium (CM) which was prepared by inducing an artificial wound in hUCMSCs in a separate experiment. Treated fibroblasts were analyzed for the gene expression profile of healing mediators involved in wound closure. Findings revealed enhanced cell migration and increased levels of healing mediators in the treated fibroblasts relative to the untreated group. Cold-induced burn wounds were developed in Wistar rats, followed by a single injection of hUCMSCs. Wound healing pattern was examined based on the healing phases: hemostasis/inflammation (Days 1, 3), cell proliferation (Day 7), and remodeling (Day 14). Findings exhibited enhanced wound closure in the treated wound. Gene expression, histological, and immunohistochemical analyses further confirmed enhanced wound regeneration after hUCMSC transplantation. Temporal gene expression profile revealed that the level of corresponding cytokines was substantially increased in the treated wound as compared with the control, indicating improvement in the processes of angiogenesis and remodeling, and a substantial reduction in inflammation. Histology revealed significant collagen formation along with regenerated skin layers and appendages, whereas immunohistochemistry exhibited increased neovascularization during remodeling. Leukocyte infiltration was also suppressed in the treated group. Overall findings demonstrate that a single dose of hUCMSCs enhances wound healing in vivo, and their secreted growth factors accelerate cell migration in vitro.

Alpha terpineol preconditioning enhances regenerative potential of mesenchymal stem cells in full thickness acid burn wounds.

Regeneration of full thickness burn wounds is a significant clinical challenge. Direct stem cell transplantation at the wound site has a promising effect on wound regeneration. However, stem cell survival within the harsh wound environment is critically compromised. In this regard, preconditioning of stem cells with cytoprotective compounds can improve the efficiency of transplanted cells. This study evaluated the possible effect of alpha terpineol (αT) preconditioned mesenchymal stem cells (αT-MSCs) in full thickness acid burn wound. An optimized concentration of 10 µM αT was used for MSC preconditioning, followed by scratch assay analysis. A novel rat model of full thickness acid burn wound was developed and characterized via macroscopic and histological examinations. Treatment (normal and αT-MSCs) was given after 48 h of burn wound induction, and the healing pattern was examined till day 40. Skin tissues were harvested at the early (day 10) and late (day 40) wound healing phases and examined by histological grading, neovascularization, and gene expression profiling of healing mediators. In scratch assay, αT-MSCs exhibited enhanced cell migration and wound closure (scratch gap) compared to normal MSCs. In vivo findings revealed enhanced regeneration in the wound treated with αT-MSCs compared to normal MSCs and untreated control. Histology revealed enhanced collagen deposition with regenerated skin layers in normal MSC- and αT-MSC treated groups compared to the untreated control. These findings were correlated with enhanced expression of α-SMA as shown by immunohistochemistry. Additionally, αT-MSC group showed reduced inflammation and oxidative stress, and enhanced regeneration, as witnessed by a decrease in IL-1ß, IL-6, TNF-α, and Bax and an increase in BCL-2, PRDX-4, GPX-7, SOD-1, VEGF, EGF, FGF, MMP-9, PDGF, and TGF-ß gene expression levels at early and late phases, respectively. Overall findings demonstrated that αT exerts its therapeutic effect by mitigating excessive inflammation and oxidative stress while concurrently enhancing neovascularization. Thus, this study offers new perspectives on managing full thickness acid burn wounds in future clinical settings.

α-Terpineol loaded, electron beam crosslinked polyvinyl alcohol/tapioca starch hydrogel sheets; fabrication, characterization and evaluation of wound healing potential on a full thickness acid burn wound.

The multifaceted challenges in treating full-thickness acid burn wounds including impaired tissue regeneration, increased risk of infection, and the pursuit of functional and aesthetically pleasing outcomes, highlights the need for innovative therapeutic approaches for their treatment. The exceptional biochemical and mechanical properties of hydrogels, particularly their extracellular matrix-like nature and their potential to incorporate functional ingredients positions them as promising materials for wound dressings, offering a potential solution to the complexities of full-thickness burn wound management. The current study has integrated functional ingredients (starch and α-terpineol), known for their angiogenic, fibroblast-adhesive, and anti-inflammatory properties into an α-terpineol loaded, electron beam crosslinked polyvinyl alcohol/tapioca pearl starch hydrogel. The hydrogel was then explored for its efficacy in treating full-thickness acid burns. The hydrogel sheets, fabricated using a 25 kGy electron beam, were characterized for structural and functional properties. Surface morphology, gel fraction, swelling ratio, moisture retention capacity and thermal stability were also evaluated. PVA/tapioca starch hydrogel demonstrated optimal macroporosity, mechanical strength, thermal stability, water retention, and moisturizing ability, making it ideal for the intended application. In vitro skin compatibility analysis of α-terpineol-loaded hydrogel confirmed its biocompatibility, demonstrating 90% fibroblast viability. In vivo sensitivity testing on normal rat skin showed no inflammatory response. Analysis of the full-thickness rat chemical burn wounds treated with the hydrogels demonstrated that α-terpineol (AT) loaded e-beam crosslinked PVA/tapioca starch hydrogels increased the rate of wound closure, promoted re-epithelialization, facilitated collagen deposition, stimulated angiogenesis, and promoted keratin deposition, ultimately leading to healing of both thick dermal and epidermal tissues, as well as partial restoration of skin appendages over a duration of 30 days as confirmed by the histological and immunohistochemistry staining. Collectively, this study indicates that α-terpineol (AT) loaded e-beam crosslinked PVA/tapioca starch hydrogel holds promise as a cost-effective and efficient wound dressing for expediting full thickness acid burn wound healing, thus expanding the practical applications of the natural polymer based sheet hydrogel dressings.

Role of quercetin and rutin in enhancing the therapeutic potential of mesenchymal stem cells for cold induced burn wound.

Introduction: Cold burn wounds differ in their pathophysiological spectrum as compared to other types of burn wounds. These wounds have prolonged devastating effects on the body including hypertrophic scars, contracture, and necrosis. Mesenchymal stem cells (MSCs) are considered promising candidates for the complete regeneration of burn wounds. However, transplanted MSCs face the challenge to survive under the harsh tissue conditions. Preconditioning of MSCs with bioactive compounds may enhance their survival and regenerative potential for use in clinical applications. Bioactive compounds of Melia azedarach are well known for their potential role in treating different types of skin wounds due to their anti-inflammatory, anti-viral, anti-cytotoxic, and anti-oxidative properties. This study aims to evaluate the synergistic effects of human umbilical cord derived MSCs (hUC-MSCs) after preconditioning them with bioactive compounds of M. azedarach (quercetin and rutin) for cold induced burn wounds. Method: Human umbilical cord MSCs (hUC-MSCs) were characterized based on their specific cell surface markers and treated with 20 µM of quercetin or rutin. In vitro scratch assay was performed to measure cell migration and wound closure. In vivo cold burn wound model was developed via direct exposure of the dorsal rat skin to liquid nitrogen. hUC-MSCs were subcutaneously transplanted next day of burn wound induction and wound was examined at different time points corresponding to the wound healing phases (days 3, 7, and 14). The regenerative potential of preconditioned hUC-MSCs was assessed in different groups; control (treated only with hUC-MSCs), and treated groups (quercetin or rutin treated hUC-MSCs). Healing potential and wound closure were evaluated by histological, gene expression, and immunohistochemical analyses of the wound tissues before and after treatment. Results: Scratch assay exhibited enhanced cell migration towards wound closure in the treated groups as compared to the control. Macroscopic examination of the wound revealed scab formation at day 14 in control, whereas scab was detached and the wound tissue was remarkably remodeled in the treated groups. Comparison between the treated groups showed that burn wound treated with quercetin significantly increased healing potential than the rutin treated MSCs. Histological findings showed enhanced regeneration of skin layers along with hair follicles in the quercetin group, while increased neovascularization was noted in both treatment groups. Gene profile of wound healing mediators illustrated significant upregulation of IL-5, IL-4, GPX-7, TXNRD-2, PRDX, VEGF, and FGF and downregulation of inflammatory cytokines IL-1ß and IL-6. Conclusion: In conclusion, synergistic effect of hUC-MSCs and bioactive compounds of M. azedarach enhances wound healing by reducing the inflammation, mitigating oxidative stress and enhancing neovascularization. The study findings will aid in designing more effective treatment options for cold burn wounds.

Potential of stem cell seeded three-dimensional scaffold for regeneration of full-thickness skin wounds.

Hypoxic wounds are tough to heal and are associated with chronicity, causing major healthcare burden. Available treatment options offer only limited success for accelerated and scarless healing. Traditional skin substitutes are widely used to improve wound healing, however, they lack proper vascularization. Mesenchymal stem cells (MSCs) offer improved wound healing; however, their poor retention, survival and adherence at the wound site negatively affect their therapeutic potential. The aim of this study is to enhance skin regeneration in a rat model of full-thickness dermal wound by transplanting genetically modified MSCs seeded on a three-dimensional collagen scaffold. Rat bone marrow MSCs were efficiently incorporated in the acellular collagen scaffold. Skin tissues with transplanted subcutaneous scaffolds were histologically analysed, while angiogenesis was assessed both at gene and protein levels. Our findings demonstrated that three-dimensional collagen scaffolds play a potential role in the survival and adherence of stem cells at the wound site, while modification of MSCs with jagged one gene provides a conducive environment for wound regeneration with improved proliferation, reduced inflammation and enhanced vasculogenesis. The results of this study represent an advanced targeted approach having the potential to be translated in clinical settings for targeted personalized therapy.

Umbilical cord-derived mesenchymal stem cells preconditioned with isorhamnetin: potential therapy for burn wounds.

BACKGROUND: Impaired wound healing can be associated with different pathological states. Burn wounds are the most common and detrimental injuries and remain a major health issue worldwide. Mesenchymal stem cells (MSCs) possess the ability to regenerate tissues by secreting factors involved in promoting cell migration, proliferation and differentiation, while suppressing immune reactions. Preconditioning of MSCs with small molecules having cytoprotective properties can enhance the potential of these cells for their use in cell-based therapeutics. AIM: To enhance the therapeutic potential of MSCs by preconditioning them with isorhamnetin for second degree burn wounds in rats. METHODS: Human umbilical cord MSCs (hU-MSCs) were isolated and characterized by surface markers, CD105, vimentin and CD90. For preconditioning, hU-MSCs were treated with isorhamnetin after selection of the optimized concentration (5 µmol/L) by cytotoxicity analysis. The migration potential of these MSCs was analyzed by the in vitro scratch assay. The healing potential of normal, and preconditioned hU-MSCs was compared by transplanting these MSCs in a rat model of a second degree burn wound. Normal, and preconditioned MSCs (IH + MSCs) were transplanted after 72 h of burn injury and observed for 2 wk. Histological and gene expression analyses were performed on day 7 and 14 after cell transplantation to determine complete wound healing. RESULTS: The scratch assay analysis showed a significant reduction in the scratch area in the case of IH + MSCs compared to the normal untreated MSCs at 24 h, while complete closure of the scratch area was observed at 48 h. Histological analysis showed reduced inflammation, completely remodeled epidermis and dermis without scar formation and regeneration of hair follicles in the group that received IH + MSCs. Gene expression analysis was time dependent and more pronounced in the case of IH + MSCs. Interleukin (IL)-1ß, IL-6 and Bcl-2 associated X genes showed significant downregulation, while transforming growth factor ß, vascular endothelial growth factor, Bcl-2 and matrix metallopeptidase 9 showed significant upregulation compared to the burn wound, showing increased angiogenesis and reduced inflammation and apoptosis. CONCLUSION: Preconditioning of hU-MSCs with isorhamnetin decreases wound progression by reducing inflammation, and improving tissue architecture and wound healing. The study outcome is expected to lead to an improved cell-based therapeutic approach for burn wounds.