Amniotic epithelial cells accelerate diabetic wound healing by protecting keratinocytes and fibroblasts from high-glucose-induced senescence.

Human amniotic epithelial cells (hAECs), one of the stem cells identified from the human placenta, possess numerous advantages and have been considered as an attractive and available cell source for regenerative medicine. Accumulating evidence has showed that cellular senescence was one of the pathogenic hubs of diabetic wound chronicity. Keratinocytes and fibroblasts are the primary cells involved in wound healing. Therefore, in this study, we aimed to investigate the anti-senescence effects of hAECs on keratinocytes and fibroblasts in diabetic wounds. Sustained hyperglycemia impaired cell function and accelerated senescence in vitro. However, this phenotype was rescued by hAECs-conditioned medium (hAECs-CM), with increased migration and proliferation in keratinocytes and fibroblasts and enhanced collagen synthesis and α-smooth muscle actin (α-SMA) production in fibroblasts. In addition, hAECs-CM dramatically inhibited intracellular reactive oxygen species (ROS) and senescence-associated ß-galactosidase (SA-ß-gal) in keratinocytes and fibroblasts under high-glucose (HG) condition. Moreover, hAECs-CM could downregulate the increased RAGE and P21 induced by continuous HG stimulation. Intradermal injection of hAECs in diabetic wounds promoted re-epithelialization and granulation tissue formation, accompanied by decreased P21+ cells and increased PCNA+ cells in epidermis and dermis, as well as promoted collagen deposition and α-SMA expression. Furthermore, CM-Dil-labeled hAECs survived to Day 5 but disappeared by Day 10 in diabetic wounds. These findings indicated that hAECs could inhibit diabetes-induced premature senescence and enhance the function of keratinocytes and fibroblasts via paracrine effects, partly by inhibiting RAGE/P21 signaling pathway. Thus, hAECs targeting cellular senescence induced by a hyperglycemic environment may be a new strategy for the treatment of diabetic wounds.

Ablative fractional carbon dioxide laser improves quality of life in patients with extensive burn scars: A nested case-control study.

BACKGROUND AND OBJECTIVES: Ablative fractional carbon dioxide laser (CO2 -AFL) for small-area burn scar management shows encouraging outcomes. Few studies, however, focused on comprehensive outcomes following CO2 -AFL treatment for extensive burn scars. This study evaluated whether CO2 -AFL surgery improved the quality of life (QoL) for burn survivors with extensive hypertrophic scars. METHODS: A retrospective nested case-control study was initiated to analyze the efficacy of CO2 -AFL treatment for patients with large-area burn scars. Patients with extensive burn scars (≥30% total body surface area [TBSA]) were registered in our hospital from March 2016 to October 2018. Patients undergoing CO2 -AFL surgery were divided into CO2 -AFL group, and patients undergoing conventional surgery were matched in a 1:1 ratio as the conventional surgery group according to the burned area. The questionnaires were collected and followed up. The 36-Item Short Form Health Survey (SF-36) and Burns Specific Health Scale-Brief (BSHS-B) were the primary parameters. Secondary parameters included the Pittsburgh Sleep Quality Index (PSQI), University of North Carolina "4P" Scars Scale (UNC4P), Patient Scars Assessment Scale for Patient (POSAS-P), and Douleur Neuropathique 4 questions (DN4). RESULTS: 23 patients (55.96 ± 21.59% TBSA) were included in CO2 -AFL group and 23 patients (57.87 ± 18.21% TBSA) in conventional surgery group. Both the BSHS-B total score (CO2 -AFL vs. conventional surgery: 115.35 ± 29.24 vs. 85.43 ± 33.19, p = 0.002) and the SF-36 total score (CO2 -AFL vs. conventional surgery: 427.79 ± 118.27 vs. 265.65 ± 81.66, p < 0.001) for the CO2 -AFL group were higher than those for the conventional surgery group. Parameters for the CO2 -AFL group were lower than those for the conventional surgery group in all of the following comparisons: PSQI total score (CO2 -AFL vs. conventional surgery: 7.70 ± 3.74 vs. 12.26 ± 4.61, p = 0.001), POSAS-P total score (CO2 -AFL vs. conventional surgery: 26.48 ± 6.60 vs. 33.04 ± 4.56, p < 0.001), UNC4P total score (CO2 -AFL vs. conventional surgery: 5.57 ± 1.97 vs. 7.26 ± 1.81, p = 0.004), and DN4 score (CO2 -AFL vs. conventional surgery: 3 [2-5] vs. 5 [4-8], p = 0.004). CONCLUSIONS: Compared to conventional surgery, whole scar CO2 -AFL surgery dramatically improved physical and mental health as well as QoL for people with extensive burn scars. Additionally, CO2 -AFL enhanced the evaluation of scars including their appearance, pain, itching, and a host of other symptoms.

Netrin-1 co-cross-linked hydrogel accelerates diabetic wound healing in situ by modulating macrophage heterogeneity and promoting angiogenesis.

Diabetic wounds, characterized by prolonged inflammation and impaired vascularization, are a serious complication of diabetes. This study aimed to design a gelatin methacrylate (GelMA) hydrogel for the sustained release of netrin-1 and evaluate its potential as a scaffold to promote diabetic wound healing. The results showed that netrin-1 was highly expressed during the inflammation and proliferation phases of normal wounds, whereas it synchronously exhibited aberrantly low expression in diabetic wounds. Neutralization of netrin-1 inhibited normal wound healing, and the topical application of netrin-1 accelerated diabetic wound healing. Mechanistic studies demonstrated that netrin-1 regulated macrophage heterogeneity via the A2bR/STAT/PPARγ signaling pathway and promoted the function of endothelial cells, thus accelerating diabetic wound healing. These data suggest that netrin-1 is a potential therapeutic target for diabetic wounds.

Clarifying sleep characteristics and analyzing risk factors of sleep disorders to promote a predictive, preventive, and personalized medicine in patients with burn scars.

Purpose: This study assessed sleep quality in patients with burn scars and investigated risk factors of sleep disorders to guide clinical therapy. From the strategy of predictive, preventive, and personalized medicine (PPPM/3PM), we proposed that risk assessment based on clinical indicators could prompt primary prediction, targeted prevention, and personalized interventions to improve the management of sleep disorders present in patients with burn scars. Methods: This retrospective study recruited patients with burn scars and healthy volunteers from the Shanghai Burn Treatment Center between 2017 and 2022. Relevant information and data, including demographic characteristics, scar evaluation, and sleep quality, were obtained through the hospital information system, classical scar scale, and self-report questionnaires. Sleep quality was assessed using the Pittsburgh Sleep Quality Index (PSQI) and monitored using a cardiopulmonary-coupled electrocardiograph. Pain and pruritus were assessed using the visual analog scale (VAS). Scar appearance was assessed using the modified Vancouver scar scale (mVSS). Results: The sample was comprised of 128 hypertrophic scar (HS) patients, with 61.7% males, a mean age of 41.1 ± 11.6 years, and burn area of 46.2 ± 27.9% total body surface area (TBSA). Patients with PSQI ≥ 7 accounted for 76.6%, and the global PSQI score was 9.4 ± 4.1. Objective sleep data showed that initial enter deep sleep time, light sleep time, awakening time, light sleep efficiency, and sleep apnea index were higher but deep sleep time, sleep efficiency, and deep sleep efficiency were lower in HS patients than that in healthy controls. Preliminary univariate analysis showed that age, hyperplasia time of scar, narrow airway, microstomia, VAS for pain and pruritus, and mVSS total (comprised of pigmentation, vascularity, height and pliability) were associated with the PSQI score (p < 0.1). Multivariable linear regression showed narrow airway, VAS for pain and pruritus, and mVSS specifically height, were the risk factors for PSQI score (p < 0.1). Conclusions: This study model identified that narrow airway, pain, pruritus and scar appearance specifically height may provide excellent predictors for sleep disorders in HS patients. Our results provided a basis for the predictive diagnostics, targeted prevention, and individualized therapy of somnipathy predisposition and progression of HS patients in the setting of PPPM/3PM health care system, which contributed to a paradigm shift from reactive cure to advanced therapy.

Acceleration of burn wound healing by micronized amniotic membrane seeded with umbilical cord-derived mesenchymal stem cells.

Umbilical cord-derived mesenchymal stem cells (UC-MSC) are promising candidates for wound healing. However, the low amplification efficiency of MSC in vitro and their low survival rates after transplantation have limited their medical application. In this study, we fabricated a micronized amniotic membrane (mAM) as a microcarrier to amplify MSC in vitro and used mAM and MSC (mAM-MSC) complexes to repair burn wounds. Results showed that MSC could live and proliferate on mAM in a 3D culture system, exhibiting higher cell activity than in 2D culture. Transcriptome sequencing of MSC showed that the expression of growth factor-related, angiogenesis-related, and wound healing-related genes was significantly upregulated in mAM-MSC compared to traditional 2D-cultured MSC, which was verified via RT-qPCR. Gene ontology (GO) analysis of differentially expressed genes (DEGs) showed significant enrichment of terms related to cell proliferation, angiogenesis, cytokine activity, and wound healing in mAM-MSC. In a burn wound model of C57BL/6J mice, topical application of mAM-MSC significantly accelerated wound healing compared to MSC injection alone and was accompanied by longer survival of MSC and greater neovascularization in the wound.

JAM-A Overexpression in Human Umbilical Cord-Derived Mesenchymal Stem Cells Accelerated the Angiogenesis of Diabetic Wound By Enhancing Both Paracrine Function and Survival of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) is promising in promoting wound healing mainly due to their paracrine function. Nonetheless, the transplanted MSCs presented poor survival with cell dysfunction and paracrine problem in diabetic environment, thus limiting their therapeutic efficacy and clinical application. JAM-A, an adhesion molecule, has been reported to play multi-functional roles in diverse cells. We therefore investigated the potential effect of JAM-A on MSCs under diabetic environment and explored the underlying mechanism. Indeed, high-glucose condition inhibited MSCs viability and JAM-A expression. However, JAM-A abnormality was rescued by lentivirus transfection and JAM-A overexpression promoted MSCs proliferation, migration and adhesion under hyperglycemia. Moreover, JAM-A overexpression attenuated high-glucose-induced ROS production and MSCs apoptosis. The bio-effects of JAM-A on MSCs under hyperglycemia were confirmed by RNA-seq with enrichment analyses. Moreover, Luminex chip results showed JAM-A overexpression dramatically upregulated PDGF-BB and VEGF in the supernatant of MSCs, which was verified by RT-qPCR and western blotting. The supernatant was further found to facilitate HUVECs proliferation, migration and angiogenesis under hyperglycemia. In vivo experiments revealed JAM-A overexpression significantly enhanced MSCs survival, promoted wound angiogenesis, and thus accelerated diabetic wound closure, partially by enhancing PDGF-BB and VEGF expression. This study firstly demonstrated that JAM-A expression of MSCs was inhibited upon high-glucose stimulation. JAM-A overexpression alleviated high-glucose-induced MSCs dysfunction, enhanced their anti-oxidative capability, protected MSCs from hyperglycemia-induced apoptosis and improved their survival, thus strengthening MSCs paracrine function to promote angiogenesis and significantly accelerating diabetic wound healing, which offers a promising strategy to maximize MSCs-based therapy in diabetic wound.

Analysis of the predictors of hypertrophic scarring pain and neuropathic pain after burn.

INTRODUCTION: Burn hypertrophic scarring pain is a common and perennial complaint which not only affects patients' quality of life, but also their recovery and reintegration. Physical therapy and medicine regimens are all available for the treatment of hypertrophic scarring pain. Unfortunately, the efficacy of clinical practice is not very satisfactory and the management of hypertrophic scarring pain remains challenging. Therefore, it is of utmost importance to explore the risk factors for hypertrophic scarring pain and further identify whether it is neuropathic pain, aiming to guide the clinical therapy and help patients live a pain-free life. METHODS: This retrospective study enrolled patients with postburn hypertrophic scarring pain between 2017 and 2020 in a burn center in Shanghai. Research objects were included strictly according to the inclusion criteria and every enrolled patient was included in the study only once. Demographic information, burn and scar characteristics, and pain scores were collected through the Changhai Hospital Medical Information System, patient questionnaire and physician assessment. Using SPSS 26.0 software, the data were first processed by descriptive statistics, and linear and logistic regression analyses were further employed to explore the significant factors. RESULTS: The sample involving 123 patients was consisted of 56.9% males, 79.7% caused by fire with a median age 40.5 years, total body surface burn-area (TBSA) 44.4%, wound healing time of target scar 57.9 days, hyperplasia time 9.3 months and the scar location mainly in the limbs (55.3%). Of all the included objects, the modified Vancouver Scar Scale (mVSS) total, visual analogue scale (VAS) score, brief pain inventory (BPI) total and the percentage of neuropathic pain were 9.6, 3.3, 36.0 and 74.8%, respectively. Integrating covariates with a P value of <0.10 through preliminary univariate analysis, multivariable linear regression showed sex (P = 0.049), age (P = 0.020), target scar location (P = 0.017, P = 0.254), and pliability (P = 0.016) were linked with severe VAS score; and burn depth of target scar (P = 0.023), hyperplasia time (P = 0.027, P = 0.001), vascularity (P = 0.028), and pliability (P = 0.001) were associated with higher BPI score. Adjusting for potential confounders, hyperplasia time (P = 0.005, P = 0.039) was found to be the only independent risk factor for hypertrophic scarring neuropathic pain in the multivariate logistic regression analysis, with mVSS total of P = 0.062. CONCLUSIONS: The model in our study has clarified that sex, age, target scar location, burn depth of target scar, hyperplasia time, and vascularity, especially pliability, may provide excellent prediction of hypertrophic scarring pain outcome; for neuropathic pain, only hyperplasia time has further prospects, with mVSS total as a potential forecast. In an era increasingly aware of life quality, this work may contribute to the elaboration of strategies to hypertrophic scarring pain management, provide an individualized therapy, and help patients live a pain-free life.

In situ-formed adhesive hyaluronic acid hydrogel with prolonged amnion-derived conditioned medium release for diabetic wound repair.

Hydrogels have long been used for encapsulating stem cell-derived conditioned mediums to achieve skin regeneration after wounding. However, inappropriate mechanical strength, low adhesion and low elasticity limit their clinical application. To address these challenges, we engineered a hyaluronic acid-based hydrogel grafted with methacrylic anhydride and N-(2-aminoethyl)-4-[4-(hydroxymethyl)-2-methoxy-5-nitrophenoxy]-butanamide (NB) groups to encapsulate a lyophilized amnion-derived conditioned medium (AM-CM). This hydrogel can photopolymerize in situ within 3 s by photo-initiated free-radical crosslinking between methacrylate moieties. Meanwhile, the formed o-nitrosobenzaldehyde groups by photo-irradiation could covalently bond with the amino groups of tissue surface, which allowed strong tissue adhesion. Furthermore, the hydrogel possessed excellent mechanical properties, high elasticity, favorable biocompatibility and prolonged AM-CM release. Our further vitro and in vivo studies showed that the hydrogel significantly accelerated diabetic wound healing by regulating macrophage polarization and promoting angiogenesis. The engineered hydrogel with AM-CM release has high potential to treat chronic wounds in clinics.

Metabolically Specific In Situ Fluorescent Visualization of Bacterial Infection on Wound Tissues.

The ability to effectively detect bacterial infection in human tissues is important for the timely treatment of the infection. However, traditional techniques fail to visualize bacterial species adhered to host cells in situ in a target-specific manner. Dihydropteroate synthase (DHPS) exclusively exists in bacterial species and metabolically converts p-aminobenzoic acid (PABA) to folic acid (FA). By targeting this bacterium-specific metabolism, we have developed a fluorescent imaging probe, PABA-DCM, based on the conjugation of PABA with a long-wavelength fluorophore, dicyanomethylene 4H-pyran (DCM). We confirmed that the probe can be used in the synthetic pathway of a broad spectrum of Gram-positive and negative bacteria, resulting in a significantly extended retention time in bacterial over mammalian cells. We validated that DHPS catalytically introduces a dihydropteridine group to the amino end of the PABA motif of PABA-DCM, and the resulting adduct leads to an increase in the FA levels of bacteria. We also constructed a hydrogel dressing containing PABA-DCM and graphene oxide (GO), termed PABA-DCM@GO, that achieves target-specific fluorescence visualization of bacterial infection on the wounded tissues of mice. Our research paves the way for the development of fluorescent imaging agents that target species-conserved metabolic pathways of microorganisms for the in situ monitoring of infections in human tissues.

ABT-263 Reduces Hypertrophic Scars by Targeting Apoptosis of Myofibroblasts.

Background: Inhibiting proliferation and inducing apoptosis of myofibroblasts is becoming one of the promising and effective ways to treat hypertrophic scar. ABT-263, as an orally bioavailable BCL-2 family inhibitor, has showed great antitumor characteristics by targeting tumor cell apoptosis. The objective of this study was to explore whether ABT-263 could target apoptosis of overactivated myofibroblasts in hypertrophic scar. Methods: In vivo, we used ABT-263 to treat scars in a rabbit ear scar model. Photographs and ultrasound examination were taken weekly, and scars were harvested on day 42 for further Masson trichrome staining. In vitro, the expression levels of BCL-2 family members, including prosurvival proteins, activators, and effectors, were detected systematically in hypertrophic scar tissues and adjacent normal skin tissues, as well as in human hypertrophic scar fibroblasts (HSFs) and human normal dermal fibroblasts (HFBs). The roles of ABT-263 in apoptosis and proliferation of HSFs and HFBs were determined by annexin V/PI assay, CCK-8 kit, and cell cycle analysis. Mitochondrial membrane potential was evaluated by JC-1 staining and the expression of type I/III collagen and α-SMA was measured by PCR, western blotting, and immunofluorescence staining. Furthermore, immunoprecipitation was performed to explore the potential mechanism. Results: In vivo, ABT-263 could significantly improve the scar appearance and collagen arrangement, decrease scar elevation index (SEI), and induce cell apoptosis. In vitro, the expression levels of BCL-2, BCL-XL, and BIM were significantly higher in scar tissues and HSFs than those in normal skin tissues and HFBs. ABT-263 selectively induced HSFs apoptosis by releasing BIM from binding with prosurvival proteins. Moreover, ABT-263 inhibited HSFs proliferation and reduced the expression of α-SMA and type I/III collagen in a concentration- and time- dependent manner. Conclusion: HSFs showed increased mitochondrial priming with higher level of proapoptotic activator BIM and were primed to death. ABT-263 showed great therapeutic ability in the treatment of hypertrophic scar by targeting HSFs.

Exosomes derived from human amniotic epithelial cells accelerate diabetic wound healing via PI3K-AKT-mTOR-mediated promotion in angiogenesis and fibroblast function.

BACKGROUND: Diabetic wounds are one of the most common and serious complications of diabetes mellitus, characterized by the dysfunction of wound-healing-related cells in quantity and quality. Our previous studies revealed that human amniotic epithelial cells (hAECs) could promote diabetic wound healing by paracrine action. Interestingly, numerous studies demonstrated that exosomes derived from stem cells are the critical paracrine vehicles for stem cell therapy. However, whether exosomes derived from hAECs (hAECs-Exos) mediate the effects of hAECs on diabetic wound healing remains unclear. This study aimed to investigate the biological effects of hAECs-Exos on diabetic wound healing and preliminarily elucidate the underlying mechanism. METHODS: hAECs-Exos were isolated by ultracentrifugation and identified by transmission electron microscopy, dynamic light scattering and flow cytometry. A series of in vitro functional analyses were performed to assess the regulatory effects of hAECs-Exos on human fibroblasts (HFBs) and human umbilical vein endothelial cells (HUVECs) in a high-glycemic microenvironment. High-throughput sequencing and bioinformatics analyses were conducted to speculate the related mechanisms of actions of hAECs-Exos on HFBs and HUVECs. Subsequently, the role of the candidate signaling pathway of hAECs-Exos in regulating the function of HUVECs and HFBs, as well as in diabetic wound healing, was assessed. RESULTS: hAECs-Exos presented a cup- or sphere-shaped morphology with a mean diameter of 105.89 ± 10.36 nm, were positive for CD63 and TSG101 and could be internalized by HFBs and HUVECs. After that, hAECs-Exos not only significantly promoted the proliferation and migration of HFBs, but also facilitated the angiogenic activity of HUVECs in vitro. High-throughput sequencing revealed enriched miRNAs of hAECs-Exos involved in wound healing. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology analyses have shown that the target genes of the top 15 miRNAs were highly enriched in the PI3K-AKT pathway. Further functional studies demonstrated that the PI3K-AKT-mTOR pathway was necessary for the induced biological effects of hAECs-Exos on HFBs and HUVECs, as well as on wound healing, in diabetic mice. CONCLUSIONS: Our findings demonstrated that hAECs-Exos represent a promising, novel strategy for diabetic wound healing by promoting angiogenesis and fibroblast function via activation of the PI3K-AKT-mTOR pathway.

Cuprous oxide nanoparticles reduces hypertrophic scarring by inducing fibroblast apoptosis.

BACKGROUND: Less apoptosis and excessive growth of fibroblasts contribute to the progression of hypertrophic scar formation. Cuprous oxide nanoparticles (CONPs) could have not only inhibited tumor by inducing apoptosis and inhibiting proliferation of tumor cells, but also promoted wound healing. The objective of this study was to further explore the therapeutic effects of CONPs on hypertrophic scar formation in vivo and in vitro. METHODS: In vivo, a rabbit ear scar model was established on New Zealand albino rabbits. Six full-thickness and circular wounds (10 mm diameter) were made to each ear. Following complete re-epithelization observed on postoperative day 14, an intralesional injection of CONPs or 5% glucose solution was conducted to the wounds. The photo and ultrasonography of each wound were taken every week and scars were harvested on day 35 for further histomorphometric analysis. In vitro, the role of CONPs in human hypertrophic scar fibroblasts (HSFs) apoptosis and proliferation were evaluated by Tunnel assay, Annexin V/PI staining, cell cycle analysis, and EdU proliferation assay. The endocytosis of CONPs by fibroblasts were detected through transmission electron microscopy (TEM) and the mitochondrial membrane potential and ROS production were also detected. RESULTS: In vivo, intralesional injections of CONPs could significantly improve the scar appearance and collagen arrangement, and decreased scar elevation index (SEI). In vitro, CONPs could prominently inhibit proliferation and induce apoptosis in HSFs in a concentration-dependent manner. In addition, CONPs could be endocytosed into mitochondria，damage the mitochondrial membrane potential and increase ROS production. CONCLUSION: CONPs possessed the therapeutic potential in the treatment of hypertrophic scar by inhibiting HSFs proliferation and inducing HSFs apoptosis.