Screening and analysis of differentially expressed circRNAs and miRNAs in chronic diabetic extremity wounds.

Increasing studies have shown that circular RNAs (circRNAs) and microRNAs (miRNAs) are related to the development of endocrine and metabolic diseases. However, there are few reports on the expression of circRNAs and miRNAs and their related co-expression and the expression of competitive endogenous RNA (ceRNA) in diabetic chronic refractory wounds. In this study, we compared the differential expression of circRNAs and miRNAs in diabetes chronic refractory wounds and normal skin tissues by high-throughput gene sequencing, and screened the differentially expressed circRNAs and miRNAs. Five abnormally expressed circRNAs and seven abnormally expressed miRNAs were detected by reverse transcription quantitative polymerase chain reaction PCR (RT-qPCR)to verify the results of RNA sequencing. We applied gene ontology (GO) to enrich and analyze dysregulated genes and elucidated their main functions via the Kyoto encyclopedia of genes and genomes analysis (KEGG). We constructed coding noncoding gene co-expression networks and ceRNA networks based on significantly abnormally expressed genes. According to the results of coding noncoding gene co-expression network analysis, hsa_circRNA_104175, hsa_circRNA_ 001588, hsa_circRNA_104330, hsa_circRNA_ 100141, hsa_circRNA_103107, and hsa_ circRNA_102044 may be involved in the regulation of the chronic intractable wound healing process in diabetes mellitus. This is particularly true in the regulation of vascular smooth muscle contraction-related pathways and the actin cytoskeleton, which affect the healing of chronic intractable wounds in diabetes. MiR-223-5p, miR-514a-3p, miR-205-5p, and miR-203-3p, which each have a targeting relationship with the above circRNAs, regulate the metabolism of nitrogen compounds in wound tissue by regulating NOD-like receptor signaling pathways, signaling pathways regulating the pluripotency of stem cells, microRNAs in cancer, and ECM-receptor interaction. This study showed circRNAs, miRNAs, and their network are associated with the development of chronic intractable wounds in diabetes, and our research identified the goals for new molecular biomarkers and gene therapy.

Epidemiological and Clinical Characteristics of 5,569 Pediatric Burns in Central China From 2013 to 2019.

Background: Pediatric burns of all the ages are prevalent worldwide, posing a severe health risk to children. This study aims to examine pediatric burns' clinical characteristics and epidemiology in central China. Methods: The pediatric patients of the Burn Research Center, Department of the First People's Hospital of Zhengzhou City from 2013 to 2019 were retrospectively studied and the relevant data were collected from the hospitalized medical records [e.g., demographic, etiology, length of stay (LOS), age, gender, burn area and depth, number of surgeries, cost, and outcome]. Results: A total of 5,569 pediatric burn patients were included, accounting for 43.9% of the total burn population. Electric burns represented a relatively small proportion (1.17%) but were more likely to lead to disabilities or death than scalds (90.63%) and flames (5.12%). The median age was 2 years [interquartile range (IQR): 1-4] and the boys/girls ratio ranged from 1.3:1 to 1.6:1. The most commonly burnt anatomic sites were the limbs (38.3%), with a median %TBSA (total body surface area) of 6 (IQR: 4-10). The complications of shock and pneumonia accounted for 7.6 and 19.2%, respectively. The peak months of pediatric burns included January, May, and August and the rural/urban ratio reached 1.61:1. The percentage of burn wounds treated surgically increased considerably from 2013 to 2019 (3.8 vs. 37.8%). The median hospital LOS was 15 days (IQR: 8-28 days), with the three high-risk factors (e.g., more surgeries, more %TBSA, full-thickness skin burns). The median cost of hospitalization was 1,511 USD (IQR: 848-2,648 USD) and the main risk factors consisted of full-thickness burns, more %TBSA, longer LOS, and more surgical procedures. Among all the patients, LA50 was 78.63% (95% CI = 75.12-83.45) and the overall mortality reached 0.1% since seven deaths were recorded. Conclusion: Scalds, flames, contact, and chemicals are the main causes of burns among children aged 1-5 years in central China. Accordingly, various prevention strategies should be employed depending upon the cause of the burn.

Reconstruction of Giant Defects Due to Electrical and Radiation Burns in the Lower Leg with Free Anterolateral Thigh Flaps.

The purpose of this study was to introduce reconstruction of giant soft tissue defects of the lower leg caused by high-voltage electrical burns and radiation burns using the free anterolateral thigh (ALT) flap. From March 2017 to January 2018, 6 patients who sustained high-voltage electrical burns and 2 patients who sustained ulcerated radiation burns were reconstructed using the free ALT flap. The mean size of the defects was 19 cm × 32 cm (range, 18 cm × 22 cm to 30 cm × 36 cm). The mean size of the flaps was 22 cm × 34 cm (range, 20 cm × 24 cm to 32 cm × 38 cm). All flaps survived completely. The mean preoperative Functional Analysis Technique Evaluation score was 62 (range, 43 to 74). The mean follow-up period was 16 months (range, 12 to 18 months). At the final follow-up, the mean postoperative score was 90 (range, 86 to 94). The mean improvement was 33% (range, 17% to 54%) with 4 excellent and 4 good results. For extensive, high-voltage electrical, and radiation burns encompassing the lower leg, early treating the giant soft tissue defects with a free ALT flap produces good functional outcomes without significant complications.

Direct reprogramming of epidermal cells toward sweat gland-like cells by defined factors.

Several studies have reported inducing adult cells into sweat gland-like cells; however, slow transition and low efficiency limit the potential for cell-based treatment. Here, we show that overexpression of the transcription factor FoxC1 was sufficient to reprogram epidermal cells to induced functional sweat gland-like cells (iSGCs). The iSGCs expressing secreting-related genes, had a global gene expression profile between fetal SGCs (P5) and adult SGCs (P28). Moreover, iSGCs transplanted into the burn mice model facilitated wound repair and sweat gland regeneration. We further demonstrated that the Foxc1 upregulated BMP5 transcription and BMP5 is responsible for the cell-type transition. Collectively, this study shows that lineage reprogramming of epidermal cells into iSGCs provides an excellent cell source and a promising regenerative strategy for anhidrosis and hypohidrosis.

Tuning Alginate-Gelatin Bioink Properties by Varying Solvent and Their Impact on Stem Cell Behavior.

Bioink optimization is considered as one of main challenges in cell-laden 3D bioprinting. Alginate-Gelatin (Alg-Gel) hydrogel have been extensively used as bioink. However, its properties could be influenced by various parameters, and little is known about the evidence featuring the impact of solvent. Here we investigated four Alg-Gel bioink by varying solvent ionic strength (named B-1, B-2, B-3 and B-4). Mechanical properties and printability of bioink samples and their impacts on behaviors of encapsulated epidermal stem cells (ESCs) were tested. Bioink with increased ionic strength of solvent showed decreased stiffness and viscosity, and increased swelling and degradation by printability and mechanical property tests. Due to the increased swelling and degradation was associated with shape-maintenance of post-printing constructs, B-3 and B-4 were hardly observable after 14 days. Cellular behaviors were assessed through viability, proliferation, aggregation and differentiation tests. B-2 with optimal properties resulted in higher viability and proliferation of ESCs, and further facilitated cellular aggregation and lineage differentiation. We demonstrated that the solvent can be tuned by ionic strength to control the properties of Alg-Gel bioink and post-printing constructs, which represented a promising avenue for promotion of therapeutic stem cell behaviors in 3D bioprinting.

A novel model of humanised keloid scarring in mice.

Treatments for keloid scarring are a major challenge to scientists and physicians for their unknown aetiology. Although several models, including monolayer cell culture to tissue-engineered models, were developed, further research on keloid has more or less been hindered by the lack of appropriate animal models. Because these aberrant scars are specific to humans, we obtained human normal and keloid skin tissues and isolated dermal fibroblasts from them. Cell morphology, growth and immunohistochemical staining of myofibroblastmarker α-SMA were examined, and the cell medium of 2-hour culture and 24-hour culture was implanted on the back of nude mice. The cell medium of 2-hour culture and 24-hour culture was also analysed by a protein array for the detection of distinction in inflammatory factors. We showed that keloid fibroblasts had similar morphology and growth compared to normal skin fibroblasts, but the α-SMA expression was obviously up-regulated. After 6 weeks, mice of the 2-hour keloid-derived culture medium group exhibited keloid-like hypertrophic nodules macroscopically, while mice of 24-hour keloid-derived culture medium group were similar to normal skin. Histological findings confirmed that the reconstituted skin tissues had the typical features of human keloids. The protein array data revealed that RANTES were involved in humanised fibrotic occurrence in mice, also suggesting they were important modulators of this inflammatory event. This novel model might help to understand the key events that result in the formation of these abnormal scars and provide new therapeutic options.

Skin appendage-derived stem cells: cell biology and potential for wound repair.

Stem cells residing in the epidermis and skin appendages are imperative for skin homeostasis and regeneration. These stem cells also participate in the repair of the epidermis after injuries, inducing restoration of tissue integrity and function of damaged tissue. Unlike epidermis-derived stem cells, comprehensive knowledge about skin appendage-derived stem cells remains limited. In this review, we summarize the current knowledge of skin appendage-derived stem cells, including their fundamental characteristics, their preferentially expressed biomarkers, and their potential contribution involved in wound repair. Finally, we will also discuss current strategies, future applications, and limitations of these stem cells, attempting to provide some perspectives on optimizing the available therapy in cutaneous repair and regeneration.

Identification of a new sweat gland progenitor population in mice and the role of their niche in tissue development.

Sweat gland cells are responsible for the regulation of body temperature and are critical for wound repair. Furthermore, they have the regenerative potential in response to injury, and show a substantial turnover during both wound healing and homeostasis. However, as a usual research model of sweat gland, mice have not too much glandular cells for experiments. In this study, we identify previously unreported sweat gland progenitor population in mice and characterize them. The progenitor characteristics of sweat gland were confirmed using cellular immunofluorescence assay and quantitative real-time PCR assay. K8 and K18 expression was barely detected in the early stage of skin development (Embryo 17.5d) and increased to a high level at P5d (postnatal 5d), then showed reduction at adult stage (P28d). Further investigation of K8 and K18 positive cells using tissue immunofluorescence revealed the presence of sweat gland progenitors in back epidermis of mice at early stage of sweat gland development and continuous reduction during the developmental process. In vivo transplantation assay with animal models elucidated that sweat gland specific niche in paw pads was critical for the development of sweat gland cells. Although the relationship between new sweat gland progenitors and their niche still needs to be further investigated, the presence of these cells implicates that there is more source ascribed to sweat glands in addition to serving as progenitors in mice.

3D bioprinting matrices with controlled pore structure and release function guide in vitro self-organization of sweat gland.

3D bioprinting matrices are novel platforms for tissue regeneration. Tissue self-organization is a critical process during regeneration that implies the features of organogenesis. However, it is not clear from the current evidences whether 3D printed construct plays a role in guiding tissue self-organization in vitro. Based on our previous study, we bioprinted a 3D matrix as the restrictive niche for direct sweat gland differentiation of epidermal progenitors by different pore structure (300-µm or 400-µm nozzle diameters printed) and reported a long-term gradual transition of differentiated cells into glandular morphogenesis occurs within the 3D construct in vitro. At the initial 14-day culture, an accelerated cell differentiation was achieved with inductive cues released along with gelatin reduction. After protein release completed, the 3D construct guide the self-organized formation of sweat gland tissues, which is similar to that of the natural developmental process. However, glandular morphogenesis was only observed in 300-µm-printed constructs. In the absence of 3D architectural support, glandular morphogenesis was not occurred. This striking finding made us to identify a previously unknown role of the 3D-printed structure in glandular tissue regeneration, and this self-organizing strategy can be applied to forming other tissues in vitro.

3D bioprinted extracellular matrix mimics facilitate directed differentiation of epithelial progenitors for sweat gland regeneration.

Sweat glands perform a vital thermoregulatory function in mammals. Like other skin appendages, they originate from epidermal progenitors. However, they have low regenerative potential in response to injury, and whether adult epidermal progenitors could be specified to differentiate to a sweat gland cell lineage remains largely unexplored. We used bioprinting technology to create a functional in vitro cell-laden 3D extracellular matrix mimics (3D-ECM) with composite hydrogels based on gelatin and sodium alginate because of chemical and structural similarity to ECM components. To achieve specific cell differentiation, mouse plantar dermis and epidermal growth factor were synchronously incorporated into the 3D-ECM mimics to create an inductive niche for epidermal progenitor cells obtained from mice. The biological 3D construct could maintain cell viability, thereby facilitating cell spreading and matrix formation. In vitro data by immunofluorescence and gene expression assay of key cell-surface markers demonstrated that the bioprinted 3D-ECM could effectively create a restrictive niche for epidermal progenitors that ensures unilateral differentiation into sweat gland cells. Furthermore, direct delivery of bioprinted 3D-ECM into burned paws of mice resulted in functional restoration of sweat glands. This study represents the rational design to enhance the specific differentiation of epidermal lineages using 3D bioprinting and may have clinical and translational implications in regenerating sweat glands. STATEMENT OF SIGNIFICANCE: Sweat gland regeneration after injury is of clinical importance but remains largely unsolved because of low regenerative potential and lack of a definite niche. Some studies have shown sweat gland regeneration with gene-based interventions or cell-based induction via embryonic components, but translation to clinic is challenging. The novelty and significance of the work lies in the fact that we design a 3D bioprinted extracellular matrix that provides the spatial inductive cues for enhancing specific differentiation of epidermal lineages to regenerate sweat glands, which is critical for treating deep burns or other wounds. Our studies are encouraging given the overwhelming advantages of our designed 3D bioprinting construct over other cell delivery technology in maintaining high cell proliferation; another interesting finding is that adult tissue components retain a gland lineage-inductive power as embryonic tissue, which can facilitate translation.

Age-associated changes in regenerative capabilities of mesenchymal stem cell: impact on chronic wounds repair.

Mesenchymal stem cells (MSCs) represent an ideal source of autologous cell-based therapy for chronic wounds. Functional characteristics of MSCs may benefit wound healing by exerting their multi-regenerative potential. However, cell ageing resulting from chronic degenerative diseases or donor age could cause inevitable effects on the regenerative abilities of MSCs. A variety of studies have shown the relationship between MSC ageing and age-related dysfunction, but few associate these age-related impacts on MSCs with their ability of repairing chronic wounds, which are common in the elderly population. Here, we discuss the age-associated changes of MSCs and describe the potential impacts on MSC-based therapy for chronic wounds. Furthermore, critical evaluation of the current literatures is necessary for understanding the underlying mechanisms of MSC ageing and raising the corresponding concerns on considering their possible use for chronic wound repair.

Cytokeratin Expression at Different Stages in Sweat Gland Development of C57BL/6J Mice.

Sweat glands exhibit a documented role in epidermal reepithelialization after wounding. However, the regenerative potential of sweat glands has remained underappreciated due to the absence of useful markers for the analysis of determination and differentiation processes in the developing eccrine sweat gland from epithelium. Although the current knowledge of keratin expression in most of the different origins has been described, it remains widely shared and not unified in eccrine sweat glands of C57BL/6J mice that are commonly used as animal models for sweat gland and wound healing studies, both at the molecular and cellular levels. Aiming to answer this question, we have investigated the changes in cytokeratin expression patterns during the embryonic, neonatal, juvenile, and young adult stages (E12.5, E17.5, P0.5, P5, and P28). In this article, we demonstrate that the morphology of murine sweat gland progenitor cells are similar to epidermal stem cells before birth (E12.5 and E17.5); at postnatal stages, the duct formed gradually and curled to glob. K8 and K19 were expressed in the eccrine sweat gland cells at all times and highly expressed after birth at both gene and protein levels. Also, histological results revealed K8 and K19 positive cells localized in the secretary portion of glands. Meanwhile, K14 strongly expressed both in vivo and in vitro at E12.5, while it weakly expressed at other stages. Moreover, K10 was rarely detected before birth, but it expressed positively in vivo and in vitro only at the protein level after birth. These data indicate the pattern of main cytokeratin expression at different stages during murine sweat gland development and might provide an efficient tool for sweat gland research and exciting potential for developing targeted therapies for wound healing.

Mesenchymal stromal cells enhance wound healing by ameliorating impaired metabolism in diabetic mice.

BACKGROUND AIMS: Mesenchymal stromal cells (MSCs) have been documented to improve delayed wound healing in diabetes, but the underlying mechanism remains obscure. We aimed to investigate whether the therapeutic effects on wounds was associated with metabolic alterations by paracrine action of MSCs. METHODS: MSCs from mice with high-fat diet/streptozotocin-induced diabetes or wild-type C57BL/6 mice were evaluated for their paracrine potential in vitro using enzyme-linked immunosorbent assay and immunohistochemical staining assay. MSCs were then evaluated for their therapeutic potential in vivo using an excisional cutaneous wound model in mice with diabetes. Metabolic alterations and glucose transporter four (GLUT4) as well as PI3K/Akt signaling pathway expression after wounding were also examined. RESULTS: MSCs from normal mice expressed even more insulin-like growth factor-1 (IGF-1) than mice with diabetes, suggesting putative paracrine action. Furthermore, compared with IGF-1 knockdown MSCs, normal MSCs markedly accelerated wound healing, as revealed by higher wound closure rate and better healing quality at 21 days post-wound. By contrast, MSCs administration increased the level of insulin as well as GLUT4 and PI3K/Akt signaling pathway expression but repressed the biochemical indexes of glucose and lipid, resulting in obvious metabolic improvement. CONCLUSIONS: These findings suggest that IGF-1 is an important paracrine factor that mediates the therapeutic effects of MSCs on wound healing in diabetes, and the benefits of MSCs may be associated with metabolism improvements, which would provide a new target for treatment.

MSC attenuate diabetes-induced functional impairment in adipocytes via secretion of insulin-like growth factor-1.

The function of subcutaneous adipocytes in promoting wound healing is significantly suppressed in diabetic wounds. Recent studies have demonstrated the ability of mesenchymal stem cell (MSC) to ameliorate impaired diabetic wound healing. We hypothesized that MSC function may involve subcutaneous adipocytes. The abnormal function of subcutaneous adipocytes from STZ induced diabetic mice including glucose uptake and free fatty acid (FFA) secretion level were assessed. Then these cells were co-cultured with MSC via a transwell system to observe the changes of metabolic index and glucose transporter four (GLUT4) as well as phosphoinositide 3-kinase/protein kinase (PI3K/AKT) signaling pathway expression. The results of metabolic index suggest that MSC obviously attenuated the diabetes-induced functional impairment. Both mRNA and protein expression analyses showed that PI3K/AKT insulin signaling pathway and GLUT4 expression were up-regulated. These changes were substantially associated with a increased level of insulin-like growth factor-1 (IGF-1) secretion from MSC. These findings suggest that MSC could attenuate abnormal function of diabetic adipocytes by IGF-1secretion, which was more or less associated with the beneficial effects of MSC on improving diabetic wound healing.