Tetrahedral framework nucleic acids promote diabetic wound healing via the Wnt signalling pathway

ObjectivesTo determine the therapeutic effect of tetrahedral framework nucleic acids (tFNAs) on diabetic wound healing and the underlying mechanism.Materials and MethodsThe tFNAs were characterized by polyacrylamide gel electrophoresis (PAGE), atomic force microscopy (AFM), transmission electron microscopy (TEM), dynamic light scattering (DLS) and zeta potential assays. Cell Counting Kit‐8 (CCK‐8) and migration assays were performed to evaluate the effects of tFNAs on cellular proliferation and migration. Quantitative polymerase chain reaction (Q‐PCR) and enzyme‐linked immunosorbent assay (ELISA) were used to detect the effect of tFNAs on growth factors. The function and role of tFNAs in diabetic wound healing were investigated using diabetic wound models, histological analyses and western blotting.ResultsCellular proliferation and migration were enhanced after treatment with tFNAs in a high‐glucose environment. The expression of growth factors was also facilitated by tFNAs in vitro. During in vivo experiments, tFNAs accelerated the healing process in diabetic wounds and promoted the regeneration of the epidermis, capillaries and collagen. Moreover, tFNAs increased the secretion of growth factors and activated the Wnt pathway in diabetic wounds.ConclusionsThis study indicates that tFNAs can accelerate diabetic wound healing and have potential for the treatment of diabetic wounds.

Tetrahedral framework nucleic acids (tFNAs) can facilitate the proliferation and migration of HaCaTs, HDFs and HUVECs in a high‐glucose environment. tFNAs can accelerate diabetic wound healing by promoting epithelialization, vascularization, collagen synthesis and the secretion of growth factors via the Wnt pathway.     

Functional extracellular matrix hydrogel modified with MSC‐derived small extracellular vesicles for chronic wound healing

ObjectivesDiabetic wound healing remains a global challenge in the clinic and in research. However, the current medical dressings are difficult to meet the demands. The primary goal of this study was to fabricate a functional hydrogel wound dressing that can provide an appropriate microenvironment and supplementation with growth factors to promote skin regeneration and functional restoration in diabetic wounds.Materials and MethodsSmall extracellular vesicles (sEVs) were bound to the porcine small intestinal submucosa‐based hydrogel material through peptides (SC‐Ps‐sEVs) to increase the content and achieve a sustained release. NIH3T3 cell was used to evaluate the biocompatibility and the promoting proliferation, migration and adhesion abilities of the SC‐Ps‐sEVs. EA.hy926 cell was used to evaluate the stimulating angiogenesis of SC‐Ps‐sEVs. The diabetic wound model was used to investigate the function/role of SC‐Ps‐sEVs hydrogel in promoting wound healing.ResultsA functional hydrogel wound dressing with good mechanical properties, excellent biocompatibility and superior stimulating angiogenesis capacity was designed and facilely fabricated, which could effectively enable full‐thickness skin wounds healing in diabetic rat model.ConclusionsThis work led to the development of SIS, which shows an unprecedented combination of mechanical, biological and wound healing properties. This functional hydrogel wound dressing may find broad utility in the field of regenerative medicine and may be similarly useful in the treatment of wounds in epithelial tissues, such as the intestine, lung and liver.

Schematic illustration showing synthesis of the SC‐Ps scaffold dressing and nanoscale sEVs loaded SC‐Ps scaffold dressing and the potential application of the dressings in diabetic wound healing and skin reconstruction.     

Advanced glycation end products delay corneal epithelial wound healing through reactive oxygen species generation

Delayed healing of corneal epithelial wounds is a serious complication in diabetes. Advanced glycation end products (AGEs) are intimately associated with the diabetic complications and are deleterious to the wound healing process. However, the effect of AGEs on corneal epithelial wound healing has not yet been evaluated. In the present study, we investigated the effect of AGE-modified bovine serum albumin (BSA) on corneal epithelial wound healing and its underlying mechanisms. Our data showed that AGE-BSA significantly increased the generation of intracellular ROS in telomerase-immortalized human corneal epithelial cells. However, the generation of intracellular ROS was completely inhibited by antioxidant N-acetylcysteine (NAC), anti-receptor of AGEs (RAGE) antibodies, or the inhibitor of NADPH oxidase. Moreover, AGE-BSA increased NADPH oxidase activity and protein expression of NADPH oxidase subunits, p22phox and Nox4, but anti-RAGE antibodies eliminated these effects. Furthermore, prevention of intracellular ROS generation using NAC or anti-RAGE antibodies rescued AGE-BSA-delayed epithelial wound healing in porcine corneal organ culture. In conclusion, our results demonstrated that AGE-BSA impaired corneal epithelial wound healing ex vivo. AGE-BSA increased intracellular ROS generation through NADPH oxidase activation, which accounted for the delayed corneal epithelial wound healing. These results may provide better insights for understanding the mechanism of delayed healing of corneal epithelial wounds in diabetes.     

Combination of chemically modified SDF‐1α mRNA and small skin improves wound healing in diabetic rats with full‐thickness skin defects

ObjectivesDiabetes mellitus is associated with refractory wound healing, yet current therapies are insufficient to accelerate the process of healing. Recent studies have indicated chemically modified mRNA (modRNA) as a promising therapeutic intervention. The present study aimed to explore the efficacy of small skin engineered to express modified mRNAs encoding the stromal cell‐derived factor‐1α (SDF‐1α) facilitating wound healing in a full‐thickness skin defect rat model. This study, devised therapeutic strategies for diabetic wounds by pre‐treating small skin with SDF‐1α modRNA.Materials and MethodsThe in vitro transfection efficiency was evaluated using fluorescence microscopy and the content of SDF‐1α in the medium was determined using ELISA after the transfection of SDF‐1α into the small skin. To evaluate the effect of SDF‐1α modRNA and transplantation of the small skin cells on wound healing, an in vivo full‐thickness skin defect rat model was assessed.ResultsThe results revealed that a modRNA carrying SDF‐1α provided potent wound healing in the small skin lesions reducing reduced scar thickness and greater angiogenesis (CD31) in the subcutaneous layer. The SDF‐1α cytokines were significantly secreted by the small skin after transfection in vitro.ConclusionsThis study demonstrated the benefits of employing small skin combined with SDF‐1α modRNA in enhancing wound healing in diabetic rats having full‐thickness skin defects.

An illustration of how the small skin was transfected with the SDF‐1α modRNA and the design of the in vivo experiments.     

Treatment effect of DNA framework nucleic acids on diffuse microvascular endothelial cell injury after subarachnoid hemorrhage

ObjectivesThe purpose of this study was to investigate the treatment effect and molecular mechanism of tetrahedral framework nucleic acids (tFNAs), novel self‐assembled nucleic acid nanomaterials, in diffuse BMEC injury after SAH.Materials and MethodstFNAs were synthesized from four ssDNAs. The effects of tFNAs on SAH‐induced diffuse BMEC injury were explored by a cytotoxicity model induced by hemin, a breakdown product of hemoglobin, in vitro and a mouse model of SAH via internal carotid artery puncture in vivo. Cell viability assays, wound healing assays, transwell assays, and tube formation assays were performed to explore cellular function like angiogenesis.ResultsIn vitro cellular function assays demonstrated that tFNAs could alleviate hemin‐induced injury, promote angiogenesis, and inhibit apoptosis in hemin cytotoxicity model. In vivo study using H&E and TEM results jointly indicated that the tFNAs attenuate the damage caused by SAH in situ, showing restored number of BMECs in the endothelium layer and more tight intercellular connectivity. Histological examination of SAH model animals confirmed the results of the in vitro study, as tFNAs exhibited treatment effects against diffuse BMEC injury in the cerebral microvascular bed.ConclusionsOur study suggests the potential of tFNAs in ameliorating diffuse injury to BMECs after SAH, which laid theoretical foundation for the further study and use of these nucleic acid nanomaterials for tissue engineering vascularization.     

SETD2 epidermal deficiency promotes cutaneous wound healing via activation of AKT/mTOR Signalling

ObjectivesCutaneous wound healing is one of the major medical problems worldwide. Epigenetic modifiers have been identified as important players in skin development, homeostasis and wound repair. SET domain–containing 2 (SETD2) is the only known histone H3K36 tri‐methylase; however, its role in skin wound healing remains unclear.Materials and MethodsTo elucidate the biological role of SETD2 in wound healing, conditional gene targeting was used to generate epidermis‐specific Setd2‐deficient mice. Wound‐healing experiments were performed on the backs of mice, and injured skin tissues were collected and analysed by haematoxylin and eosin (H&E) and immunohistochemical staining. In vitro, CCK8 and scratch wound‐healing assays were performed on Setd2‐knockdown and Setd2‐overexpression human immortalized keratinocyte cell line (HaCaT). In addition, RNA‐seq and H3K36me3 ChIP‐seq analyses were performed to identify the dysregulated genes modulated by SETD2. Finally, the results were validated in functional rescue experiments using AKT and mTOR inhibitors (MK2206 and rapamycin).ResultsEpidermis‐specific Setd2‐deficient mice were successfully established, and SETD2 deficiency resulted in accelerated re‐epithelialization during cutaneous wound healing by promoting keratinocyte proliferation and migration. Furthermore, the loss of SETD2 enhanced the scratch closure and proliferation of keratinocytes in vitro. Mechanistically, the deletion of Setd2 resulted in the activation of AKT/mTOR signalling pathway, while the pharmacological inhibition of AKT and mTOR with MK2206 and rapamycin, respectively, delayed wound closure.ConclusionsOur results showed that SETD2 loss promoted cutaneous wound healing via the activation of AKT/mTOR signalling.     

The advanced glycation end-products (AGEs)/ROS/NLRP3 inflammasome axis contributes to delayed diabetic corneal wound healing and nerve regeneration

Diabetic keratopathy (DK) is an important diabetic complication at the ocular surface. Chronic low-grade inflammation mediated by the NLRP3 inflammasome promotes pathogenesis of diabetes and its complications. However, the effect of the NLRP3 inflammasome on DK pathogenesis remains elusive. Wild-type (WT) and Nlrp3 knockout (KO) C57BL/6 mice were used to establish a type I diabetes model by intraperitoneal injection of streptozotocin. The effect of the NLRP3 inflammasome on diabetic corneal wound healing and never regeneration was examined by a corneal epithelial abrasion model. Western blot, immunofluorescence staining, enzyme-linked immunosorbent assay (ELISA) and pharmacological treatment were performed to investigate the regulatory mechanism of advanced glycation end products (AGEs) on NLRP3 inflammasome activation and corneal wound healing in vivo. The cultured mouse corneal epithelial cells (TKE2) were used to evaluate the effect and mechanism of AGEs on NLRP3 inflammasome activation in vitro. We revealed that NLRP3 inflammasome-mediated inflammation and pyroptosis contributed to DK pathogenesis. Under physiological conditions, the NLRP3 inflammasome was required for corneal wound healing and nerve regeneration. However, under a diabetic scenario, sustained activation of the NLRP3 inflammasome resulted in postponed corneal wound healing and impaired nerve regeneration. Mechanistically, the accumulated AGEs promoted hyperactivation of the NLRP3 inflammasome through ROS production. Moreover, genetically and pharmacologically blocking the AGEs/ROS/NLRP3 inflammasome axis significantly expedited diabetic corneal epithelial wound closure and nerve regeneration. Our results revealed that AGEs-induced hyperactivation of the NLRP3 inflammasome resulted in delayed diabetic corneal wound healing and impaired nerve regeneration, which further highlighted the NLRP3 inflammasome as a promising target for DK treatment.     

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells

ObjectivesDelivery systems that provide time and space control have a good application prospect in tissue regeneration applications, as they can effectively improve the process of wound healing and tissue repair. In our experiments, we constructed a novel micro‐RNA delivery system by linking framework nucleic acid nanomaterials to micro‐RNAs to promote osteogenic differentiation of mesenchymal stem cells.Materials and MethodsTo verify the successful preparation of tFNAs–miR‐26a, the size of tFNAs–miR‐26a were observed by non‐denaturing polyacrylamide gel electrophoresis and dynamic light scattering techniques. The expression of osteogenic differentiation‐related genes and proteins was investigated by confocal microscope, PCR and western blot to detect the impact of tFNAs–miR‐26a on ADSCs. And finally, Wnt/β‐catenin signaling pathway related proteins and genes were detected by confocal microscope, PCR and western blot to study the relevant mechanism.ResultsBy adding this novel complex, the osteogenic differentiation ability of mesenchymal stem cells was significantly improved, and the expression of alkaline phosphatase (ALP) on the surface of the cell membrane and the formation of calcium nodules in mesenchymal stem cells were significantly increased on days 7 and 14 of induction of osteogenic differentiation, respectively. Gene and protein expression levels of ALP (an early marker associated with osteogenic differentiation), RUNX2 (a metaphase marker), and OPN (a late marker) were significantly increased. We also studied the relevant mechanism of action and found that the novel nucleic acid complex promoted osteogenic differentiation of mesenchymal stem cells by activating the canonical Wnt signaling pathway.ConclusionsThis study may provide a new research direction for the application of novel nucleic acid nanomaterials in bone tissue regeneration.

MiR‐26a‐tetrahedral framework nucleic acids mediated osteogenesis of adipose‐derived mesenchymal stem cells.     

Exosomes from adipose‐derived stem cells and application to skin wound healing

Skin wound healing is an intractable problem that represents an urgent clinical need. To solve this problem, a large number of studies have focused on the use of exosomes (EXOs) derived from adipose‐derived stem cells (ADSCs). This review describes the mechanisms whereby ADSCs‐EXOs regulate wound healing and their clinical application. In the wound, ADSCs‐EXOs modulate immune responses and inflammation. They also promote angiogenesis, accelerate proliferation and re‐epithelization of skin cells, and regulate collagen remodelling which inhibits scar hyperplasia. Compared with ADSCs therapeutics, ADSCs‐EXOs have highly stability and are easily stored. Additionally, they are not rejected by the immune system and have a homing effect and their dosage can be easily controlled. ADSCs‐EXOs can improve fat grafting and promote wound healing in patients with diabetes mellitus. They can also act as a carrier and combined scaffold for treatment, leading to scarless cutaneous repair. Overall, ADSCs‐EXOs have the potential to be used in the clinic to promote wound healing.     

In vitro glycation of a tissue-engineered wound healing model to mimic diabetic ulcers

Diabetic foot ulcers are a major complication of diabetes that occurs following minor trauma. Diabetes-induced hyperglycemia is a leading factor inducing ulcer formation and manifests notably through the accumulation of advanced glycation end-products (AGEs) such as N-carboxymethyl-lysin. AGEs have a negative impact on angiogenesis, innervation, and reepithelialization causing minor wounds to evolve into chronic ulcers which increases the risks of lower limb amputation. However, the impact of AGEs on wound healing is difficult to model (both in vitro on cells, and in vivo in animals) because it involves a long-term toxic effect. We have developed a tissue-engineered wound healing model made of human keratinocytes, fibroblasts, and endothelial cells cultured in a collagen sponge biomaterial. To mimic the deleterious effects induced by glycation on skin wound healing, the model was treated with 300 µM of glyoxal for 15 days to promote AGEs formation. Glyoxal treatment induced carboxymethyl-lysin accumulation and delayed wound closure in the skin mimicking diabetic ulcers. Moreover, this effect was reversed by the addition of aminoguanidine, an inhibitor of AGEs formation. This in vitro diabetic wound healing model could be a great tool for the screening of new molecules to improve the treatment of diabetic ulcers by preventing glycation.     

Procyanidin B2 improves endothelial progenitor cell function and promotes wound healing in diabetic mice via activating Nrf2

One of the major reasons for the delayed wound healing in diabetes is the dysfunction of endothelial progenitor cells (EPCs) induced by hyperglycaemia. Improvement of EPC function may be a potential strategy for accelerating wound healing in diabetes. Procyanidin B2 (PCB2) is one of the major components of procyanidins, which exhibits a variety of potent pharmacological activities. However, the effects of PCB2 on EPC function and diabetic wound repair remain elusive. We evaluated the protective effects of PCB2 in EPCs with high glucose (HG) treatment and in a diabetic wound healing model. EPCs derived from human umbilical cord blood were treated with HG. The results showed that PCB2 significantly preserved the angiogenic function, survival and migration abilities of EPCs with HG treatment, and attenuated HG-induced oxidative stress of EPCs by scavenging excessive reactive oxygen species (ROS). A mechanistic study found the protective role of PCB2 is dependent on activating nuclear factor erythroid 2-related factor 2 (Nrf2). PCB2 increased the expression of Nrf2 and its downstream antioxidant genes to attenuate the oxidative stress induced by HG in EPCs, which were abolished by knockdown of Nrf2 expression. An in vivo study showed that intraperitoneal administration of PCB2 promoted wound healing and angiogenesis in diabetic mice, which was accompanied by a significant reduction in ROS level and an increase in circulating EPC number. Taken together, our results indicate that PCB2 treatment accelerates wound healing and increases angiogenesis in diabetic mice, which may be mediated by improving the mobilization and function of EPCs.     

Cell Hypertrophy and MEK/ERK Phosphorylation are Regulated by Glyceraldehyde-Derived AGEs in Cardiomyocyte H9c2 Cells

Diabetic cardiomyopathy has been shown to promote hypertrophy, leading to heart failure. Recent studies have reported a correlation between diabetic cardiomyopathy and oxidative stress, suggesting that the accumulation of advanced glycation end products (AGEs) induces the production of reactive oxygen species (ROS). In a clinical setting, AGEs have been shown to increase the risk of cardiovascular disease; however, the relationship between AGEs and cardiac hypertrophy remains unclear. This study sought to identify the role of AGEs in cardiac hypertrophy by treating H9c2 cells with glyceraldehyde-derived AGEs (200 μg/ml) or H₂O₂ (50 μM) for 96 h. Our results demonstrate that AGEs significantly increased protein levels and cell size. These effects were effectively blocked with PD98059 (10 μM; MEK/ERK inhibitor) pretreatment, suggesting that AGEs caused cell hypertrophy via the MEK/ERK pathway. We then treated cells with AGEs and H₂O₂ for 0–120 min and employed the Odyssey infrared imaging system to detect MEK/ERK phosphorylation. Our results show that AGEs up-regulated MEK/ERK phosphorylation. However, this effect was blocked by NAC (5 mM; ROS inhibitor), indicating that AGEs regulate MEK/ERK phosphorylation via ROS. Our findings suggest that glyceraldehyde-derived AGEs are closely related to cardiac hypertrophy and further identify a molecular mechanism underlying the promotion of diabetic cardiomyopathy by AGEs.     

Periodontitis‐compromised dental pulp stem cells secrete extracellular vesicles carrying miRNA‐378a promote local angiogenesis by targeting Sufu to activate the Hedgehog/Gli1 signalling

ObjectivesPreviously, our investigations demonstrated robust pro‐angiogenic potentials of extracellular vesicles secreted by periodontitis‐compromised dental pulp stem cells (P‐EVs) when compared to those from healthy DPSCs (H‐EVs), but the underlying mechanism remains unknown.Materials and methodsHere, circulating microRNAs (miRNAs) specifically found in P‐EVs (compared with H‐EVs) were identified by Agilent miRNA microarray analysis, and the roles of the candidate miRNA in P‐EV‐enhanced cell angiogenesis were confirmed by cell transfection and RNA interference methods. Next, the direct binding affinity between the candidate miRNA and its target gene was evaluated by luciferase reporter assay. CCK‐8, transwell/scratch wound healing and tube formation assays were established to investigate the proliferation, migration, and tube formation abilities of endothelial cells (ECs). Western blot was employed to measure the protein levels of Hedgehog/Gli1 signalling pathway components and angiogenesis‐related factors.ResultsThe angiogenesis‐related miRNA miR‐378a was found to be enriched in P‐EVs, and its role in P‐EV‐enhanced cell angiogenesis was confirmed, wherein Sufu was identified as a downstream target gene of miR‐378a. Functionally, silencing of Sufu stimulated EC proliferation, migration and tube formation by activating Hedgehog/Gli1 signalling. Further, we found that incubation with P‐EVs enabled the transmission of P‐EV‐contained miR‐378a to ECs. Subsequently, the expressions of Sufu, Gli1 and vascular endothelial growth factor in ECs were significantly influenced by P‐EV‐mediated miR‐378a transmission.ConclusionsThese data suggest that P‐EVs carrying miR‐378a promote EC angiogenesis by downregulating Sufu to activate the Hedgehog/Gli1 signalling pathway. Our findings reveal a crucial role for EV‐derived miR‐378a in cell angiogenesis and hence offer a new target for modifying stem cells and their secreted EVs to enhance vessel regenerative potential.     

Antimycotic ciclopirox olamine in the diabetic environment promotes angiogenesis and enhances wound healing

Diabetic wounds remain a major medical challenge with often disappointing outcomes despite the best available care. An impaired response to tissue hypoxia and insufficient angiogenesis are major factors responsible for poor healing in diabetic wounds. Here we show that the antimycotic drug ciclopirox olamine (CPX) can induce therapeutic angiogenesis in diabetic wounds. Treatment with CPX in vitro led to upregulation of multiple angiogenic genes and increased availability of HIF-1α. Using an excisional wound splinting model in diabetic mice, we showed that serial topical treatment with CPX enhanced wound healing compared to vehicle control treatment, with significantly accelerated wound closure, increased angiogenesis, and increased dermal cellularity. These findings offer a promising new topical pharmacologic therapy for the treatment of diabetic wounds.     

Advanced glycation end‐products activate extracellular signal‐regulated kinase via the oxidative stress‐EGF receptor pathway in renal fibroblasts

Advanced glycation end‐products (AGEs), epidermal growth factor receptor (EGFR), reactive oxygen species (ROS), and extracellular signal‐regulated kinases (ERK) are implicated in diabetic nephropathy (DN). Therefore, we asked if AGEs‐induced ERK protein phosphorylation and mitogenesis are dependent on the receptor for AGEs (RAGE)–ROS–EGFR pathway in normal rat kidney interstitial fibroblast (NRK‐49F) cells. We found that AGEs (100 µg/ml) activated EGFR and ERK1/2, which was attenuated by RAGE short‐hairpin RNA (shRNA). AGEs also increased RAGE protein and intracellular ROS levels while RAGE shRNA and N‐acetylcysteine (NAC) attenuated AGEs‐induced intracellular ROS. Hydrogen peroxide (5–25 µM) increased RAGE protein level while activating both EGFR and ERK1/2. Low‐dose hydrogen peroxide (5 µM) increased whereas high‐dose hydrogen peroxide (100 µM) decreased mitogenesis at 3 days. AGEs‐activated EGFR and ERK1/2 were attenuated by an anti‐oxidant (NAC) and an EGFR inhibitor (Iressa). Moreover, AGEs‐induced mitogenesis was attenuated by RAGE shRNA, NAC, Iressa, and an ERK1/2 inhibitor (PD98059). In conclusion, it was found that AGEs‐induced mitogenesis is dependent on the RAGE–ROS–EGFR–ERK1/2 pathway whereas AGEs‐activated ERK1/2 is dependent on the RAGE–ROS–EGFR pathway in NRK‐49F cells. J. Cell. Biochem. 109: 38–48, 2010. © 2009 Wiley‐Liss, Inc.     

SHED aggregate exosomes shuttled miR‐26a promote angiogenesis in pulp regeneration via TGF‐β/SMAD2/3 signalling

ObjectivesPulp regeneration brings big challenges for clinicians, and vascularization is considered as its determining factor. We previously accomplished pulp regeneration with autologous stem cells from deciduous teeth (SHED) aggregates implantation in teenager patients, however, the underlying mechanism needs to be clarified for regenerating pulp in adults. Serving as an important effector of mesenchymal stem cells (MSCs), exosomes have been reported to promote angiogenesis and tissue regeneration effectively. Here, we aimed to investigate the role of SHED aggregate‐derived exosomes (SA‐Exo) in the angiogenesis of pulp regeneration.Materials and MethodsWe extracted exosomes from SHED aggregates and utilized them in the pulp regeneration animal model. The pro‐angiogenetic effects of SA‐Exo on SHED and human umbilical vein endothelial cells (HUVECs) were evaluated. The related mechanisms were further investigated.ResultsWe firstly found that SA‐Exo significantly improved pulp tissue regeneration and angiogenesis in vivo. Next, we found that SA‐Exo promoted SHED endothelial differentiation and enhanced the angiogenic ability of HUVECs, as indicated by the in vitro tube formation assay. Mechanistically, miR‐26a, which is enriched in SA‐Exo, improved angiogenesis both in SHED and HUVECs via regulating TGF‐β/SMAD2/3 signalling.ConclusionsIn summary, these data reveal that SA‐Exo shuttled miR‐26a promotes angiogenesis via TGF‐β/SMAD2/3 signalling contributing to SHED aggregate‐based pulp tissue regeneration. These novel insights into SA‐Exo may facilitate the development of new strategies for pulp regeneration.     

Thymosin beta 4 improves dermal burn wound healing via downregulation of receptor of advanced glycation end products in db/db mice

BackgroundThe delay of dermal burn wound healing caused by vascular disorders is a critical problem for many diabetic patients. Thymosin β₄ (Tβ₄), identified by subtractive cloning of endothelial cells on plastic versus basement membrane substrates, has been found to promote angiogenesis and dermal wound repair in rats, aged mice, and db/db diabetic mice. However, previous studies involving the role of Tβ₄ in wound repair were limited to mechanical damage and dermal impairment. Thus, this study aimed to evaluate the improvement of healing of burn wounds by Tβ₄ in relation to advanced glycation end products (AGE), which are pathological factors in diabetes.MethodsWe adapted a dermal burn wound in vivo model in which the dorsal skin of db/db mice was exposed for 10 s to 100 °C heated water to produce a deep second-degree burn 10 mm in diameter. Five mg/kg of Tβ₄ was then injected intradermally near the burn wound twice a week for 2 weeks.ResultsAfter treatment, Tβ₄ improved wound healing markers such as wound closure, granulation, and vascularization. Interestingly, Tβ₄ reduced levels of receptor of AGE (RAGE) during the wound healing period.ConclusionsTβ₄ exerts effects to remedy burn wounds via downregulation of RAGE.General significanceOur results suggest the potential importance of Tβ₄ as a new therapy for impaired burn wound healing that is associated with diabetes.     

Proliferation and wound healing of vascular cells trigger the generation of extracellular reactive oxygen species and LDL oxidation

Cell proliferation of vascular cells is a key feature in vascular biology, wound healing, and pathophysiological processes such as atherosclerosis and restenosis. In atherosclerotic intima, cell proliferation colocalizes with oxidized LDL that indicate a local oxidative stress. This study aims to investigate whether cell proliferation is causally related with extracellular ROS generation and subsequent LDL oxidation. Sparse proliferating endothelial and smooth muscle cells generate higher levels of extracellular ROS (O₂^•− and H₂O₂) and LDL oxidation than confluent contact-inhibited cells. During wound healing of confluent cell layer, cell proliferation associated with healing also induced enhanced extracellular ROS generation and LDL oxidation. Proliferation-associated extracellular ROS generation is mediated through mitogenic signaling pathways, involving ERK1/2 and PKC, but is independent of de novo DNA synthesis, gene expression and protein synthesis. Data obtained with inhibitors of oxidases suggest that proliferation-associated extracellular ROS are not generated by a single ROS-generating system and are not essential for cell proliferation. In conclusion, our data show that proliferating vascular cells (in sparse culture or during wound healing) generate high levels of extracellular ROS and LDL oxidation through regulation of ROS-generating systems by mitogenic signaling. This constitutes a link between proliferative events and oxidative stress/LDL oxidation in atherosclerotic lesions and restenosis.     

Ameliorative effects of clindamycin - nanoceria conjugate: A ROS responsive smart drug delivery system for diabetic wound healing study

BackgroundIncreased incidence of antibiotic-resistant species calls for development of new types of nano-medicine that can be used for healing of bacteria-caused wounds, such as diabetic foot ulcer. As diabetic patients have inefficient defense mechanism against reactive oxygen species (ROS) produced in our body as a by-product of oxygen reduction, the process of wound healing takes longer epithelialisation period. Ceria nanoparticles (CNPs) are well-known for their antibacterial and ROS-scavenging nature. Yet till now no significant effort has been made to conjugate ceria nanoparticles with drugs to treat diabetic wounds.MethodsIn this experiment, CNPs were synthesized in-house and clindamycin hydrochloride was loaded onto it by physical adsorption method for reactive oxygen species responsive drug delivery. Various physico-chemical characterisations such as Transmission electron microscopy, Fourier transform infrared spectroscopy, X-ray diffraction, Energy dispersive X-ray, Thermogravimetric study etc. were performed to affirm the formation of both nanoceria along with drug encapsulated nanoceria.ResultsBoth of these as-prepared formulations inhibited the growth of Gram-positive as well as Gram-negative bacteria confirmed by Disk diffusion study; exhibiting their antibacterial effect. In-vitro drug release study was carried out in physiological environment both in absence and presence of hydrogen peroxide solution to test the reactive ROS-responsiveness of the drug loaded nanocomposites. It also exhibited faster wound healing in diabetes-induced rats. Therefore, it could successfully lower the amount of serum glucose level, inflammation cytokines, hepatotoxic and oxidative stress markers in diabetic rats as confirmed by various ex vivo tests conducted.ConclusionThus, drug loaded ceria nanoparticles have the potential to heal diabetic wounds successfully and can be considered to be useful for the fabrication of appropriate medicated suppositories beneficial for diabetic foot ulcer treatment in future.     

Human platelet lysate‐derived extracellular vesicles enhance angiogenesis through miR‐126

ObjectivesExtracellular vesicles (EVs) are key biological mediators of several physiological functions within the cell microenvironment. Platelets are the most abundant source of EVs in the blood. Similarly, platelet lysate (PL), the best platelet derivative and angiogenic performer for regenerative purposes, is enriched of EVs, but their role is still too poorly discovered to be suitably exploited. Here, we explored the contribution of the EVs in PL, by investigating the angiogenic features extrapolated from that possessed by PL.MethodsWe tested angiogenic ability and molecular cargo in 3D bioprinted models and by RNA sequencing analysis of PL‐derived EVs.ResultsA subset of small vesicles is highly represented in PL. The EVs do not retain aggregation ability, preserving a low redox state in human umbilical vein endothelial cells (HUVECs) and increasing the angiogenic tubularly‐like structures in 3D endothelial bioprinted constructs. EVs resembled the miRNome profile of PL, mainly enriched with small RNAs and a high amount of miR‐126, the most abundant angiogenic miRNA in platelets. The transfer of miR‐126 by EVs in HUVEC after the in vitro inhibition of the endogenous form, restored angiogenesis, without involving VEGF as a downstream target in this system.ConclusionPL is a biological source of available EVs with angiogenic effects involving a miRNAs‐based cargo. These properties can be exploited for targeted molecular/biological manipulation of PL, by potentially developing a product exclusively manufactured of EVs.

A high amount of small‐size extracellular vesicles (EVs) can be isolated from platelet lysate (PL)‐based preparations. When endothelial cells (HUVEC) are cultured in presence of EVs of platelet origin, they are able to significantly enhance the formation of angiogenic tubularly‐like structures in 3D endothelial bioprinted constructs (2A,B). PL‐derived EVs reflect a similar angiogenic microRNA profile (3A). Accordingly, EVs are mainly enriched with miR‐126, also known as angio‐miRNA, the most expressed miRNA by platelets in the blood. Hence, the silencing of the endogenous levels of miR‐126 in HUVEC and the retransferring of the same through PL‐derived EVs, restore angiogenesis in endothelial cells (3B). Images were created with the Biorender software.