Trypsin Potentiates Human Fibrocyte Differentiation

Trypsin-containing topical treatments can be used to speed wound healing, although the mechanism of action is unknown. To help form granulation tissue and heal wounds, monocytes leave the circulation, enter the wound tissue, and differentiate into fibroblast-like cells called fibrocytes. We find that 20 to 200 ng/ml trypsin (concentrations similar to those used in wound dressings) potentiates the differentiation of human monocytes to fibrocytes in cell culture. Adding trypsin inhibitors increases the amount of trypsin needed to potentiate fibrocyte differentiation, suggesting that the potentiating effect is dependent on trypsin proteolytic activity. Proteases with other site specificities such as pepsin, endoprotease GluC, and chymotrypsin do not potentiate fibrocyte differentiation. This potentiation requires the presence of albumin in the culture medium, and tryptic fragments of human or bovine albumin also potentiate fibrocyte differentiation. These results suggest that topical trypsin speeds wound healing by generating tryptic fragments of albumin, which in turn potentiate fibrocyte differentiation.     

Peripheral blood fibrocytes: differentiation pathway and migration to wound sites

Fibrocytes are a distinct population of blood-borne cells that display a unique cell surface phenotype (collagen I+/CD11b+/CD13+/CD34+/CD45RO+/MHC class II+/CD86+) and exhibit potent immunostimulatory activities. Circulating fibrocytes rapidly enter sites of tissue injury, suggesting an important role for these cells in wound repair. However, the regulatory processes that govern the differentiation of blood-borne fibrocytes and the mechanisms that underlie the migration of these cells to wound sites are currently not known. We report herein that ex vivo cultured fibrocytes can differentiate from a CD14+-enriched mononuclear cell population and that this process requires contact with T cells. Furthermore, we demonstrate that TGF-beta1 (1-10 ng/ml), an important fibrogenic and growth-regulating cytokine involved in wound healing, increases the differentiation and functional activity of cultured fibrocytes. Because fibrocytes home to sites of tissue injury, we examined the role of chemokine/chemokine receptor interactions in fibrocyte trafficking. We show that secondary lymphoid chemokine, a ligand of the CCR7 chemokine receptor, acts as a potent stimulus for fibrocyte chemotaxis in vitro and for the homing of injected fibrocytes to sites of cutaneous tissue injury in vivo. Finally, we demonstrate that differentiated, cultured fibrocytes express alpha smooth muscle actin and contract collagen gels in vitro, two characteristic features of wound-healing myofibroblasts. These data provide important insight into the control of fibrocyte differentiation and trafficking during tissue repair and significantly expand their potential role during wound healing.     

Inhibition of fibrocyte differentiation by serum amyloid P

Wound healing and the dysregulated events leading to fibrosis both involve the proliferation and differentiation of fibroblasts and the deposition of extracellular matrix. Whether these fibroblasts are locally derived or from a circulating precursor population is unclear. Fibrocytes are a distinct population of fibroblast-like cells derived from peripheral blood monocytes that enter sites of tissue injury to promote angiogenesis and wound healing. We have found that CD14(+) peripheral blood monocytes cultured in the absence of serum or plasma differentiate into fibrocytes within 72 h. We purified the factor in serum and plasma that prevents the rapid appearance of fibrocytes, and identified it as serum amyloid P (SAP). Purified SAP inhibits fibrocyte differentiation at levels similar to those found in plasma, while depleting SAP reduces the ability of plasma to inhibit fibrocyte differentiation. Compared with sera from healthy individuals and patients with rheumatoid arthritis, sera from patients with scleroderma and mixed connective tissue disease, two systemic fibrotic diseases, were less able to inhibit fibrocyte differentiation in vitro and had correspondingly lower serum levels of SAP. These results suggest that low levels of SAP may thus augment pathological processes leading to fibrosis. These data also suggest mechanisms to inhibit fibrosis in chronic inflammatory conditions, or conversely to promote wound healing.     

Fibrocytes and activated fibrocytes in the healing of an open skin wound

The microscopic, electron microscopic and immunohistochemical observation of biopsy specimens taken at an early stage, at close and regular intervals (every 4 hours), from open skin wounds created in the pig and the monkey, together with quantitative analysis of the various cell types in the granulation tissue, supports the conception that the activated fibrocyte (fibroblast) originates from the fibrocyte of the wound edges and thus completes some earlier experimental studies. We describe here the various stages of the differentiation of the wound edge fibrocyte into an activated fibrocyte and its proliferation and migration from the edges to the site of the wound. This does not exclude the possibility that local mesenchymal cells take part in the formation of activated fibrocytes. The activated fibrocyte build the collagen of the granulation tissue and then remodel and ensure wound contraction by becoming fibroclasts and myofibroblasts. This article defines the signification of the terms fibrocyte, activated fibrocyte, fibroblast and activated fibroblast.     

Fibrocytes can be reprogrammed to promote tissue remodeling capacity of dermal fibroblasts

Fibroblasts play a pivotal role in wound healing process participating in both tissue fibrosis and remodeling. However, it remains unclear which factors activate such diversity of fibroblast responses and how this decision-making process is made. Previous reports have demonstrated that wound milieu stimulates the transformation of circulating precursor cells into fibrocytes. These pro-fibrogenic cells promote the collagen production by resident fibroblasts. Conversely, recruited cells with anti-fibrogenic profile that can compete with fibrocytes have not been identified. This report describes a novel transdifferentiation process of fibrocytes induced by changing culture conditions. The reprogrammed fibrocytes markedly increased cell proliferation and MMP-1 expression in dermal fibroblasts. The MMP-1 up-regulation was directly related to the number of fibrocytes that followed this cell transformation. In vitro and in vivo results have confirmed that TGF-β deprivation plays an important role in this novel fibrocyte differentiation pathway. Our findings demonstrate that, changing the fibrocyte commitment, it is possible to exponentially stimulate the tissue remodeling capacity of dermal fibroblasts. These results will open new research approaches to understand the role of cell transdifferentiation and local environment not only in the wound healing process of skin, but also in several other fibrocyte-associated diseases such as lung fibrosis, asthma, liver cirrhosis, chronic pancreatitis, and atherosclerosis.     

负压创面治疗肝源性糖尿病并发糖尿病足的研究

目的:讨论创面负压治疗促进肝硬化合并糖尿病并发糖尿病足的临床疗效。方法:将30例慢性肝硬化合并糖尿病并发糖尿病足患者随机分为创面负压治疗组和敷料包扎组,观察两组创面细菌负荷及创面微循环血流量变化。结果:清创即刻,负压创面治疗组和敷料包扎组细菌负荷与创面微循环血流量无显著差异(P>0.01);创后3天、6天和9天时,负压创面组创面细菌负荷低于敷料包扎组(P<0.01),创面微循环血流量高于敷料包扎组(P<0.01)。负压创面组创面愈合时间少于敷料包扎组(P<0.01)。结论:负压创面治疗有助于慢性肝硬化并发糖尿病合并糖尿病足的创面愈合。     

Application of platelet derived growth factor-BB and diabetic wound healing: the relationship with oxidative events

The reasons that cause delay in wound healing in diabetes are a decrease in the level of growth factors secretion, an increase in the destruction of growth factors and in oxidative stress. Platelet derived growth factor (PDGF) is one of the important growth factors that play a role in all phases of wound healing. This study investigates time-dependent effects of topically PDGF-BB administration on oxidative events on the healing of dorsolateral-excisional wounds in diabetic rats. Forty-two female Wistar-albino rats with streptozotocin-induced diabetes were divided into four groups: control group, untreated group, chitosan-treated group, chitosan?+?PDGF-BB-treated group. Two identical full-thickness excisional skin wounds were made under anaesthesia in all rats except for the control group. In the PDGF-BB-treated and chitosan-treated groups, the wounds were treated topically PDGF-BB (7?ng/mL, single daily dose) and blank chitosan gel (equal amount) after wounding, respectively. After these administrations, on day 3 and day 7 of wound healing, rats were sacrificed. Thiobarbituric acid reactive substances, glutathione, nitric oxide, ascorbic acid levels, and superoxide dismutase activity in wound tissues were spectrophotometrically measured. PDGF-BB administration significantly increased TBARS levels and non-enzymatic antioxidant levels in early phase of diabetic wound healing. PDGF-BB dramatically reduced NO_x levels on day 3 and sharply increased NO_x levels on day 7 of wound healing. Consequently, PDGF-BB administration can be effective on oxidative balance in the early phase of diabetic wound healing.     

Dynamic multiphoton imaging of acellular dermal matrix scaffolds seeded with mesenchymal stem cells in diabetic wound healing

Significantly effective therapies need to be developed for chronic nonhealing diabetic wounds. In this work, the topical transplantation of mesenchymal stem cell (MSC) seeded on an acellular dermal matrix (ADM) scaffold is proposed as a novel therapeutic strategy for diabetic cutaneous wound healing. GFP‐labeled MSCs were cocultured with an ADM scaffold that was decellularized from normal mouse skin. These cultures were subsequently transplanted as a whole into the full‐thickness cutaneous wound site in streptozotocin‐induced diabetic mice. Wounds treated with MSC‐ADM demonstrated an increased percentage of wound closure. The treatment of MSC‐ADM also greatly increased angiogenesis and rapidly completed the reepithelialization of newly formed skin on diabetic mice. More importantly, multiphoton microscopy was used for the intravital and dynamic monitoring of collagen type I (Col‐I) fibers synthesis via second harmonic generation imaging. The synthesis of Col‐I fibers during diabetic wound healing is of great significance for revealing wound repair mechanisms. In addition, the activity of GFP‐labeled MSCs during wound healing was simultaneously traced via two‐photon excitation fluorescence imaging. Our research offers a novel advanced nonlinear optical imaging method for monitoring the diabetic wound healing process while the ADM and MSCs interact in situ. Schematic of dynamic imaging of ADM scaffolds seeded with mesenchymal stem cells in diabetic wound healing using multiphoton microscopy. PMT, photo‐multiplier tube.      

Effects of umbilical cord blood stem cells on healing factors for diabetic foot injuries

The use of stem or progenitor cells from bone marrow, or peripheral or umbilical cord blood is becoming more common for treatment of diabetic foot problems. These cells promote neovascularization by angiogenic factors and they promote epithelium formation by stimulating cell replication and migration under certain pathological conditions. We investigated the role of CD34 + stem cells from human umbilical cord blood in wound healing using a rat model. Rats were randomly divided into a control group and two groups with diabetes induced by a single dose of 55 mg/kg intraperitoneal streptozocin. Scarred areas 5 mm in diameter were created on the feet of all rats. The diabetic rats constituted the diabetes control group and a diabetes + stem cell group with local injection into the wound site of 0.5 × 106 CD34 + stem cells from human umbilical cord blood. The newly formed skin in the foot wounds following CD34 + stem cell treatment showed significantly improvement by immunohistochemistry and TUNEL staining, and were closer to the wound healing of the control group than the untreated diabetic animals. The increase in FGF expression that accompanied the local injection of CD34 + stem cells indicates that FGF stimulation helped prevent apoptosis. Our findings suggest a promising new treatment approach to diabetic wound healing.     

Connexin 43 mimetic peptide Gap27 reveals potential differences in the role of Cx43 in wound repair between diabetic and non‐diabetic cells

During early wound healing (WH) events Connexin 43 (Cx43) is down‐regulated at wound margins. In chronic wound margins, including diabetic wounds, Cx43 expression is enhanced suggesting that down‐regulation is important for WH. We previously reported that the Cx43 mimetic peptide Gap27 blocks Cx43 mediated intercellular communication and promotes skin cell migration of infant cells in vitro. In the present work we further investigated the molecular mechanism of Gap27 action and its therapeutic potential to improve WH in skin tissue and diabetic and non‐diabetic cells. Ex vivo skin, organotypic models and human keratinocytes/fibroblasts of young and old donors and of diabetic and non‐diabetic origin were used to assess the impact of Gap27 on cell migration, proliferation, Cx43 expression, localization, phosphorylation and hemichannel function. Exposure of ex vivo WH models to Gap27 decreased dye spread, accelerated WH and elevated cell proliferation. In non‐diabetic cell cultures Gap27 decreased dye uptake through Cx hemichannels and after scratch wounding cells showed enhanced migration and proliferation. Cells of diabetic origin were less susceptible to Gap27 during early passages. In late passages these cells showed responses comparable to non‐diabetic cells. The cause of the discrepancy between diabetic and non‐diabetic cells correlated with decreased Cx hemichannel activity in diabetic cells but excluded differences in Cx43 expression, localization and Ser368‐phosphorylation. These data emphasize the importance of Cx43 in WH and support the concept that Gap27 could be a beneficial therapeutic to accelerate normal WH. However, its use in diabetic WH may be restricted and our results highlight differences in the role of Cx43 in skin cells of different origin.     

Low-dose photodynamic therapy effect on closure of scratch wounds of normal and diabetic fibroblast cells: An in vitro study

Chronic wounds such as diabetic ulcers are a serious public health problem. Extensive research is needed to find new alternatives for wound treatment. Photodynamic therapy (PDT) is a non-invasive method, which has been studied for several decades to treat cancer, infections, and other diseases. PDT involves the administration of a photosensitizer compound followed by irradiation with using light at specific wavelength to produce reactive oxygen species (ROS) using molecular oxygen. It is possible that low dose photodynamic therapy (LDPDT) could improve wound healing and stimulates the cell repair process. This study we explored the effect of LDPDT on wound healing in vitro using normal and diabetic cellular wound models. The effects of different concentrations of 5-ALA and different energy densities (dark or light) on the cell viability of human fibroblast cells were studied using the MTT assay. After ascertaining the optimum parameters, a scratch wound assay was performed on both normal and diabetic cells and then cells treated with 1 and 5 μg/mL of 5-ALA at 1 J/cm² energy density. ROS production and morphological alteration of the cells were studied. The mortality of normal fibroblast cells increased with increasing 5-ALA concentration and also increasing energy density (up to 3 J/cm²). However, in diabetic cells, the mortality rate did not decrease. Diabetic cells showed increased migration and closure of the scratch compared to normal cells under similar conditions. A low concentration of 5-ALA (5 μg/mL) and low energy density of 1 J/cm² in both normal and diabetic cells gave a small increase in ROS levels compared to controls. This may explain the positive effects of LDPDT on wound healing. The findings of this study suggest that LDPDT may have a potential effect on the wound healing of diabetic wounds.     

Activity of mesenchymal stem cells in therapies for chronic skin wound healing

Chronic or non-healing skin wounds present an ongoing challenge in advanced wound care, particularly as the number of patients increases while technology aimed at stimulating wound healing in these cases remains inefficient. Mesenchymal stem cells (MSCs) have proved to be an attractive cell type for various cell therapies due to their ability to differentiate into various cell lineages, multiple donor tissue types, and relative resilience in ex-vivo expansion, as well as immunomodulatory effects during transplants. More recently, these cells have been targeted for use in strategies to improve chronic wound healing in patients with diabetic ulcers or other stasis wounds. Here, we outline several mechanisms by which MSCs can improve healing outcomes in these cases, including reducing tissue inflammation, inducing angiogenesis in the wound bed, and reducing scarring following the repair process. Approaches to extend MSC life span in implant sites are also examined.     

Gr‐1+CD11b+ myeloid cells efficiently home to site of injury after intravenous administration and enhance diabetic wound healing by neoangiogenesis

Vascularization is an important factor that affects diabetic wound healing. There is increasing evidence that myeloid cell lineages play a role in neovascularization. In this study, the efficiency of Gr‐1+CD11b+ myeloid cells to home to the site of injury and enhance diabetic wound healing by neoangiogenesis after intravenous administration was investigated. Gr‐1+CD11b+ myeloid cells were injected into tail vein after establishment of dorsal window chamber, hindlimb ischaemia and ear‐punch injury in diabetic or non‐diabetic mice. The Gr‐1+CD11b+ myeloid cells efficiently homed to the site of injury after intravenous administration and increased neoangiogenesis. The chemokine receptor type 4 (CXCR4) is robustly expressed by Gr‐1+CD11b+ myeloid cells. Inhibition of CXCR4 decreases the homing ability of Gr‐1+CD11b+ myeloid cells to the site of injury, which indicates that the CXCR4/SDF‐1 axis plays an important role in the homing of Gr‐1+CD11b+ myeloid cells to the site of injury. In addition, Gr‐1+CD11b+ myeloid cells were found to improve blood flow recovery of ischaemic limb and enhance wound healing in diabetic mice by neoangiogenesis after intravenous administration. Taken together, the results of this study suggest that Gr‐1+CD11b+ myeloid cells may serve as a potential cell therapy for diabetic wound healing.     

Effects of Senescence in Wound Healing

With the advent of aging society, the effects of aging on all aspects of the body are attracting more and more attention. Among them, the increasing incidence of chronic wounds in the elderly not only affects the quality of the elderly’s life significantly, but also brings a heavy medical burden on society. Delayed and poor wound healing increases the possibility of severe infections. To find out a solution for infection and chronic wounds, it is necessary to clarify the specific mechanisms of wound healing and possible intervention targets. Wound healing is a complex physiological process in the human body, which involves the coordinated activation of multiple cell types and signaling pathways. The role of senescent cells in wound healing is causing growing attention in recent years. It was thought that wound healing needs to take a longer time in elder people. In recent years, it has been found that senescent cells promote wound healing. So far, the effects of senescent cells on the efficiency and quality of wound healing and its specific mechanism have not been fully clarified. What is certain is that different types of senescent cells and even different subtypes of the same senescent cells play different roles in fast and chronic wound healing. It is not only the heterogeneity of the senescent cell itself, but also the difference in the surrounding microenvironment that determines the effect of senescent cells on wound healing. The study of its mechanism is helpful to find a way to promote the healing of wounds. It is worth noting that senescent cells themselves may also induce poor wound prognosis, such as chronic wounds, inflammation and decreased anti-infection ability. Therefore, the ideal treatment strategy to apply senescent cells will be a comprehensive plan that maximizes the efficiency and quality of wound healing, while minimizing the risk of senescent cells itself becoming an inducement for chronic wounds.     

Priming of mononuclear cells with a combination of growth factors enhances wound healing via high angiogenic and engraftment capabilities

Recently, we demonstrated that a specific combination of growth factors enhances the survival, adhesion and angiogenic potential of mononuclear cells (MNCs). In this study, we sought to investigate the changes of the angiogenic potential of MNCs after short‐time priming with a specific combination of growth factors. MNCs were isolated using density gradient centrifugation and incubated with a priming cocktail containing epidermal growth factor (EGF), insulin‐like growth factor (IGF)‐1, fibroblast growth factor (FGF)‐2, FMS‐like tyrosine kinase (Flt)‐3L , Angiopoietin (Ang)‐1, granulocyte chemotactic protein (GCP)‐2 and thrombopoietin (TPO) (all 400 ng/ml) for 15, 30 and 60 min. Wounds in nonobese diabetic‐severe combined immune deficiency (NOD‐SCID) mice were created by skin excision followed by cell transplantation. We performed a qRT‐PCR analysis on the growth factor–primed cells. The angiogenic factors vascular endothelial growth factor (VEGF)‐A, FGF‐2, hepatocyte growth factor (HGF), platelet‐derived growth factor (PDGF) and interleukin (IL)‐8 and the anti‐apoptotic factors IGF‐1 and transforming growth factor‐β1 were significantly elevated in the MNCs primed for 30 min. (T30) compared with the non‐primed MNCs (T0). The scratch wound assay revealed that T30‐ conditioned media (CM) significantly increased the rate of fibroblast‐mediated wound closure compared with the rates from T0‐CM and human umbilical vein endothelial cells (HUVEC)‐CM at 20 hrs. In vivo wound healing results revealed that the T30‐treated wounds demonstrated accelerated wound healing at days 7 and 14 compared with those treated with T0. The histological analyses demonstrated that the number of engrafted cells and transdifferentiated keratinocytes in the wounds were significantly higher in the T30‐transplanted group than in the T0‐transplanted group. In conclusion, this study suggests that short‐term priming of MNCs with growth factors might be alternative therapeutic option for cell‐based therapies.     

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.     

Monotropein promotes angiogenesis and inhibits oxidative stress‐induced autophagy in endothelial progenitor cells to accelerate wound healing

Attenuating oxidative stress‐induced damage and promoting endothelial progenitor cell (EPC) differentiation are critical for ischaemic injuries. We suggested monotropein (Mtp), a bioactive constituent used in traditional Chinese medicine, can inhibit oxidative stress‐induced mitochondrial dysfunction and stimulate bone marrow‐derived EPC (BM‐EPC) differentiation. Results showed Mtp significantly elevated migration and tube formation of BM‐EPCs and prevented tert‐butyl hydroperoxide (TBHP)‐induced programmed cell death through apoptosis and autophagy by reducing intracellular reactive oxygen species release and restoring mitochondrial membrane potential, which may be mediated viamTOR/p70S6K/4EBP1 and AMPK phosphorylation. Moreover, Mtp accelerated wound healing in rats, as indicated by reduced healing times, decreased macrophage infiltration and increased blood vessel formation. In summary, Mtp promoted mobilization and differentiation of BM‐EPCs and protected against apoptosis and autophagy by suppressing the AMPK/mTOR pathway, improving wound healing in vivo. This study revealed that Mtp is a potential therapeutic for endothelial injury‐related wounds.     

Extracellular vesicle‐enclosed miR‐486‐5p mediates wound healing with adipose‐derived stem cells by promoting angiogenesis

Adipose‐derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC‐secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC‐based therapies. Considering its effects, we aimed to investigate the role of ASC‐EVs in the cutaneous wound healing accompanied with the study on the specific cargo‐medicated effects on wound healing. Two full‐thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α‐SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co‐cultured with EVs isolated from ASC (ASC‐EVs), respectively, followed by the evaluation of their viability and mobility using CCK‐8, scratch test and transwell migration assays. Matrigel‐based angiogenesis assays were performed to evaluate vessel‐like tube formation by HMECs in vitro. ASC‐EVs accelerated the healing of full‐thickness skin wounds, increased re‐epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR‐486‐5p antagomir abrogated the ASC‐EVs‐induced effects. Intriguingly, miR‐486‐5p was found to be highly enriched in ASC‐EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC‐EVs‐secreted miR‐486‐5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR‐486‐5p released by ASC‐EVs could be a critical mediator to develop an ASC‐based therapeutic strategy for wound healing.