Porcine acellular dermal matrix accelerates wound healing through miR-124-3p.1 and miR-139-5p.

BACKGROUND AIMS: Cutaneous wound management is a major health problem and imposes a huge economic burden worldwide. Previous studies have demonstrated that wound healing is a highly coordinated process including epithelialization, angiogenesis, remodeling and scarring. This progression requires self-renewal, preservation and repair properties of stem cells. However, our understanding of the detailed internal regulatory mechanism following injury and the means to accelerate wound healing are limited. METHODS: Our previous research revealed that porcine acellular dermal matrix (ADM) effectively promotes wound healing and scar formation through epidermal stem cells (ESCs), and this process is relevant to the alteration of internal miRNA levels. In this study, we investigated the regulatory function of porcine ADM treatment on miRNAs in ESCs. RESULTS: We report that the treatment of porcine ADM reduced the levels of miR-124-3p.1 and miR-139-5p in wounds. MiR-124-3p.1 and miR-139-5p inhibited the expression of JAG1 and Notch1, respectively, by directly targeting miRNAs in ESCs. CONCLUSIONS: This work demonstrates that porcine ADM induced down-regulation of miR-124-3p.1/139-5p in wounds and up-regulation of JAG1/Notch1 in ESCs, thus enhancing cutaneous wound healing.

Role of caveolin-1 in epidermal stem cells during burn wound healing in rats.

Local transplantation of stem cells has therapeutic effects on skin damage but cannot provide satisfactory wound healing. Studies on the mechanisms underlying the therapeutic effects of stem cells on skin wound healing will be needed. Hence, in the present study, we explored the role of Caveolin-1 in epidermal stem cells (EpiSCs) in the modulation of wound healing. We first isolated EpiSCs from mouse skin tissues and established stable EpiSCs with overexpression of Caveolin-1 using a lentiviral construct. We then evaluated the epidermal growth factor (EGF)-induced cell proliferation ability using cell counting Kit-8 (CCK-8) assay and assessed EpiSC pluripotency by examining Nanog mRNA levels in EpiSCs. Furthermore, we treated mice with skin burn injury using EpiSCs with overexpression of Caveolin-1. Histological examinations were conducted to evaluate re-epithelialization, wound scores, cell proliferation and capillary density in wounds. We found that overexpression of Caveolin-1 in EpiSCs promoted EGF-induced cell proliferation ability and increased wound closure in a mouse model of skin burn injury. Histological evaluation demonstrated that overexpression of Caveolin-1 in EpiSCs promoted re-epithelialization in wounds, enhanced cellularity, and increased vasculature, as well as increased wound scores. Taken together, our results suggested that Caveolin-1 expression in the EpiSCs play a critical role in the regulation of EpiSC proliferation ability and alteration of EpiSC proliferation ability may be an effective approach in promoting EpiSC-based therapy in skin wound healing.

Curcumin promotes burn wound healing in mice by upregulating caveolin-1 in epidermal stem cells.

We aimed to explore the effect of curcumin on epidermal stem cells (ESCs) in regulating wound healing and the underlying molecular mechanism. We treated mouse ESCs isolated from skin tissues with curcumin, and then assessed the proliferation ability of cells induced by epidermal growth factor using cell counting kit-8 assay. The pluripotency of ESCs was evaluated as well through examination of Nanog expression in ESCs. Further, mice with skin burns were treated with ESCs with or without curcumin pretreatments. Histological evaluations were then preformed to determine wound scores, cell proliferation, reepithelialization, and capillary density in wounds. Curcumin treatment promoted the proliferative ability of ESCs and conditioned medium from curcumin-treated ESCs enhanced human umbilical vein endothelial cell (HUVEC) tube formation. We also found curcumin treatment elevated caveolin-1 expression in ESCs, which was required for the beneficial effect of curcumin on ESC proliferation and HUVEC tube formation. Next, using a mouse model of burn wound healing, curcumin-treated ESCs exhibited enhanced wound closure, which also required caveolin-1 expression. Our current study demonstrates the beneficial effect of curcumin on burn wound healing in mice, which is mediated by upregulating caveolin-1 in ESCs, and supports the potential therapeutic role of curcumin in ESC-based treatment against skin wound healing.

Effects of porcine acellular dermal matrix treatment on wound healing and scar formation: Role of Jag1 expression in epidermal stem cells.

Skin wound healing involves Notch/Jagged1 signaling. However, little is known how Jag1 expression level in epidermal stem cells (ESCs) contributes to wound healing and scar formation. We applied multiple cellular and molecular techniques to examine how Jag1 expression in ESCs modulates ESCs differentiation to myofibroblasts (MFB) in vitro, interpret how Jag1 expression in ESCs is involved in wound healing and scar formation in mice, and evaluate the effects of porcine acellular dermal matrix (ADM) treatment on wound healing and scar formation. We found that Jag1, Notch1 and Hes1 expression was up-regulated in the wound tissue during the period of wound healing. Furthermore, Jag1 expression level in the ESCs was positively associated with the level of differentiation to MFB. ESC-specific knockout of Jag1 delayed wound healing and promoted scar formation in vivo. In addition, we reported that porcine ADM treatment after skin incision could accelerate wound closure and reduce scar formation in vivo. This effect was associated with decreased expression of MFB markers, including α-SMA Col-1 and Col-III in wound tissues. Finally, we confirmed that porcine ADM treatment could increase Jag1, Notch1 and Hesl expression in wound tissues. Taken together, our results suggested that ESC-specific Jag1 expression levels are critical for wound healing and scar formation, and porcine ADM treatment would be beneficial in promoting wound healing and preventing scar formation by enhancing Notch/Jagged1 signaling pathway in ESCs.

EGFL7-overexpressing epidermal stem cells promotes fibroblast proliferation and migration via mediating cell adhesion and strengthening cytoskeleton.

Epidermal growth factor (EGF)-like family members mediate a wide range of biological activities including cell proliferation and migration. Increasing evidence indicated that EGF plays an important role in the process of wound healing by stimulating fibroblast motility. The aim of this study was to see whether EGF-like domain 7 (EGFL7)-overexpressing epidermal stem cells (EGFL7-ESCs) would promote fibroblast proliferation and migration. We found that mRNA and protein levels of EGFL7 expression were significantly increased in EGFL7-ESCs. The protein expression of EGFL7 was significantly elevated in conditioned media (CM) of EGFL7-ESCs compared to ESCs CM or vector-ESCs CM. The cell count and cell viability of EGFL7-ESCs CM-treated fibroblasts were also significantly increased compared to control. In addition, EGFL7-ESCs CM-treated fibroblasts showed elevated migration compared with control. Moreover, the expressions of ß1-integrin, ß-tubulin, ß-actin, and Vimentin were increased, while that of E-cadherin was decreased in EGFL7-ESCs CM-treated fibroblasts. These results indicate that EGFL7-ESCs contribute towards promoting fibroblast migration through enhancing cell adhesion, strengthening cytoskeleton, and reducing intercellular aggregation. These findings suggest that the stimulating effect of EGFL7-ESCs on fibroblast proliferation and migration may provide a useful strategy for wound healing.

Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway.

Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro Importantly, Jag1 overexpression improves diabetic wound healing in vivo These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound.

The effect of porcine ADM to improve the burn wound healing.

To study the effect of porcine acellular dermal matrix (ADM) on the burn wound healing. Seventy healthy Wistar rats were inflicted with 2 cm second degree burn and divided into 2 groups; one group was treated with porcine ADM and the other with Povidone Iodine Cream. Biopsies were taken on day 1, 3, 5, 7, 10, 14, 21 for histopathological and biochemical analysis to test PCNA, K19, Integrin-ß1, PDGF, EGF and FGF. The results revealed relatively better and faster regeneration after treatment of porcine ADM, along with greatly increased synthesis in collagen in the experimental group. PCNA, K19, Integrin-ß1 had an increase and then tapered down, and were stronger in the experimental group than in the contrast group during 21 days after burns. PDGF, EGF and FGF levels increased on day 3, peaked on day 5 and then started to decrease, while significantly enhanced expression of relevant growth factors were observed in the experimental group. Porcine ADM stimulate collagen synthesis, stem cells proliferation and differentiation, and the expression of relevant growth factors and ultimately improve the burn wound healing.

An improved method for the isolation and culture of rat epidermal stem cells.

The management of burns and injuries using novel treatment strategies involving epidermal stem cells (ESC) requires a better understanding of the biology of these cells, in particular, their isolation and the maintenance of their unique characteristics in culture. The purpose of this study was to describe an improved method for isolating putative ESC from fetal rat skin and to maintain them long term in culture. Single ESC suspensions were obtained from fetal rat skin by enzyme digestion containing 0.5% neutral protease. The target cells were harvested by rapid adherence on type IV collagen plates and were cultured in complex DMEM. After primary isolation, cells were continuously cultured in K-serum free medium. After reaching 70-80% confluence, the cells were digested with 0.25% trypsin at 37°C for 5-10 minutes, and passaged at a ratio of 1:2. The cultured ESC showed good growth, resulting in cell viability of over 98%. Four days later, clones containing 100-200 cells were detected, showing cobblestone-like characteristics. The rapidly adherent cells were positive for keratin 15, 19 and P63. Eighty three percent of cells expressed ß1 integrin. The growth-curve showed that the rapidly adherent cells were in the exponential growth phase. The protocol described in this paper provides a simplified and effective method to isolate and maintain long-term culture of epidermal stem cells from fetal rat skin. This method should be valuable for isolating and studying ESC from various transgenic rat lines that are currently available.

Application of acellular dermal xenografts in full-thickness skin burns.

The aim of this study was to explore the clinical value of the porcine acellular dermal xenograft (ADX) in combination with autologous split-thickness skin and pure autologous split-thickness skin grafting applied in deep full-thickness burns and scar wounds. A total of 30 patients with deep burns were randomly divided into experimental and control groups following escharectomy. The patients were separately treated with porcine acellular dermal xenograft (ADX) in combination with autologous split-thickness skin and pure autologous split-thickness skin graft. The wound healing was observed routinely and the scores were evaluated using Vancouver scar scale at different times following transplant surgery. The samples of cograft regions and the control group (pure transplant split-thickness skin autograft) were observed using light microscopy and electron microscopy, and the follow-up results were recorded. No conspicuous rejections on the cograft wound surface were observed. Compared with the control group, the cograft wounds were smooth, presented no scar contracture and exhibited good skin elasticity and recovery of the joint function. The cografted skin combined well and displayed a clear and continuous basal membrane, as well as gradually combined skin structure, a mature stratum corneum, downward extended rete pegs, a mainly uniform dermal collagen fiber structure, regular alignment, and fewer blood capillaries. Clear desmosome cograft regions were identified among heckle cells, as well as a clear and continuous basal membrane. The cografted skin of the combined split-thickness autograft and the acellular heterologous (porcine) dermal matrix showed an improved shape and functional recovery compared with the pure split-thickness skin autograft. The combination of the meshed ADX and the split-thickness skin autograft applied in deep full-thickness burns and scar wounds may induce tissue regeneration via dermis aiming. This method also has superior shape and functional recovery, and has an extensive clinical application value.

[Clinical application of xenogenic (porcine) acellular dermal atrix (ADM) in scar treatment].

OBJECTIVE: To investigate the role of xenogenic (porcine) ADM as dermal substitute in scar treatment. METHODS: After scar excision, the wounds were covered with composite grafts of DR procine ADM and autologous thin split-thickness grafts in one stage or in two stages. RESULTS: 22 out of 47 cases were treated in two-staged procedure. After the ADMs were applied to the wound, the autologous thin split-thickness grafts were implanted 7 days later. 25 cases were treated in one-staged procedure. The survival rates of composite grafts were (88.3 +/- 3.7)% for subcutaneous recipient bed and (89.7 +/- 3.4)% for deep fascia recipient bed in group with two-staged procedure, compared with (92.5 +/- 4.1)% and (93.2 +/- 5.2)%, respectively, in group with one-staged procedure. Early after grafts taken, the grafts had a pink colour and smooth surface. The patients were followed up for 90 days at most. The survived composite grafts were durable, elastic, smooth and soft with good function and appearance like normal skin. They could even be pinched up. The scar along the edge of the grafts was slightly hypertrophic. CONCLUSIONS: The survival rate of composite graft is higher in patients with one-staged procedure. The elasticity and textural of the taken grafts are better on subcutaneous recipient bed than on deep fascia recipient bed, though the function has no difference. Xenogenic (porcine) ADM can be an optimal dermal substitute for wound coverage after scar excision.

The study of inhibiting systematic inflammatory response syndrome by applying xenogenic (porcine) acellular dermal matrix on second-degree burns.

OBJECTIVE: To investigate the influence of xenogenic (porcine) acellular dermal matrix on the systematic inflammatory reaction syndrome (SIRS), and the reaction of burn patients to tissue damage upon application to second-degree burn wounds. METHOD: Seventy-two cases of patients with acute second-degree burns were enrolled in the study. According to the total burn surface area (TBSA) and the treatment methods, we randomly divided the patients into four groups. Group A (treatment group): patients with less than 30% TBSA covered with xenogenic acellular dermal matrix. Group B (control group): patients with less than 30% TBSA covered with betadine ointment gauzes. Group C (treatment group): patients with more than 30% TBSA covered with porcine acellular dermal matrix. Group D (control group): patients with more than 30% TBSA covered with betadine ointment gauzes. Serum level of C-reactive protein (CRP) was measured by single radial immunodiffusion method on 1, 4, 7 and 14 days postburn. RESULTS: The serum level of CRP in group A was significantly less than that of in group B (P<0.05) on days 4, 7 and 14. The serum level of CRP in group C increased slowly, descended quickly and was significantly less than that of in group D on days 4, 7 and 14. CONCLUSION: The application of xenogenic (porcine) acellular dermal matrix on second-degree burn wound can decrease serum level of CRP of the patients, which may play an important role in reducing SIRS and sepsis incidence.

[Treatment of deep partial thickness burns by a single dressing of porcine acellular dermal matrix].

OBJECTIVE: To explore the effect of one dressing of porcine acellular dermal matrix on deep partial thickness burns. METHODS: From January 1997 to January 2004, sixty-seven cases of deep partial thickness total burned surface area (TBSA) from 50% to 90% burn wound were treated by a single dressing of porcine acellular dermal matrix (the porcine acellular dermal matrix group). Ten cases of deep partial thickness burned patients with the same TBSA treated by exposure method served as the exposure method group. The healing time of the wound was observed. The patients were followed up for 3 months to 2 years, and the scar proliferation was observed. RESULTS: The deep partial-thickness wound would be healed without dressing change in the porcine acellular dermal matrix group, and the average healing time was (12.2 +/- 2.6) days. The average healing time of the exposure method group was (27.4 +/- 3.5) days. Follow up of the patients within 3 months to 2 years showed that scar proliferation in the porcine acellular dermal matrix group was much less than that in the exposure method group, even no scar proliferation was observed in some patients. CONCLUSION: Without tangential excision, autografting and dressing change, a single dressing of porcine acellular dermal matrix on deep partial thickness burn wound could shorten the healing time and inhibit scar proliferation.

Control of hypertrophic scar from inception by using xenogenic (porcine) acellular dermal matrix (ADM) to cover deep second degree burn.

OBJECTIVE: We have spent 7 years to investigate the method of applying porcine acellular dermal matrix (ADM) on deep partial thickness burn wound until the wound heals without dressing change. Known as "Feng's pig skin method" by our hospital, the method appears to encourage rapid re-epithilization with minimum scarring. METHOD: The deep partial thickness burn wound was rinsed cleanly under anesthesia when the patient admitted. ADM was applied on the wound after the detached epidermis was thoroughly removed, wrapped and fixed by sterile gauze and bandages. The dressing was removed within two weeks and the wound completely healed. The outcome of the treatment was analyzed by using the modified Vancouver Burn Scar Assessment Scale. RESULT: All the wounds healed with one dressing within 2 weeks, and the time of wound re-epithelialization shortened to 7-12 days. Scar hyperplasia did not occur, or it was greatly ameliorated compared with traditional treatment after a followed-up period of 3 months to 2 years. The Scar Index was significant lower than that of the traditional exposure method. CONCLUSION: Using ADM to cover deep second degree burn can preserve maximally residual dermal tissue and epithelium, help accelerate the regeneration of epithelial and stem cells, thus shorten the healing time, remodel the skin structure, and consequently has the effect of controlling hypertrophic scar at inception.

[A clinical study on composite transplantation of meshed split-thickness autograft and heterologous dermal matrix].

OBJECTIVE: To investigate the feasibility of the composite transplantation of 1:3 meshed split-thickness autograft and acellular heterologous (porcine) dermal matrix. METHODS: 9 inpatients with full thickness skin burn or hypertrophic scar were selected in this study. After the eschar or scar was excised, the wound was covered with acellular heterologous dermal matrix. Then the meshed (1:3) split-thickness autologous skin sheet was grafted on the dermal matrix. Before dressing up, the radiated pigskin was placed on the composite transplants. RESULTS: The composite transplantation was successfully used in 9 cases. The meshed split-thickness autograft was expanded 3 times and covered the dermal matrix tightly. The clinical results of the composite transplantation were similar to that of intermediate split thickness skin graft or full thickness skin graft. CONCLUSION: The composite transplantation of meshed (1:3) split-thickness autograft and acellular heterologous (porcine) dermal matrix allowed the expansion of the autologous skin sheet to 3 times. The clinical results were similar to that of intermediate split thickness skin graft or full thickness skin graft.

[Porcine acellular dermal matrix in the treatment of deep partial-thickness burns in human].

OBJECTIVE: To investigate the possibility of treating deep partial-thickness burns by closed dressing of the wounds with porcine acellular dermal matrix (ADM) and evaluate the therapeutic effects. METHODS: We conducted a retrospective review of 128 cases of burn patients who received treatment with porcine ADM within the period from January 1998 to January 2002 in our hospital. Different procedures were adopted according to the degree of the burn injury. As for "fairly superficial" deep partial-thickness skin burns, after removing the necrotic epidermis and washing with 0.1 % benzalkonium bromide, the wound was covered with porcine ADM pretreated with povidone-iodine and then bandaged with cotton gauze and bandages. In cases of "fairly deep" deep partial skin thickness burns, eschar excisions as deep as to expose parabiotic lamina were performed prior to dressing the wounds in the same manner as described above. RESULTS: All the patients were successfully treated with satisfactory clinical results. CONCLUSIONS: Porcine ADM is feasible as an efficient dressing material for deep partial-thickness burns, which may promote epithelialization in the wounds and help stabilize the patients' condition during burn shock stage to reduce the complications and shorten the treatment courses.