Skin Wound Healing Process Research Articles

Delayed wound healing in aged skin rat models after thermal injury is associated with an increased MMP-9, K6 and CD44 expression

Age-related differences in wound healing have been documented but little is known about the wound healing mechanism after burns. Our aim was to compare histological features and immunohistochemical expression of matrix metalloproteinase-9 (MMP-9), collagen IV, K6 and CD44 in the burn wound healing process in aged and young rats.Following burns the appearance of the wound bed in aged rats had progressed but slowly, resulting in a delayed healing process compared to the young rats.At 21 days after injury, epithelial K6, MMP-9 and CD44 expression was significantly increased in aged rats with respect to young rats; moreover, in the aged rat group we observed a not fully reconstituted basement membrane. K6, MMP-9 and CD44 expression was significantly increased in wounded skin compared to unwounded skin both in young and aged rats.We hypothesise that delayed burn skin wound healing process in the aged rats may represent an age dependent response to injury where K6, MMP-9 and CD44 play a key role. It is therefore possible to suggest that these factors contribute to the delayed wound healing in aged skin and that modulation could lead to a better and faster recovery of skin damage in elderly.

Quantitative proteomics reveals altered expression of extracellular matrix related proteins of human primary dermal fibroblasts in response to sulfated hyaluronan and collagen applied as artificial extracellular matrix

Fibroblasts are the main matrix producing cells of the dermis and are also strongly regulated by their matrix environment which can be used to improve and guide skin wound healing processes. Here, we systematically investigated the molecular effects on primary dermal fibroblasts in response to high-sulfated hyaluronan [HA] (hsHA) by quantitative proteomics. The comparison of non- and high-sulfated HA revealed regulation of 84 of more than 1,200 quantified proteins. Based on gene enrichment we found that sulfation of HA alters extracellular matrix remodeling. The collagen degrading enzymes cathepsin K, matrix metalloproteinases-2 and -14 were found to be down-regulated on hsHA. Additionally protein expression of thrombospondin-1, decorin, collagen types I and XII were reduced, whereas the expression of trophoblast glycoprotein and collagen type VI were slightly increased. This study demonstrates that global proteomics provides a valuable tool for revealing proteins involved in molecular effects of growth substrates for further material optimization.Electronic supplementary materialThe online version of this article (doi:10.1007/s10856-012-4760-x) contains supplementary material, which is available to authorized users.

The CD100 Receptor Interacts with Its Plexin B2 Ligand to Regulate Epidermal γδ T Cell Function

γδ Tcells respond rapidly to keratinocyte damage, providing essential contributions to the skin wound healing process. The molecular interactions regulating their response are unknown. Here, we identify a role for interaction of plexin B2 with the CD100 receptor in epithelial repair. Invitro blocking of plexin B2 or CD100 inhibited γδ Tcell activation. Furthermore, CD100 deficiency invivo resulted in delayed repair of cutaneous wounds due to a disrupted γδ Tcell response to keratinocyte damage. Ligation of CD100 in γδ Tcells induced cellular rounding via signals through ERK kinase and cofilin. Defects in this rounding process were evident in the absence of CD100-mediated signals, thereby providinga mechanistic explanation for the defective wound healing in CD100-deficient animals. The discovery of immune functions for plexin B2 and CD100 provides insight into the complex cell-cell interactions between epithelial resident γδ Tcells and the neighboring cells they support.

Quantification of MSCs involved in wound healing: use of SIS to transfer MSCs to wound site and quantification of MSCs involved in skin wound healing

Mesenchymal stem cells (MSCs) are known to be effective in wound healing, but not much has been reported on quantitative correlations between MSCs injected into the wound site and MSCs that actually participate in wound healing. This study traced MSCs participating in wound healing by using small intestinal submucosa (SIS) as a cell carrier, identified their moving path and calculated the number of MSCs involved in wound healing. First, MSCs were isolated from the nude mouse and 1 × 10(6) cells were seeded onto the centre of the SIS. MSC-seeded SIS complexes were injected onto full-thickness skin wounds made on the dorsum of nude mice. Tracing of MSC-seeded SIS complex transplanted to the wound site revealed that 27.6% of the MSCs were migrated to the wound site at the first attempt. Second, repeated injection of additional MSCs did not increase the number of MSCs participating in wound healing beyond a certain constant maximum amount. The number of MSCs present in the wound site remains constant in the range 2-3 × 10(5) from day 1 to day 10. The expression of skin regeneration-related growth factors was confirmed by real-time polymerase chain reaction (PCR) and enzyme-linked immunosorbent assay (ELISA). MSCs participating in wound healing were found not only to suppress inflammation of the wound but also to increase the skin regeneration-related growth factors that enable the recovery of the skin. An optimal number of about 3 × 10(5) MSCs injected into the site was found to adapt themselves to the skin wound-healing process effectively.

Myostatin-null mice exhibit delayed skin wound healing through the blockade of transforming growth factor-β signaling by decorin

Myostatin (Mstn) is a secreted growth and differentiation factor that belongs to the transforming growth factor-β (TGF-β) superfamily. Mstn has been well characterized as a regulator of myogenesis and has been shown to play a critical role in postnatal muscle regeneration. Herein, we report for the first time that Mstn is expressed in both epidermis and dermis of murine and human skin and that Mstn-null mice exhibited delayed skin wound healing attributable to a combination of effects resulting from delayed epidermal reepithelialization and dermal contraction. In epidermis, reduced keratinocyte migration and protracted keratinocyte proliferation were observed, which subsequently led to delayed recovery of epidermal thickness and slower reepithelialization. Furthermore, primary keratinocytes derived from Mstn-null mice displayed reduced migration capacity and increased proliferation rate as assessed through in vitro migration and adhesion assays, as well as bromodeoxyuridine incorporation and Western blot analysis. Moreover, in dermis, both fibroblast-to-myofibroblast transformation and collagen deposition were concomitantly reduced, resulting in a delayed dermal wound contraction. These decreases are due to the inhibition of TGF-β signaling. In agreement, the expression of decorin, a naturally occurring TGF-β suppressor, was elevated in Mstn-null mice; moreover, topical treatment with TGF-β1 protein rescued the impaired skin wound healing observed in Mstn-null mice. These observations highlight the interplay between TGF-β and Mstn signaling pathways, specifically through Mstn regulation of decorin levels during the skin wound healing process. Thus we propose that Mstn agonists might be beneficial for skin wound repair.

The bright and the dark sides of activin in wound healing and cancer

Activin was initially described as a protein that stimulates release of follicle stimulating hormone from the pituitary, and it is well known for its important roles in different reproductive functions. In recent years, this multifunctional factor has attracted the attention of researchers in other fields, as new functions of activin in angiogenesis, inflammation, immunity, fibrosis and cancer have been discovered. Studies from our laboratory have identified activin as a crucial regulator of wound healing and skin carcinogenesis. On the one hand, it strongly accelerates the healing process of skin wounds but, on the other hand, it enhances scar formation and the susceptibility to skin tumorigenesis. Finally, results from several laboratories have revealed that activin enhances tumour formation and/or progression in some other organs, in particular through its effect on the tumour microenvironment, and that it also promotes cancer-induced bone disruption and muscle wasting. These findings provide the basis for the use of activin or its downstream targets for the improvement of impaired wound healing, and of activin antagonists for the prevention and treatment of fibrosis and of malignant tumours that overexpress activin. Here, we summarize the previously described roles of activin in wound healing and scar formation and discuss functional studies that revealed different functions of activin in the pathogenesis of cancer. The relevance of these findings for clinical applications will be highlighted.

New Hope for Wound Healing after Bowel Resection

See Article on Page 132-139 The anastomotic leakage is a crucial complication after colorectal surgery and has a variable incidence, occurring in up to 1.8 to 12% of cases [1-3]. The importance of this potential complication is emphasized to colorectal surgeons, especially in the case of lower rectal surgery, because it is a major cause of surgical morbidity and mortality, early local recurrence for cancer surgery and the need for advertent additional surgery. Also, leakage increases both the hospital stay and the medical cost. The stem cell engineering technique is an emerging field of medicine and has been put on the stage because of the hidden potential of stem cells. Collawn et al. [4] recently reported the effectiveness of adipose-derived stromal cells in the skin wound healing process. Adipose tissue is a major reservoir of mesenchymal stem cells, which can be differentiated into endothelial progenitors, adipocytes, chondrocytes, etc. This article showed very promising results for the use of adipose stem cells in the bowel wall healing process. Rat adipose tissue harvested from the suprascapular and inguinal areas was used. After 14 days of organ culture, the outgrown cells in the fibrin matrix were recovered by selective degradation of the fibrin matrix, plated on tissue culture dishes, and propagated using a conventional two-dimensional culture. In vitro and in vivo experiments revealed that the experimental group showed significantly favorable histopathologic features, including less inflammation and more microvascular density. However, this feature does not confirm stem cell effects because vascular endothelial growth factor was used to induce endothelial differentiation during the cell manipulation stage. If this new technique is to be applied to clinical fields, an easier and more conventional process without additional pharmacological treatment would be expected.

Anti-Epidermal Growth Factor/Epidermal Growth Factor Receptor Therapeutic Anti-cancer Drugs and the Wound Healing Process

Cutaneous wound healing is a complex process involving blood clotting, inflammation, tissue formation, and tissue remodeling. Many experimental and clinical studies have demonstrated varied, but in most cases beneficial, effects of exogenous growth factors on the healing process. The use of targeted anti-cancer agents is increasing. It is common to utilize a multi-modal treatment approach towards solid tumors, often including surgical resection, and it has become apparent that some targeted agents can impair wound healing or cause increasing risk of perioperative complications. There are limited data regarding the wound healing process of anti-cancer target drugs blocking the EGF/EGFR system. The aim of this paper is to review and to comment the effects of anti- EGF/EGFR drugs on the skin wound healing process after programmed or emergency surgical procedures. A review of the current literature, including our own results, was undertaken. We included the monoclonal antibodies cetuximab, panitumumab, nimotuzumab; the small tyrosine kinase molecules erlotinib and gefitinib; and the EGFbased cancer vaccine; CIMAvax and the EGFR-based cancer vaccine; HER-1 vaccine. Apparently, there are no deleterious effects of the anti-EGF/EGFR drugs in the wound healing post-operative process. Taking into account that treatment with anti-EGF/EGFR drugs inhibits tumor cell proliferation, and the lack of deleterious effects of these EGF/EGFR specific inhibitors in the wound healing post-operative process; we suggest that these kinds of drugs could be maintained and their effects tested, with very special surveillance during the post-surgical period.

Characterization of the cytokine profile of platelet rich plasma (PRP) and PRP-induced cell proliferation and migration: Upregulation of matrix metalloproteinase-1 and -9 in HaCaT cells

BackgroundThe underlying rationale of platelet rich plasma (PRP) therapy is that an injection of concentrated PRP at the site of injury may promote tissue repair via cytokine release from platelets. The molecular mechanisms of PRP therapy in the skin wound healing process are not well understood at present, and would benefit from clarification.MethodsPRP was stimulated with angonists for 5 min, and cytokine profile analysis was performed. To investigate the wound healing activity of PRP, cell proliferation and migration analyses were performed in skin cells. The effects of PRP were analyzed on the expression and activity of matrix metalloproteinase (MMP)-1, -2, -9, and the activation of transcription factors.ResultsThrombin was found to be a strong stimulator of PRP activation to release growth factors and chemokines. PRP induced cell proliferation and migration in HUVECs, HaCaT cells, and HDFs, as well as MMP-1and MMP-9 expression in HaCaT cells, but PRP did not have a significant effect on the expression or activity of MMPs in HDFs. The transcription factors, including signal transducer and activator of transcription-3 (STAT-3) were found to be phosphorylated following PRP treatment in HaCaT cells.ConclusionIn this study, we have identified the cytokine profile of activated PRP after agonist stimulation. We have shown that PRP plays an active role in promoting the proliferation and migration of skin cells via the regulation of MMPs, and this may be applicable to the future development of PRP therapeutics to enhance skin wound healing.

Effects of low-power laser irradiation (LPLI) at different wavelengths and doses on oxidative stress and fibrogenesis parameters in an animal model of wound healing

Gallium-arsenide (GaAs) and helium-neon (HeNe) lasers are the most commonly used low-energy lasers in physiotherapy for promoting wound healing and pain modulation. The aim of this study was investigate the effect of low-power laser irradiation (LPLI) at different wavelengths and doses on oxidative stress and fibrogenesis parameters in an animal model of wound healing. The animals were randomly divided into five groups (n=6): Controls (skin injured animals without local or systemic treatment), skin injury treated with HeNe 1 J/cm(2) (two seg); skin injury treated with HeNe 3 J/cm(2) (six seg); skin injury treated with GaAs 1 J/cm(2) (three seg); skin injury treated with GaAs 3 J/cm(2) (nine seg). A single circular wound measuring 8 mm in diameter was surgically created on the back of the animal. The rats were irradiated at 2, 12, 24, 48, 72, 96, and 120 h after skin injury. The parameters, namely hydroxyproline content, activities of the antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and lipid (TBARS) and protein oxidation (carbonyl groups) measurements were assessed. In addition, wound size regression was also analyzed. The results showed an improvement in the wound healing reflected by the reduction in wound size and increased collagen synthesis. Moreover, a significant reduction in TBARS levels, carbonyl content, and SOD and CAT activities were observed after laser irradiation, particularly with the treatments HeNe laser 1 and 3 J/cm(2) dose and GaAs 3 J/cm(2) dose. The data strongly indicate that LPLI therapy is efficient in accelerating the skin wound healing process after wounding, probably by reducing the inflammatory phase and inducing collagen synthesis.

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.

Intracellular delivery of adenosine triphosphate enhanced healing process in full-thickness skin wounds in diabetic rabbits

Background Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery and were tested for diabetic skin wounds in rabbits. Methods Diabetes was induced by alloxan. The mean peak blood glucose concentration was 505 mg/dL. One ear was made ischemic and 80 full-thickness wounds were created in 10 animals. ATP-vesicles or saline was used and healing was compared. Results On the non-ischemic ears, mean closure time for ATP-vesicles–treated wounds was 13.7 days versus 16.4 days for saline-treated wounds ( P < .05). On the ischemic ears, mean closure time for ATP-vesicles–treated wounds was 15.3 days versus 19.3 days for saline-treated wounds ( P < .01). Histological study indicated better healing and re-epithelialization in the ATP-vesicles–treated wounds. Conclusions Intracellular delivery of ATP accelerated the healing process of diabetic skin wounds on ischemic and non-ischemic rabbit ears. The mechanisms deserve further study but may be related to improved cellular energy supplies.

The effects of vitamin E and selenium on blood flow to experimental skin burns in rats using the 133Xe clearance technique

Abstract The aim of the present experimental study was to determine and to compare the effects of vitamin E and of selenium on the blood flow to a wound region in rats, using the xenon-133 (133Xe) clearance technique. Burn wounds were made on the right thighs of rats followed by oral administration of vitamin E or selenium for a period of 10 days. The effect of vitamin E and of selenium individually on blood flow in the wound region was assessed before and after oral administration using the 133Xe clearance technique. Both vitamin E and selenium significantly increased the blood flow in the wound region (P&lt;0.05). Our results in rats suggest that both vitamin E and selenium have beneficial effects on the skin wound healing process by increasing the skin blood flow.

Matrix Metalloproteinase-3 Accelerates Wound Healing following Dental Pulp Injury

Matrix metalloproteinases (MMPs) are implicated in a wide range of physiological and pathological processes, including morphogenesis, wound healing, angiogenesis, inflammation, and cancer. Angiogenesis is essential for reparative dentin formation during pulp wound healing. The mechanism of angiogenesis, however, still remains unclear. We hypothesized that certain MMPs expressed during pulp wound healing may support recovery processes. To address this issue, a rat pulp injury model was established to investigate expression of MMPs during wound healing. Real-time RT-PCR analysis showed that expression MMP-3 and MMP-9 (albeit lower extent) was up-regulated at 24 and 12 hours after pulp injury, respectively, whereas expression of MMP-2 and MMP-14 was not changed. MMP-3 mRNA and protein were localized in endothelial cells and/or endothelial progenitor cells in injured pulp in vivo. In addition, MMP-3 enhanced proliferation, migration, and survival of human umbilical vein endothelial cells in vitro. Furthermore, the topical application of MMP-3 protein on the rat-injured pulp tissue in vivo induced angiogenesis and reparative dentin formation at significantly higher levels compared with controls at 24 and 72 hours after treatment, respectively. Inhibition of endogenous MMP-3 by N-Isobutyl-N-(4-methoxyphenylsulfonyl)-glycylhydroxamic acid resulted in untoward wound healing. These results provide suggestive evidence that MMP-3 released from endothelial cells and/or endothelial progenitor cells in injured pulp plays critical roles in angiogenesis and pulp wound healing.

Peroxisome Proliferator-Activated Receptor-α Is a Functional Target of p63 in Adult Human Keratinocytes

p63 is a master switch in the complex network of signaling pathways controlling the establishment and maintenance of stratified epithelia. We provide evidence that peroxisome proliferator-activated receptor-alpha (PPARalpha), a ligand-activated nuclear receptor that participates in the skin wound healing process, is a target of p63 in human keratinocytes. Silencing of p63 by RNA interference and transient transfections showed that p63 represses PPARalpha through a functional region of promoter B. Chromatin immunoprecipitation analyses indicate that p63 is bound to this region, in the absence of a recognizable p63-binding motif, suggesting that it acts through interactions with other transcription factors (TFs). Distinct PPARalpha transcripts are differentially regulated by p63, indicating a bimodal action in promoter and/or transcription start specification. PPARalpha repression is consistent with lack of expression in the interfollicular epidermis under physiological conditions. Furthermore, we show that PPARalpha is a negative regulator of DeltaNp63alpha levels and that it also binds to a functional region of the DeltaNp63 promoter that lacks PPRE motifs. Therefore, the reciprocal regulation is exerted either through binding to non-consensus sites or through interactions with other DNA-bound TFs. In conclusion, our data establish a link between two TFs intimately involved in the maintenance of skin homeostatic conditions.

Morphology of lymphatic regeneration in rat incision wound healing in comparison with vascular regeneration

In the wound healing process, angiogenesis is involved in the recovery of vasculature, and its process has been investigated. On the other hand, the reconstruction of lymphatic vessels in the injured subcutaneous tissue has not been studied in detail. We studied the recovery of lymphatic vessels using podoplanin immunohistochemistry in the paraffine section microscopy of the rat skin incision wound. Our result indicates a novel finding that subcutaneous tissue of the incised skin area does not show any recovery of lymphatic vessels up to 84 days after the skin incision. As the wound area shrunk, the surrounding subcutaneous tissue covered with the normal skin epithelial cells approached toward the center of the wound, and the lymphatic vessels in the surrounding tissue gradually reached the incision wound area. On the other hand, the regeneration of the vasculature occurred within the wound area as assessed by CD31 and von Willebrand Factor (vWF) immunohistochemistry. This difference was confirmed by the morphometric quantification of podoplanin- or vWF-positive vessels. Our results show that there is a clear difference in the recovery pattern of vascular and lymphatic vessels in the skin wound healing process.

The plasmid encoding HSP47 enhances collagen expression and promotes skin wound healing in an alloxan‐induced diabetic model

The objective of this work was initially to investigate the effects on skin wound healing process by local injection of HSP47 recombinant plasmid in an alloxan-induced diabetic rat model and assess the possibility and utility of gene therapy based on HSP47 plasmid to improve the diabetic skin wound healing. Rats were injected intraperitoneally with alloxan (120mg/kg) to induce diabetes. The fragment containing the rat 47kDa heat shock protein (HSP47) gene lacking its own promoter was cloned into plasmids containing a promoter and green fluorescent protein (GFP). The resulting gene constructs were first tested in vitro using 3T3 fibroblast cell line and subsequently in vivo after inducing wounds with alloxan in diabetic rats. Immunohistochemistry, quantitative fluorescent RT-PCR, and Western blotting 3-5 days after plasmid injection were performed to measure the expression changes of HSP47 and collagen I. The results demonstrate an increase of HSP47 levels in vitro in 3T3 fibroblast cells and in vivo in diabetic rat after treatment with plasmids expressing HSP47. The level of collagen I around the wound during the repair process was higher in the treated group than that in the control group, indicating that the constructs may have use in human gene therapy in cases of impaired skin wound healing in diabetes.

The dose effect of human bone marrow-derived mesenchymal stem cells on epidermal development in organotypic co-culture

A wealth of evidences have shown the participation and benefits of bone marrow-derived mesenchymal stem cells (BM-MSCs) in wound healing and skin tissue repair in vivo. However, their role in epidermal development and reconstitution is not clearly investigated. Here we examine the quantitative effect of human BM-MSCs on epidermal regeneration in vitro. Human keratinocytes and BM-MSCs are cultured at ratios from 0% to 100% on top of a fibroblast-embedded collagen gel in a three-dimensional organotypic co-culture model at an air-liquid interface up to 20 days and analyzed by histochemical and immunochemical staining of filaggrin, involucrin and keratin 10 on days 14 and 20. Human BM-MSCs were tracked with quantum dots in organotypic co-cultures. It was found that epidermal development is strongly influenced by the percentage of co-cultured BM-MSCs. A strong chemotactic effect between keratinocytes and MSCs was seen in the group with 50% of BM-MSCs, which resulted in an impaired epidermal development, whereas at a low percentage of BM-MSCs (10%), a stratified epidermal structure resembling native skin was established on day 14 of culture. Moreover, the immunostaining studies revealed that BM-MSCs in the low percentage (10%) participated in the basal periphery of reconstructed epidermis and a similar pattern characteristic of native epidermis was demonstrated in this experimental group, which was superior to all other experimental groups in terms of the thickness of stratum corneum and the expression profile of epidermal differentiation markers. This study indicates the advantage of using a new skin equivalent model incorporating a small fraction of MSCs to develop biologically useful tissues for maintaining homeostasis during skin regeneration and wound healing process.

Effect of Visible and Infrared Polarized Light on the Healing Process of Full-Thickness Skin Wounds: An Experimental Study

Polarized light has already been experimentally and clinically used in an effort to promote wound healing, but the findings have been equivocal. The aim of this study was to evaluate the effect of visible and infrared polarized light of a specific range of wavelength (580-3400 nm) on the secondary healing of full-thickness skin wounds in rats. Forty male Wistar rats were used, divided in two groups of 20 animals each. A standardized open full-thickness skin wound was created on the back of each animal. In the first group the rats were exposed to polarized light (40 mW/cm(2) and 2.4 J/cm(2)) for 7 min on a daily basis (total daily dose = 16.8 J/cm(2)), while the second group acted as controls. Clinical and histological evaluation of wound healing were performed on days 5, 10, 15, and 20 post-wound. The size of the wounds was measured with the use of planimetry, whereas epithelialization, inflammatory response, neovascularization, and collagen formation were histologically assessed. According to our findings, the group exposed to light therapy showed statistically significantly faster epithelialization seen on days 10 and 15 post-wound compared to controls, as well as better quality of the healing process (although not statistically significantly) at all time points. In conclusion, this specific fraction of polarized light seems to have beneficial effects on wound healing, leading to faster epithelialization and qualitatively better wound healing.

Intracellular Adenosine Triphosphate Delivery Enhanced Skin Wound Healing in Rabbits

Small unilamellar lipid vesicles were used to encapsulate adenosine triphosphate (ATP-vesicles) for intracellular energy delivery. This technique was tested in full-thickness skin wounds in 16 adult rabbits. One ear was rendered ischemic by using a minimally invasive surgery. The other ear served as a normal control. Four circular full-thickness wounds were created on the ventral side of each ear. ATP-vesicles or saline was used and the wounds were covered with Tegaderm (3M, St. Paul, MN). Dressing was changed and digital photos were taken daily until all the wounds were healed. The mean healing times of ATP-vesicles-treated wounds were significantly shorter than that of saline-treated wounds on ischemic and nonischemic ears. Histologic study indicated better-developed granular tissue and reepithelialization in the ATP-vesicles-treated wounds. The wounds treated by ATP-vesicles exhibited extremely fast granular tissue growth. More CD31 positive cells were seen in the ATP-vesicles-treated wounds. This preliminary study shows that direct intracellular delivery of ATP can accelerate the healing process of skin wounds on ischemic and nonischemic rabbit ears. The extremely fast granular tissue growth was something never seen or reported in the past.