Effect of subatmospheric pressure on the acute healing wound

PURPOSE: Vacuum-assisted closure (VAC), originally developed as an adjunct to wound care, has gained popularity in managing complex, chronic wounds. This study was designed to compare VAC with traditional saline-wet-to-dry (WD) dressings on acute wound healing in a pig model. METHODS: Nine animals were divided into groups of 3. Three rows of 2, 4-cm diameter circular defects were excised on each animal. Vacuum-assisted closure therapy was applied to 2 adjacent wound beds, WD dressings were applied to 2 adjacent wound beds, and ventilated transparent dressing covered the 2 remaining wounds as controls. Random members from each group had their wounds harvested on postoperative days (POD) number 4, 7, and 9, respectively. The specimens were histopathologically evaluated and graded with regard to immature granulation tissue, mature granulation tissue, necropurulent surface crust, proliferating cell nuclear antigen (PCNA), and collagen deposition. RESULTS: The WD-treated wounds had less necropurulent material on the surface compared with the VAC and control groups (p < 0.05). Day 9 specimens demonstrated increased immature collagen in the VAC and WD groups compared with control. No other statistically significant variations existed between the treatment groups. CONCLUSIONS: Under the conditions of this study, the histopathologic observations do not support more rapid wound healing for the acutely injured VAC-treated wound compared with the WD-treated wound in young healthy pigs.     

Quantitative and reproducible murine model of excisional wound healing

The goal of animal wound healing models is to replicate human physiology and predict therapeutic outcomes. There is currently no model of wound healing in rodents that closely parallels human wound healing. Rodents are attractive candidates for wound healing studies because of their availability, low cost, and ease of handling. However, rodent models have been criticized because the major mechanism of wound closure is contraction, whereas in humans reepithelialization and granulation tissue formation are the major mechanisms involved. This article describes a novel model of wound healing in mice utilizing wound splinting that is accurate, reproducible, minimizes wound contraction, and allows wound healing to occur through the processes of granulation and reepithelialization. Our results show that splinted wounds have an increased amount of granulation tissue deposition as compared to controls, but the rate of reepithelialization is not affected. Thus, this model eliminates wound contraction and allows rodents' wounds to heal by epithelialization and granulation tissue formation. Given these analogies to human wound healing, we believe that this technique is a useful model for the study of wound healing mechanisms and for the evaluation of new therapeutic modalities.     

Collagen-embedded platelet-derived growth factor DNA plasmid promotes wound healing in a dermal ulcer model

BACKGROUND: Gene therapy has shown limited efficacy for treating congenital diseases, partly due to temporary gene expression and host immune responses. Such results suggest that gene therapy is ideal for chronic wound treatment where limited duration of target gene expression is required. This study tested the wound healing effects of topically applied platelet-derived growth factor (PDGF)-A or -B chain DNA plasmids embedded within a collagen lattice. MATERIALS AND METHODS: Four 6-mm dermal ulcer wounds were created in the ears of young adult New Zealand White rabbits made ischemic by division of the central and rostral arteries. Wounds were treated with lyophilized collagen containing PDGF-B DNA (1.0-3.0 mg), PDGF-A DNA (1.0 mg), irrelevant DNA (1.0 mg), or collagen alone. Wounds were dressed and harvested after 10 days for measurement of granulation tissue formation, epithelialization, and wound closure. Results were evaluated with a paired two-tailed Student t test, with P values < 0.05 considered significant. RESULTS: PDGF-B DNA increased new granulation tissue (NGT) formation up to 52% and epithelialization 34% compared with controls. Wound closure was increased up to threefold. At 1.0 mg DNA, PDGF-A and PDGF-B stimulated similar responses. No difference in NGT or epithelialization was seen between control groups. CONCLUSIONS: PDGF DNA gene therapy is effective at accelerating wound healing in ischemic dermal ulcers and provides a viable alternative to peptide growth factor therapy.     

Influence of platelet-derived growth factor on microcirculation during normal and impaired wound healing

The aim of the current study was to evaluate the influence of platelet-derived growth factor (PDGF) on skin microcirculation during normal and impaired wound healing. Secondary healing wounds were created on the ears of hairless mice and treated once with 3 microg of PDGF-BB immediately after wound creation. Intravital fluorescence microscopy was used to quantify reepithelialization, revascularization, vessel diameters, vascular permeability, and leukocyte-endothelium interactions up to 24 days after wound creation. Microvascular perfusion was assessed by laser Doppler flowmetry. Wound healing was studied in normal (n = 15) and ischemic skin tissue (n = 15) as well as in mice (n = 17) rendered hyperglycemic by an intravenous injection of streptozotocin 7 days prior to wound creation. Treatment with PDGF accelerated reepithelialization and reduced the time for complete wound closure in ischemic skin from 14.9 +/- 2.5 (control) to 12.3 +/- 1.8 days (p < 0.03), and in hyperglycemic animals from 15.0 +/- 2.4 (control) to 12.0 +/- 3.0 days (p < 0.04). Revascularization of these wounds was also significantly enhanced after PDFG application. No other parameters were influenced by the treatment. Normal wound healing was not affected. This study confirms the positive influence of PDGF on wound healing under pathophysiological conditions. The effects in this model seem to be primarily due to the mitogenic potency of PDGF on keratinocytes and endothelial cells. A significant effect on leukocyte activation during the inflammatory process was not observed.     

Impaired cutaneous wound healing in transforming growth factor‐β inducible early gene1 knockout mice

Transforming growth factor‐β inducible early gene (TIEG) is induced by transforming growth factor‐β (TGF‐β) and acts as the primary response gene in the TGF‐β/Smad pathway. TGF‐β is a multifunctional growth factor that affects dermal wound healing; however, the mechanism of how TGF‐β affects wound healing is still not well understood because of the complexity of its function and signaling pathways. We hypothesize that TIEG may play a role in dermal wound healing, with involvement in wound closure, contraction, and reepithelialization. In this study, we have shown that TIEG1 knockout (TIEG1^–/–) mice have a delay in wound closure related to an impairment in wound contraction, granulation tissue formation, collagen synthesis, and reepithelialization. We also found that Smad7 was increased in the wounds and appeared to play a role in this wound healing model in TIEG1^–/– mice.     

Delayed wound healing in Mac-1–deficient mice is associated with normal monocyte recruitment

The Mac-1 integrin is an important mediator of migration and inflammatory activation of neutrophils and monocytes. However, the role of Mac-1 in modulating macrophage emigration and activation and its subsequent impact on cutaneous wound healing have not been fully elucidated. To examine the significance of Mac-1 to murine wound healing, we measured epithelialization and granulation tissue formation in partial-thickness ear wounds and full-thickness head wounds, respectively, in Mac-1-deficient mice. Wounds were histologically analyzed at postwounding days 3, 5, and 7. The gap measured between the leading edges of inward-migrating granulation tissue was significantly increased in knockout mice compared with control animals at day 5 (3.8+/-0.3 vs. 2.6+/-0.5 mm; p<0.001) and day 7 (2.2+/-0.4 vs. 0.96+/-0.73 mm; p=0.005). Epithelial gap measurements were also increased in knockout mice vs. wild-type controls at days 3 (0.62+/-0.02 vs. 0.54+/-0.07 mm; p<0.05) and 5 (0.58+/-0.06 vs. 0.39+/-0.08 mm; p<0.001). Immunohistochemistry showed equal numbers of macrophages in knockout and control wounds. These findings show that Mac-1 is required for normal wound healing but that the attenuation in the deposition of granulation tissue and wound epithelialization in Mac-1 knockout mice is not associated with decreased monocyte migration into the wound.     

The effect of sepsis on wound healing.

BACKGROUND: Normal wound healing is a regulated sequence of events that successfully restore tissue integrity. Previous studies have suggested that wound healing is impaired in a septic host. The current study examines the effect of sepsis on the inflammatory and proliferative phases of wound healing at a remote site of secondary injury. METHODS: Polyvinyl alcohol sponges, either inoculated with a standard dose of Pseudomonas aeruginosa (experimental) or soaked in normal saline (control), were placed subcutaneously in the anterior abdominal region of male B6D2F1 mice. Immediately following sponge placement, full thickness excisional dermal wounds were created on the dorsum. Wound healing was examined at days 3, 5, and 7 postinjury. The infiltration of neutrophils and macrophages into wounds was quantified, and the reepithelialization rate and collagen content were measured. RESULTS: Peripheral neutrophil counts were significantly elevated in infected mice, yet neutrophil content of the remote wound of infected animals was significantly reduced (5% of control, P < 0.05). Wounds of infected mice also showed a 30% reduction in the macrophage content. Wounds of infected animals exhibited delayed reepithelialization (76 +/- 3 vs 97 +/- 3% at day 5, P < 0.05) and collagen synthesis (55.3 +/- 9.5 vs 105 +/- 13.0 microg/wound, P < 0.05). CONCLUSION: Systemic infection alters both the inflammatory and the proliferative processes at remote sites of injury. Multiple factors seem likely to contribute to the increased incidence of wound complications in septic patients.     

Hyperbaric oxygen therapy promotes wound repair in ischemic and hyperglycemic conditions,increasing tissue perfusion and collagen deposition

The treatment of chronic wounds remains inconsistent and empirical. Hyperbaric oxygen therapy (HBOT) is a promising method to improve wound repair but there is still a lack of understanding of its mechanisms of action and its indications are not yet clearly defined. We studied the effects of HBOT in four different wound conditions by inflicting bilateral wounds on the dorsal aspect of the feet of nonischemic or ischemic limbs in normoglycemic or hyperglycemic rats. To create an ischemic condition, arterial resection was performed unilaterally. Forty‐four animals received HBOT five times a week until complete wound closure. Wound repair was compared with 44 rats receiving standard dressing only. HBOT increased blood flow and accelerated wound closure in ischemic and hyperglycemic wounds, most significantly when the two conditions were combined. Wound contraction and reepithelialization were similarly stimulated by HBOT. The acceleration of wound contraction was not associated with increased myofibroblasts expression, nor fibroblast recruitment or higher cell count in the granulation tissue. Of note, we observed a significant increase in collagen deposition in early time points in ischemic wounds receiving HBOT. This data emphasizes that an early application of HBOT might be crucial to its efficacy. We concluded that wounds where ischemia and hyperglycemia are combined, as it is often the case in diabetic patients, have the best chance to benefit from HBOT.     

145Biological Effects of Calreticulin on Wound Repair

Calreticulin (CRT) is a major classic Ca‐binding chaperone protein of the endoplasmic reticulum. Recently, CRT has been recognized to have widespread extracellular effects as well. In the current study we show that CRT increases both epithelial migration and granulation tissue formation in models of porcine and murine wound repair. Partial thickness wounds were created on the paravertebral area of pigs (n = 4) and 0.1% and 0.5% CRT, and PDGF (positive control) applied for 4 consecutive days. In wounds harvested at 5 days, CRT induced a 28 and 22% greater extent of reepithelialization than PDGF and Tris/Ca buffer control, respectively (% healed = 56/CRT; 40.5/PDGF; 44/control). In addition, CRT stimulated earlier granulation tissue formation in a dose‐dependent manner (cumulative dermal depth, microns: 1615/CRT; 1250/PDGF; 1325/control). A similar granulation tissue inducing effect of CRT was also observed in a steroid‐impaired pig model. As a diabetic model of wound repair, two 5 mm circular full‐thickness wounds were created on the dorsum of db/db mice; the wounds were splinted open with silicone rings and covered with occlusive dressing (n = 24). After 5 days of treatment with 0.1, 0.5, and 5% CRT, a dose‐dependent 8‐, 4.5‐, and 2‐fold increase in granulation tissue formation was observed (p < 0.05). However, there was no apparent effect on wound closure. Tissue sections showed a highly cellular dermis in the CRT treated wounds. In addition, CRT (50 pg/ml) stimulated wound closure in a scratch plate assay using fibroblasts by 45%, in 48 hrs, compared to 2% for the control. Therefore, CRT may be a novel agent for wound healing by acting as a chemoattractant for cells involved in wound remodeling and in epithelial migration. Suppported by Calretex, LLC. NJ, USA (LIG) and the Alumni Fund, Alumni Association Downstate College of Medicine (MJC).     

Polyunsaturated phosphatidylcholine protects against wound contraction in experimental skin burn injury.

Polyunsaturated phosphatidylcholine (PP) is a soybean lecithin, whose oral supplementation prevents fibrosis and strictures in animal models. The purpose of the present study was to investigate the effect of PP on wound contraction using a model of skin burn injury. Thirty male Sprague-Dawley rats were randomly divided into three groups: control (C), just burn (JB), and burn treated with PP (BPP). Burns were induced on the dorsum of the rats by a metal plate with a 4-cm(2) contact surface. Ten percent emulsion of PP was given orally for 21 days in BPP group. The effects of PP on burns were evaluated histologically on day 21, and wound contraction was measured by planimetry weekly. Microscopic evaluation included the amount and density of myofibroblasts and collgen type III, the existence of mature collagen fibers in the regenerated dermis, and thickness measurements of the regenerated tissue. There was a significant decrease in mean wound size index in group JB when compared to BPP on days 14 and 21 (p <.05 and p <.01, respectively). After 3 weeks, the granulation tissue was more cell dense, containing high numbers of myofibroblasts in group JB when compared to BPP (p <.05). The wounds had more mature collagen bundles, but less collagen type III in group BPP when compared to JB. Thus, polyunsaturated phospatidylcholine protected against wound contraction and modulated wound healing in the rat model of skin burn injury.     

The murine excisional wound model: Contraction revisited

Rodent models of healing are considered limited because of the perception that rodent wounds heal by contraction while humans heal by reepithelialization The purpose of this report is to present evidence that simple murine excisional wounds provide a valid and reproducible wound model that heals by both contraction and reepithelialization. Previous studies have shown that, although rodent wounds contract by up to 80%, much of this contraction occurs only after epithelial closure. To confirm these previous findings, we measured re‐epithelialization and contraction in three separate mouse strains, (BALB/c, db/+, and db/db); reepithelialization and contraction each accounted for ～40 to 60% of the initial closure of full thickness excisional wounds. After closure, the wound continues to contract and this provides the impression of dominant closure by contraction. In conclusion, the simple excisional rodent wound model produces a well defined and readily identifiable wound bed over which the process of reepithelialization is clearly measurable.     

Importance of defining experimental conditions in a mouse excisional wound model

The murine dorsum dermal excisional wound model has been widely utilized with or without splint application. However, variations in experimental methods create challenges for direct comparison of results provided in the literature and for design of new wound healing studies. Here, we investigated the effects of wound location and size, number of wounds, type of adhesive used for splint fixation on wound healing using splinted or unsplinted dorsum excisional full thickness wound models. One or two 6‐ or 8‐mm full thickness wounds were made with or without splinting in genetically diabetic but heterozygous mice (Dock7^m + / + Lepr^db). Two different adhesives: tissue adhesive and an over the counter cyanoacrylate adhesive (OTCA) “Krazy glue” were used to fix splints. Wound contraction, wound closure, and histopathological parameters including reepithelialization, collagen deposition and inflammation were compared between groups. No significant effect of wound number (1 vs. 2), side (left vs. right and cranial vs. caudal) or size on wound healing was observed. The OTCA group had a significantly higher splint success compared to the tissue adhesive group that resulted in significantly higher reepithelialization and collagen deposition in the OTCA group. Understanding the outcomes and effects of the variables will help investigators choose appropriate experimental conditions for the study purpose and interpret data.     

Topical vascular endothelial growth factor reverses delayed wound healing secondary to angiogenesis inhibitor administration

The prevention of new blood vessel growth is an increasingly attractive strategy to limit tumor growth. However, it remains unclear whether anti-angiogenesis approaches will impair wound healing, a process thought to be angiogenesis dependent. Results of previous studies differ as to whether angiogenesis inhibitors delay wound healing. We evaluated whether endostatin at tumor-inhibiting doses delayed excisional wound closure. C57/BL6J mice were treated with endostatin or phosphate-buffered solution 3 days prior to the creation of two full-thickness wounds on the dorsum. Endostatin was administered daily until wound closure was complete. A third group received endostatin, but also had daily topical vascular endothelial growth factor applied locally to the wound. Wound area was measured daily and the wounds were analyzed for granulation tissue formation, epithelial gap, and wound vascularity. Endostatin-treated mice showed a significant delay in wound healing. Granulation tissue formation and wound vascularity were significantly decreased, but reepithelialization was not effected. Topical vascular endothelial growth factor application to wounds in endostatin-treated mice resulted in increased granulation tissue formation, increased wound vascularity, and wound closure approaching that of control mice. This study shows that the angiogenesis inhibitor endostatin delays wound healing and that topical vascular endothelial growth factor is effective in counteracting this effect.     

Titanium wound chambers for wound healing research

Standardized and reproducible animal models are crucial in medical research. Rodents are commonly used in wound healing studies since, they are easily available, affordable and simple to handle and house. However, the most significant limitation of rodent models is that the wounds heal by contraction while in humans the primary mechanisms of healing are reepithelialization and granulation tissue formation. The robust contraction results in faster wound closure that complicates the reproducibility of rodent studies in clinical trials. We have developed a titanium wound chamber for rodent wound healing research. The chamber is engineered from two pieces of titanium and is placed transcutaneously on the dorsum of a rodent. The chamber inhibits wound contraction and provides a means for controlled monitoring and sampling of the wound environment in vivo with minimal foreign body reaction. This technical report introduces two modalities utilizing the titanium chambers in rats: (1) Wound in a skin island model and, (2) Wound without skin model. Here, we demonstrate in rats how the “wound in a skin island model” slows down wound contraction and how the “wound without skin” model completely prevents the closure. The titanium wound chamber provides a reproducible standardized models for wound healing research in rodents.     

Modulation of scar tissue formation using different dermal regeneration templates in the treatment of experimental full-thickness wounds

The recovery of skin function is the goal of each burn surgeon. Split-skin graft treatment of full-thickness skin defects leads to scar formation, which is often vulnerable and instable. Therefore, the aim of this study was to analyze wound healing and scar tissue formation in acute full-thickness wounds treated with clinically available biopolymer dermal regeneration templates. Full-thickness wounds (3 x 3 cm) on both flanks of Gottingen mini pigs (n= 3) were treated with split-thickness skin graft alone or in combination with a 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide (EDC) cross-linked-collagen scaffold, Integra, or a polyethyleneglycol terephthalate-polybutylene terephthalate (PEGT/PBT) scaffold. The wounds (n= 12 per group) were examined weekly for six weeks to evaluate graft take, contraction (planimetry), and cosmetic appearance. Histologic samples taken after one and six weeks were used to assess scaffold angiogenesis, biocompatibility, and scar tissue quality. In all wounds, one week postwounding graft take was between 93 and 100 percent. The control wound, treated with split-skin graft, showed little granulation tissue formation, whereas the EDC-collagen treated wounds showed two to three times more granulation tissue formation. The collagen scaffold was completely degraded within one week. The Integra and PEGT/PBT scaffolds showed angiogenesis only through two-thirds of the scaffold, which resulted in loss of integrity of the epidermis. Only basal cells survived, proliferated, and regenerated a fully differentiated epidermis within three weeks. Granulation thickness was comparable to collagen scaffold-treated wounds. After six weeks, control wounds showed a wound contraction of 27.2 +/- 6.1 percent, Integra-treated wounds 34.6 +/- 6.4 percent, collagen scaffold-treated wounds 38.1 +/- 5.0 percent, and PEGT/PBT scaffold-treated wounds 54.5 +/- 3.9 percent. The latter wounds had significantly more contraction than wounds of other treatment groups. Microscopically, the control and collagen scaffold-treated wounds showed an immature scar tissue that was two times thicker in the EDC-collagen treated wounds. The Integra-treated wounds showed nondegraded collagen scaffold fibers with partly de novo dermal tissue formation and partly areas with giant cells and other inflammatory cells. The PEGT/PBT scaffold was almost completely degraded. Scaffold particles were phagocytosized and degraded intracellularly by clusters of macrophages. The scar tissue was in the early phase of ECM remodeling. In conclusion, this study showed that the rate of dermal tissue formation and scarring is influenced by the rate of scaffold angiogenesis, degradation, and host response induced by the scaffold materials.     

Acceleration of wound healing with topically applied deoxyribonucleosides.

HYPOTHESIS: We hypothesized that a topical mixture of purified deoxyribonucleosides would accelerate wound healing in an open wound model. DESIGN: Full-thickness 6-mm wounds were made on the ears of young adult rabbits. In some experiments, 2 of the 3 arteries in each ear were divided to induce wound ischemia. INTERVENTIONS: An equiweight mixture containing all 4 of the major deoxyribonucleosides (deoxyadenosine, deoxycytidine, deoxyguanosine, and thymidine), designated PN105, or other subgroups of deoxyribonucleosides, or vehicle (saline) was applied to wounds on 1 ear every 2 days, with the other ear serving as a control. MAIN OUTCOME MEASURES: Wound tissue was processed for histological examination 7 days after the initial wounding. Granulation tissue formation and epithelialization were measured in histological cross sections of wounds. RESULTS: Treatment of wounds with PN105 resulted in a 191% increase in total new granulation tissue (P<.05) and a higher incidence of complete wound reepithelialization (67% vs 37%; P<.05) when compared with controls, and a similar increase under ischemic conditions on day 7. Wound ischemia markedly impairs healing; PN 105 treatment resulted in a 242% increase in the amount of new granulation tissue formed by day 7 in ischemic wounds, relative to the appropriate controls (P<.05). All 4 of the major deoxyribonucleosides were required for optimum activity; mixtures with 3 or 2 were less active or inactive. CONCLUSIONS: Topically applied deoxyribonucleosides reproducibly accelerate wound healing in normal and ischemic wounds, and to a magnitude equivalent to that of recombinant growth factors such as platelet-derived growth factor, previously studied in this model. In view of their safety, availability, and efficacy, deoxyribonucleosides hold considerable promise for improving healing of chronic wounds.     

Histologic characterization of vaginal vs. abdominal surgical wound healing in a rabbit model

We aimed to compare the histologic characteristics of vaginal vs. abdominal surgical wound healing in the rabbit. Bilateral 6 mm full-thickness circular segments were excised from the vagina and abdominal skin in 34 New Zealand white female rabbits. Animals were euthanized on the day of and 4, 7, 10, 14, 21, 28, and 35 days after wounding, and their wounds were evaluated using a modified scoring system. The inter- and intraobserver agreements of the scoring system were good (weighted kappa 0.63 and 0.71, respectively). A transient fibrinous crust was evident in 75% of the abdominal and in none of the vaginal wound specimens on days 4-7 after wounding (p=0.01). Acute inflammation peaked at day 4 in both the vaginal and abdominal wounds, while chronic inflammation peaked at days 4-7 and 14-21 in the abdomen and vagina, respectively. Both neovascularization and the amount of granulation tissue peaked at days 4 and 7 in the vagina and abdomen, respectively. Maturation of granulation tissue and collagen deposition increased persistently in both tissues until postwounding day 35. Reepithelialization increased after wounding, and was completed by day 14 in both tissues. The surgical wound-healing process in both the vagina and abdomen includes transient acute and chronic inflammation, fibroblast proliferation, and neovascularization, as well as progressive maturation of granulation tissue, reepithelialization, and collagen deposition. A transient fibrinous crust forms in the abdomen but not in the vagina 4-7 days after wounding. The modified histologic scoring system described here was found to be reliable and reproducible.     

Exogenous calreticulin improves diabetic wound healing

A serious consequence of diabetes mellitus is impaired wound healing, which largely resists treatment. We previously reported that topical application of calreticulin (CRT), an endoplasmic reticulum chaperone protein, markedly enhanced the rate and quality of wound healing in an experimental porcine model of cutaneous repair. Consistent with these in vivo effects, in vitro CRT induced the migration and proliferation of normal human cells critical to the wound healing process. These functions are particularly deficient in poor healing diabetic wounds. Using a genetically engineered diabetic mouse (db/db) in a full‐thickness excisional wound healing model, we now show that topical application of CRT induces a statistically significant decrease in the time to complete wound closure compared with untreated wounds by 5.6 days (17.6 vs. 23.2). Quantitative analysis of the wounds shows that CRT increases the rate of reepithelialization at days 7 and 10 and increases the amount of granulation tissue at day 7 persisting to day 14. Furthermore, CRT treatment induces the regrowth of pigmented hair follicles observed on day 28. In vitro, fibroblasts isolated from diabetic compared with wild‐type mouse skin and human fibroblasts cultured under hyperglycemic compared with normal glucose conditions proliferate and strongly migrate in response to CRT compared with untreated controls. The in vitro effects of CRT on these functions are consistent with CRT's potent effects on wound healing in the diabetic mouse. These studies implicate CRT as a potential powerful topical therapeutic agent for the treatment of diabetic and other chronic wounds.     

The bone marrow-derived endothelial progenitor cell response is impaired in delayed wound healing from ischemia

OBJECTIVE: Vasculogenesis relies on the recruitment of bone marrow-derived endothelial progenitor cells (BMD EPCs) and is stimulated by tissue-level ischemia. We hypothesized that the BMD EPC response is impaired in ischemic wounds and studied the relationship between BMD EPCs and wound healing. METHODS: We used transgenic Tie-2/LacZ mice, which carry the beta-galactosidase (beta-gal) reporter gene under Tie-2 promoter control. Wild-type mice were lethally irradiated and reconstituted with Tie-2/LacZ bone marrow. Four weeks later, the mice underwent unilateral femoral artery ligation/excision and bilateral wounding of the hindlimbs. Ischemia was confirmed and monitored with laser Doppler imaging. A subset of mice received incisional vs excisional nonischemic bilateral hindlimb wounds, without femoral ligation. Excisional wound closure was measured by using daily digital imaging and software-assisted calculation of surface area. RESULTS: Ischemia resulted in significantly delayed wound healing and differentially affected the number of BMD EPCs recruited to wound granulation tissue and muscle underlying the wounds. At 3 days postwounding, the granulation tissue of the wound base contained significantly fewer numbers of BMD EPCs in ischemic wounds compared with the nonischemic wounds (P < .05). In contrast, significantly more BMD EPCs were present in the muscle underlying the ischemic wounds at this same time point compared with the muscle under the nonischemic wounds (P < .05). In ischemic wounds, eventual wound closure significantly correlated with a delayed rise in BMD EPCs within the wound granulation tissue (Kendall's correlation, -.811, P = .0005) and was significantly associated with a gradual recovery of hindlimb perfusion (P < .0001). By 7 days postwounding, BMD EPCs were incorporated into the neovessels in the granulation tissue. At 14 days and 75 days, BMD EPCs were rarely observed within the wounds. CONCLUSIONS: Granulation tissue of excisional ischemic wounds showed significantly less BMD EPCs 3 days postwounding, in association with significantly delayed wound closure. However, the number of BMD EPCs were increased in ischemic hindlimb skeletal muscle, consistent with the notion that ischemia is a powerful signal for vasculogenesis. To our knowledge, this is the first report identifying a deficit in BMD EPCs in the granulation tissue of ischemic skin wounds and reporting the key role for these cells in both ischemic and nonischemic wound healing.