Maria B. Witte

GENERAL PRINCIPLES OF WOUND HEALING

Wound healing is a complex process involving different biologic and immunologic systems. Despite improvements in diagnostics and therapy, wound failures remain a clinical problem. The approach to a nonhealed wound is an interdisciplinary challenge that should not be underestimated. Better understanding of the complex wound-healing cascade helps our approach to wound healing and its possible failure. Manipulations of the involved immunologic features offer future therapeutic strategies.

Role of nitric oxide in wound repair

After injury, wound healing is essential for recovery of the integrity of the body. It is a complex, sequential cascade of events. Nitric oxide (NO) is a small radical, formed from the amino acid L-arginine by three distinct isoforms of nitric oxide synthase. The inducible isoform (iNOS) is synthesized in the early phase of wound healing by inflammatory cells, mainly macrophages. However many cells participate in NO synthesis during the proliferative phase after wounding. NO released through iNOS regulates collagen formation, cell proliferation and wound contraction in distinct ways in animal models of wound healing. Although iNOS gene deletion delays, and arginine and NO administration improve healing, the exact mechanisms of action of NO on wound healing parameters are still unknown. The current review summarizes what is known about the role of NO in wound healing and points out path for further research.

Nitric Oxide and Wound Healing

Nitric oxide is a short-lived free radical that acts at the molecular, cellular, and physiologic level. Since its discovery almost 20 years ago it has proven itself as an important element in wound healing. This review highlights many of the important aspects of nitric oxide in wound healing, including a review of the basic biology of nitric oxide, its role as part of the cytokine cascade and as a promoter of angiogenesis, as well as its more recently elucidated role in apoptosis.

A new wound-based severity score for diabetic foot ulcers: A prospective analysis of 1,000 patients.

OBJECTIVE—Several well-accepted classification systems are available for diabetic foot ulcers. However, there are only a few and scientifically not validated severity scores. The aim of this study was to establish a new wound-based clinical scoring system for diabetic foot ulcers suitable for daily clinical practice anticipating chances for healing and risk of amputation. RESEARCH DESIGN AND METHODS—Four clinically defined parameters, namely palpable pedal pulses, probing to bone, ulcer location, and presence of multiple ulcerations, were prospectively assessed in 1,000 consecutive patients. In the next step, a new diabetic ulcer severity score (DUSS) was created from these parameters. Palpable pedal pulses were categorized by the absence (scored as 1) or presence (scored as 0) of pedal pulses, while probing to bone was defined as yes (scored as 1) or no (scored as 0). The site of ulceration was defined as toe (scored as 0) or foot (scored as 1) ulcer. Patients with multiple ulcerations were graded as 1 compared with those with single ulcers (scored as 0). The DUSS was calculated by adding these separate gradings to a theoretical maximum of 4. Wounds were followed-up for 365 days or until healing or amputation if earlier. Probability of healing and risk of amputation were calculated by the Kaplan-Meier method. RESULTS—Uni- and multivariate analyses showed a significantly higher probability of healing for patients with palpable pulses, no probing to bone, toe ulcers, and absence of multiple ulcerations. When patients were divided into subgroups with the same DUSS, we found significantly different probabilities for healing. We showed a decreasing probability of healing for ulcers with a high DUSS, concurrent with increasing amputation rates. An increase in the DUSS by one score point reduced the chance for healing by 35%. Similarly, the higher the ulcer score, the larger the initial wound area, the longer the wound history, and the more likely the need for surgery or hospitalization. CONCLUSIONS—The DUSS categorizes different ulcers into subgroups with specific severity and similar clinical outcome. Using this score, the probabilities for healing, amputation, need for surgery, and hospitalization are predictable with high accuracy. This might be useful for the anticipation of health care costs and for comparison of subgroups of patients in clinical studies.

Supplemental L-arginine enhances wound healing in diabetic rats

L‐arginine has been shown to enhance wound strength and collagen deposition in rodents and humans. Diabetes mellitus, which impairs wound healing, is accompanied by a reduction in nitric oxide at the wound site. The amino acid L‐arginine is the only substrate for nitric oxide synthesis. We sought to determine whether supplemental L‐arginine can restore the impaired wound healing of diabetic rats. Fifty‐six male Lewis rats were used in this study, of which twenty‐nine rats were rendered diabetic 7 days prior to surgery with intraperitoneal streptozotocin. Twenty‐seven untreated rats served as controls. Animals underwent a dorsal skin incision with implantation of polyvinyl‐alcohol sponges. Sixteen diabetic and 14 normal rats received 1 g/kg/day of L‐arginine by injection, while the remainder received saline injections only. Animals were euthanized 10 days postwounding, and their wounds were analyzed for breaking strength. The wound sponges were assayed for total hydroxyproline and nitrite/nitrate content. Plasma and wound fluid concentrations of L‐arginine, ornithine, and citrulline were determined. Wound sponge RNA was extracted and subjected to Northern blot analysis for procollagen I and III. Diabetic wounds had greatly decreased breaking strengths compared with controls. L‐arginine significantly enhanced wound breaking strengths in both control (+23%) and diabetic animals (+44%), and also increased wound hydroxyproline levels in both diabetic (+40%) and control animals (+24%) as compared to their saline‐treated counterparts. mRNA for procollagen I and III were elevated by L‐arginine treatment in both diabetic rats and controls. Treatment with L‐arginine significantly increased wound fluid nitrite/nitrate levels in diabetic animals. The data show that the impaired healing of diabetic wounds can be partially corrected by L‐arginine supplementation, and that this effect is accompanied by enhanced wound nitric oxide synthesis. (WOUND REP REG 2003;11:198–203)

Repair of full-thickness bowel injury.

ObjectiveColon surgery is more and more often performed in complex situations such as after trauma, under immunosuppression, or in the elderly. Even under optimal conditions, anastomosis fails in certain situations. The objective of this study was to demonstrate the normal phases of bowel healing and to review the local and systemic factors affecting healing with special attention to critical care variables such as major surgery, acute hemorrhage, and infections. Data SourceMEDLINE cited and/or published articles. DesignReview analysis. ResultsColon healing is a structured cascade of different phases that can be affected by a multitude of local (infection, ischemia) and systemic (diabetes, malnutrition, anemia, hypothermia, trauma) factors. The normal phases of repair, the resulting bursting pressure as an experimental index of healing, and the available published data on local and systemic factors affecting healing are summarized. ConclusionSeveral local and systemic factors negatively affect bowel healing; there is still a small portion of patients who fail to heal, suggesting that intrinsic factors need to be analyzed.

Expression and function of inducible nitric oxide synthase during rat colon anastomotic healing.

Nitric oxide plays a significant but incompletely understood role in fibroblast function and cutaneous wound collagen synthesis; however, the participation of inducible nitric oxide synthase (iNOS) in gastrointestinal anastomotic healing has not been studied. Male Sprague-Dawley rats underwent single-layer left colonie anastomosis. Animals were killed at 24-hour intervals postoperatively and the anastomosis was excised. Parallel uninjured colon tissue samples were also analyzed. Reverse transcriptasepolymerase chain reaction confirmed the absence of iNOS messenger RNA in control colon and expression of the gene in anastomotic tissue on all study days. Northern hybridization demonstrated maximal iNOS messenger RNA transcription on day 1 with decreased levels on days 3 and 5. iNOS enzyme activity, measured biochemically by the conversion of [3H]-arginine to [3H]-citrulline ex vivo, was also maximal on day 1 (7.35 ±1.34 pmol/mg protein/min [± standard error of the mean], n = 10) and decreased on days 3 (4.37 ±2.32 pmol/mg protein/min; n = 6) and 5 (2.80 ± 0.92 pmol/mg protein/min; n = 6). Immunohistochemical staining demonstrated that (1) iNOS expression is confined to a discrete cell population in the region of the anastomosis containing inflammatory cells; (2) those cells assume a highly conserved position on the luminal edge of the proliferating scar; and (3) the iNOS-expressing cells are present throughout the fibroplastic phase of healing. To functionally assess the role of iNOS in colonie healing, rats were treated with a continuous intravenous infusion of S-methylisothiourea (a selective inhibitor of iNOS) at a dosage of 200 mg/kg/day for 5 days after anastomosis. There was a significantly reduced anastomotic bursting pressure in rats treated with the inhibitor as compared to rats treated with intravenous normal saline solution (108.4 ±13.2 mm Hg vs. 148.4 ±10.3 mm Hg; P <0.05). These results suggest that iNOS gene expression is induced during colonie anastomotic healing, that it is present through all phases of healing but is maximal through the inflammatory phase, and that iNOS activity is required for optimal anastomotic healing.

The Route of Nutrition Support Affects the Early Phase of Wound Healing

BACKGROUND Nutrition support via the enteral route has been shown to be superior to parenteral administration in maintaining immune function, decreasing septic complications, and increasing survival after severe trauma and surgical injury. Whether the route of nutrition support affects wound healing, another important determinant of outcome following injury, is not known. METHODS Forty-nine Sprague-Dawley rats, 290 to 360 g body wt, underwent identical surgical manipulation consisting of central venous catheterization, fashioning of gastrostomy and dorsal skin incision, and placement of polyvinyl alcohol sponges into subcutaneous pockets. Identical infusates of 25% dextrose, 4.25% amino acids, and vitamins were given, half the animals receiving the infusion via the gastrostomy and the other half via the venous catheter. Animals were killed on day 5, 7, or 10. Wound breaking strength, sponge hydroxyproline content (an index of wound collagen deposition), and types I and III collagen gene expression were measured. RESULTS There were no nutritional differences between the two groups in terms of energy intake, body weight gain, and plasma levels of albumin, total protein, or urea nitrogen. On day 5 wound breaking strength was significantly higher in the enterally supported group (89.3 +/- 90.7 vs 64.9 +/- 40.2 g for the parenteral group, p < .05). This was paralleled by enhanced wound collagen accumulation (182 +/- 19 vs 132 +/- 13 microg, p < .05). Gene expression of type I, but not type III, collagen also was increased in the enterally fed group. There were no differences noted between the two groups in wound healing parameters 7 and 10 days after injury. CONCLUSIONS The data demonstrate that the route of nutrition administration can influence wound healing. The beneficial effect of the enteral feeding route is limited to the early phases of healing.

M.A.I.D.: a prognostic score estimating probability of healing in chronic lower extremity wounds.

Objective:To evaluate a wound-based prognostic score for chronic lower extremity wounds suitable for daily routine use capable of predicting long-term healing. Summary Background Data:The main obstacle in the treatment of chronic wounds is to estimate long-term clinical outcome. For diabetic foot ulcers, several ulcer, and nonulcer-related risk factors associated with impaired healing have been described in the past. Methods:A new chronic lower extremity ulcer score (M.A.I.D.) was created out of 4 clinically defined parameters, namely palpable pedal pulses (I), wound area (A), ulcer duration (D), and presence of multiple ulcerations (M). Palpable pedal pulses were categorized by the absence (scored as 1) or presence (scored as 0) of pedal pulses, while wounds >4 cm2 were scored as 1 and wounds ≤4 cm2 as 0. Ulcers lasting more than 130 days were categorized as 1 and wounds with a duration of <130 days as 0. Patients with multiple ulcerations were graded as 1 (=1) compared with those with single ulcers (=0). M.A.I.D. was calculated by adding these separate scores to a theoretical maximum of 4. Results:Two thousand nineteen consecutive patients with 4004 wounds were included. When patients were divided into subgroups with the same M.A.I.D., we showed a decreasing probability of healing for ulcers with higher M.A.I.D. scores. An increase in the M.A.I.D. by 1 score-point reduced the chance for healing by 37%. Similarly, the higher the ulcer score, the larger the initial wound area, the longer the wound history, and the more likely the occurrence of soft-tissue infection during follow-up. Conclusions:This new chronic lower extremity ulcer score is capable of anticipating long-term probability of healing by combining 4 clinically assessable parameters. However, adequate and standardized wound care is an indispensable prerequisite for M.A.I.D. to be a valid diagnostic tool in daily clinical routine.

Experimental ischemic wounds: correlation of cell proliferation and insulin-like growth factor I expression and its modification by different local IGF-I release systems.

We investigated cell proliferation and local insulin‐like growth factor‐I (IGF‐I) expression in ischemic wounds after topical application of IGF‐I through different delivery systems. IGF‐I dressings were fabricated from an IGF‐I containing polyvinyl alcohol film placed on a standard hydrogel dressing. In vitro, the release of IGF‐I from this dressing was assessed by enzyme‐linked immunosorbent assay. For animal experiments, a standardized ischemic skin flap containing a full‐thickness wound was created on the back of male Sprague‐Dawley rats. An identical wound outside the flap served as control. We initially investigated intracutaneous pO2 (ptiO2), cell proliferation, and local IGF‐I expression. In a second setting, wounds were treated either with IGF‐I dissolved in methylcellulose gel or with an IGF‐I dressing, and ulcer size and cell proliferation were assessed. In vitro, approximately 60% of IGF‐I was released from the IGF‐I dressing, compared to a 97% release from methylcellulose gel. In vivo, ischemic wounds showed less cell proliferation and decreased IGF‐I expression than nonischemic wounds. A lower local ptiO2 correlated with larger wound size, less cell proliferation, and decreased IGF‐I expression. Ulcer size was reduced after treatment with either IGF‐I dressing or methylcellulose gel. However, cell proliferation only increased after treatment with IGF‐I dressing, but not after methylcellulose gel treatment. We conclude that IGF‐I expression is decreased in ischemic wounds and correlates with low cell proliferation. This can be reversed by local IGF‐I application, but the efficacy of treatment depends on the delivery system.