David R. Knighton

Classification and treatment of chronic nonhealing wounds: successful treatment with autologous platelet-derived wound healing factors (PDWHF)

Previous animal data showed that platelets contain growth factors that stimulate capillary endothelial migration (angiogenesis), fibroblast proliferation and migration, and collagen synthesis. This study utilized autologous platelet-derived wound healing factors (PDWHF) to treat 49 patients with chronic nonhealing cutaneous ulcers. Patients were classified on the basis of 20 clinical and wound status parameters to generate a wound severity index. Forty-nine patients--58% diabetic (20% with renal transplants); 16% with trauma, vasculitis, etc.; 14% with decubitus ulcers; and 6% each with venous stasis or arterial insufficiency--with a total of 95 wounds had received conventional wound care for an average of 198 weeks (range: 1-1820 weeks). After informed consent was obtained, patients received autologous PDWHF. Mean 100% healing time for all patients was 10.6 weeks. There was no abnormal tissue formation, keloid, or hypertrophic scarring. A multivariant analysis showed a direct correlation to 100% healing with initial wound size and the initiation of PDWHF therapy. This is the first clinical demonstration that locally acting growth factors promote healing of chronic cutaneous ulcers.

Definitions and guidelines for assessment of wounds and evaluation of healing

Chronic wounds represent a worldwide problem. For laboratory and clinical research to adequately address this problem, a common language needs to exist. This language should include a system of wound classification, a lexicon of wound descriptors, and a description of the processes that are likely to affect wound healing and would healing end points. The report that follows defines wound, acute wound, chronic wound, healing and forms of healing, wound assessment, wound extent, wound burden, and wound severity. The utility of these definitions is demonstrated as they relate to the healing of a skin wound, but these definitions are broadly applicable to all wounds.

Wound healing agents derived from platelets

Platelet enriched plasma is produced from blood. The platelets are activated by thrombin which causes the release of platelet derived growth and angiogenesis factors. A carrier such as a microcrystalline collagen is added to produce a wound treating salve. The compound is applied directly to wounds and initiates healing in non-healing wounds as well as accelerating normal wound healing by increasing vascularization, stimulating fibroblast mitosis and migration and increasing collagen synthesis by fibroblasts. The process of treatment involves the use of a composition containing the materials released by platelets during the platelet release reaction.

Wound healing angiogenesis : indirect stimulation by basic fibroblast growth factor

Basic fibroblast growth factor (bFGF) was tested for its ability to stimulate angiogenesis in vivo using the rabbit corneal assay. Basic FGF (50-1,000 ng) was incorporated into 10% Hydron, and 50-500 ng of bFGF were incorporated into 10% Elvax. Human serum albumin (HSA) (10 ng) and 50 ng of transforming growth factor-beta (TGF-beta) served as negative and positive controls. Pellets of the polymers containing test compounds were implanted in the rabbit cornea, examined daily, and after 7 days corneal angiogenesis was scored on a graded scale [(-) for no response and +4 for a maximum response]. Histologic analysis of the corneas was performed on days 2 and 7. Basic FGF (50-500 ng) in Hydron failed to stimulate significant angiogenesis, though it did induce angiogenesis accompanied by inflammation at the 1,000-ng dose. Basic FGF in Elvax elicited inflammation-associated +3 to +4 responses at all doses tested. New blood vessels did not form in response to HSA in Hydron or Elvax, while TGF-beta induced +4 angiogenesis accompanied by vigorous inflammation. In vivo release kinetics for bFGF in Hydron and Elvax were compared, and the release of bioactive bFGF from Hydron and Elvax was demonstrated in vitro. These results suggest that the bFGF and Elvax combination incites an inflammatory response which stimulates indirect angiogenesis, while the same concentrations of bFGF delivered in Hydron produced no inflammation or angiogenesis. Although bFGF alone is a potent mitogen for endothelial cells, it does not appear to directly stimulate in vivo angiogenesis.

Identification and partial characterization of angiogenesis bioactivity in the lower respiratory tract after acute lung injury.

Survival after acute lung injury (ALI) depends on prompt alveolar repair, a process frequently subverted by the development of granulation tissue within the alveolar airspace. Immunohistochemical examination of the intraalveolar granulation tissue confirmed that capillaries as well as myofibroblasts were the principal cellular constituents. We therefore hypothesized that angiogenesis factors would be present on the air-lung interface after ALI. To evaluate this hypothesis, bronchoalveolar lavage fluid from patients with ALI (n = 25) and patient controls (n = 8) was examined for angiogenesis bioactivity by its ability of induce endothelial cell migration. While lavage fluid from controls had no bioactivity, lavage fluid from 72% of patients with ALI promoted endothelial cell migration. Heparin affinity, ion exchange, and gel filtration chromatography resolved the bioactivity into at least two moieties. One appeared identical to the well characterized endothelial cell growth factor, basic fibroblast growth factor. The other was a 150-kD non-heparin binding protein that mediated endothelial cell migration and attachment in vitro, and the growth of new vessels in vivo. These data are consistent with the hypothesis that the growth of capillaries into the alveolar airspace results from angiogenesis factors present on the alveolar surface of the lung after ALI.

Amputation prevention in an independently reviewed at-risk diabetic population using a comprehensive wound care protocol

An independent review panel composed of an orthopedic surgeon, a vascular surgeon, and an endocrinologist was convened to conduct a case history review. The 71 patients reviewed had 124 wounds on 81 limbs and participated in the comprehensive wound management program of the University of Minnesota. Based on their expertise, the review panel classified the wounds by severity and identified the limbs risk for amputation. The resulting scores were then compared with the patients actual outcome. The review panel predicted 65 (80%) of the limbs would be salvaged and 16 (20%) would be amputated. The actual outcome was that 75 (93%) of the limbs were salvaged and 6 (7%) were amputated (p less than 0.005). The universitys wound management program was highly successful, compared with the predictions of the reviewers.

The deleterious effect of reduced pH and hypoxia on neutrophil migration in vitro

The effect of altered microenvironment on human neutrophil locomotion was investigated. Reduced pH impaired both random and chemotactic migration using both the under-agarose and Boyden chamber techniques. Using the under-agarose technique, migration was progressively inhibited below pH 6.5, achieving significance at pH 5.5 (P less than 0.01 vs pH 7.5 for chemotactic and random migration). A similar pattern was noted using the Boyden chamber technique. At pH 7.40, extreme hypoxia (less than 30 mm Hg) caused a small (15-25%) but significant reduction in chemotactic migration. An additive deleterious effect of low pH and hypoxia on PMN migration was also found. These studies suggest that altered local microenvironment may contribute to the failure of host leukocytes to resolve infection.

Migration of myogenic cells in the rat extensor digitorum longus muscle studied with a split autograft model.

SummaryThe ability of myogenic cells to migrate perpendicular to the long axis of freely autografted muscles was examined. Rat extensor digitorum longus muscles were divided, and one half was devitalized by repeated freezing in liquid nitrogen while the other half was kept viable in physiologic saline. The halves were reunited with sutures and grafted back into the original muscle bed. At intervals between 5 and 25 days the grafts were removed and examined histologically for the presence of myotubes within the devitalized region. Myotubes were first seen in the devitalized half 10 days postgrafting with the maximum number of myotubes observed after 12 to 15 days. These results indicate that myogenic cells are capable of migration perpendicular to the long axis of the muscle fibers in an autograft.

Hypoxia and endotoxin induce macrophage-mediated suppression of fibroblast proliferation.

Cellular mechanisms and environmental factors contributing to wound failure following shock and wound contamination are unclear. The activation of macrophages by exposure to hypoxia (pO2 less than 20) and/or lipopolysaccharide (10 micrograms/ml) in vitro was investigated for its effect on macrophage regulation of fibroblast proliferation. The effect on fibroblast proliferation of conditioned medium from activated murine macrophages or co-culture with activated macrophages was tested by measuring 3T3 fibroblast incorporation of 3H-thymidine and culture DNA content. Unstimulated macrophages produced growth factors that increase fibroblast proliferation (proliferation index (PI) = 1.4 +/- 0.15, p less than 0.05 vs. control). Activation by hypoxia alone had little effect on macrophage regulation of fibroplasia (PI = 1.55 +/- 0.28, N.S. vs. unstimulated macrophages). LPS activated macrophages suppressed fibroplasia and the combination of hypoxia with LPS augmented the suppression (PI = 0.5 +/- 0.11, LPS alone, p less than 0.05 and 0.25 +/- 0.05, LPS + hypoxia, p less than 0.01). In addition, hypoxia + LPS treated co-cultures had reduced DNA contents, suggesting reduced cell numbers (12.5 +/- 2.6 micrograms vs. 8.2 +/- 2.0 micrograms). We screened several macrophage cytokines for their direct effect on 3T3 proliferation and found that mr-Tumor Necrosis Factor-alpha (150 units) also suppressed proliferation. Conditioned supernatants from LPS activated macrophages contained 12 +/- 2 units of mrTNF as measured by L929 cytolysis; however, this was significantly less than required to induce suppression of proliferation by direct addition. The regulatory role of the macrophage appears to be dependent on its level of activation. Activation by hypoxia and LPS altered macrophage regulation of fibroblast proliferation from stimulation to suppression.(ABSTRACT TRUNCATED AT 250 WORDS)

Alloxan diabetes alters the rabbit transarterial wall oxygen gradient

PURPOSE Atherosclerotic vascular occlusive disease is the most common complication of diabetes mellitus and accounts for 75% of deaths in diabetic patients. Determining the initiator and continuing stimulus for the cellular events in the formation of atherosclerotic lesions in diabetic patients could lead to the prevention of this common and deadly complication. Diabetes-induced arterial wall hypoxia is proposed as an initiator and continuing stimulus for atherosclerotic vascular occlusive disease. METHODS Transarterial wall oxygen gradient measurements were performed on the infrarenal aorta with an oxygen microelectrode 14 to 16 weeks after the induction of alloxan diabetes in rabbits. RESULTS Both insulin-treated and untreated alloxan diabetic rabbits revealed significantly decreased oxygen tensions throughout the arterial wall compared with control rabbits. There was no significant difference in the transarterial wall oxygen gradient between the two groups of diabetic rabbits. This effect was noted despite no difference in the partial pressure of oxygen in arterial blood or visual evidence of atherosclerotic lesion formation in the three groups. CONCLUSIONS These findings suggest that diabetes induces arterial wall hypoxia independent of insulin therapy and before the formation of atherosclerotic lesions. Diabetes-induced arterial wall hypoxia may contribute to the formation of atherosclerotic lesions.