Platelet-derived growth factor-BB accelerates wound closure in a new mesentery culture model without macrophages.

Platelet-derived growth factor-BB has multiple effects in vitro and has been demonstrated to accelerate wound healing in several animal models, but its precise mechanisms of action in vivo have not been fully explained. A rat mesentery culture model was developed, and platelet-derived growth factor-BB (100 ng/ml) was added to the medium containing 2% fetal calf serum for examining its closure effects. The mesentery specimens were stained by routine hematoxylin and eosin and immunohistochemical staining for macrophages. A significant improvement in closure rate and complete closure rate was seen when platelet-derived growth factor-BB was added to the medium compared with the controls (p < 0.05). There are significantly fewer macrophages in the in vitro specimen than in the in vivo specimen. In conclusion, platelet-derived growth factor-BB can contribute to wound closure of a mesenteric defect even without macrophages, suggesting a second mechanism in which it can act directly on mesenchymal cells during wound healing. This tissue-culture model is useful for gaining further insights into growth factor interactions and wound healing.

Modulation of wound healing response in chronic irradiated tissues.

Radiation wounds represent some of the most difficult wounds to treat. Historically, there have been few nonsurgical options. Recent research suggests future potential of a therapeutic role for growth factors. Hyperbaric oxygen is currently widely used, but its addition to growth factors may give much greater benefits in the future.

Enhancement of wound healing by the alkaloid taspine defining mechanism of action.

Taspine (mol wt 369,000) is an alkaloid extracted from trees of Croton (family Euphorbiaceae) of the western Amazon region that has been used by natives and others as a vulnerary agent. Taspine was purified from tree sap to test its healing properties using different topical concentrations in the paired rat surgical incision model. Wound tensile strength and histology were evaluated. Samples treated with 250 micrograms, but not those treated with 50 micrograms or 10 micrograms, had significant higher values for MBS than paired controls (26%, P < 0.005, and 30%, P < 0.001, by Days 5 and 7, respectively). Taspine did not modify MBS at Day 12. Sample treated with 250 micrograms had significantly greater mononuclear cellular infiltration at Days 5 and 7 but not at Day 12. To better understand the effect of taspine as an enhancer of wound healing, we conducted in vitro studies in cell cultures. Taspine stimulated chemotaxis for fibroblasts. Taspine did not have an effect on specific assays for macrophage chemotaxis, neutrophil activation, fibroblast proliferation, or matrix assembly. Taken together, the data suggest that taspine promotes early phases of wound healing in a dose-dependent manner with no substantial modification thereafter. Its mechanism of action is probably related to its chemotactic properties on fibroblasts and is not mediated by changes in extracellular matrix.

Occurrence and localization of transforming growth factor-beta (TGF-beta 1, beta 2) during rabbit skin development.

The expression of (TGF beta-1) and (TGF beta-2) in adult, fetal, and neonatal rabbit skin has been analyzed by immunohistochemistry. Samples were collected from the backs of fetal rabbits (23 and 30 days of gestation), newborn rabbits (1 day post-partum) and adult pregnant and nonpregnant rabbits. Forty samples were studied. To identify TGF-beta two antibodies were used: (RAB4) and (CL-B1/29). TGF-beta 1 and TGF-beta 2 displayed distinct distribution and intensity of staining at different stages of development. TGF-beta 1 was present in fetal skin (23 days of pregnancy) expressing abundant staining in cells within epidermis, dermis and hypodermis. Fetal skin (30 days of pregnancy) exhibited weak staining for TGF-beta 1; only isolated dermal cells in close proximity to hair follicles and sweat glands expressed intracellular staining. Newborn rabbit skin showed low expression for TGF-beta 1, around hair follicles and sweat glands. In contrast adult pregnant and non-pregnant rabbit skin exhibited abundant staining for TGF-beta 1 in epidermis, papillary dermis, around hair follicles, sweat glands, and blood vessels, no difference was observed among pregnant and non-pregnant rabbits. TGF-beta 2 was detected in all specimens and the distribution and intensity of staining were similar to those found for TGF-beta 1 in all groups. Our findings demonstrate that the expression of TGF-beta 1 and TGF-beta 2 is intense during early stages of rabbit skin development when morphogenesis is taking place and skin appendages are being formed; and less intense when these structures are formed and skin growth predominates. These results suggest an important role for TGF-beta 1 and TGF-beta 2 in rabbit skin development, probably regulating morphogenesis and growth.

Acceleration of tissue repair by transforming growth factor beta 1: identification of in vivo mechanism of action with radiotherapy-induced specific healing deficits.

BACKGROUND: Transforming growth factor beta 1 (TGF-beta 1) is an effective accelerator of soft tissue repair in both normal and impaired healing models; however, its in vivo mechanism of action remains unclear. Modern radiation techniques can create unique healing deficits, allowing for a more specific definition of tissue response to growth factor therapy. In the rat linear skin incision model, cobalt 60 photon beam total body irradiation (TBI), 800 rads, causes a marked depression of circulating monocytes and largely spares the skin tissue. Megavoltage electron beam surface irradiation (SI), 2500 rads, markedly impairs surface healing while sparing the bone marrow. With these models of selective healing deficits, the ability of TGF-beta 1 to accelerate tissue repair directly in the absence of circulating macrophage precursors (TBI) or in the presence of damaged dermal fibroblasts (SI) was evaluated. METHODS: Adult male Sprague-Dawley rats were randomly assigned to groups of TBI, SI, or nonirradiated sham controls and received radiation 2 days before wounding. Paired linear full-thickness skin incisions were created and a single dose of TGF-beta 1 (2 micrograms/wound) or vehicle control was applied to each wound. RESULTS: Both radiation techniques produced a marked healing deficit when assessed on postwounding days 7 and 12. TBI treatment was characterized by severe monocytopenia, confirmed by a tissue macrophage-specific immunohistochemical technique. On days 7 and 12 after wounding, TGF-beta 1 significantly accelerated soft tissue repair and wound-breaking strength in the TBI-treated rats, demonstrating an ability to directly promote the induction of collagen synthesis in the absence of monocytes/macrophages. In contrast, TGF-beta 1 was unable to reverse the SI-induced healing deficit characterized by impaired function of dermal fibroblasts. CONCLUSIONS: These in vivo observations provide further evidence for a direct mechanism of action by TGF-beta 1 on collagen synthesis by wound fibroblasts during soft tissue repair.

Acceleration of soft tissue repair by a thrombin-derived oligopeptide.

Augmentation of thrombin-modulated chemotaxis and mitogenic activity within the early phase of soft tissue repair is now possible. Identification of high-affinity thrombin receptor binding domains within thrombin has enabled the synthesis of a family of peptides which interact with thrombin receptors and enhance in vitro mitogenesis. A single (5.0 micrograms/wound) application of the thrombin receptor-activating peptide (P517-30) significantly increased wound breaking strength from Day 5 (31% over controls) to Day 12. Two models of impaired healing created by radiotherapy (RT) were used to elucidate possible mechanisms of P517-30 action. Although P517-30 did not completely overcome the RT-induced healing impairments, it increased breaking strength under conditions of penetrating whole body RT-induced pancytopenia by 22% and of nonpenetrating surface RT-induced dermal cell damage by 42%. This suggests that P517-30 directly stimulates resident endothelial cells, fibroblasts, or other cells to overcome dermal and circulating monocytic deficits. These results suggest a method to accelerate wound healing with potential clinical applications and emphasize the activity of thrombin as a growth factor.

Platelet-activating factor: improvement in wound healing by a chemotactic factor.

Platelet-activating factor (PAF) is chemotactic for inflammatory cells and activates macrophages but, unlike growth factors, which have been demonstrated to accelerate healing, it has no effects on cell proliferation. The possible role of PAF in wound healing has not been explored previously. We examined the effect of different topical concentrations of PAF on the paired rat surgical incision model. Samples harvested on different days after wounding were evaluated for tensiometry and histology. Samples treated with 1 microgram, but not with 0.1 or 10 micrograms, showed an increase in maximal breaking strength (33.2%, 25.6%, and 4.9% by days 5, 7, and 12, respectively), reaching significance on days 5 and 7. Samples treated with 1 microgram PAF demonstrated a greater cellular infiltration (fibroblasts and monocytes) on day 7. Further histochemical studies revealed an increase of macrophages by day 7. Treatment with PAF receptor antagonist blocked the response to PAF but had no effect on normal wound healing, suggesting that, although PAF can accelerate healing, its endogenous production does not play a key role in normal wound repair. Our results demonstrate promotion of wound healing by PAF, a phospholipid chemotactic factor not previously shown to have wound-healing properties. This study gives new insights into how cytokines act to promote healing and suggests a strategy for improving wound repair with a monocyte chemotactic factor.

Essential fatty acids are not required for wound healing.

Rats with essential fatty acid deficiency (EFAD) exhibit mild body growth retardation, diminished leukocyte influx in certain models of inflammation, and skin lesions characterized by ulceration, thinning and decreased pigmentation. In the present study we examined the role of EFAD in cutaneous wound healing, a process in which the inflammatory response and the macrophage play a central role. We reproduced the EFAD condition in Lewis rats (n = 35), and examined its effects in wound healing using the paired rat surgical incision model. Rats were compared with weight-matched controls, receiving standard chow diet. Skin samples harvested at days 5, 7, 14 and 21 post-wounding were evaluated for tensiometry and histology. EFAD rats exhibited all the characteristics of this condition, and the typical alteration of liver lipids. Skin samples harvested at different days post-wounding did not show difference in maximal breaking strength when compared to weight-matched controls. Histological evaluation of skin samples showed no difference in the cellular inflammatory infiltration in either EFAD rats or in weight-matched controls. Immunohistochemical studies revealed no difference in the influx of macrophages in the different groups of rats. Fatty acid supplementation of EFAD rats (n = 7), successfully reversed the EFAD state as assessed by the macroscopic skin and liver changes and liver fatty acid content, without modifying either tensile strength or cellular inflammatory infiltration. Our results suggest that EFAD does not alter the normal course of the cutaneous wound repair in rats, despite all the cutaneous alterations produced by this condition. We conclude that essential fatty acids (EFAs) are not essential for cutaneous wound repair.

Collagenase production at the border of granulation tissue in a healing wound: macrophage and mesenchymal collagenase production in vivo.

We demonstrated the cells producing collagenase and the time course of collagenase-production at early stages of wound healing, using histology and two immunohistochemical procedures on cross sections of rat skin harvested 0, 3, 5, 7 and 12 days after full-thickness incisions. A monospecific rabbit polyclonal antibody to neutral collagenase purified from rat myometrial cells was used to demonstrate collagenase production. Specificity of this reaction was confirmed by blocking the reaction with excess homogeneously purified antigen. Macrophages were simultaneously labelled using a mouse anti-rat monoclonal antibody recognizing exclusively mature macrophages. Intracellular collagenase was not reliably detectable at day 0, but was prominent at days 3 and 5 and thereafter declined. Double labeling technique showed occasional macrophages producing collagenase in the developing granulation tissue, but most cells labeled as macrophages were negative for collagenase. Most activity was found in fibroblasts adjacent to granulation tissue elements. Since the granulation tissue parallels revascularization in a dendritic pattern, a cross section at three days typically shows an annulus of collagenase-positive cells surrounding a branch of the active granulation tissue. At days 5, 7 and 12 after wounding the pattern of collagenase expression became indistinct as more tissue was involved in the granulation process. However, double-labelling for macrophages and collagenase showed the dichotomy between collagenase expression and presence of macrophages to persist. The finding that collagenase is produced in connective tissue adjacent to granulation tissue suggests an inductive process, possibly due to diffusion of cytokines produced by granulation tissue elements.

Current concepts in wound healing: growth factor and macrophage interaction.

Growth factors are potent wound healing promoters which accelerate incisional wound repair by distinct mechanisms. Transforming growth factor-beta (TGF-beta), a chemotactic factor, increases synthesis of extracellular matrix and stimulates granulation tissue. We demonstrated that a single topical dose of TGF-beta increased the wound breaking strength in normal models of tissue repair as well as in models of impaired wound repair, characterized by severe monocytopenia. PDGF, a chemotactic agent for inflammatory cells, with mitogenic activity, activates monocytes and stimulates collagen production, significantly increased the wound breaking strength with effects that lasted for up to 47 days. In contrast to TGF-beta, PDGF was only active in normal models of wound healing and its effects were dependent upon the presence of macrophages. PAF is a glycerophospholipid which chemotaxes and activates macrophages, but differs from growth factors in lacking mitogenic activity. A single topical dose of PAF significantly increased the wound breaking strength and promoted macrophage migration.