Robert F. Diegelmann

WOUND HEALING: AN OVERVIEW OF ACUTE, FIBROTIC AND DELAYED HEALING

Acute wounds normally heal in a very orderly and efficient manner characterized by four distinct, but overlapping phases: hemostasis, inflammation, proliferation and remodeling. Specific biological markers characterize healing of acute wounds. Likewise, unique biologic markers also characterize pathologic responses resulting in fibrosis and chronic non-healing ulcers. This review describes the major biological processes associated with both normal and pathologic healing. The normal healing response begins the moment the tissue is injured. As the blood components spill into the site of injury, the platelets come into contact with exposed collagen and other elements of the extracellular matrix. This contact triggers the platelets to release clotting factors as well as essential growth factors and cytokines such as platelet-derived growth factor (PDGF) and transforming growth factor beta (TGF-beta). Following hemostasis, the neutrophils then enter the wound site and begin the critical task of phagocytosis to remove foreign materials, bacteria and damaged tissue. As part of this inflammatory phase, the macrophages appear and continue the process of phagocytosis as well as releasing more PDGF and TGF beta. Once the wound site is cleaned out, fibroblasts migrate in to begin the proliferative phase and deposit new extracellular matrix. The new collagen matrix then becomes cross-linked and organized during the final remodeling phase. In order for this efficient and highly controlled repair process to take place, there are numerous cell-signaling events that are required. In pathologic conditions such as non-healing pressure ulcers, this efficient and orderly process is lost and the ulcers are locked into a state of chronic inflammation characterized by abundant neutrophil infiltration with associated reactive oxygen species and destructive enzymes. Healing proceeds only after the inflammation is controlled. On the opposite end of the spectrum, fibrosis is characterized by excessive matrix deposition and reduced remodeling. Often fibrotic lesions are associated with increased densities of mast cells. By understanding the functional relationships of these biological processes of normal compared to abnormal wound healing, hopefully new strategies can be designed to treat the pathological conditions.

Wound Healing: Biochemical and Clinical Aspects

Presents comprehensive up-to-date discussions of both the clinical and basic science aspects of wound healing. This text enables the reader to obtain information from several different scientific disciplines and clinical specialities in one place. Additionally, future directions are identified to help the reader focus on important research in the upcoming decade.

Ability of chronic wound fluids to degrade peptide growth factors is associated with increased levels of elastase activity and diminished levels of proteinase inhibitors

The stability of peptide growth factors exposed to fluids from healing surgical wounds and from nonhealing chronic wounds was examined in vitro. 125I‐Labeled transforming growth factor‐β1 or platelet‐derived growth factor‐BB was incubated with fluids from healing surgical wounds and fluids from venous stasis or pressure ulcers. Fluids from healing surgical wounds had no appreciable effect on the level of 125I corresponding to intact growth factor. In contrast, incubation with fluids from several venous stasis or pressure ulcers resulted in significant degradation of these growth factors. Degradation was blocked by broad‐spectrum serine proteinase inhibitors and by specific inhibitors of neutrophil elastase. Levels of elastase activity in wound fluids correlated with the ability to degrade peptide growth factors. Further comparisons showed qualitative and quantitative differences in the endogenous proteinase inhibitors, α2‐macroglobulin and α1‐antiproteinase. These results could explain, in part, the variable growth factor levels which have been found in chronic wounds. More importantly, the ability of some chronic nonhealing wounds to rapidly degrade exogenously added growth factors has important implications with regard to past and future clinical attempts to use peptide growth factors to treat these types of problem wounds.

The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis.

Keloids are characterized by an overabundant deposition of collagen, and they recur frequently following excision. Fibroblasts isolated from keloid tissue and maintained in cell culture continue to express an increased capacity to produce collagen. In an effort to define the mechanisms responsible for keloid formation, the potential of exogenous transforming growth factor beta 1 (TGF-beta 1) to differentially affect DNA synthesis and collagen expression in cultured human fibroblasts derived from keloid or normal dermis was investigated. In this study, TGF-beta 1 at a concentration of 5.0 ng/ml was found to stimulate DNA synthesis of keloid-derived fibroblasts to a greater extent than fibroblasts derived from normal dermis. With a microassay to measure levels of collagenase-digestible radiolabeled proteins, TGF-beta 1 was found to elicit a greater increase in absolute collagen synthesis in keloid-derived fibroblasts compared with fibroblasts derived from normal dermis. Examination of tRNA(pro) pool-specific activities indicated that these observed differences in rates of collagen synthesis were not the result of unequal rates of proline transport or pool size. Likewise, TGF-beta 1 did not alter the uptake of vitamin C, an essential cofactor and mediator needed for maximal collagen expression. The increase in collagen synthesis by keloid-derived fibroblasts treated with TGF-beta 1 was accompanied by a corresponding increase in procollagen type I mRNA levels, indicating that the differential response of keloid and normal dermal fibroblasts to this growth factor is occurring primarily at a pretranslational level. These results suggest a unique sensitivity of keloid fibroblasts to TGF-beta 1 and thus a possible role for this mediator in keloid pathogenesis.

Transforming growth factor beta (TGF-β) induces fibrosis in a fetal wound model†**

The adult cellular response to tissue injury is characterized by acute inflammation followed eventually by fibroblast proliferation and collagen synthesis. Fetal tissue responses to injury differ markedly from those of the adult; an early acute inflammatory response is absent, few fibroblasts participate, and no collagen is deposited. The object of the present study was to analyze the effects of transforming growth factor beta (TGF-β), an important regulatory molecule in adult healing events, on the fetal tissue response following wounding. Fetal cellular and extracellular matrix responses to injury were evaluated by placing subcutaneous wound implants containing TGF-β (0.01 to 10 ng) in fetal rabbits at 24 days gestation (term =31 days). Histologic responses one to seven days later were compared with fetal and adult control implants without TGF-β. The histology of the adult implant was characterized by an early acute inflammatory response: by day 7 fibroblasts and collagen were predominant. In contrast, control implants removed from fetal rabbits had no histologic evidence of acute inflammation or fibroblast penetration and no collagen was deposited. When implants containing 1.0 ng TGF-β were removed from fetal rabbits at seven days, a grossly fibrotic reaction was observed: histology confirmed marked fibroblast penetration with collagen deposition. Fetal implants containing 0.01 ng or 10 ng TGF-β showed few fibroblasts but had increased numbers of inflammatory cells compared with controls. These observations demonstrate that the fetal response becomes adultlike with fibroblast proliferation and collagen accumulation when TGF-β is added, thus documenting the responsiveness of the fetal system to adult repair signals. Such responsiveness thus suggests a critical difference in the fetal wound environment. Fetal repair may proceed in the absence of trophic factors like TGF-β, thus accounting for optimal “healing” in the absence of excessive fibrosis.

Cleavage of Type I Procollagen by Human Mast Cell Chymase Initiates Collagen Fibril Formation and Generates a Unique Carboxyl-terminal Propeptide

The ability of human mast cell chymase and tryptase to process procollagen was examined. Purified human intestinal smooth muscle cell procollagen was incubated with human mast cell tryptase or human mast cell chymase. Purified chymase, but not tryptase, exhibited procollagen proteinase activity in the presence of EDTA. Addition of purified porcine heparin over a range of 0.1-100 μg/ml did not affect either the rate or the products of procollagen chymase cleavage. The cleavage site of chymase on the pro-α1(I) collagen carboxyl terminus was found to be in the propeptide region at Leu-1248-Ser-1249. Cleavage at this site suggested that the collagen products would form fibrils and confirmed the production of a unique carboxyl-terminal propeptide. Turbidometric fibril formation assay demonstrated de novo formation of chymase-generated collagen fibrils with characteristic lag, growth, and plateau phases. When observed by dark field microscopy, these fibrils were similar to fibrils formed by the action of procollagen proteinases. Thus, mast cell chymase, but not tryptase, exhibits procollagen peptidase-like activity as evidenced by its ability to process procollagen to fibril-forming collagen with concurrent formation of a unique carboxyl-terminal propeptide. These data demonstrate that mast cell chymase has a potential role in the regulation of collagen biosynthesis and in the pathogenesis of fibrosis.

Fetal response to injury in the rabbit

Fetal, neonatal, and adult tissue response to a standardized injury was studied using subcutaneous wound implants, linear incisions, and punch wounds in New Zealand white rabbits. In the fetus, sutured incisions healed by primary intention without antecedent inflammation. However, neither contraction nor healing by secondary intention was seen in punch or unsutured wounds. Healing both by primary and secondary intention following inflammatory infiltration was observed uniformly in neonatal and adult rabbits. Wound implants were extensively infiltrated with collagen in the adults studied; however, no collagen was seen in fetal implants and collagen hydroxyproline content could not even be detected by high performance liquid chromatography techniques; rather, a matrix rich in hyaluronic acid was found. The fetal tissue response to injury differs from the adult, proceeding in the absence of a classical inflammatory stimulus and lacking contractile capabilities. The deposition of extracellular matrix rich in hyaluronic acid but devoid of collagen suggests that the fetal response to injury may be a process more closely resembling regeneration or growth rather than repair by scar deposition.

Quantitation of Collagen Types I and III during Wound Healing in Rat Skin

Summary The collagen Types synthesized during early wound healing have not been quantitatively analyzed. Therefore, open wounds were made on the backs of Sprague-Dawley rats and analyzed at various times for newly synthesized collagen Types I and III. Biopsies were incubated in tissue culture medium with [3H]proline and collagen was extracted and purified prior to separation on an A5-M column. Type III collagen was increased significantly at 10 hr compared to all other times through Day 12. By 24 hr, the percentage Type III collagen returned to a normal skin value of 20%. The early appearance of Type III collagen is associated with an early increase in collagen synthesis and may function in providing initial wound structure and support for subsequent healing events.

Hyaluronic acid modulates proliferation, collagen and protein synthesis of cultured fetal fibroblasts

Fetal wound healing is characterized by minimal inflammation, mild fibroplasia and rapid, but organized collagen deposition such that scarring is not apparent. The matrices of fetal wounds differ greatly from adult wounds in that fetal wounds are persistently enriched with hyaluronic acid (HA). It has been shown that a reduction in fetal rabbit wound HA results in an adult-like healing response with increased fibroplasia and neovascularization. These observations suggest that HA can modulate cellular activity in fetal repair. Therefore, this study was designed to define the effect of HA on fetal fibroblast function. Fibroblasts from the skin of fetal rabbits were isolated and maintained in culture medium containing either no HA (controls), 1 microgram/ml, 10 micrograms/ml or 100 micrograms/ml of HA (n = 6 for each group). Fibroblast proliferation was quantitated by DNA content in each culture, and collagen and noncollagen protein synthesis were analyzed by incorporation of [3H] proline into collagenase-digestible and collagenase-nondigestible protein, respectively. At all concentrations tested, HA significantly inhibited fetal fibroblast proliferation (p < 0.02), but stimulated collagen (p < 0.002) and noncollagen protein (p < 0.005) synthesis. These findings provide further evidence that HA affects the function of fetal fibroblasts. Moreover, this study in conjunction with previous in utero findings suggests that HA may have a regulatory influence in scarless fetal healing by affecting cellular function during the repair process.

Excessive neutrophils characterize chronic pressure ulcers.

Although it is well recognized that pressure‐induced ischemia initiates the formation of pressure ulcers, the many complex mechanisms responsible for the pathogenesis of these ulcers remain poorly understood. It has been reported that chronic ulcers contain an elevated level of proteolytic enzymes, especially neutrophil‐derived matrix metalloproteinase‐8 and elastase. This evidence suggests that neutrophils are a major component in the pathogenesis of chronic pressure ulcers. Therefore, this study characterized the cellular components of chronic pressure ulcers. Three‐millimeter biopsies (6 mm deep) from granulation tissue in pressure ulcers were obtained from 11 patients. A total of 14 biopsies were obtained from these 11 patients for analysis. A portion of each specimen was fixed in formalin for routine histology. Other portions of biopsies were frozen for analysis of myeloperoxidase activity. In addition, cells on the surfaces of the ulcers were collected by lavage for histologic characterization. Routine histologic analysis of all 14 biopsies of the pressure ulcers showed regions near the surface of each that contained dense neutrophil infiltration associated with edema and apparent marked matrix dissociation. In the deeper regions there was an increased density of blood vessels, and many contained rounded endothelial cells surrounded by migrating neutrophils. Cells collected by lavage from the ulcer surface were prepared by Cytospin and found to be greater than 95% neutrophils with occasional large macrophages actively phagocytosing depleted neutrophils. In addition, there was a significant correlation of myeloperoxidase activity with actual neutrophil counts in the ulcer biopsies further confirming the dense presence of neutrophils. These studies directly show that there is extensive neutrophil infiltration in chronic pressure ulcer granulation tissue. Furthermore, the persistence of neutrophils and their destructive enzymes appears responsible for the extensive matrix dissociation and thus contributes to the chronicity of these ulcers. (WOUND REP REG 2003;11:490–495)