Modified cotton gauze dressings that selectively absorb neutrophil elastase activity in solution.

Dressings for chronic human wounds have been aimed at protection, removal of exudate, and improved appearance. However since the time of ancient Greece wound care and dressing strategies have primarily relied on empiricism. Recent studies have shown that chronic wounds contain high levels of tissue and cytokine destroying proteases including collagenase and neutrophil elastase. Therefore we sought to develop an effective wound dressing that could absorb elastase through affinity sequestration. Cotton gauze was modified by oxidation, phosphorylation, and sulfonation to enhance elastase affinity by ionic or active site uptake. Type VII absorbent cotton gauze was oxidized to dialdehyde cotton which was subsequently converted in part to the bisulfite addition product. Gauze preparations were also phosphorylated and carboxymethylated. Modified cotton gauzes were compared with untreated gauze for reduction of elastase activity in buffered saline. Solutions of elastase that were soaked in oxidized, sulfonated, and phosphorylated cotton gauze showed reduced elastase activity. The initial velocities (v(o)) and turnover rates of elastase showed significant decreases compared with solutions taken from untreated gauze. The reduction in enzyme activity with dialdehyde cotton gauze was confirmed in solution by determining elastase inhibition with dialdehyde starch. The dialdehyde cotton gauze also decreased elastase activity in human wound fluid in a dose response relation based on weight of gauze per volume of wound fluid. Absorbency, pH, air permeability and strength properties of the modified gauze were also compared with untreated cotton gauze. This report shows the effect of reducing elastase activity in solution with cotton containing aldehydic or negatively charged cellulose fibers that may be applicable to treatment modalities in chronic wounds.

Inhibition of elastase by a synthetic cotton-bound serine protease inhibitor: in vitro kinetics and inhibitor release.

A cotton-bound serine protease inhibitor of elastase (fiber-inhibitor) has been formulated for in vitro evaluation in chronic wound fluid. As a model to understand the properties of the inhibitor in wound dressings, the kinetic profile and in vitro release of the fiber-inhibitor formulation have been examined. The elastase inhibitor N-Methoxysuccinyl-Ala-Ala-Pro-Val-chloromethylketone was modified onto cotton cellulose fibers and assayed as a colloidal system. Amino acid analysis and reversed phase high performance liquid chromatography were compared as semiquantitative methods to assess elastase inhibitor release from the cotton fibers. The kinetics of inhibition was assessed on treated fibers of synthetic dressings such that a colloidal suspension of the fiber-inhibitor and elastase was employed as an assay. A dose-response relationship was observed in the kinetics of substrate hydrolysis catalyzed by three elastases: porcine pancreatic elastase, which was employed to model this approach; human leukocyte elastase; and elastase in human chronic wound fluid. Both freely dissolved and fiber-bound inhibitors were studied. The initial rates of substrate hydrolysis were inversely linear with freely dissolved inhibitor dose. The apparent first order rate constants, kobs, for the elastase-inhibitor complex were calculated from the kinetic profiles. The kobs for inhibitor bound enzyme varied as a function of inhibitor vs. enzyme concentration and based on the order of mixing of substrate, inhibitor and enzyme in the assay. Enzyme inhibition by the fiber-inhibitor was measured as inhibitor concentration at 50% inhibition (I50). I50 values measured from the colloidal assay with fiber-released inhibitor were within the same range to those for freely dissolved inhibitor. Inhibition of elastase activity in chronic wound fluid was observed with 1-5 mg of fiber-inhibitor formulation. This approach constitutes an in vitro assessment of synthetic serine protease inhibitors on fibers and may be employed to evaluate structure vs. function of elastase inhibition in the modified fibers of wound dressing composites.

MMP-8 is the predominant collagenase in healing wounds and nonhealing ulcers.

BACKGROUND: The initial cleavage of collagen by collagenase represents the rate-limiting step in the degradation of this central extracellular matrix protein. Chronic nonhealing ulcers, especially pressure ulcers, typically contain elevated levels of collagenolytic activity. However, there have been no detailed attempts to identify the source of these collagenases and their activity either in normal healing wounds or in chronic nonhealing ulcers. MATERIALS AND METHODS: Levels of the matrix metalloproteinases, MMP-1 and MMP-8, and the tissue inhibitor of matrix metalloproteinases, TIMP-1, were measured in fluids and tissues of healing human wounds and nonhealing ulcers by ELISA. Relative MMP-1 and MMP-8 levels were also analyzed by substrate preference in a functional assay. RESULTS: The patterns of the collagenases MMP-1 and MMP-8 in healing wounds were distinct, with MMP-8 appearing in significantly greater amounts than MMP-1. Chronic nonhealing ulcers were characterized by significantly higher levels of MMP-1 and MMP-8, and lower levels of TIMP-1, than in healing wounds. Levels of both MMP-1 and MMP-8 varied greatly in chronic ulcers, although MMP-8 was always the predominant collagenase present in these wounds. Interestingly, these collagenases were present almost exclusively in their inactive forms in healing wounds, whereas nonhealing ulcers possessed significant levels of the active forms of these enzymes. CONCLUSIONS: These results clearly demonstrate that the neutrophil-derived MMP-8 is the predominant collagenase present in normal healing wounds and suggest that overexpression and activation of this collagenase may be involved in the pathogenesis of nonhealing chronic ulcers. In addition, excessive collagenolytic activity in chronic ulcers is made possible, partly because of the reduced levels of the inhibitor, TIMP-1.

Dynamics of the matrix metalloproteinases MMP-1 and MMP-8 in acute open human dermal wounds.

Extracellular matrix degradation during dermal wound healing involves multiple levels of regulation by several enzymes of the matrix metalloproteinase family, their activators, and their inhibitors. This study tested the hypothesis that a temporal pattern of interstitial collagenase appearance occurs during normal dermal wound healing, with matrix metalloproteinase-8 originating from neutrophils appearing earlier than the fibroblast-derived matrix metalloproteinase-1. Open (6 mm) full-thickness dermal wounds, which were covered by transparent occlusive dressings, were made in healthy human volunteers (n = 20). Wound fluids from under the dressings were collected daily through day 8, and wound tissue biopsies were obtained on days 0, 2, 4, 14, and 28. Collagenases were extracted from homogenized tissue biopsies for analysis. Samples were analyzed for the presence of matrix metalloproteinase-1 and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays and by collagenase activity assays using purified types I and III collagen as substrates. In addition, tissue inhibitor of metalloproteinases-1 and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes in wound fluids were measured. Results showed a differential temporal pattern of matrix metalloproteinase-1 and matrix metalloproteinase-8 in wound exudates with peak levels of matrix metalloproteinase-8 occurring on day 4 and matrix metalloproteinase-1 peak levels on day 7. Maximal levels in tissue for both enzymes occurred on day 2. At all time points examined, levels of matrix metalloproteinase-8 were statistically higher than matrix metalloproteinase-1 (100-fold to 200-fold). Tissue inhibitor of metalloproteinases-1 levels declined over time, whereas levels of matrix metalloproteinase-1/tissue inhibitor of metalloproteinase-1 complexes increased to a plateau on day 7. This study provides new evidence implicating matrix metalloproteinase-8 as a major collagenase in healing human dermal wounds. It also shows a temporal pattern in the appearance of the matrix metalloproteinases, tissue inhibitor of metalloproteinase-1, and matrix metalloproteinase-1/tissue inhibitor of metalloproteinases-1 complexes, suggesting that a tightly regulated pattern of expression of matrix metalloproteinases and their inhibitors is essential for normal wound healing in humans.

Interleukin-1alpha and collagenase activity are elevated in chronic wounds.

Interleukin-1-alpha (IL-1alpha) is a member of a family of proinflammatory polypeptide mediators that has been shown in vitro to stimulate collagenase production. Collagenase is a proteolytic enzyme classified as one of the matrix metalloproteinases (MMP-1) that specifically recognizes and cleaves collagen. Therefore, the objective of this study was to compare the levels of these two proteins in chronic wounds as possible factors in the pathogenesis of chronic wounds. Fluids from 10 chronic wounds were collected before and after a 1-week treatment with a hydroactive dressing (Cutinova cavity). In addition, fluids were collected from 20 acute wounds for comparison. IL-1alpha and MMP-1 levels were quantified using sandwich ELISA. Collagenase activity was measured using a radiolabeled collagen as substrate. Clinically, the chronic wounds showed decreased area (-21.0 cm2) and reduced volume (-134.5 cm3) by 4 weeks after treatment with the hydroactive dressing. There were no significant differences in the protein concentrations between acute wound fluids (21.0 +/- 3.0 mg/ml) and chronic wound fluids before and after treatment with the hydroactive dressing (18.3 +/- 5.5 and 25.2 +/- 7.6 mg/ml, respectively). Levels of IL-1alpha in the acute wound fluids were low (0.019 pg/mg), whereas in the chronic wound fluid before treatment they had been significantly elevated (44.9 + 21.8 pg/mg). Following treatment with the hydroactive dressing, the IL-1alpha levels dropped to 10.3 + 3.3 pg/mg (p < 0.05). Collagenase activity was not detectable in acute wound fluid, elevated in pretreatment chronic wounds (12.9 + 3.4 units), and decreased in chronic wounds after treatment (11.4 + 3.3 units). This study correlated clinical healing of chronic wounds with biochemical changes in the ulcer microenvironment. As the chronic wounds began to heal, there was a significant decrease in the IL-1alpha levels and collagenase activity, thus suggesting that these two proteins may contribute to the lack of healing characteristic of chronic wounds.

Physiology of the chronic wound.

There is as yet no unified mechanism that explains the pathophysiology of every nonhealing wound. This is largely because many of the basic biological investigations in this area have only been undertaken seriously in the past few years and many phenomena remain unexplained. As new data become available, these theoretical models undoubtedly will continue to evolve. However, a clear understanding of the facts presently available should provide the clinician with a good scientific basis for rational clinical interventions. There is an urgent need to bridge the gap that often exists between laboratory research and clinical practice. A number of wound care practices, which have been shown to be ineffective or harmful, are still widely used. This includes the application of toxic wound cleansing agents, inappropriate use of topical antibiotics, and the practice of wet-to-dry dressings. The past 2 decades have witnessed an unprecedented proliferation of wound care products, few of which have be proven to be consistently superior to simpler and more cost-effective measures. For the foreseeable future, the search for the magic wound 'portions and lotions' probably will continue to revolve around topical growth factor and antiprotease therapy. However, such efforts will only come to fruition when we truly understand the pathophysiologic basis for abnormal wound healing.

Endoscopic otoplasty in the rabbit model: effect of mechanical abrasion on ear cartilage deformation.

The purpose of this study was (1) to introduce an endoscopic technique for performing otoplasty; (2) to compare the durability of folding with scoring, sutures, or abrasion; and (3) to study the histologic changes associated with three techniques. Thirteen adult New Zealand White rabbits (26 ears) were divided into three otoplasty groups: bent cartilage (nine ears), cut cartilage (nine ears), or endoscopic abrasion of cartilage (eight ears). In all three groups, the ears were folded using external mattress sutures, with a transverse 180-degree fold. Sutures were removed at 1 to 6 weeks, and the ears followed for 4 weeks postoperatively. Ear-folding angles were followed weekly from suture removal to 4 weeks postoperatively. After sacrifice, the ear cartilage was harvested for histologic analysis. The ears were maintained in a 180-degree ventral fold by splinting sutures, which were removed at various times. Four weeks after suture removal, the mean ear angles were 13 degrees in the bent (control) group, 84 degrees in the cut group, and 132 degrees in the abraded group. The differences between the abraded and cut versus bent groups were significant (p = 0.0001 and 0.0073, respectively). There was no significant difference between the abraded versus cut groups. Histologic analysis showed perichondrial thickening on the convex surface in all of the groups. Fibrocartilage was produced in the cut and abraded groups. Based on histologic observation, the repair cartilage in the cut group was more fibrous, whereas that in the endoscopic group was more abundant with marked degenerative changes in the central zone. A new method of percutaneous endoscopic otoplasty in the rabbit model is described. This study showed that endoscopic otoplasty produced folding that was well maintained during the study period.

Hyaluronic acid inhibits fetal platelet function: implications in scarless healing.

Platelets are important for the initiation of inflammation in adults, but the role of fetal platelets in fetal wound healing is unclear because fetal dermal wounds heal with a minimal inflammatory response and lack of excessive scarring. Because fetal tissue is abundant in glycosaminoglycans (GAGs), predominantly hyaluronic acid (HA), this study was designed to test the hypothesis that HA inhibits the reactivity of platelets and thus contributes to the minimal scarring characteristic of fetal tissue repair. Platelets were isolated from 10 fetal pigs at day 80 of gestation (term, 115 days) and exposed to 0.5 mg/mL of arachidonic acid, an agent shown in prior studies to evoke maximal aggregation and degranulation of fetal platelets. The ability of HA at 0.1 and 0.5 mg/mL to inhibit this response was determined. The presence of HA resulted in a dose-dependent reduction in platelet aggregation at 180 seconds (control, 99.7 +/- 0.3%; HA [0.1 mg/mL] 91.7 +/- 3.8%; and HA [0.5 mg/mL] 48.5 +/- 9.0%; P < .005 v control). The onset of aggregation was also significantly delayed by 0.5 mg/mL of HA (13.5 +/- 2.5 seconds) compared to control (2.9 +/- 0.7 seconds), P < .05. No significant diminution of platelet aggregation could be achieved by the addition of other GAGs at similar concentrations. HA also significantly impaired the release of platelet-derived growth factor (PDGF)-AB from fetal platelets. The authors conclude that HA, the predominant GAG in fetal dermal matrix, inhibits platelet aggregation and cytokine release. This inhibition of platelet aggregation and resultant inflammatory response may explain, in part, the minimal inflammation and scarless healing characteristic of fetal dermal repair.

Ultrastructural analysis of fetal rabbit wounds.

Fetal wound healing proceeds rapidly with minimal inflammation and fibroplasia and little or no scar formation. These observations have led to the hypothesis that fetal wound healing more closely resembles regeneration rather than adult wound repair. To test this hypothesis, this study used ultrastructural analysis of fetal and adult fibroblasts and collagen to gain greater insight into differences in the healing processes. Full-thickness, primarily closed linear incisions were created dorsally on 24-day gestational age fetal rabbits (n = 9). The fetuses were killed 5 days later, and the wounds were excised and evaluated with transmission electron microscopy. Similarly, uninjured fetal skin of the same gestational age was obtained and analyzed. Adult rabbit dermal wounds were analyzed after 8 days of healing. Resting adult dermal fibroblasts had features of quiescent, inactive cells, whereas adult wound fibroblasts were highly active and filled with secretory vesicles. In contrast, both fetal normal dermal and wound fibroblasts appeared highly active and contained numerous secretory vesicles. In the adult wound, collagen fibril diameter was only 45% of the diameter of normal dermal collagen. However, fetal wound collagen fibrils were basically the same as normal dermal collagen, having a diameter that was 82% of the size of dermal collagen. These observations suggest that fetal wound fibroblasts do not require activation from an inactivated state and that fetal wound collagen deposition undergoes more rapid organization and maturation. These findings have significance in extending our understanding of the rapidity and functional superiority of fetal wound healing compared with adult wound healing.

Collagen induces cytokine release by fetal platelets: implications in scarless healing.

In previous studies the authors demonstrated that unlike adult platelets, fetal platelets respond poorly to collagen. When platelets make contact with the exposed collagen at the site of injury, the result is activation, aggregation, and degranulation with the release of cytokines and growth factors. This sequence of events is well characterized in adult wounds, which heal with an acute inflammatory response and dense scar formation. In sharp contrast, fetal dermal wounds heal without an acute inflammatory response and minimal scar formation. Therefore, the aim of this study was to test the hypothesis that collagen, abundant at the site of dermal injury, is a poor inducer of cytokine release by fetal platelets. This could explain, in part, the minimal inflammation accompanying fetal dermal wound healing. Platelet suspensions from six fetal Yorkshire swine at day 80 of gestation (term, 114 days) were exposed to either arachidonic acid, 0.5 mg/mL, collagen, 0.19 mg/mL, or saline. The release into plasma of transforming growth factor-beta (TGF-beta 1), and platelet-derived growth factor (PDGF)-AB effected by these agents was determined by enzyme-linked immunosorbent assays. Transmission electron microscopy (TEM) was used to correlate the biochemical findings with ultrastructural changes and showed that arachidonate-treated platelets were aggregated and devoid of granules. In contrast, collagen-treated platelets had undergone conformational changes but showed only a moderate change in the quantity and homogeneity of their secretory granules compared with saline-treated controls. There was a significant increase in TGF-beta 1 release into plasma after treatment with collagen (6.64 +/- 0.36 ng/mL) and arachidonate (7.64 +/- 0.77 ng/mL) compared with saline (4.74 +/- 0.36 ng/mL), P < .05. Likewise, PDGF-AB release was significantly higher after collagen (0.22 +/- 0.02 ng/mL) and arachidonate treatment (0.44 +/- 0.04 ng/mL) compared with saline (0.09 +/- 0.02 ng/ mL), P < .05. The authors conclude that fetal platelets actually do release cytokines in response to contact with collagen despite poor aggregation. Therefore, impaired aggregation to collagen cannot solely explain the minimal inflammation after fetal wounding.

In vitro analysis of fetal fibroblast collagenolytic activity.

Tissue repair in the rabbit fetus is remarkably rapid and occurs without significant inflammation or excessive collagen deposition. The objective of this study was to compare the ability of rabbit fetal and adult fibroblasts to express the matrix metalloproteinases which are thought to be critical to scar tissue remodeling. In vitro, both fetal and adult rabbit fibroblasts express procollagenase messenger RNA in a constitutive manner. Mechanical disruption of fetal fibroblast monolayers caused a twofold increase in procollagenase mRNA. In contrast, the adult rabbit fibroblast procollagenase mRNA remained unchanged. The mRNA data correlated well with enzyme protein levels. Quantitation by immunoprecipitation showed a 2.3-fold increase in fetal fibroblast procollagenase protein after mechanical injury, whereas the level in adult rabbit fibroblasts remained unchanged. However, it was noted that the constitutive levels of procollagenase mRNA and protein were higher in adult fibroblasts. Analysis of enzyme activity, by means of a fluorogenic substrate, showed that adult fibroblasts had 2.2 times more collagenase activity compared with fetal cells. After mechanical injury, the fetal fibroblast collagenase activity increased 1.3-fold compared with 1.7-fold in the adult fibroblasts. In contrast, fetal fibroblast gelatinase activity was 1.25 times greater than in adult cells and increased 1.4-fold after mechanical injury, whereas the adult profile remained unchanged. Immunolocalization studies indicated that 1 hour after mechanical injury, procollagenase was produced primarily by fibroblasts along the mechanical injury ridge. By 4 hours after injury, the ridge cells began to migrate out into the open area and procollagenase was noted in adjacent cells of both adult and fetal origin. By 12 and 18 hours, all cells throughout the monolayer were expressing procollagenase. These findings show that in vitro fetal fibroblasts actually had lower levels of procollagenase, but higher levels of gelatinase compared with adult fibroblasts. The increased gelatinase expression may explain why fetal wounds do not have excessive collagen accumulation and heal without a visible scar.

Wound fluids from human pressure ulcers contain elevated matrix metalloproteinase levels and activity compared to surgical wound fluids.

Fluid from acute surgical wounds and from nonhealing pressure ulcers was examined for the presence of several matrix metalloproteinases. Gelatin zymography demonstrated the presence of two major gelatinases with apparent molecular masses of 72 kDa and 92 kDa and two minor gelatinases with apparent mobilities of 68 kDa and 125 kDa. Antigen-specific sera identified the 72-kDa protein as matrix melloproteinase-2. The same sera also reacted with the 68-kDa protein, which is consistent with it being an activated form of matrix metalloproteinase-2. Antigen-specific sera identified the 92-kDa and 125-kDa proteins as matrix metalloproteinase-9. Levels of matrix metalloproteinase-2 and matrix metalloproteinase-9 were elevated more than 10-fold and 25-fold, respectively, in fluids from pressure ulcers compared with fluids from healing wounds. Examination of total potential and actual collagenolytic activity revealed that fluid from pressure ulcers contained significantly greater levels of both total and active collagenase compared with that of acute surgical wounds. In addition, an enzyme-linked immunosorbent assay demonstrated that fluids from pressure ulcers contained significantly more collagenase complexed with the inhibitor, tissue inhibitor of metalloproteinases. Together, these observations suggest that an imbalance exists between levels of matrix metalloproteinases and their inhibitors in the fluids of pressure ulcers and that this is primarily the result of elevated levels of the matrix metalloproteinases. The presence of excessive levels of activated forms of matrix-degrading enzymes at the wound surface of pressure ulcers may impede the healing of these wounds and may be relevant to the development of new rationales for treatment.

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.

Commentaries on venous leg ulcers diagnostic and treatment draft guideline.

A guideline on venous leg ulcer diagnosis and treatment was developed by a research team from the University of Pennsylvania School of Medicine, Philadelphia, Pa., in collaboration with an inter-disciplinary panel of wound care clinicians. Working from a consensus statement based on a literature review, the authors developed preliminary algorithms, which were reviewed by a national advisory panel. The draft guideline was prepared, and the authors now seek national peer review to address whether it is clinically relevant, useful and represents current practice. The entire diagnostic draft guideline was published in the April issue of Ostomy/Wound Management; the entire treatment draft guideline in the May issue. After peer review and pilot testing, the guideline will be modified and validated in prospective clinical trials.

Pneumothorax and wound dehiscence related to collagenase deregulation: treatment with diphenylhydantoin.

BACKGROUND: Wound dehiscence is an uncommon complication of operation, usually related to a recognized risk factor. A clinical dilemma arises when dehiscence has no identifiable cause or treatment. METHODS: We describe the case of a previously healthy 45-year-old man in whom recurrent spontaneous pneumothoraces developed followed by multiple dehiscences of thoracotomy, diaphragmatic, and abdominal wounds. Analysis over several years of laboratory investigation of cultured tissue from test incisions was initially unsuccessful. The patient was supported symptomatically until a remarkable laboratory finding enabled us to develop an effective treatment plan. RESULTS: Cultured patient fibroblasts were ultimately found to express abnormally elevated levels of collagenase, which could be inhibited by diphenylhydantoin (phenytoin) in vitro. Treatment of the patient with a course of diphenylhydantoin allowed adequate healing of test incisions and subsequent definitive surgical treatment with successful wound healing. CONCLUSIONS: This report of the rigorous application of the scientific method to the investigation and treatment of an enigmatic case of wound dehiscence might serve as a guide to surgeons faced with similar healing problems.

Analysis of the effects of chitosan on inflammation, angiogenesis, fibroplasia, and collagen deposition in polyvinyl alcohol sponge implants in rat wounds.

Chitosan is a large molecular weight, positively charged polysaccharide extracted and purified from the chitin of crab shells. This compound has been shown to have hemostatic activity and has been suggested for use as a topical agent in tissue repair. The objective of this study was to analyze the effect of chitosan on the wound healing response to a standardized injury in the rat. The polyvinyl alcohol sponge implant model was used as a means to deliver either chitosan or its vehicle to a standardized subcutaneous wound on the backs of Sprague-Dawley rats. On days 8 and 14, the chitosan-treated implants contained primarily polymorphonuclear leukocytes compared with the vehicle controls which contained mainly macrophages, fibroblasts, collagen, and new blood vessels. High-performance liquid chromatography analysis for hydroxy-L-proline deposited in the sponge implants showed significantly lower amounts on both days 8 and 14 in the chitosan treatment group. These histologic and biochemical studies suggest that chitosan modulates wound healing by first reducing the influx of activated tissue macrophages, which in turn reduces the subsequent events of angiogenesis, fibroplasia, and connective tissue deposition.

Fetal wound healing: an overview.

The ability of fetal tissues to heal without scarring has prompted extensive research into the biochemical and molecular differences between fetal and postnatal wound healing. A thorough understanding of the basic mechanisms of fetal wound repair may to lead to approaches to correct or prevent the clinical problems encountered in abnormal adult wound healing and fetal surgery. This article contrasts the normal healing response in adults with fetal repair in animal models, highlighting investigations of extracellular matrix expression, cytokine profiles, and cellular dynamics.

Lower cytokine release by fetal porcine platelets: a possible explanation for reduced inflammation after fetal wounding.

Fetal dermal wound healing is unique because of its rapidity, minimal inflammation, and lack of scarring. Cytokines such as transforming growth factor beta (TGF-beta) and platelet-derived growth factor (PDGF) evoke an inflammatory response and scarring when applied to fetal wounds. Because adult and fetal platelet counts are comparable, the aim of this study was to test the hypothesis that the minimal inflammatory response seen in the fetus is attributable to differences in the serum content of cytokines released by fetal platelets. Using Yorkshire swine, blood was collected from 10 adults and 10 fetuses at day 60 of gestation (fullterm, 114 days). Platelets were isolated from anticoagulated blood and examined by transmission electron microscopy. Serum was analyzed for PDGF-AB and TGF-beta 2 by enzyme-linked immunosorbent assay (ELISA), and TGF-beta 1 by 125I radioimmunoassay. TGF-beta samples were assayed with and without prior acid activation to determine the total TGF-beta and the biologically active form of the cytokine. Electron microscopy of adult and fetal platelets showed no gross structural differences. Alpha granules, which contain cytokines as well as procoagulant factors, were present in similar quantities and with the same degree of homogeneity. The cytokines analyzed were present in all the adult and fetal sera tested. However, PDGF-AB was present in significantly lower concentrations in the fetus (383 +/- 72 pg/mL v 972 +/- 185 pg/mL in the adult; P<.05). In addition, the fetal samples contained lower amounts of TGF-beta 1 (13,895 +/- 1,770 v 29,864 +/- 5,050 pg/mL; P < .05) and TGF-beta 2 (6,758 +/- 734 v 13,407 +/- 1,395 pg/mL; P < .05). The majority of TGF-beta was in latent form; the adult sera contained significantly more active TGF-beta 1 and active TGF-beta 2 than the fetal sera. The ratios of active TGF-beta 1 to active TGF-beta 2 were similar for the adult (22.3) and fetus (18.5). However the ratio of total TGF-beta 1 to total TGF-beta 2 was significantly lower for the fetus (2.26 v 7.69). The authors conclude that although no gross differences in platelet ultrastructure were noted, fetal porcine platelets release lower quantities of cytokines into serum. This lower serum cytokine content and the relative concentrations of TGF-beta 1 of TGF-beta 2 may explain, in part, the minimal inflammation and sparse fibrosis characteristic of fetal wounds. These observations provide further insight into the unique fetal response to wounding and may offer alternative avenues to modulate the postnatal wound healing response.

Aggregatory characteristics and expression of the collagen adhesion receptor in fetal porcine platelets.

Fetal wound healing differs significantly from that of the adult by its rapidity, the paucity of an inflammatory response, and the lack of scarring. In the adult, activation and aggregation of platelets at the site of injury result in the release of cytokines and inflammatory mediators that stimulate wound healing by initiating an acute inflammatory response. The aim of this study was to characterize the activity of midtrimester (day 60) and third-trimester (day 95) fetal porcine platelets (full term, 114 days) compared with that of adults in an attempt to understand the lack of inflammation in fetal wounds. The aggregatory capabilities of adult and fetal platelets were analyzed after exposure to adenosine diphosphate (ADP) concentrations of 10 mumol/L and 40 mumol/L concentrations, collagen of 0.19 mg/mL, and arachidonic acid of 0.5 mg/mL. Expression of the alpha 2 subunit of the collagen receptor (alpha 2 beta 1) was evaluated by Western blot analysis. The aggregation of day-60 fetal platelets when exposed to ADP (10 mumol/L and 40mumol/L) and collagen was significantly lower than that of the adult. The aggregation of third-trimester platelets to 10 mumol/L of ADP was similar to that of the adult and significantly greater than that of midtrimester fetuses at higher concentrations (40 mumol/L). Both fetal groups responded suboptimally to collagen, and the response was significantly less than that of adults. In contrast, arachidonic acid caused rapid and complete aggregation of both fetal platelet groups, suggesting that both mid- and late-trimester fetal platelets possessed the ability to fully aggregate with the appropriate stimulus. The different aggregatory responses to collagen could not be explained by differences in collagen receptor expression, because these were found to be similar in adults and midtrimester fetuses. It is concluded that although fetal platelets have the potential to aggregate effectively, they aggregate poorly to collagen and exhibit improved aggregation to ADP with increasing maturity. There is a transition to "adultlike" platelet aggregatory activity in the third trimester, which correlates with the period of transition to adultlike wound healing in utero. Similar expression of the alpha 2 beta 1 collagen receptor in the fetus and adult cannot explain the differences observed in their responses to collagen.