Showing papers in "Wound Repair and Regeneration in 1999"
TL;DR: The general physicochemical and biological properties of hyaluronan, and how these properties may be utilized in the various processes of wound healing: inflammation, granulation and reepithelization, are presented.
Abstract: Hyaluronan is a major carbohydrate component of the extracellular matrix and can be found in skin, joints, eyes and most other organs and tissues. It has a simple, repeated disaccharide linear copolymer structure that is completely conserved throughout a large span of the evolutionary tree, indicating a fundamental biological importance. Amongst extracellular matrix molecules, it has unique hygroscopic, rheological and viscoelastic properties. Hyaluronan binds to many other extracellular matrix molecules, binds specifically to cell bodies through cell surface receptors, and has a unique mode of synthesis in which the molecule is extruded immediately into the extracellular space upon formation. Through its complex interactions with matrix components and cells, hyaluronan has multifaceted roles in biology utilizing both its physicochemical and biological properties. These biological roles range from a purely structural function in the extracellular matrix to developmental regulation through effects of cellular behavior via control of the tissue macro- and microenvironments, as well as through direct receptor mediated effects on gene expression. Hyaluronan is also thought to have important biological roles in skin wound healing, by virtue of its presence in high amounts in skin. Hyaluronan content in skin is further elevated transiently in granulation tissue during the wound healing process. In this review, the general physicochemical and biological properties of hyaluronan, and how these properties may be utilized in the various processes of wound healing: inflammation, granulation and reepithelization, are presented.
1,041 citations
TL;DR: The elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers, which parallels the processes observed in normally healing acute wounds and supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.
Abstract: To assess the differences in proteolytic activity of acute and chronic wound environments, wound fluids were collected from acute surgical wounds (22 samples) and chronic wounds (25 samples) of various etiologies, including mixed vessel disease ulcers, decubiti and diabetic foot ulcers. Matrix metalloproteinase (MMP) activity measured using the Azocoll assay was significantly elevated by 30 fold in chronic wounds (median 22.8 microg MMP Eq/ml) compared to acute wounds (median 0.76 microg MMP Eq/ml) (p < 0.001). The addition of the matrix metalloproteinase inhibitor Illomostat decreased the matrix metalloproteinase activity by approximately 90% in all samples, confirming that the majority of the activity measured was due to matrix metalloproteinases. Gelatin zymograms indicated predominantly elevated matrix metalloproteinase-9 with smaller elevations of matrix metalloproteinase-2. In addition tissue inhibitor of metalloproteinase-1 levels were analyzed in a small subset of acute and chronic wounds. When tissue inhibitor of metalloproteinase-1 levels were compared to protease levels there was an inverse correlation (p = 0.02, r = - 0.78). In vitro degradation of epidermal growth factor was measured by addition of 125I labelled epidermal growth factor to acute and chronic wound fluid samples. There was significantly higher degradation of epidermal growth factor in chronic wound fluid samples (mean 28.1%) compared to acute samples (mean 0.6%). This also correlated to the epidermal growth factor activity of these wound fluid samples (p < 0. 001, r = 0.64). Additionally, the levels of proteases were assayed in wound fluid collected from 15 venous leg ulcers during a nonhealing and healing phase using a unique model of chronic wound healing in humans. Patients with nonhealing venous leg ulcers were admitted to the hospital for bed rest and wound fluid samples were collected on admission (nonhealing phase) and after 2 weeks (healing phase) when the ulcers had begun to heal as evidenced by a reduction in size (median 12%). These data showed that the elevated levels of matrix metalloproteinase activity decreased significantly as healing occurs in chronic leg ulcers (p < 0.01). This parallels the processes observed in normally healing acute wounds. This data also supports the case for the addition of protease inhibitors in chronic wounds in conjunction with any treatments using growth factors.
867 citations
TL;DR: It is concluded that treatment with becaplermin gel at a dose of 100 μg/g once daily, in conjunction with good ulcer care, is effective and well tolerated in patients with full thickness lower extremity diabetic ulcers.
Abstract: The results of a combined analysis and separate analyses of four multicenter, randomized, parallel group studies that evaluated the effects of once-daily topical administration of becaplermin gel for the treatment of chronic, full thickness, lower extremity diabetic ulcers are presented. The four studies included a total of 922 patients with nonhealing lower extremity diabetic ulcers of at least 8 weeks' duration. Following initial complete sharp debridement of the ulcer, patients were randomized to receive a standardized regimen of good ulcer care alone, good ulcer care plus placebo gel, or good ulcer care plus becaplermin gel-30 microg/g, or good ulcer care plus becaplermin gel-100 microg/g, with various combinations of regimens used in the four studies. Safety was assessed by monitoring adverse events and by clinical laboratory evaluations. Meta-analytic statistical techniques were used in the combined analysis to establish homogeneity of treatment comparisons across studies. Based on an analysis of patients with baseline ulcer area common to all trials (
436 citations
TL;DR: The current understanding of the proteolytic enzymes found in chronic wounds is discussed and attempts to relate this information to the abundant presence of neutrophils.
Abstract: A consistent feature of chronic leg and pressure ulcers is chronic inflammation associated with an elevated infiltration of neutrophils. Neutrophils and their proteases have been implicated in mediating the tissue damage associated with a variety of chronic inflammatory diseases. This review discusses our current understanding of the proteolytic enzymes found in chronic wounds and attempts to relate this information to the abundant presence of neutrophils. In addition, the implications that the proteolytic environment may have for current and future treatment strategies of chronic nonhealing wounds are discussed.
388 citations
TL;DR: Before the role of matrix metalloproteinases in ulceration and disease is understood, it is necessary to determine the function these enzymes serve in normal tissues and repair.
Abstract: During repair, many different matrix metalloproteinases are produced by multiple cell types residing in various compartments within the wound environment. This diversity of enzymes, coupled with discreet cellular expression, implies that different matrix metalloproteinases serve different functions, acting on a variety of substrates, during wound healing. With few exceptions, however, the actual function and spectrum of functions of matrix metalloproteinases in vivo is not known. Even with the advent of genetically defined animal models, few studies have rigorously addressed the substrates and role of matrix metalloproteinases in wound repair. Before we can understand the role of matrix metalloproteinases in ulceration and disease, we need to determine the function these enzymes serve in normal tissues and repair.
372 citations
TL;DR: Analysis with multivariate regression methods showed that patients treated with Graftskin were twice as likely to achieve complete wound closure by 6 months and over 60% more effective in achieving wound closure than active control.
Abstract: The efficacy of a bilayered, living skin construct (APLIGRAF(R) [Graftskin]) was evaluated in patients (n = 120) with hard- to-heal venous leg ulcers of greater than 1 year's duration. The study was prospective, randomized, and controlled. Patients received Graftskin plus compression therapy, or standard compression therapy (active control). Patients were evaluated for frequency and time to complete (100%) wound closure. Treatment with Graftskin was significantly more effective than active control in the percentage of patients healed by 6 months (47% vs. 19%; p < 0.005) and the median time to complete wound closure (p < 0.005). Analysis with multivariate regression methods, adjusting for factors generally thought to influence wound healing (duration, baseline area, depth, location, fibrinous wound bed, and infection), showed that patients treated with Graftskin were twice as likely to achieve complete wound closure by 6 months (p < 0.005), and over 60% more effective in achieving wound closure than active control (p < 0.01). These data indicate that Graftskin is an effective treatment for venous ulcers of greater than 1 year's duration.
363 citations
TL;DR: Results show that curcumin enhanced wound repair in diabetic impaired healing, and could be developed as a pharmacological agent in such clinical settings.
Abstract: Tissue repair and wound healing are complex processes that involve inflammation, granulation and tissue remodeling. Interactions of different cells, extracellular matrix proteins and their receptors are involved in wound healing, and are mediated by cytokines and growth factors. Previous studies from our laboratory have shown that curcumin (diferuloylmethane), a natural product obtained from the rhizomes of Curcuma longa, enhanced cutaneous wound healing in rats and guinea pigs. In this study, we have evaluated the efficacy of curcumin treatment by oral and topical applications on impaired wound healing in diabetic rats and genetically diabetic mice using a full thickness cutaneous punch wound model. Wounds of animals treated with curcumin showed earlier re-epithelialization, improved neovascularization, increased migration of various cells including dermal myofibroblasts, fibroblasts, and macrophages into the wound bed, and a higher collagen content. Immunohistochemical localization showed an increase in transforming growth factor-β1 in curcumin-treated wounds compared to controls. Enhanced transforming growth factor-β1 mRNA expression in treated wounds was confirmed by in situ hybridization, and laser scan cytometry. A delay in the apoptosis patterns was seen in diabetic wounds compared to curcumin treated wounds as shown by terminal deoxynucleotidyl transferase–mediated deoxyuridyl triphosphate nick end labeling analysis. Curcumin was effective both orally and topically. These results show that curcumin enhanced wound repair in diabetic impaired healing, and could be developed as a pharmacological agent in such clinical settings.
313 citations
TL;DR: Once‐daily application of becaplermin gel is efficacious in the treatment of chronic full thickness pressure ulcers, compared with that of placebo gel.
Abstract: Pressure ulcers are associated with significant rates of morbidity and mortality, particularly in the geriatric and spinal cord-injured populations. Newer pharmacologically active therapies include the use of topically applied recombinant human platelet-derived growth factor-BB (becaplermin), the active ingredient in REGRANEX) (becaplermin) Gel 0.01%, which has been approved in the United States for treatment of lower extremity diabetic neuropathic ulcers that extend into the subcutaneous tissue or beyond and have an adequate blood supply. In this study, the efficacy of becaplermin gel in the treatment of chronic full thickness pressure ulcers was compared with that of placebo gel. A total of 124 adults (>/= 18 years of age) with pressure ulcers were assigned randomly to receive topical treatment with becaplermin gel 100 microg/g (n = 31) or 300 microg/g (n = 32) once daily alternated with placebo gel every 12 hours, becaplermin gel 100 microg/g twice daily (n = 30), or placebo (sodium carboxymethylcellulose) gel (n = 31) twice daily until complete healing was achieved or for 16 weeks. All treatment groups received a standardized regimen of good wound care throughout the study period. Study endpoints were the incidence of complete healing, the incidence of >/= 90% healing, and the relative ulcer volume at endpoint (endpoint/baseline). Once-daily treatment of chronic pressure ulcers with becaplermin gel 100 microg/g or 300 microg/g significantly increased the incidences of complete and >/= 90% healing and significantly reduced the median relative ulcer volume at endpoint compared with that of placebo gel (p < 0.025 for all comparisons). Becaplermin gel 300 microg/g did not result in a significantly greater incidence of healing than that observed with 100 microg/g. Treatment with becaplermin gel was generally well tolerated and the incidence of adverse events was similar among treatment groups. In conclusion, once-daily application of becaplermin gel is efficacious in the treatment of chronic full thickness pressure ulcers.
189 citations
TL;DR: Although electrical stimulation produces a substantial improvement in the healing of chronic wounds, further research is needed to identify which electrical stimulation devices are most effective and which wounds respond best to this treatment.
Abstract: The purpose of this meta-analysis was to quantify the effect of electrical stimulation on chronic wound healing. Fifteen studies, which included 24 electrical stimulation samples and 15 control samples, were analyzed. The average rate of healing per week was calculated for the electrical stimulation and control samples. Ninety-five percentage confidence intervals were also calculated. The samples were then grouped by type of electrical stimulation device and chronic wound and reanalyzed. Rate of healing per week was 22% for electrical stimulation samples and 9% for control samples. The net effect of electrical stimulation was 13% per week, an increase of 144% over the control rate. The 95% confidence intervals of the electrical stimulation (18‐26%) and control samples (3.8‐14%) did not overlap. Electrical stimulation was most effective on pressure ulcers (net effect = 13%). Findings regarding the relative effectiveness of different types of electrical stimulation device were inconclusive. Although electrical stimulation produces a substantial improvement in the healing of chronic wounds, further research is needed to identify which electrical stimulation devices are most effective and which wounds respond best to this treatment. (WOUND REP REG 1999;7:495‐503) Electrical stimulation (ES) is a largely unknown and poorly understood treatment modality for chronic wound healing. Appreciation for the potential contribution of ES in promoting chronic wound healing has been limited by the scientific community’s failure to collectively consider the entire body of clinical research in this area. Instead, attention has focused on the limited data available for each specific type of ES device and the unanswered questions regarding the optimal ES dose-response. 1 Examination of ES based on the entire body of evidence, regardless of the ES device or dose parameters, can provide valuable information about the merits of using this adjunctive therapy in practice and the utility of pursuing further research in this area. ES is believed to restart or accelerate the wound
179 citations
TL;DR: Tests have shown that measures commonly used to reduce costs, e.g., disease and multi discliplinary management strategies, have been shown to help prevent the occurrence of diabetic ulcers.
Abstract: Complications secondary to diabetes, such as diabetic foot ulcers, continue to be a major worldwide health problem. At the same time, health care systems are changing rapidly, causing concern about the quality of patient care. While the ultimate effect of current changes on health care professionals and patient outcomes remain uncertain, measures commonly used to reduce costs, e.g., disease and multi- disciplinary management strategies, have been shown to help prevent the occurrence of diabetic ulcers. In addition, utilizing a multi- disciplinary approach, the principles of off-loading and optimal wound care, the vast majority of diabetic foot ulcers can be expected to heal within 12 weeks of treatment. Education of primary care providers and patients is paramount.
160 citations
TL;DR: The mechanisms by which leukocyte proteinases can contribute to physiologic processes occurring during wound healing, as well as their roles in pathologic processes are discussed.
Abstract: Leukocytes express a number of proteinases which play critical roles in physiologic processes during wound healing. However, if the activity of these proteinases is uncontrolled, they can contribute to devastating tissue injury that can affect most organ systems. Until recently, little was known about the mechanisms by which leukocytes retain the activity of their proteinases within the extracellular space which contains highly effective proteinase inhibitors. Studies of the cell biology of leukocyte proteinases have begun to identify the mechanisms by which proteinases can circumvent the effects of physiologic proteinase inhibitors. Herein, we will review the cell biology of leukocyte proteinases, and we will discuss the mechanisms by which leukocyte proteinases can contribute to physiologic processes occurring during wound healing, as well as their roles in pathologic processes.
TL;DR: It is concluded that granulocyte‐macrophage colony stimulating factor injected perilesionally may be a useful drug for the treatment of chronic venous leg ulcers.
Abstract: Chronic venous leg ulcers are a common ailment with no ideal treatment. Recent reports have shown granulocyte- macrophage colony stimulating factor to be of use in the healing of these chronic wounds. Therefore, we conducted a double-blind, randomized, placebo-controlled study which enrolled 60 patients with chronic venous leg ulcers, whom we treated with placebo or with 200 or 400 microg of granulocyte-macrophage colony stimulating factor by perilesional injections of the drug in four weekly treatment episodes. Observations were conducted at each treatment visit, at weeks 5, 9, 13, and six months after the inclusion in the protocol. The number of healed wounds in the placebo and the treated arms were significantly different (p = 0.05), with 4 of 21 (19%) in the first group having healed at week 13, as compared to 12 of 21 (57%) and 11 of 18 (61%), in the 200 microg and the 400 microg groups, respectively. There were only minor side-effects attributable to the treatment, and the reobservation at 6 months showed that none of the treated ulcers recurred during that period. We conclude that granulocyte-macrophage colony stimulating factor injected perilesionally may be a useful drug for the treatment of chronic venous leg ulcers.
TL;DR: It is concluded that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase‐9 expression suggesting the presence of a proteolytic cascade initiated by the plasmine activator/plasmin system during wound healing leading to the activation of matrix metaloproteinases‐9.
Abstract: The plasminogen activator/plasmin system is known to initiate a proteolytic cascade resulting in the activation of matrix metalloproteinases in vitro leading to the degradation of extracellular matrix. To investigate whether or not this cascade is present during delayed wound healing and contributes to the pathophysiological basis of impaired healing we examined the temporal expression of urokinase plasminogen activator, plasminogen activator inhibitor-1 and gelatinase-B in fluid collected from chronic venous leg ulcers compared to acute surgical mastectomy wounds. Using a chromogenic substrate assay, levels of active urokinase plasminogen activator in chronic wounds were found to be about five fold higher compared to sera and two fold higher compared to mastectomy wounds. Levels of active plasminogen activator inhibitor-1 in chronic wounds were four times higher than those found in sera and two times higher than those found in mastectomy wound fluid. Using a fibrin overlay system and reverse zymography, we found that when the wound was not healing, the expression of urokinase plasminogen activator in chronic wound fluid was initially detected in the active forms (50 and 33 kDa), but that as the wound healed and decreased in size, was detected as an inhibitor- bound urokinase plasminogen activator-plasminogen activator inhibitor-1 complex ( congruent with 80-116 kDa). When the expression of active urokinase plasminogen activator was highest, no plasminogen activator inhibitor-1 was detectable. In contrast, urokinase plasminogen activator was always detected in the inhibitor bound form as a urokinase plasminogen activator-plasminogen activator inhibitor-1 complex in blood- and plasma-derived serum and mastectomy wound fluid. Plasminogen activator inhibitor-1 was detected in blood-derived serum and mastectomy wound fluid but not in plasma derived serum. Expression of matrix metalloproteinase-9 in chronic wound fluids, analyzed by gelatin zymography, showed that when urokinase plasminogen activator was detected in the active forms, matrix metalloproteinase-9 was overexpressed by approximately twice that found in mastectomy wounds and approximately 30 times that detected in blood-derived sera. When urokinase plasminogen activator appeared almost entirely as an enzyme- inhibitor complex, the level of expression of matrix metalloproteinase-9 was similar to that seen in mastectomy wound fluid. We conclude that the switch in urokinase plasminogen activator expression from an active to inhibitor bound form correlates with the decrease seen in matrix metalloproteinase-9 expression suggesting the presence of a proteolytic cascade initiated by the plasminogen activator/plasmin system during wound healing leading to the activation of matrix metalloproteinase-9. In addition, expression of urokinase plasminogen activator and matrix metalloproteinase-9 appear to be useful biomarkers to determine clinical wound healing status.
TL;DR: Pentoxifylline is effective in accelerating healing of leg ulcers and complete wound closure occurred at least 4 weeks earlier in the majority of patients treated with pentoxifiers by comparison to placebo.
Abstract: Several small studies have indicated that the systemic administration of pentoxifylline may accelerate healing of venous leg ulcers. The goal of this study was to further evaluate these findings in a larger scale placebo controlled trial and to explore the effect of the dose of pentoxifylline on healing. The study used a prospective, randomized, double-blind, parallel group placebo controlled design in a multicenter outpatient setting. Patients with one or more venous ulcer were enrolled, with all patients receiving standardized compression bandaging for treatment for their ulcers. Patients were also randomized to receive either pentoxifylline 400 mg, pentoxifylline 800 mg (two 400 mg tablets), or placebo tablets three times a day for up to 24 weeks. The main outcome measure was time to complete healing of all leg ulcers, using life table analysis. The study was completed as planned in 131 patients. Patients receiving 800 mg three times a day of pentoxifylline healed faster than placebo (p = 0.043, Wilcoxon test). The median time to complete healing was 100, 83, and 71 days for placebo, pentoxifylline 400 mg, and pentoxifylline 800 mg three times a day, respectively. Over half of all patients were ulcer free at week 16 (placebo) and at week 12 in both pentoxifylline groups. Whereas the placebo group had only achieved complete healing in half of the cases by week 16, all of the subjects remaining in the group receiving the high dose of pentoxifylline had healed completely. Treatment with pentoxifylline was well tolerated with similar drop-out rates in all three treatment groups. Complete wound closure occurred at least 4 weeks earlier in the majority of patients treated with pentoxifylline by comparison to placebo. A higher dose of pentoxifylline (800 mg three times a day) was more effective than the lower dose. We conclude that pentoxifylline is effective in accelerating healing of leg ulcers.
TL;DR: This model of delayed healing in rats shares many of the key biochemical, molecular and mechanistic characteristics found in human chronic wounds, namely elevated tumor necrosis factor‐α and protease levels and will likely prove to be useful in chronic wound research, particularly in developing novel therapeutics.
Abstract: The purpose of this study was to provide molecular and mechanistic evaluation of an ischemic wound model in rats to determine if it is a valid model for human chronic wounds Compared to acute wounds, human chronic wounds contain markedly elevated levels of proinflammatory cytokines and matrix metalloproteinases, while matrix metalloproteinase inhibitors and growth factor activity are diminished Accordingly, tissue from ischemic and normal rat wounds were analyzed for cytokine, proteases and growth factor levels Dorsal full thickness punch wounds were created in rats using a reproducible template The ischemic wound group (n = 10) had six uniformly placed wounds within a bipedicled dorsal flap The control group (n = 10) had the same wounds created without elevation of a flap On postwound days 3, 6 and 13 wounds were excised and analyzed Protein levels for tumor necrosis factor-alpha were determined with a rat-specific enzyme-linked immunosorbent assay, while mRNA was determined by RNase protection assay Matrix metalloproteinases and serine protease detection was done using gelatin and casein zymography, respectively Significant delay in healing was achieved in the ischemic group: 50% healing for control wounds was at 7 days and 11 days for ischemic wounds (p < 0001) No significant differences between wound groups were found for interleukin-1beta, and mRNA for tumor necrosis factor-alpha and interleukin-1beta However, at day 13 ischemic wounds contained significantly more tumor necrosis factor-alpha than controls and normal skin (586 +/- 106 pg/biopsy vs 79 +/- 7 pg/biopsy vs 52 +/- 2 pg/biopsy; p < 0 001) Zymography showed substantially greater quantities of matrix metalloproteinase-2, matrix metalloproteinase-9, and serine proteases in ischemic wounds This model of delayed healing in rats shares many of the key biochemical, molecular and mechanistic characteristics found in human chronic wounds, namely elevated tumor necrosis factor-alpha and protease levels As such, this model will likely prove to be useful in chronic wound research, particularly in developing novel therapeutics
TL;DR: The data suggest that hypoxia may modulate extracellular matrix production by human mesothelial cells via a transforming growth factor‐β1 dependent mechanism.
Abstract: Overexpression and accumulation of extracellular matrix is central to peritoneal adhesion formation following surgically induced tissue trauma. Transforming growth factor-beta1 and hypoxia have been implicated in tissue fibrosis and postoperative adhesion formation. To extend this observation we examined whether transforming growth factor-beta1 and/or hypoxia regulate the expression of type I and III collagen in human peritoneal mesothelial cells. Cultured human mesothelial cells were maintained under hypoxia (2% oxygen), or treated with transforming growth factor-beta1 (1 ng/ml) or a combination of hypoxia and transforming growth factor-beta1. Total cellular RNA from treated and untreated cells were collected and subjected to multiplex reverse transcription/polymerase chain reaction to quantitate collagen I and III mRNA levels in response to these treatments. The results indicate that 6 hours of hypoxia increased collagen III mRNA by 7.2 fold which was further increased to 9.4 fold following transforming growth factor-beta1 treatment; in contrast collagen I mRNA decreased by 0.42 fold which was further decreased by 0.3 fold following transforming growth factor-beta1 treatment. Transforming growth factor-beta1 treatment under normal conditions resulted in an 8.4-fold increase and a 0.3-fold decrease in collagen III and I mRNA levels, respectively. Hypoxia treatment also resulted in a 1.9-fold increase in transforming growth factor-beta1 mRNA level compared with control. The ratio of type III/I collagen was increased in response to transforming growth factor-beta1 treatment under hypoxic condition. In conclusion, the data suggest that hypoxia may modulate extracellular matrix production by human mesothelial cells via a transforming growth factor-beta1 dependent mechanism.
TL;DR: Data show the potential of markers of collagen biochemistry as predictors of repair in venous ulcers; in particular pro‐matrix metalloproteinase‐9 and neutrophil elastase were found to be accurate prognostic indicators of subsequent healing.
Abstract: A 25 patient study was conducted into the relationship between markers of collagen metabolism in venous ulcer exudates and healing status, and their prognostic value in predicting healing performance. Wounds were sampled on at least 5 occasions over 12 months, the frequencies of which were determined by the need for clinic attendance. Specimens were taken from several sites on each ulcer using sterile preweighed filters. Wound margins were traced and sites recorded for each collection. Sample sites were evaluated for severity as improving, static, or deteriorating according to subsequent wound progression. Specimens were analyzed for levels of proenzyme and active forms of matrix metalloproteinases 2 and 9, neutrophil elastase, and type I collagen C propeptide. There was an overall trend of greater expression of all markers with increasing severity of wound site, this being highly significant for pro-matrix metalloproteinase-9 (p = 0.006). For samples collected simultaneously from improving and deteriorating regions of the same wound, paired data analysis showed statistically significant differences for pro-matrix metalloproteinase-9 (p < 0.001), neutrophil elastase (p < 0.005) and activated matrix metalloproteinase-9 (p < 0.05). Taken overall, these data show the potential of markers of collagen biochemistry as predictors of repair in venous ulcers; in particular pro-matrix metalloproteinase-9 and neutrophil elastase were found to be accurate prognostic indicators of subsequent healing.
TL;DR: It is concluded that little matrix metalloproteinase‐9 activity (the gelatinase involved in early tissue repair) is present in keloids and hypertrophic scars, while matrix meetallop proteinase‐2 activity ( the gelatin enzyme involved in prolonged tissue remodeling) ispresent in donor skin and is significantly increased in hypertrophic scar levels.
Abstract: Keloids and hypertrophic scars are characterized by excessive deposition of collagen, which may result from insufficient protein degradation. Little is known about the levels of two gelatinases, matrix metalloproteinase-2 (72 kD type IV collagenase) and matrix metalloproteinase-9 (matrix metalloproteinase-9; 92 kD type IV collagenase) in these abnormal scars. The purpose of this study was to determine levels of these proteinases in tissue from hypertrophic scars, keloids, and donor skin. Ten hypertrophic scar samples, 9 keloid samples, and 10 donor skin samples were frozen, pulverized, homogenized, clarified by centrifugation, and analyzed for matrix metalloproteinases by quantitative zymography. Identity of matrix metalloproteinases was determined using a conditioned media reference standard, molecular weight ladders, and Western blotting. Levels of matrix metalloproteinase-9 activity were very low or undetectable in all samples. However, matrix metalloproteinase-2 activity was significantly elevated in keloids and hypertrophic scars vs. donor samples: 2.6 and 3.9-fold increases for latent matrix metalloproteinase-2, 7.8 and 6.9-fold increases for active matrix metalloproteinase-2, respectively. We conclude that little matrix metalloproteinase-9 activity (the gelatinase involved in early tissue repair) is present in keloids and hypertrophic scars, while matrix metalloproteinase-2 activity (the gelatinase involved in prolonged tissue remodeling) is present in donor skin and is significantly increased in hypertrophic scars and keloids.
TL;DR: The results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue.
Abstract: A critical stage of cutaneous wound healing is the development and maturation of the epidermis. In the aged, and in certain pathologies, this repair process is compromised due to a variety of deficiencies, one of which is tissue oxygenation. Several phases of wound healing are dependent on adequate tissue oxygen levels, and hyperbaric oxygenation has been shown to transiently elevate these levels. The use of human cell monolayers, dermal equivalents and human skin equivalents provide excellent opportunities for studying wound healing using in vivo relevant models. The goal of this study was to examine the effect of hyperbaric oxygen on cell proliferation, differentiation, and matrix biosynthesis in monolayer cultures and epidermopoiesis in the developing skin equivalent. Normal human dermal fibroblasts, keratinocytes and melanocytes, dermal equivalents and skin equivalents were exposed to hyperbaric oxygen at pressures up to three atmospheres, for up to 10 consecutive daily treatments lasting 90 minutes each. Increase in fibroblast proliferation (cf., 30% at 1 atmosphere after 10 days treatment), was observed without a significant effect on proliferation of normal human melanocytes and glycosaminoglycan synthesis. Stimulation of collagen synthesis after two days of treatment was only significant at 1 atmosphere (about 20% increase) but this differential was not observed after 5 days of treatment. Hyperbaric oxygenation above 2 atmospheres, inhibited proliferation of fibroblasts and keratinocytes in cell monolayer cultures (e.g., a 10 day treatment at 3 atmospheres appeared cytostatic to keratinocytes). In contrast, hyperbaric treatment up to 3 atmospheres dramatically enhanced keratinocyte differentiation, and epidermopoiesis in the complete human skin equivalent. These results support the importance of hyperbaric oxygen therapy in wound healing, and should provide an insight into oxygen utilization during repair of peripheral human tissue. The results also show the utility of the human skin equivalent as a model for evaluation of parameters involved in wound healing. (WOUND REP REG 1999;7:53–64)
TL;DR: It is indicated that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor‐β1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.
Abstract: Tissue injury and pelvic inflammation often results in peritoneal scar tissue formation. The objective of this study was to determine whether mesothelial cells which line the peritoneal cavity express matrix metalloproteinases (MMPs) and tissue inhibitors of matrix metalloproteinases (TIMPs), and if their expression is regulated by transforming growth factor-beta1, a key regulator of tissue fibrosis. For this purpose we used Met-5A cells, a cell line derived from human normal mesothelial cells, and for comparative analysis we used U-937 cells, a human monocytic/macrophage cell line. The cells were treated with transforming growth factor-beta1 (1 ng/ml) for various time periods and the levels of MMP and TIMP mRNA and protein expression were determined using quantitative reverse transcription-polymerase chain reaction and enzyme-linked immunosorbent assay, respectively. The results indicate that the mesothelial cells and macrophages express MMP-1 (collagenase-1), MMP-3 (stromelysin-1), TIMP-1 and TIMP-2 mRNA and protein at various levels, with significantly higher TIMPs than MMPs, and higher MMP-1 than MMP-3 (p < 0.001). The mesothelial cells express significantly less MMP-1, higher MMP- 3 and similar levels of TIMP mRNA compared to macrophages. In a time-dependent manner, treatment of the mesothelial cells with transforming growth factor-beta1 resulted in a significant decrease in the expression of MMP-1, while increasing the expression of TIMP-1 mRNA (p = 0.05). In contrast, MMP-3 and TIMP-2 expression was unaffected in mesothelial cells and in macrophages, compared to untreated controls. There was a significant increase in secreted MMP-1 and TIMP-2 by mesothelial cells following transforming growth factor-beta1 treatments in a time-dependent manner (p = 0.05 and p = 0.01), without affecting the secretion of these proteins by macrophages. A major portion of MMP-1 in the culture conditioned media of both cell types was found in complex with TIMP-1. The ratios of MMP-1/TIMPs production were significantly higher than MMP-3/TIMPs in mesothelial cells and macrophages, and progressively decreased following transforming growth factor-beta1 treatments (p < 0.05). In conclusion, these results indicate that mesothelial cells express MMP and TIMP mRNA and protein, and their expression is differentially regulated by transforming growth factor-beta1, a mechanism that in part may influence the outcome of peritoneal tissue repair and adhesion formation.
TL;DR: Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons, which could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.
Abstract: Biochemical, biomechanical and ultrastructural properties of the connective tissue matrix were investigated during the early remodeling phase of tissue repair in experimentally tenotomized and repaired rabbit Achilles tendons. Sterile surgical tenotomy was performed on the right Achilles tendons of 14 rabbits and allowed to heal for 15 days. The animals were euthanized and the Achilles tendons excised from both limbs. The left contralateral Achilles tendon of each rabbit was used as a control in the experiments. Prior to biochemical analysis, both intact and healing tendons were tested for their biomechanical integrity. The results revealed that the healing tendons had regained some of their physicochemical characteristics, but differed significantly from the intact left tendons. The healing tendons regained 48% tensile strength, 30% energy absorption, 20% tensile stress, and 14% Young’s modulus of elasticity of intact tendons. In contrast, biochemical analysis showed that the healing tendons had 80% of the collagen and 60% of the collagen crosslinks (hydroxypyridinium) of normal tendons. Sequential extraction of collagen from the tissues yielded more soluble collagen in the healing tendons than intact tendons, suggesting either an increase in collagen synthesis and/or enhanced resorption of mature collagen in healing tendons compared to intact tendons. Electron microscopic studies revealed remarkable differences in the ultrastructure between intact and healing tendons. These observations could explain, in part, the connective tissue response to healing during the early phases of tissue remodeling.
TL;DR: Heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells is investigated, finding that normal skin fibroblast cultures were found to have a two‐fold higher percentage of apoptotic cells than did keloids fibro Blast cultures.
Abstract: The purpose of this study was to determine if aberrant apoptosis plays a role in pathologic wound healing as manifested by hypertrophic scarring and keloid formation. Apoptosis has recently been found to participate in the transition between granulation tissue and the development of definitive scar. The question that remains to be answered is what stimuli initiate apoptosis during wound healing. Hitherto, regulatory factors and pathways involved have been largely undefined. We investigated heterogeneity among fibroblasts derived from normal skin and keloid scar, by examining apoptotic profiles and pathways for these cells. Quantitative analysis of apoptotic cells using an Annexin-V-FITC binding assay showed that normal skin fibroblast cultures were found to have a two-fold higher percentage of apoptotic cells than did keloid fibroblast cultures. To study apoptotic pathways and related death-associated genes, a ribonuclease protection assay was performed for fibroblasts exposed to anti-Fas antibody and tumor necrosis factor-alpha to activate the Fas/TNF receptor apoptotic pathway. Compared with normal skin fibroblasts, keloid fibroblasts exhibited decreased expression of apoptosis-associated genes.
TL;DR: The results of this study are significant because the specific integrins affected are intimately involved in two events that have been shown to be important to intrasynovial flexor tendon healing, namely fibronectin deposition as part of the provisional matrix and angiogenesis/revascularization.
Abstract: Integrins are important players in soft tissue healing as molecules that mediate communication between cells and extracellular matrix. Thus, the regulation of the expression of these molecules would be important during wound repair. To explore the regulatory roles of specific growth factors on integrin expression by intrasynovial flexor tendon cells, the present study assessed the in vitro effects of basic fibroblast growth factor and platelet derived growth factor-BB on expression of the alpha5beta1 and alpha(v)beta3 integrins in these cells. Analyses were carried out at the transcriptional (reverse transcription-polymerase chain reaction) and translational (immunohistochemistry) levels of cellular metabolism. Both types of analyses revealed increased expression of alpha5beta1 and alpha(v)beta3 by tendon cells exposed to either basic fibroblast growth factor or platelet-derived growth factor-BB over a wide range of growth factor concentrations employed in the study. Semiquantitative reverse transcription-polymerase chain reaction showed that, relative to control, basic fibroblast growth factor and platelet-derived growth factor-BB increased the expression of alpha(v) mRNA by 2-and 3-fold, respectively. Alpha 5 mRNA expression was also increased 3-fold by basic fibroblast growth factor, and 2-fold by platelet-derived growth factor-BB. We believe the results of this study are significant because the specific integrins affected are intimately involved in two events that have been shown to be important to intrasynovial flexor tendon healing, namely fibronectin deposition (alpha5beta1) as part of the provisional matrix and angiogenesis/revascularization (alpha(v)beta3).
TL;DR: A mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor‐β pathway is presented.
Abstract: Fibrosis is a consequence of injury which is characterized by accumulation of excess collagen and other extracellular matrix components, resulting in the destruction of normal tissue architecture and function. Transforming growth factor-beta, a potent wound healing agent, has also been shown to be an agent that can produce fibrosis because it is a potent stimulator of collagen synthesis. Both glucocorticoids and bleomycin have recently been shown to affect collagen synthesis in opposite directions, by utilizing a common pathway of involving transforming growth factor-beta activator protein binding to the transforming growth factor-beta element. This article presents a mechanistic overview of collagen synthesis regulation by glucocorticoids and bleomycin through the transforming growth factor-beta pathway.
TL;DR: The above observations indicate that GK rat fibroblast proliferation is suppressed when the cells are cultured in high glucose containing media, and protein kinase C and hyaluronic acid might play a role as modulators of fibroblasts proliferation during the diabetic state.
Abstract: Intact fibroblast function is required for normal wound healing. Although healing is generally accepted to be disturbed in non-insulin dependent diabetes mellitus, the signals modulating this disturbance are not fully understood. Therefore, we studied dermal fibroblasts from the GK rat, a non-insulin dependent diabetes mellitus model, and the Wistar rat (control) regarding growth characteristics, and L-lactate production at 5.5 mM and 25.5 mM glucose in the absence or presence of protein kinase C-inhibition, or alpha-tocopherol acetate. In addition, growth and L-lactate responses to hyaluronic acid were assessed under normal glucose conditions. At 5.5 mM glucose, the fibroblasts from the GK rat showed a lower proliferation rate during the first 24 hours, measured as DNA content, as compared to Wistar rats, i.e. at 8 hours GK was 57% of control, p < 0.01, at 24 hours GK was 60% of control, p < 0.01. The GK rat fibroblasts accumulated higher L-lactate levels in the media at 24-96 hours. Addition of glucose at a concentration of 25.5 mM decreased the total DNA content in GK rat fibroblast cultures to 74% (p < 0.05) and in control to 87% (p < 0.05), and increased L-lactate levels, measured at 48 hours. A protein kinase C-inhibitor, bisindolylmaleimide IX, increased DNA content and decreased L-lactate in both cell types during culture in high glucose, but only affected GK rat fibroblasts during normal glucose. Hyaluronic acid, increased DNA content in both types of fibroblasts, GK: 139% (p < 0.05), control: 127% (p < 0.05) and reduced L-lactate production. The above observations indicate that GK rat fibroblast proliferation is suppressed when the cells are cultured in high glucose containing media. In addition, protein kinase C and hyaluronic acid might play a role as modulators of fibroblast proliferation during the diabetic state.
TL;DR: Results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro, and that they reform epidermal barrier more rapidly after grafting than culturedskin substitutes in saturated humidity.
Abstract: Cultured skin substitutes have been used successfully for adjunctive treatment of excised burns and chronic skin wounds. However, limitations inherent to all models of cultured skin include deficient barrier function in vitro, and delayed keratinization after grafting in comparison to native skin autografts. Experimental conditions for incubation of skin substitutes were tested to stimulate barrier development before grafting, and measure responses in function and stability after grafting. Cultured skin substitutes consisted of human keratinocytes and fibroblasts attached to collagen-glycosaminoglycan biopolymer substrates. Parallel cultured skin substitutes were incubated at the air-liquid interface in ambient (48-61%) or saturated (79-91%) relative humidity, and grafted to athymic mice on culture day 14. Additional cultured skin substitutes were incubated in the experimental conditions for a total of 28 days. Cadaveric human skin and acellular biopolymer substrates served as controls. Epidermal barrier was evaluated as the change in surface hydration by surface electrical capacitance with the NOVA Dermal Phase Meter. Cultured skin substitutes and cadaveric skin incubated in ambient humidity had lower baseline surface electrical capacitance and less change in surface electrical capacitance than parallel samples incubated in saturated humidity at all time points in vitro. Data from healing cultured skin substitutes at 2, 4, 8 and 12 weeks after grafting showed an earlier return to hydration levels comparable to native human skin, and more stable engraftment for skin substitutes from ambient humidity. The data indicate that cultured skin substitutes in ambient humidity have lower surface electrical capacitance and greater stability in vitro, and that they reform epidermal barrier more rapidly after grafting than cultured skin substitutes in saturated humidity. These results suggest that restoration of functional epidermis by cultured skin substitutes is stimulated by incubation in reduced humidity in vitro.
TL;DR: A review of data strongly indicates that the phylogenetic age and biological differences between different species should be taken into account before extrapolation of regenerative properties of nonprimate tissues on the regenerative responses in the primates.
Abstract: Most of the available information regarding the regenerative potential and compensatory remodeling of mammalian tissues has been obtained from nonprimate animals, mainly rodent experimental models. The increasing use of transgenic mice for studies of the mechanisms controlling organogenesis and regeneration also requires a clear understanding of their applicability as experimental models for studies of similar processes in humans and other mammals. Application of modern cell biology methods to studies of regenerative processes has provided new insights into similarity and differences in cellular responses to injury in the tissues of different mammalian species. During more than 200-million years of progressive divergent evolution of mammals, cellular mechanisms of tissue regeneration and compensatory remodeling evolved together with increasingly adaptive functional specialization and structural complexity of mammalian tissues and organs. Rodents represent a phylogenetically ancient order of mammals that has conservatively retained a number of morphofunctional characteristics of early representatives of this class, which include enhanced regenerative capacity of tissues. A comparative analysis of regenerative processes in skeletal and cardiac muscle, as well as in several other mammalian tissues, shows that time courses and intensities of regeneration in response to the same type of injury vary even within taxonomically related species (e.g., rat, mouse, and hamster). The warm bloodedness of mammals facilitated the development of more complex mechanisms of metabolic, immune, and neurohumoral regulation, which resulted in a stronger dependence of regenerative processes on vascularization and innervation. For this reason, interspecies modifications of regenerative responses are limited by the capacity of the animal to resorb rapidly the foci of necrosis and to revascularize and reinnervate the volume of the regenerating tissue. These differences, among other factors, result in significantly lower rates of reparative regeneration in mammals possessing larger body sizes than rodents. A review of these data strongly indicates that the phylogenetic age and biological differences between different species should be taken into account before extrapolation of regenerative properties of nonprimate tissues on the regenerative responses in the primates.
TL;DR: Keratinocyte growth factor‐2 may be useful in accelerating healing in wounds healing mainly by the process of epithelialization such as venous stasis ulcers, partial thickness burn wounds, and skin graft donor sites and might also accelerate the gain in incisional wound strength in acute surgical or traumatic wounds.
Abstract: Human keratinocyte growth factor-2 exerts a proliferative effect on epithelial cells and mediates keratinocyte migration. It has also been shown to increase both deposition of granulation tissue and collagen and maturation of collagen. Because these properties should affect the healing trajectory of wounds, this study set out to investigate the effects of keratinocyte growth factor-2 on the healing of three different types of wounds. Human meshed skin grafts explanted to athymic "nude" rats, surgical incisions in Sprague-Dawley rats, and acute excisional rat wounds inoculated with Escherichia coli were used. Two concentrations of recombinant human keratinocyte growth factor-2 were compared to a vehicle control and keratinocyte growth factor-1. Keratinocyte growth factor-2 significantly accelerated the rate of epithelialization in the meshed skin graft model and effected a modestly more rapid gain in breaking strength of surgical incisions than keratinocyte growth factor-1 or the vehicle control treatment. Neither keratinocyte growth factors accelerated wound closure by contraction of the excisional wounds. Based on these data, keratinocyte growth factor-2 may be useful in accelerating healing in wounds healing mainly by the process of epithelialization such as venous stasis ulcers, partial thickness burn wounds, and skin graft donor sites. It might also accelerate the gain in incisional wound strength in acute surgical or traumatic wounds.
TL;DR: Adenoviral mediated gene transfer is a promising efficient means of growth factor delivery to chronic wounds, however, selection of the proper transgene with appropriate biologic activity in wound healing may be essential to overcome the potential adverse effects of adenovirus infection.
Abstract: Chronic nonhealing wounds represent a large clinical problem resulting in severe disabilities and large healthcare expenditures. Despite the scope of this problem, effective new therapies are lacking. The deficiency of growth factors in chronic wounds has brought attention to the topical application of growth factors, but initial clinical trials have resulted in only modest improvements in healing despite large, repetitive doses. The modest improvement in healing observed in these trials show that growth factors can improve chronic wound healing, but a better means of growth factor delivery is needed. We hypothesized that gene therapy using a recombinant adenoviral vector could be used to induce transgene production directly by cells in the wound. An adenovirus containing the beta-galactosidase reporter transgene (Ad-LacZ) was used in the ischemic rabbit ear model to test this hypothesis. Ad-LacZ resulted in efficient transgene delivery to cells participating in the wound healing response, with expression up to 2 weeks. However, wound reepithelialization was impaired in Ad-LacZ treated wounds compared to vehicle control wounds. Adenoviral mediated gene transfer is a promising efficient means of growth factor delivery to chronic wounds. However, selection of the proper transgene with appropriate biologic activity in wound healing may be essential to overcome the potential adverse effects of adenoviral infection.
TL;DR: Examination of the effect of incubation of platelet‐derived growth factor with chronic wound fluid from leg ulcers on the in vitro growth of human dermal fibroblasts showed that, in standard culture conditions, Chronic wound fluid always stimulated fibroblast proliferation, and, in most cases, incubating platelet-derived growth factors with chronic wounds fluid increased the stimulation.
Abstract: The fate of biologically active proteins applied to chronic wounds is almost totally unknown. Growth factors may be degraded by proteases, which are produced by both inflammatory and skin cells and by resident bacteria. However, there has been little work on the effect of chronic wound fluid on the activity of growth factors. A bioassay method has been chosen to examine the effect of incubation of platelet-derived growth factor with chronic wound fluid from leg ulcers on the in vitro growth of human dermal fibroblasts. Human dermal fibroblasts were cultured in serum-free medium, and a dose-response curve for proliferation in response to platelet-derived growth factor was obtained. Wound fluid was collected under occlusive dressings from five patients with chronic leg ulcers. Platelet-derived growth factor was incubated with chronic wound fluid at 37 degrees C for 4 hours, and the reactions arrested by snap freezing. The resultant solutions were tested for their ability to promote fibroblast proliferation. A colorimetric assay was used to monitor changes in the platelet-derived growth factor mitogenicity. The results showed that, in our standard culture conditions, chronic wound fluid always stimulated fibroblast proliferation, and, in most cases, incubation of platelet-derived growth factor with chronic wound fluid increased the stimulation compared with that produced by platelet-derived growth factor or chronic wound fluid alone.