Ulcer Bed Research Articles

4476 Differential expansion of mucosal t cells in intestinal ulceration tissues of patients with inflammatory bowel disease.

[Backgrounds and Aims] Ulcerative colitis (UC) and Crohn's disease (CD) were associated with chronic continuous inflammation of the intestinal tract with undefined pathogenesis. Impaired immune responses have been thought to be closely associated with both disorders. Although endoscopic features of UC and CD differed, e.g. geographic shallow ulceration or diffuse erosions in UC and distinct longitudinal ulcers in CD, underlined immunological features of intestinal inflammation in each disease remain unclarified. The aim of this study was to determine immunologic differences between UC and CD, focused on activated lymphocytes in the intestinal mucosal. [Subjects and Methods] Biopsy or surgical specimens of intestinal tissues from patients with UC and CD, who had underwent colonoscopy were promptly fixed with periodate-lysine-2% paraformaldehyde, and offered for single and double immunohistochemistry using monoclonal antibodies (Ki67 for proliferating cells; CD3, CD4 and CD8 for T cells; CD19 and surface immunoglobulin for B cells; CD45RA and CD45RO for naïve and memory cells). [Results and Discussion] In normal intestinal mucosa, Ki67-immunostaining was observed mainly in lower part of crypt epithelium, and lamina propria lymphocytes (LPL) stained for Ki67 were only scatteredly observed. In geographical ulcer of UC, Ki67-positive cells were increased and distributed throughout ulcer bed tissue. Most frequent phenotype was CD3-, CD19+, Ki67+and CD45RA+, which showed predominant expansion of naïve type B cells. In Crohn's disease, on the contrary, most frequently observed proliferating cells was CD3+, CD4+ and either CD45RA+ or CD45RO+, which showed predominant expansion of memory or naïve type T cells. In UC, mucosal barrier break, which is represented by shallow ulceration, allows excessive invasion of luminal contents including bacterial microflora, and stimulate LPL for B cell predominant manner. On the contrary, in distinct longitudinal ulcer often seen in CD, overactivation of memory T cells recruited from systemic lymphocytes pool was exerted. Further characterization about pathways of antigen invasion and immune reaction in each type of ulcer formation will prvide us with important information in the pathogenesis of both diseases. [Conclusion] These differences in mucosal immune responses may closely relate to the manifestation of tissue damage in both diseases. [Backgrounds and Aims] Ulcerative colitis (UC) and Crohn's disease (CD) were associated with chronic continuous inflammation of the intestinal tract with undefined pathogenesis. Impaired immune responses have been thought to be closely associated with both disorders. Although endoscopic features of UC and CD differed, e.g. geographic shallow ulceration or diffuse erosions in UC and distinct longitudinal ulcers in CD, underlined immunological features of intestinal inflammation in each disease remain unclarified. The aim of this study was to determine immunologic differences between UC and CD, focused on activated lymphocytes in the intestinal mucosal. [Subjects and Methods] Biopsy or surgical specimens of intestinal tissues from patients with UC and CD, who had underwent colonoscopy were promptly fixed with periodate-lysine-2% paraformaldehyde, and offered for single and double immunohistochemistry using monoclonal antibodies (Ki67 for proliferating cells; CD3, CD4 and CD8 for T cells; CD19 and surface immunoglobulin for B cells; CD45RA and CD45RO for naïve and memory cells). [Results and Discussion] In normal intestinal mucosa, Ki67-immunostaining was observed mainly in lower part of crypt epithelium, and lamina propria lymphocytes (LPL) stained for Ki67 were only scatteredly observed. In geographical ulcer of UC, Ki67-positive cells were increased and distributed throughout ulcer bed tissue. Most frequent phenotype was CD3-, CD19+, Ki67+and CD45RA+, which showed predominant expansion of naïve type B cells. In Crohn's disease, on the contrary, most frequently observed proliferating cells was CD3+, CD4+ and either CD45RA+ or CD45RO+, which showed predominant expansion of memory or naïve type T cells. In UC, mucosal barrier break, which is represented by shallow ulceration, allows excessive invasion of luminal contents including bacterial microflora, and stimulate LPL for B cell predominant manner. On the contrary, in distinct longitudinal ulcer often seen in CD, overactivation of memory T cells recruited from systemic lymphocytes pool was exerted. Further characterization about pathways of antigen invasion and immune reaction in each type of ulcer formation will prvide us with important information in the pathogenesis of both diseases. [Conclusion] These differences in mucosal immune responses may closely relate to the manifestation of tissue damage in both diseases.

Treatment of venous leg ulcers with clobetasol propionate ointment

BACKGROUND: The use of topical steroids should be minimized in the treatment of venous leg ulcers. However, as a consequence of venous insufficiency poorly understood inflammatory processes may occur. These may impair ulcer healing, so in cases of severe inflammatory changes steroids may be required to permit healing. METHODS: A 71-year-old female had chronic venous ulceration of the right lower leg. Histology showed eczema and ulceration and the patient complained of a 'pain' rather than 'itching'. Treatment commenced with betamethasone to eczematous skin but improvement was slow. After developing acute erythema and exudation of most of the skin of the right lower leg, daily treatment with clobetasol propionate ointment was commenced to the inflamed skin and ulcer beds. RESULTS: The exudation ceased, the 'pain' reduced and the ulcers began to heal. CONCLUSION: This isolated case suggests that a minority of patients with venous leg ulcers may benefit from topical steroid treatment to the ulcer beds. This hypothesis merits to be tested in a doubleblind, vehicle-controlled study. (J Dermatol Treat (2000) 11:53–55)

Effect of basic fibroblast growth factor on gastric ulcer healing and its own mRNA expression

The application of an acid-stable mutein of basic fibroblast growth factor (bFGF) called CS23 results in acceleration of ulcer healing. The modes by which this cytokine exerts these effects are not yet completely understood. To describe the pattern of bFGF-mRNA expression during ulcer healing and to examine the effects of exogenously applied CS23 on gastric ulcer healing in an animal model. The speed of healing of gastric ulcers, expression of extracellular matrix gene mRNAs such as pro alpha(I) collagen (by non-radioactive in situ hybridization), cellular proliferation evidenced by the display of PCNA (by immunohistochemistry), angiogenesis, and the feedback of this growth factor on its own mRNA expression pattern were used to evaluate the effects of CS23 on rat gastric ulcer healing in an animal model. CS23 accelerates gastric ulcer healing at 7, 14 and 21 days after ulcer induction. We found an increase in connective tissue beneath the ulcer bed in treated animals in comparison to controls. The expression of PCNA as well as pro alpha(I) collagen mRNA was markedly increased in ulcers, yet there was no distinct difference between treatment arms. In contrast, the density of microvessels was significantly increased in the submucosa of ulcers by CS23 application. bFGF-mRNA expression is up-regulated in the submucosa during early ulcer healing; this increase diminishes within days but can be restituted by the exogenous application of CS23. CS23 speeds gastric ulcer healing and significantly increases the density of microvessels in the ulcerated tissue without affecting the numbers of proliferating cells or the transcription of collagen mRNA. In addition, it augments the expression of bFGF-mRNA during the later stages of healing, suggesting a positive feedback loop.

Bleeding ulcer: inject or clip?

The authors retrospectively compared endoscopic injection therapy with endoscopic hemoclipping by examining 113 patients over a 10-yr period between 1985 and 1995 who had gastrointestinal bleeding and who received one or the other procedure. Endoscopic injection using ethanol alone, epinephrine plus ethanol, or aethoxysclelol plus ethanol was performed in 87 cases. Endoscopic hemoclipping was performed in 26 cases from 1993 to 1995. The permanent hemostasis rate of endoscopic injection was 84% and that of endoscopic hemoclipping was 100%. Complications associated with endoscopic injection included enlargement of the ulcer bed, bleeding caused by exposed vessels within the expanded ulcer and one case of perforation. No complications were noted among patients treated with endoscopic hemoclipping.

Patterns of matrix metalloproteinase and TIMP expression in chronic ulcers.

Controlled proteolysis is needed for cell migration, angiogenesis, and matrix remodeling during normal wound repair. Our objective has been to investigate how chronic leg ulcers differ from normally healing wounds (pinch graft donor sites) with respect to their metalloproteinase expression patterns. Using in situ hybridization and immunohistochemistry, we found that collagenase-1 (MMP-1), stromelysin-1 (MMP-3) and stromelysin-2 (MMP-10) were expressed in keratinocytes bordering both acute and chronic wounds. Unlike MMP-1, signal for collagenase-3 (MMP-13) was not detected in keratinocytes but exclusively in fibroblasts deep in the ulcer bed of chronic wounds, suggesting that while MMP-1 production is important for migration, MMP-13 plays a role in matrix remodeling. Tissue inhibitor of metalloproteinase (TIMP)-1 was not detected in the epidermis of any chronic wound sample while it was expressed in keratinocytes bordering normally healing wounds. TIMP-3 was abundantly expressed in stromal fibroblast- and macrophage-like cells surrounding vessels and sweat glands in both types of wounds. Our results suggest that there are no qualitative differences in the expression of MMPs-1, -3 and -10 in the epidermis of chronic vs normally healing wounds. However, the number of stromal cells expressing MMP-1 and MMP-3 was greater in chronic vs acute wounds, whereas MMP-10 was never detected in the dermis. TIMP-1 expression near the basement membrane in acute, but not in chronic, wounds suggests that the balance between MMPs and their inhibitors may be altered in poorly healing wounds. Analogous to chronic cutaneous wounds, MMP-1 and -3 are abundantly expressed in chronic small and large bowel ulcers, while the migrating surface epithelium is negative for TIMP-1 expression.

Ulcer bed infection

We report a case of ulcer bed infection in an enlarging venous leg ulcer without clinical signs of cellulitis in the surrounding tissues. Signs of infection in the leg ulcer were: 1) cocci-like structures and bacteria-like rods around vessel walls in the viable ulcer bed, 2) vasculitis-like inflammation of deeply situated vessels of the viable tissue, 3) Pseudomonas aeruginosa-specific antibodies in the serum (other than against exotoxin A), 4) extensive epidermolysis of normal human skin by the wound exudate in vitro, and 5) P. aeruginosa exotoxin A in the wound exudate (23 ng/ml). In an in vitro cell assay, the wound exudate was cytotoxic and rabbit antibodies to exotoxin A, but not a serine proteinase inhibitor, inhibited this cytotoxicity. P. aeruginosa exotoxin A might contribute to the pathogenesis of the ulcer enlargement. The ulcer improved after the third skin graft, probably mainly due to effective treatment with a long-stretch compression bandage.

Limb-salvage in reconstruction of recalcitrant pressure sores using the inferiorly based rectus abdominis myocutaneous flap.

Pressure sore closure is frequently a reconstructive challenge. This challenge is particularly evident in cases of multiply recurrent sores. In such settings, there are often opportunities to manage the recurrent wounds either by repeated advancement of previous flaps or by design of alternative ones. However, these interventions are not always feasible, and limb amputation with total thigh flap closure must be considered. A review of operative experience with seven such complex pressure sores in seven patients is presented. Each patient had previously suffered a permanent thoracic-level spinal cord injury. Prior attempts at wound closure were unsuccessful. Despite consideration of all described locoregional flaps, no limb-sparing procedure could be designed satisfactorily. As an alternative to either hip disarticulation and total thigh flap coverage or distant free-tissue transfer, we reconstructed the debrided ulcer beds with inferiorly based rectus abdominis myocutaneous flaps. Six of the seven wounds healed primarily, whereas one required repeated debridement and the addition of a gracilis muscle flap to achieve complete closure. Postoperative follow-up has ranged from 6 to 45 months. Each patient has returned to his baseline preoperative activity level with no clinical compromise of abdominal wall function. All wounds have healed. Successful application of the inferiorly based vertical rectus abdominis myocutaneous flap for cases of both recalcitrant ischial and trochanteric pressure sores is demonstrated and its consideration is advocated if no reconstructive options short of extremity amputation and total thigh flap coverage exist for such challenging sores.

Expression of intestinal trefoil factor in early repairing process of acute gastric mucosal damage induced, by indomethacin in rat

Backeround & Aims; Trefoil peptides such as pS2, PSP, and intestinal trefoil factor (ITF) are a relatively new group of peptides which have been shown to prevent and/or ameliorate gastric mucosal injury induced by ethanol and by NSAIDs. They may also serve to reseal mucosal wounds, influence cell proliferation, and accelerate the migration of proliferating epithelial cells in humans and mice. All are considered to be tissue-specific, and ITF is shown to be produced by goblet ceils in the normal intestinal mucosa and to be delivered with mucin glycoprotein. However, it has not been fully examined whether ITF is expressed constitutively in the stomach. Moreover, a possible role of ITF expression in repairing process of gastric mucosal injury remains undetermined. In the present study, a model of acute gastric mucosal damage in the rat was used to examine the expression of ITF which may play an active part in the healing response. Methods: Acute g~/stric mucosal damage was induced by intragastric administration of indomethacin (20 mg/kg). The expression of ITF was monitored over the next 72 hours using immunohistochernistry, and was assessed quantitatively by measuring proportion of ITF-positive area (%) to the background on the samples with immunohistochemical staining. The expression of proliferating cell nuclear antigen (PCNA) was also examined. Re$ult$: (1) In the normal gastroduodenal rnucosa, ITF immunoreactivity was present strongly at the glandular bases of the gastric body, weakly at the glandular necks, and most strongly within the duodenal glands (linked with mucin glycoprntein). No immunostaining was seen in the bulk of the gastric surface epithelium. (2) Administration of indomethacin induced hemorrhagic lesions over the body after 3 hrs, erosions and ulcerations over the antrum after 24 hrs, and reepithelialization of the ulcerations after 72 hrs. (3) Three hours after ulcer induction, enhanced immunoreactivity of ITF was not detected. Twenty four hours or later after induction, immunoreactive ITF was enhanced not only in the mucous neck cells, but also at the glandular bases. ITF-positive area at the ulcer beds was 0% after 3 and 24 hrs, and 0.8% after 72 hrs. In contrast, ITF-positive area at the ulcer edges increased in a time-related fashion (0%, 3.3% and 8.4% after 3, 24 and 72 hrs, respectively), whereas it did not change at non-ulcerated regions during the period. (4) PCNA labeling index at the ulcer edge increased, corresponding with enhanced immunoreactivity of ITF. Conclusions: The localization of ITF to the normal gastric epithelium, particularly the bases of fundic glands, raises the possibility that ITF may act as a regulator of secretory function. The pronounced expression of ITF around the ulcerations after induction of gastric injury suggests that ITF peptide may play a significant role in the repair-accelerating mechanisms, and also in the later ulcer-healing processes in the stomach.

Mechanisms of Ulcer Healing and Effects of Nonsteroidal Anti-inflammatory Drugs

Ulceration of the gastroduodenal mucosa occurs frequently in humans, particularly in patients with a history of peptic ulcer disease. In order for healing to occur, mucosal damage stimulates secretion of growth factors in the adjacent mucosa and ulcer bed. Peptic ulcer healing is accomplished by the filling of the mucosal defect with cells that migrate from the ulcer margin and by connective tissue, including microvessels originating from granulation tissue. Peptic ulcer healing is accelerated both in humans and experimental models by gastric acid inhibition, which enhances cell migration and maturation of the granulation tissue. In experimental models, peptic ulcer healing can also be accelerated by oral administration of basic fibroblast growth factor, which increases angiogenesis in the ulcer bed, or by nitric oxide-releasing compounds, which improve gastric blood flow. Clinical and experimental data indicate that traditional nonsteroidal anti-inflammatory drugs (NSAIDs) delay the healing of peptic ulcers by interfering with the action of growth factors, decreasing epithelial cell proliferation in the ulcer margin, decreasing angiogenesis in the ulcer bed, and slowing maturation of the granulation tissue. In order to reduce the gastroduodenal side-effects of NSAIDs, selective cyclo-oxygenase (COX)-2 inhibitors have been developed, which inhibit the inducible COX-2 isoform in inflammatory tissue but have only limited effect on the constitutive COX-1 isoform in the stomach. It has been reported that the selective COX-2 inhibitor L-745,337 has a reduced liability for gastrointestinal ulceration. In our chronic experimental gastric ulcer model in rats, however, delay of gastric ulcer healing with L-745,337 was comparable to that with ordinary NSAIDs. It has also been reported that nitric oxide-releasing NSAIDs have a low relative risk of gastrointestinal ulceration but, again, in our chronic gastric ulcer model, nitric oxide did not reverse NSAID-induced deleterious effects on ulcer healing. In contrast, the proton pump inhibitor omeprazole has been shown to reverse NSAID-induced deleterious effects on gastric ulcer healing in our model. Comparable results have also been reported in humans. Histologic analysis has shown that omeprazole reverses the effects of NSAIDs on cell proliferation, angiogenesis, and maturation of the granulation tissue. In conclusion, only highly effective gastric acid inhibition reliably reverses NSAID-induced delay of gastric ulcer healing.

Effects of inhibition of prostaglandin endoperoxide synthase-2 in chronic gastro-intestinal ulcer models in rats.

1. In the stomach, prostaglandins protect the gastric mucosa against injuries. One rate-limiting step in prostaglandin synthesis is mediated by prostaglandin endoperoxide synthase (PGHS), the target enzyme of non-steroidal anti-inflammatory drugs (NSAIDs). Two isoforms of PGHS exist: a constitutive (PGHS-1) and an inducible (PGHS-2) enzyme. PGHS-1 is the major source of gastric prostaglandins under physiological conditions. Inhibition of prostaglandin synthesis by traditional NSAIDs such as indomethacin and diclofenac which non-selectively inhibit both PGHS-1 and PGHS-2, causes gastric and intestinal ulceration and delays gastric ulcer healing in chronic models. It has been shown that selective PGHS-2 inhibitors such as L-745,337 (5-methanesulphonamide-6-(2,4-difluorothio-phenyl)-1-inda none) are not ulcerogenic and do not inhibit gastro-intestinal prostaglandin synthesis. However, minimal information is available on the long-term effects of PGHS-2 inhibitors on the healing of previously established gastric injuries. We assessed the cellular localization and expression of PGHS-1 and PGHS-2 during gastric ulcer healing and assessed the effects of L-745,337 on previously established cryoulcers in the rat gastric stomach. 2. PGHS-1 and PGHS-2 were located and quantified by immunohistochemistry during experimental gastric ulcer healing. PGHS-2 immunoreactivity was only negligible in the normal gastric wall, but after gastric ulcerations, it was strongly detected in monocytes, macrophages, fibroblasts and endothelial cells below and between the regenerative glands. PGHS-1 immunoreactivity detected in normal gastric mucosa, disappeared after gastric ulceration in the mucosa adjacent to the ulcer crater. However, it reappeared in the regenerative glands from day 5 onwards. Thus, PGHS-1 and PGHS-2 were located at different sites and their maximal expression followed a different time-sequence. 3. We assessed the effects of L-745,337, indomethacin and diclofenac on gastric ulcer healing and histological healing parameters in rats. L-745,337, indomethacin and diclofenac dose-dependently decreased the healing of gastric ulcers. L-745,337, indomethacin and diclofenac decreased epithelial cell proliferation in the ulcer margin and microvessel density in the ulcer bed on day 8 and increased the thickness of the granulation tissue below the ulcer crater and the gap between both edges of the muscularis mucosae on day 15. Indomethacin and diclofenac, but not L-745,337, decreased synthesis of 6-keto-PGF1alpha and PGE2 in tissue fragments from the stomach and terminal ileum and decreased platelet thromboxane B2 synthesis in clotting whole blood. 4. Dose-response curves for the inhibition of chronic gastric ulcer healing by L-745,337 (administered twice daily intragastrically) showed an ID50 value of 1.7 mg (4.3 micromol) kg(-1). Dose-response curves for the inhibition of PGE2 synthesis in inflammatory exudates in the acute carrageenin sponge rat model, showed ID50 values of 1.1 mg (3.1 micromol) kg(-1) and 1.3 (3.3 micromol) mg kg(-1) for indomethacin and L-745,337, respectively. Thus, inhibition of chronic gastric ulcer healing by L-745,337 occurs within a potentially therapeutic dose-range. 5. In summary, PGHS-2 is markedly accumulated after gastric ulceration in monocytes, macrophages, fibroblasts and endothelial cells in regions of maximal repair activity. Selective inhibition of PGHS-2 by L-745,337 delayed gastric ulcer healing though interference with epithelial cell proliferation, angiogenesis and maturation of granulation tissue in a potentially therapeutic dose range. PGHS-2-derived prostaglandins seem to have an important role in gastric ulcer healing.

Fibroblast senescence in pressure ulcers.

Pressure ulcers appear as localized chronic wounds in the middle of normal functioning skin. This study focuses on pressure ulcer fibroblasts cultured from the ulcer bed, ulcer margin, and normal, nonulcerated skin adjacent to the wound. From these areas we show that pressure ulcer fibroblasts become prematurely senescent. Verification of senescence was based on failure of cell populations to undergo a 0.5 population doubling after 1 week in culture, light microscopic appearance of late-passage senescent cells in culture, light microscopic verification of senescence in vivo and in vitro by anti-terminin staining, and ultrastructural identification of senescent fibroblasts by anti-terminin colloidal gold labeling. Although not all ulcer fibroblasts are senescent, there is a range of proliferative ability. The senescent phenotype of ulcer fibroblasts remains intact in vitro as these cells remain viable but are unable to complete DNA synthesis. Fibroblast senescence is not uniform in chronic wounds but varies within areas of the ulcer bed and from patient to patient. Although ulcer fibroblasts exhibit limited proliferative ability, fibroblasts from the ulcer margin and adjacent normal skin show a continued ability to divide. However, in all areas of the wound, the mean generation time of the fibroblast population is longer than commonly observed for cultured human diploid fibroblasts. At first passage, the mean generation time for fibroblasts from all patients is significantly different (p = .001, analysis of variance) among fibroblasts from the ulcer bed (4.2 2.2 days), ulcer margin (2.9 +/- 1.3 days), and adjacent normal skin (1.9 +/- 0.6 days). Analysis of the three cell groups by the post hoc Tukey test, using corrected p values shows that the difference between mean generation times among fibroblast populations from adjacent normal skin and ulcer bed are significantly different (p < 0.05), whereas the difference between fibroblasts from adjacent normal skin and ulcer margin and ulcer margin and ulcer bed are not significantly different (p > 0.05). These data may help to explain the poor response of certain pressure sores to aggressive medical treatment.

Chemokine and inflammatory cytokine changes during chronic wound healing

Wound healing is a complex process resulting from an interplay of processes including coagulation, inflammation, angiogenesis, and epithelialization. The chemokine family has been shown to contain members that are potent regulators of many of these pathways. Because we have previously shown that chemokines "pool" in biologic wound dressings, we studied the levels of CXC and CC chemokines, along with key inflammatory mediators, serially from a group of patients undergoing therapy for chronic venous leg ulcers. After 8 weeks, all patients had marked clinical healing of their ulcers (median 63.3% reduction in size) with two of 10 completely healed. Wound fluids extracted from dressings showed high levels of platelet factor-4 and interferon-gamma-inducible protein, with a trend toward increases in the ratio of the sums of the angiogenic versus angiostatic CXC chemokines (p = 0.082) in the tissues collected from the center of the ulcers during wound closure. Neutrophil-activating peptide-2 and interleukin-8 accounted for the most changes in wound fluid angiogenic chemokines, with significant differences both as compared with baseline levels and with patients' plasma level noted at various time points between weeks 0 and 8. The level of angiostatic chemokines, interferon-y inducible protein 10 and platelet-activating-4, fell most significantly between weeks 0 and 3 as compared with plasma levels. The observed shift toward angiogenic CXC chemokines suggests that effective healing in chronic venous insufficiency ulcers appears to "move" the ulcer bed toward a state more conducive to epithelialization,characteristic of the proliferative phase of wound healing. CC chemokines were also elevated at baseline in the wound fluid samples as compared with the patients' plasma levels. Macrophage inflammatory protein-1 (3 and regulated on activation, normal T expressed and secreted (RANTES) levels decreased with healing, whereas there were significant increases in the tissue levels of monocyte chemoattractant protein-1 and macrophage inflammatory protein-1 a over the first 4 weeks of therapy (p< or = 0.05 for both). Coincident with these changes was a steady increase in the ratio of interleukin-1 R/interleukin-1 receptor antagonist protein in the ulcer center tissues, which also correlated with healing (p < 0 .05) as compared with a decreasing ratio at the ulcer edge, and a biphasic response in the wound fluids. These findings suggest that advanced wound care techniques help move the ulcer from a chronic inflammatory state into one more characteristic of the late inflammatory/early proliferative phase of wound healing. Chemokines may play a critical role in the pathogenesis of chronic venous ulcers through their effects on angiogenesis and/or the progression of inflammatory reactions at the site of injury.

Pressure ulcer accelerated healing with local injections of granulocyte macrophage-colony stimulating factor

This is the first report of granulocyte macrophage-colony stimulating factor (GM-CSF) inducing accelerated healing of a sacral pressure ulcer in a bedridden patient with bilateral hemiplegia. GM-CSF was diluted and injected locally around and into the ulcer bed every 2-3 days for 2 weeks, then weekly for 4 weeks until complete healing occurred. A new firm granulation tissue was noted within a few days. The ulcer showed 85% healing within 2 weeks and 100% by 2 months. Healing started from the periphery and from within the ulcer bed at sites of GM-CSF injections. It was slower at areas where there was complete necrosis and detachment of skin from underlying tissue. The ulcer remained closed until the patient's sudden death 9 months later. A biopsy of granulation tissue showed inflammatory cells and reactive fibroblasts. The potential role of GM-CSF and growth factors in pressure ulcer therapy and wound healing are discussed.

Distinct Populations of Stromal Cells Express Collagenase-3 (MMP-13) and Collagenase-1 (MMP-1) in Chronic Ulcers but Not in Normally Healing Wounds

Proteolysis is an intrinsic component of cutaneous wound repair and several matrix metalloproteinases have been shown to participate in various stages of this process. Therefore, we investigated the expression of a novel metalloproteinase, collagenase-3 (MMP-13), in normally healing cutaneous wounds and chronic venous ulcers. MMP-13 was expressed abundantly by fibroblasts deep in the chronic ulcer bed but was not detected in epidermis and all the acute wounds. The spatial expression of MMP-13 differed from that of collagenase-1 (MMP-1), which was prominently expressed by migrating keratinocytes and dermal cells located just beneath the wound surface. Northern blot hybridization did not reveal expression of MMP-13 by fibroblasts cultured on tissue culture plastic. In accordance with our in vivo findings, however, fibroblasts grown in a collagen gel produced MMP-13 mRNA abundantly. Our results suggest that MMP-13 can be induced in skin during wound repair after altered cell-matrix interactions. Although both MMP-1 and MMP-13 have the unique ability to degrade fibrillar collagens, their regulation and role during wound repair seem different. Collagenase-1 is critical for re-epithelialization, and MMP-13 most likely plays a role in the remodeling of collagenous matrix in chronic wounds.

Split-thickness skin grafting for lower extremity ulcerations.

Leg ulcers are often refractory to conservative treatment, often mandating the use of skin grafting. This review article discusses skin grafts, with special emphasis on split-thickness grafts for lower extremity ulcerations. Historical background, proposed mechanisms of action, biology of skin grafts, techniques for skin grafting, and results after grafting are discussed separately. Skin grafting has been performed for centuries. However, how skin grafts work, whether solely as tissue replacement or, additionally, as a stimulus for healing, is still not fully known. After placement, the grafted skin proceeds through a series of phases by which nutrients are supplied and neovascularization occurs. Adherence to the ulcer bed through interactions between the graft and the ulcer bed appear critical. When meshed split-thickness skin grafts are properly performed, success rates from 50% to 75% have been reported for refractory venous ulcers. Better understanding of the biologic and clinical aspects of skin grafting should lead to improved patient care. After studying this article, participant should be able: 1. To understand the various types of skin grafts. 2. To learn the potential mechanisms of action of how skin grafts work. 3. To appreciate the benefit of skin grafts for lower extremity ulcerations.

Role of endogenous basic fibroblast growth factor in the healing of gastric ulcers in rats.

Recently, it has been pointed out that growth factors play an important role in the healing of gastrointestinal ulcers. In the present study, we examined the role of endogenous basic fibroblast growth factor (bFGF) in the healing of gastric ulcers in the rat. In male SD rats, gastric ulcers were induced in the antrum by injection of acetic acid. Time-dependent changes in the area and bFGF content in the ulcerated area and distribution of bFGF in the ulcerated mucosa were examined. Effects of bFGF mutein CS23 (TGP-580) and a monoclonal antibody for bFGF (MAb 3H3) on the healing of the gastric ulcers and angiogenesis in the ulcer bed were also examined. The content of bFGF in the ulcerated area increased with time as the ulcer healed and reached a maximum 7 days after ulcer formation. In the gastric ulcer bed, many cells such as fibroblasts and macrophages were positively stained immunohistochemically by anti-bFGF antiserum. MAb 3H3 (0.1 mg/rat/day, i.v.) inhibited angiogenesis in the ulcer bed and significantly delayed ulcer healing, while TGP-580 (0.001-0.1 mg/kg x 2/day, p.o.) increased the number of microvessels in the ulcer bed and accelerated the healing. These results suggest that endogenous bFGF may play an important role in the healing of gastric ulcers in the rat and that the angiogenic properties of bFGF (TGP-580) may be involved in its effect on ulcer healing.

Epidermal Growth Factor in Gastric Ulcer Healing by Nocloprost, a Stable Prostaglandin E2 Derivative

Background: The gastroprotective and ulcer-healing properties of prostaglandins, especially in gastric ulcers induced by non-steroidal anti-inflammatory drugs, are well established. Ulcer healing is an active process of filling the mucosal defect with migrating and proliferating epithelial cells combined with angiogenesis in granulation tissue at the ulcer bed. Growth factors, especially epidermal growth factor (EGF) and transforming growth factor alpha (TGFa) are crucial in the regulation of the reconstruction of damaged mucosal structures. Methods: In this double-blind, randomized, prospective study 40 patients with gastric ulcer were treated with nocloprost, a stable prostaglandin E2 derivative, or with ranitidine. All subjects underwent endoscopy before and after 4 and 8 weeks of anti-ulcer therapy. During endoscopy mucosal biopsies were performed for determination of EGF content in gastric mucosa at the ulcer margin and in the intact mucosa. Additionally, EGF output in saliva and its plasma concentrations were determined in all subjects before and during the treatment. Results: The gastric ulcer healing rate after 4 weeks was significantly higher in patients treated with nocloprost than in those treated with ranitidine (63% versus 39%, respectively). At initial examination the EGF content in the gastric mucosa obtained from the ulcer edge was significantly higher than that in the intact mucosa. There was a significant increase in the EGF content in both the ulcer margin and the intact mucosa in subjects treated with nocloprost but not in patients under treatment with ranitidine. Similarly, patients treated with nocloprost had significantly higher EGF output in saliva and higher EGF concentration in plasma throughout the anti-ulcer therapy. Conclusion: Nocloprost is superior to ranitidine in the treatment of chronic gastric ulcers, and these effects could be due, at least in part, to higher expression and mucosal content of EGF in the ulcer area.

Sucralfate impedes intestinal epithelial sheet migration in a Caco-2 cell culture model.

Sucralfate, which binds to the matrix of the ulcer bed, is theoretically advantageous for duodenal ulcer therapy, but has not fulfilled its promise clinically. We examined the effects of sucralfate and related compounds in a human intestinal epithelial (Caco-2) cell culture model of restitution on sheet migration across and adhesion to collagen I. Migration was quantitated across a collagen I matrix treated with sucralfate or related compounds and correlated with cell adhesion. Caco-2 motility was significantly and dose-responsively inhibited by sucralfate at therapeutic luminal concentrations. Sucrose octaacetate, the sucralfate backbone, and lactose octaacetate exhibited similar effects while the beta-bonded disaccharide maltose octaacetate had little effect. Sucrose itself slightly stimulated motility. Adhesion effects paralleled motility. Thus, sucralfate may inhibit intestinal epithelial motility by sterically interfering with adhesion to collagen I. A sucralfate analog with a lactose octaacetate backbone might retain growth factor binding without inhibiting enterocyte motility, perhaps improving its clinical efficacy.

Acceleration of wound healing in gastric ulcers by local injection of neutralising antibody to transforming growth factor beta 1.

Application of neutralising antibodies (NAs) to transforming growth factor beta 1 (TGF beta 1) improves wound healing in experimental glomerulonephritis and dermal incision wounds. TGF beta 1 has been detected in the stomach, but despite the fact that this cytokine plays a central part in wound healing no information is available to determine if modulation of the TGF beta 1 profile influences the healing of gastric ulcers. This study examines gastric ulcer healing in the rat after local injection of NAs to TGF beta 1. Chronic gastric ulcers were induced in Wistar rats by the application of 100% acetic acid to the serosal surface of the stomach. Immediately after ulcer induction and on day 2, NAs to TGF beta 1 (50 micrograms), TGF beta 1 (50 ng), saline or control antibodies (IgG; 50 micrograms) were locally injected into the subserosa. Controls received no subserosal injections. Animals were killed on day 5 or 11, the ulcer area was measured planimetrically, sections were embedded in paraffin wax, and stained with trichrome or haematoxylin and eosin. Depth of residual ulcer was assessed on day 11 by a scale of 0-3, the percentage of connective tissue was determined by a semiquantitative matrix score and granulocytes and macrophages in the ulcer bed were also assessed. The application of NAs to TGF beta 1 led to a significant acceleration of gastric ulcer healing on day 11 (0.6 (SD 0.8) v 3.7 (SD 2.6) mm2), a reduction in macrophages (23.7 (SD 22.6) v 38 (26) per 40 x power field) and granulocytes (8.5 (SD 5.6) v 20 (10) per 40 x power field), fewer histological residual ulcers (mean 1 (SD 0.9) v 2 (1.1)), a reduced matrix score, and a regenerative healing pattern. Excessive scarring was seen in the TGF beta 1 treated group. Further treatment of gastric ulcers may induce a new treatment modality by local injection of NA to TGF beta 1 in an attempt to accelerate and improve ulcer healing.

Skin grafts as pharmacological agents: pre-wounding of the donor site

Initially thought to act as tissue replacement, cultured epithelial allografts are now known to work by providing a potent stimulus for healing. In a similar fashion, we believe that traditional autografts may also provide a stimulus to help heal chronic wounds, thus acting as pharmacological agents for healing. We attempted to assess the possibility of augmenting the stimulatory properties of donor skin by initiating the healing process in the donor region prior to grafting. This was accomplished by pre-wounding the donor area 3 days prior to harvesting the donor skin. We compared these 'pre-wounded' grafts to those harvested immediately. Two patients underwent punch grafting for chronic leg ulceration. Half of the ulcer was grafted with donor skin harvested from an area that was pre-wounded and the other half from freshly harvested skin. We evaluated each for improvement of granulation tissue and degree of edge effect (migration of the previously dormant wound edges). All the grafts did well. There was marked improvement in granulation tissue in the ulcer bed after grafting, and the obvious presence of an edge effect. The edge effect was increased on the site where the pre-wounded grafts were placed. It may be possible to augment the growth stimulatory properties of donor skin. This may offer therapeutic options in patients with chronic wounds.