Modifications to the rate of wound contraction by allopurinol

The effect of allopurinol (50 mg/kg) on the rate of full thickness excisional wound contraction in the Hooded Lister rat was assessed by planimetric and histological examinations. Compared with control animals, those treated with allopurinol showed a significantly lower coefficient of wound area contraction for days 0-7 (p < 0.05) than those of control animals. Histologically, in the allopurinol treated wounds the granulation tissue was less cellular but appeared to contain more collagen. This inhibition by allopurinol of the contraction phase of wound healing associated with an increased quantity of granulation tissue suggests that mediation of the process may involve a complex interaction between the fibroblasts/myofibroblasts and free radicals.     

Cellular retinol-binding protein-1 is transiently expressed in granulation tissue fibroblasts and differentially expressed in fibroblasts cultured from different organs

We have reported that cellular retinol-binding protein-1 (CRBP-1) is transiently expressed by arterial smooth muscle cells during experimental intimal repair (P. Neuville, A. Geinoz, G. Benzonana, M. Redard, F. Gabbiani, P. Ropraz, G. Gabbiani: Am J Pathol 1997, 150:509-521). We have examined here the expression of CRBP-1 during wound healing after a full-thickness rat skin wound. CRBP-1 was transiently expressed by a significant proportion of fibroblastic cells including myofibroblasts. Expression started 4 days after wounding, reached a maximum at 12 days, and persisted up to 30 days when a scar was formed. After wound closure, most CRBP-1-containing fibroblastic cells underwent apoptosis. We have further investigated CRBP-1 expression in rat fibroblasts cultured from different organs. CRBP-1 was abundant in lung and heart fibroblasts and was detected in decreasing amounts in muscle, tendon, subcutaneous tissue, and granulation tissue fibroblasts. Dermis fibroblasts contained no detectable levels of CRBP-1. All-trans retinoic acid and transforming growth factor-beta1 inhibited cell proliferation and increased CRBP-1 expression in fibroblastic populations except dermis fibroblasts. We demonstrate that during granulation tissue formation a subpopulation of fibroblastic cells express CRBP-1 de novo. We also demonstrate that CRBP-1 expression by fibroblasts is regulated in vitro by retinoic acid and transforming growth factor-beta1. Our results suggest that CRBP-1 and possibly retinoic acid play a role in the evolution of granulation tissue.     

Discordant growth in twin pregnancy--value of Doppler ultrasound

Fibroblast accumulation in a cutaneous wound requires phenotypic modulation of fibroblasts. In response to injury, resident fibroblasts in the surrounding tissue proliferate for the first 3 days and then at day 4 migrate into the wounded site. Once within the wound, they produce type I procollagen as well as other matrix molecules and deposit these extracellular matrix molecules in the local milieu. By day 7, abundant extracellular matrix has accumulated and fibroblasts switch to a myofibroblast phenotype replete with actin bundles along the cytoplasmic face of the plasma membrane. Wound contraction occurs as these myofibroblast gather in the wound extracellular matrix by extending pseudopodia, attaching to extracellular matrix molecules, such as fibronectin and collagen, then retracting the pseudopodia. Once these processes have been accomplished, the fibroblasts appear to undergo apoptosis. Therefore, during cutaneous wound repair, fibroblasts appear to progress through four phenotypes: first proliferating, second migrating, third synthesizing extracellular matrix molecules, and fourth expressing thick actin bundles as myofibroblasts.     

Centralized echocardiogram quality control in a multicenter study of regression of left ventricular hypertrophy in hypertension

Deep skin wounds in the adult mammal close spontaneously by epithelialization, wound contraction, and scar synthesis. In previous wound healing studies, it has been unsuccessfully attempted to separate from each other the natural processes that close wounds. In this study, we attempted to isolate skin regeneration from spontaneous processes of wound closure using "island" grafts. A porous analog of the extracellular matrix, composed of a graft copolymer of type I collagen and chondroitin 6-sulfate, was seeded with uncultured autologous keratinocytes and served to induce regeneration of the dermis and the epidermis. Grafts of the copolymer, measuring 1 x 2 cm, were placed in the center of 5 x 6-cm wounds in guinea pigs. By day 14, the edges of the island grafts were clearly separated from the host epidermis and dermis by a distinct bed of granulation tissue. Histologic study of island grafts on day 14 showed that the copolymer grafts had largely degraded and that a new epidermis and dermis had been synthesized in its place. The thickness of the new epidermis increased as the density of cells seeded into the graft increased. No synthesis of epidermis or dermis was observed in the granulation tissue outside the perimeter of the island grafts. We conclude that island grafting allows the study of early events in skin regeneration in isolation from epithelialization, contraction, and scar synthesis.     

Macrophage-fibroblast interaction and its possible role in regulating collagen metabolism during wound healing

The role of macrophages in the resorption of denatured collagen in the wound tissue was demonstrated by transmission and scanning electron microscopy investigation. Tight cell contacts between the macrophages and fibroblasts in the granulation tissue preceded active collagen synthesis and fibrillogenesis. A hypothesis was suggested according to which these contacts played an important role in the transfer of specific signals for the initiation of collagen biosynthesis; collagen decomposition products were considered as the actual signals, thus connecting the collagen catabolism and biosynthesis. Such a feed-back mechanism probably served as an integral part of the general hemostasis regulating the growth of connective tissue.     

Role of serum nonsuppressible insulin-like activity (NSILA) in wound healing. I. Influence of thyroparathyroidectomy on serum NSILA and wound healing in the rat

Serum contains a 90,000 molecular weight glycoprotein that exhibits insulin-like activity on adipocytes, skeletal muscle, and fibroblasts in tissue culture; however, this protein is physicochemically and immunochemically distinct from insulin and presently is termed "nonsuppressible insulin-like activity ("nsilA)." This study was designed to assess the response of serum NSILA to thyroparathyroidectomy (TPTHX) and to associate this response with tissue repair processes in the injured rat. It was postulated that NSILA modulates the fibroblastic response in wound healing. TPTHX decreased NSILA to 40 percent of control levels (p less than 0.001); 90 male animals subsequently were wounded by either a 10 percent third-degree burn or skin excision. Following injury, NSILA significantly increased in both control and TPTHX groups, but this acute-phase response was attenuated in TPTHX animals. Light microscopy of granulation tissue demonstrated a diminution in the fibroblastic response in TPTHX animals. Hydroxyproline analysis of granulation tissue revealed a significant decrease (p less than 0.025) in collagen content in TPTHX animals with low NSILA levels. The results suggest that serum NSILA levels are controlled, in part, by thyroid hormone and that NSILA may modulate the fibroblastic response of connective tissue repair processes.     

Contractile events during inflammation

Contractile events during wound healing. During granulation tissue contraction, fibroblasts develop characteristics typical of smooth muscle; (1) they contain an extensive cytoplasmic fibrillar system, (2) they show immunofluorescent labeling of anti-actin antibodies, (3) there are cell and cell to stroma attachments, (4) strips of granulation tissue, when tested pharmacologically in vitro, behave similarly to smooth muscle. These data support the view that under certain conditions, fibroblasts can differentiate into a cell type structurally and functionally similar to smooth muscle and this cell, the 'myofibroblast', plays an important role in connective tissue contraction. During epithelialization, epidermal cells develop an extensive cytoplasmic contractile apparatus which has morphological and immunological characteristics similar to those of myofibroblasts. Such apparatus disappears as soon as epithelialization is completed. It is proposed that such a contractile apparatus plays a role in cell motility enabeling individual cells to rearrange themselves in an appropriate pattern. In conclusion, significant amounts of contractile proteins may be synthetized by fibroblasts and epithelial cells during wound healing and may play an important role in this process.     

Electron microscopic observations on sperm penetration in cytochalasin-treated eggs of the rose bitterling

The influence of Aloe vera (L.) Burman f. on the glycosaminoglycan (GAG) components of the matrix in a healing wound was studied. Wound healing is a dynamic and complex sequence of events of which the major one is the synthesis of extracellular matrix components. The early stage of wound healing is characterized by the laying down of a provisional matrix, which is then followed by the formation of granulation tissue and synthesis of collagen and elastin. The provisional matrix or the ground substance consists of GAGs and proteoglycans (PGs), which are protein GAG conjugates. In the present work, we have studied the influence of Aloe vera on the content of GAG and its types in the granulation tissue of healing wounds. We have also reported the levels of a few enzymes involved in matrix metabolism. The amount of ground substance synthesized was found to be higher in the treated wounds, and in particular, hyaluronic acid and dermatan sulphate levels were increased. The levels of the reported glycohydrolases were elevated on treatment with Aloe vera, indicating increased turnover of the matrix. Both topical and oral treatments with Aloe vera were found to have a positive influence on the synthesis of GAGs and thereby beneficially modulate wound healing.     

Promotion of second intention wound healing by emu oil lotion: comparative results with furasin, polysporin, and cortisone

Previous studies showed that twice-daily application of emu oil lotion (mixture of emu oil/fat, vitamin E, and botanical oil) immediately after creation of full-thickness skin defects delayed wound healing 6 days later, perhaps owing to its antiinflammatory actions. If administration was delayed for 48 hours, a two-fold promotion of wound contraction, epithelialization, and infiltration of organized granulation tissue was observed. In the present study, emu oil lotion was applied to full-thickness skin defects in rodents 24 hours after surgery. Six days postoperatively, wound contraction and infiltration of fronts of epithelialized and granulation tissue were assessed. Results indicated a two-fold promotion of all of the above parameters with emu oil lotion. No such effects were exerted by pure emu oil, furasin, cortaid, or polysporin. Data obtained indicate promise for emu oil lotion as an aid in treating full-thickness skin defects if applied after the major postinflammatory stages of wound healing have transpired.     

TGF-beta1 influences early gingival wound healing in rats: an immunohistochemical evaluation of stromal remodelling by extracellular matrix molecules and PCNA

The effect of topically applied transforming growth factor beta1 (TGF-beta1) on the rat gingival wound healing process after flap surgery was evaluated by immunohistochemistry for extracellular matrix molecules (ECM), such as tenascin, heparan sulfate proteoglycan (HSPG) and type IV collagen, and for proliferating cell nuclear antigen (PCNA) in fibroblasts. TGF-beta1 solution was applied to the surgical wound experimental sites. Two microg/microl were applied at the time of the operation, and 1 microg/microl at days 1 and 2 after surgery, with contralateral control sites receiving the vehicle alone. Periodontal tissues were histologically examined at 3 and 7 days post-surgery. Tenascin was found to be more strongly stained in the granulation tissue from experimental sites at 3 days post-surgery. At 7 days postsurgery, HSPG-positive areas in granulation tissue had become smaller and there was a prominent proliferation of PCNA-positive fibroblast-like cells and type IV collagen-positive blood vessels. These results suggest that TGF-beta1 applied to surgical wounds influences early proliferation of gingival fibroblast-like cells, the formation of blood vessels, and ECM remodelling. In conclusion, TGF-beta1 application appears to promote granulation tissue formation in periodontal wound healing.     

Nitric oxide, an autocrine regulator of wound fibroblast synthetic function

Nitric oxide (NO) is synthesized in wounds, but its exact role and cellular source are not known. Wound fibroblasts (WF) are phenotypically characterized by increased collagen synthesis and contractility. We hypothesized that WF may be also phenotypically altered during wound healing to synthesize NO. WF were isolated from polyvinyl alcohol sponges implanted in male Lewis rats and harvested 10 days later. Proliferation in response to 10% fetal bovine serum was assessed by [3H]thymidine incorporation in a microculture system. A fibroblast-populated collagen lattice was used for assaying contractility. Collagen synthesis was determined by measuring the collagenase-sensitive fraction of protein-incorporated [3H]proline. Fibroblasts were incubated in the presence or the absence of 0.5 mM S-methyl-isothio-uronium or 0.5 mM N-monomethyl-L-arginine, both competitive inhibitors of NO synthase. WF spontaneously synthesize and release NO (4.60 +/- 0.29 nmol nitrite/microg DNA/48 h). Normal dermal fibroblasts do not synthesize NO. WF NO synthesis was limited to the first and second passages postharvest and was inhibitable by S-methyl-isothio-uronium (96%) and N-monomethyl-L-arginine (84%). In vivo iNOS expression by WF was confirmed by in situ hybridization and immunohistochemistry. Inhibition of endogenous NO synthesis had no effect on fibroblast proliferation. However, fibroblast-mediated collagen contraction was enhanced (p < 0.01), and collagen synthesis was significantly decreased (p < 0.05) by inhibiting NO synthase. The data show that WF are phenotypically altered during the healing process to synthesize NO, which, in turn, regulates their collagen synthetic and contractile activities.     

Ultrastructural evidence of myofibroblasts in circumscribed fibromatosis

The myofibroblast, which exhibits ultrastructural features of both smooth muscle cells and fibroblasts, is the predominant cell type in the granulation tissue of healing wounds. Similar cells were identified in an ultrastructural study of a case of circumscribed fibromatosis. Myofibroblasts have been described in several apparently unrelated conditions, suggesting that the acquisition of smooth muscle-like features is one of a limited number of ways in which fibroblasts may react to abnormal stimuli.     

Necrotizing fasciitis: case study of a nursing dilemma

Necrotizing fasciitis usually manifests as a low grade cellulitis that quickly deteriorates to a limb and life threatening soft tissue infection. Immediate surgical debridement is essential, after which wound management becomes the nurse's primary concern. Case #1 reports on a 72 year old female who, upon presenting to the ER with a "sore bottom," subsequently had these diagnoses: (1) anal-rectal abscess, (2) Fournier's gangrene, (3) ulcerative enterocolitis, (4) chronic blood loss/anemia, and (5) protein caloric malnutrition. After debridement, her anal-rectal wound extended from labia to left buttocks. Care was multidisciplinary and included applying a water based aloe gel and saline soaked gauze twice a day. After 45 days, the wound exhibited a pink base with granulation tissue and contraction of the wound edges. Case #2 reports on a 48 year old male with seroma of the left leg secondary to a crush injury. Within three days he developed deep vein thrombosis in that leg as well as two large seroma cavities on either side of the thigh. Care included packing with the aloe gel and saline soaked sponges. Two weeks after admission, the anterior wound was covered with a split thickness skin graft while partial closure of the lateral cavity was attempted unsuccessfully with retention sutures. After five weeks, healing was complete for the anterior wound and 95 percent complete for the posterior wound.     

Morphometric studies of collagen and fibrin lattices contracted by human gingival fibroblasts; comparison with dermal fibroblasts

Cell shape variations and substratum re-organization during contraction of floating collagen and fibrin lattices seeded with human gingival fibroblasts were determined by computerized image analysis of light and scanning electron microscopic images. Data were compared with those obtained with lattices populated with human dermal fibroblasts. The extent of collagen lattice contraction was similar with both cell types, resulting in a two-fold decrease in the area fractions occupied by collagen fibers. Fibroblasts exhibited a rounded shape (form factors equal to 0.8 and 0.7 for gingival and dermal cells, respectively) at day 1 of culture; they possessed a more elongated appearance (with form factors equal to 0.3 and 0.15 for gingival and dermal cells, respectively) at day 7. Continuous (gingival) and discontinuous (dermal) layers of cells were evidenced at the cortex of lattices. Contractions were associated with a significant reduction of the diameters of collagen fibers. Re-organization of substratum, as analyzed by the "Rose of Directions" technique, was evidenced only at the vicinity of filopodia where fibers ran parallel to these protrusions. Several lysed matrix cavities were observed when fibrin lattices were populated with gingival but not dermal fibroblasts at day 5 of culture. Although cells in fibrin lattices exhibited morphometric parameters comparable with those in collagen lattices, no fibroblast layers could be demonstrated at gel peripheries. Fibrin matrices consisted of an isotropic network of entangled fibrin filaments from the start of culture, and only a slight reduction of the diameters of fibrin fibers could be evidenced in dermal fibroblast-populated lattices. Fibrinolysis at the vicinity of gingival fibroblasts led to an entire re-organization of substratum toward the formation of larger fibers. The differential behavior of gingival vs. dermal fibroblasts inside fibrin but not collagen matrices could therefore partly explain the increased rate of remodeling of gingiva as compared with dermis.     

Ultrastructure of collagen thermally denatured by microsecond domain pulsed carbon dioxide laser

BACKGROUND: Clinical improvement in photodamaged skin after carbon dioxide (CO2) laser resurfacing is thought to result in part from thermal collagen shrinkage. The presence of such collagen has not been unequivocally demonstrated. To identify and characterize the morphological features of collagen after CO2 laser exposure, we irradiated ex vivo human facial skin and bovine calcaneus tendon with microsecond domain pulsed CO2 laser energy and examined specimens for histopathological and ultrastructural changes in collagen. OBSERVATIONS: In dermis and tendon, 3 zones of collagen structure were apparent on electron microscopy. The first, most superficial zone demonstrated loss of collagen structure. The second zone consisted of admixed normal collagen fibers and thickened collagen fibers. Zone 3 consisted of normal-appearing collagen fibers. CONCLUSIONS: Ultrastructural examination of irradiated collagen revealed distinct morphological zones of denatured collagen fibers. Partially denatured fibers had an increased diameter consistent with lineal shrinkage. Zonal distinction was undetectable by light microscopy. Ultrastructurally, the zones of denatured collagen located above the normal fibers correlated with the zone of altered material seen on light microscopy. These findings suggest that collagen fiber shrinkage does occur after pulsed CO2 laser irradiation and that this phenomenon contributed, at least in part, to the immediate tissue contraction observed clinically.     

Development of new wound dressing composed of spongy collagen sheet containing dibutyryl cyclic AMP

Although cyclic AMP has been considered to regulate cell proliferation, the mechanism of this function is largely unknown. Recent studies suggest that cyclic AMP promotes the proliferation of skin cells in a dose-dependent manner. An ointment containing dibutyryl cyclic AMP has been used in the treatment of skin ulcers and found to be effective in promoting tissue repair. To search more efficacious wound management, the authors developed a new wound dressing composed of a spongy atelo-collagen sheet containing dibutyryl cyclic AMP. This wound dressing was evaluated in two types of animal tests. One is the application of the wound dressing to a full-thickness skin defect in order to evaluate the granulation tissue formation and the wound size reduction. The wound dressing was found to promote the granulation tissue formation and naturally reduce the wound size. The other test was the application of the wound dressing to the full-thickness skin defect, leaving behind a skin island in a central portion, in order to evaluate the epithelialization. This skin island left in a full-thickness skin defect was extremely enlarged. The enlargement of the skin island seems to be related to the epithelialization from the margin of the skin island as well as by the expansion of a skin island induced by contraction of the developed granulation tissue in the surrounding wound area. These results suggest that an atelo-collagen spongy sheet containing dibutyryl cyclic AMP is effective in promoting the granulation tissue formation and epithelialization.     

L-2-Hydroxyglutaric aciduria: neuropathological correlations and first report of severe neurodegenerative disease and neonatal death

Chromolaena odorata (formerly Eupatorium odoratum) is used as a traditional medicine in Vietnam (Nghiem, 1992), where its Vietnamese common name is "co hoi." While it has been widely considered a weed by agriculturalists (Holm et al., 1991), the aqueous extract and the decoction from the leaves of this plant have been used throughout Vietnam for the treatment of soft tissue wounds, burn wounds, and skin infections. A number of clinical studies done by Vietnamese as well as foreign medical workers has demonstrated the efficacy of this extract on the wound-healing process. In this article, the effect of the Eupolin extract on hydrated collagen lattice contraction by human dermal fibroblasts, an in vitro model of wound contraction, is described. The significant inhibition of collagen gel contraction by Eupolin extract at 50 to 200 micrograms/ml is demonstrated in various concentrations of collagen. When the extract at 50 to 150 micrograms/ml was washed out of the lattices and replaced by fresh medium without Eupolin, the contraction of collagen by cells was resumed. The visualization of cells in the lattices by incubation in a tetrazolium salt for 2 h showed live cells at 50 to 150 micrograms/ml of extract. In contrast, all cells were killed in the higher extract doses of 300 or 400 micrograms/ml. These preliminary results showing the inhibitory effect of Eupolin extract on collagen contraction suggest that a clinical evaluation of its effect on wound contraction and scar quality should be made. This work illustrates that traditional remedies that are used by folk practitioners to improve healing can be examined in a scientific manner using in vitro wound-healing models. It could be that the synergistic properties of components of the natural extract contribute to the positive effects demonstrated on various wound-healing mechanisms.     

Delayed effect of denervation on wound contraction in rat skin

Neuronal supply in soft tissues may be an important part of cutaneous wound healing. In order to observe the effect of denervation on wound contraction, rectangular full-thickness skin defects were created on the dorsum of two groups of Wistar rats. In the experimental group (n = 20), spinal nerves corresponding to the area of the open wound (T11 to L2) were isolated and divided bilaterally. In the control group (n = 20), the same pairs of spinal nerves were dissected but left intact. Limits of denervation were verified by the pinprick test. Wound healing, which is primarily in the form of wound contraction in this model, was evaluated by tracing wound margins onto millimetric paper weekly. Wound contraction was delayed significantly in the experimental group (p < 0.05) at all follow-up periods when compared with the controls. Loss of neuropeptide secretion from the nerve endings in denervated tissues may be responsible for the retarded wound contraction, since neuropeptides are thought to exert trophic effects on skin wound healing.     

Normoxic wound fluid contains high levels of vascular endothelial growth factor

OBJECTIVE: To examine the temporal integration of vascular endothelial growth factor (VEGF), which has been shown to be present in wound fluid, with the putatively related processes of wound fluid oxygen content, wound angiogenesis, and granulation tissue formation. SUMMARY BACKGROUND DATA: During cutaneous wound repair, new tissue formation starts with reepithelialization and is followed by granulation tissue formation, including neutrophil and macrophage accumulation, fibroblast ingrowth, matrix deposition, and angiogenesis. Because angiogenesis and increased vascular permeability are characteristic features of wound healing, VEGF may play an important role in tissue repair. METHODS: A ventral hernia, surgically created in the abdominal wall of female swine, was repaired using silicone sheeting and skin closure. Over time, a fluid-filled wound compartment formed, bounded by subcutaneous tissue and omentum. Ultrasonography was performed serially to examine the anatomy and dimensions of the subcutaneous tissue and wound compartment. Serial wound fluid samples, obtained by percutaneous aspiration, were analyzed for PO2, PCO2, pH, and growth factor concentrations. RESULTS: Three independent assays demonstrate that VEGF protein is present at substantially elevated levels in a wound fluid associated with the formation of abdominal granulation tissue. However, the wound fluid is not hypoxic at any time. Serial sampling reveals that transforming growth factor beta-1 protein appears in the wound fluid before VEGF. CONCLUSIONS: The results suggest that VEGF is a prominent regulator of wound angiogenesis and vessel permeability. A factor other than hypoxia, perhaps the earlier appearance of another growth factor, transforming growth factor beta-1, may positively regulate VEGF appearance in the wound fluid.