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Vacuum-assisted closure: a new method for wound control and treatment: animal studies and basic foundation.

A series of basic animal studies using a new subatmospheric pressure technique (The V.A.C.) to expedite wound healing are presented. The technique entails placing an open-cell foam into the wound, sealing the site with an adhesive drape, and applying subatmospheric pressure (125 mmHg below ambient) that is transmitted to the wound in a controlled manner. Utilizing a pig model, four studies were undertaken to determine the effect of subatmospheric pressure on laser Doppler-measured blood flow in the wound and adjacent tissue (N = 5), rate of granulation tissue formation (N = 10), clearance of bacteria from infected wounds (N = 5), and measurement of nutrient flow by random-pattern flap survival (N = 5). Blood flow levels increased fourfold when 125 mmHg subatmospheric pressure was applied. Significantly increased rates of granulation tissue formation (p < or = 0.05) occurred with both continuous (63.3 +/- 26.1%) and intermittent (103% +/- 35.3%) application. Tissue bacterial counts significantly decreased (p < or = 0.05) after 4 days of application. Random-pattern flap survival significantly increased (p < or = 0.05) by 21% compared to controls. We determined that the application of controlled subatmospheric pressure creates an environment that promotes would healing.

Animals↗

[Microspectrophotometric study of the lipid content in regenerating connective tissue].

An experimental quantitative histological investigation was performed to study lipids in cellular elements of the regenerated connective tissue. Flap wounds were cut on the back of white mice and granulation tissue dissected 1, 3, 7, 14 30 and 60 days after the operation was investigated. Lipids and cellular elements were revealed with Sudan III-IV black B and Nile blue. Lipid quantity was estimated by means of a modified scanning integrating microphotometer. The greatest amount of lipids in the cellular elements of the granulation tissue was revealed on the 3d day of the experiment, total optic density (TOD) of lipids in leucocytes was 0.83, TOD in histiocytes--0.6. On the 7th day the amount of lipids decreased, their TOD in leucocytes was 0.6, in histiocytes--0.4. By the 14th day only phospholipids were revealed, in histiocytes their TOD was 0.3. In subsequent days phospholipid contents continued decreasing and by the 30th day their TOD was 0.2. By the 60th day, when a scar was formed in the wound, in fact, there were no lipids in the cellular elements. The microphotometric investigation performed demonstrated qualitative and quantitative characteristics of lipids in the cellular elements of the granulation tissue in dynamics and their close dependence on the time of wound healing. It was also revealed that the regenerative process was accompanied by decreasing lipid amount in the cellular elements, and this phenomenon could be used as a peculiar test to prognosticate the process of wound healing.

Animals↗

Effect of Hepatogard--an indigenous formulation on dexamethasone induced antihealing effects in male albino rats.

Hepatogard, is a multi-ingredient phytopharmaceutical product containing crude powders of eleven plants. Its effect on the different parameters of wound healing was assessed alone and in the presence of dexamethasone. The parameters chosen for the study were the breaking strength of incised wound, breaking strength of granulation tissue and hydroxyproline content. The result showed that Hepatogard increased the breaking strength of granulation tissue but not of incised wound. It reversed the dexamethasone induced decrease in breaking strength in both incised wound and granulation tissue. Even though it had no effect of its own on hydroxyproline concentration, it reversed the dexamethasone induced decrease in the hydroxyproline content of granulation tissue. Thus, Hepatogard has the potential for antagonizing the antihealing effect of steroids in patients receiving steroid therapy.

Animals↗

Expression of matrix metalloproteinase-2 and -9 during early human wound healing.

BACKGROUND: During wound healing, keratinocytes detach from the basement membrane and migrate to cover the exposed connective tissue. Subsequently, the wound clot is degraded gradually and replaced by the epithelial cells and the granulation tissue. Both of these processes are likely to be affected by matrix-modifying enzymes. Type IV collagenases are members of the matrix metalloproteinase family (MMP), which are known to degrade several matrix components. The aim of this study was to investigate the expression of MMP-2 and MMP-9 (72-kd and 92-kd type IV collagenases, respectively) during early human wound healing. EXPERIMENTAL DESIGN: Experimental wounds were created in human oral mucosa and biopsies were taken 1, 3, and 7 days after wounding. In situ hybridization on paraffin sections was used for the detection of messenger RNAs coding for MMP-2 and MMP-9, and the secretion of MMPs into the oral cavity after wounding was followed by zymography. Regulation of MMP-2 and MMP-9 expression by cytokines was studied using cultured mucosal keratinocytes, gingival fibroblasts, and wound granulation tissue fibroblasts. RESULTS: By in situ hybridization, the expression of MMP-2 was localized in the connective tissue fibroblasts and endothelial cells during all phases of wound healing. Mucosal epithelium was practically negative for MMP-2 expression. MMP-9 messenger RNA was found in mucosal epithelium on days 1, 3, and 7. A strong signal was localized in basal and suprabasal cell layers in the nonwounded area, while only the basal cell layer was MMP-9 positive in the migrating epithelial sheet. Seven days after wounding, granulation tissue exhibited an unusually strong signal for MMP-9 messenger RNA. Wound fluid contained mainly MMP-9, the amount of which was highest in two- to four-day-old secretions. None of the cytokines tested (transforming growth factor beta-1, interleukin-1 beta, basic fibroblast growth factor, tumor necrosis factor-alpha, interferon-gamma) were able to regulate MMP-2 expression in cultured wound fibroblasts. However, keratinocyte MMP-9 production was enhanced by interleukin-1 beta, transforming growth factor beta-1, and tumor necrosis factor-alpha. CONCLUSIONS: During wound healing, MMP-9 is suggested to be involved in keratinocyte migration and granulation tissue remodelling. Expression of MMP-2 remains stable during wound healing.

Cells, Cultured↗

Stimulation of wound healing, using brain extract with fibroblast growth factor (FGF) activity. II. Histological and morphometric examination of cells and capillaries.

Reported in this paper are studies by which evidence was produced to increased formation of granulation tissue in rats, aged two and six months, seven days after repeated localised administration of brain extract from cattle with FGF activity (fibroblast growth factor). Such increased formation of granulation tissue was attributable to the formation in the same granulation tissue of larger amounts of capillaries, which actually provided conditions for better blood supply. The above increase was associated with stimulation of the synthetic function of fibroblasts and myofibroblasts in the granulation tissue. Comprehensive morphometric tests, including differential counting, appeared to show that additional effects had to be assumed, in particular on macrophages and lymphocytes. Such increase in angiogenesis seemed to suggest that in the in vivo model studied FGF proved to be, first of all, a factor of angiogenesis rather than a factor of fibroblast growth. The above results, as obtained from rats which differed in age, exhibited a certain variation in response to FGF. This seems to underline the importance of age-dependent examination also in the context of pharmacological studies.

Animals↗

[A growing skull fracture: a case report].

A case of growing skull fracture was reported with some considerations on the literature. At the age of 1 year, the patient fell down stairs and struck the right occipital region, but did not receive any treatment at that time. At the age of 19 years, he suffered from several epileptic attacks and was admitted to our ward. Plain craniograms showed a right occipital bone defect measuring 7 x 3 cm in size. CT scan demonstrated a cystic lesion just beneath a membranous substance existing at the bone defect. MRI revealed cystic encephalomalacia underlying the cyst very clearly. At the operation the bone defect was found to be replaced by the granulation tissue which was examined microscopically. Under the granulation tissue, a cyst filled with yellowish fluid and surrounding gliotic brain was found. The dural tear was noticed to be much wider than the bone defect. A dural plasty with LYODURA and a cranioplasty with methacrylic resin were performed. Microscopic examination revealed the granulation tissue to be a fibroglial cicatrix. According to these findings, we concluded that the contused and swollen brain tissue which had herniated through the dural tear had enlarged the skull fracture and formed a fibroglial cicatrix with subcutaneous tissue. It is emphasized that MRI combined with CT scan is an indispensable procedure for diagnosis of growing skull fracture, and microscopic examination contributes to an understanding of its pathogenesis.

Adult↗

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.

Abdominal Muscles↗

The linoleic acid metabolite 9DS-hydroxy-10,12(E,Z)-octadecadienoic acid is a strong proinflammatory mediator in an experimental wound healing model of the rat.

Locally administered 9Ds-hydroxy-10,12(E,Z)-octadecadienoic acid (9-HODE) caused a drastic inflammatory response in the experimental model of granulation tissue formation of the rat according to RUDAS (Arzneimittelforsch. 10,226-229, 1960). Three days after implantation of the polyvinyl chloride rings the granulation tissue became inhomogeneous with proliferation islets surrounded by edematous regions containing a diminished number of cells. The number of polymorphonuclear leukocytes and of macrophages was greatly enhanced in the whole tissue, whereas the number of lymphocytes was reduced. After seven days the whole granulation tissue was loosened, and its mass was twice as high as in the control animals. The number of fibroblasts per area unit and the hydroxyproline content were diminished. Linoleic acid and 13Ls-hydroxy-9,11(Z,E)-octadecadienoic acid (13-HODE) caused also some changes in the formation of granulation tissue, but in a different manner, in particular, without accumulation of polymorphonuclear leukocytes and macrophages, indicating the specificity of the effect of 9-HODE. The recruitment of leukocytes was not due to a direct chemotactic action of 9-HODE as shown in an agarose diffusion test comparing the effects of 9-HODE and leukotriene B4. The possible biological importance of the proinflammatory effect of 9-HODE is discussed.

Animals↗

A novel role for erythropoietin during fibrin-induced wound-healing response.

In this study, we investigated the role of the hematopoietic cytokine erythropoietin (EPO) during wound healing, the physiological response to tissue injury. We used an in vivo wound-healing assay (fibrin Z-chambers) consisting of fibrin-filled chambers implanted subcutaneously in rats. The fibrin inside the chambers is replaced by granulation tissue consisting of new blood vessels, macrophages and fibroblasts as part of the wound-healing response. Local, exogenous recombinant EPO administration into the fibrin matrix significantly increased granulation tissue formation in a dose-dependent manner. To investigate the physiological role of endogenous EPO during wound healing, we used soluble EPO receptor or anti-EPO monoclonal antibodies to neutralize EPO and observed dose-dependent inhibition of granulation tissue formation, consistent with an important role for EPO in the wound-healing cascade. The ability of recombinant EPO to promote wound healing was associated with a proangiogenic effect during granulation tissue formation. We also found abundant expression of EPO receptor protein in macrophages, cells that play a pivotal role during wound healing. Modulation of wound healing because of administration of recombinant EPO or inhibition of endogenous EPO-EPO receptor correlated with changes in levels of inducible nitric oxide synthase protein in granulation tissue. These data demonstrate a novel function for EPO by providing in vivo evidence for a physiological role during fibrin-induced wound healing.

Animals↗

Repair of molluscan tissue injury: role of PDGF and TGF-beta1.

Various cellular and humoral activities of the wound repair process and the effects of PDGF-AB and TGF-beta1 on tissue repair mechanisms in the mollusc Limax maximus are studied by histological and immunocytochemical procedures. Histological examination at different times after the wound production demonstrates that tissue repair is the result of successive and related activities distinguishable by different morphological pictures. In the first hours, an infiltration phase is activated 24 h after the incision, hemocytes stratify at wound margins and actively phagocitize cell debris and damaged tissue in the surrounding area. Moreover, the cells are immunoreactive to anti-IL-1alpha, IL-8 and TNF-alpha antibodies. After 24-72 h, granulation tissue rich in small blood lacunae is formed and the provisional matrix is synthesized and deposited on the base of the new tissue. In histological sections 72 h after injury, the incision is filled with granulation tissue, and at the wound base, a layer of fibrous connective tissue is formed. Hemocytes present in the newly formed blood lacunae and fibroblasts are involved in the synthesis and deposit of extracellular matrix components, i.e. fibronectin, reticular and collagen fibres. Ninety-six h after wound production, the repair process continues and the granulation tissue is more developed. At 192 h, re-epithelialization begins, and this is more evident in the histological sections after 14 days. Hemocytes are immunoreactive to the cytokines at all the times examined. Exogenous administration of PDGF-AB and TGF-beta1 stimulates the tissue healing process through a general acceleration of the activities involved. A larger closing area of clumped hemocytes and a smaller damaged tissue area are observed 24 h after treatment of the wound. At 72 h, the granulation tissue is more developed and more extracellular matrix components are deposited than in the control incision. A larger number of cells express cytokine-like molecules, and re-epithelialization of the wound is accelerated, as 14 days after growth factor treatments almost all the wound area is covered by a layer of cubic epithelial cells, and the alcianophilic cell coat is restored. No differences in the responses of the two growth factors are observed.

Animals↗

[Repair of the connective tissue in a non-sutured skin wound].

During the healing of a second intention wound, granulation tissue is built up during the inflammatory and detersive phase which commences in the wound edges as soon as the loss of substance is created. Granulation tissue becomes scar tissue via connective and vascular proliferation and epidermization takes place concurrently. The origin of the fibroblasts composing the granulation tissue is debated: monocytes or fibroblasts from the wound edges? Ultrastructural and immunohistochemical data from studies of experimental wounds in the pig and the monkey make it possible to determine their origin and follow their progress. These cells originate from the fibroblasts of the wound edges and become undifferentiated cells which multiply and migrate to reach the loss of substance. There they become activated fibroblasts and contribute to repairing the loss of substance by secreting fibronectin and type I and type III collagen. As differentiated fibroblasts and myofibroblasts, they remodel and contract scar tissue.

Acute-Phase Reaction↗

Bronchial foreign bodies in adults.

Aspiration of foreign bodies into the bronchial tree is infrequently seen in adults. We reviewed 40 such cases during an 8-year period at Chang Gung Memorial Hospital, Kaohsiung. Only six patients had underlying conditions that contributed to the aspiration of foreign bodies. A positive history of aspiration was found in 18 patients (48%). Clinical manifestations were nonspecific. Chest roentgenograms were useful in 11 of the 40 patients. Bronchoscopic findings were classified into three groups: foreign bodies in the bronchial tree without granulation tissue, foreign bodies in the bronchial tree with marked granulation tissue and foreign bodies embedded in the granulation tissue. The diagnoses and removal of foreign bodies were successful in 37 patients (92%) using grasping forceps or biopsy forceps and in one patient using basket type grasping forceps. Complications of bronchoscopy were rare and not serious. The diagnosis of occult foreign bodies is often difficult and demands a high index of suspicion. Removal of endobronchial foreign bodies is usually possible with a flexible fiberoptic bronchoscope and has a high success rate.

Adolescent↗

[Identification of periodontopathic bacteria DNA from periodontitis-involved gingival tissue using a PCR method].

The purpose of this study was to determine the presence of periodontopathic bacteria DNA in the granulation tissue of periodontally involved gingival tissue. Forty periodontitis patients were examined. Subgingival plaque and saliva samples were collected from the patients before surgery. Then, granulation tissue was also collected during surgery. Another 20 patients, who had more than 4 mm in probing pocket depth, were also examined as a control. They received only initial treatment without periodontal surgery. Plaque and saliva samples were also collected from them. Seven periodontopathic bacteria, Porphyromonas gingivalis, Bacteroides forsythus, Toreponema denticola, Campylobacter rectus, Actinobacillus actinomycetemcomitans, Prevotella intermedia, and Prevotella nigrescens, were detected by a PCR method from these samples of all patients. As clinical parameters, probing pocket depth, clinical attachment level, and bleeding on probing were recorded. One year after surgery, sampling of subgingival plaque and saliva, and measurement of clinical parameters were performed on the patients. The DNA of periodontopathic bacteria was detected in the granulation tissue of 29 out of 40 patients. P. gingivalis, B. forsythus, T. denticola, C. rectus, A. actinomycetemcomitans, P. intermedia and P. nigrescens were detected in 23, 11, 12, 9, 3, 2 and 1 subject(s) respectively. The sites with undetectable level of any periodontal pathogens at one year after surgery showed a clinical attachment gain of 2.44 mm, while those that showed detectable pathogen levels had only a 0.77 mm gain. Our results indicated that the removal of granulation tissue contributed to eliminating periodontal pathogens and to avoiding re-infection by those bacteria. Complete eradication of periodontopathic bacteria would be necessary for successful periodontal treatment.

Aggregatibacter actinomycetemcomitans↗

[Effect of autoskin grafting in fault hypodermis wound of granulation excision on full-thickness burn healing].

OBJECTIVE: To explore the mechanism of full-thickness burn wound healing with autoskin grafting in fault hypodermis wound of granulation excision and to evaluate its effect. METHODS: By the techniques of clinical observation, histopathology, immunohistochemistry, TEM and FCM, we observed changes of the activity and structure of grafted skin and the granulation tissue, collagnous fiber,microvessels,the ultramicrostructure of fibroblasts and the expression of basic fibroblast growth factor (bFGF) in the base of autoskin grafting in fault hypodermis wound in burned adult minipigs (Group A), and compared with traditional method of autoskin grafting on the basilar fibrous tissue wound of scraped partly granulation being (Group B) and control group (Group C, without treatment except de-fur). RESULTS: The grafted skin survived after 3 days of operation, and it had less injury and higher proliferative index (PI) in group A than in group B. The hyperplasia of granulation tissue and vascular endothelial and the expression of bFGF were more evident in group A. After 5 days, the proliferation of endothelial cells and granulation and the protein synthesis of fibroblasts were more active in group A, and at this moment, fresh collagen appeared and proliferated more actively in group B. After 7-14 days, epidermic structure and dermic microvascular density became normal gradually, the granulation on grafting base matured and transformed into fibrous connective tissue in group A. The same change deferred about 2 days in group B. After 21 days, the above pathologic change in group A was less than that in group B. After 30-60 days of operation, Group A achieved much less contraction and transfiguration than Group B, and the grafted skin was tender and movable. CONCLUSION: Autoskin grafting in fault hypodermis wound of granulation excision has a better effect than traditional operation.

Animals↗

Participance of fibronectin and various collagen types in the formation of fibrous extracellular matrix in cardiosclerosis.

Investigation of the extracellular matrix composition of the left heart ventricle was carried out on autopsy material of subjects, aged from 60 to 70 years, in a number of cases, including: (1) tissue without cardiosclerosis; (2) granulation tissue formed 2 weeks after infarction; (3) post-infarctial fibrous scars; (4) diffuse cardiosclerosis in consequence of stenotic coronary atherosclerosis. Cryostat sections treated with highly specific antibodies to fibronectin and types I, III, IV and V collagens were examined by the indirect immunofluorescence technique. Fibronectin and the mentioned collagenous proteins were detected in the extracellular matrix of granulation tissue. In contrast, fibronectin and collagen type IV were not revealed in post-infarctial fibrous scars. Collagen types III and V were diffusely distributed in fibrous tissue, whereas collagen type I was demonstrated to accumulate preferentially in the deeper regions of post-infarctial scars. Fibronectin and collagen types I, III, V, but never type IV, were also found in the connective tissue in diffuse cardiosclerosis. The significance of type V collagen in the extracellular matrix is discussed.

Aged↗

[Histomorphological and spectroscopic studies on the skeleton following alloplastic joint substitution (author's transl)].

We report on our morphological and spectrometric findings with complete acetabuloplastics. The histomorphological investigation revealed the formation of a narrow cicatricial coat round the prothesis. The collagenous tissue borders on a zone of granulation tissue of varying width. Granular foreign matter can be found in cicatrical tissue especially in the extracellular space and in granulation tissue in macrophages. Only a few partigles turned out to be iron in the Prussian blue reaction. Spectral analysis proved the hip and thigh-bones to contain more than 0.001 p. c. of cobalt and chrome. Toxicological problems inherent in the use of the cobalt alloy "Vitallium" are discussed. The dangers of metal poisoning are dealt with in addition to those of the development of osteomyelitis and the formation of a sarcoma. At last we discuss our macroscopic findings concerning the fit of the prosthesis, the metal demonstration by electron micrsocopy, and the scanning electron microscopic investigation of the metal surfaces.

Chromium↗

[Gd-DTPA-enhanced MR imaging of cholesteatoma].

It is sometimes difficult to differentiate a cholesteatoma from an accompanying cholesterol granuloma, mucosa, fluid and, especially, granulation tissue. This study was designed to assess the reliability with which cholesteatoma can be differentiated from such accompanying lesions by Gd-DTPA-enhanced MR imaging. Twenty-one patients with cholesteatoma were studied with magnetic resonance (MR) imaging. The cholesteatoma had intermediate to high signal intensities similar to those of granulation tissue, on both T1- and T2-weighted images. On Gd-enhanced images, however, there was marked enhancement of all granulation tissues while the cholesteatoma showed no such enhancement. Gd-DTPA-enhanced images distinguished cholesteatoma from granulation tissues and permitted determination of the exact extent of the cholesteatoma. Cholesterol granulomas showed high signal intensities on both T1- and T2-weighted images. Cholesterol granuloma can, therefore, be distinguished from cholesteatoma and granulation tissues on both T1- and T2-weighted images. Gd-DTPA-enhanced MR imaging is a valuable tool in the evaluation and management of cholesteatoma.

Adolescent↗

Tympanic membrane and middle ear pathologic correlates in mucoid otitis media.

OBJECTIVE: The goal of this study was to correlate tympanic membrane (TM) and middle ear (ME) pathologies in mucoid otitis media (MOM). METHODS AND MATERIAL: Forty ears with MOM and 56 control ears were retrospectively evaluated for TM and ME pathologies. Comparisons of TM thicknesses in MOM versus control ears were correlated with the Student t test; chi(2) analysis was used to correlate pathologic findings of the TM and ME. RESULTS: Thicknesses in all quadrants except the umbo were increased in MOM because of infiltration of inflammatory cells and fibrosis. The most common ME pathologies were granulation tissue and fibrosis. Significant correlations included (1) TM retraction and ME granulation tissue and fibrosis and (2) pars flaccida, posterosuperior, and anteroinferior thickness and ME granulation tissue and fibrosis. CONCLUSION: TM changes are likely to occur in patients with otitis media with effusion (MOM), and their presence is a strong indication of underlying ME pathology.

Adolescent↗