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A method for simultaneous and quantitative determination of Cr, Mn, Fe, Co, Cu, Zn, Se and Cd elements in the subcellular fractions of nuclei, mitochondria, lysosome, microsome and cytosol of wound granulation tissue of severe burn patients by octopole reaction system (ORS) inductively coupled plasma mass spectrometry (ICP-MS) was established. Using differential centrifugation, the sample is separated into different subcellular fractions. The subcellular fraction was digested by HNO3 + H2O2 with microwave digestion followed by dilution with ultrapure water then the above 8 trace elements in the solution were analyzed directly by ICP-MS. In the presented method, using ORS eliminates the polyatomic interferences caused by the matrixes. Rh as internal standard element was used to compensate matrix effect and signal drift. The detection limits of the 8 elements are in the range of 0.72-33.05 ng x L(-1), and the RSD is less than 8.4%. The results showed that the levels of some elements in subcellular fractions of wound granulation tissues were significantly different from those of normal skin tissues. ORS-ICP-MS is a useful tool for simultaneous determination of multi-elements in wound granulation tissue of severe burn patients, and could be widely used in other biological samples analysis.

Accurate and rapid assessment of the healing status of a wound in a simple and noninvasive manner would enable clinicians to diagnose wounds in real time and promptly adjust treatments to hasten the resolution of nonhealing wounds. Histologic and biochemical characterization of biopsied wound tissue, which is currently the only reliable method for wound assessment, is invasive, complex to interpret, and slow. Here we demonstrate the use of Raman microspectroscopy coupled with multivariate spectral analysis as a simple, noninvasive method to biochemically characterize healing wounds in mice and to accurately identify different phases of healing of wounds at different time-points. Raman spectra were collected from "splinted" full thickness dermal wounds in mice at 4 time-points (0, 1, 5, and 7 days) corresponding to different phases of wound healing, as verified by histopathology. Spectra were deconvolved using multivariate factor analysis (MFA) into 3 "factor score spectra" (that act as spectral signatures for different stages of healing) that were successfully correlated with spectra of prominent pure wound bed constituents (i.e., collagen, lipids, fibrin, fibronectin, etc.) using non-negative least squares (NNLS) fitting. We show that the factor loadings (weights) of spectra that belonged to wounds at different time-points provide a quantitative measure of wound healing progress in terms of key parameters such as inflammation and granulation. Wounds at similar stages of healing were characterized by clusters of loading values and slowly healing wounds among them were successfully identified as "outliers". Overall, our results demonstrate that Raman spectroscopy can be used as a noninvasive technique to provide insight into the status of normally healing and slow-to-heal wounds and that it may find use as a complementary tool for real-time, in situ biochemical characterization in wound healing studies and clinical diagnosis.

Vascular lesions are commonly encountered in routine pathologic practice and often pose diagnostic challenges owing to their morphologic diversity. Although WT-1 expression was reported in some vascular tumors, little is known about its staining patterns in a spectrum of vascular lesions from various locations. We examined WT-1 immunostain in 95 cases of vascular lesions including angiosarcomas (AS, 19 cases), hemangioendotheliomas (HE, 5), Kaposi's sarcomas (KS, 4), cavernous hemangiomas (CVH, 12), capillary hemangiomas (CPH, 7), pyogenic granulomas (PG, 4), lymphangiomas (LA, 4), hemangiopericytomas (HP, 5), glomus tumors (GT, 8), vascular malformation (VM, 13) and granulation tissue (GRT, 14). Strong WT-1 cytoplasmic stain was invariably observed in all cases of malignant and borderline vascular tumors including AS (19/19), KS (4/4) and HE (5/5). WT-1 was also consistently expressed in CPH (7/7), PG (4/4), and GRT (14/14), while it became weaker in VM (10/13) and often negative in CVH (2/12) and LA (0/4). WT1 stain was not demonstrated in HP (0/5) and rarely in GT (2/8). We conclude that consistent and diffuse WT-1 cytoplasmic stain in AS, HE and KS can be useful in distinguishing these tumors from poorly differentiated tumors with mimicking features. On the other hand, reliable WT-1 stain in CPH, PG and GRT may help in differential diagnosis with non-endothelial vascular tumors such as GT and HP. Recognizing the WT-1 cytoplasmic stain in a broad spectrum of benign and neoplastic tissues is critical in formulating appropriate immunohistochemical panels and avoiding misinterpretation of results.

Introduction: Apical periodontitis is an inflammatory disease of the periradicular tissues caused by the host's immune response to infection of the root canal system. MicroRNAs (miRNAs) have been shown to play an important role in the regulation of inflammation and the immune response; however, their role in the pathogenesis of endodontic periapical disease has not been explored. The purpose of this study was to examine the differential expression of miRNAs in diseased periapical tissues as compared with healthy controls., Methods: We first compared miRNA profiles in diseased periapical tissues collected from patients undergoing endodontic surgery with those of healthy pulps by using microarray analyses. The target genes of the differentially expressed miRNAs were identified by using miRWalk and PubMed. Selected miRNAs linked to inflammation and the immune response were then confirmed in a separate cohort of diseased and healthy tissues by using quantitative reverse transcription-polymerase chain reaction. Healthy pulps and periodontal ligaments were used as controls. Data were normalized to the level of SNORD 44, which served as an endogenous control., Results: Of the 381 miRNAs identified by using microarray, 24 miRNAs were down-regulated in diseased periapical tissues compared with controls (n = 13) (P < .003). The down-regulation of 7 miRNAs was confirmed from 9 selected miRNAs by using quantitative real-time polymerase chain reaction (n = 19) (P < .05). Target genes of these miRNAs include key mediators in the immune and inflammatory response such as interleukin-6, matrix metalloproteinase-9, and transforming growth factor-β., Conclusions: These findings offer new insight into the pathogenesis of endodontic disease and have the potential to impact the development of new methods for prevention, diagnosis, and treatment of apical periodontitis., (Copyright © 2013 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.)

Carica papaya L. (Linn) (Caricaceae) is traditionally used to treat various skin disorders, including wounds. It is widely used in developing countries as an effective and readily available treatment for various wounds, particularly burns. This study evaluated the wound-healing and antimicrobial activity of C. papaya seed extract. Ethanol extract of C. papaya seed (50 mg/kg/day) was evaluated for its wound-healing activity in Sprague-Dawley rats using excision wound model. Animals were randomly divided into four groups of six each (group 1 served as control, group 2 treated with papaya seed extract, group 3 treated with a standard drug mupirocin and papaya seed extract (1:1 ratio) and group 4 treated with a mupirocin ointment. Rate of wound contraction and hydroxyproline content were determined to assess the wound-healing activity of the seed extract. The group 2 animals showed a significant decrease in wound area of 89% over 13 days when compared with groups 1 (82%), 3 (86%) and 4 (84%) respectively. The hydroxyproline content was significantly higher with the granulation tissue obtained from group 2 animals which were treated with C. papaya seed extract. Histological analysis of granulation tissue of the group 2 animals showed the deposition of well-organized collagen. The extract exhibited antimicrobial activity against Salmonella choleraesuis and Staphylococcus aureus. Our results suggest that C. papaya promotes significant wound healing in rats and further evaluation for this activity in humans is suggested., (© 2012 The Authors. © 2012 Blackwell Publishing Ltd and Medicalhelplines.com Inc.)

Background: The receptor activator for nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) are the key factors controlling the osteoclast and osteoblast action in the bone., Purpose: The study objective was to investigate the expression level of RANKL and OPG in cholesteatoma and granulation tissue, and to assess the relationship between their expression levels and osteolysis., Material and Methods: Patients with chronic otitis media with cholesteatoma (n = 28) and without cholesteatoma (n = 24) treated surgically at the Department of Otolaryngology of the Medical University of Gdańsk were included in the study. RANKL and OPG expressions were analyzed by immunohistochemistry and Western blot., Results: RANKL and OPG were expressed in all cholesteatoma and granulation tissues. RANKL expression was mainly observed in cholesteatoma subepithelial stroma, whereas OPG-positive cells originated from the epithelium. The number of OPG-positive cells in the normal skin was significantly higher than in cholesteatoma tissues. The RANKL protein level in cholesteatoma tissues was 1.8- and 1.5-fold higher than in the auditory canal skin and granulation tissues, respectively. The number of RANKL-positive cells in cholesteatoma tissues was significantly higher than in the normal skin. No substantial differences were found in average OPG protein levels between cholesteatoma tissues and the normal auditory canal skin. The ratio of RANKL/OPG was significantly higher in cholesteatoma tissues (2.93 ± 0.79) than in the skin samples (1.36 ± 0.34)., Conclusions: Altered ratio of RANKL/OPG protein level in cholesteatoma tissues suggests that these proteins might be somehow involved in the pathogenesis of cholesteatoma. However, to resolve this issue a study on a larger group of patients should be conducted., (Copyright © 2010 Elsevier Inc. All rights reserved.)

The objective of our study is to investigate the wound healing activity of the bark extract of Carapa guianensis in rats using three different wound models. The animals were randomly divided into two groups of six each in all the models. Test group animals were treated topically with the bark extract (200 mg/kg of body weight), and the controls were treated with petroleum jelly in the excision wound model. In the incision and dead space wound models the test group animals were treated with the extract of C. guianensis (200 mg/kg/day) orally by mixing in drinking water, and the control group animals were maintained with plain drinking water. Healing was assessed by the rate of wound contraction, period of epithelialization, skin breaking strength, granulation tissue weight, and hydoxyproline content. Antimicrobial activities of the extract against the microorganisms were also assessed. On day 15 extract-treated animals exhibited 99% reduction in the wound area compared to controls (93%). The extract-treated wounds were found to epithelialize faster than controls (P < .02). The skin breaking strength was significantly higher in extract-treated animals compared to controls (P < .001). The weight and hydroxyproline content of granulation tissue were significantly increased compared to controls. These observations support the use of C. guianensis is in the management of wound healing.

Background: Frog skin has been sequentially and scientifically evaluated by our group for its wound healing efficiency. Owing to the complex structure of skin, attempts were being made to analyse the role of individual constituents in different phases of healing. Our earlier papers have shown the significance of frog skin not only in wound healing but also enhancing the proliferating activity of the epidermal and dermal cells which are instrumental for normal healing process. We also have identified for the first time novel antimicrobial peptides from the skin of Rana tigerina and thereby reduce the complications involved in the sepsis., Purpose of the Study and Results: The current study envisages the role of frog skin lipids in the inflammatory phase of wound healing. The lipid moiety of the frog skin dominated by phospholipids exhibited a dose dependent acceleration of healing irrespective of the mode of application. The efficiency of the extract is attributed partially to the anti-inflammatory activity as observed by the histochemical and immunostimulatory together with plethysmographic studies., Conclusions: Thus, frog skin for the first time has been demonstrated to possess lipid components with pharmaceutical and therapeutic potential. The identification and characterization of such natural healing molecules and evaluating their mechanism of action would therefore provide basis for understanding the cues of Nature and hence can be used for application in medicine.

We prepared full thickness skin defects in rats fed on a protein-free diet as a hypoproteinaemia model, then switched the animals to a diet containing a normal protein level 1, 6 or 12 days after wounding (inflammatory, granulation and rearrangement phases of the wound healing process) to examine whether improvement in the low-protein state promotes subsequent wound healing. The interval until wound healing in rats fed on a normal protein diet was significantly shorter, whereas that in rats continuously fed on a protein-free diet was significantly longer than those of other groups. Early correction tended to accelerate wound healing. Although wound contraction in groups receiving a protein-corrected or protein-free diet remained similar until 15 days after wounding, thereafter the duration of the rearrangement phase was significantly longer in the protein-free group than in the other groups. The collagen level per unit of granulation tissue area during wound healing was significantly lower in the protein-free group than in the other groups. These findings indicate that protein correction at any time after wounding accelerates wound healing, although early correction is more effective, and reduces the duration of the rearrangement phase more than those of the inflammatory and granulation phases because of the deposit of collagen.

The wound healing potential of the aqueous, alcoholic extracts, and the butanolic fraction of the alcoholic extract obtained from the bark of Schrebera swietenioides were evaluated in the dexamethasone suppressed wound healing model. The work was conducted on rodents using incision, excision, and dead space wound models. The extracts of S swietenioides enhanced the breaking strength of incision wounds significantly (P < .05). Faster epithelization and contraction of excision wounds were observed in the treated groups (P < .05). Dead space wound model demonstrated an increase in breaking strength of granulation tissue and weight of dried granulation tissue after treatment with the extracts.The extracts attenuated the effect of dexamethasone on healing.The total RNA isolated from the granulation tissues of the extract-treated animals was significantly higher than in both dexamethasone and normal groups, (P < 0.05). It was observed that the DNA was intact in all the groups. These findings suggest that dexamethasone suppresses wound healing, possibly through an inappropriate transcription rather than causing DNA damage.The S swietenioides extracts have the capacity to reverse this effect.

Object: In this paper the authors' goal was to identify histological and immunohistochemical differences between cervical disc hemrniation and spondylosis., Methods: A total of 500 cervical intervertebral discs were excised from 364 patients: 198 patients with disc herniation and 166 patients with spondylosis. We examined en bloc samples of endplate-ligament-disc complexes. Types of herniation and graded degrees of disc degeneration on MR images were examined histologically and immunohistochemically., Results: The herniated discs showed granulation tissue, newly developed blood vessels, and massive infiltration of CD68-positive macrophages, which surrounded the herniated tissue mainly in the ruptured outer layer of the anulus fibrosus. The vascular invasion was most significant in uncontained (extruded)-type herniated discs. Chondrocytes positive for matrix metalloproteinase (MMP)-3, tumor necrosis factor (TNF)-alpha, basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF) were abundant in both herniated and spondylotic discs. Free nerve fibers, positive for nerve growth factor (NGF), neurofilament 68, growth-associated protein (GAP)-43, and substance P, were strongly apparent in and around the outer layer of uncontained (extruded)-type herniated discs, with enhanced expression of NGF. The authors observed that herniated discs showed more advanced degeneration in the outer layer of the anulus fibrosus around the granulation tissue than spondylotic discs. On the other hand, spondylotic discs showed more advanced degeneration in the cartilaginous endplate and inner layer of the anulus fibrosus than herniated discs. Spondylotic discs also had thicker bony endplates and expressed TNFalpha and MMP-3 more diffusely than herniated discs, especially in the inner layer of the anulus fibrosus., Conclusions: The authors' results indicate that herniated and spondylotic intervertebral discs undergo different degenerative processes. It is likely that TNFa, MMP-3, bFGF, and VEGF expression is upregulated via the herniated mass in the herniated intervertebral discs, but by nutritional impairment in the spondylotic discs. Macrophage accumulation around newly formed blood vessels in the herniated disc tissues seemed to be regulated by MMP-3 and TNFalpha expression, and both herniated and spondylotic discs exhibited marked neoangiogenesis associated with increased bFGF and VEGF expression. Nerve fibers were associated with NGF overexpression in the outer layer of the anulus fibrosus as well as in endothelial cells of the small blood vessels.

The aim of the study was to observe the effects of dibutyrylchitin (DBC) on the repair processes and to explain the mechanisms of its action in comparison with other dressing materials made of butyrylchitin (BC), regenerated chitin (RC), and chitosan. The results showed that DBC implanted subcutaneously to the rats increased weight of the granulation tissue. Increased cell number isolated from the wound and cultured on the DBC films was also revealed. The DBC was proved to reduce also the necrotic cells number in the culture. DBC elevates the glycosaminoglycans (GAG) level in the granulation tissue. The total collagen content in the wound was not influenced by all applied dressing materials. However, a low level of the poorly polymerized soluble collagen in the wounds treated with DBC and BC indicated better polymerization of the remaining part of that protein. Both DBC and chitosan increased the weight of granulation tissue. However, chitosan contrary to DBC lowered GAG content and increased water capacity in the wound. The study documents the beneficial influence of DBC on the repair, which could be explained by the modification of the extracellular matrix and cells number. The best effects were observed after application of DBC with [eta] DBC-1 = 1.75 dL/g.

The experimental data indicate that melatonin actively influences time-causes of changes of lipid content in rats tissue during the inflammation process. Its effect depends on a dose, modes of administration (intraperitonial, hypodermic or local) and duration of treatment. A single dose intraperitonial administration of melatonin (4 mg/kg) did not influence lipid content in the granular-fibrose tissue, while repeated injections of this hormone limited the increase in contents of lipids and phospholipids at the 5th and 8th days of regeneration. Long-term subcutaneous injections of melatonin caused distinct changes of lipids: at the dose of 0.3 mg/kg it prevented, and at the dose of 4 mg/kg it promoted the increase of lipid content in the granular-fibrose tissue. Local application of a melatonin solution (1.5 mg/ml) at early periods of regeneration caused insignificant changes of total lipids and total phospholipids in the granular-fibrose tissue. However, the higher concentration (15 mg/ml) of melatonin caused the decrease of total lipids due to reduced content of cholesterol and triglycerides and the increase of total phospholipids and some of their fractions.

The present investigation was undertaken to determine the efficacy of topical administration of flavone of sea buckthorn (Hippophae rhamnoides L.) on cutaneous wound healing in rats. Four full-thickness excision wounds were created on the back of rat and 1.0% w/v flavone prepared in propylene glycol was applied topically. Control animals received the vehicle alone in an identical manner. The healing of the wound was assessed by the rate of wound contraction, period of epithelialization, hydroxyproline, hexosamine, antioxidants estimation and histopathology of the granulation tissue. The sea buckthorn flavone promoted the wound healing activity as indicated by improved rate of wound contraction, decreased time taken for epithelialization (16.3 days versus 24.8 days in controls) and significant increase in hydroxyproline (26.0%) and hexosamine (30.0%) content. These findings were also confirmed by histopathological examinations. In addition, it was observed that sea buckthorn flavone possesses potent antioxidant properties as evidenced by significant increase in reduced glutathione (55.0%), vitamin C (70.0%) and catalase (20.0%) activities in wound granulation tissue. The flavone treatment also resulted in significant decrease in lipid peroxide levels (39.0%). The results suggest that the sea buckthorn flavone promotes wound healing activity.

The aim of this study is to determine whether and to which extent pentosidine (PEN) is influenced in the case of advanced osteoarthritis (OA) in urine and in some tissues. Reverse phase HPLC method for PEN determination with fluorescence monitoring was utilized. Urinary pentosidine (U-PEN) and pyridinoline (U-PD) were determined in the group (N=67, age 67.1+/-11.7 years) in patients with OA before and after surgery (knee or hip replacement), and in healthy age-matched controls (N=18, age 67.0+/-6.0 years). Cartilage, synovial membrane and granulation tissue were also treated. In OA group significantly higher (P<0.001) U-PEN concentrations before (7.5+/-5.1 nmol/mmol creat.) and after (4.9+/-2.6 nmol/mmol creat.) surgery were found. U-PD was not significantly different between the groups (62.5+/-33.0 and 55.0+/-29.51 nmol/mmol creat., respectively), but differed significantly (P<0.0002 and P<0.01) from healthy subjects (40.2+/-16.5 nmol/mmol creat.). In the newly formed granulation tissue, PEN concentrations are significantly lower than in cartilage and in synovial tissues.

Hippophae rhamnoides L. (family Elaeagnaceae), commonly known as seabuckthorn, is a wild shrub growing at high altitude (1200-4500 meters) in adverse climatic conditions. The aim of the present study was to evaluate healing potential of seabuckthorn leaves in a preclinical study on rats using a cutaneous excision-punch wound model. Four full-thickness excision-type wounds of 8.0 mm diameter were created on the dorsal surface of rats under aseptic conditions. The aqueous lyophilized extract of seabuckthorn leaves, at doses of 0.5%, 1.0%, and 1.5% w/v prepared in propylene glycol, were applied topically twice daily for 7 days. Control animals received the vehicle alone in an identical manner. Wound granulation tissues were excised on eighth day postwounding, and the hydroxyproline, hexosamine, total protein content, and antioxidant levels were determined. Wound surface area was also measured on the eighth day before wound excision to determine wound contraction. Topical application of 1.0% seabuckthorn leaf extract statistically significantly augmented the healing process, as evidenced by increases in the content of hydroxyproline and protein as well as the reduction in wound area when compared with similar effects in response to treatment using povidone-iodine ointment (standard care). The reduced glutathione, vitamin C, superoxide dismutase, catalase, and glutathione peroxidase activities showed significant increases in seabuckthorn leaf extract-treated wounds as compared to controls. The lipid peroxide levels were significantly decreased in leaf extract-treated wounds. The results suggest that aqueous leaf extract of seabuckthorn promotes wound healing, which may be due to increased antioxidant levels in the granulation tissue.

Wound healing occurs as a fundamental response to tissue injury. Several natural products have been shown to accelerate the healing process. The present investigation was undertaken to determine the efficacy of topical administration of an alcoholic bark extract of Butea monosperma (B. monosperma) on cutaneous wound healing in rats. Full-thickness excision wounds were made on the back of rat and B. monosperma extract was administered topically. The granulation tissue formed on days 4, 8, 12 and 16 (post-wound) was used to estimate total collagen, hexosamine, protein, DNA and uronic acid. The extract increased cellular proliferation and collagen synthesis at the wound site, as evidenced by increase in DNA, total protein and total collagen content of granulation tissues. The extract treated wounds were found to heal much faster as indicated by improved rates of epithelialization and wound contraction, also confirmed by histopathological examinations. Also, the tensile strength of drug-treated wounds was increased significantly. In addition, we show that B. monosperma possesses antioxidant properties, by its ability to reduce lipid peroxidation. The results clearly substantiate the beneficial effects of the topical application of B. monosperma in the acceleration of wound healing.

The subcellular localization of cerium and lanthanum in the intestinal mucosa was studied after oral administration of cerium chloride or lanthanum chloride or lanthanum chloride followed 30 minutes after of cerium chloride to young adults Wistar rats. Two methods of observation and microanalysis were used. The transmission electron microscopy revealed the presence of dense electron granulations in the lysosmes of the duodenum enterocyte, when these elements were administrated simultaneously. The ion mass microanalysis permits to detect the presence of La and Ce as bright points outlining the intestinal villi. These points correspond to the lysosomes containing the granulations previously described. These granulations are formed by the cerium and the lanthanum associated to the phosphor and forming probably insoluble salts of Ce/La phosphate.

Background: We investigated the effect of preoperative rectal irrigation with short-chain fatty acids on irradiated colonic anastomosis in rats., Methods: Sixty male Wistar rats were divided into four groups. Group I (control group, n = 15) underwent left colon resection and primary anastomosis. Group II (Short-chain fatty acids pretreatment group, n = 15) had short-chain fatty acids rectal irrigation for five days preoperatively. Group III (preoperative radiotherapy group, n = 15) underwent irradiation to the whole pelvis eight and four days before the operation, for a total dose of 20 Gy. Group IV (preoperative radiotherapy group + short-chain fatty acids pretreatment group, n = 15) had rectal irrigation with short-chain fatty acids for five days after the second irradiation. Within each group, animals were anesthetized to assess the clinical, mechanical, histologic, and biochemical parameters of anastomotic healing on either the third or seventh postoperative days., Results: The mean bursting pressure was significantly low in Group III on Day 3 and was significantly high in Group IV on Day 7 (P = 0.001, P = 0.021). The burst occurred at the anastomoses in all animals tested on the third postoperative day, and outside of the anastomoses in all animals tested on the seventh postoperative day. The histologic parameters of anastomotic healing, such as epithelial regeneration and formation of granulation tissue, were significantly improved by use of preoperative rectal irrigation with short-chain fatty acids on Day 7. The amount of total and salt-soluble collagen concentrations significantly increased in Group IV compared with the control group on Day 3 (P = 0.008, P = 0.004)., Conclusion: Some mechanical and histologic aspects of colonic anastomotic healing can be adversely affected by preoperative radiotherapy, but rectal irrigation with short-chain fatty acids may improve anastomotic healing.

In this study, we investigated the role of nerve growth factor (NGF)-incorporated collagen on wound healing in rats. Full-thickness excision wounds were made on the back of female rats weighing about 150-160 g. Topical application of NGF-incorporated collagen, at a concentration of 1 microg/1.2 mg collagen/cm(2), once a day, for 10 days resulted in complete healing of wounds on the 15th day. The concentrations of collagen, hexosamine and uronic acid in the granulation tissue were determined. The NGF-incorporated collagen-treated rats required shorter duration for the healing with an increased rate of wound contraction. Histological and electron microscopical evaluations were also performed, which reveal the activation of fibroblasts and endoplasmic reticulum and therefore increased level of collagen synthesis due to NGF application. These results clearly indicate that the topical application of NGF-incorporated collagen enhanced the rate of healing of excision wounds.

Background: During wound repair, fibroblasts orchestrate replacement of the provisional matrix formed during clotting with tenascin, cellular fibronectin and collagen III. These, in turn, are critical for migration of endothelial cells, keratinocytes and additional fibroblasts into the wound site. Fibroblasts are also important in the deposition of collagen I during scar formation. The CXC chemokine chicken Chemotactic and Angiogenic Factor (cCAF), is highly expressed by fibroblasts after wounding and during development of the granulation tissue, especially in areas where extracellular matrix (ECM) is abundant. We hypothesized that cCAF stimulates fibroblasts to produce these matrix molecules., Results: Here we show that this chemokine can stimulate precocious deposition of tenascin, fibronectin and collagen I, but not collagen III. Studies in culture and in vivo show that tenascin stimulation can also be achieved by the N-terminal 15 aas of the protein and occurs at the level of gene expression. In contrast, stimulation of fibronectin and collagen I both require the entire molecule and do not involve changes in gene expression. Fibronectin accumulation appears to be linked to tenascin production, and collagen I to decreased MMP-1 levels. In addition, cCAF is chemotactic for fibroblasts and accelerates their migration., Conclusions: These previously unknown functions for chemokines suggest that cCAF, the chicken orthologue of human IL-8, enhances healing by rapidly chemoattracting fibroblasts into the wound site and stimulating them to produce ECM molecules, leading to precocious development of granulation tissue. This acceleration of the repair process may have important application to healing of impaired wounds.

The influence of long term parenteral melatonin administration on the biochemical composition of granulation tissue of surgical wounds in rats during healing was investigated. Physiological solution and solutions containing various concentrations of melatonin were subcutaneously injected to the animals within 3 weeks. Control and injected animals were wounded. Samples of gramilation tissue were investigated on the 5-th and the 8-th day of healing. The contents of oxyprolin, uronic acids, hexosamines, total lipids and their fractions, fractional composition of glycosaminoglicans and proteins composition of salt extracts were determined in the se samples. Repeated injections during three weeks caused the changes in biochemical composition of researched samples which were characteristic for stressful reaction of connective tissue. The specific changes are most expressed at long term introduction of a physiological solution to animals. The introduction of melatonin during similar period cansed protective effect, partially defending biochemical composition of granulation tissue from changes, which were induced by stressful situation.

The aims of this study were to investigate the expression of pro-inflammatory, anti-inflammatory and immune-related cytokines present in periapical lesions. We investigated the expression of cytokines: namely interleukins IL-2, IL-4, IL-6, IL-10 and interferon-gamma (IFN-gamma) in formalin-fixed, paraffin-embedded sections of periapical granulation tissue. The study samples were biopsies from 24 patients with periapical lesions: 12 with periapical granulomas and 12 patients with radicular cysts. Immunohistochemistry was also performed on tonsillar tissue which served as a control. We utilised a set of specific monoclonal antibodies and polyclonal monospecific antibodies to detect cells that expressed the different cytokines within the tissues. We also considered the nature of the periapical immune response by investigation of the T-helper 1 (Th-1) and T-helper 2 (Th-2) lymphocyte subsets using their cytokine profile, i.e., Th-1: IL-2 and IFN-gamma and Th-2: IL-4, IL-5 and IL-6. Only a few cells were weakly positive for the IL-2 protein in each of the tissue sections. Cells that expressed IL-4 or IL-6 were far more numerous than cells that expressed either IL-2 or IFN-gamma. Thus, we demonstrated a greater number of Th-2 cells in periapical lesions. This relative ratio of the T-cell subsets underlines the importance of the anti-inflammatory mechanisms taking place in the diseased tissue manifested by the wide array of IL-10-expressing cells: B cells, T suppressor cells (CD8 (+)) and tissue macrophages. The numbers of inflammatory cells expressing the anti-inflammatory molecules far outnumbered the cells that expressed pro-inflammatory cytokines. Thus, the downregulation of the inflammatory response and the predominant Th-2 or humoral immune response in periapical periodontitis may be important features that dictate the outcome of the disease process in the periapical lesion.

Wound healing is a finely controlled biological process involving a series of complex cellular interactions. Following inflammation, the wound bed matrix is gradually replaced by granulation tissue followed by the long slow process where collagen accumulates and restores tensile strength. The studies revealed that human granulation tissue varied in many aspects in comparison with normal skin. In granulation tissue the molecular organization of collagen showed an increased amount of type III collagen resembling embryonic tissue. The presence of type V collagen with three distinct chains was the characteristic feature of granulation tissue. The physicochemical properties of collagen extracted from granulation tissue showed the influence of proteoglycans during collagen aggregation and these proteoglycans from the major non-collagenous proteins during the proliferative phase of healing.

Wounds with a covering of eschar require debridement before optimal wound healing can proceed. There are several different methods available but these have been derived empirically with no direct evidence of the structure or composition of the tissue they are designed to remove, or of the potential autolytic mechanisms which are the targets for some of these treatments. The aim of this study therefore was to determine the composition of chronic wound eschar and hence identify potential targets for the induction of autolytic debridement. Chronic wound eschar was removed by surgical debridement and analysed using immunohistochemistry, polyacrylamide gel electrophoresis (PAGE) and gelatin zymography. Immunohistochemistry using antibodies specific for extracellular matrix (ECM) proteins revealed a definite tissue structure, consisting of many fibrous regions and fine fibrillar elements separated by areas of tissue which were of a more irregular and amorphous nature. An antibody specific for all leucocytes revealed the presence of leucocytes in the region of tissue closest to the wound bed. The presence of this leucocyte population correlated to elevated levels of gelatinase activity as identified by gelatin zymography. PAGE analysis identified various protein species in the range 3.5-60 kDa molecular weight. These data indicate that wound debridement is likely to require multiple enzyme specificities to degrade the eschar and that these enzymes may be supplied by inflammatory leucocytes infiltrating the eschar from the wound bed. The various protein species demonstrated by PAGE may represent ECM proteins, those with lower molecular weight possibly representing the degradation products of autolytic debridement.

A neutrophil chemotactic factor has been purified from the homogenate of rat granulation tissues. The purified chemoattractant was a basic protein with heparin-binding site and gave a single band corresponding to a molecular mass of 16 kDa on SDS-PAGE under reducing conditions. The chemoattractant was treated with lysylendopeptidase and the resulting peptides were isolated by reversed-phase HPLC. Amino acid sequences of the peptides were almost identical with the sequence of N-terminal fibronectin type III domain of human collagen type XIV, suggesting that the purified chemoattractant consists mainly of N-terminal fibronectin type III domain and the adjacent heparin-binding site of rat collagen type XIV. The 16-kDa fragment of collagen type XIV dose dependently attracted rat neutrophils and transiently increased the intracellular free Ca2+ concentration of neutrophils. The results suggest that the novel chemoattractant plays a role in neutrophil recruitment in rat inflammation., (Copyright 1999 Academic Press.)

This study examined potential regional differences in the deposition of granulation tissue in cellulose sponges placed in the dorsum of rats. Four cellulose sponge implants, 10 x 10 x 5 mm in size, two in cranial and two in caudal positions, were used to induce granulation tissue formation in the back of the rat. A cranio-caudal difference in connective tissue formation was assessed from 1 to 24 weeks after implantation. Granulation tissue ingrowth, measured histomorphometrically, was enhanced at 2 weeks and the surrounding capsule was thinner from 1 to 3 weeks in implants located in the cranial part of the back. In the cranial position, the number of fibroblasts, assessed semiquantitatively, was higher and the ratio of inflammatory cells to fibroblasts lower at 2-3 weeks. Also, the ratio of hydroxyproline content to total nitrogen content was higher after the first week in cranial specimens. Thus, a distinct cranio-caudal difference in the proliferative phase was observed. These results show the obvious practical considerations underlying the need for randomization or comparison between implants from exactly corresponding cranio-caudal locations.

Nine cholesteatomas, seven middle ear granulations and five deep meatal skin specimens were analysed for gelatinase activity at molecular weights corresponding to those of matrix metalloproteinase-1 (MMP-1) using SDS PAGE zymography. Gelatinase activity at 41-43 kDa and 45-47 kDa was investigated. Western blotting was employed using a primary monoclonal antibody to MMP-1 to provide a qualitative assessment of MMP-1. Western blotting was also used with a monoclonal antibody to MMP-3 to discover if MMP-3 gelatinase activity occurring around the molecular weight of MMP-1 may have contributed to the results. A significantly higher expression of activity was recorded in cholesteatoma and middle ear granulations at 45-47 kDa in comparison with deep meatal skin. Western blotting indices were to be present in all of the cholesteatoma specimens tested. Only one of the specimens (cholesteatoma) tested showed any MMP-3 presence.

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.

During cutaneous wound healing a number of migratory and remodeling events occur that require the action of matrix metalloproteinases (MMPs) and their natural inhibitors (TIMPs). In this study, we analyzed the temporal and spatial expression patterns of these molecules during the healing of murine excisional skin wounds. Our data imply that defined phases of repair rely on distinct repertoires of MMP activity and TIMP counterregulation. Reepithelialization was found to be associated with active production of collagenase, 92-kDa gelatinase, and stromelysins-1 and -2 by distinct subpopulations of keratinocytes at the migrating border. Notably, no TIMP transcripts were expressed in the epidermis, but TIMP-1 expression in the wound colocalized with expression of collagenase, 92-kDa gelatinase, and stromelysin-1, albeit in distinct cells. Concomitant with the formation of an extensive hyperproliferative epithelium, TIMP-1 transcripts accumulated at the mesenchymal/epidermal border of the granulation tissue. During later phases of wound repair, we observed an increase in 72-kDa gelatinase and MT1-MMP expression, whereby the transcripts of these colocalizing MMPs were detected exclusively and at high levels in the granulation tissue. At completion of reepithelialization, the expression levels of the MMPs and TIMP-1 seen in epidermal and dermal compartments declined to near-basal levels, whereas the macrophage-specific metalloelastase (MME) reached maximum expression. In reepithelialized wound tissue, MME transcripts were detected in deep layers of reconstituted dermis and seemed to cluster around vascular structures. Systemic glucocorticoid treatment, which is known to result in impaired wound healing, led to a nearly complete shut-off of MME expression. These observations imply an additional role of macrophage-related proteolysis, independent of its classical roles during earlier, inflammatory phases of cutaneous wound repair.

Wound healing is a fundamental response to tissue injury that results in restoration of tissue integrity. This end is achieved mainly by the synthesis of the connective tissue matrix. Collagen is the major protein of the extracellular matrix, and is the component which ultimately contributes to wound strength. In this work, we report the influence of Aloe vera on the collagen content and its characteristics in a healing wound. It was observed that Aloe vera increased the collagen content of the granulation tissue as well as its degree of crosslinking as seen by increased aldehyde content and decreased acid solubility. The type I/type III collagen ratio of treated groups were lower than that of the untreated controls, indicating enhanced levels of type III collagen. Wounds were treated either by topical application or oral administration of Aloe vera to rats and both treatments were found to result in similar effects.

The aim of this study was to study innervation and angiogenesis in response to grafts of dermis and cultured keratinocytes using immunohistochemical techniques. In a porcine model, fresh autologous de-epidermalized dermis and cultured autologous keratinocytes were combined using a two-stage technique, to produce keratodermal grafts. Wounds were encased within skin graft chambers that prevented the influence of the surrounding skin. As grafts contracted, a peripheral rim of granulation tissue became exposed, allowing us to compare the wound bed beneath grafts with that beneath the raw granulating surface. Grafts were studied for 6 weeks. Angiogenesis was studied using antisera to von Willebrand factor to detect endothelial cells. Nerve growth was studied using antisera to S-100, a Schwann cell marker, and to four axonal markers: protein gene product 9.5, C-flanking peptide of neuropeptide Y, calcitonin gene-related peptide, and vasoactive intestinal peptide. In kerato-dermal grafts (n = 28), organization of blood vessels and nerve growth occurred only beneath areas with epidermal cover as compared with the surrounding granulation tissue. Initially, the immunoreactivity to von Willebrand factor was high, but in areas with epidermal cover it assumed a more orderly pattern with fewer blood vessels. Innervation was first detected by S-100 immunoreactivity seen at 1 to 2 weeks, closely followed by that to protein gene product 9.5 and much later to calcitonin gene-related peptide. C-flanking peptide of neuropeptide Y and vasoactive intestinal peptide immunoreactivity were detected in the wound depth surrounding large blood vessels at 4 to 6 weeks. In control wounds that had been either grafted with de-epidermalized dermis alone (n = 10) or allowed to granulate (n = 10), persistently there was high immunoreactivity to von Willebrand factor but minimal immunoreactivity to the neural markers. In conclusion, kerato-dermal grafts become innervated, and beneath their surface there is also vascular organization to resemble normal skin. Keratinocytes themselves may influence angiogenesis and innervation, as both processes failed to occur beneath granulating areas.

Pathology of a round window membrane rupture was demonstrated in a human temporal bone from a case in which labyrinthotomy had been performed through the round window membrane. Proliferation of mesothelial cells was seen in the inner layer of the membrane, and it appeared to be reinforced from the inside by these reactive cells. The middle layer of the membrane was thickened by increased collagen and elastin. The pathologic changes which take place during healing of the ruptured round window membrane are discussed.

To evaluate the spatial and temporal expression of type V collagen in a wound healing model, subcutaneously implanted viscose cellulose sponges in rats were used to induce granulation tissue formation. Analyses on granulation tissue were carried out on days 3, 5, 8, 14, 21, 30, 59 and 84. Acid soluble collagens were extracted and the relative amount of type V collagen was quantified by SDS-PAGE. Specific antibodies to type I, III and V collagens were used in immunohistochemistry and specific RNA probes to proalpha1(I), proalpha1(III) and proalpha1(V) collagen in in situ hybridization. Type V collagen content increased relative to type I and III collagens up to day 8 and remained at the same level for up to the three months. Type V collagen was expressed strongly in blood vessel walls as seen in immunohistochemistry. In situ hybridization showed that all of the three types of collagen were expressed mostly in fibroblast-like cells and also in rounded cells, especially type V collagen. In conclusion, type V collagen was seen in the wound healing model in increasing amounts from day 3 onwards, its localization being highly associated with blood vessels in granulation tissue and it was synthesized by fibroblast-like and rounded cells.

Reports on the histological and biochemical nature of periprosthetic fibrous/granulomatous tissue has, to date, been largely limited to frozen tissue sections. This study reports the cytokine and matrix metalloproteinase profiles found in periprosthetic interface tissues in THA which have failed due to aseptic loosening and in capsular tissues obtained at primary surgery. The study employs immunohistochemistry, in situ hybridization, and color video image analysis on formalin fixed and paraffin embedded sections.

Tenascin-C (TN) is an extracellular matrix glycoprotein expressed during embryogenesis. Its distribution is restricted in normal adult tissues and is upregulated in tumors and inflammatory conditions. Twenty-five specimens were studied, including 7 reactive vascular lesions (6 cases of granulation tissue and 1 case of bacillary angiomatosis), and 18 vascular tumors (6 angiosarcomas, 7 hemangioendotheliomas, and 5 AIDS-related nodular type Kaposi's sarcomas). Formalin fixed-paraffin-embedded tissues were stained with monoclonal antibody to TN (DAKO) and with MIB-1 (AMAC). Heterogeneous expression of TN immunoreactivity was seen in all cases, with a diffuse pattern in bacillary angiomatosis and most granulation tissue cases and a focal pattern in angiosarcoma and most hemangioendothelioma cases. Kaposi's sarcoma cases showed both a focal and diffuse pattern of distribution. In most cases proliferation indices (PI) did not correlate with TN expression. Electron microscopy demonstrated active angiogenesis in bacillary angiomatosis and granulation tissue and vasculogenesis in angiosarcoma and hemangioendothelioma. The study demonstrated positive TN expression in reactive lesions with angiogenesis (granulation tissue and bacillary angiomatosis) and neoplastic lesions showing vasculogenesis (angiosarcoma and hemangioendothelioma), although with a different pattern of distribution. These results suggest that TN might be an important extracellular matrix glycoprotein in angiogenesis and vasculogenesis.

Basic fibroblast growth factor (bFGF) is thought to play an important role in wound healing. However, its histological localization, both in normal and pathological conditions in the oral mucosa, has not been well documented. We have studied the immunolocalization of bFGF in normal gingiva and gingival epulis specimens corresponding to different organizing stages. In normal gingiva, bFGF was detected in subpopulations of macrophages, mast cells and most endothelial cells in the lamina propria. Granulation tissue in epulides was histopathologically classified into six organizing stages. In stages 1 and 2, a small number of bFGF-positive macrophages was seen at the periphery of ulcer bases. In stages 3 and 4, histologically characterized by prominent capillary proliferation, large numbers of bFGF-positive macrophages and mast cells were located within granulation tissue. A positive reaction for bFGF was also found in some endothelial cells and in myxoedematous stroma that was rich in heparan sulfate proteoglycan. In stages 5 and 6, when fibrosis was accelerated, bFGF-positive macrophages and mast cells decreased in number and were localized only at the periphery of the fibrous tissue. These findings suggest that maximum amounts of bFGF are synthesized and released from some macrophages and mast cells into the extracellular matrix during neovascularization of granulation tissue.

The pathophysiology of rotator cuff tears can be elucidated by examining the tendinous insertion of the supraspinatus muscle. As seen by light microscopy, the granulation tissue around the insertion of a torn supraspinatus tendon appears to induce osteochondral destruction by means of multinucleated giant cells and chemical mediators. The purpose of this study was to examine the contribution of certain chemical mediators to osteochondral destruction using immunohistochemical analysis of interleukin-beta, cathepsin D, and matrix metalloprotease-1. Sixteen supraspinatus insertions with portions of the greater tuberosity, including eight complete-thickness tears and eight incomplete-thickness tears, were obtained during surgery. Six fresh cadaveric supraspinatus tendons without grossly evident tears served as normal controls. Strong immunoreactivity was found in all 16 torn supraspinatus insertions but not in the six insertions of apparently intact tendons. Macrophages and multinucleated giant cells, which showed immunoreactivity for all three chemical mediators, were often found at the interface between the osteochondral margin of the enthesis and the granulation tissue, suggesting that they may be involved in osteochondral destruction. We therefore concluded that, in addition to repetitive subacromial impingement, this granulation tissue may contribute to the development of rotator cuff tears by weakening the insertion.

The three-dimensional structure of the collagen fibers in the outer membrane of recurrent chronic subdural hematoma was studied by scanning electron microscopy (SEM). Specimens obtained at surgery were treated with NaOH at room temperature to digest away all cellular components and expose the collagen fibers. SEM observation of the dural side of the outer membrane showed the collagen fibers were woven into a compact feltwork with a dense arrangement. The fiber bundles had a honeycomb structure framed by the collagen fibers. Observation of the hematoma side found the collagen bundles had a sparse wavy appearance. The arrangement of the collagen fibers on the dural side is different from that on the hematoma side. The thick outer membrane may be formed by granulation resulting from inflammatory reaction. Collagen fibrillar networks are not fragile, and may reinforce the outer membrane of the recurrent hematoma.

Cholesterol granuloma is a histological term used to describe a non-specific inflammatory reaction produced by the presence of cholesterol crystals, which may originate a foreign-body reaction. Cholesterol granuloma of the temporal bone is silent for years and not preceded by a history of otitis media. The diagnosis is made by CT and MRI, which reveal an expansive, isodense lesion brain tissue and increased signal intensity on both T1 and T2-weighted images respectively. A case of cholesterol granuloma of the petrous apex is presented. The granuloma was excised completely using an infra-retrolabyrinthine approach.

During early granulation tissue formation of wound repair, new capillaries invade the fibrin clot, a process that undoubtedly requires an interaction of vascular cells with the wound provisional matrix composed mainly of fibrin, fibronectin, and vitronectin. Integrin alphaVbeta3 is the vascular cell receptor for these wound-associated adhesive proteins. Therefore, we investigated the expression of this receptor on new capillaries of healing full-thickness cutaneous porcine wounds. During granulation tissue formation, alphaVbeta3 was expressed specifically on capillary sprouts invading the central fibrin clot whereas the closely related integrin alphaVbeta5 failed to localize to these cells. Cyclic peptides or antibody antagonists of alphaVbeta3 specifically inhibited granulation tissue formation in a transient manner during the period of invasive angiogenesis. Immunolocalization studies revealed that alphaVbeta3 became aggregated and lost from sprouting vessels after treatment with a peptide antagonist. In contrast, beta 1 integrins were not modulated by this treatment. Once granulation tissue filled the wound and invasive angiogenesis terminated, the alphaVbeta3 showed little or no expression in the granulation tissue microvasculature. These data demonstrate that integrin alphaVbeta3 plays a fundamental, but transient, role during invasive angiogenesis and granulation tissue formation in a healing wound.

Activins are members of the transforming growth factor beta (TGF beta) superfamily, which comprises a growing group of dimeric proteins. TGF beta and several other members of this superfamily are known to play an important role in wound healing. However, expression of activin during wound healing has not been demonstrated so far. In this study we have analyzed the expression pattern of activin and activin receptors in normal and wounded skin. We found a large induction of activin A and a minor induction of activin B mRNA expression 1 day after skin injury and high expression levels of activin A and B were found within the first 7 days after wounding. At 13 days after injury, expression of activin A mRNA had returned to the basal level, whereas high levels of activin B persisted. In situ hybridization studies revealed expression of activin A in the granulation tissue below the wound and activin B in the hyperproliferative epithelium at the wound edge and in the migrating epithelial tongue. All known types of activin receptors as well as the activin binding protein follistatin were expressed in normal and wounded skin. However, no significant induction of receptor gene expression was seen during the repair process. The distribution of activins and activin receptors in the wound suggests multiple autocrine and paracrine activities of the ligands during wound healing. Our data provide evidence for a novel function of activin and indicate that--besides TGF beta s themselves--other members of this superfamily might also play an important role in tissue repair.

The pineal gland hormone was shown to be involved in the regulation of repair and connective tissue accumulation in a wound. The hypothesis that melatonin injection could reverse the pinealectomy-induced elevation of the collagen content in scar tissue was verified. The effect of various melatonin doses (3, 30 and 100 micrograms/100 g body wt) on soluble, insoluble and total collagen content in the granulation tissue of a wound was investigated. The collagen level was estimated in pinealectomized rats, as well as in pinealectomized animals treated with melatonin. Ivalon sponges inserted subcutaneously were applied as a wound model. After 4 weeks the collagen content was measured as a hydroxyproline concentration in the dry tissue of the wound. The results showed that melatonin at the dose of 30 micrograms/100 g body wt significantly decreased both total and insoluble collagen content in the wounded tissue (p < 0.05), but other doses were ineffective. Pinealectomy increased the total (p = 0.05) and insoluble collagen level (p < 0.05) in the granulation tissue. Melatonin suppressed pinealectomy-induced elevation of the total and insoluble collagen content in the wounds (p < 0.05). No influence of the pineal gland on the soluble collagen content was observed. The results showed that melatonin was involved in the inhibitory control of the collagen content.

Objective: To evaluate the effect of a porous bovine-derived collagen membrane (PBCM) on the rates of wound healing, cellular events, presence of granulation tissue, and appearance at termination of the study in surgically created full-thickness cutaneous wounds of the distal portion of the extremities of horses., Design: Treated wounds (n = 12) received a PBCM dressing and control wounds were covered with a nonadherent dressing. Forelimbs and hind limbs were cross paired; the right forelimb and left hind limb always received the same dressing application, as did the left forelimb and right hind limb. Limbs pairs were then randomly assigned to 1 of the 2 dressings., Animals: Six healthy male horses (3 sexually intact, 3 geldings) ranging from 2 to 10 (mean, 6.5) years and weighing between 278 and 568 (mean, 408.5) kg were studied., Procedure: Full-thickness skin wounds (6.25 cm2) were created on the dorsal metatarsi and metacarpi of the experimental animals. A preformed PBCM dressing was evaluated in half the wounds (n = 12). Control wounds (n = 12) were dressed with a nonadherent gauze dressing. At each bandage change, wounds were subjectively assessed and were photographed, and measurements of horizontal and vertical wound dimensions were documented. Wound biopsy specimens obtained on days 2, 5, 7, 10, 21, and 31 were evaluated for presence of collagen, fibrin, inflammation, epithelium, and cellular elements of healing. Planar morphometry was used to determine total wound area and granulation area from the wound photographs. Percentage of contraction and epithelialization were calculated from these values. Linear regression analysis of the square root of the total wound area and the granulation area was performed. Wound area measurements were analyzed, using ANOVA for repeated measures. Regressions were compared, using covariance analysis and ANOVA. Significance was considered at P < 0.05., Results: Fibrin score, neutrophil score, and degree of inflammation were significantly greater in the PBCM-treated wounds. No significant differences in the total wound, epithelialized, or contraction areas were detected between the PBCM-treated and control (nonadherent-treated) wounds. Rates of wound healing were not statistically different between the 2 treatment groups, though they were significantly slower in the hind limbs, compared with the forelimbs. Scabs were formed more frequently in the PBCM-treated wounds., Conclusions: Application of a porous collagen bandage was not detrimental to full-thickness cutaneous wound healing in horses.

To clarify the origin of the myofibroblasts appearing in the healing process of the acetic acid-induced ulcer and effect of basic fibroblast growth factor (bFGF) on these myofibroblasts, we conducted an immunohistochemical study using antibody to intermediate filaments, desmin and vimentin. The binding sites of bFGF on the regenerative tissues were also studied by the radioautographic study of soluble compounds. As a result, the binding sites of bFGF were accumulated on the fibroblasts and myofibroblasts as well as on endothelial cells. The effect of CS23, acid-stable human recombinant bFGF was shown on distribution of myofibroblasts and regeneration of the microvascular system in the mucosal and submucosal layers.

We investigated the expression of proteoglycans (PGs) and hyaluronan (HA) during healing of human mucosal wounds. Biopsy specimens of experimental wounds were taken 1, 3, and 7 days after wounding. Frozen sections were used for immunolocalization of CD44, syndecan-1, basement membrane-associated heparan sulfate proteoglycan (BM-HSPG), decorin, and biglycan. HA was localized in paraffin sections with a specific HA-binding probe. Epithelium showed first signs of migration on Day 1, more progressive migration on Day 3, and epithelial sheets confronted on Day 7. CD44 surrounded migrating keratinocytes at all stages of wound healing. In epithelium, CD44 and HA remarkably localized to the same region. Expression of syndecan-1 was switched from the suprabasal cell layer of unwounded epithelium to the basal cell layer of the migrating wound epithelium. BM-HSPG was absent under migrating keratinocytes. It started to reappear at the basement membrane zone on Day 7. The area under the wound epithelium containing newly synthesized collagen fibers first became positive for decorin on Day 7, whereas staining of biglycan was negative. Granulation tissue was also strongly positive for CD44 and hyaluronan. Our results indicate that migrating keratinocytes express both CD44 and syndecan-1 but not BM-HSPG. During differentiation of keratinocytes, expression of CD44 preceded that of syndecan-1. The results suggest that different HSPGs have multiple functions in keratinocyte migration and differentiation during reepithelialization.

Application of zinc oxide has been shown to accelerate the healing of both chronic and acute wounds, but the mechanisms are unknown. We quantified the gene expression (mRNA) for one important growth factor, insulin-like growth factor-1 (IGF-1) in 12 full-thickness wounds in each of three domestic pigs treated with or without topical zinc oxide. We used a RNAase protection/solution hybridisation technique to measure IGF-1 mRNA concentrations, which were 50% higher in the granulation tissue in wounds treated with zinc oxide compared with control wounds on days 3-4 (p < 0.05), but not thereafter (up to postoperative day 11). Topical zinc oxide increased the healing rate of wounds compared to the control group (p < 0.01). The cell composition of the granulation tissue was similar in the two groups. The increased gene expression of IGF-1 may be one mechanism by which topical zinc oxide enhances wound healing.

Contractile and phenotypic properties of human fibroblasts from healing wounds were compared to those of dermal fibroblasts using in vitro models. Wound fibroblasts were recovered from implants, made of a polyvinyl alcohol sponge threaded into a perforated silicone tube, 12 days after their subcutaneous implantation in human volunteers. Dermal fibroblasts were isolated from the skin of healthy subjects. Two morphologically different fibroblast populations were observed in cells cultured from implants. In order to characterize these fibroblast populations, intracellular alpha-actin expression was studied by immunofluorescence labeling of cells cultured in monolayer. This protein was detected in less than 1% of the dermal fibroblasts. By contrast, 30 to 40% of wound fibroblasts were labeled and contained fiber networks of alpha-actin. These results confirm the presence of myofibroblasts in human wound healing tissues. The contractile property of fibroblasts and myofibroblasts was evaluated using a three-dimensional cell culture model (fibroblast populated collagen gels). Cells were incorporated in a collagen matrix and cultured for 14 days. The surface area of collagen gels was measured every day. Our results show that wound fibroblasts strongly contract collagen gels during the first 24 hr (surface area at 24 hr = 20-55% of initial surface area) in comparison to dermal fibroblasts (surface area at 24 hr = 70-75% of initial surface area). This superior level of contraction was observed until the fifth day of culture.(ABSTRACT TRUNCATED AT 250 WORDS)

Immunohistochemical study of tissues from purulent wounds in rats after treatment with the collagenase isolated from the King crab Paralithodes camtschatica was undertaken. The enzymotherapy resulted in a rapid and efficient removal of necrotic debris. It was accompanied by fibrin elimination from the wound bed and subsequent formation of new capillaries. Cellular fibronectin with ED-A sequence was identified in the newly formed granulation tissue, which points to its active synthesis in situ. Polyclonal antibodies against two isozymes of the crab collagenolytic protease were obtained. By their use it was shown that, after application of the collagenase, both isozymes accumulated in fibrin deposits at the wound bed but did not penetrate adherent granulation tissue.

Two neutrophil chemotactic factors have been purified from the exudate of chronic-phase of rat carrageenin-induced inflammation. Both the factors were derived from the third component of complement (C3); one was an NH2-terminal fragment (C3a) of alpha chain of C3 and another chemoattractant was a COOH-terminal fragment of beta chain, which was referred to as C3 beta c, on the basis of their molecular masses and NH2-terminal amino acid sequences. C3a exhibits potent chemotactic activity for rat neutrophils, and C3 beta c is a novel basic neutrophil chemoattractant with molecular mass of about 11 kDa, suggesting that both chemoattractants play a role in the neutrophil infiltration into inflammatory site of carrageenin-induced inflammation in rats.