An in vivo model of human proliferative scar.

BACKGROUND: Many aspects related to the biology and the effective therapy of proliferative scars have remained undefined, in part due to a lack of an accurate and reproducible animal model with which to systematically study them. This report describes a new model for investigating the pathophysiology and manipulation of human proliferative scars. MATERIALS AND METHODS: Human proliferative scars (n = 86) were explanted into flaps based on isolated vascular pedicles in congenitally athymic rats. Serial analysis of the structural and functional integrity of the explanted scars was performed by microscopy and by measurement of human procollagen type III peptide (PIIIP) production, human factor VIII immunostaining, and in vitro cellular proliferation. RESULTS: By these methods, both fibroblastic and epithelial components of explanted scar specimens retained the histologic characteristics of original human scar specimens, for up to 12 months. Over the same duration, scar explants continued to have high levels of human PIIIP, comparable to those found in original surgical specimens. The microvasculature of scar explants demonstrated a double basement membrane, with no staining of human factor VIII in the inner capillary endothelial layer, suggesting that host vessels were growing into ghost vessels of the human donor scar. Human factor VIII staining decreased over time. Fibroblasts cultured from explanted scar demonstrated less aggressive growth characteristics than those from original surgical specimens. CONCLUSIONS: This new model is the first to allow such long-term maintenance and serial evaluation of human proliferative scar on an accessible, isolated vasculature. It may prove useful in further defining the biology and therapy of this widespread pathologic process.

Mild hypothermia protects against ischaemia-reperfusion injury in rabbit skeletal muscle.

In three groups of rabbits, the rectus femoris muscle was subjected to 4 hours of total ischaemia. In Group 1 (normothermia, n = 5) the core temperature was maintained within the range 36-38 degrees C for the duration of ischaemia. In Group 2 (total hypothermia, n = 5) the core temperature was allowed to fall to 31.5-33.5 degrees C. In Group 3 (muscle only hypothermia, n = 5) core temperature was maintained as in Group 1 but the muscle temperature was allowed to fall to 29.5-31.5 degrees C. After 24 hours of reperfusion the muscles were harvested and measurements made of muscle viability, oedema and myeloperoxidase content. The mean (s.e.m.) muscle viability of Group 1, 19.5 (3.8)%, was significantly less than that of both Group 2, 86.0 (2.0)%, and Group 3, 87 (4.1)%, (P < 0.001). Muscle oedema and myeloperoxidase levels were elevated in all experimental groups, but differences were not significant. These findings indicate that ischaemia-reperfusion injury in skeletal muscle in this model is highly temperature-sensitive, small reductions in muscle temperature during ischaemia providing significant protection against ischaemia-reperfusion injury.

Effects of low dose intra-arterial monoclonal antibodies to ICAM-1 and CD11/CD18 on local and systemic consequences of ischaemia-reperfusion injury in skeletal muscle.

The aim of this study was to investigate the effects of intra-arterial infusion of low doses of monoclonal antibodies (Mabs) against adhesion molecules (the neutrophil CD18 integrins, and the endothelial adhesion molecule, ICAM-1) on reperfusion injury in skeletal muscle. The rabbit rectus femoris muscle was rendered ischaemic for 2 1/2 hours. Mabs were infused (approximately 0.5 mg/kg) commencing 20 minutes before the end of ischaemia and for the first hour of reperfusion. 24 hours after reperfusion, the muscle was assessed for viability, oedema and neutrophil infiltration (myeloperoxidase (MPO) levels). The results of the viability assessment (control--20.9 (0-47.5)% [median (range)], anti-CD18--30.5 (3.0-89.4)%, anti-ICAM-1--27.9 (7.8-78.1)% and anti-CD18 combined with anti-ICAM-1--45.2 (15.6-92.3)%) showed no significant differences between groups, while analysis of MPO in the postischaemic muscle showed that the anti-ICAM-1 Mab reduced neutrophil infiltration significantly. Furthermore, in contralateral unoperated muscles MPO levels were elevated 24 hours after ischaemia in the contralateral muscle. This increased neutrophil infiltration was prevented by pretreatment with anti-ICAM-1. These results suggest that low doses of anti-CD18 and anti-ICAM-1 Mabs do not reduce reperfusion injury in skeletal muscle but may help to protect against systemic effects of severe trauma. The evidence suggests that reperfusion injury in this skeletal muscle model may be largely independent of neutrophil involvement.

A comparison of a new campylobacter selective medium (CAT) with membrane filtration for the isolation of thermophilic campylobacters including Campylobacter upsaliensis.

The newly developed CAT campylobacter selective medium employing the blood-free charcoal-based agar containing cefoperazone (8 mg l-1), amphotericin (10 mg l-1) and teicoplanin (4 mg l-1) was compared with the membrane filtration culture technique for isolation of Campylobacter spp. including Camp. upsaliensis. Nine hundred and fifty human, 275 dog and 65 cat faeces (in which modified CCDA medium was also compared) were tested. In addition, the recovery of Camp. upsaliensis from pure cultures and from spiked human faeces was examined after membrane filtration. A 50-fold reduction in recovery after filtration using the 0.65 micron filters and a 150-fold reduction using the 0.45 micron filters was found. Recovery of Camp. upsaliensis from spiked faeces was considerably improved using the CAT medium compared with filtration, especially with the lower concentration of organisms (approx. 10(4) cfu mL-1). Campylobacter upsaliensis was recovered from 91 specimens of animal faeces, with CCDA recovering 26 isolates (29%), CAT recovering 76 isolates (84%) and membrane filtration (0.65 microns) recovering 82 isolates (90%). CAT selective agar was found to be a suitable medium for the isolation of thermophilic campylobacters including Camp. upsaliensis from faecal samples.

The role of cellular adhesion molecules in surgery.

The purpose of this review is to detail the roles played by the cellular adhesion molecules (CAM) in inflammatory and immunological reactions relevant to surgery. The interactions between leucocytes and endothelial cells which are mediated by CAM are central to the development of ischaemia/reperfusion injury (IRI) as occurs when blood flow is restored after an ischaemic period; for example, following revascularization of replanted digits and microvascular tissue transfers, angioplasty and tourniquet procedures. Cellular adhesion molecules are also important in wound healing and other inflammatory processes. In addition, the immunological response to organ allograft transplantation is mediated by cellular interactions mediated by CAM. This review details the functions and regulation of the various CAM involved in inflammation and allograft rejection and summarizes the results of previous surgical studies in which various techniques have been used to block the interactions mediated by CAM in an attempt to improve surgical outcomes.

Cervicofacial lymphatic malformation: clinical course, surgical intervention, and pathogenesis of skeletal hypertrophy.

This is a retrospective review of the clinical course and long-term soft-tissue/skeletal problems in 17 patients with large cervicofacial lymphatic malformations. Morbidity included infection (71 percent), airway compromise requiring tracheostomy (65 percent), poor dental health with aggressive caries (53 percent), abnormal articulatory patterns (47 percent), and episodic bleeding (35 percent). All patients underwent soft-tissue excision (mean four procedures per patient). Damage to facial nerve (76 percent) and hypoglossal nerve (24 percent) were common postoperative sequelae. Contour resection did not alter the progression of skeletal hypertrophy. Overgrowth most commonly occurred in the mandibular body, manifesting as anterior open bite deformity and class III occlusion (65 percent). Early mandibular body ostectomy was done in four children with grotesque hypertrophy. Jaw osteotomy was required in 71 percent of the patients to improve the maxillary/mandibular relationship. Histologic examination revealed intraosseous lymphatic malformation in areas of skeletal overgrowth in two-thirds of surgical specimens. The complexity of managing cervicofacial lymphatic malformation underscores the need for an interdisciplinary program in every major referral center.

Congenital fibrosarcoma masquerading as lymphatic malformation: report of two cases.

Two infants presented with a congenital cervicothoracic mass; both were initially diagnosed as having lymphatic malformation. A biopsy specimen for one child and excision for the other showed that both lesions were congenital fibrosarcomas. Postoperative chemotherapy was administered to both children. One died within 6 months of incisional biopsy from widespread metastatic disease; the other is still being treated. Congenital fibrosarcoma can be confused in its clinical presentation, radiographic findings, and histopathology with lymphatic malformation (cystic hygroma).

Nitric oxide synthase inhibitors improve skin flap survival in the rat.

The ability of nitric oxide (NO) synthase inhibitors to reduce ischemia-induced skin flap necrosis was assessed using a modified McFarlane flap in the rat. Flap survival was significantly improved in L-NIO treated (86 +/- 2%), L-NAME-treated (84 +/- 2%), and aminoguanidine-treated (76 +/- 2%) animals compared to the saline-treated group (54 +/- 2%), P < 0.005. Inhibition of NO synthase significantly decreased the hyperemia and edema within the flaps at 24 hours post-elevation. These findings suggest that endogenous NO production contributes to ischemic necrosis and that inhibition of NO synthase may prove useful in extending survival of tissues subjected to ischemia.

Training model for microvessel anastomosis.

In the early stages of microsurgical training a model is required which is accessible, inexpensive, and approximates human tissue. In this research centre initial training exercises utilize cold stored vessels harvested from sacrificed animals used in other experiments. This model serves as an initial training tool prior to the progression to live animal research which maximizes use of animal specimens and approximates the in vivo qualities of blood vessels. The costs involved are minimal and a large number of vessels can be efficiently harvested and stored using basic equipment. As a training model, cold stored vessels have balanced cost with practicality, given an excellent approximation to living tissue and have markedly reduced our usage of live animals for teaching. This latter effect is significant at a time of increased scrutiny of the ethical use of laboratory animals.

Effects of granulocyte-macrophage colony-stimulating factor on wound contraction.

The effect of topical recombinant murine and human GM-CSF, 1 or 10 micrograms/cm2 for one to ten days, on the contraction and healing of acute and chronic granulating wounds infected with Escherichia coli was studied in Sprague-Dawley rats. Bacterial contamination of wounds produced significant inhibition of wound contraction. Application of GM-CSF at either dose level to infected wounds markedly increased the rate of wound closure compared to the rate in infected untreated controls. Ten days treatment was found to be more effective than a single application. An advanced stage of wound healing was observed at ten days in the GM-CSF-treated rats compared with controls. Bacterial counts decreased in the GM-CSF-treated wounds which may suggest bactericidal activity. Topical treatment with GM-CSF was shown to effectively inhibit the retardation of wound closure produced by bacterial contamination and may therefore be useful in the management of patients with infected wounds.

Heterotopic vascularized growth plate transfer in juvenile dogs.

Prior animal studies of vascularized epiphyseal transfers placed growth plates in 2 bone systems where the independent growth of epiphyses and their response to altered stresses has been difficult to assess. This study assessed growth of vascularized ulnar epiphyses transferred to the ipsilateral humerus of 12-week-old puppies. Growth was permitted by a specially designed extensible plate. Control groups showed that humeral dissection, osteotomy and ostectomy alone do not stimulate growth. In 4 puppies initial growth of the transferred epiphysis was seen but late collapse and formation of bridging callus occurred so that overall humeral length at maturity was not significantly different from control humeri. Physical forces inherent in heterotopic transfer may preclude long term growth of transferred epiphyses particularly in sites of higher relative load. The extensible plate used here may be a useful device in the fixation of transferred epiphyses with growth potential.

Selective medium for thermophilic campylobacters including Campylobacter upsaliensis.

AIMS: To develop a selective supplement for the recovery of thermophilic Campylobacter spp, including Campylobacter upsaliensis from faeces, using campylobacter blood free selective agar base as the growth medium. METHODS: Minimum inhibitory concentrations (MICs) of cefoperazone and of teicoplanin were determined for 51 strains of C upsaliensis, 159 strains of other thermophilic Campylobacter spp, and for 66 Enterobacteriaceae (cefoperazone only). From these results a medium using campylobacter blood free selective agar base incorporating cefoperazone (8 mg/l), amphotericin (10 mg/l), and teicoplanin (4 mg/l)--CAT medium--was formulated and compared with the commercially available campylobacter blood free selective medium (modified CCDA) for isolation of Campylobacter spp from 7000 human faecal specimens. The two media were also compared for the recovery of C upsaliensis from 45 spiked human faeces. RESULTS: Isolation rates of Campylobacter spp other than C upsaliensis were similar for both media, but the CAT medium alone recovered four of the five strains of C upsaliensis from the faecal samples examined. From the spiked faeces specimens, recovery of C upsaliensis was increased by between 35% and over 200-fold on the CAT medium compared with the modified CCDA. CONCLUSIONS: CAT selective agar was a suitable alternative medium to modified CCDA for the growth of thermophilic Campylobacter spp, including C upsaliensis from faeces.

Newly recognized autosomal dominant disorder with craniosynostosis.

We report a family with a newly recognized form of autosomal dominant craniosynostosis. The disorder has high penetrance and variable expression with respect to sutural involvement and cranial abnormalities, ranging from fronto-orbital recession to clover-leaf skull deformity. Associated problems included headache, poor vision, and seizures; intelligence, however, is normal. Assignment of a well-described syndromic designation, e.g., Crouzon, Pfeiffer, Saethre-Chotzen, or Jackson-Weiss, is precluded based upon the absence of characteristic changes, i.e., midfacial hypoplasia, orbital hypertelorism, blepharoptosis, hand anomalies, or foot anomalies. The large size of this family and high penetrance of the disorder suggests that this may be an excellent candidate for positional cloning of a locus important in craniofacial development.

Surface area measurement of pressure sores using wound molds and computerized imaging.

OBJECTIVE: To develop a method to measure wound surface area. DESIGN: Validity study. SETTING: Inpatient service. PARTICIPANTS: Four patients with decubitus ulcers. MEASUREMENTS: A new imaging process estimating wound surface area and volume by NMR spectroscopy of a mold of the pressure ulcer and a comparison measurement of volume of the mold by water displacement. Measurements made serially totalled 17. RESULTS: Measurement of the volume of the mold by the computer system correlated strongly with measurement of the volume by water displacement. Surface area of the pressure sore mold correlated strongly with volume of the mold raised to the two-thirds power. CONCLUSIONS: It is possible for the first time to measure surface area of decubitus ulcers. This may provide a way of determining accurately the dose of newly proposed topical treatments.

The effect of denervation on soft-tissue infection pathophysiology.

Pressure is the sine qua non in the etiology of pressure sores; however, ischemia, denervation, edema, and infection also have been implicated. The role of denervation in tissue infection was studied in an isolated in vivo ovine flap model. Twenty-six adult ewes, divided into three groups, had 29 island pedicle flaps raised on their buttocks. In group I, the cutaneous nerve remained intact, while group II had its nerve divided acutely. Group III had prolonged denervation, where the nerve was divided 7 days before flap elevation. All flaps received intradermal inoculations of 10(7) Staphylococcus aureus. Ninety-six hours later, quantitative bacteriology showed counts of 10(7), 10(7), and 10(9) colony-forming units (CFU) per gram of tissue in groups I, II, and III, respectively. Septic foci were larger in group III, and there was a significant increase in tissue edema between groups I and III. A 25-fold increase in bacterial counts seen in the prolonged denervation group may help explain why neurologically injured patients are more susceptible to infection and pressure ulcerations.

The safety and effect of topically applied recombinant basic fibroblast growth factor on the healing of chronic pressure sores.

The first randomized, blinded, placebo-controlled human trials of recombinant basic fibroblast growth factor (bFGF) for pressure sore treatment were performed. Three different concentrations of bFGF in five dosing schedules were tested for safety using hematology, serum chemistries, urinalysis, absorption, antibody formation, and signs of toxicity. Efficacy was evaluated by wound volumes, histology, and photography. No toxicity, significant serum absorption, or antibody formation occurred. In six of eight subgroups, there was a trend toward efficacy with bFGF treatment. When all subgroups were combined, comparison of the slopes of the regression curves of volume decrease over initial pressure sore volume demonstrated a greater healing effect for the bFGF-treated patients (p < 0.05). Histologically, bFGF-treated wound sections demonstrated increased fibroblasts and capillaries. More patients treated with bFGF achieved > 70% wound closure (p < 0.05). Blinded observers were able to distinguish differences in visual wound improvement between bFGF and placebo groups. These data suggest that bFGF may be effective in the treatment of chronic wounds.

Pseudomonas aeruginosa exotoxin A: its role in retardation of wound healing: the 1992 Lindberg Award.

Bacterial concentrations greater than 10(5) colony-forming units/gm of tissue prevent wound healing. However, it has not been determined whether it is the number of bacteria or a toxin produced by these organisms that impedes the wound healing process. Pseudomonas aeruginosa (PSAR), a burn wound pathogen, produces a dermonecrotic toxin, exotoxin A. Studies have indicated a role for exotoxin A in the pathogenicity of PSAR. We investigated the role of exotoxin A in the retardation of contraction. Acute granulating wounds were created on 90 Sprague-Dawley rats. The animals were equally divided into six groups and were treated topically as follows: group 1, sham: no infection, no treatment; group 2, exotoxin A; group 3, exotoxin A and antiexotoxin; group 4, autoclaved PSAR 10(6); group 5, 10(6) viable PSAR inoculated in the wound; group 6, 10(6) viable PSAR and antiexotoxin. Wound contraction was measured with the use of planimetry twice a week. Serial biopsies were performed on all wounds. Contraction rates revealed significantly (p < 0.05) retarded closure in the animals treated with exotoxin A and in the viable PSAR group when compared with the rates of the noninfected control groups. Animals treated with exotoxin A plus antiexotoxin A and those treated with live PSAR and antiexotoxin showed contraction rates identical to the control groups. These data suggest that exotoxin A in PSAR infections retards wound healing and that neutralization of the toxin restores the normal healing process.

Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.

To better define thrombin-receptor interactions, we synthesized human thrombin peptides and identified binding-domain peptides that bind thrombin receptors and activate mitogenic signals (Glenn, K.C., G.H. Frost, J.S. Bergmann, and D.H. Carney. 1988. Pept. Res. 1:65-73). Treatment of full dermal dorsal incisions with a single topical application of thrombin receptor-activating peptide (TRAP-508) or human alpha-thrombin in saline enhances 7-d incisional breaking strength in normal rats up to 82% or 55% over saline-treated controls, respectively. Control wounds require approximately 11.5 d to achieve breaking strength equivalent to TRAP-treated wounds at day 7. Thus, a single application of TRAP accelerates healing, shifting the time course forward by up to 4.5 d. Histological comparisons at day 7 show more type I collagen, less evidence of prolonged inflammation, and an increase in number and maturity of capillaries in TRAP- and thrombin-treated incisions. Angiograms also show 50-65% more functional vascularization going across thrombin- and TRAP-treated surgical incisions. Thus, alpha-thrombin and thrombin peptides, such as those released following injury, appear to initiate or enhance signals required for neovascularization and wound healing. The ability to accelerate normal wound healing events with synthetic peptides representing receptor binding domains of human thrombin may offer new options for management of wound healing in man.

Exercise stress testing for the pediatric patient with burns: a preliminary report.

There is no literature concerning postburn exercise tolerance among pediatric patients. In an effort to quantify the endurance capabilities of pediatric burn victims, stress testing of 59 patients was carried out with a modified Bruce protocol. This treadmill test consists of eight 3-minute stages with incremental increases in speed and incline. Fifty-nine patients (37 boys and 22 girls) were tested. The mean burn surface area was 46%; an average of 33% consisted of full-thickness burns. The average time since burn injury at which patients were tested was 2.9 +/- 1.9 years. The average age of the patients at the time of test was 11.4 +/- 3.9 years. No differences in exercise tolerance were found among these children irrespective of the presence of inhalation injury, method of excision, or burn size. If these trends continue as more data are accumulated, the long-term prognosis for the child with severe burns will be encouraging.