Intraoperative and delayed wound approximation in closure of skin defects in different areas.

OBJECTIVE: Wound approximation device is an interesting reconstructive option but not well popularised. In this study we present a simple device that can be used for immediate or delayed closure of large dermal wounds in different anatomical areas. METHOD: Patients with acute and chronic wounds were recruited and underwent immediate intra-operative wound approximation and/or delayed wound approximation, with a home-made wound approximation device. RESULTS: Approximation time in the immediate closure group ranged from 20-140 minutes. Satisfactory scars were obtained in 19 patients (76%) and adherent scars developed in 6 patients. Delayed wound approximation was used successfully in closure of 9 defects. CONCLUSION: This simple dermal wound approximation device can be used intraoperatively to successfully close large difficult wounds, located on the trunk and thigh, with minimal complications. The device can also be used to approximate delayed wounds located in regions where closure is particularly problematic, like the lower leg, foot, and scalp. Some modifications of the device are needed to improve its safety and efficacy. Wound tension is detrimental to adequate wound healing and tensile strength, another basic principle that should not be overlooked to avoid wound dehiscence. Wound approximation is adding to reconstructive options, not replacing them, and they must always be considered.

Novel pyrazole derivatives as anticancer and radiosensitizing agents.

The present article describes the synthesis of some novel pyrrole, pyrazolo[4,3-d]oxazole, pyrrolo[2,3-b]pyridine, 1,2,3-triazole and oxoazetidin derivatives incorporating pyrazole moiety, the structures of which were confirmed by elemental analyses and spectral data. All the target compounds were subjected to in-vitro antitumor activity against liver and colon human tumor cell lines (HEPG2 and HCT), furthermore, the most potent compounds were evaluated for their ability to enhance the cell killing effect of γ-radiation (radiosensitizing evaluation). The results of in-vitro anticancer evaluation showed that compounds 3 and 16a were the most potent compounds on HEPG2 (IC50=2.6 and 4.2 µg/ml) and compounds 2 and 10 were the most potent on HCT (IC50=2.7 and 3.9 µg/ml) compared to vinblastine (IC50=4.6 on HEPG2 and 2.6 µg/ml on HCT), while, the activity of the most potent compounds increased after combination with γ-radiation and they showed no toxicity on normal hepatocytes and colon cells at their effective concentrations.

Potential of pottery materials in manufacturing radioactive waste containers.

Various pottery materials were evaluated for possible use in manufacturing containers for radioactive waste. Their potential was examined from the viewpoints of the effectiveness of disposal and the changes induced in them by gamma rays. Samples of these materials were irradiated with high-energy neutrons and gamma rays in a reactor near its core. the physical and mechanical properties of the materials before and after gamma irradiation (in a 60Co gamma cell) were compared. The study showed that pottery materials are resistant to radiation. Therefore, they were proposed for manufacturing drums for disposal of radioactive waste of high gamma activity.

Utilization of colorimetric and atomic absorption spectrometric determination of aztreonam through ion pair complex formation.

Three rapid and sensitive, colorimetric and atomic absorption spectrometric methods were developed for the determination of aztreonam. The proposed methods depend upon the reaction of cobaltthiocyanate (I) or reineckate (II) ions with the drug to form stable ion-pair complexes which extractable with chloroform. The greenish blue and pink color complexes are determined either colorimetrically at lambda(max) 625 and 525 nm for I and II reagents, respectively, or by atomic absorption spectrometry, directly using the organic extracted complex, or indirectly, using the supernatant. The three procedures are applied for the determination of aztreonam in pure and in pharmaceutical dosage forms applying the standard additions technique and the results obtained agreed well with those obtained by the official method.

Transforming growth factor beta 1 inhibits the proliferative effect of insulin on human infragenicular vascular smooth muscle cells.

PURPOSE: The distribution of atherosclerotic arterial disease in diabetes mellitus characteristically involves the infragenicular arterial tree including the anterior tibial, posterior tibial, and peroneal arteries. The proliferation of vascular smooth muscle cell (VSMC) is essential in the development of the atherosclerotic lesion. It has long been held that insulin plays a causative role in the formation of the atherosclerotic lesion in diabetes. We studied the role played by insulin in the proliferation of these cells in culture and the interaction of insulin with transforming growth factor beta 1 (TGF beta 1), a factor known for its possible inhibitory effects. METHODS: We have grown and characterized a line of VSMC harvested from atherosclerotic infragenicular arteries of human subjects undergoing below-knee amputation. The cultures were defined as being of VSMC origin by immunohistochemical staining with alpha-smooth muscle actin. Confluent cultures of passages 4 through 7 were seeded into six well plates at a density of 5000 cells/well. After serum deprivation the cells were exposed to insulin (100 ng/ml) alone or in combination with TGF beta 1 (6 ng/ml). RESULTS: Our findings indicate that a 48-hour incubation with insulin augments the proliferation of human infragenicular VSMC, producing a 207% increase in cell number when compared with control cells (11,328 +/- 686, n = 56 vs 3682 +/- 182, n = 87; p < 0.0001). The addition of TGF beta 1 in combination with insulin abolished the accelerated growth rate seen in test groups treated with insulin alone (3614 +/- 247, n = 32 vs 11,328 +/- 686, n = 56; p < 0.0001). CONCLUSION: These results strongly suggest that insulin is a potent stimulant of human infragenicular VSMC proliferation. The mitogenic effect of insulin is inhibited by TGF beta 1, producing proliferation rates comparable to those observed in control cells incubated with serum-free media.

Proliferation and extracellular matrix production by human infragenicular smooth muscle cells in response to interleukin-1 beta.

PURPOSE: Atherosclerotic peripheral vascular disease commonly involves the infragenicular arterial tree. Our study evaluated the effect of interleukin (IL)-1 beta on the proliferation of vascular smooth muscle cells (VSMCs) derived from atherosclerotic infragenicular arteries of human subjects who underwent below-knee amputation, as well as the role of IL-1 beta in VSMCs' production of extracellular matrix components, substances that are important in the transformation of VSMCs from the contractile to the synthetic phenotype. This transformation to the synthetic phenotype is an important step in the formation of the atherosclerotic lesion. METHODS: Cultures were identified as being of smooth muscle origin through staining with the cytoskeletal marker, alpha-smooth muscle actin. Proliferation assays were performed by seeding confluent cultures of passages 4 to 7 into six-well plates at 10,000 cells per well. After serum starvation, samples were incubated with IL-1 beta (1 ng/ml). Cell number was determined on a daily basis. To study extracellular matrix production, cells were propagated in tissue culture chamber slides in the absence or presence of growth media containing IL-1 beta. After fixation with 100% methanol, each sample was stained with a primary antibody specific for an extracellular matrix component. After staining with the fluorescein-tagged secondary antibody, each sample was examined using immunofluorescent microscopic examination. RESULTS: The results of our proliferation assays showed that IL-1 beta caused a significant increase in the proliferation of VSMCs at 24, 48, 72, and 96 hours (p < or = 0.003 when comparing IL-1 beta-treated samples with control specimens at each time period using unpaired t test). The number of IL-1 beta-treated cells at 96 hours was double the number present in the control samples (16,033 +/- 238 vs 8102 +/- 824). When compared with control samples, IL-1 beta was found to affect the production of extracellular matrix proteins by infragenicular VSMCs. IL-1 beta caused an increase in the production of fibronectin, a decrease in the production of laminin, and no change in the production of collagen type IV. CONCLUSIONS: These results suggest that interleukin-1 beta acts as a potent stimulant of the proliferation of human infragenicular VSMCs. IL-1 beta also acts to augment the production of fibronectin by these cells. Fibronectin has been implicated in the phenotypic transformation of VSMCs from the contractile to the synthetic state. Therefore, IL-1 beta may serve as an important regulatory factor in the development of atherosclerosis by stimulating the proliferation of VSMCs and their transformation to the synthetic state, two important steps in the formation of the atherosclerotic lesion.

Phenotypic characterization of human smooth muscle cells derived from atherosclerotic tibial and peroneal arteries.

PURPOSE: The vascular smooth muscle cell plays a pivotal role in the development of atherosclerosis. The objectives of this study were to characterize smooth muscle cells from the human atherosclerotic tibial artery to determine their phenotypic properties and to examine the contractile reactions of these cells to physiologic and pharmacologic stimuli. METHODS: After below-knee amputations were performed, vascular smooth muscle cells were harvested and cultivated from tibioperoneal source. Characterization was done with transmission electron microscopy and immunocytochemistry. The contractile properties were determined by observing the response to various stimuli. In addition, segments of vessels harvested were submitted to electron microscopy studies for comparison with the cultured cells. RESULTS: Immunofluorescent labeling was positive for alpha-smooth muscle actin. Electron microscopy revealed the presence of a thickened basal laminae and large intracellular lipid vacuoles. The earlier passages revealed cells with a large number of microfilaments characteristic of a contractile cell. As later passages were examined, there was a notable change in character with an increasing amount of rough endoplasmic reticulum and Golgi complexes. The increased thickness of the basal lamina in the cultured cells resembled that found in vessel segments studied by electron microscopy. A rapid contraction response was seen when the cells were incubated with angiotensin II, bradykinin, or endothelin. No response was seen with the addition of isoproterenol, nitroglycerin, or nitroprusside, known smooth-muscle relaxants. CONCLUSION: This model demonstrates the apparent inability of these smooth muscle cells from atherosclerotic tibial arteries to relax to pharmacologic and physiologic stimuli. In addition, as seen by transmission electron microscopy, these cells maintain their atherosclerotic phenotype after multiple passages.

Use of modern craniofacial techniques for comprehensive reconstruction of the acromegalic face.

The severe acromegalic patient poses a difficult reconstructive dilemma to the craniofacial surgeon. Significant facial deformities can include frontal bossing, prominent supraorbital ridges, malar flatness, maxillary hypoplasia, mandibular prognathism with class III malocclusion, and macrogenia. Reports on the correction of these deformities are rare. Prior publications describe long hospital stays, weeks of intermaxillary fixation, requirement for a tracheostomy, as well as the need for multiple, staged procedures and interdisciplinary teams. In an effort to extend the advances of modern craniofacial techniques to this group of patients, we performed an extensive reconstruction on a 28-year-old acromegalic patient using a one-stage procedure without the use of intermaxillary fixation and without the added morbidity of a tracheostomy. The procedure addressed the skeletal deformities of the upper face, the midface, and the lower face. The operation was performed by a single plastic surgery team and the patient was extubated in 36 hours and discharged in 6 days. We believe that the use of rigid fixation and the judicious application of modern craniofacial principles can allow a complex yet safe one-stage procedure to reconstruct the acromegalic face. Such an approach showed decreased perioperative morbidity and provided an excellent functional and aesthetic result.

Effect of growth factors on cell proliferation and epithelialization in human skin.

The failure of chronic wounds to heal remains a major medical problem. Recent studies have suggested an important role for growth factors in promoting wound healing. We investigated the mitogenic effect of basic fibroblast growth factor (FGF), insulin-like growth factor-1 (IGF-1), and epidermal growth factor (EGF), comparing their effects with those of media alone (MEM) in a human skin explant model. A stable organ culture system for maintaining the histologic structure of human epidermis for 10 days in vitro was developed. DNA synthesis was measured on Days 1, 3, and 7 of organ culture using [3H]thymidine ([3H]thy) uptake and expressed as cpm/mg dry weight (mean +/- SEM). FGF, IGF-1, and EGF were each capable of stimulating [3H]thy uptake on Day 1 of culture (2372 +/- 335 FGF, 2226 +/- 193 IGF-1, 4037 +/- 679 EGF vs 1108 +/- 70 MEM, P < 0.05). IGF-1 and EGF also stimulated [3H]thy uptake on Days 3 and 7 of culture. The organ culture system was further employed to observe epidermal outgrowth. Longest keratinocyte outgrowth from the explant periphery (simulating epithelial regeneration from the wound edge) was observed on Day 7. EGF resulted in maximum stimulation of epithelial outgrowth (440 +/- 80 microns), followed by FGF (330 +/- 56 microns), IGF-1 (294 +/- 48 microns), and MEM (189 +/- 50 microns). We postulate, therefore, that FGF, IGF-1, and EGF are important mitogens for wound healing and that EGF in particular is capable of stimulating epithelialization.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenine nucleotide depletion in cryopreserved human cardiac valves: the "stunned" leaflet interstitial cell population.

Preparation protocols for human cardiac valves are intended to minimize cytotoxicity because it has been thought that viable leaflet interstitial cells may enhance homograft durability. Preimplantation factors influencing the status of these cells at the time of transplantation include ischemia, disinfection, and cryopreservation freezing programs. In these experiments, adenine nucleotide quantitation was undertaken to assess metabolic consequences of preparation; preharvest ischemia served as an independent variable to examine the relationship between time of procurement (postmortem) and high-energy phosphate status of the cryopreserved leaflets at thaw. Nucleotides were measured using high-performance liquid chromatography performed on extracts of semilunar cusps from 25 cryopreserved human valves with documented ischemic times. Results indicate total adenine nucleotides (TAN; [ATP] + [ADP] + [AMP], in nmol TAN/mg leaflet protein) are higher (P < 0.05) after < 2 h of harvest ischemia (1.16 +/- 0.36) than with ischemic times of 3-6 h (undetected), 7-12 h (0.18 +/- 0.07), and 13-20 h (0.06 +/- 0.06). Depletion of ATP was similar, with many leaflets devoid of detectable levels. Net utilization of leaflet energy stores demonstrates time dependency when assayed after completed processing. However, relatively elevated catabolites, even with brief ischemia, and infrequently identified ATP, ADP, and AMP, suggest a consumption so accelerated that the following cryopreservation it is virtually independent of procurement-associated ischemia. We conclude resumption of a functional cell population obligates significant de novo phosphoanhydride boned reformation or a repopulation of dead/dying interstitial cells from a subset surviving the apparently severe rigors of valve preparation.

Dual structural and functional phenotypes of the porcine aortic valve interstitial population: characteristics of the leaflet myofibroblast.

The cellular properties of semilunar cardiac valve leaflets may be more complex than previously assumed. In particular, the cells of the leaflet matrix which are likely critical to proper cusp function are a poorly described population to date. We hypothesized that, similar to the matrix cells of atrioventricular valves, aortic valve leaflet interstitial cells (AoLIC's) possess characteristics of both fibroblasts (matrix secretion) and smooth muscle cells (contraction). Porcine AoLIC's were structurally examined for contractile and stress fiber protein assemblies using transmission electron microscopy and immunocytochemistry. Contractile function in response to vasoactive stimuli was directly assessed using AoLIC's cultured on flame-polymerized silicone, with cell contraction identified by the appearance of wrinkles in the substratum after challenge with each agent. The structural analyses showed cellular microfilaments were often organized into various contractile arrangements including polygonal networks, and that AoLIC's are rich in smooth muscle-specific alpha-actin. Incomplete basal laminae often associated with myofibroblasts were observed. Contraction experiments indicated a responsivity of similar latency, but variable peak and duration to 10(-7) M L-epinephrine, 3.2 x 10(-7) M angiotensin II, 110 microM carbachol, 50 mM KCl, 3.2 x 10(-7) M bradykinin, 110 microM isoproterenol, and 5 x 10(-7) M endothelin I. Soluble and insoluble matrix secretion was confirmed with FITC-conjugated monoclonal antibodies to chondroitin sulfate, fibronectin, and prolyl-4-hydroxylase. These data show that the AoLIC's are best designated as myofibroblasts. The unusual features of the myofibroblast may be central to lifelong aortic leaflet durability.

Inhibition of adenosine deaminase and nucleoside transport. Utility in a model of homograft cardiac valve preimplantation processing.

Human cardiac valves are increasingly used in the reconstruction of ventricular outflow tracts and offer performance advantages over porcine and mechanical prostheses; the durability of these replacements has been associated with leaflet interstitial cell viability and a presumed sustained function after implantation. Preimplantation tissue preparation entails sequential steps that are potentially cytotoxic and may therefore affect functional cell survival at thaw. We defined the metabolic consequences of each interval using semilunar cusps from 118 porcine valves to model a homograft preparation with 40 minutes of fixed cadaveric (harvest) ischemia. Fifty-eight valves served as controls and were first processed according to standard cryopreservation protocol; nucleosides were extracted at the end of each step to differentiate independent contributions to high-energy phosphate depletion. Sixty simultaneously harvested leaflets were administered the nucleoside transport inhibitor p-nitrobenzy-thionosine (NBMPR) and the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) at procurement, to attempt adenosine salvage and restitution of processing-incurred adenine nucleotide losses. High-performance liquid chromatography was used to compare adenosine triphosphate, diphosphate, and monophosphate and diffusible nucleopurines of the control and EHNA/NBMPR-treated groups. Control results indicate that disruption of the adenosine triphosphate-diphosphate cycle occurs independently with antibiotic disinfection and cryopreservation. However, throughout all preparation steps, adenine nucleotides were maintained at harvest (baseline) concentrations in the EHNA/NBMPR valves. This suggests that salvage therapy may protect a significant number of cells from net high-energy phosphate catabolism. If, with further study, the durability of transplanted valves is concluded to benefit from retained leaflet interstitial cell viability, such enhancement of metabolic tolerance to the obligatory processing may facilitate functional recovery.

High energy phosphate depletion in leaflet matrix cells during processing of cryopreserved cardiac valves.

Preimplantation preparation of cardiac valves includes three major steps: (1) harvesting with accompanying ischemia (warm time from cessation of donor heart beat), (2) antibiotic disinfection, and (3) controlled-rate cryopreservation. To define the interdependent injury effects of these manipulations on leaflet matrix cells and specifically the potential for prolonged harvest-related ischemia to predispose greater injury by the subsequent steps, 96 semilunar valves were harvested from pigs in a manner analogous to human heart valve retrievals and randomly allocated to study groups as follows: 48 control valves were exposed to increasing harvested-related ischemic times, (2, 6, 12, 24 hr) and immersed in liquid nitrogen to arrest metabolic activity (i.e., prior to cryopreservation) and conclude the ischemia; another 48 were similarly harvested, subjected to identical ischemic times, then disinfected in 4 degrees C RPMI medium with standard antibiotics for 24 hr and dimethylsulfoxide cryopreserved at -1 degrees C/min to -170 degrees C (i.e., formal cryopreservation protocol). At thawing, each valve was extracted in 12% trichloroacetic acid and assayed by high performance liquid chromatography for components of the adenine nucleotide pool including ATP, lower energy nucleotides (total adenine nucleotides, [TAN] = [ATP] + [ADP] + [AMP]), adenosine, and the diffusible purines. Results are reported as nanomoles metabolite/milligram of leaflet cell protein (Lowry) and reflect a maintenance of total high energy phosphates in the control groups (5.41 +/- 0.29 nmole TAN at 2 hr; 8.34 +/- 0.67 nmole TAN at 24 hr), which fell significantly in all cryopreserved groups (1.27 +/- 0.33 nmole TAN at 2 hr; 0.34 +/- 0.22 nmole TAN at 24 hr).(ABSTRACT TRUNCATED AT 250 WORDS)