In vitro functional properties of the rat bladder regenerated by the bladder acellular matrix graft

PURPOSE: To assess the response of rat urinary bladder regenerated by the homologous bladder acellular matrix graft (BAMG) to in vitro electrical and pharmacologic stimuli. MATERIALS AND METHODS: In Sprague-Dawley rats, partial cystectomy (>50%) was performed, followed by BAMG augmentation cystoplasty. After 4 months, organ bath studies of tissue strips in 10 were used to compare the contractility of the BAMG regenerates and the corresponding host detrusor smooth muscle. RESULTS: The BAMG regenerates exhibited contractile activity to electrical field stimulation and a qualitatively identical pattern of response to muscarinic, purinergic, alpha- and beta-adrenergic drug administration and nitric oxide. At 4 months after surgery, the maximum forces of contraction of the BAMG regenerates to carbachol stimulation amounted to close to 80% of the host bladder response. With electrical field stimulation, they equaled 44% and 62% of the host bladder response after 2.5 and 4 months, respectively. Histological and immunohistochemical studies confirmed the presence of receptors for neurotransmitters that these functional in vitro studies implied. CONCLUSIONS: The present study provides further evidence that augmentation cystoplasty with the BAMG leads to functional regeneration of the rat bladder detrusor smooth muscle.     

Artificial ureteral replacement for ureteral necrosis after renal transplantation: report of 3 cases

PURPOSE: We applied a new minimally invasive technique of artificial ureteral replacement for renal transplant ureteral necrosis. MATERIALS AND METHODS: Artificial ureteral replacement was performed in 3 renal transplant recipients with ureteral necrosis (complete in 1 and distal in 2) after failure of primary endoscopic treatment. Under fluoroscopic guidance a percutaneous tract is created and progressively dilated. The ureteral silicone polytetrafluoroethylene bonded tube is introduced into the pyelocaliceal renal graft cavities, tracked subcutaneously down to the suprapubic area and introduced into the bladder via a short incision. RESULTS: There were no immediate postoperative complications except for transient postoperative acute prostatitis in 1 patient. No secondary complications were observed with a mean followup of 2.5 years. All grafts have good late function and all tubes are patent with no evidence of encrustation or obstruction. The tubes are well tolerated underneath the skin. Reflux was present in all 3 cases with no clinical manifestation. An asymptomatic episode of lower urinary tract infection was observed in the female patient. CONCLUSIONS: In select cases of ureteral necrosis after renal transplantation artificial ureteral replacement by subcutaneous pyelovesical bypass offers a possible alternative to open ureteral reconstruction.     

Effects of radial wall motion and flow waveform on the wall shear rate distribution in the divergent vascular graft

Among the hemodynamic factors influencing intimal hyperplasia in the anastomotic region of a vascular graft, wall shear rate is believed to be one of the most important. We would like to study the effects radial wall motion on the wall shear rate distribution in the end-to-end anastomosis model of an artery and a divergent graft. Rigid and elastic models are constructed and the wall shear rate distributions are measured along the anastomosis using photochromic flow visualization method for carotid and femoral flow waveform. The mean and peak of shear rate decrease along the divergent graft, and the decreases are more significant in the elastic model. The shear rate waves are decomposed using the Fourier transform in order to separate the effects of radial wall motion and geometry. The percentage reductions of mean wall shear rates compared to steady shear rates at mean flow are calculated, and additional 8% (carotid) and 22% (femoral) reductions are observed in the elastic models near the end of the divergent graft. Also radial wall motion decreases the amplitudes of higher harmonics of wall shear rates in the elastic models. Since radial wall motion may affect the flow field differently for different geometry, wall elasticity should be considered in studying arterial hemodynamics.     

Free fibular graft still has a place in the treatment of bone defects

We report on 27 patients illustrating the use of non-vascularized single fibular strut graft, augmented with a corticocancellous bone graft to bridge bone defects. The indications were varied and included infection, fracture with bone loss, non-union, bone tumour, bone cyst and congenital pseudarthrosis. Primary union was achieved in 92 per cent. Stress fracture occurred in 26 per cent and no significant fibular graft hypertrophy occurred. The aim of this paper is to show that the non-vascularized single fibular graft, if augmented with corticocancellous bone graft along its whole length, is a simple procedure that is still valid to bridge bone defects.     

The biologic concept for the use of enamel matrix protein: true periodontal regeneration

Guided tissue regeneration procedures have been used successfully to reestablish periodontal attachment. However, this new attachment reportedly differs from the original attachment in strength and continuity. Enamel matrix proteins secreted by Hertwig's epithelial sheath play an important role in cementogenesis on roots and in the development of the periodontal attachment apparatus. Enamel matrix protein harvested from developing porcine teeth, or enamel matrix derivative (Emdogain), is reported to induce true periodontal regeneration (the attachment of new, acellular cementum to the underlying dentin surface). The results of experimental and clinical trials of Emdogain are reviewed, and the procedure for application of the material is described.     

Extracellular matrix remodeling at the early stages of liver regeneration in the rat

Previous studies have shown that activity of urokinase-type plasminogen activator (u-PA) increases very rapidly (within 1 minute) after partial hepatectomy. In view of the well-recognized roles of u-PA as one of the major initiators of the matrix proteolysis cascade and as an activator of plasminogen and hepatocyte growth factor (HGF), we studied matrix degradation in liver shortly after partial hepatectomy. The activation of plasminogen to plasmin following partial hepatectomy was examined by Western blot analysis, and a small increase in plasmin at approximately 15 minutes followed by a large elevation at approximately 3 to 6 hours after partial hepatectomy was detected. In addition, we found that fibrinogen, the major substrate for plasmin, begins to be degraded at approximately 15 to 30 minutes following partial hepatectomy. Using immunohistochemical staining, we detected that the distribution of fibrinogen in normal liver is localized to the perisinusoidal space surrounding the periportal region. A decreased distribution of fibrinogen in the periportal region was found by 15 minutes and continued through 24 hours following partial hepatectomy. In addition, the distribution of fibronectin in normal liver was localized to the perisinusoidal space surrounding the periportal and the pericentral regions. A strikingly decreased distribution of fibronectin in the periportal region was found at 5 minutes after partial hepatectomy. Furthermore, we observed that the protein levels of laminin, entactin, and fibronectin in an extracellular matrix (ECM)-enriched preparation decreased shortly after partial hepatectomy, and were restored later. No changes were observed with either vitronectin or the integrin chain alpha(v). In contrast to the protein levels of the ECM components, the messenger RNA (mRNA) levels of fibronectin, integrin chain beta1, and integrin chain alpha(v) gradually increased over 18 hours and then decreased thereafter. Taken together, these results suggest that rapid reorganization of selected ECM components are important for hepatocyte proliferation at the early stages of liver regeneration.     

Early events of liver regeneration in rats: a multiparametric analysis

CONCLUSION: Production of excited oxygen species is earlier in the liver than in the pancreas and could contribute to damage in a reflux model. Treatment with SOD could attenuate 59% light emission in pancreas, but did not modify serum enzyme levels or pancreatic edema, resulting as an insufficient isolated therapy. Unexpectedly, it was found an increased plasma antioxidant capacity that was related to total bilirubin levels, and declined at late stages probably denoting other circulating antioxidant consumption. BACKGROUND: Oxidative stress has been shown to play a role in different models of acute pancreatitis, although it has not been studied in the severe necrohemorrhagic model produced by closed duodenal loop pancreatitis. METHODS: We studied Sprague Dawley female rats in two groups: a closed duodenal loop pancreatitis group and a control, sham-operated group. In order to evidence the oxygen excited species production, in situ spontaneous chemiluminescence from living and naturally perfused pancreas and liver was measured at 0, 0.5, 1.5, 3, 6, 12, and 24 h after the duodenal ligature. Blood pancreatic amylase and aminotransferases levels were determined as expression of tissue damage in pancreas and liver. At the same time, plasma antioxidant capacity was measured by the peroxyl radical trapping capability of plasma samples compared to that of Trolox (synthetic analog of vitamin E), and results are expressed as Trolox equivalence. Bovine superoxide dismutase (SOD) was administered to attenuate oxygen free radicals activity at the beginning of the peroxidation chain and also as a therapeutic tool. RESULTS: The experimental procedure induced a severe pancreatitis, as evidenced by pancreatic enzymes that rose markedly in the early hours of disease and remained heightened throughout the experiment. The results show early light emission from the liver at 3 h and peak levels at 12 h, whereas in the pancreas, luminescence increased at 6 h and doubled later at 12 h, both returning to control levels at 24 h.     

A biomechanical study of replacement of the posterior cruciate ligament with a graft. Part II: Forces in the graft compared with forces in the intact ligament

To evaluate the allelic frequency and genetic diversity of alpha-thalassemia defects in Sicily, both epidemiological and patient-oriented studies were carried out. For the epidemiological study, phenotypic data were collected on more than 1000 Sicilian individuals. Among them, 427 were explored at the molecular level for nine alpha-thalassemic variants known to be common in the Mediterranean region. Our data reveal an allele frequency of 4.1% for alpha(+)-thalassemia matching that of beta-thalassemia in this region. The presence of alpha0-thalassemia (--MEDI and --CAL) was observed only in the group of referred patients. Newly acquired nucleotide sequence data on the deletional breakpoint of --CAL allowed us to design a simple PCR-based procedure for exploring this allele. The data also provide additional information concerning the genetic mechanisms involved in such large deletions.     

Glial cell extracellular matrix: boundaries for axon growth in development and regeneration

The validation of a simple quantitative clinical test of personal neglect is described in this study of 17 right brain damaged CVA patients with extrapersonal neglect, 14 without unilateral extrapersonal neglect, 13 left brain damaged CVA patients and 17 age-matched controls. The test had a high reliability and clearly differentiated neglect patients from all other groups. Furthermore the test identified a much higher incidence of personal neglect among extrapersonal neglect patients (59%) than has previously been found. Moreover this study confirms earlier findings by showing a double dissociation between personal and extrapersonal neglect. Seven patients with extrapersonal neglect showed no personal neglect while five patients showing no extrapersonal neglect did show personal neglect on this test.     

The role of connective tissue matrix in thermomechanical reactions of the aorta wall

A tomato peroxidase gene, TPX2, that is only weakly expressed in the roots of young tomato seedlings is highly expressed in tomato suspension cells adapted to high external NaCl concentration. The protein encoded by this gene, with an isolectric point value of approximately 9.6, is found in the culture medium of the growing cells. Our data suggest that the expression of TPX2 in the salt-adapted cells is not the result of the elicitation imposed by the in vitro culture or the presence of high NaCl concentration in the medium.     

A clinical evaluation of guided tissue regeneration with a bioabsorbable matrix membrane combined with an allograft bone graft. A series of case reports

THE PURPOSE OF THIS STUDY was to evaluate the clinical effectiveness of a surgical technique in treating periodontal defects. The technique combined tetracycline treatment of a root planed root, grafting of the osseous defect with a demineralized freeze-dried bone allograft combined with tetracycline and the placement of a bioabsorbable matrix membrane, made of polylactic acid softened with citric acid ester. Thirty defects were treated in 27 patients. Statistically significant changes, as a result of the surgical procedure, were observed in marginal recession (mean: 0.5 mm), probing depth reductions (mean: 5.7 mm), and attachment level gain (mean: 5.2 mm). No statistically significant difference existed between the results in the furcation and non-furcation groups. The defects with probing depths > or = 10 mm had a greater mean probing depth reduction (7.4 mm) and mean attachment level improvement (7.2 mm) than the defects with < 10 mm probing depths (probing depth reduction 4.5 mm and attachment level gain 3.9 mm). The proposed surgical procedure seemed to be an effective method to treat periodontal defects.     

Antibiotic-induced release of bacterial cell wall components in the pathogenesis of sepsis and septic shock: a review

This article reviews the new criteria for selecting the proper antimicrobial agent and dosage regimen for standard treatment of severe sepsis, with the intention of preventing septic shock. After introducing new concepts on the pathogenesis of sepsis and septic shock, the authors analyze the parameters of betalactam antibacterial activity, the antibiotic-induced release of bacterial endotoxin and the interrelationships between pharmacokinetics and pharmacodynamics of antibiotics in the search for an optimum dosage regimen of antimicrobial mono- or polytherapy for severely ill septic patients admitted to the intensive care unit.     

Migration of eosinophils through basement membrane components in vitro: role of matrix metalloproteinase-9

In general, inflammatory cells cross basement membranes by producing proteinases. To investigate the role of proteinases in eosinophil basement membrane migration, we studied peripheral blood eosinophils in Matrigel-coated chemotaxis chambers. Electron microscopy showed degradation of the Matrigel layer when eosinophils, added to the upper chamber, transmigrated the membrane in the presence of both platelet-activating factor (PAF) in the lower chamber and interleukin (IL)-5 in both chambers. In the absence of either or both PAF and IL-5, no changes occurred in the Matrigel layer. Matrigel transmigration of eosinophils induced by PAF and IL-5 was inhibited by 1,10-phenanthroline, batimastat, 3,4-dichloroisocoumarin, chymostatin, and a neutralizing antibody for the matrix metalloproteinase (MMP)-9, indicating that serine proteinase(s) and MMP, specifically MMP-9, were involved in the transmigration of eosinophils through Matrigel. In contrast, eosinophil migration through a bare membrane was not affected by batimastat. Using gelatin zymography and immunoblotting, MMP-9 was detected in the migration upper chamber supernatant of the eosinophil transmigration assay and in the conditioned medium of eosinophils. Release of MMP-9 by eosinophils was increased by IL-5, PAF, or both, but the substrate-degrading activity of MMP-9 was increased only in the presence of both IL-5 and PAF, indicating that the releasing and activating mechanisms of MMP-9 are involved in eosinophil basement membrane migration. This study implicates MMP-9 in basement membrane migration of eosinophils and suggests its involvement in inflammatory diseases where tissue eosinophilia plays a role.     

Stimulation of extracellular matrix components in the normal brain by invading glioma cells

Malignant gliomas are characterized by an extensive invasion of tumor cells into the normal brain parenchyma. A substantial amount of data indicates that cell movement in general is regulated by specific interactions between extracellular matrix components and specific cell-surface receptors. In the present work, multicellular spheroids from 4 human glioma cell lines (U-373Mg, A-172Mg, U-251Mg and HF-66) were confronted with normal rat brain cell aggregates in vitro, which resulted in a progressive invasion of tumor cells into the brain aggregates. The co-cultures were then sectioned and immuno-stained for specific extracellular matrix components (laminin, fibronectin and collagen type IV) and for specific cell-surface receptors which bind to these components (integrins beta1, beta4, alpha3, alpha6). In addition, flow-cytometric measurements and Northern blot analyses showed expression of several different integrins within the cell lines. The alpha3 subunit was expressed strongly in all cell lines. Whereas the beta1 subunit was expressed weakly in exponentially growing monolayer cultures, it showed a pronounced expression in multicellular spheroids, indicating that the integrin expression may vary depending on the micro-environment within a tumor. Furthermore, normal brain tissue was able to produce laminin when confronted with the glioma cells, which also was observed for fibronectin and collagen type IV. The relevance of our observations to the in vivo situation was investigated further by immuno-staining 5 human glioma biopsy samples for laminin. In some areas of the tumors, specific deposits of laminin were observed. In conclusion, we have shown that normal brain tissue has the ability to produce extracellular matrix components, such as laminin, collagen type IV and fibronectin, when confronted with invading glioma cells. Our results show that the glioma cells express specific integrins which can interact with these extracellular matrix components. Such interactions may facilitate tumor cell migration and invasion.     

Treatment of bacteria-biofilm graft infection by in situ replacement in normal and immune-deficient states

PURPOSE: Complications of grafts infected with coagulase-negative staphylococci can be eradicated by antibiotic administration, tissue debridement, and in situ graft replacement, but successful treatment may be diminished in a setting of altered immune function. METHODS: In a canine model of an established aortic graft infection from Staphylococcus epidermidis, outcomes after in situ replacement were compared between normal dogs and animals made immune-deficient by administration of azathioprine (50 mg/day) and prednisone (10 mg/day). In situ replacement of an infected infrarenal aortic graft with either antibiotic-bonded (silver-ciprofloxacin: Ag-cipro) or conventional polytetrafluoroethylene (PTFE) grafts was performed in 17 control and 18 immune-deficient animals. RESULTS: Four weeks after implantation of a Dacron graft colonized with a biofilm of S. epidermidis, all study animals demonstrated a bacterial biofilm infection with perigraft inflammation or abscess, and in immune-suppressed dogs the incidence of perianastomotic aortitis was increased (p < 0.05). Six weeks after in situ replacement both the Ag-cipro and conventional PTFE grafts were healed without signs of infection in controls, but anatomic evidence of persistent infection and increased S. epidermidis recovery was observed in immune-suppressed animals that underwent in situ replacement of a standard (five of seven) versus antibiotic-bonded (one of 11) PTFE graft (p < 0.006). Overall in situ replacement with an antibiotic-bonded graft yielded a lower frequency of S. epidermidis recovery (two of 19 Ag-cipro graft biofilm with positive culture results versus nine of 16 conventional graft biofilm with positive culture results; (p < 0.003). CONCLUSIONS: This study supports the efficacy of in situ replacement for low-grade graft infections caused by S. epidermidis in normal hosts and demonstrates superiority of antibiotic-bonded grafts in immune-deficient hosts.