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ABSTRACTMesenchymal cells (MSCs) are considered to be cellular populations of common embryological origin. For clinical research applications, MSCs are expanded and increased with cells obtained from a primary culture. By extracting cells from tissue and encouraging them to reproduce, the stem cell population ends up dominating the culture due to a high proliferation rate and self-renewal. The first subcultures between the third and sixth are chosen in order to obtain the maximum number of cells with optimal differentiation capacity. However, few studies have reported long-term cultivation of MSCs. The objective of this study was to advance the knowledge on the characteristics of MSCs by assessing their capacity for self-renewal and phenotypic maintenance beyond 50 cell subcultures, which is defined as the normal limit for cellular survival. Rat subcutaneous adipose tissue was the source of mesenchymal adipose stem cells (MASCs) cultured over 175 subcultures. Early 1 to 5 and late 25 to 30 subcultures were used to induce cellular differentiation to become adipogenic, chondrogenic and osteogenic connective tissue cells. MASCs characteristics were studied using flow cytometry, transmission electron microscopy (TEM), and immunohistochemical and reverse transcription polymerase chain reaction (RT-qPCR) assays. The MASCs maintained cell differentiation capacity for more than 30 subcultures but lost potentiality starting at 60 up to 175 subcultures. MASCs showed the embryonic phenotypes OCT3/4 and Nanog indefinitely, and developed compensatory mechanisms, such as autophagy, to achieve cell survival over a long time period. Therefore, long-term subcultures showed that MASCs could maintain their potential for clinical research use.

Wound healing is a complex process that can be severely impaired due to pathological situations such as diabetes mellitus. Diabetic foot ulcers are a common complication of this pathology and are characterized by an excessive inflammatory response. In this work, the effects of local treatment with recombinant human epidermal growth factor (rhEGF) were studied using a full-thickness wound healing model in streptozotocin-induced diabetic rats. Wound healing process was assessed with different concentrations of rhEGF (0.1, 0.5, 2.0 and 8.0 µg/mL), placebo and both diabetic and non-diabetic controls (n= 53). The macroscopic healing observed in treated diabetic rats was affected by rhEGF concentration. Histologically, we also observed an improvement in the epithelialization, granulation tissue formation and maturation in treated groups, finding again the best response at doses of 0.5 and 2.0 µg/mL. Afterwards, the tissue immune response over time was assessed in diabetic rats using the most effective concentrations of rhEGF (0.5 and 2.0 µg/mL), compared to controls. The presence of macrophages, CD4+T lymphocytes and CD8+T lymphocytes, in the reparative tissue was quantified, and cytokine expression was measured by quantitative real-time PCR. rhEGF treatment caused a reduction in the number of infiltrating macrophages in the healing tissue of diabetic, as well as diminished activation of these leukocytes. These findings show that local administration of rhEGF improves the healing process of excisional wounds and the quality of the neoformed tissue in a dose-dependent manner. Besides, this treatment reduces the local inflammation associated with diabetic healing, indicating immuno-modulatory properties.

Human umbilical cord Wharton's jelly stem cells (HWJSCs) are gaining attention as a possible clinical source of mesenchymal stem cells for cell therapy and tissue engineering due to their high accessibility, expansion potential, and plasticity. We employed a combination of highly sensitive techniques to determine the average cell viability levels and proliferation capabilities of 10 consecutive cell passages of cultured HWJSCs and then used RNA microarrays to identify genes associated with changes in cell viability levels. We found an initial decrease in cell viability from the first to the third cell passage followed by an increase until the sixth passage and a final decrease from the sixth to tenth cell passages. The highest cell viability levels corresponded to the fifth and sixth passages. The intracellular ionic contents of potassium, sodium, and chlorine suggest that the lower cell viability levels at passages 2, 3, and 8–10 may be associated with apoptotic cell death. In fact, gene expression analysis revealed that the average cell viability was significantly associated with genes with a function in apoptotic cell death, especially pro-apoptotic FASTKD2, BNIP3Lgenes and anti-apoptotic TNFAIP8and BCL2L2genes. This correlation with both pro-apoptotic and anti-apoptotic genes suggests that there may be a complex live-death equilibrium in cultured HWJSCs kept in culture for multiple cell passages. In this study, the highest cell viability levels corresponded to the fifth and sixth HWJSC passages, suggesting that these passages should be preferentially employed in cell therapy or tissue engineering protocols using this cell type.

This study was designed to evaluate the behavior of a new composite polypropylene-polyurethane (PL-PU99) when placed in direct contact with the visceral peritoneum during the repair of an abdominal wall defect. Full-thickness abdominal wall defects (7×5 cm) were created in 36 anaesthetized white New Zealand rabbits. The defects were repaired with polypropylene prostheses or PL-PU99 prostheses (comprised of PL and a polyurethane sheet glued to the PL with acrylic adhesive) to establish two study groups (n=18 each). Animals were sacrified 14, 30, or 90 days after implantation and prosthesis/surrounding tissue specimens were subjected to light and electron microscopy and morphometric analysis of the newly formed peritoneum. Immunohistochemical analysis was performed using the rabbit specific monoclonal antibody RAM-11. The biomechanical strength of the implants was also assessed. Firm adhesions were detected in the PL implants, whereas adhesions were practically non-existent in the PL-PU99 implants. The surface area covered by adhesions was greater (p<0.01) in the PL group (7.36 vs. 0.11 cm2). The neoperitoneum formed after the implantation of a PL prosthesis was disorganized in structure, whereas that formed at the interface with the PL-PU99 prosthesis was structurally similar to the host peritoneum. The excellent performance of the PL-PU99 prosthesis shown in this study warrants further investigation into its use for the repair of abdominal wall defects when the prosthetic patch needs to be placed in contact with the intestinal loops. Le but de cette étude a été d’évaluer le comportement biologique d’une nouvelle prothèse composite (PL-PU99) placée directement en contact avec les viscères lors de la réparation pariétale. Sous anesthésie, on a créé des pertes de substance complètes de la paroi abdominale (7×5 cm) chez 36 lapins blancs New Zealand. Les defects ont été réparés avec soit du polypropylène (PL) (n=18), soit la prothèse PL-PU99 (composée de PL et une feuille de polyuréthane collée sur la feuille de PL par une adhesive acrylique) (n=18). Les animaux ont été sacrifiés 14, 30 et 90 jours après l’insertion de la prothèse et des prélèvements tissulaires comprenant la prothèse et les tissus environnants ont été examinés en microscopie classique et électronique; le néopéritoine a été analysé morphométriquement. L’analyse immunohistochimique a été réalisée par des anticorps monoclonaux spécifiques du lapin RAM-11. La solidité bio-mécanique des implants a été évaluée. De fortes adhérences ont été détectées en ce qui concerne les plaques PL, alors qu’il n’y en avait pratiquement pas en cas de prothèses PL-PU99. La surface couverte par des adhérences était plus grande (p<0.01) dans le groupe PL (7.36 vs. 0.11 cm2). La structure du néopéritoine était désorganisée au niveau de la prothèse PL alors que celle à l’interface avec la prothèse PL-PU99 ainsi que l’étude de la fonction étaient similaires au péritoine natif. L’excellent comportement de la prothèse PL-PU99 démontré dans cette étude mérite d’autres investigations en ce qui concerne son utilisation pour la réparation des defects de la paroi abdominale lorsqu’il y a besoin de poser une prothèse au contact des anses intestinales. Se realiza este estudio para evaluar el comportamiento de una nueva prótesis mixta (PL-PU99) al colocarla en contacto directo con el peritoneo visceral durante la reparación de un defecto de la pared abdominal. Se realizaron, bajo anestesia, defectos de la totalidad de la pared abdominal de 7×5 cm en 36 conejos blancos de Nueva Zelanda. Los defectos se repararon con prótesis de polipropileno (PL) o de PL-PU99 (se trata de una prótesis mixta constituida por PL y una hoja de poliuretano pegada al PL por un adhesivo acrílico). Los animales se distribuyeron en dos grupos, cada uno de 18 conejos. Se sacrificaron a los 14, 30 ó 90 días tras la implantación; la prótesis y tejidos circundantes fueron estudiados mediante microscopía de luz y electrónica. Se analizó la morfometría del neoperitoneo formado. También se efectuaron análisis inmunohistoquímicos empleando el anticuerpo monoclonal RAM-11, específico del conejo. Así mismo se evaluó la resistencia biomecánica de los implantes. En los implantes de PL se detectaron firmes adherencia, mientras que éstas fueron prácticamente inexistentes en el grupo con prótesis PL-PU99. La superficie cubierta con adherencias fue mucho mayor (p<0.01) en el grupo PL (7.36 vs 0.11 cm2). El neoperitoneo formado tras el implante de PL aparecía estructuralmente desorganizado mientras que, el formado en contacto con la superficie de la prótesis PL-PU99 era estructural y funcionalmente semejante al peritoneo del huésped. El excelente comportamiento demostrado en este estudio de la prótesis PL-PU99 justifica nuevas investigaciones por lo que a su empleo en el tratamiento de los defectos de la pared abdominal se refiere, sobre todo cuando el implante debe colocarse en contacto con las asas intestinales.

This study was designed to evaluate the behavior of a new composite polypropylene-polyurethane (PL-PU99) when placed in direct contact with the visceral peritoneum during the repair of an abdominal wall defect. Full-thickness abdominal wall defects (7×5 cm) were created in 36 anaesthetized white New Zealand rabbits. The defects were repaired with polypropylene prostheses or PL-PU99 prostheses (comprised of PL and a polyurethane sheet glued to the PL with acrylic adhesive) to establish two study groups (n=18 each). Animals were sacrified 14, 30, or 90 days after implantation and prosthesis/surrounding tissue specimens were subjected to light and electron microscopy and morphometric analysis of the newly formed peritoneum. Immunohistochemical analysis was performed using the rabbit specific monoclonal antibody RAM-11. The biomechanical strength of the implants was also assessed. Firm adhesions were detected in the PL implants, whereas adhesions were practically non-existent in the PL-PU99 implants. The surface area covered by adhesions was greater (p<0.01) in the PL group (7.36 vs. 0.11 cm2). The neoperitoneum formed after the implantation of a PL prosthesis was disorganized in structure, whereas that formed at the interface with the PL-PU99 prosthesis was structurally similar to the host peritoneum. The excellent performance of the PL-PU99 prosthesis shown in this study warrants further investigation into its use for the repair of abdominal wall defects when the prosthetic patch needs to be placed in contact with the intestinal loops.

Prosthetic materials currently used to repair abdominal wall defects occasionally must be placed in direct contact with the visceral peritoneum. The prosthesis–peritoneum interface is the site of several possible problems, including the formation of adhesions and erosion of the intestinal loops, which may lead to the formation of fistulas. This investigation was designed to compare the behavior of two prosthetic biomaterials in composite form at the level of the peritoneum. Defects (7 × 5 cm) were created in the abdominal wall of 18 white New Zealand rabbits weighing approximately 2500 g. The defects (involving aponeurotic and muscular planes and the parietal peritoneum) were repaired with polypropylene (PL) + ePTFE (Preclude dura substitute) or Parietex composite (PC) prostheses. The prostheses were secured to the edges of the defect by continuous PL sutures interrupted at the corners of the implant. Three study groups were established according to the type of implant: group I (n= 6) (controls)—PL; group II (n= 6)—PL + ePTFE; and group III (n= 6)—PC. The animals were sacrificed 14 days after implant, and the prostheses were examined by light microscopy and scanning electron microscopy (SEM). The formation of adhesions at the prosthesis–visceral peritoneum interface were quantified according to a protocol previously described by us. The biomechanical resistance of the implant was evaluated using strips comprising prosthetic material and anchorage tissue. The Mann-Whitney U-test was used to compare data corresponding to each group. There was no postimplant mortality. No infection or rejection of the prosthesis was observed in any of the animals. Firm adhesions were detected in the PL implants, whereas in the PL + ePTFE and PC implants the adhesions were loose. The mean prosthetic surface areas covered by adhesions were 7.67, 0.10 and 0.19 cm2for groups I, II, and III, respectively, showing a significant difference between values corresponding to groups I and II and to groups I and III (p< 0.05). Comparison of values recorded for groups II and III yielded no significant difference (p> 0.05). In groups II and III, the neoperitoneum was homogeneous and composed of organized and vascularized connective tissue covered by a mesoendothelium that was interrupted by accumulations of fibroblasts and white blood cells. In contrast, a disorganized neoperitoneum of rough texture was observed in the group I specimens. At times, areas of hemorrhage and necrosis corresponding to the sites of adhesion formation could be observed. Resistance to traction of composite implants (mean ± SD: 15.72 ± 1.32 and 15.89 ± 2.73) was similar to that of the PL implants (15.03 ± 2.92) (Mann-Whitney U-test, p< 0.05). It may be concluded that (1) composite prostheses show optimum behavior in terms of adhesion formation at the prosthesis–visceral peritoneum interface; (2) the neoperitoneum formed after the implant of a composite prosthesis almost physically and functionally replaces the normal peritoneum; (3) a significantly greater degree of peritoneal regeneration is achieved after implant of a PC prosthesis; and (4) there was no significant difference regarding biomechanical resistance between PL prostheses and PL + ePTFE and Parietex composites.

Prosthetic materials currently used to repair abdominal wall defects occasionally must be placed in direct contact with the visceral peritoneum. The prosthesis–peritoneum interface is the site of several possible problems, including the formation of adhesions and erosion of the intestinal loops, which may lead to the formation of fistulas. This investigation was designed to compare the behavior of two prosthetic biomaterials in composite form at the level of the peritoneum. Defects (7 × 5 cm) were created in the abdominal wall of 18 white New Zealand rabbits weighing approximately 2500 g. The defects (involving aponeurotic and muscular planes and the parietal peritoneum) were repaired with polypropylene (PL) + ePTFE (Preclude dura substitute) or Parietex composite (PC) prostheses. The prostheses were secured to the edges of the defect by continuous PL sutures interrupted at the corners of the implant. Three study groups were established according to the type of implant: group I (n= 6) (controls)—PL; group II (n= 6)—PL + ePTFE; and group III (n= 6)—PC. The animals were sacrificed 14 days after implant, and the prostheses were examined by light microscopy and scanning electron microscopy (SEM). The formation of adhesions at the prosthesis–visceral peritoneum interface were quantified according to a protocol previously described by us. The biomechanical resistance of the implant was evaluated using strips comprising prosthetic material and anchorage tissue. The Mann-Whitney U-test was used to compare data corresponding to each group. There was no postimplant mortality. No infection or rejection of the prosthesis was observed in any of the animals. Firm adhesions were detected in the PL implants, whereas in the PL + ePTFE and PC implants the adhesions were loose. The mean prosthetic surface areas covered by adhesions were 7.67, 0.10 and 0.19 cm2for groups I, II, and III, respectively, showing a significant difference between values corresponding to groups I and II and to groups I and III (p< 0.05). Comparison of values recorded for groups II and III yielded no significant difference (p> 0.05). In groups II and III, the neoperitoneum was homogeneous and composed of organized and vascularized connective tissue covered by a mesoendothelium that was interrupted by accumulations of fibroblasts and white blood cells. In contrast, a disorganized neoperitoneum of rough texture was observed in the group I specimens. At times, areas of hemorrhage and necrosis corresponding to the sites of adhesion formation could be observed. Resistance to traction of composite implants (mean ± SD: 15.72 ± 1.32 and 15.89 ± 2.73) was similar to that of the PL implants (15.03 ± 2.92) (Mann-Whitney U-test, p< 0.05). It may be concluded that (1) composite prostheses show optimum behavior in terms of adhesion formation at the prosthesis–visceral peritoneum interface; (2) the neoperitoneum formed after the implant of a composite prosthesis almost physically and functionally replaces the normal peritoneum; (3) a significantly greater degree of peritoneal regeneration is achieved after implant of a PC prosthesis; and (4) there was no significant difference regarding biomechanical resistance between PL prostheses and PL + ePTFE and Parietex composites.

Objectives Chronic venous disease (CVeD) is a multifactorial and debilitating condition that has a high prevalence in Western countries and an important associated socioeconomic burden. Varicose veins (VVs) are the most common manifestations of CVeD. Pathologically, many morphological and functional changes have been described in VVs, which most notably affect venous wall integrity. Previous studies have found several molecular alterations that negatively affect normal cell signaling pathways. Insulin receptor substrate (IRS)-4 is a central adaptor protein that is closely related to insulin/insulin-like growth factor-1 signaling upstream, phosphatidylinositol 3-kinase/Akt or mitogen-activated protein kinases downstream, and other proteins. These molecular pathways have been implicated in CVeD pathogenesis. Thus, the aim of our study was to identify the role of IRS-4 in VV tissue.Methods We conducted a histopathological study to analyze IRS-4 protein expression in CVeD patients compared with healthy controls.Results Our results demonstrate a significant increase in IRS-4 expression in VV tissue.Conclusions IRS-4 may be implicated in CVeD development and progression. Therefore, IRS-4 could be a potential diagnostic or therapeutic target for patients with this condition.

An investigation was made into the effect of blood flow on endothelial cells (EC) and mesothelial cells (MC) seeded on a vascular expanded polytetrafluoroethylene (ePTFE) prosthesis coated with a fibroblastic matrix. Endothelial cells were obtained from the external jugular vein and MC from the omentum. To test the performance of prostheses, a custom designed, femoral “ex vivo”circuit was developed in mongrel dogs. Four study groups were established: a control group, A1, where prostheses were uncoated and seeded with EC; a second control group, A2, where prostheses were uncoated and seeded with MC; group B1where prostheses were coated with a fibroblastic matrix and seeded with EC; and group B2where coated prostheses were seeded with MC. All cells were labeled with 111Indium oxine (10 µCi/mL) before seeding. After the seeded cells had formed a monolayer on the ePTFE prostheses (which took approximately 24 h) the prostheses were placed in the “ex vivo”circuit. The rates of blood flow to which prostheses were exposed were measured at the point of inflow (117.5 ± 12.50 mL/min, mean ± SD) and outflow (72.6 ± 14.3 mL/min). MC showed a greater baseline radionuclide uptake than did EC. The cells of groups B1and B2adhered sufficiently to the fibroblastic matrix and covered enough of the prosthetic surface to be positioned in the “ex vivo”circuit (76.90 ± 8.24% surface covered in EC-seeded prostheses and 71.65 ± 6.23% in MC-seeded prostheses). After exposure to blood flow the quantity of radionuclide-labeled cells and the prosthetic surface covered by them were greatly reduced though the fibroblast-coated prostheses showed greater cell retention. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 39, 32–39, 1998.

Pharmacologic modulation by an inhibitor of angiotensin-converting enzyme (IACE: cilazapril) of vascular proliferative response to a full-thickness arterial injury (autograft) was studied in rats. An arterial autograft 5 mm long was made in the right common iliac artery of 50 female Sprague-Dawley rats (weight 250-300 g) by micro-surgical techniques. The animals were divided into two study groups: group I (controls), 20 animals that underwent arterial autograft but received no other treatment; and group II (cilazapril-treated), 20 rats that underwent arterial autograft and received cilazapril (Roche), 10 mg/day orally (p.o.) in an excipient of 2% arabic gum, for 4 days before operation. Animals were killed on postoperative days 7, 14, 21, 30, and 50, and grafts were studied by light microscopy, scanning and transmission electron microscopy, and morphometry. In the control group, the hyperplasic response had begun by postoperative day 14 and was established by postoperative day 50. In the medial layer, the muscle cells changed in the phenotype from contractile to secretory cells. The adventitia had a highly proliferative appearance. In the cilazapril-treated group, fibrin deposits and platelets formed a layer on the internal elastic lamina. This layer appeared to evolve toward an intimal hyperplasia that became quantifiable by postoperative day 21. The medial layer was clearly thinned and showed intense accumulation of lipid microvacuoles, elastic degeneration, and vacuolized cells. Our results suggest that the use of an inhibitor of ACE modified the origin of the intimal hyperplasia in the arterial autograft model. Enhancement of the thrombogenicity of the luminal surface favors myointimal development by thrombus reorganization.