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Introducción: La proteína relacionada con la parthormona (PTHrP), un factor abundante en el tejido óseo, es un importante modulador de la formación ósea. Se ha demostrado que la PTHrP (107-111), conocida como osteostatina, cargada en el material cerámico mesoporoso SBA-15, ejerce acciones osteogénicas in vitro. Objetivo: Comprobar si dicho material y una versión funcionalizada del mismo (C8-SBA-15) promueven la reparación ósea en un modelo de defecto cavitario en el fémur del conejo. Material y métodos: Para ello se han llevado a cabo estudios histológicos e inmunohistoquímicos y de microtomografía computerizada (µCT). Resultados: Tras la implantación de los biomateriales no se observaron niveles significativos de inflamación ni resorción ósea (4 y 8 semanas). A las 8 semanas las biocerámicas no cargadas con la osteostatina se encontraban separadas de la médula ósea por una cápsula fibrosa, que disminuía de forma significativa en presencia del péptido. Se observó (por µCT) un aumento de la neo-formación ósea a diferentes distancias de los biomateriales, principalmente en los cargados con la osteostatina. Además, estos resultados fueron confirmados por inmunohistoquímica de marcadores osteoblásticos. Conclusión: Nuestros hallazgos sugieren que estas biocerámicas cargadas con la osteostatina son una buena estrategia para acelerar la regeneración ósea.

The foam properties of base wines made from red autochthonous varieties (Trepat and Monastrell) were studied. Four wines of each variety were elaborated (fermented off skins at industrial scale in two consecutive harvests) and blended at different proportions with the white traditional variety (Macabeo, Xarel.lo, and Parellada) wines to elaborate Cava (closed-bottle-fermented sparkling wine). When crescent amounts of Trepat were added to the traditional white blend, the foamability and the color intensity (CI) of the wine increased polinomically. The increase of the CI depended on the year of harvest. Thus, oenologists could decide the blend proportion most suitable to elaborate either a “blanc de noirs” sparkling wine or a new type of Cava. Keywords: Trepat; Monastrell; foam; sparkling wine; Cava; red variety; color intensity

White grapes of three different varieties (Macabeo, Xarel.lo and Parellada), which are used in Spanish sparkling wines production (O.A.C. Cava), were treated with a pectic-enzyme preparation on an industrial scale. Foam capacity (foamability (HM), Bickerman coefficient (Σ), foam stability time (TS) and surface tension (ST)) of nine musts from pectic enzyme treated grapes and their corresponding wines were compared to their respective control samples. The HM, Σ, and TS values decreased and ST increased in most of the treated samples. This could be the case, because samples from enzyme-treated grapes had lower protein and polysaccharide contents and higher values of polyphenols than untreated samples: HM and Σ were positively correlated with proteins and polysaccharides and negatively with ST; polyphenol contents had a negative relationship with TS values.

BACKGROUND: Elastin expression is higher in tissues where elastic fibres are essential for the correct maintenance of function such as blood vessels. Elastin expression usually diminishes with age, however, it may be re-expressed in response to injury or repair processes. Some authors attribute the characteristic loss of elasticity of the varicose vein to a drop in the population of smooth muscle cells in the media layer. A reduction in elastin has been observed in chronic venous insufficiency, but little is known about some of the factors involved in elastin synthesis such as lysyl oxidases. The aim of this study was to examine the in vivo expression of the elastin precursor, tropoelastin (TE), and lysyl oxidase-like 1 (LOXL1), a cross-linking enzyme responsible for elastin polymer deposition. The effects of age on these expression patterns were also evaluated. METHODS: Saphenous vein segments were obtained during surgery from organ donors (controls, n=20) and subjects with venous insufficiency (varicose veins, n=20). Both these groups were subdivided according to subject age into or=50 years (n=10). Control and varicose vein tissue specimens were immunolabelled using anti-tropoelastin and anti-LOXL1 antibodies and also subjected to Western blot analysis. RESULTS: Our results indicate that the levels of these markers of elastin synthesis (LOXL/tropoelastin) in the vein wall diminish in a significant way (por=50 years (n=10). Control and varicose vein tissue specimens were immunolabelled using anti-tropoelastin and anti-LOXL1 antibodies and also subjected to Western blot analysis. RESULTS: Our results indicate that the levels of these markers of elastin synthesis (LOXL/tropoelastin) in the vein wall diminish in a significant way (p

This study explores the possibility of a regulatory role for cytokine IL-10 in platelet aggregation as an active vascular repair mechanism. Endothelial cells from human umbilical cord vein were cultured in the presence of different IL-10 concentrations (0-100 ng/ml). Platelet-rich plasma was then added to these cultures and allowed to act for 30 minutes. To rule out blood plasma involvement, washed platelets were also incubated with IL-10 (0-100 ng/ml). Changes in endothelial cell morphology were observed depending on the IL-10 concentration used; apoptotic cells appearing at the highest IL-10 concentration. Greatest platelet adhesion was noted at the highest IL-10 concentration. It was concluded that, in this in vitro model, low IL-10 levels do not affect cell viability or the pattern of platelet adhesion, but at high doses, this cytokine induces cell death and enhances platelet deposition.

The occurrence of intimal hyperplasia after vascular surgery is an ongoing concern in current clinical practice. Arnong the many factors involved in the development of this pathology, platelet adhesion and myointimal proliferation play a major role. Both these processes are mediated by integrins (mainly avP3 integrins). Over the past years, severa1 substances have been designed to delay or inhibit the cell proliferation that leads to hyperplasia and mainly include monoclonal antibodies directed against integrins. The aim of the present study was to evaluate the effects of an antibody denoted P37 (anti B3 integrin) on human smooth muscle cells (SMC) and its role in blocking the 83 subunit. To this end, SMC from human umbilical artery were cultured in the presence or absence of the cell substrate vitronectin (VN) and incubated with P37. After 4 days of treatment, determination was made of cell proliferation and migration. Smooth muscle cells grown on VN showed increased proliferation and migration compared to control VN-free cultures. However, the presence of P37 in the culture medium inhibited proliferation and reduced migration. Combined treatment with VN and P37 led to improved proliferation but VN was unable to reverse the effects on migration observed in the former cultures. Results suggest that in vitro, P37 is capable of blocking human SMC 83 integrins and thus impedes cell proliferation and migration These findings may have clinical implications related to modulation of the development of hyperplasia.

The role of Sandimmun Neoral® (S-n) and the immune response in transplant-associated coronary vasculopathy (TACV) was evaluated in a Lewis (Lew)- to-Fischer-344 (F344) rat abdominal heterotopic heart transplant model. Some of the transplant recipients were treated with S-n (5mg/ kg/ day) for 14 days posttransplant, or until sacrifice. Grafts were subjected to immunohistochemical (ED1, CD4, CD8 and a-actin+ cells) anal ysis from day 7 to 100 post-transplant. Singenic controls did not develop TACV, irrespective of whether they had received the drug or not. TACV was detected in Lew-F344 transplants regardless of S-n administration with participation of ED1+, CD8+ and aactin+ cells, although its incidence was lower in animals receiving prolonged S-n treatment. In this model, accelerated arteriosclerosis of the graft appeared to be related more to the rejection effect than to the action of the immunosuppressive agent.

This study was designed to evaluate the role of the smooth muscle cell and the apoptosis in the pathogenesis of the varicose vein. Segments of saphenous vein were obtained from healthy subjects and from those with varicose veins. The vein specimens were subdivided according to subject age (younger or older than 50 years) and according to the varicose vein source (distal or proximal). Morphological, ultrastructural, cell proliferation (anti- PCNA method) and cell death (TUNEL method) analysis were performed. The walls of healthy, control vein specimens acquired a more collagenous and papilomatous appearance with age. A slight increase in the number of TUNEL-positive cells was also observed in specimens from older subjects. The proportion of apoptotic cells was much greater in the varicose veins than in control specimens. Most cellular alterations were seen in proximal varicose segments obtained from young subjects. These specimens showed hypertrophic areas with a high degree of cellularity (both in the media and in the thickened intima). The highest proportion of apoptotic cells and collagenisation were also observed in these areas. The enhanced number of apoptotic cells in varicose veins observed mainly in proximal/young vein specimens could be responsible, at least in part, for the acceleration of the final fibrosclerotic process characteristic of the varicose vein wall.

Cytotoxic T cells may induce myocardial apoptosis by histiocyte activation during rejection following allogenic heart transplant. The aim of the present investigation was to evaluate the macrophage response and its relationship to the programmed death of cardiomyocytes in rejection and during cyclosporin-A (CsA) treatment. An abdominal, heterotopic heart transplant rat model was used establishing two groups: singenic (ST) and allogenic QAL 'tran splant. 5mglkglday (s.c.) CsA (Sandimun ) was administered to half of the animals in each group. Morphological and structural analysis was performed 7, 14,21,30,50 and 100 days post-transplant. Macrophages were detected using the monoclonal antibody (ED1). The TUNEL method was used to visualise apoptotic cells. Two weeks after ST in animals without immunosuppressive treatment, the transplanted myocardium had been extensively infiltrated by inflammatory cells, many of which were ED1-positive. At 21 days follow-up, the number of labelled cells had fallen. In animals treated with CsA the amount of ED1-positive cells was lower than that seen in the anterior group. Only a few isolated cells of the infiltrate were TUNEL-positive. In the AT group, rejection took place between 9-15 days in the untreated animals. The myocardium was highly infiltrated by mononuclear cells. Some were ED1- positive. Small groups of apoptotic cells were visible in the infiltrate and in some vessel lumens. Rejection was resolved in animals treated with CsA. The macrophage response diminished during follow-up in a similar way to that occurring in the ST. Few cells showed TUNEL positivity. It may be concluded that: a) CsA treatment diminishes the amount of infiltrated macrophages; b) animals receiving ST or AT, show a low level of Offprint requests to: Prof. Julia BujAn, PhD., Department of Morphological Sciences and Surgery, Faculty of Medicine, University of AlcalA de Henares, Crta. Madrid-Barcelona Km. 33,600. 28871- AlcalA de Henares (Madrid). Spain. Fax: 34.91885-48-85. e-mail: cmmjb@ cirug.alcala.es apoptosis; c) in the present model, the apoptosis of cardiomyocytes does not appear to be induced by macrophages; and d) in this model it is not possible to relate apoptosis and rejection.

The present study investigates the effects on the cardiac muscle cell of two of the determining factors for the success of organ transplant; ischaemia-perfusion and immunosuppressive treatment with cyclosporin-A (CsA). To this end an abdominal, heterotopic heart transplant model in singenic Sprague-Dawley rats was employed. Three study groups were established: Group I (control, n=15) animals undergoing heart transplant without treatment; Group I1 (n=15) animals undergoing heart transplant and subjected to a daily dose of CsA in a cremophor vehicle (sandimunB) (5 mg/kg/sc); Group I11 (n=15): animals undergoing heart transplant and administered a daily dose of pure CsA (5 mg/kg/sc). Recipient animals were sacrificed 7, 14, 21, 30 and 50 days after transplant. During the post-operative period, heart function was assessed by daily abdominal palpation. Graft specimens obtained at each follow-up period were subjected to light and transmission electron microscopy. Immunohistochemical analysis of specimens was performed using the rat macrophagespecific monoclonal antibody MCA-341. The ischaemia/reperfusion process induced considerable alteration to cardiac muscle cells of control animals. Effects, apparent after the first week of transplant, included mitochondrial swelling and loss of cristae, hypertrophy of the sarcoplasmic reticulum and structural changes to sarcomeres. Two weeks after transplant, the myocardium was infiltrated by inflammatory cells. These effects diminished 30 days post-transplant. Cardiac tissues of treated animals (groups I1 and 111) showed similar behaviour although, in the latter group, mitochondrial damage was greater and intense myocardial fibrosis took place. Infiltration of cardiac muscle by white blood cells did not take place until 3 weeks post-implant. These results indicate: a) The ultrastructural changes detected in cardiac fibres of animals of the three study groups were attributable to the ischaemialreperfusion process rather than to treatment with CsA; b) CsA appears to augment mitochondrial damage and myocardial fibrosis; c) the inflammatory response was delayed and reduced by the immunosupressant; and d) the cremophor administration vehicle did not seem to exert an independent toxic effect on the myocardium.

The possibility of resuscitating livers after warm ischaemia has been recently suggested. The aim of the present investigation was to analyse the effects of anoxia on the morphology of hepatic cells, to determine whether these effects are reversible after providing a resuscitation period between warm ischaemia (WI) and cooling, and to study the behaviour of the resuscitated liver in the recipient organism. Ten female, Large-White pigs acted as donors for 10 recipient animals of the same kind who received an orthotopic liver graft. Recipients were divided into two groups depending on whether the livers they received had undergone a resuscitation period (Group 1 (n=5) where animal livers were subjected to 5 min warm ischaemia (WI) without resuscitation, and Group 11 (n=5) where the livers were subjected to 5 min WI followed by 5 min resuscitation). Morphological and ultrastructural studies of liver cells were performed using light and electron microscopy. ATP, ADP and AMP levels were determined in liver biopsies by high performance liquid chromatography (HPLC). Plasma AST and bilirubin levels in the two groups were compared 24 h after transplantation. After 5 min of anoxia, hepatocytes showed two morphological pattems in response to WI. Some were appreciably condensed with dark mitochondria, peroxisomes and some cytoplasmic vacuoles. Others showed electronlucent organelles, inflamed mitochondria with broken cristae and disorganized endoplasmic reticulum. Hepatocytes showed globular microvilli and bleb formation with migration towards the sinusoids. One hour after the revascularisation of the resuscitated livers, the hepatocytes showed nearly normal morphological characteristics. However, the hepatocytes of nonresuscitated organs continued to show alterations. Kupffer cells were activated in the livers of both experimental groups. Ultrastructural changes and total tissue adenine nucleotide (TAN) levels recovered completely in resuscitated livers soon after transplant. These results suggest that when short WI periods are followed by equivalent periods of resuscitation, the hepatocytes of transplanted livers recover from the effects of anoxia.

Currently one of the most widely used prosthetic materials in the repair of abdominal wall defects, is expanded polytetrafluoroethylene (ePTFE). It has been suggested that its behavior with respect to the reparative process may depend on its structure. The aim of the present study was to evaluate the effect of the structure of 3 ePTFE prostheses on the scarring process in an abdominal-wall-defect experimental model. The prostheses employed were the Soft Tissue Patch (STP) which is laminar in structure, Mycro Mesh (MM) which is multilaminar with perforations, and the Dual Mesh (DM) prosthesis which has one non-porous surface. Abdominal wall defects (7x5cm) were created in 36 New Zealand rabbits and repaired using fragments of STP, MM and DM. Follow-up periods were 14, 30, 60 and 90 days post-implant. At these times prostheses were macroscopically examined for the presence of infection andlor rejection and the formation of adhesians to abdominal viscera. Specimens were also taken for microscopic analysis (optical and scanning electron) and for immunohistochemical analysis using the rabbit macrophage-specific monoclonal antibody RAM-11. Labelled macrophage counts were performed at each follow-up session. No cases of infection or rejection were found. Loose adhesions between prosthesis and underlying viscera were observed in 2 of the STP, 4 of the MM and 2 of the DM implants. STP and DM implants were progressively encapsulated by organized connective tissue on both peritoneal and subcutaneous surfaces. Cellular colonization was observed on both STP surfaces and on the porous surface of the DM although no more than a third of the biomaterial was penetrated by cells in either case. Colonization was very slight at prosthesis anchorage points. MM implants differed only in the formation of connective tissue bridges in perforated areas, and cellular infiltration in interlaminar spaces. Macrophage response was similar in Offprint requests 10: Prof. J. Buján, Department of Morphological Sciences and Surgery, Faculty of Medicine, University of Alcalá de Henares, Crta. Madrid-Barcelona, km 33.600, 28871 -Alcalá de Henares, Madrid, Spain the 3 prostheses with a reduction in RAM-11 labelled cells (p

A combination of vascular pathologies and other complicating factors results in chronic wounds which constitute a serious burden for both patients and national health systems, due to prolonged hospital stays, high costs, and prolonged nursing staff dedication. Here we investigate whether proadrenomedullin N-terminal 20 peptide (PAMP), a naturally occurring peptide of the skin with antimicrobial and proangiogenic properties, either alone or in combination with autologous skeletal muscle stem/progenitor cells, acts as a wound healing factor. The rabbit ear was chosen as a test system, since it offers a reliable model for normoxic and ischemic wounds. Topical treatments with PAMP, stem/progenitor cells, or a combination of both, resulted in significant improvements of healing, when compared to untreated wounds. PAMP was very effective in promoting reepithelialization and angiogenesis, whereas treatment with stem/progenitor cells alone resulted in less wound contraction. Interestingly, the combination of PAMP and stem/progenitor cells, while maintaining angiogenic potency, reverted to the contraction levels observed in the untreated controls. Under ischemic conditions, generalized necrosis of the dermis and the underlying cartilage was observed in untreated wounds. Treatments of these wounds with PAMP or stem/progenitor cells allowed a timely recovery. In conclusion, PAMP either alone or in combination with autologous stem/progenitor cells may provide a useful tool for improving wound healing both in normoxic and ischemic conditions.

This paper describes a polymer site-specific delivery system containing human growth hormone in an in vivo model of scarring in the diabetic state. Copolymer discs with the hormone were introduced into incisions made in rats previously injected with streptozotocin in order to induce diabetes. Tissue specimens for evaluation were obtained at 3, 7 or 10 days after the procedure. Study groups were healthy rats and diabetic rats untreated or treated with/without the hormone. Histological sections were prepared for light microscopy examination of wound zones. Three and 7 days after surgery, polymer remains could be observed in the subcutaneous tissue. These remnants induced a moderate foreign body reaction. The number of macrophages detected was directly related to neovessel formation and metalloelastase expression. The CD4+/CD8+ ratio was low during the initial follow up stages (3 and 7 days) in untreated diabetic rats, yet an increased ratio corresponding to areas around the polymer remains was noted in the animals treated with copolymer loaded with the growth hormone. Copolymer is biodegradable in vivo and may be used as a vehicle for the slow release of active substances. The presence of the hormone at the site of skin injury induces cell proliferation and enhances the repair process.

Introduction. The viability and immunological response induced by cryopreserved arterial allografts remain unclear. This study examines the post-graft behaviour of this type of vessel substitute. Materials and methods. Both iliac arteries were extracted from Lewis rats (donors) and used to establish groups of allogeneic fresh (group I) or cryopreserved (group II) grafts in Fisher-344 rats (recipients). Cryopreserved segments for grafting were prepared by automated controlled freezing at a cooling rate of 1°C/min followed by storage in liquid nitrogen vapour at -145°C for 30 days. Before grafting, the vessels were slowly thawed. Animals were sacrificed at 14, 30, 90 and 180 days post-surgery when graft specimens were obtained for light and electron microscopy and immunohistochemical detection of inflammatory cells (CD45, ED1, CD4, CD8). Results. After surgery, 85.71% of the grafts in group I and 82.14% in group II were patent. Following long-term implant, both the fresh and cryopreserved allografts showed complete loss of the muscle compartment of the media. Inflammatory or CD45-positive cells (mainly macrophages and CD8 T-lymphocytes) were detected at earlier time points in suture zones and adventitia. In the fresh allografts, the number of immunolabelled cells steadily increased until they were seen to occupy the entire adventitia at 90 days, with high numbers persisting at 6 months. In the cryopreserved allografts, this adventitial inflammatory infiltrate was significantly reduced. Conclusions. The cryopreservation/slow thawing protocol used diminished the immune response induced by fresh arterial allografts improving their behaviour after grafting.

Cytotoxic T cells may induce myocardial apoptosis by histiocyte activation during rejection following allogenic heart transplant. The aim of the present investigation was to evaluate the macrophage response and its relationship to the programmed death of cardiomyocytes in rejection and during cyclosporin-A (CsA) treatment. An abdominal, heterotopic heart transplant rat model was used establishing two groups: singenic (ST) and allogenic (AL) transplant. 5 mg/kg/day (s.c.) CsA (Sandimun) was administered to half of the animals in each group. Morphological and structural analysis was performed 7, 14, 21, 30, 50 and 100 days post-transplant. Macrophages were detected using the monoclonal antibody (ED1). The TUNEL method was used to visualise apoptotic cells. Two weeks after ST in animals without immuno-suppressive treatment, the transplanted myocardium had been extensively infiltrated by inflammatory cells, many of which were ED1-positive. At 21 days follow-up, the number of labelled cells had fallen. In animals treated with CsA the amount of ED1-positive cells was lower than that seen in the anterior group. Only a few isolated cells of the infiltrate were TUNEL-positive. In the AT group, rejection took place between 9-15 days in the untreated animals. The myocardium was highly infiltrated by mononuclear cells. Some were ED1-positive. Small groups of apoptotic cells were visible in the infiltrate and in some vessel lumens. Rejection was resolved in animals treated with CsA. The macrophage response diminished during follow-up in a similar way to that occurring in the ST. Few cells showed TUNEL positivity. It may be concluded that: a) CsA treatment diminishes the amount of infiltrated macrophages; b) animals receiving ST or AT, show a low level of apoptosis; c) in the present model, the apoptosis of cardiomyocytes does not appear to be induced by macrophages; and d) in this model it is not possible to relate apoptosis and rejection.

Subendothelial invasion by leukocytes is a sign of intimal thickening in arteriosclerosis and in the response of a vessel to mechanical damage. Our study was designed to establish whether these cells are implicated in the formation of a neointima in an autologous arterial graft model in the rat and to evaluate the effects of cyclosporin A (CsA). Three study groups were established according to whether the animals were treated with CsA-Cp (Sandimmun)®, CsA-Et (ethanol vehicle) or received no treatment (control group). Both drug forms were administered (5 mg/kg/day, s.c.) from 4 days prior to surgery until the time of sacrifice. Antibodies specific for lymphocytes (CD4, CD8), monocytes/macrophages-ED1, smooth muscle a-actin and the von Willebrand factor (vWF) were used to identify the cells in the grafted arterial wall. In control grafts, the neointima had formed by 2 weeks postimplant. However, the cells comprising this layer generally presented no positivity whatsoever towards the antibodies employed. At 50 days, the new layer was observed to be formed by a vWF-positive endothelium and a-actin-positive cells. In all three groups, several polymorphonuclear (PMN) cells adhered to the denuded luminal surface from 7 days onwards. In the treated animals, neutrophils and monocytes were seen to infiltrate intimal and medial layers during the later postimplant stages. Around the third week post-implant, the neointima had reached the grafted segment from the distal portion of the recipient artery, and by 50 days it was similar to that seen in control specimens. Our findings suggest that: a) neutrophils play a role in neointimal thickening in this arterial autograft model; and b) CsA promotes the adhesion and infiltration of neutrophils in the injured arterial wall.

Background. Despite their degradation in the host organism, the benefits of collagen bioprostheses remain unclear. This study addresses the absorption and long-term host tissue incorporation of several collagen biomeshes. Material and methods. Partial ventral hernial defects created in the abdominal wall of rabbits were repaired using the crosslinked meshes Permacol® or CollaMend®, or the non-crosslinked Surgisis®, Tutomesh® or Strattice®. After 90 and 180 days of implant, morphological studies and morpho-metric analysis of the thickness of the meshes were performed. Immunofluorescence confocal microscopy combined with differential interference contrast (DIC) imaging was used to distinguish newly formed collagen from that comprising the mesh. The macrophage response was examined by immunohisto-chemistry. Results. At 90 days, the thinner non-crosslinked biomeshes Tutomesh and Surgisis were more fully degraded with much of their collagen replaced with loose connective tissue. By 180 days, both implants had been practically fully absorbed. In contrast, in Strattice only the outermost third was infiltrated by neoformed tissue. On both surfaces of the crosslinked meshes, a fibrous capsule with host cells lining its perimeter was observed at both time points, though at 180 days these cells had penetrated the mesh interior. At both implant times, Strattice showed the higher expression of collagen type I while collagen III expression was similar for all the meshes. The non-crosslinked materials elicited lower macrophage counts at both time points, significantly so for Strattice. The macrophage response decreased over time for all the meshes but Surgisis. Conclusions. Strattice, the thicker, more compacted non-crosslinked mesh showed the best balance between tissue incorporation and absorption while eliciting a minimal foreign-body reaction in the long-term.

This study was designed to evaluate the role of the smooth muscle cell and the apoptosis in the pathogenesis of the varicose vein. Segments of saphenous vein were obtained from healthy subjects and from those with varicose veins. The vein specimens were subdivided according to subject age (younger or older than 50 years) and according to the varicose vein source (distal or proximal). Morphological, ultrastructural, cell proliferation (anti-PCNA method) and cell death (TUNEL method) analysis were performed. The walls of healthy, control vein specimens acquired a more collagenous and papillomatous appearance with age. A slight increase in the number of TUNEL-positive cells was also observed in specimens from older subjects. The proportion of apoptotic cells was much greater in the varicose veins than in control specimens. Most cellular alterations were seen in proximal varicose segments obtained from young subjects. These specimens showed hypertrophic areas with a high degree of cellularity (both in the media and in the thickened intima). The highest proportion of apoptotic cells and collagenisation were also observed in these areas. The enhanced number of apoptotic cells in varicose veins observed mainly in proximal/young vein specimens could be responsible, at least in part, for the acceleration of the final fibrosclerotic process characteristic of the varicose vein wall.

Background. While lightweight (LW) polypropylene (PP) meshes are been used for hernia repair, new prosthetic meshes also of low-density and with large pores have recently been introduced composed of other polymer materials. This study compares the behavior in the short-term of two macroporous LW prosthetic materials, PP and non-expanded PTFE. Methods. Partial defects were created in the lateral wall of the abdomen in New Zealand White rabbits and then repaired using a LW PP mesh or a new monofile, LW PTFE mesh. At 14 days postimplant, shrinkage and tissue incorporation, gene and protein expression of neocollagens (qRT-PCR/immunofluorescence), macrophage response (immunohistochemistry) and biomechanical strength were determined. Results. Both meshes induced good host tissue ingrowth, yet the macrophage response was significantly greater for the PTFE implants (p

The terms construct or tissue equivalent refer to neotissue produced by tissue engineering techniques. The elements forming the construct are scaffolds on which cells are “recreated” to form an enginnered-tissue sensitive to certain cell signals. The ability of the cells to expand and differentiate on the scaffold is determined by properties such as fixation, adhesion, proliferation and migration. Among the cell types that seem to be most promising for designing constructs are tissue-residing, or adult, stem cells, which show two main features: a capacity to differentiate into many cell lineages and the power of self-renewal. These features make them good candidates for cell replacement therapies. Here, we report the identification, isolation and culture of muscle stem cells aimed at establishing the ideal culture in terms of defining when the cultured cell population would show optimal characteristics for transfer to the scaffold to obtain a particular construct. Stem cells harvested from the dorsal muscle of white New Zealand rabbits were cultured in vitro and characterized 5 to 14 days after the start of culture. Fibroblasts obtained from the same experimental animal served as controls. The stem cells were examined by light and scanning electron microscopy. For stem cell identification, we used the antibodies anti-m-cadherin, anti-CD34 and anti-Myf-5. The markers of muscle differentiation used were: antivimentin, anti-a-actin, anti-desmin and anti-myosin. The expression profiles of the different markers of muscle differentiation and TGFß1 in the cell cultures were confirmed by Western blotting. Proliferation rates were determined by monitoring tritiated thymidine incorporation. The thymidine incorporation rate was substantially higher for the population of undifferentiated cells than for control fibroblasts obtained from the same animal. During the first five days of culture, most cells were negative for all the markers examined, with the exception of m-cadherin, CD34 and Myf-5, although discrete signs of vimentin expression started to emerge. After 14 days of culture, the adult stem cells showed vimentin (94.2%) and desmin (33.8%) expression yet scarce labeling for myosin (16.2%) and a-actin (8.3%). Control fibroblasts showed intense labeling for vimentin (99.3%) and a-actin (62.2%), while less than 2% of the population expressed myosin (0.9%) and desmin (1.6%). After two weeks of culture, muscle-derived stem cells show good proliferative and adhesion properties as they initiate differentiation. These conditions seem ideal for obtaining the desired construct.

The present study investigates the effects on the cardiac muscle cell of two of the determining factors for the success of organ transplant; ischaemia-perfusion and immunosuppressive treatment with cyclosporin-A (CsA). To this end an abdominal, heterotopic heart transplant model in singenic Sprague-Dawley rats was employed. Three study groups were established: Group I (control, n = 15) animals undergoing heart transplant without treatment; Group II (n = 15) animals undergoing heart transplant and subjected to a daily dose of CsA in a cremophor vehicle (Sandimun) (5 mg/kg/sc); Group III (n = 15): animals undergoing heart transplant and administered a daily dose of pure CsA (5 mg/kg/sc). Recipient animals were sacrificed 7, 14, 21, 30 and 50 days after transplant. During the post-operative period, heart function was assessed by daily abdominal palpation. Graft specimens obtained at each follow-up period were subjected to light and transmission electron microscopy. Immunohistochemical analysis of specimens was performed using the rat macrophage-specific monoclonal antibody MCA-341. The ischaemia/reperfusion process induced considerable alteration to cardiac muscle cells of control animals. Effects, apparent after the first week of transplant, included mitochondrial swelling and loss of cristae, hypertrophy of the sarcoplasmic reticulum and structural changes to sarcomeres. Two weeks after transplant, the myocardium was infiltrated by inflammatory cells. These effects diminished 30 days post-transplant. Cardiac tissues of treated animals (groups II and III) showed similar behaviour although, in the latter group, mitochondrial damage was greater and intense myocardial fibrosis took place. Infiltration of cardiac muscle by white blood cells did not take place until 3 weeks post-implant. These results indicate: a) The ultrastructural changes detected in cardiac fibres of animals of the three study groups were attributable to the ischaemia/reperfusion process rather than to treatment with CsA; b) CsA appears to augment mitochondrial damage and myocardial fibrosis; c) the inflammatory response was delayed and reduced by the immunosupressant; and d) the cremophor administration vehicle did not seem to exert an independent toxic effect on the myocardium.