Darío Fernández Zoppino | Universidad de Burgos (original) (raw)

Papers by Darío Fernández Zoppino

The cells involved in spermatogenesis are germ-cells, called spermatogonia, classified as: type A... more The cells involved in spermatogenesis are germ-cells, called spermatogonia, classified as: type A-undifferentiated, type A-intermediate and type B. During the spermatogenesis, more than 75% of the germ-cells undergo apoptosis and most of them are phagocyted by Sertoli cells. Peritubular macrophages in adult mouse testis are macrophages that both stimulate the proliferation and differentiation of undifferentiated spermatogonia in the wall of the seminiferous tubule. They have long processes and ramified appearance that squished between the lateral sides of neighbor myoid cells. We show, that a population of peritubular macrophages, grouped in pairs and activated, phagocyted undifferentiated spermatogonia in apoptosis. In adult mouse testis, 3.3x 105undifferentiated spermatogonia are in the germinal epithelium and 8,250 of them are in apoptosis. We counted in the testis 2,634.2 ±160 peritubular macrophages with phagocytic activity. If each one phagocyted one undifferentiated spermatog...

Frontiers in Medicine, 2021

This manuscript presents findings from the first dichotomous data pooling analysis on clinical tr... more This manuscript presents findings from the first dichotomous data pooling analysis on clinical trials (CT) regarding the effectiveness of binding potassium. The results emanated from pairwise and network meta-analyses aiming evaluation of response to commercial potassium-binding polymers, that is, to achieve and maintain normal serum potassium (n = 1,722), and the association between this response and an optimal dosing of renin-angiotensin-aldosterone system inhibitors (RAASi) needing individuals affected by heart failure (HF) or resistant hypertension, who may be consuming other hyperkalemia-inducing drugs (HKID) (e.g., β-blockers, heparin, etc.), and frequently are affected by chronic kidney disease (CKD) (n = 1,044): According to the surface under the cumulative ranking area (SUCRA), sodium zirconium cyclosilicate (SZC) (SUCRA >0.78), patiromer (SUCRA >0.58) and sodium polystyrene sulfonate (SPS) (SUCRA <0.39) were different concerning their capacity to achieve normokale...

World Journal of Stem Cells, 2019

Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to the... more Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to their plasticity and easiness of sourcing. MSC-based treatments are generally considered a safe procedure, however, the long-term results obtained up to know are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and osteogenic differentiation. Many studies have proposed modifications to improve MSC engraftment and osteogenic differentiation of the transplanted cells. Several strategies are aimed to improve cell resistance to the hostile microenvironment found in the recipient tissue and increase cell survival once transplanted. These strategies could range from a simple modification of the culture conditions, known as cell-preconditioning, to the genetic modification of the cells to avoid cellular senescence. Many efforts have also been done in order to enhance the osteogenic potential of the transplanted cells and induce bone formation, mainly by the use of bioactive or biomimetic scaffolds, although alternative approaches will also be discussed. This review aims to summarize several of the most recent approaches, providing an up-to-date view of the main developments in MSCbased regenerative techniques.

Sociedad Española de Bioquímica y Biología Molecular, 2010

Trabajo presentado en el XXXIII Congreso de la Sociedad Española de Bioquímica y Biología Molecul... more Trabajo presentado en el XXXIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Córdoba, del 14 al 17 de septiembre de 2010Peer reviewe

Biology of Reproduction, 2008

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cell... more In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth musclealpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMCmyosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 48C, but did at 208C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1. male reproductive tract, myosin, peritubular myoid cells, seminiferous tubule, testis

Journal of Clinical and Experimental Dentistry, 2021

Background: In the dental clinic impacted teeth are frequent findings, especially upper and lower... more Background: In the dental clinic impacted teeth are frequent findings, especially upper and lower third molars, leading to their exodontia. Among surgical techniques piezosurgery is advantageous for delicate structures in the oral cavity. Extracted teeth, usually discarded, have been revalued as biological material, providing living tissues with possible applications in regenerative dentistry. The aim was to compare cross-section methods of upper included third molars by ultrasonic piezoelectric technique to obtain dental pulp, with diamond-coated tip (DT) against titanium nitride-coated tip (TN), according to the pulp tissue cell viability and the section surface characteristics.

Material and methods: Patients attending dental consultation were evaluated. Upper third molars (n= 24) were avulsed from 15 patients with exodontia indication, age 18-26 years old, who agreed to participate of the study. Third molars were cross-sectioned at amelocemental junction level with piezoelectric device using DT or TN inserts. Pulps were mechanic and enzymatically treated, and tissue viability determined by Trypan Blue test. Sectioned teeth were visualized using Scanning Electron Microscope (SEM). Ethical principles of biomedical research were respected; all patients gave their informed consent.

Results: Viability of pulp tissue was 84.71% not associated to sex (p= 0.141) nor to teeth position, upper right third molar or upper left third molar (p= 0.580). According to the insert used, pulp tissue viability was 85.21% with TN, similar to 84.00% with DT (p= 0.611). By SEM, cut performed by TN insert showed smooth and uniform surfaces, while DT insert surfaces were irregular, porous, with fissures.

Conclusions: Piezosurgery applied to cross-section upper third molars with both types of inserts showed differences in the cut surfaces but similar effectiveness regarding preservation of pulp tissue with high viability, thus, they could be allocated for further cellular developments. Key words:Impacted teeth, third molars, piezosurgery, regenerative dentistry.

Dentistry, 2018

reconstructive dentistry resorts to its substitution, by placing prosthetic devices using surgica... more reconstructive dentistry resorts to its substitution, by placing prosthetic devices using surgical procedures, in order to seek the functional repair of the affected area. In order to carry out these surgeries, standard prostheses are frequently used, which often fail to correct the impairments. The use of this type of prosthetic devices requires the adjustment of the implant piece to the anatomy of the patient in situ, prolonging the surgical length. Due to the difficulties implicit in these adjustments, patients may suffer sequelae such as decreased speech ability or difficulties to swallow, and must undergo successive surgical procedures as to restore these functions. On the other hand, the surgical reconstruction approach that involves placing personalized prostheses to the patients allows the surgeon to restore their facial symmetry and normal oral-maxillofacial functioning. These devices must be designed taking into consideration the anatomical parameters of the patient related to shape, size, weight and other features from preoperative computerized and imaging studies. An anatomic model with the defect is then used to create the implant for reconstruction and surgical placement. Customized designs may improve not only the aesthetic conditions, but also morphological and functional issues regarding the movements of the area under treatment. As it has been described, this method of reconstruction is rapid, exact and reduces operative time significantly in these defects [1]. The development of surgical protocols aimed to improve the approach and rehabilitation of patients suffering from these health

En 1970 Michael y colaboradores (Michael, Blau y Vernier 1970) mostraron que la cara luminal de l... more En 1970 Michael y colaboradores (Michael, Blau y Vernier 1970) mostraron que la cara luminal de los podocitos o celulas epiteliales del glomerulo renal, estaban recubiertas de una superficie que podia ser digerida con neuraminidasa a la cual denominaron “polianion epitelial” por su afinidad por colorantes cationicos. Posteriormente, otros autores (Kerjaschki, Sharkey y Farquhar 1984), aislaron una sialoproteina glomerular de unos 140 kDa, tingible por colorantes cationicos, digerible por neuraminidasa y capaz de fijarse a aglutinina de germen de trigo. Estos autores concluyeron que esta proteina era el equivalente bioquimico del polianion del epitelio glomerular, descrito por medios histoquimicos, y la denominaron podocalicina (PODXL) dada su localizacion en el glicocalix de los podocitos glomerulares. Trabajos posteriores demostraron que su carga negativa era debida a su alto contenido en sulfato y acido sialico (Dekan, Gabel y Farquhar 1991). Se ha considerado que la podocalicina o, para ser mas precisos, su carga superficial, es esencial para mantener la estructura de las celulas epiteliales (podocitos) de los glomerulos renales e impedir la oclusion de las rendijas epiteliales, tambien denominadas espacios urinarios (Charest y Roth 1985, Kerjaschki y col. 1984, Michael y col. 1970). La importancia patologica de la podocalicina se apoya en los hallazgos de la disminucion del contenido glomerular de acido sialico, distorsion de la estructura de los podocitos y obliteracion de las rendijas epiteliales en casos de nefrosis humana de “cambios minimos” o en nefrosis experimentales (Caulfield, Reid y Farquhar 1976, Seiler y col. 1977). CD34, podocalicina y endoglicano se agrupan como una familia de proteinas (familia CD34) en base a la conservacion de la organizacion de sus dominios asi como de su organizacion genomica (Doyonnas y col. 2001, Nielsen y col. 2002, Li y col. 2001).

Thrombosis Research, 2008

El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido ... more El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido facilitado todavía por el investigador a cargo del archivo del mismo.

Thrombosis Research, 2010

Podocalyxin (PODXL) is a 145 KDa sialoprotein abundantly expressed in the glycocalix of the intra... more Podocalyxin (PODXL) is a 145 KDa sialoprotein abundantly expressed in the glycocalix of the intraglomerular kidney epithelial cells, essential in maintaining a normal renal function. PODXL is also found in vascular endothelial cells, megakaryocytes and platelets. The function of PODXL in platelets is ignored; however, its surface exposure upon platelet activation suggests its participation in controlling the hemostasis. We have generated mice (prαIIb-PODXL) overexpressing PODXL specifically in megakaryocytes , either alone or as a fusion protein with green fluorescent protein. The transgenic mice showed a phenotype characterized by decreased bleeding time, mild rebleeding and enhanced platelets aggregation upon agonist stimulation. The cytohematological exams as well as the prothrombin time (PT) and (APTT) tests did not differ from the control group. The biochemical analysis showed only a discrete hyperlipemia and a rise in plasma uric acid levels in the transgenic mice. The present data seem to indicate that PODXL may act as a costimulator of agonists in the activation of platelets and formation of a stable thrombus.

Molecular and Cellular Neuroscience, 2010

is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial c... more is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl floxed/floxed mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl −/− mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.

Biology of Reproduction, 2012

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (ST... more In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2011

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells... more Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.

PLoS ONE, 2011

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in... more Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.

Biochemical and Biophysical Research Communications, 2013

Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells... more Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell–cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.

Journal of Inorganic Biochemistry

Thrombosis and Haemostasis, 2010

The availability of mice with tissue-specific expression of recombinase Cre is the limiting step ... more The availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the Cre-cDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of

The cells involved in spermatogenesis are germ-cells, called spermatogonia, classified as: type A... more The cells involved in spermatogenesis are germ-cells, called spermatogonia, classified as: type A-undifferentiated, type A-intermediate and type B. During the spermatogenesis, more than 75% of the germ-cells undergo apoptosis and most of them are phagocyted by Sertoli cells. Peritubular macrophages in adult mouse testis are macrophages that both stimulate the proliferation and differentiation of undifferentiated spermatogonia in the wall of the seminiferous tubule. They have long processes and ramified appearance that squished between the lateral sides of neighbor myoid cells. We show, that a population of peritubular macrophages, grouped in pairs and activated, phagocyted undifferentiated spermatogonia in apoptosis. In adult mouse testis, 3.3x 105undifferentiated spermatogonia are in the germinal epithelium and 8,250 of them are in apoptosis. We counted in the testis 2,634.2 ±160 peritubular macrophages with phagocytic activity. If each one phagocyted one undifferentiated spermatog...

Frontiers in Medicine, 2021

This manuscript presents findings from the first dichotomous data pooling analysis on clinical tr... more This manuscript presents findings from the first dichotomous data pooling analysis on clinical trials (CT) regarding the effectiveness of binding potassium. The results emanated from pairwise and network meta-analyses aiming evaluation of response to commercial potassium-binding polymers, that is, to achieve and maintain normal serum potassium (n = 1,722), and the association between this response and an optimal dosing of renin-angiotensin-aldosterone system inhibitors (RAASi) needing individuals affected by heart failure (HF) or resistant hypertension, who may be consuming other hyperkalemia-inducing drugs (HKID) (e.g., β-blockers, heparin, etc.), and frequently are affected by chronic kidney disease (CKD) (n = 1,044): According to the surface under the cumulative ranking area (SUCRA), sodium zirconium cyclosilicate (SZC) (SUCRA >0.78), patiromer (SUCRA >0.58) and sodium polystyrene sulfonate (SPS) (SUCRA <0.39) were different concerning their capacity to achieve normokale...

World Journal of Stem Cells, 2019

Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to the... more Mesenchymal stem cells (MSCs) are promising candidates for bone regeneration therapies due to their plasticity and easiness of sourcing. MSC-based treatments are generally considered a safe procedure, however, the long-term results obtained up to know are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and osteogenic differentiation. Many studies have proposed modifications to improve MSC engraftment and osteogenic differentiation of the transplanted cells. Several strategies are aimed to improve cell resistance to the hostile microenvironment found in the recipient tissue and increase cell survival once transplanted. These strategies could range from a simple modification of the culture conditions, known as cell-preconditioning, to the genetic modification of the cells to avoid cellular senescence. Many efforts have also been done in order to enhance the osteogenic potential of the transplanted cells and induce bone formation, mainly by the use of bioactive or biomimetic scaffolds, although alternative approaches will also be discussed. This review aims to summarize several of the most recent approaches, providing an up-to-date view of the main developments in MSCbased regenerative techniques.

Sociedad Española de Bioquímica y Biología Molecular, 2010

Trabajo presentado en el XXXIII Congreso de la Sociedad Española de Bioquímica y Biología Molecul... more Trabajo presentado en el XXXIII Congreso de la Sociedad Española de Bioquímica y Biología Molecular, celebrado en Córdoba, del 14 al 17 de septiembre de 2010Peer reviewe

Biology of Reproduction, 2008

In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cell... more In the mammalian testis, peritubular myoid cells (PMCs) surround seminiferous tubules. These cells are contractile, express the cytoskeletal markers of true smooth musclealpha-isoactin and F-actin-and participate in the contraction of seminiferous tubules during the transport of spermatozoa and testicular fluid to the rete testis. Myosin from PMCs (PMCmyosin) was isolated from adult rat testis and purified by cycles of assembly-disassembly and sucrose gradient centrifugation. PMC-myosin was recognized by a monoclonal anti-smooth muscle myosin antibody, and the peptide sequence shared partial homology with rat smooth muscle myosin-II, MYH11 (also known as SMM-II). Most PMC-myosin (95%) was soluble in the PMC cytosol, and purified PMC-myosin did not assemble into filaments in the in vitro salt dialysis assay at 48C, but did at 208C. PMC-myosin filaments are stable to ionic strength to the same degree as gizzard MYH11 filaments, but PMC-myosin filaments were more unstable in the presence of ATP. When PMCs were induced to contract by endothelin 1, a fraction of the PMC-myosin was found to be involved in the contraction. From these results we infer that PMCs express an isoform of smooth muscle myosin-II that is characterized by solubility at physiological ionic strength, a requirement for high temperature to assemble into filaments in vitro, and instability at low ATP concentrations. PMC-myosin is part of the PMC contraction apparatus when PMCs are stimulated with endothelin 1. male reproductive tract, myosin, peritubular myoid cells, seminiferous tubule, testis

Journal of Clinical and Experimental Dentistry, 2021

Background: In the dental clinic impacted teeth are frequent findings, especially upper and lower... more Background: In the dental clinic impacted teeth are frequent findings, especially upper and lower third molars, leading to their exodontia. Among surgical techniques piezosurgery is advantageous for delicate structures in the oral cavity. Extracted teeth, usually discarded, have been revalued as biological material, providing living tissues with possible applications in regenerative dentistry. The aim was to compare cross-section methods of upper included third molars by ultrasonic piezoelectric technique to obtain dental pulp, with diamond-coated tip (DT) against titanium nitride-coated tip (TN), according to the pulp tissue cell viability and the section surface characteristics.

Material and methods: Patients attending dental consultation were evaluated. Upper third molars (n= 24) were avulsed from 15 patients with exodontia indication, age 18-26 years old, who agreed to participate of the study. Third molars were cross-sectioned at amelocemental junction level with piezoelectric device using DT or TN inserts. Pulps were mechanic and enzymatically treated, and tissue viability determined by Trypan Blue test. Sectioned teeth were visualized using Scanning Electron Microscope (SEM). Ethical principles of biomedical research were respected; all patients gave their informed consent.

Results: Viability of pulp tissue was 84.71% not associated to sex (p= 0.141) nor to teeth position, upper right third molar or upper left third molar (p= 0.580). According to the insert used, pulp tissue viability was 85.21% with TN, similar to 84.00% with DT (p= 0.611). By SEM, cut performed by TN insert showed smooth and uniform surfaces, while DT insert surfaces were irregular, porous, with fissures.

Conclusions: Piezosurgery applied to cross-section upper third molars with both types of inserts showed differences in the cut surfaces but similar effectiveness regarding preservation of pulp tissue with high viability, thus, they could be allocated for further cellular developments. Key words:Impacted teeth, third molars, piezosurgery, regenerative dentistry.

Dentistry, 2018

reconstructive dentistry resorts to its substitution, by placing prosthetic devices using surgica... more reconstructive dentistry resorts to its substitution, by placing prosthetic devices using surgical procedures, in order to seek the functional repair of the affected area. In order to carry out these surgeries, standard prostheses are frequently used, which often fail to correct the impairments. The use of this type of prosthetic devices requires the adjustment of the implant piece to the anatomy of the patient in situ, prolonging the surgical length. Due to the difficulties implicit in these adjustments, patients may suffer sequelae such as decreased speech ability or difficulties to swallow, and must undergo successive surgical procedures as to restore these functions. On the other hand, the surgical reconstruction approach that involves placing personalized prostheses to the patients allows the surgeon to restore their facial symmetry and normal oral-maxillofacial functioning. These devices must be designed taking into consideration the anatomical parameters of the patient related to shape, size, weight and other features from preoperative computerized and imaging studies. An anatomic model with the defect is then used to create the implant for reconstruction and surgical placement. Customized designs may improve not only the aesthetic conditions, but also morphological and functional issues regarding the movements of the area under treatment. As it has been described, this method of reconstruction is rapid, exact and reduces operative time significantly in these defects [1]. The development of surgical protocols aimed to improve the approach and rehabilitation of patients suffering from these health

En 1970 Michael y colaboradores (Michael, Blau y Vernier 1970) mostraron que la cara luminal de l... more En 1970 Michael y colaboradores (Michael, Blau y Vernier 1970) mostraron que la cara luminal de los podocitos o celulas epiteliales del glomerulo renal, estaban recubiertas de una superficie que podia ser digerida con neuraminidasa a la cual denominaron “polianion epitelial” por su afinidad por colorantes cationicos. Posteriormente, otros autores (Kerjaschki, Sharkey y Farquhar 1984), aislaron una sialoproteina glomerular de unos 140 kDa, tingible por colorantes cationicos, digerible por neuraminidasa y capaz de fijarse a aglutinina de germen de trigo. Estos autores concluyeron que esta proteina era el equivalente bioquimico del polianion del epitelio glomerular, descrito por medios histoquimicos, y la denominaron podocalicina (PODXL) dada su localizacion en el glicocalix de los podocitos glomerulares. Trabajos posteriores demostraron que su carga negativa era debida a su alto contenido en sulfato y acido sialico (Dekan, Gabel y Farquhar 1991). Se ha considerado que la podocalicina o, para ser mas precisos, su carga superficial, es esencial para mantener la estructura de las celulas epiteliales (podocitos) de los glomerulos renales e impedir la oclusion de las rendijas epiteliales, tambien denominadas espacios urinarios (Charest y Roth 1985, Kerjaschki y col. 1984, Michael y col. 1970). La importancia patologica de la podocalicina se apoya en los hallazgos de la disminucion del contenido glomerular de acido sialico, distorsion de la estructura de los podocitos y obliteracion de las rendijas epiteliales en casos de nefrosis humana de “cambios minimos” o en nefrosis experimentales (Caulfield, Reid y Farquhar 1976, Seiler y col. 1977). CD34, podocalicina y endoglicano se agrupan como una familia de proteinas (familia CD34) en base a la conservacion de la organizacion de sus dominios asi como de su organizacion genomica (Doyonnas y col. 2001, Nielsen y col. 2002, Li y col. 2001).

Thrombosis Research, 2008

El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido ... more El artículo seleccionado no se encuentra disponible por ahora a texto completo por no haber sido facilitado todavía por el investigador a cargo del archivo del mismo.

Thrombosis Research, 2010

Podocalyxin (PODXL) is a 145 KDa sialoprotein abundantly expressed in the glycocalix of the intra... more Podocalyxin (PODXL) is a 145 KDa sialoprotein abundantly expressed in the glycocalix of the intraglomerular kidney epithelial cells, essential in maintaining a normal renal function. PODXL is also found in vascular endothelial cells, megakaryocytes and platelets. The function of PODXL in platelets is ignored; however, its surface exposure upon platelet activation suggests its participation in controlling the hemostasis. We have generated mice (prαIIb-PODXL) overexpressing PODXL specifically in megakaryocytes , either alone or as a fusion protein with green fluorescent protein. The transgenic mice showed a phenotype characterized by decreased bleeding time, mild rebleeding and enhanced platelets aggregation upon agonist stimulation. The cytohematological exams as well as the prothrombin time (PT) and (APTT) tests did not differ from the control group. The biochemical analysis showed only a discrete hyperlipemia and a rise in plasma uric acid levels in the transgenic mice. The present data seem to indicate that PODXL may act as a costimulator of agonists in the activation of platelets and formation of a stable thrombus.

Molecular and Cellular Neuroscience, 2010

is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial c... more is a type I membrane mucin-protein of the CD34 family abundantly expressed in kidney epithelial cells (podocytes) where it plays a crucial functional role. Podxl is also expressed in tissues other than kidney, like in brain, but its function is ignored. To investigate the functional role of podocalyxin (Podxl) in brain we produced the specific brain-ablation of the Podxl gen in mice by crossing Podxl floxed/floxed mice, generated in our laboratory, to mice with pan-neural expression of recombinase Cre (Cre3). Podxl −/− mice show no apparent behavioral phenotype but their brains showed enlargement of ventricular volumes detected in vivo by MR imaging. The pattern of brain vasculature was of normal appearance but the thickness of the main carotid artery was significantly increased. Moreover, the histological analysis showed increased number of choroidal capillaries lining the ventricular spaces. These findings are analyzed in the light of the role likely played by podocalyxin in cell migration and cell-cell recognition during brain development and also on the consistent findings of increased ventricular spaces in human pathological disorders like schizophrenia.

Biology of Reproduction, 2012

In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (ST... more In the mammalian testis, peritubular myoid cells (PM cells) surround the seminiferous tubules (STs), express cytoskeletal markers of true smooth muscle cells, and participate in the contraction of the ST. It has been claimed that PM cells contain bundles of actin filaments distributed orthogonally in an intermingled mesh. Our hypothesis is that these actin filaments are not forming a random intermingled mesh, but are actually arranged in contractile filaments in independent layers. The aim of this study is to describe the organization of the actin cytoskeleton in PM cells from adult rat testes and its changes during endothelin-1-induced ST contraction. For this purpose, we isolated segments of ST corresponding to the stages IX-X of the spermatogenic cycle (ST segments), and analyzed the actin and myosin filament distribution by confocal and transmission electron microscopy. We found that PM cells have actin and myosin filaments interconnected in thick bundles (AF-MyF bundles). These AF-MyF bundles are distributed in two independent layers: an inner layer toward the seminiferous epithelium, and an outer layer toward the interstitium, with the bundles oriented perpendicularly and in parallel to the main ST axis, respectively. In endothelin-1 contracted ST segments, PM cells increased their thickness and reduced their length in both directions, parallel and perpendicular to the main ST axis. The AF-MyF bundles maintained the same organization in two layers, although both layers appeared significantly thicker. We believe that this is the first time this arrangement of AF-MyF bundles in two independent layers has been shown in smooth muscle cells, and that this organization would allow the cell to generate contractile force in two directions.

Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2011

Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells... more Podocalyxin (PODXL) is a type I membrane mucoprotein abundantly presented in the epithelial cells (podocytes) of kidney glomeruli where it plays an important role in maintaining the plasma filtration. PODXL is also expressed in other types of cells but its function is ignored. A recombinant soluble fragment of the PODXL ectodomain modifies the signaling of the membrane bound PODXL. Based on this antecedent, we aimed at investigating whether PODXL could be cleaved and released into the extracellular space as a soluble peptide. In this study, we used a fusion protein of human PODXL and green fluorescent protein expressed in CHO cells (CHO-PODXL-GFP) and a human tumor cell (Tera-1) inherently expressing PODXL. PODXL was detected by wide-field microscopy in the Golgi, the plasma membrane and in a vesicular form preferentially located at the leading edges of the cell and also progressing along the filopodium. We detected PODXL in the insoluble and soluble fractions of the extracellular medium of CHO-PODXL-GFP cells. Stimulation of protein kinase C (PKC) by Phorbol-12-myristate-13-acetate (PMA) enhanced the release of PODXL to the extracellular space whereas this effect was prevented either by inhibitors of PKC or specific inhibitors of matrix metalloproteinases. It is concluded that intact PODXL is released to the extracellular space as a cargo of microvesicles and also as a soluble cleaved fragment of ectodomain.

PLoS ONE, 2011

Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in... more Podocalyxin (Podxl) is a type I membrane sialoprotein of the CD34 family, originally described in the epithelial glomerular cells of the kidney (podocytes) in which it plays an important function. Podxl can also be found in megakaryocytes and platelets among other extrarenal places. The surface exposure of Podxl upon platelet activation suggested it could play some physiological role. To elucidate the function of Podxl in platelets, we generated mice with restricted ablation of the podxl gene in megakaryocytes using the Cre-LoxP gene targeting methodology. Mice with Podxl-null megakaryocytes did not show any apparent phenotypical change and their rates of growth, life span and fertility did not differ from the floxed controls. However, Podxl-null mice showed prolonged bleeding time and decreased platelet aggregation in response to physiological agonists. The number, size-distribution and polyploidy of Podxl-null megakaryocytes were similar to the floxed controls. Podxl-null platelets showed normal content of surface receptors and normal activation by agonists. However, the mice bearing Podxl-null platelets showed a significant retardation in the ferric chloride-induced occlusion of the carotid artery. Moreover, acute thrombosis induced by the i.v. injection of sublethal doses of collagen and phenylephrine produced a smaller fall in the number of circulating platelets in Podxl-null mice than in control mice. In addition, perfusion of uncoagulated blood from Podxl-null mice in parallel flow chamber showed reduced adhesion of platelets and formation of aggregates under high shear stress. It is concluded that platelet Podxl is involved in the control of hemostasis acting as a platelet co-stimulator, likely due to its pro-adhesive properties.

Biochemical and Biophysical Research Communications, 2013

Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells... more Podocalyxin (PODXL) is a type I membrane sialomucin, originally described in the epithelial cells (podocytes) of kidney glomeruli. PODXL is also found in extra-renal tissues and in certain aggressive tumors, but its precise pathophysiological role is unknown. Expression of PODXL in CHO cells enhances their adhesive, migratory and cell–cell interactive properties in a selectin and integrin-dependent manner. We aimed at defining the PODXL domains responsible for those cell responses. For this purpose we have analyzed the cell adhesion/migration responses to deletion mutants of human PODXL, and the correlation with the activities of Rac1 and Cdc42 GTPases. The results obtained indicate that integrity of the PODXL ectodomain is essential for enhancing cell adhesion but not migration, while the integrity of the cytoplasmic domain is required for both adhesion and migration. Deletion of the carboxy-terminal DTHL domain (PODXL-ΔDTHL) limited only cell adhesion. The activities of Rac1 and Cdc42 GTPases parallel the PODXL-induced variations in cell adhesion and migration. Moreover, silencing the rac1 gene virtually abolished the effect of PODXL in enhancing cell adhesion.

Journal of Inorganic Biochemistry

Thrombosis and Haemostasis, 2010

The availability of mice with tissue-specific expression of recombinase Cre is the limiting step ... more The availability of mice with tissue-specific expression of recombinase Cre is the limiting step for a successful gene targeting by the Cre-LoxP methodology. This work aimed at generating transgenic mice with restricted expression of recombinase Cre in megakaryocytes and platelets, driven by the promoter of the αIIb gene (mαIIb-cre). Mice oocytes were microinjected with a 4.1 Kb construct comprising a 2.7 Kb promoter fragment of the glycoprotein αIIb gene, linked to the Cre-cDNA and followed by the polyA tail of the SV40. We found four mice with positive DNA genotype and three probable sites of genomic integration of the transgene. Only two of the founders showed presence of Cre-mRNA and production of Cre protein, restricted to megakaryocytes. The activity of Cre in mediating gene targeting was assessed by crossing mαIIb-cre mice to Cre-reporter mice (ROSA26-lacZ). The activity of

XXII Congress of the International Society on Thrombosis and Haemostasis. Kyoto, Japan. 23 – 28/07/2011, 2011

The restricted deletion of the podocalyxin gene in megakaryocytes perturbs the control of hemosta... more The restricted deletion of the podocalyxin gene in megakaryocytes perturbs the control of hemostasia.

Podocalyxin (Podxl) is an adhesive sialoprotein of the CD34 family,
expressed in platelets and exposed at the membrane surface upon stimulation
by physiological agonists, suggesting a regulatory role in
the platelet function(s). This work aimed at investigating the role of
Podxl in platelet function and hemostasia.
To elucidate the role of Podxl in platelets we used the Cre-LoxP gene
targeting methodology to generate mice with a restricted ablation of
the podxl gene in megakaryocytes. Mice with Podxl-null megakaryocytes
did not produce any apparent phenotypical changes, showing a
normal life span and fertility, and normal blood cells count. However,
the bleeding time was retarded and the aggregation of platelets
in response to physiological agonists was diminishsed in Podxl-null
platelets. The number, size-distribution and polyploidy were similar
in nul-Podxl megakaryocytes to the floxed controls. Podxl-null platelets
exhibited normal surface receptors content and normal stimulation
by agonists. However, the ferric chloride-induced occlusion of
the carotid artery was significantly retarded in the platelet Podxl-null
mice. Moreover, perfusion of uncoagulated blood from Podxl-null
mice in parallel flow chamber showed reduced platelet adhesion and
formation of aggregates.
To conclude, megakaryocyte/platelets Podxl is involved in the regulation
of hemostasis, likely acting as a platelet co-stimulator due to its
proadhesive properties.