Progenitor-derived endothelial cell response, platelet reactivity and haemocompatibility parameters indicate the potential of NaOH-treated polycaprolactone for vascular tissue engineering.

The haemocompatibility of NaOH-treated poly(ε-caprolactone) (PCL) has been evaluated in vitro by analysing several parameters, including plasma recalcification time, whole blood clotting time and platelet adhesion/activation. NaOH-treated PCL films showed a significant decrease in the clot formation speed and a reduced number of adhered platelets, which mainly exhibited non-activated morphologies. Furthermore, mature endothelial cells derived from peripheral endothelial progenitor cells were cultured on the polymer to investigate the effects of the endothelial lining on polymer haemocompatibility. Interestingly, cells cultured on NaOH-treated PCL films showed a significant stimulation of NO production. Although further research is required, NaOH treatment could be an interesting and simple strategy to modify PCL-based materials in order to enhance endothelial NO production, where compromised, and provide a better interaction of the scaffold with the blood components. In conclusion, these results reinforce the use of NaOH-treated PCL as a haemocompatible polymer for vascular tissue-engineering applications.

Nitric oxide production by endothelial cells derived from blood progenitors cultured on NaOH-treated polycaprolactone films: A biofunctionality study.

Poly(epsilon-caprolactone) (PCL) is a biodegradable polyester whose biocompatibility has been widely demonstrated both in vivo and in vitro. In the last few years, our group has confirmed that NaOH-treated PCL films can serve as a suitable biomaterial for vascular tissue engineering by supporting the culture of primary vascular cells and, more recently, endothelial-like EC(2) cells derived from endothelial progenitor cells (EPC). In the present study, NO production in basal conditions and after stimulation with different agents has been evaluated and related to the reactive oxygen species (ROS) content and the intracellular calcium levels on EC(2) cells cultured on NaOH-treated PCL films. The results obtained demonstrate that EC(2) seeded on NaOH-treated PCL films enhance the basal NO levels and show a faster, more intense response to physiological stimuli such as VEGF, bradykinin and thrombin than vein endothelial cells (ECv). This result could be indicative of a better capacity of EC(2) cells to maintain their endothelial functionality when seeded on polymers. On the other hand, the culture of both EC(2) and ECv cells on NaOH-treated PCL films induces a significant increase in both ROS content and intracellular calcium that is balanced out through the stimulation of NO production in these cells. In conclusion, these results demonstrate the ability of NaOH-treated PCL films to support endothelial cell production of nitric oxide and reinforce the idea of considering the endothelial-like EC(2) cells derived from blood progenitors as an adequate source of endothelial cells to functionalize vascular grafts. Furthermore, NaOH-treated PCL films could be considered as a promising cellular NO production-inducing biomaterial for vascular tissue engineering applications.

L929 fibroblast and Saos-2 osteoblast response to hydroxyapatite-betaTCP/agarose biomaterial.

Biphasic calcium phosphate, a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has been successfully used as an excellent bone graft substitute because of the HA capacity for direct interaction with bone and the beta-TCP resorption properties. Agarose has been recently mixtured with ceramics as natural biodegradable binder to increase the biomaterial flexibility facilitating its placement into the bone defect. In this study, the behavior of L929 fibroblasts and Saos-2 osteoblasts cultured on hydroxyapatite-betaTCP/agarose disks has been evaluated. Both cell types adhere and proliferate on the biomaterial surface maintaining their characteristic morphology. Transitory changes on cell cycle, size, and complexity are observed. The biomaterial induces apoptosis in Saos-2 osteoblasts but not in fibroblasts. A transitory stimulation of fibroblast mitochondrial activity is observed. This effect remains in osteoblasts after 9 days of culture showing a higher sensitivity of this cell type. However, the intracellular reactive oxygen species content and the lactate dehydrogenase release of Saos-2 osteoblasts indicate that hydroxyapatite-betaTCP/agarose does not induce oxidative stress in this cell type and confirm the integrity of the osteoblast plasma membrane. These results underline the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.

Biocompatibility markers for the study of interactions between osteoblasts and composite biomaterials.

Biphasic calcium phosphate (BCP), a mixture of hydroxyapatite (HA) and beta-tricalcium phosphate (beta-TCP), has attracted attention as an excellent bone graft substitute. Mixtures of ceramics with agarose, as natural biodegradable binder, have been recently performed in order to increase the flexibility of the ceramic component and to facilitate the biomaterial preparation. In previous studies we have evaluated the response of both L929 fibroblasts and Saos-2 osteoblasts to hydroxyapatite-betaTCP/agarose disks observing a higher sensitivity of osteoblasts to this biomaterial. In the present study, the use of specific fluorescent probes and antibodies has allowed to evaluate different cell function parameters as biocompatibility markers for the cell/biomaterial interaction of Saos-2 osteoblasts cultured for 7 days on hydroxyapatite-betaTCP/agarose disks. The cell cycle subG(1) fraction, the exposition of phosphatidylserine on the outside surface of the plasma membrane and the analysis of plasma membrane integrity versus cell size, indicate that the interaction with the biomaterial induces a light increase of apoptosis in osteoblasts without producing cell necrosis. The high percentage of viable cells on the biomaterial and the preservation of endothelial nitric oxide synthase (eNOS) expression, eNOS activity and mitochondrial membrane potential (Deltapsi(m)), demonstrate the good biocompatibility of hydroxyapatite-betaTCP/agarose disks and its potential utility for bone substitution and repair.

In vitro positive biocompatibility evaluation of glass-glass ceramic thermoseeds for hyperthermic treatment of bone tumors.

A new kind of magnetic thermoseed for bone tissue engineering has been synthesized. The materials used are specially designed to restore bone tissue after tumor extirpation, because they exhibit bioactive behavior and the ability to act as thermoseeds for cancer treatment using hyperthermia. The L929 cell line of mouse fibroblasts has been used in a wide biocompatibility study concerning cell proliferation and morphology studies, mitochondrial function determination, lactate dehydrogenase measurement, and flow cytometry studies, including cell cycle analysis, cell size and complexity, and intracellular reactive oxygen species content. The results presented in this work indicate that these bioactive magnetic materials are highly biocompatible and show greater cell response for thermoseeds with a higher magnetic phase content. There were no significant alterations detected in the cell cycle, and the interaction between fibroblasts and the different mixtures did not induce significant apoptosis.

High grade B-cell gastric lymphoma with complete pathologic remission after eradication of Helicobacter pylori infection: report of a case and review of the literature.

BACKGROUND: Treatment of primary gastric diffuse large B-cell lymphoma is still controversial. The treatment of localized disease was based on surgery alone, or followed by chemotherapy and/or radiotherapy. High-grade gastric lymphomas are generally believed to be Helicobacter pylori (HP)-independent growing tumors. However a few cases of regression of high-grade gastric lymphomas after the cure of Helicobacter pylori infection had been described. CASE PRESENTATION: We report here a case with primary diffuse large B-cell lymphoma that showed a complete pathologic remission after HP eradication and we reviewed the literature. A computerized literature reach through Medline, Cancerlit and Embase were performed, applying the words: high grade gastric lymphoma, or diffuse large B cell, MALT gastric lymphoma, DLBCLL (MALT) lymphoma and Helicobacter. Articles and abstracts were also identified by back-referencing from original and relevant papers. Selected for the present review were papers published in English before January 2007. CONCLUSION: Forty two cases of primary high grade gastric lymphoma that regressed with anti HP treatment were found. There were anecdotal cases reported and patients belonging to prospective studies; four trials studied the effect of eradication of Helicobacter pylori as first line therapy in high grade gastric lymphoma: 22 of a total of 38 enrolled patients obtained complete remission. Depth of gastric wall infiltration and clinical stage were important factors to predict the response to antibiotic therapy. Our case and the review of the literature show that high-grade transformation is not necessarily associated with the loss HP dependence. In early stage, for high-grade B-cell HP-positive gastric lymphomas, given the limited toxicity of anti-HP therapy, this treatment may be considered as one of the first line treatment options.

Endothelial cells derived from circulating progenitors as an effective source to functional endothelialization of NaOH-treated poly(epsilon-caprolactone) films.

Biomaterials have been widely used to prepare synthetic vascular grafts over the past thirty years, but the inherent thrombogenicity of their surface can lead to graft failure. Endothelial progenitor cells (EPC) are circulating premature cells able to differentiate in either myocardial or endothelial cells (EC). The therapeutic potential of these cells and its easy obtaining technique are important reasons why these cells could be used to improve the performance of vascular grafts. In this study, two different stages of differentiation of EC derived from EPC (EC(1) and EC(2)) were characterized and cultured on poly(epsilon-caprolactone) (PCL) films treated with NaOH (PCL-NaOH). We investigated by immunolabeling the expression of CD31, von Willebrand factor (vWF), and endothelial nitric oxide synthase (eNOS) in these cells during the differentiation process. The proliferation, cell cycle, and mitochondrial function of EC(2) cultured on PCL-NaOH were evaluated at different times. The effect of this biomaterial on the nitric oxide (NO) content was also measured. The mature EC obtained from circulating progenitor cells (EC(2)) showed an appropriate growth and functionality on NaOH-treated films. They conserved their capacity to define vessel-like structures in culture and increased their basal NO production. These results underline the potential usefulness of these EC(2) to get a functional endothelialization of polymers with applications in vascular tissue engineering.

Ingeniería de tejidos: aplicaciones en el diseño de implantes cardiovasculares / Tissue engineering: designing cardiovascular grafts

El desarrollo de implantes biodegradables para Cirugía Cardiovascular medianteIngeniería de tejidos es una de las áreas actualmente más prometedoras dentrode la investigación Biomédica para reparar patologías cardiovasculares congénitas o adquiridas en pacientes neonatos y adultos. Estos implantes deberán estar formadospor un material biodegradable adecuado recubierto de células cultivadasque permitan una sustitución progresiva y completa del tejido dañado. El diseñoy la obtención de implantes biodegradables funcionales requiere una aproximaciónmultidisciplinar y una investigación coordinada en las áreas de Cirugía Cardiovascular,Biomateriales, Bioquímica, Biología Celular y Bioingeniería.Los grupos de investigación Biomédica de las Universidades Complutense(UCM), Rey Juan Carlos (URJC), Politécnica (UPM) y el Instituto Pediátrico delCorazón (IPC) - Cirugía Cardiaca Infantil del Hospital Universitario «12 de Octubre» de Madrid, desarrollan un proyecto coordinado y multidisciplinar para laobtención de implantes biodegradables autólogos y no trombogénicos, mediantetécnicas de Ingeniería de tejidos, que puedan cumplir todas las característicasrequeridas desde el punto de vista de adecuación del soporte, funcionalidad bioquímicay resistencia mecánica a las técnicas quirúrgicas habituales y que presentenuna capacidad de crecimiento acorde con el desarrollo del paciente, evitandolas reoperaciones que se requieren en la actualidad al utilizar prótesis artificiales.El biopolímero poli(ε-caprolactona) (PCL), modificado para mejorar la adhesióny proliferación celular, se ha seleccionado como soporte para el cultivo dedistintas poblaciones celulares (endotelio, músculo liso vascular, HUVEC y célulasmesenquimales) realizándose estudios de biocompatibilidad, biofuncionalidad invitro y pruebas de resistencia mecánica para comprobar la viabilidad de los bioimplantes

The development of biodegradable grafts for Cardiovascular Surgery by TissueEngineering techniques is at present a promising research field in Biomedicalresearch for repairing both congenital and acquired cardiovascular diseases inneonatal and adult patients. These grafts should be constituted by a suitable biodegradablematerial covered with cultured cells that allows a progressive and completesubstitution of the damaged tissue. The design and preparation of functionalbiodegradable grafts requires a multidisciplinary approach and a coordinated researchin Cardiovascular Surgery, Biomaterials, Biochemistry, Cell Biology andBioengineering fields. The biomedical research groups of the Universidad Complutense(UCM), Rey Juan Carlos (URJC), Politécnica (UPM) and the Instituto Pediátricodel Corazón (IPC) – Cirugía Cardiaca Infantil of the Hospital Universitario«12 de Octubre» de Madrid, develop a multidisciplinar coordinated project forobtaining autologue, non trombogenic and biodegradable grafts, by Tissue Engineeringtechniques, that fulfill specific characteristics, from the point of view ofscaffold’s adequacy, biochemical function and mechanical resistance to the usualsurgical techniques with growth capacity in agreement with the patient development avoiding the successive operations that are necessary nowadays when artificialprosthesis are used.The biopolymer poli(ε-caprolactona) (PCL), modified to improve the cell adhesionand proliferation, has been selected as scaffold for culturing different cells(endothelial, vascular smooth muscle, HUVEC and mesenchymal cells) carryingout in vitro biocompatibility, biofunctionality studies and mechanical assays toevaluate the grafts’ viability

Osteonecrosis of the jaw. A newly emerging site-specific osseous pathology in patients with cancer treated with bisphosphonates. Report of five cases and review of the literature.

BACKGROUND: Bisphosphonates are commonly used as standard care in the management of patients with advanced-stage cancer involving bone. There has recently been growing concern that the use of bisphosphonates is associated with osteonecrosis of the jaw (ONJ). METHODS: Between 2001 and 2005, five patients with ONJ associated with pamidronate and zoledronate therapy were diagnosed at our department. The patients had breast cancer, renal carcinoma, mesothelioma, and multiple myeloma, all involving bone. The literature was reviewed. RESULTS: The duration of bisphosphonate therapy before presentation of ONJ ranged from 21 to 36 months. The lesions were localized to the mandible (n=3) and maxilla (n=2). All of the patients presented with pain and exposed bone; in two of them, symptoms began after tooth extraction. A review of the literature through March 2006 identified more than 250 reported cases of ONJ. CONCLUSIONS: The findings in our patients, combined with the literature review, suggest that: (1) the most common clinical presentation of ONJ is pain and exposed bone of the mandible or maxilla; (2) for patients who develop ONJ, conservative, non-surgical treatment is strongly recommended; (3) clinical dental examination and a panoramic jaw radiograph should be performed before patients begin bisphosphonate therapy; (4) dental treatment and other oral procedures should be completed before initiating bisphosphonate therapy; (5) patients should be informed and instructed on the importance of maintaining good oral hygiene and having regular dental assessment; and (6) the medical community needs to be aware of the association between bisphosphonate usage and ONJ so that unnecessary and harmful surgical procedures can be avoided.

Mitochondrial membrane potential and reactive oxygen species content of endothelial and smooth muscle cells cultured on poly(epsilon-caprolactone) films.

A transitory but significant stimulation of mitochondrial activity, increase of reactive oxygen species (ROS) and oxidative stress were previously observed in L929 fibroblasts cultured on poly(epsilon-caprolactone) (PCL) films. ROS, mainly formed in mitochondria, play a physiological role but an excessive production can promote endothelial dysfunction, cause oxidative injury to vascular cells, oxidize lipoproteins and accelerate atherothrombogenesis. On the other hand, mitochondria have a crucial position in programmed cell death control and are responsible for ATP synthesis through the coupling of oxidative phosphorylation to respiration. This coupling requires the existence of a mitochondrial membrane potential (Deltapsi(m)). The aim of the present study was to evaluate by flow cytometry the ROS content and Deltapsi(m) of both endothelial (EC) and smooth muscle cells (SMC) cultured on PCL films as a potential substrate for vascular graft development. Cell size, internal complexity and cell cycle were also analyzed to detect the possible appearance of the subG(1) cell fraction, characteristic of apoptotic cells. The effect of treating PCL films with NaOH before culture was also studied. PCL decreases the ROS content of EC during the culture but produces an increase of these levels in SMC after 7 days. PCL also induces variations of Deltapsi(m) which show a significant parallelism with the changes observed in ROS levels proving the importance and sensitivity of these measurements as indicators of the mitochondrial function. The treatment of PCL with NaOH decreases these effects demonstrating the benefits of increasing the surface hydrophilicity before cell culture which improves cell adhesion and proliferation and reduces oxidative stress. Since no important changes have been detected in subG(1) fraction of EC and SMC cultured on either PCL or PCL-NaOH, the changes of Deltapsi(m) observed in the present study cannot be related to apoptosis. These results confirm the potential utility of PCL as a suitable scaffold in Vascular Tissue Engineering.

Alkaline-treated poly(epsilon-caprolactone) films: degradation in the presence or absence of fibroblasts.

In the first stage, we observed the study of the degradation behavior of alkaline-treated poly(epsilon-caprolactone) (PCL) in two biologically-related media: phosphate buffered saline (PBS) and Dulbecco's modified Eagle's medium (DMEM) for 18 months, finding a much accelerated degradation in the last one. As expected, the degradation in the presence of cells is much pronounced even considering that the study is limited to 6 months. The characterization of the degraded substrates by chemiluminescence (CL) allows to explain the modifications of the substrate and their relations with transitory oxidative stress phenomena described in the fibroblasts seeded onto the PCL membranes.

Transitory oxidative stress in L929 fibroblasts cultured on poly(epsilon-caprolactone) films.

Poly(epsilon-caprolactone) (PCL) is considered as a potential substrate for wide medical applications. In previous studies we carried out the in vitro biocompatibility assessment of PCL films using L929 mouse fibroblasts, obtaining good cell behaviour but a transitory stimulation of mitochondrial activity and cell retraction. Reactive oxygen species (ROS), mainly formed in mitochondria, can impair the function of several cellular components and produce cell oxidative stress by changing the normal red-ox status of the major cell antioxidants as glutathione. The aim of this study was to measure intracellular ROS production and glutathione content of L929 fibroblasts cultured on PCL films. Cell size, internal complexity, cell cycle and lactate dehydrogenase release were also evaluated. The films were treated with NaOH before culture to improve the cell-polymer interaction. PCL induces a transitory but significant oxidative stress in L929 fibroblasts. The treatment of PCL films with NaOH reduces this effect. PCL also induces transitory changes on cell size and complexity. Nevertheless, after 7 days in culture, cells reach control levels for all the studied parameters. Neither cell cycle nor membrane integrity appears affected by this oxidative stress respect to control cells at any culture time. These results underline the cytocompatibility of PCL films and, therefore, its potential utility as a suitable scaffold in tissue engineering.

Vascular endothelial and smooth muscle cell culture on NaOH-treated poly(epsilon-caprolactone) films: a preliminary study for vascular graft development.

Tissue engineering offers the potential of providing vessels that can be used to replace diseased and damaged native blood vessels. The endothelization of a synthetic vascular graft minimizes the failures associated with blood clotting and platelet activation. The aim of this study was to culture vascular-derived endothelial and smooth muscle cells on both untreated and NaOH-treated poly(epsilon-caprolactone) (PCL) films, a biocompatible and bio-resorbable polymer, and to evaluate the behavior of both cell types as a preliminary study for vascular graft development. PCL films were prepared by hot pressing; characterized by DSC, IR, SEM, and scanning force microscopy; and treated with NaOH to increase the surface hydrophilicity before cell culture. Endothelial and smooth muscle cells, isolated from pig cava vein, were characterized by immunofluorescence and confocal microscopy studies of endothelial nitric oxide synthase and alpha-smooth muscle actin. Good adhesion, growth, viability and morphology of both the endothelial and smooth muscle cells on PCL films were obtained, but a light stimulation of mitochondrial activity was observed during short culture times. NaOH treatment improved the adhesion and enhanced the proliferation in both cell types. This verified the possible use of this modified polymer as a support in the preparation of a synthetic vascular graft. [Diagram: see text] SEM micrograph of smooth muscle cells cultured on NaOH-treated PCL film. (Original magnification: 1000x).