Paulo Cortez

Characterization and preliminary in vivo evaluation of a novel modified hydroxyapatite produced by extrusion and spheronization techniques

A glass-reinforced hydroxyapatite (HA) composite, recently registered as Bonelike®, was developed for bone grafting. This biomaterial is composed of a modified HA matrix with α- and β-tricalcium phosphate secondary phases and ionic species that mimic the chemical composition of human bone. Several in vitro and in vivo studies have confirmed the benefits of these properties. However, these studies were all executed with Bonelike® polygonal granules obtained by crushing. In this study, Bonelike® pellets were produced through a patented process, which required the use of techniques such as extrusion and spheronization. The final product presented a homogeneous size, a 55.1% global porosity and a spherical shape. This spherical shape permitted a better adaptation to the implantation site and improved injectability. Additionally, it also may contribute to formation of macropores as pellets packaging leaves open spaces. After implantation of Bonelike® polygonal granules and Bonelike® pellets in monocortical defects in sheep for 8 and 12 weeks, light microscopy and scanning electron microscopy showed extensive osteointegration simultaneously with bone regeneration for both presentations. Histomorphometric analysis did not reveal statistically significant differences between defects treated with Bonelike® polygonal granules and Bonelike® pellets, which suggests similar in vivo performances.

A glass-reinforced hydroxyapatite and surgical-grade calcium sulfate for bone regeneration: in vivo biological behavior in a sheep model

A glass-reinforced hydroxyapatite (HA) composite (Bonelike®) was developed for bone grafting. This biomaterial is composed of a modified HA matrix with α- and β-tricalcium phosphate secondary phases, resulting in higher solubility than single HA type of materials. Several in vitro and in vivo studies demonstrated that Bonelike® has a highly bioactive behavior, which was also confirmed by employing granular forms of this biomaterial in orthopedics and dental applications. However, a fast consolidation vehicle was needed to promote the fixation of Bonelike® granules if applied in larger defects or in unstable sites. Surgical-grade calcium sulfate (CS), which is widely recognized as a well-tolerated and inexpensive bone graft material, was the chosen vehicle to improve the handling characteristics of Bonelike® as it can be used in the form of a powder that is mixed with a liquid to form a paste that sets in situ. After application in non-critical monocortical defects in sheep, histological, and scanning electron microscopy evaluations demonstrated that Bonelike® associated to CS functioned as a very satisfactory scaffold for bone regeneration as it achieved synchronization of the ingrowing bone with biomaterial resorption and subsequent preservation of the bone graft initial volume. Therefore, our results indicate that CS is an effective vehicle for Bonelike® granules as it facilitates their application and does not interfere with their proven highly osteoconductive properties. In the opposite way, the incorporation of Bonelike® improves the bone regeneration capabilities of CS.

Morphology effect of bioglass-reinforced hydroxyapatite (Bonelike(®) ) on osteoregeneration.

In the last decades, the well-known disadvantages of autografts and allografts have driven to the development of synthetic bone grafts for bone regeneration. Bonelike(®) , a glass-reinforced hydroxyapatite (HA) composite was developed and registered for bone grafting. This biomaterial is composed by a modified HA matrix, with α- and β-tricalcium phosphate secondary phases. Aiming to improve the biological characteristics of Bonelike(®) , new spherical pelleted granules, of different shape and size, were developed with controlled micro and macrostructure. In the present study, it was compared the physicochemical properties and in vivo performance of different Bonelike(®) granule presentations-Bonelike(®) polygonal (500-1000 µm size) and Bonelike spherical (250-500 µm; 500-1000 µm size). For the in vivo study, Bonelike(®) was implanted on sheep femurs, with various implantation times (30 days, 60 days, 120 days, and 180 days). X-ray diffraction analysis revealed that the phase composition of different granules presentations was similar. Bonelike(®) spherical 500-1000 µm was the most porous material (global porosity and intraporosity) and Bonelike(®) polygonal 500-1000 µm the less porous. Considering the in vivo study, both polygonal and spherical granules presented osteoconductive proprieties. The spherical granules showed several advantages, including easier medical application through syringe and improved osteointegration, osteoconduction, and degradation, by the presence of larger pores, controlled micro- and macrosctructure and suitable particle format that adapts to bone growth. Bonelike(®) spherical 500-1000 µm showed improved new bone invasion throughout the materials structure and Bonelike(®) spherical 250-500 µm appeared to induce faster bone regeneration, presenting less unfilled areas and less lacunae in the histological analysis.

The in vivo performance of an alkali-free bioactive glass for bone grafting, FastOs(®) BG, assessed with an ovine model.

Although bioactive glasses are successfully used as bone substitutes, recent studies have revealed that the high alkali content in these glasses leads to fast in vivo degradation rates that may not match the rate of new bone ingrowth. This prompted us to design and develop novel bioactive glasses that are devoid of alkali but still demonstrate high bioactivity in vitro. This article describes the in vivo performance of an alkali-free bioactive glass with the following composition (Wt %): 13.03 MgO-33.98 CaO-13.37 P2 O5 -38.84 SiO2 -0.77 CaF2 (labelled as FastOs® BG). An animal model was used to assess the in vivo performance of FastOs® BG, using 45S5 Bioglass® as control. The evaluation was performed through implantation of FastOs® BG and 45S5 Bioglass® , during one month, in femoral bone defects in sheep. Subcutaneous implantation of both glasses was also performed in order to assess tissue response through a standardized method. Histological and scanning electron microscopy assessment of retrieved subcutaneous and bone samples demonstrated that FastOs® BG is biocompatible, osteoconductive, that it can be osteointegrated, and that it is more slowly resorbed than 45S5 Bioglass® . These features suggest that FastOs® BG is a potential candidate for bone grafting.

Caveolin-1 in Diagnosis and Prognosis of Canine Mammary Tumours: Comparison of Evaluation Systems

Caveolin-1 (Cav-1) is a major structural protein of caveolae, plasma membrane invaginations related to several cellular processes including regulation of signal transduction. In recent years there has been some controversy regarding the distribution of Cav-1 in normal and neoplastic mammary cell types, which may be attributed to different scoring systems adopted in different studies. The present study compares Cav-1 immunoexpression in normal (n=17) and neoplastic (n=79) canine mammary tissues assessed by two different scoring methods (previously reported by others with conflicting results) and associates Cav-1 expression with metastasis and overall survival (OS). Results obtained with both scoring methods were similar, revealing absence of immunoreactivity in normal luminal epithelium and in benign neoplasms and clearly associating Cav-1 expression with malignant transformation. The data suggest that Cav-1 expression is associated with highly malignant subtypes of mammary tumours (i.e. basal-like carcinoma), invasion and metastasis, thus supporting the hypothesis that it may play a major role in the epithelial-mesenchymal transition process. Furthermore, one of the scoring systems employed associated Cav-1 expression with unfavourable prognosis in canine mammary carcinomas, showing a strong correlation between Cav-1-positive carcinomas and shorter OS.

Coccidioidomycosis in a cat imported from the USA to Portugal

COCCIDIOIDOMYCOSIS is a mycotic infection caused by the dimorphic fungus Coccidioides species ( C immitis and C posadasii ) that affects both human beings and domestic animals (Gross and others 2005, Barker and others 2007, Caswell and Williams 2007, Parish and Blair 2008). Inhalation of airborne arthroconidia is responsible for a systemic disease in animals characterised by fever, anorexia, depression, cough and dyspnoea (Gross and others 2005, Caswell and Williams 2007). Neurological, ophthalmological, cardiac and osseous manifestations have also been reported (Caswell and Williams 2007, Shubitz 2007, Graupmann-Kuzma and others 2008). Less commonly, local inoculation of Coccidioides species may produce a solitary cutaneous lesion, usually without systemic involvement (Plotnick and others 1997, Gross and others 2005). Coccidioidomycosis is an endemic zoonosis in the south-western USA, northern Mexico and some areas of Central and South America, and remains very rare outside endemic regions (Caswell and Williams 2007, Parish and Blair 2008). However, at a time when the movement of people and their companion animals is increasingly common, it is important to be aware that they can carry a variety of infectious agents from endemic to disease-free regions, thus contributing to the emergence of imported diseases. In fact, during the past decade, some cases of coccidioidomycosis have been reported in European and Asian citizens who had travelled to endemic areas (Chandesris and others 2008, Hombach and others 2008, Indhirajanti and others 2009, Kwok and others 2009). However, to date there are no documented cases of coccidioidal infection in animals outside endemic regions. This short communication describes a case of coccidioidomycosis in Europe, imported by a cat travelling from the south-western USA. A four-year-old male domestic shorthair cat, born and resident in the state of Texas, was brought to …

Hybrid Chitosan Membranes Tested in Sheep for Guided Tissue Regeneration

Previous in vitro studies confirmed an improved cytocompatibility of chitosan-silicate hybrid membranes over chitosan membranes. The main goal of this study was to assess the in vivo histocompatibility of both membranes through subcutaneous implantations at different time periods, 1 week, 1, 2 and 3 months, using a sheep model. Chitosan membranes elicited an exuberant inflammatory response and were consequently rejected. The hybrid chitosan membranes were not rejected and the degree of inflammatory response decreased gradually until the third month of implantation. Histological evaluation also showed that these membranes can be resorbed in vivo. This study demonstrates that the incorporation of silicate into the chitosan solution improves its histocompatibility, indicating that the hybrid chitosan-silicate membranes are suitable candidates to be used in clinical applications.

Assessing in vivo digestibility and effects on immune system of sheep fed alfalfa hay supplemented with a fixed amount of Ulva rigida and Gracilaria vermiculophylla

Ruminants could be the most suitable domestic animals to be supplemented with seaweeds as the rumen ecosystem might provide the animal the ability to use these feed resources by breaking down the complex polysaccharides. The objective of the present in vivo study was to determine the digestibility and the effects on the immune system of one green (Ulva rigida) and one red (Gracilaria vermiculophylla) seaweed cultivated in an integrated multitrophic aquaculture system (IMTA) and included in the diet of sheep at a supplementing level up to 25%. Both seaweeds showed lower dry matter digestibilitity than alfalfa hay, the organic matter digestibility of U. rigida being higher than that of G. vermiculophylla. The studied seaweeds had similar fiber and energy digestibility. Seaweed supplementation did not influence hematological parameters, reactive oxygen species production by neutrophils, nor lymphocytic response to T and B cells mitogens. The low fiber digestibility of selected seaweeds would be the major constraint to their use in high amounts in ruminant diets. Dietary seaweed supplementation has no deleterious effect on the immune function of cells mediating innate and acquired immunity.

The Benefit Of Bone Marrow Concentrate In Addition To A Glass‐Reinforced Hydroxyapatite For Bone Regeneration: An In Vivo Ovine Study

This study evaluates the ability of a Glass Reinforced Hydroxyapatite Composite (GRHC), in a new microporous pellet formulation with autologous bone marrow concentrate (BMC), to enhance bone regeneration and new bone formation. Ninety non‐critical sized bone defects were created in the femurs of nine Merino breed sheep and randomly left unfilled (group A), filled with GRHC pellets alone (group B) or filled with GRHC pellets combined with BMC (group C). The sheep were sacrificed at 3 weeks (three sheep), 6 weeks (three sheep) and 12 weeks (three sheep) and histological analysis (Light Microscopy‐LM), scanning electron microscopy (SEM) and histomorphometric analysis (HM) were performed. At 3, 6, and 12 weeks, HM revealed an average percentage of new bone of 48, 72, 83%; 25, 73, 80%, and 16, 38, 78% for Groups C, B and A respectively (significantly different only at 3 weeks p < 0.05). LM and SEM evaluation revealed earlier formation of well‐organized mature lamellar bone in Group C. This study demonstrates that the addition of a bone marrow concentrate to a glass reinforced hydroxyapatite composite in a pellet formulation promotes early bone healing.