A new sheep model with automatized analysis of biomaterial-induced bone tissue regeneration.

Presently, several bone graft substitutes are being developed or already available for clinical use. However, the limited number of clinical and in vivo trials for direct comparison between these products may complicate this choice. One of the main reasons for this scarcity it is the use of models that do not readily allow the direct comparison of multiple bone graft substitutes, due especially to the small number of implantation sites. Although sheep cancellous bone models are now well established for these purposes, the limited availability of cancellous bone makes it difficult to find multiple comparable sites within a same animal. These limitations can be overcome by the monocortical model here proposed as it consists in 5-6 holes (5 mm Ø), in the femoral diaphysis, with similar bone structure, overlying soft tissue and loading pattern for all defects. Associated to this model, it is also described a fast histomorphometric analysis method using a computer image segmentation test (Threshold method) to assess bone regeneration parameters. The information compiled through the experimental use of 45 sheep in several studies allowed determining that this ovine model has the potential to demonstrate differences in bone-forming performance between various scaffolds. Additionally, the described histomorphometric method is fast, accurate and reproducible.

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 ß-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 material's structure and Bonelike(®) spherical 250-500 µm appeared to induce faster bone regeneration, presenting less unfilled areas and less lacunae in the histological analysis.

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 ß-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.

In vitro and in vivo chitosan membranes testing for peripheral nerve reconstruction.

Tissue regeneration over a large defect with a subsequent satisfactory functional recovery still stands as a major problem in areas such as nerve regeneration or bone healing. The routine technique for the reconstruction of a nerve gap is the use of autologous nerve grafting, but still with severe complications. Over the last decades several attempts have been made to overcome this problem by using biomaterials as scaffolds for guided tissue regeneration. Despite the wide range of biomaterials available, functional recovery after a serious nerve injury is still far from acceptable. Prior to the use of a new biomaterial on healing tissues, an evaluation of the host's inflammatory response is mandatory. In this study, three chitosan membranes were tested in vitro and in vivo for later use as nerve guides for the reconstruction of peripheral nerves submitted to axonotmesis or neurotmesis lesions. Chitosan membranes, with different compositions, were tested in vitro, with a nerve growth factor cellular producing system, N1E-115 cell line, cultured over each of the three membranes and differentiated for 48h in the presence of 1.5% of DMSO. The intracellular calcium concentrations of the non-differentiated and of the 48h-differentiated cells cultured on the three types of the chitosan membranes were measured to determine the cell culture viability. In vivo, the chitosan membranes were implanted subcutaneously in a rat model, and histological evaluations were performed from material retrieved on weeks 1, 2, 4 and 8 after implantation. The three types of chitosan membranes were a viable substrate for the N1E-115 cell multiplication, survival and differentiation. Furthermore, the in vivo studies suggested that these chitosan membranes are promising candidates as a supporting material for tissue engineering applications on the peripheral nerve, possibly owing to their porous structure, their chemical modifications and high affinity to cellular systems.

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.

Jaw avascular osteonecrosis after treatment of multiple myeloma with zoledronate.

PURPOSE: Multiple myeloma, the second most common haematopoietic cancer, which represents the collection of plasma-cell neoplasms that invariably becomes fatal when self-renewing myeloma cells begin unrestrained proliferation. The major clinical manifestation of multiple myeloma is related to the loss of bone through osteolysis. This can lead to pathologic fractures, spinal cord compression, hypercalcaemia, and pain. It is also a major cause of morbidity and mortality in these patients, who frequently require radiation therapy, surgery and analgesic medications. Bisphosphonates are specific inhibitors of osteoclastic activity, and are currently used to prevent bone complications and to treat malignant hypercalcaemia in patients with multiple myeloma, or bone metastases from breast and prostate cancers. Hence, osteonecrosis of the mandible has been reported in three patients from Centro Hospitalar de Vila Nova de Gaia (CHVNG) with multiple myeloma treated for over 18-48 months with intravenous zoledronate, commonly prescribed for multiple myeloma therapy. Although, this report alerts clinicians about the potential complication of bone necrosis in patients receiving bisphosphonate therapy, many questions remain concerning the underlying pathogenesis of this process. PATIENTS AND METHODS: The medical and dental records of three patients with multiple myeloma, who were treated in CHVNG in the past 4 years, were reviewed. These three patients presented exposed bone and osteonecrosis of the mandible, and shared one common clinical feature: all of them were treated with bisphosphonate zoledronate, administered intravenously for long periods. Sequential orthopantomograms (OPGs) and histological evaluation have been analysed from the biopsies of the non healing dental extraction sites of these patients. RESULTS: After a routine dental extraction, these patients developed avascular osteonecrosis of the mandible and secondary bone infection with Actinomyces israelii (actinomycotic osteomyelitis), with no evidence of metastatic disease evaluated by biopsy. In these three described clinical cases, surgical debridment without flap elevation, intensive antibiotherapy and the suspension of the zoledronate treatment allowed a partial recovery of the patients. The purpose of this clinical report is to point out that patients suffering from multiple myeloma can develop bone osteonecrosis induced by treatment with bisphosphonates. Research to determine the mechanism of this dental phenomenon is needed to fully validate and substantiate the possible link between bisphosphonate treatment of multiple myeloma or other cancer diseases and avascular osteonecrosis of the jaws. Until then, clinicians involved in the care of patients at risk should consider this possible complication.