Papers by Author: José Mauro Granjeiro

Abstract: Incorporation of bioceramics on the surface of dental implants has been utilized in an attempt to increase biological response of bone to materials. This paper reports the in vitro biological evaluation of Ca/P-based nanothickness bioceramic coated alumina-blasted/acid-etched titanium implants (AB/AE nanotite implant) and compare its performance to the untreated and uncoated implants, Ca/P-based nanothickness bioceramic coated untreated implants (untreated nanotite implant), alumina-blasted/acid-etched titanium implants (AB/AE implant) and hydroxyapatite plasma-sprayed implants (PSHA Implant). Balb/c 3T3 fibroblasts were used to asses the cytocompatibility of implant materials according to ISO-10993-5 protocols. Osteoblasts from Balb/c femurs seeded onto different implant surfaces showed the effect of surface topography and chemistry on cell adhesion. The results showed that all implants were not cytotoxic and that PSHA and AB/AE nanotite implants favored osteoblasts adhesion.

Abstract: The aim of this paper was to evaluate the usefulness of coupling digital image analysis with immunohistochemistry and histomorphometry data to the study of tissue response to hydroxyapatite in a model of critical size bone defect in calvaria of rats. A transosseous defect measuring 8 mm in diameter was performed with a surgical trephine in the parietal bone of 40 rats and divided into two experimental groups according to the treatment: group I (blood clot, control), group II (HA) and killed 1, 3, 6 and 9 months after implantation (n=5/group/period). The skullcaps with overlaying skin were collected and processed for paraffin embedding. The specimens were cut in the laterolateral direction into 5-µm thick semi-serial sections and stained with hematoxylin-eosin for identification and counting of polymorphonuclears cells, mastocytes, and multinucleated giant cells, MNG, or immunolabeled with anti- lysozyme, -factor VIII and –PCNA. Digital images were obtained and analyzed with the ImagePro-Plus® software for cell couting (polymorphonuclears cells, mastocytes, macrophages and MNG) and microvessel density. Image segmentation of anti-PCNA immunostaining was used for cell proliferation analysis. The digital images obtained allowed clear identification of cells of interest by through morphological aspects or immunostaining. Data recording and analysis was facilitated by the use of specific software for image processing and graphical and statistical analysis. It can be concluded that the techniques applied were usefull to identify and count cells, structures and process of interest making easier the effectiveness of hydroxyapatite in the critical size defect in rat calvaria model.

Abstract: The aim of this study was verify the biological efficacy of the use of a xenograft for bone loss therapy. Blood clot, particulate autogenous bone or anorganic bovine xenograft filled critical size defects (CSD) in rat calvaria (8mm diameter). After 0, 7, 30 and 90 days the animals were killed and macroscopic, radiographic and histopathological analysis were conducted. Although no treatment promoted the total closure of bone defect, autogenous bone group had better bone repair after 90 days, followed by xenograft group that exhibited direct bone neoformation onto, and around, the particles confirming its osteoconductivity. In conclusion, the xenograft tested in vivo showed biocompatibility, biodegradability and osteoconductive properties in rat calvaria CSD.

Abstract: The study aimed the evaluation of the effect of osseous repair in the synthetic hydroxyapatite cylinder (HA) implanted in rabbit’s tibia. Fifteen adult animals, weighing around 2.5 and 3.0 Kg, sourced by the Fluminense Federal University Animal Lab were anesthetized with spinal anesthesia, and two perforations were created in right tibia and filled with cylinders (2 x 6 mm) of HA. The animals were killed after 28 days for evaluating the HA cylinder through attenuated total reflection infrared microscopy (ATR-FTIR). The two fragments of tibia with the cylinder were collected and fixed in alcohol 70° prior to dehydration in successive alcohol solutions and then impregnated and embedded in methyl-methacrylate. Not stained neither pasted to lamina, sections of 200 µm thickness were analyzed in a Shimadzu IRPrestige-21 Automatic Infrared Microscope (AIM-8800) with ATR Objective Mirror (ATR-8800M). Prior to implantation, HA cylinder was characterized by X-Ray diffraction (XRD), Attenuated Total Reflection- Infra Red microscopy (ATR-FTIR) and X-Ray Fluorescence (XRF). After the implantation, the spectrum of the ATR-FTIR at the interface to neoformed bone was acquired. The hydroxyapatite synthesized was crystalline and stoichiometric with Ca/P ratio of 1.66, becoming carbonated after 28 days of implantation. We conclude that attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) is a useful tool for biomaterial evaluation and able to detect the modification of the chemical HA cylinder pattern occurred probably by ions migration in the biological environment.

Abstract: Biomaterials for treatment of bone defects have been studied for a long time. Alloplastic materials, mainly hydroxyapatite (HA), are under intense investigation due to its biocompatibility and osteoconductive properties. The HA can be modified by the incorporation of bivalent cations as Zn2+ known as a positive effectors for bone repair. The purpose of this study was to evaluate comparatively the effectiveness of 5% zinc-containing hydroxyapatite (ZnHA) in the treatment of critical size defect (CSD) in rat’s calvaria. CSD (8mm diameter) created in the skull of forty-five Wistar rats were filled with autogenous bone, HA and ZnHA. Skulls harvested after 30, 90 and 180 days were submitted to histological processing for paraffin embedding. Sections of 5 µm-thick stained with hematoxylin and eosin (HE) allowed histomorphometric analysis. The area of neoformed bone increased (p<0.001) from 30 to 180 days irrespective to treatment groups. ZnHA and the control group showed a large at 180 days but no significant difference compared to HA. Therefore, we concluded that both biomaterials are biocompatibles and osteoconductors, promoting new bone formation and apposition of bone on the surface throughout the periods and the addition of zinc improved the osteogenesis.

Abstract: Porous granules without organic residues were produced from bovine medular anorganic bone. Sample showed carbonate content and crystalline order similar to natural bone. The cytotoxicity of anorganic bovine xenograft (Ossĕus®) was valuated from fibroblasts (1.5x104/cm2) cultured in serial diluted extract (0 – 100%) of Ossĕus plus 10% of fetal bovine serum (FBS). Pure extract (100%) reduced in 15% the number of viable cells (p<0.05, ANOVA, Tukey test) indicating very low cytotoxicity. Samples were implanted in the subcutaneous tissue of mice in order to evaluate tissue reaction. An organized connective tissue in contact to the granules was observed nine weeks after implantation. The anorganic bovine xenograft (Ossĕus®) was biocompatible and its behavior and osteoconduction potential should be evaluated in bony defects.

Abstract: In order to improve implant-bone attachment, porous titanium has been used to achieve the ingrowth of bone tissue within the porous structure. Although this biomaterial has shown efficient bone adhesion for orthopedic and dental implants, the ideal surface must have chemical bonds at the implant-bone interface. In this work, samples of pure porous titanium were produced by powder metallurgy technique and submitted to biomimetic process in order to evaluate the material’s bioactivity and to enhance its osteoconductivity. The samples were immersed in modified simulated body fluid (mSBF) which induces the nucleation and growth of a calcium phosphate bioactive film and a chemical bond with titanium. SEM, EDX and FTIR analyses showed that a calcium phosphate deposition occurred without the need of pre-treatments to increase the surface bioactivity. As a result, this research revealed the potential for obtaining a bonelike apatite film on this porous titanium by biomimetic method.

Abstract: Hydroxyapatite (HA), widely used as bone graft, can be modified by the incorporation of bivalent cations (Mg2+ and Zn2+) and its gradual release could favor the bone repair. The purpose of this research was to evaluate the effect of the HA and zinc-containing hydroxyapatite (Zn-HA) in the bone repair in rat calvaria in comparison to autogenous bone. Critical size defect in the calvaria was filled with the graft material and the samples were harvested at the 30, 90 and 180 days. The light microcopy observations showed the biocompatibility of the graft materials. In the Zn-HA group the area of neoformed bone was larger than in the HA group, but smaller than in the autograft. A fibrous connective tissue was more evident around HA granules. It could be conclude that the presence of zinc ions in HA crystal accelerated the osteogenesis and increased the area of newly formed bone in relation to HA.

Abstract: The study was carried out aiming the evaluation of the effect of hydroxyapatite (HA) and zinc-substituted hydroxyapatite (ZnHA) on osseous repair of rabbit’s tibia. For the study, 15 adult animals, weighing around 2.5 and 3.0 Kg, sourced by the Fluminense Federal University Animal Lab were acquired. Two perforations were created in each tibia and filled with cylinders (2x6 mm) of HA (group 1, right) or ZnHA (group 2, left). The animals were killed after 7, 14, and 28 days for evaluating the histological aspects of the interface site and bone repair. No sign of inflammatory reaction surrounding the cylinders area were observed, neither giant cells. Osteogenesis was evidently accelerated in all healing periods for the ZnHA group in regards to the HA group. Randomly dispersed areas of neoformed bone among wide areas of fibrous connective tissue was observed in the HA group. We conclude that both biomaterials are biocompatible, but zinc-containg HA enhanced and accelerated the osteogenesis in relation to HA.

Abstract: Hydroxyapatite (HA) and apatite based biomaterials are important for bone replacement. Different apatites could be produced by substituting calcium, phosphate or hydroxyl groups, resulting in new materials with different physical, chemical and biological properties. In this work we investigate the biological compatibility of apatites modified by divalent cations in cultured mammalian cells using Balb/c 3T3 fibroblasts cell line. Modified apatites, Ca9.5M0.5(PO4)6(OH)2 (M=Fe, Zn, Cu, Co, Sr, V e Pb) and Ca10(VO4)6(OH)2 were produced and characterized by FTIR, XRD and XRF. Extracts of each metal-modified apatites (0.1mg/mL of Dulbeco Modified Eagle Medium – DMEM without serum) were obtained. Cells (3x104) were exposed for 24 h/37C to the pure extract (100%). Afterwards, the number of viable cells was determined in a hemocitometer. The number of viable cells in the absence of any extract was taking as 100%. SHA didn’t present any toxicity while phenol killed 82% of the cells. In the presence of pure extract of Fe-HA or Sr- HA an increase of 70% and 20%, respectively, in the number of relative cells was observed. On the other hand, the number of residual cells after treatment with the pure extract of Pb+2, Zn+2, Co+2, and (VO4)3- was73, 65, 48, and 21%, respectively. In conclusion, cells response was strongly dependent on the metal that substitutes calcium or phosphate. Further studies are required to better understand the biological effect of these substitutions.