Gustavo Vicentis de Oliveira Fernandes

A randomized controlled clinical trial to evaluate a new xenograft for alveolar socket preservation

OBJECTIVE The aim of this clinical trial was to compare the effect of Bio-Oss(®) and a new bovine xenograft (Osseus(®) ) in alveolar sockets after a 24-week healing period. MATERIALS AND METHODS A total of 20 adult volunteers ages 30-60 were subjected to single tooth extraction. A tooth extraction was performed at the baseline. All sites were randomly allocated to two test groups (TG1: grafted using a new bovine xenograft, Osseus(®) , and TG2: grafted using commercially available bovine xenograft-Bio-Oss(®) ). Six months later, a sample of the grafted area was obtained and implants were inserted in the same site. Histological sections were examined focusing on the presence of fibrous connective tissue (CT), and newly formed bone in direct contact with the graft. The HE-stained sections were subjected to histomorphometrical evaluation using Image Pro-Plus(®) software (Release 7.0). The definitive crown was placed 3 months later. RESULTS Upon completion of the study, no patients were removed from the study and all inserted implants (10 in each group) were eventually integrated. After 6 months, in the TG1, the mean value of new bone formation was 33.7 (± 7.1), for CT was 32.3 (± 8.9) and for the remaining biomaterial was 10.7 (± 16.2). In the TG2, the mean value of new bone formation was 19.3 (± 22.6), of the CT was 49.9 (± 14.1) and of the remaining biomaterial was 22.6 (± 7.9). CONCLUSIONS No statistically significant difference was observed between TG1 and TG2 after 6 months (P > 0.05), and both biomaterials afforded a more favorable implant position.

Cytocompatibility of Porous Biphasic Calcium Phosphate Granules With Human Mesenchymal Cells by a Multiparametric Assay

This work aims to evaluate the cytocompatibility of injectable and moldable restorative biomaterials based on granules of dense or porous biphasic calcium phosphates (BCPs) with human primary mesenchymal cells, in order to validate them as tools for stem cell-induced bone regeneration. Porous hydroxyapatite (HA) and HA/beta-tricalcium phosphate (β-TCP) (60:40) granules were obtained by the addition of wax spheres and pressing at 20 MPa, while dense materials were compacted by pressing at 100 MPa, followed by thermal treatment (1100°C), grinding, and sieving. Extracts were prepared by 24-h incubation of granules on culture media, with subsequent exposition of human primary mesenchymal cells. Three different cell viability parameters were evaluated on the same samples. Scanning electron microscopy analysis of the granules revealed distinct dense and porous surfaces. After cell exposition to extracts, no significant differences on mitochondrial activity (2,3-bis(2-methoxy-4-nitro-5-sulfophenly)-5-[(phenylamino) carbonyl]-2H-tetrazolium hydroxide) or cell density (Crystal Violet Dye Elution) were observed among groups. However, Neutral Red assay revealed that dense materials extracts induced lower levels of total viable cells to porous HA/β-TCP (P < 0.01). Calcium ion content was also significantly lower on the extracts of dense samples. Porogenic treatments on BCP composites do not affect cytocompatibility, as measured by three different parameters, indicating that these ceramics are well suited for further studies on future bioengineering applications.

Effect of Hydroxyapatite and Zinc-Containing Hydroxyapatite on Osseous Repair of Critical Size Defect in the Rat Calvaria

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.

Osteoblast Adhesion Dynamics : A Possible Role for ROS and LMW-PTP

Reactive oxygen species (ROS) modulate a variety of intracellular events, but their role in osteoblast adhesion and spreading remains unclear. ROS is a very‐known physiological modulators of Protein Tyrosine Phosphatases activities, mainly to low molecular weight protein tyrosine phosphatase (LMW‐PTP) activity. As this biological mechanism is not clear in osteoblast adhesion, we decided to investigate ROS levels and phosphorylations of FAK and Src, identifying these proteins as potential substrates to LMW‐PTP activity. Our results showed that during osteoblast adhesion/spreading (30 min and 2 h of seeding) the intracellular ROS content (hydrogen peroxide) is finely regulated by an effective anti‐oxidant system [catalase and Superoxide Dismutase (SOD) activities were evaluated]. During the first 30 min of adhesion, there was an increase in ROS production and a concomitant increase in focal adhesion kinase (FAK) activity after its phosphorylation at Tyrosine 397 (Y397). Moreover, after 2 h there was a decrease in ROS content and FAK phosphorylation. There was no significant change in LMW‐PTP expression at 30 min or 2 h. In order to validate our hypothesis that LMW‐PTP is able to control FAK activity by modulating its phosphorylation status, we decided to overexpress and silence LMW‐PTP in this context. Our results showed that FAK phosphorylation at Y397 was increased and decreased in osteoblasts with silenced or overexpressed LMW‐PTP, respectively. Together, these data show that ROS modulate FAK phosphorylation by an indirect way, suggesting that a LMW‐PTP/FAK supra‐molecular complex is involved in transient responses during osteoblast adhesion and spreading. J. Cell. Biochem. 115: 1063–1069, 2014.

Histomorphometric Analysis of Bone Repair in Critical Size Defect in Rats Calvaria Treated with Hydroxyapatite and Zinc-Containing Hydroxyapatite 5%

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.

Exploring anorganic bovine bone granules as osteoblast carriers for bone bioengineering: a study in rat critical-size calvarial defects

It is known that current trends on bone bioengineering seek ideal scaffolds and explore innovative methods to restore tissue function. In this way, the objective of this study was to evaluate the behavior of anorganic bovine bone as osteoblast carrier in critical-size calvarial defects. MC3T3-E1 osteoblast cells (1x10(5) cells/well) were cultured on granules of anorganic bovine bone in 24-well plates and after 24 h these granules were implanted into rat critical-size calvarial defects (group Biomaterial + Cells). In addition, other groups were established with different fillings of the defect: Blood Clot (negative control); Autogenous Bone (positive control); Biomaterial (only granules) and Cells (only MC3T3-E1 cells). After 30 days, the animals were euthanized and the calvaria were technically processed in order to allow histological and morphometric analysis. It was possible to detect blood vessels, connective tissue and newly formed bone in all groups. Particularly in the Biomaterial + Cells group, it was possible to observe a profile of biological events between the positive control group (autogenous bone) and the group in which only anorganic bovine granules were implanted. Altogether, the results of the present study showed that granules of anorganic bovine bone can be used as carrier to osteoblasts and that adding growth factors at the moment of implantation should maximize these results.

Magnesium incorporation into β-TCP reduced its in vivo resorption by decreasing parathormone production†

Beta-tricalcium phosphate (β-TCP), one of the most widely used bioresorbable materials for bone therapy, can be doped with magnesium ions, generating β-TCMP. The objectives of this work were to evaluate, on a murine dental alveolus grafting model, the biocompatibility of β-TCP and β-TMCP granules by histomorphometric analysis, as well as the impact on plasmatic levels of receptor activator of nuclear factor κB ligand (RANK-L), osteoprotegerin (OPG), osteocalcin, osteopontin, and parathormone (PTH) during bone repair, using Luminex multiplexing technology. After grafting for 42 days, β-TCP grafted group presented higher bioresorption and induced more newly formed bone than β-TCMP (p < 0.05). β-TCP grafting also induced higher plasmatic levels of RANK-L, compared to β-TCMP and control (blood clot) groups at 21st day (p < 0.05). PTH, which remained at low levels in control group, presented a time-dependent increase in grafted groups, attaining significantly higher levels with β-TCP by the 42nd day (p < 0.05). RANK-L/OPG ratio increased on β-TCP group and attained a peak on the 21st day. In conclusion, β-TCP granules were more bioresorbable and osteogenic than β-TCMP granules, and the resorption of both materials might have been affected by osteoclastogenesis modulated by changes in the plasmatic levels of PTH and RANK-L.

Quality and intensity of the tissue response to two synthetic granular hydroxyapatite implanted in critical defects of rat calvaria

The objective of the present study was to evaluate the quality and intensity of the tissue response to two synthetic hydroxyapatites implanted in critical defects in the skulls of rats. Sixty animals were divided into three experimental groups: I (control), II (HA-1 = HA with 28% crystallinity) and III (HA-2 = HA with 70% crystallinity). They were sacrificed 1, 3, 6, and 9 months after implantation (n = 5 individuals per group/period). Histomorphometric analysis included i) counting of polymorphonuclear leucocytes, mast cells, macrophages and foreign body multinucleated giant cells stained with anti-lysozyme; ii) microvascular density stained with anti-Factor VIII and iii) degree of cell proliferation stained with anti-PCNA. There were no significant differences between the experimental groups in either the quality or quantity of cells in the inflammatory infiltrate, or the degree of angiogenesis and cell proliferation. We conclude that HA-1 and HA-2 are biocompatible and that the physico-chemical differences of these biomaterials did not affect cellular response.

Evaluation of the in vivo biocompatibility of hydroxyapatite granules incorporated with zinc ions

The incorporation of zinc onto biomaterials may improve their biological properties, since it is an essential trace element, involved on important steps of bone remodeling. The aim of this work was to evaluate the in vivo biocompatibility of previously characterized hydroxyapatite granules incorporated with 5% zinc (ZnHA), through a subcutaneous grafting test. Granules produced from powders of ZnHA or stoichiometric HA were subcutaneously grafted onto Balb/c mice, and histomorphometrical analysis was performed at 7, 14, 21, 28 or 60 days after grafting. HA and ZnHA induced a similar foreign body reaction, with formation of granulome and the presence of a fibrous capsule. Granulocytes were virtually absent, and agranulocytes decreased with time. ZnHA treated samples presented faster reduction on multinucleated giant cells and increase on observed angiogenesis, as compared to HA. It could be concluded that ZnHA granules are biocompatible. Further study should better address its potential use for bone therapy.

Effectiveness and acceleration of bone repair in critical‐sized rat calvarial defects using low‐level laser therapy

Tissue regeneration remains a challenge for orthopedic and craniomaxillofacial surgery to treat bone loss. The use of low‐level laser therapy suggests a promise on this road with positive results for narrow defects. However, temporal and quantitative evaluations are required to understand the healing process of large injuries. The aim of this study was to investigate the repair of critical‐size bone defects in rat calvaria using a GaAlAs laser.