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Brazilian Dental Journal

Print version ISSN 0103-6440

Braz. Dent. J. vol.25 no.4 Ribeirão Preto  2014

http://dx.doi.org/10.1590/0103-6440201300067 

Original Articles

Can Inorganic Bovine Bone Grafts Present Distinct Properties?

Fernanda de Paula do Desterro 1  

Márcia Soares Sader 2  

Glória Dulce de Almeida Soares 2  

Guaracilei Maciel Vidigal Jr 3   4  

1Department of Oral Implantology, Unigranrio University, Duque de Caxias, RJ, Brazil

2Department of Metallurgical and Materials Engineering, UFRJ - Federal University of Rio de Janeiro, RJ, Brazil

3Postdoctoral in Periodontics, School of Dentistry, UERJ - State University of Rio de Janeiro, RJ, Brazil

4Department of Oral Surgery and Periodontics, USP - University of São Paulo, Ribeirão Preto, SP, Brazil

ABSTRACT

The aim of this study was to evaluate the physicochemical characteristics of 3 mineralized bovine inorganic biomaterials and correlate them with the dissolution rate. Bio-Oss(r), GenoxInorgânico(r), and Bonefill(r) were examined using field emission gun scanning electron microscopy (FEG-SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), specific surface area (BET), calcium/phosphorous molar ratio and a dissolution assay. Bio-Oss(r) showed a micro- and nanoporous structure consisting of 15-nm hydroxyapatite (HA) crystallites; Genox(r), a microporous structure composed of 39-nm HA crystallites; and Bonefill(r), micro- and nanoporous structure of indeterminable crystallite size. FTIR analysis showed that Bio-Oss(r) and Genox(r) were composed of calcium phosphate. The absorption bands of phosphate were poorly defined in Bonefill(r). By XRD, Bio-Oss(r) was shown to contain peaks related to the carbonated HA, whereas Genox(r) only contained peaks corresponding to HA. The broad bands in Bonefill(r) indicated low crystallinity. Bio-Oss(r) showed a greater surface area and calcium release rate than that of Genox(r). Although all biomaterials were of bovine origin, the different manufacturing processes result in materials with different physicochemical properties and may influence the biological and clinical response.

Key words: biomaterials; hydroxyapatite; physicochemical characterization; bovine bone mineral

RESUMO

O objetivo deste estudo foi avaliar as características físico-químicas de 3 biomateriais bovinos inorgânicos mineralizados e correlacioná-los com a taxa de dissolução. Bio-Oss(r), Genox Inorgânico(r) e Bonefill(r) foram caracterizados por microscopia eletrônica de varredura com fonte de emissão por efeito de campo (MEV-FEG), espectroscopia de infravermelho com transformada de Fourier (FTIR), difração de raios-X (DRX), área superficial específica (BET), razão molar cálcio/fósforo e análise da dissolução. Bio-Oss(r) mostrou uma estrutura micro e nanoporosa consistindo de cristalitos de hidroxiapatita (HA) de 15 nm; Genox(r), uma estrutura microporosa composta de cristalitos de HA de 39 nm e Bonefill(r), estrutura micro e nanoporosa com tamanho indeterminável de cristalito. Análises de FTIR mostraram que Bio-Oss(r) e Genox(r) eram compostos por fosfato de cálcio. As bandas de absorção de fosfato encontraram-se pouco definidas no Bonefill(r). Por DRX, Bio-Oss(r) mostrou picos relacionados à HA carbonatada, enquanto Genox(r) somente apresentou picos correspondentes à HA; as bandas alargadas no Bonefill(r) indicaram baixa cristalinidade. Bio-Oss(r) apresentou maior área de superfície e taxa de liberação de cálcio quando comparado ao Genox(r). Embora todos os biomateriais fossem de origem bovina, os diferentes processos de manufatura resultam em materiais com diferentes propriedades físico-químicas e podem influenciar a resposta biológica e clínica.

Introduction

Correct positioning is a key factor in esthetic and functional dental implant treatment, and it is essentially determined by the adequate amount of soft and hard tissues (1). Various treatment modalities, including bone grafting biomaterials, have been employed to achieve bone augmentation to allow proper implant installation. For this objective natural bone grafting biomaterials can be obtained from different origins: autogenous, allogenous, and xenogenous. Autogenous bone grafts are commonly associated with a second surgical site, increasing the treatment morbidity, and a limited amount of bone tissue (2). The allogenous bone grafts are associated with disease transmission risk (3). Xenogenous materials have been extensively researched and present a history of results. They have been successfully used to maintain the dimensions of the post-extraction alveolar ridge; in the treatment of alveolar process defects and maxillary sinus lifting ( 4 , 5 ).

Natural bone presents two major constituents: organic and inorganic. The organic bone matrix is composed of collagen, proteoglycans and glycoproteins (6). The collagen is frequently used to manufacture membrane barriers for guided bone regeneration (7). The major constituent of the inorganic part of bone is hydroxyapatite (HA) and it is used for inorganic bone grafts (5). Inorganic bone grafts should function like a bone remodeling base to be replaced by the host bone. This remodeling process is associated with the physicochemical properties of the HA. Although the inorganic phase of bone is commonly referred to as HA, it is actually a carbonated hydroxyapatite, which has crystallinity and chemical composition similar to human bone (8). The crystals are deposited on the collagen, which was first deposited, forming a composite biomaterial (6). The collagen forms long fibers and their association with the HA crystals results in bone strength (6). During the manufacture of inorganic bone grafts, thermal treatments are employed to remove the organic part of the bone (9). Temperatures above 900 °C (10) used in this process may change the atomic structure of the HA crystal (11), which may strongly affect the graft behavior ( 6 , 9 ).

Several bovine bone-derived inorganic biomaterials have been commercialized (12). However, the fact that different biomaterials have the same origin may not guarantee the same biological and clinical results (13). The differences in the manufacturing process of biomaterials may cause changes in the physicochemical properties of the bovine bone. Thus, the purpose of this study was to evaluate the physicochemical characteristics of three inorganic bovine biomaterials and determine the relationship with their dissolution rates.

Material and Methods

The analyzed inorganic bovine bone biomaterials were Bio-Oss(r) (GeistlichPharma AG, Wolhusen, Switzerland), GenoxInorgânico(r) (Baumer, São Paulo, SP, Brazil), and Bonefill(r) (Bionnovation, Bauru, SP, Brazil). The techniques employed for physicochemical characterization of the biomaterials included field emission gun scanning electron microscopy (FEG-SEM), Fourier transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), determination of calcium/phosphorus ratio, and dissolution test.

FEG-SEM

Morphological characterization of the three biomaterials was performed with a field emission gun scanning electron microscope with increasing magnification (Quanta 400F; FEI Company, Oregon, USA). The samples, in their commercial form, were mounted on stubs and sputter-coated with gold at 10 mA for 120 s (Emitech K550 Sputter Coater, Emitech Ltd., Ashford, Kent, UK) with.

FTIR

The characteristic absorption bands for calcium phosphate were identified by FTIR. To prepare the samples, 1 mg of each biomaterial was mixed with 150 mg of potassium bromide (KBr) followed by pressing at 10,000 psi with a hydraulic press (Carver laboratory press, mode C; Fred S. Carver Inc., Wabash, IN, USA). The used spectrometer was FTIR Spectrum 100 (PerkinElmer Brazil Ltd., São Paulo, SP, Brazil). The spectra were collected at room temperature and recorded from 400 to 4000 cm-1.

XRD

The crystalline phases of the crushed commercial samples were identified by XRD. The Shimadzu XRD-6000 X-ray diffractometer (Shimadzu Corporation, Chiyoda-ku, Tokyo, Japan) was used with Cu K radiation, operating at 30 kV and 30 mA. Scanning in 2 was 3° - 80°, in 0.02° steps (2). The obtained diffractograms were analyzed using Match Phase Analysis Report (Crystal Impact, Bonn, Germany). The crystallite size was calculated using the Scherrer equation (14): L = Kγ/βcosΦ.

BET Surface area analysis

The specific surface area of each biomaterial was calculated based on the amount of nitrogen required to physically adsorb to the surface sample completely dry, at different pressures (Fig. 1). The physical adsorption is caused by van der Waals forces between molecules of the gas and atoms or ions of the sample. The physisorption analyzer ASAP 2020 model (Micromeritics Instrument Corporation, Norcross/Atlanta, GA, USA) was used. The samples were subjected to pre-treatment at 200 °C, specific for hydroxyapatites.

Figure 1. Schematic representation of the physical adsorption of nitrogen to the surface sample. Increasing gas pressure will cause the beginning of multi-layer coverage. 

Calcium/Phosphorus molar ratio

For the analysis of initial calcium, 50 mg of each biomaterial powder was weighed and dissolved in 1 mL of 5% nitric acid, in a 50-mL volumetric flask using double distilled water. A 250-μL aliquot of this solution was transferred into another 50-mL volumetric flask to make a 200× dilution in double distilled water. The 0.2% potassium chloride (VETEC Fine Chemicals LTD, Duque de Caxias, RJ, Brazil) was used to suppress ionization in atomic absorption. The released calcium was measured by atomic absorption (Shimadzu, model AA 6800; SINC Brazil, São Paulo, SP, Brazil).

The same steps in the analysis of calcium were followed for the analysis of initial phosphorus, except the use of potassium chloride. The 0.1% nitric acid was used to ensure that the phosphate ion was always detectable as PO4 3-. Measurements were conducted using an ultraviolet-visible spectrophotometer (UV-VIS) (Lambda 25 Model; PerkinElmer Brazil LTD, São Paulo, SP, Brazil). One mL of sample, 1 mL of 4% ferrous sulfate, and 1 mL of 2% ammonium molybdate were used. The concentration of phosphorus was measured using a 700-nm wavelength.

Dissolution assay

The granular biomaterials, in their commercial form, were passed through a 420-μm screen (Granutest; Telastem Peneiras para Análises Ltda, São Paulo, SP, Brazil) and were retained on a 350-μm screen using the dry screen method (15). The resulting particles were separated into Falcon tubes and evaluated at different time points, namely 7, 24, 48, 72 h and 168 h. Each test was performed in triplicate, requiring a total of 45 Falcon tubes. The dissolution test was performed using 0.1 M potassium acetate buffer solution (VETEC) under air atmosphere, pH 5, and at 37 °C. The reagent was weighed (9.814 g) and adjusted to pH 5 using a 0.5 M acetic acid solution (VETEC). The particles were suspended at a ratio of approximately 0.10 g of dry powder in 2 mL of buffer solution. Quantification of the material was conducted according to ISO/FDIS 10993-14:2001 (16). At the end of each time point, the supernatant was pipetted and diluted 200× in double distilled water, as previously described. The amount of released calcium was measured by atomic absorption (Shimadzu Model AA 6800l).

Statistical analysis

The results of calcium released from samples during dissolution tests were compared using the statistical software package Origin(r) 8.0 (OriginLab, Northampton, MA, USA). Histogram normality test on each group of data was performed to determine whether it follows a normal distribution. Then, one-way ANOVA followed by the Tukey's test was used to determine statistically significant differences. The significance level was set at 0.05.

Results

FEG-SEM

FEG-SEM analysis showed micro and nano-morphological differences among the various analyzed bone grafts. At 1,000× magnification, micropores were observed within a diameter range of 40-70 μm associated with spaces of the Haversian blood vessel system (Figs. 2A and 2B). Bonefill(r) possessed a fibrous structure (Fig. 2C). Using 10,000× magnification, the morphological differences among Bio-Oss(r), Genox(r), and Bonefill(r) were more accentuated. Bio-Oss(r) showed a mineral scaffold organization similar to the collagen fibers (Fig. 3A). Genox(r) did not show the same organized structure and exhibited greater HA particle sizes. Microscopic pores, presenting polyhydric shapes, were observed among the particles (Fig. 3B). Bonefill(r) consisted of some micropores on its outer surface, with the HA particles forming a single body, thus hindering visualization of individual particles at this specific magnification stage (Fig. 3C). Figs. 4A and 5A show nanopores on the surface of Bio-Oss(r). Genox(r) had micropores (Fig. 3B) but no nanopores were visualized (Figs. 4B and 5B). Bonefill(r) also presented nanopores, similar to Bio-Oss(r), but they were observed only at 50,000× and 100,000× magnifications (Figs. 4C and 5C). Surface texture of the powder particles varied from rough (Figs. 5A and 5C) to smooth (Fig. 5B).

Figure 2. A: Bio-Oss(r). Measurement of the Haversian system: 73.10 µm. Measurement of osteoplast: 5.19 µm. B:- Genox(r). Measurement of the Haversian system: 43.33 µm. C: Bonefill(r). Notice the fibrous structure of the bone. Original magnification 1,000×. 

Figure 3. A: Bio-Oss(r). Mineral scaffold organization similar to the collagen fibers. B: Genox(r). Notice the greater HA particle size. C: Bonefill(r). Micropores on its outer surface. Original magnification 10,000×. 

Figure 4. A: Bio-Oss(r). Measurement of nanotubules: 43.5 nm and 46.9 nm. B: Genox (r). FEG-SEM image suggestive of a sintered HA in high temperature. C:- Bonefill(r). Original magnification 50,000×. 

Figure 5. A: Bio-Oss(r). Presence of nanopores. B: Genox (r). Smooth surface texture without nanopores. C: Bonefill(r). Presence of nanopores. Original magnification 100,000×. 

FTIR

Variations in the vibration modes of the functional groups were observed using the infrared spectra (Fig. 6). The higher intensities of the absorption bands for CO3 2-(v3) at 1,463 and 1,412 cm-1 and for CO3 2-(v2) at 872 cm-1 reflect a higher incorporation of carbonate in the apatite. The resolution of the absorption band of PO4 3-(v3) at 1,032 cm-1 diminished with increasing CO3 2-, as seen in the FTIR spectrum of Bio-Oss(r). The 1,463, 1,412 and 872 cm-1 bands correspond to CO3 2- replacing PO4 -3, thus characterizing the biomaterial as a calcium-deficient and carbonate-rich HA. The 1,641 band corresponds to H2O, it has higher resolution in the FTIR spectrum of Bio-Oss(r). The 964 band corresponds to PO4 3- (v1), the 568 band corresponds to PO4 3- (v4), and the 472 band corresponds to PO4 3- (v2), all present in Bio-Oss(r) and GenoxInorgânico(r). The crystalline hydroxyapatite generates two characteristic OH bands of approximately 3,562 and 630 cm-1. These bands are present in the FTIR spectrum of GenoxInorgânico(r). There is a higher resolution of the absorption band of PO4 3-(v3) at 1,032cm-1 and a decrease in the absorption band for carbonate, indicating a lower rate of replacement of PO4 -3 by CO3 2- in GenoxInorgânico(r). The 1,382 band corresponds to NO3 -, and it is present in the FTIR spectrum of Bonefill(r). However, the absorption bands of PO4 3- are poorly defined in Bonefill(r).

Figure 6. FTIR spectra (A, Bio-Oss(r); B, Genox (r); C, Bonefill(r))

XRD

X-ray diffractograms of the analyzed biomaterials were compared with diffractometric patterns of individual phases available at the International Centre for Diffraction Data (ICDD) for the various calcium phosphates. The increase in the CO3 2- content caused a reduction in crystallinity of apatites, as indicated by the peak broadening of XRD (Fig. 7). Bio-Oss(r) showed a HA crystallite size of 15 nm, lower crystallinity, and X-ray diffractograms indicative of carbonated hydroxyapatite [00-004-0697] and Whitlockite [00-006-0426]. Genox(r) consisted of HA with a crystallite size of 39 nm and higher crystallinity identified by higher and narrower diffraction peaks in [002] and [300] planes. The XRD diffractogram of Genox(r) showed main peaks that were related to hydroxyapatite (00-024-0033). For Bonefill(r), it was not possible to calculate its crystallite size because the biomaterial was highly amorphous.

Figure 7. X-ray diffractograms (A, Bio-Oss(r); B, Genox (r); C, Bonefill(r))

BET Surface area analysis

Bio-Oss(r) showed a greater surface area of approximately 86 m²/g, whereas GenoxInorgânico(r) had a specific surface area of <10 m²/g. It was not possible to determine the specific surface area of Bonefill(r).

Calcium/Phosphorus molar ratio

The average mass fractions of calcium in Bio-Oss(r), GenoxInorgânico(r) and Bonefill(r) were 33.52 ± 1.20, 35.49 ± 0.77, and 23.63% ± 0.44, respectively. The average mass fractions of phosphorus in Bio-Oss(r), GenoxInorgânico(r) and Bonefill(r) were 15.74 ± 1.41, 17.36 ± 0.53, and 17.64% ± 2.46, respectively. Consequently, the molar Ca/P ratios of Bio-Oss(r), GenoxInorgânico(r), and Bonefill(r) were 1.65, 1.58, and 1.03, respectively.

Dissolution assay

The release of calcium in an acid solution is shown in Figure 8. In the analysis of materials, Bio-Oss(r) and Bonefill(r) showed a higher rate of calcium release at all time points when compared with GenoxInorgânico(r). According to the results of the dissolution assay, the behaviors of Bio-Oss(r) and Bonefill(r) were not statistically different in the amount of released calcium, and the behavior of GenoxInorgânico(r) was statistically different from Bio-Oss(r) at 24, 72 and 168h.

No statistically significant differences were observed when the dissolution time for each material was changed.

Figure 8. Release of calcium at 7 h (A), 24 h (B), 48 h (C), 72 h (D) and 168 h (E). XX Axis: Material (A, Bio-Oss(r); B, Genox (r); C, Bonefill(r)). YY Axis: Calcium content (mgL-1). Intragroup comparisons (a x b) - different letters represent statistically significant differences (p<0.05). 

Discussion

The FEG-SEM images (Fig. 3) showed that Bio-Oss(r) was the only bovine bone inorganic biomaterial evaluated in this study, which preserved the fiber-like structure of the bone collagen to maintain the original bone morphology. This finding demonstrates that the mineral crystallites that formed within the collagen fibrils may achieve sufficient inter-crystallite bonding to retain the fibrillar structure even after dissolution of the protein template. This suggests that the manufacturing process did not alter some physicochemical properties, like crystal size. Manufacturing processes using higher temperatures can alter crystal size, producing bigger grains with higher crystallinity (17). These structural characteristics of the bone mineral phase demonstrated by Bio-Oss(r) could improve its osteoconductivity. GenoxInorgânico(r) and Bonefill(r) did not show the same organized structure. FEG-SEM facilitates visualization of small particles at a resolution that was only previously available by transmission electron microscopy (TEM), while maintaining the field depth that is the main characteristic of SEM. It also produces a greater level of brightness and smaller spot size, supplied by the field emission source (18). Due to these characteristics it was possible to study bovine inorganic bone grafts with high-resolution and without decalcification, a common step used in TEM sample preparations, enabling the visualization of the original graft morphology.

This study showed that Bio-Oss(r) presented a 15-nm crystallite size. The manufacturing of this biomaterial probably maintained the original mineral scaffold consisting of nanopores, in which the cytoplasmic processes of osteocytes were located, resulting in a higher surface area (86 m²/g), similar to the observations of another study (17). GenoxInorgânico(r), which has the same bone origin, had a larger HA crystallite size of 39 nm and a lower surface area (<10 m²/g). The high temperatures, often used to remove the organic content of bone (9), resulted in the HA crystallite growth (19). These changes could have an impact on the in vivo biomaterial behavior ( 6 , 9 ). The particle size of grafts and the spaces between the particles are important determinants of osteogenesis and bone regeneration (20). Although its label states that only the mineral portion remains after manufacturing, Bonefill(r) presented a release of substrate in the BET reactor when heated to 200°C, suggesting presence of residual organic material. Thus, it was not possible to calculate its specific area.

In biological apatites, some phosphate ions (PO4 3-) are replaced by carbonate ions (CO3 2-), as detected by FTIR (Fig. 6). The higher CO3 2- absorption band intensities indicate a high rate of incorporation of carbonate in the apatite. The resolution of the PO4 3-(v3) absorption band decreased with increasing levels of CO3 2- (Fig. 6A), featuring a type-B carbonated apatite. GenoxInorgânico(r) showed OH bands, a higher definition of the phosphate band, and a reduced carbonate absorption band, all characteristic of crystalline HA (Fig. 6B). High crystalline biomaterials tend to have a static behavior without remodeling (21). Thus, GenoxInorgânico(r) may be considered more crystalline than Bio-Oss(r) and Bonefill(r). The broader bands, observed in the FTIR spectrum of Bonefill(r), indicated low crystallinity (Fig. 6C). Moreover, the absorption bands of PO4 3- are less defined. The 1,382 band indicates the presence of NO3 - in Bonefill(r). Nitrogen is a marker for the possible presence of protein within the surface layers of biomaterials (22); therefore more studies are required to investigate the presence of organic constituents in inorganic biomaterials.

Previous studies have shown that a larger diffraction peak indicates a smaller crystallite size (23). This feature was also observed in the XRD analysis of Bio-Oss(r) (Fig. 7A). Its higher carbonate content also resulted in wider XRD peaks, lower crystallinity and smaller crystallite size (15 nm). GenoxInorgânico(r) showed larger crystallite size (39 nm) and higher crystallinity based on the sharper and narrower XRD peaks (Fig. 7B). The broadening of diffraction peaks observed in the XRD of Bonefill(r) suggested the presence of organic material (Fig. 7C).

For bone regeneration, a biomaterial must possess essential qualities such as biocompatibility, osteoconductivity, good integration and adequate resorption rate (24). Thus, a bone substitute biomaterial should be resorbable to become replaced by the native bone. In bovine-derived apatite, the dissolution was shown to be the greatest from non-sintered bone without organic matrix, followed by non-sintered bone with organic matrix, and then sintered bone. This dissolution behavior can be explained by the crystal size, which is much greater in sintered (1000°C) bone than in non-sintered bone (25). This study used the dissolution test, carried out in acidic conditions (pH 5), to simulate the acid environment during osteoclastic resorption. GenoxInorgânico(r) was more crystalline and showed the lowest rate of calcium release in the dissolution assay. The higher rate of calcium release in Bio-Oss(r) and Bonefill(r) is attributable to their physicochemical characteristics. For Bio-Oss(r), its larger surface area (86 m²/g) and lower crystallinity may facilitate a greater calcium release rate in the dissolution test using an acid environment. The associated manufacturing process largely influences these physicochemical characteristics. Bio-Oss(r) is manufactured by heating to approximately 300°C, followed by a chemical treatment using a NaOH solution ( 8 , 22 ). Bonefill(r) receives only sequential bath treatments to solubilize the organic structures. Although Bio-Oss(r) presented a stoichiometric Ca/P ratio, this biomaterial showed low crystallinity (as measured by XRD) and a larger amount of carbonate (as showed by FTIR), both of which are characteristics that contribute to a higher dissolution rate. The inorganic portion of the bovine bone has maintained its original architecture and chemical composition, resulting in the large surface area, as observed in Bio-Oss(r), resulting in its degradation when exposed to the potassium acetate solution for 7 days. Understanding the dissolution rate of various biomaterials for bone grafting is essential because this may also influence its biodegradation or bioresorption (19). Various in vivo applications may require different rates of biodegradation. A bone graft biomaterial should be resorbed and replaced by bone tissue.

This study has identified correlations among physicochemical characteristics of Bio-Oss(r), i.e. increased surface area, low crystallinity and high carbonate content, with a high rate of dissolution in acidic conditions. GenoxInorgânico(r), a biomaterial with a high degree of crystallinity showed a low dissolution rate. BET analysis and FTIR spectrum of Bonefill(r) suggests that an evaluation of organic material may be necessary. The present study evaluated biomaterials of bovine origin, and showed significant differences in their physicochemical characteristics. These differences could be related to differences in the manufacturing process and may influence the biological and clinical response.

Acknowledgements

The authors acknowledge CAPES, CNPq and FAPERJ Brazilian agencies for the support to this study.

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Received: April 17, 2014; Accepted: July 22, 2014

Correspondence: Dra. Fernanda de Paula do Desterro, Rua Conde de Bonfim, 232 sala 701 Tijuca, 20520-054 Rio de Janeiro, RJ, Brasil Tel: 55-21-99609-7215, 55-21-2284-0539. e-mail: fernandadesterro@ig.com.br

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