WO2004026781A1

WO2004026781A1 - Bioactive niobium phosphate glasses for osseointegrated applications

Info

Publication number: WO2004026781A1
Application number: PCT/BR2003/000129
Authority: WO
Inventors: Marcelo José CARBONARI; Liborío Jose FARIA, Jr.; Bruno KÖNIG, Jr.; José Roberto MARTINELLI
Original assignee: Conselho Nacional De Desenvolvimento Científico E Tecnológico - Cnpq
Priority date: 2002-09-20
Filing date: 2003-09-09
Publication date: 2004-04-01

Abstract

Bioactive glasses with different mechanical and chemical properties were produced by mixing ammonium dihydrogen phosphate, niobium oxide, calcium oxide, and sodium oxide. These materials are proposed to be used as implant and graft, combined with other metallic and polymeric materials, associated to bone morphogenetic proteins, and associated to metallic implants. The chemical and mechanical properties depend on the amount of niobium in the glass composition.

Description

BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED

APPLICATIONS

INTRODUCTION

Several glasses and glass-ceramics for biomaterial applications include silicate based-glasses such as Bioglass™, which bond with natural bone tissues. Some medical applications with bioglasses are surgical reparation of periodontal defects, reparation of hypersensitive dentin, reparation of face's bone, and reparation of tibia and frontal bone. Calcium phosphate glasses are being investigated as new bioactive materials due to their very similar composition compared to the human bones, or because they improve the neo formation of bone tissues. Phosphates are known to form glasses. However, these glasses are chemically instable. The addition of niobium and calcium promotes the chemical stability and improves the mechanical properties of these glasses while the addition of Na₂0 increases the solubility of phosphate glasses in water, so they become more susceptible to water attack. However, modifications in the chemical composition of these glasses can increase the chemical durability. OBJECTIVES OF THE INVENTION The present invention deals with bioactive glasses obtained from mixtures of phosphorus, calcium, sodium, and niobium for use as implants and grafts. The use of niobium in the biomedical materials is advantageous in the sense that Brazil has about 80% (eighty percent) of the niobium minerals reservation known globally, and new applications would be favorable. The present invention allows compositions with variable niobium concentration. The use of bioactive glasses is restricted to situations that do not require materials with high mechanical resistance. To increase the use of bioactive glasses, sometimes composites can be produced by mixing glasses with metallic and polymeric reinforcements to improve the mechanical resistance. ADVANTAGES OF THE INVENTION RELATED TO THE STATUS OF THE KNOWN TECHNIQUE

The use of variable amounts of niobium in the glass composition with the objective to produce bioactive glasses with appropriated mechanical properties and different chemical durability depending on the application in the human body is an innovation of the known technique. Brazil has about 80% (eighty percent) of the reservations of niobium known globally and the importance of this element is increasing in biomedical applications. The composition of the bioactive glasses of the present invention is based on phosphorus, calcium, sodium, and niobium, and it does not contain silicon. This fact is an innovation when compared to the commercial bioactive glasses. SUMMARIZED DESCRIPTION OF THE INVENTION Phosphate glasses were produced by melting mixtures of (NH₄)₂HP0₄, Nb₂0₅, CaO, and Na₂C0₃ in an electric furnace. Glasses are produce by weighing and mixing the precursors during 1 . This mixture was heated inside an alumina crucible at 450^gC for 30min in an electric furnace to decompose the (NH₄)₂HP0₄. The material was melted at 1400²C and kept at this temperature during 1 h for homogenization and fining. The liquid was poured into stainless steel molds previously heated at 440°-C, and kept at this temperature for 10min. The material was withdrawn from the mold and annealed in the temperature range of 570^ρC - 620^δC for 1 h and then furnace cooled. METHODOLOGY TO PRODUCE THE INVENTION AND USES

Bioactive glasses are obtained, according to the present invention, by weighing and mixing (NH₄)₂HP0₄, Nb₂0₅, CaO, and Na₂C0₃. The homogenization of this mixture is done in mixing equipment for 1 hour. After that, the mixture was heated inside an alumina crucible at 450^SC for 30min in an electric furnace to decompose the (NH₄)₂HP0₄. The material was heated to 1400^SC at 10°C/min and kept at this temperature during 1 h for homogenization and fining. The liquid was poured into stainless steel molds previously heated at 440^SC, and kept at this temperature for 10min. The material was withdrawn from the mold and annealed in the temperature range of 570⁹C - 620^QC for 1h and then cooled inside the furnace. The niobium improved the mechanical and chemical properties of the glasses. This invention allows the production of bioactive glass powders with different particle size distributions that could be associated with bone morphogenetic proteins or applied to dental implants favoring and accelerating the osseointegration of the implants.

CHARACTERISTICS OF THE BIOACTIVE GLASSES PREPARED ACCORDING TO THE INVENTION

PREPARATION OF PHOSPHATE GLASSES

Table 1 shows the glass nominal compositions. Even though only a limited number of glasses are shown in this table, glasses with niobium concentration varying from 1 to 30 mol% were also produced. Table 1 - Glass nominal composition (mol%)

CHARACTERIZATION OF PHOSPHATE GLASSES

FOURIER TRANSFORMATION INFRARED SPECTROSCOPY (FTIR) Fourier transformation infrared spectroscopy was used to evaluate the presence of water molecules in the glass structure and to help the identification of structural atomic groups. For this analysis, samples with 12.0mm in diameter and 0.5mm in thickness were prepared by compacting glass particles with medium size of 10μm and 2 weight % of dehydrated KBr powder. These samples were maintained at 100°C before the analyses to avoid the absorption of water vapor in the atmosphere. The FTIR curves shown in Figures 1 and 2 confirm that the niobium participate as a glass former and the amount of water in the glass decreases as the amount of niobium increases. RAMAN SPECTROSCOPY Raman spectroscopy allows studying changes in the glass structure caused by the addition of glass modifiers, glass intermediate elements, and glass formers. The Raman spectra in Figure 3 show that the bridging between tetrahedrons structure formed by 1 phosphorous atom and four oxygen atoms is broken and re-bridged by niobium atoms in an octahedron structure forming the skeleton -O-P-O-NB-O-NB-O-P-0-. DENSITY

The glass densities were determined by Archimedes^' method. Table 2 shows the result of density for different glasses. Table 2 - Density of different glasses

Table 2 shows that the density increases as a function of niobium concentration.

X-RAY DIFFRACTION Figure 4 shows the X-ray diffraction patterns for different glass samples. No evidence of the presence of crystalline phases can be observed by this technique.

CYTOTOXiCITY

To evaluate the toxicity of these glasses cytotoxicity assay in vitro was performed by the neutral red uptake technique. The cell line NCTC clone

L929 was used as supplied by the American Type Culture Collection (ATCC) bank and the cell culture medium was a MEM (minimum Eagle's medium from Sigma Co). The cells were maintained in complete MEM (MEM containing 10% fetal calf serum, 20 mM glutamine and 1% non-essential amino acids) in a humidified incubator with 5% C0₂ at 37 °C. A 0.2mL of

2.5x10⁵cell/mL cell suspension was seeded in a flat-bottomed 96 microplate well (Costar, Cambridge, MA, USA). The microplate was kept for 24h at 37 °C in a C0₂ humidified incubator. After that, the medium of the plate was discarded and replaced with 0.2mL of serially diluted extract of each sample (50, 25, 12.5, 6.25%). The extract was prepared with immersion of 1cm superficial area of each glass sample per 1 mL of MEM during 48h and diluted with complete MEM. Control of cell culture was replaced with complete MEM. In the same assay, positive control (0.02% Phenol solution) and negative control (atoxic Tin stabilized poly vinyl chloride, Dacarto SA Industria de Plasticos, Osasco, Sao Paulo, Brasil) were used. Samples and controls were tested in triplicate. The plate was incubated again for 24h in the same conditions. After 24h the medium and extracts were discarded and replaced with 0.2mL of complete MEM with neutral red (0.5μg/mL). After 3h of incubation at 37 °C the dye medium was discarded and the microplate was washed twice with phosphate-saline buffer and one time with 1% CaCI₂ in 0.5% formaldeid solution. The rupture of cells and neutral red release was obtained by addition of 0.2mL/well of extractant solution (50% ethanol in 1% acetic acid). After reading the absorbance in an Organon spectrophotometer for microplates with 540nm filter, the average of optical density units were used to calculate the percentage of cell viability in relation to cell control (100%). The cytotoxic effect was evaluated by the cytotoxicity index (lCs_0(%)) and it can be obtained in the graphic plotted with the cell viability percentage of each extract dilution of samples, negative control and positive control and extract concentration, showed in Figure 1. IC_50(%) is the concentration of the extract which injures or kills 50% of cell population due to toxic elements extracted from tested sample. We can observe that positive control presented toxic effect with IC₅o(%) = 33. All glass samples showed similar behavior of negative control with IC₅₀(%) > 100, indicating no cytotoxicity effect, as shown in

Figure 5. OPERATIVE PROCEDURE Glass samples with different niobium concentration (keeping the same molar ratio of the other compounds) were inserted in rabbit's tibia where they remained for eight weeks. During this healing time polyfluorochrome sequential labeling was carried out. This study follows the Rules of the Ethics Committee in Animal and Research - University of Sao Paulo, in accordance to the surgical protocol (Protocol 97/2002). After a period of housing, 8 male rabbits (Oryctolagus cunniculus) with an average weight of 3 Kg received 32 implants (8 for group). According to the surgical plan two implants were inserted in the proximal margin of each tibia under general anesthesia and antibiotic protection. The surgical protocol was the same for all the animals under general anesthesia (intramuscular injection of ketamine 20mg/Kg). The medial skin of both legs was shaved, antisepsis was performed, and local anesthesia was supplied with 1 :100,000-epinephrine vasoconstrictor. A full flap was made in the area near the surgical site and the implants were inserted in the proximal epiphysis of the tibias under saline irrigation. After the insertion, the flaps were carefully closed with a silk thread suture (Ethicon - Johnson's & Johnson's). For the evaluation of the modeling and remodeling during the healing time, the polyfluorochrome sequential labeling described in Table 3 was followed. Table 3 - Sequence of the polyfluorochrome sequential labeling and days of application.

Postop. (days) Substance Doses Injection (mg/Kg)

14 Tetracycline 60 3g/100ml + 2g NaHCO₃

21 Tetracycline 60 3g/100ml + 2g NaHCO₃

28 Alizarin 30 3g/100ml + 2g NaHCO₃

35 Alizarin 30 3g/100ml + 2g NaHCO₃

42 Calcein 10 3g/100ml + 2g NaHCO₃

49 Calcein 10 3g/100ml + 2g NaHCO₃

56 Exitus

SECTION PREPARATION After a healing period of eight weeks the animals were sacrified by an overdose of pentobarbital. The tibias were dissected and bone blocks containing the implants were removed and maintained in a 4% neutral formalin buffered solution. After the fixation the samples were washed in running water for 12h and dehydrated in different solution of ethanol from 70° to 99° staying for 24h in each solution. After dehydration the samples remained in two solutions of xylene for 24h and 48h respectively. The samples were then embedded in a methacrylate solution (Tecnovit VCL, Sigma) and after the polymerization they were processed according to the cutting-grinding technique. TOLUIDINE BLUE Histological studies were performed in slices stained with toluidine blue and observed by using a light microscope. Figure 6 shows a large number of osteoblastic cells and the bone remodeling in direct contact of the glass. Figure 7 shows the differences between the old tissues and the remodeling tissues (lamellar and osteons). SCANNING ELECTRON MICROSCOPY

The scanning electron microscopy was used to observe the contact between the glass and the bone tissues. Figure 8 allowed observing the direct contact between the glasses and the remodeling tissues. The Energy Dispersion Spectroscopy (EDS) on the surface of the glass and on the bone remodeling tissues indicates that niobium is only found in the glass. There is no contamination of niobium in the remodeling tissues even after 8 weeks. Fluorescent microscopy

The fluorescent microscopy was used to observe the periodicity of bone deposition. Figure 9 shows the implant in the rabbit's tibia. The remodeling associated to the implant can be observed in cortical region near to the bioactive glass. Figure 10 shows that the bone marked as light brown and dark brown are the ones formed in the beginning and in the middle of the bone remodeling, respectively. The bone marked as green represented the maturation of the bone tissues. This label was injected in the end of the healing period, which is represented by osteons. SIRIUS RED TECHNIQUE

The picro sirius technique allowed observing the type of collagen present near the glass. Figure 11 shows that there are a large amount of type I collagen marked as red and yellow indicating that it is a mature collagen and type III collagen marked as green is a immature collagen. The mature collagen is ready to receive the mineral part of the bone.

Claims

1. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS were produced by mixing ammonium dihydrogen phosphate, niobium oxide, calcium oxide, and sodium oxide.

2. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, as described in Claim 1 , contain 1 to 30 mol% of niobium, keeping the concentration of other elements described in Table 1.

3. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, with variable chemical resistance depending on the amount of niobium in their composition.

4. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, according to the above claims, characterized by the homogenization and mixture in mixing equipment, for a period of 1 hour.

5. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, characterized by mixing and homogenized mixtures heated at 450°C for 30min inside a alumina crucible, or until a complete decomposition of the ammonium dihydrogen phosphate.

6. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, according to the Claim 5 characterized by heating the previously heated material to 1400^ρC at 10°C/min and kept at this temperature during 1h for homogenization and fining. The liquid was poured into stainless steel molds previously heated at 440^QC, and kept at this temperature for 10min. The material was withdrawn from the mold and annealed in the temperature range of 570⁹C - 620^QC for 1 h and then cooled inside the furnace.

7. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, according to the above claims, characterized by cooling the material to room temperature, and annealing the material to remove mechanical stress caused by the fabrication process.

8. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, according to the above claims, characterized by the production of bioactive glasses in the form of powders, particles, that could be associated with bone morphogenetic proteins or applied to dental implants favoring and accelerating the osseointegration of the implants.

9. BIOACTIVE NIOBIUM PHOSPHATE GLASSES FOR OSSEOINTEGRATED APPLICATIONS, according to the above claims, characterized by their use in the production of implants and grafts, bone repair, and metallic implant coatings.