Biphasic Calcium Phosphate Materials Research Articles

Synthesis and characterization of HA/β‐TCP bioceramic powder

AbstractThe main goal of this study is to elaborate and evaluate the physicochemical properties of the synthetic biphasic calcium phosphate (BCP) powder: a combined mixture of hydroxyapatite (HA): Ca10(PO4)6(OH)2 and beta‐tricalcium phosphate (β‐TCP): Ca3(PO4)2. The BCP mixture has two advantages: high bioactivity (HA) and rapid biodegradation (β‐TCP). The obtained powders of BCP were prepared by precipitate method. XRD analysisconfirmed that the synthetic materials were comprised of both HA and β‐TCP crystalline phases. SEM images showed that the small particles of HA attached on bigger particles of β‐TCP in the structure morphology of BCP. The in‐vitro experiment was carried out in static condition by soaking of a series of 50 mg BCP powders in 100 ml simulated body fluid (SBF) solution at different periods of soaking time. The microstructure and chemical bonds after soaking were studied by XRD and SEM methods. The obtained results confirmed the bioactivity of synthetic BCP material by the formation of a new apatite layer on its surface.

Synthesis of Biphasic Calcium Phosphate and its Behaviour in Simulated Body Fluid

The main goal of this study is to elaborate and evaluate the physicochemical properties of the synthetic biphasic calcium phosphate (BCP) powder: an associate compound of hydroxyapatite (HA): Ca10(PO4)6(OH)2 and beta-tricalcium phosphate (β-TCP): Ca3(PO4)2. The new compound BCP has two advantages: high bioactivity (HA) and fast biodegradation (β-TCP). The obtained powder of BCP was prepared by the precipitate method. XRD analysis confirmed the synthetic material contained both HA and β-TCP crystalline phases. SEM images showed that the small particles of HA attached to bigger particles of β-TCP in the structure morphology of BCP. The in vitro experiment was carried out in static condition by soaking of a series of 50 mg BCP powder in 100 ml of simulated body fluid solution at different period of soaking time. The XRD and SEM methods studied the microstructureand chemical bond after soaking. The obtained results confirmed the bioactivity of synthetic BCP material by the formation of a new apatite layer on its surface.

The adsorption of bone‐related proteins on calcium phosphate ceramic particles with different phase composition and its adsorption kinetics

Calcium phosphate (CaP) ceramics, especially hydroxyapatite and biphasic calcium phosphate (BCP), have attracted significant interest in bone tissue engineering because of their compositional similarities to bone mineral and excellent biocompatibility. The purpose of this study is to investigate the effect of phase compositions of CaP ceramics on their bone morphogenetic protein‐2 (BMP‐2) adsorption behaviours in phosphate‐buffered saline. The BCP material surface probably has more adsorption sites which can adsorb more BMP‐2 molecules. Langmuir, Frundlich, and Temkin isotherm models were used to describe the adsorption of BMP‐2 onto CaP ceramics. The Langmuir isotherm model fit the equilibrium data better than the Frundlich and Temkin isotherm models. The pseudo‐first‐order kinetic model is suitable for the description of the adsorption kinetics, and 240 min is thought to be the optimal adsorption time. A possible adsorption mechanism has been proposed. The electrostatic interaction plays an important role in the adsorption processes. In addition, the BMP‐2 could be adsorbed to the surface which showed slight conformational changes analysed by Fourier transform infrared spectroscopy. Copyright © 2016 John Wiley & Sons, Ltd.

Influence of bone morphogenetic protein and proportion of hydroxyapatite on new bone formation in biphasic calcium phosphate graft: Two pilot studies in animal bony defect model

PurposeThe purpose of these two pilot studies using animal bony defect models was to evaluate the influence of bone morphogenetic protein (BMP) and proportion of hydroxyapatite (HA)/beta-tricalcium phosphate (β-TCP) in biphasic calcium phosphate (BCP) graft on new bone formation. MethodsIn this study, four kinds of synthetic osteoconductive bone materials known for bone growth scaffold, OSTEON™II(HA:β-TCP 30:70), OSTEON™III (HA:β-TCP 20:80), OSTEON™II Collagen, and OSTEON™III Collagen, were prepared as BCP graft materials. In pilot study 1, three BCP materials (OSTEON™II, OSTEON™III, and OSTEON™II Collagen) were grafted in rabbit calvarial defects after impregnating in rhBMP-2. OSTEON™II without the rhBMP-2 impregnation was included in the study as the control. The amount of new bone was examined and measured histologically at 2, 4, and 8 weeks. In pilot study 2, four BCP materials (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) were grafted in beagle dog mandibular defects after soaking in the rhBMP-2. The amount of total bone and new bone were measured three-dimensionally using microCT and healing process was examined histologically at 2, 4, and 8 weeks. ResultsIn pilot study 1, rhBMP-2 impregnated groups showed more new bone formation than the rhBMP-2 free group. In pilot study 2, increased new bone formation was observed in time-dependent manner after graft of BCP and BCP-collagen (OSTEON™II, OSTEON™III, OSTEON™II Collagen, and OSTEON™III Collagen) impregnated with rhBMP-2. Also, BCP with a higher proportion of HA (30% HA) showed more favorable result in new bone formation and space maintenance, especially at the 8 weeks. ConclusionFrom the results of the pilot studies, rhBMP-2 played positive roles in new bone formation and BCP could become a scaffold candidate for rhBMP-2 impregnation to induce new bone formation. Moreover, BCP with a higher proportion of HA (30% HA) could be considered more appropriate for rhBMP-2 carrier.

Bone tissue modelling and remodelling following guided bone regeneration in combination with biphasic calcium phosphate materials presenting different microporosity

The aim of this study was to investigate bone regeneration following application of a novel biphasic calcium phosphate (BCP I) composed of microstructured granules of 90% β-tricalcium phosphate (β-TCP)/10% hydroxyapatite (HA) compared to BCP non-microstructured biphasic calcium phosphate with a composite of 60% hydroxyapatite/40% β-TCP (BCP II) and a deproteinized bovine bone mineral (DBBM) at surgically created defects in the mandible of minipigs in a combined approach with guided bone regeneration (GBR). Sixteen minipigs were used for the study. Lower premolars P2, P3, P4 and first molar M1 were extracted. Following 3 months of healing, two defects with a width and depth of 7 mm were created bilaterally in the mandible. The different grafting materials were randomly placed in the created defects and covered by means of a collagen membrane. After 3 and 8 weeks, biopsies were sampled. All specimens were evaluated with descriptive histology and histomorphometric evaluations complemented by micro-CT scan analysis. All three biomaterials presented with higher bone volume at 8 weeks compared to 3 weeks (P < 0.0442). BCP I and DBBM demonstrated a significant higher amount of bone formation compared to BCP II at 8 weeks (P < 0.0328). BCP I also demonstrated a significant higher percentage of remaining graft volume compared to the other test groups both at 3 and 8 weeks (P < 0.0001 to P < 0.0003). Congruently, defects containing BCP I showed a significant higher amount of mineralized tissue compared to the other groups. All the three test materials performed well with regard to bone formation at 8 weeks. BCP I showed significant higher amounts of newly formed bone despite a higher remaining graft volume compared to the other groups. With regard to the regenerative outcome, all the three materials can be recommended for clinical use.

Synthesis, Characterization and &lt;i&gt;In Vitro&lt;/i&gt; Evaluation of Strontium-Containing Sol-Gel Derived Bioactive Glass/ Biphasic Calcium Phosphate Nanocomposite

In the present research, strontium containing nanobioactive glass (NBG-Sr) was synthesized by sol-gel method. The morphology was analyzed by transmission electron microscope (TEM). Different amounts (0.5 to 5 wt%) of NBG-Sr were then added to biphasic calcium phosphate (BCP). They were sintered at different temperatures, i.e., 1100, 1200 and 1300 °C and changes in physical and mechanical properties were investigated. A sharp decrease in pore volume was observed as the temperature increased. The maximum bending strength (~45 MPa) was achieved for BCP which was mixed with 3 wt% NBG-Sr and sintered at 1200 °C. This value was approximately the same when it was sintered at 1300 °C. The bending strength failed when both lower and higher amounts of 3 wt% NBG-Sr were utilized. Therefore, sintering of composites at 1200 °C was economically reasonable. The X-ray results showed that NBG-Sr additive did not change the phase composition of BCP when it was heat treated at 1200 °C. The attachment and proliferation of rat calvarium-derived osteoblasts on samples sintered at 1200 °C were also evaluated by scanning electron microscopy (SEM). Based on cell studies, all NBG-Sr-added BCPs supported attachment and proliferation of osteoblastic cells. Overall, biphasic calcium phosphate materials with improved mechanical and biological properties can be produced by using certain quantity of strontium-containing bioactive glass particles.

A histomorphometric and micro–computed tomography study of bone regeneration in the maxillary sinus comparing biphasic calcium phosphate and deproteinized cancellous bovine bone in a human split-mouth model

The gain of mineralized bone was compared between deproteinized bovine bone allograft (DBA) and biphasic calcium phosphate (BCP) for dental implant placement. Five patients with atrophic maxillae underwent bilateral sinus elevation with DBA (Bio-Oss) and BCP (Straumann BoneCeramic). After 3 to 8 months, 32 Camlog implants were placed, and biopsies were retrieved. Bone and graft volume, degree of bone mineralization, and graft degradation gradient were determined using micro-computed tomography, and bone formation and resorption parameters were measured using histomorphometry. Implant functioning and peri-implant mucosa were evaluated up to 4 years. Patients were prosthetically successfully restored. All but one of the implants survived, and peri-implant mucosa showed healthy appearance and stability. Bone volume, graft volume, degree of bone mineralization, and osteoclast and osteocyte numbers were similar, but BCP-grafted biopsies had relatively more osteoid than DBA-grafted biopsies. The BCP and DBA materials showed similar osteoconductive patterns and mineralized bone, although signs of more active bone formation and remodeling were observed in BCP- than in DBA-grafted biopsies.

Mechanical properties and in vitro cellular behavior of zinc-containing nano-bioactive glass doped biphasic calcium phosphate bone substitutes

In the present study, different amounts (0.5-5 wt%) of a sol gel-derived zinc-containing nano-bioactive glass (NBG-Zn) powder were added to biphasic calcium phosphate (BCP). The mixtures were sintered at 1,100-1,300 °C and physical characteristics, mechanical properties, phase composition and morphology of them were studied. The samples were also soaked in human blood plasma for 15 days to evaluate variations in their surface morphologies. Rat calvarium-derived osteoblastic cells were seeded on tops of various samples and cell adhesion, proliferation, and alkaline phosphatase activity were evaluated at different culturing periods. The maximum bending strength (62 MPa) was obtained for BCP containing 0.5 wt% NBG-Zn at temperature 1,200 °C. This value was approximately 80% higher than that of pure BCP. The bending strength failed when both sintering temperature and amount of added NBG-Zn increased. At 1,100 °C, NBG-Zn additive did not change the phase composition of BCP. At temperatures 1,200 and 1,300 °C, both alpha-tricalcium calcium phosphate (α-TCP) and beta-tricalcium phosphate (β-TCP and) phases were detected. However, adding higher amount of NBG-Zn to BCP resulted in elevation of β-TCP at 1,200 °C and progression of α-TCP at 1,300 °C. Based on the microscopic observations, adding 0.5 wt% NBG-Zn to BCP led to disappearance of grain boundaries, reduction of micropores and formation of a monolithic microstructure. No calcium phosphate precipitation was observed on sample surfaces after soaking in blood plasma, but some pores were produced by phase dissolution. The size and volume of these pores were directly proportional to NBG-Zn content. Based on the cell studies, both BCP and NBG-Zn-added BCP samples supported attachment and proliferation of osteoblasts, but higher alkaline phosphatase enzyme was synthesized within the cells cultured on NBG-Zn-added BCP. Overall, biphasic calcium phosphate materials with improved mechanical and biological properties can be produced by using small quantity of zinc-containing bioactive glass particles.

A novel three-dimensional analysis of standardized bone defects by means of confocal scanner and micro-computed tomography

The aim of the study was to introduce a novel three-dimensional (3D) method to quantify the relative amount of different tissue components in bone substitute-treated defects by means of integration of confocal laser imaging into micro-computed tomography (μCT) analysis. One standardized semisaddle intraosseous defect was prepared in the mandibles of six minipigs and scanned by an optical scanner to capture the surface of the fresh defect in a 3D manner. Subsequently, all the defects were filled with a biphasic calcium phosphate material. The animals were divided into two groups of three animals each, which were allowed to heal for 3 and 8 weeks, respectively. μCT analysis followed the two healing periods and was performed on all defect locations. The data from optical scanning and μCT were used for three-dimensional evaluation of bone formation, nonmineralized tissue ratio, and graft degradation. The integration of confocal laser scanning into μCT analysis through a superimposition imaging procedure was conducted using the software Amira (Mercury Computer Systems, Chelmsford, MA, USA). The feasibility of combining the confocal imaging into μCT data with regard to obtaining accurate 3D quantification was demonstrated. The amount of tissue components was identified and quantified in all the investigated samples. Quantitative analysis demonstrated that a significant increase in the amount of bone filling the defect was observed in vivo (p < 0.02) while a significant decrease in the amount of nonmineralized tissue occurred (p < 0.04). No difference in the amount of residual grafting material was detected between 3 and 8 weeks in vivo (p > 0.38). The combination of confocal imaging and micro-computed tomography techniques allows for analysis of different tissue types over time in vivo. This method has revealed to be a feasible alternative to current bone regeneration quantification methods. Assessment of bone formation in a large animal model is a key step in assessing the performance of new bone substitute materials. Reliable and accurate methods are needed for the analysis of the regenerative potential of new materials.

Evaluation of moldable, in situ hardening calcium phosphate bone graft substitutes

Moldable in situ self-stabilizing and hardening bone graft materials facilitate handling and may be suitable for membrane-free bone regeneration methods. This study aimed to compare two moldable synthetic calcium phosphate materials in a rabbit calvarial defect model. In 12 New Zealand white rabbits, four evenly distributed 6mm diameter defects were drilled in the calvarial bone. Three filler materials were randomly applied to 48 defects: an in situ hardening polylactide-coated ß-tricalcium phosphate (TCP), an in situ hardening polylactide-coated biphasic calcium phosphate (BCP), and a granular deproteinized bovine bone matrix (DBBM, positive control). One defect remained untreated and served as a negative control. Six animals were sacrificed after 4weeks, and the remaining animals were sacrificed after 16weeks. Biocompatibility, bone graft substitute integration and resorption, bone formation, defect bridging, and height of reconstructed hard tissue were assessed histologically and histomorphometrically. All tested materials showed good biocompatibility. Semi-quantitative analysis and pair-wise comparison suggested that BCP was more efficient in centripetal bone formation when compared with TCP. After 4weeks, significantly more bone had formed in the defects treated with either TCP or BCP materials compared with the untreated sites. BCP and DBBM did not show macroscopic signs of degradation, whereas the TCP material was partially resorbed after 16weeks. Otherwise, no major differences were detected between the three materials. The moldable, synthetic calcium phosphates are safe and suitable bone graft substitutes with outcomes that are comparable to the control material.

The use of Straumann® Bone Ceramic in a maxillary sinus floor elevation procedure: a clinical, radiological, histological and histomorphometric evaluation with a 6‐month healing period

In this study, we evaluated the quality and quantity of bone formation in maxillary sinus floor elevation procedure using a new fully synthetic biphasic calcium phosphate (BCP) consisting of a mixture of 60% hydroxyapatite and 40% of beta-tricalcium phosphate (Straumann Bone Ceramic). A unilateral maxillary sinus floor elevation procedure was performed in six patients using 100% BCP. Biopsy retrieval for histological and histomorphometric analysis was carried out before implant placement after a 6-month healing period. In this study, the maxillary sinus floor elevation procedure with the use of BCP showed uneventful healing. Radiological evaluation after 6 months showed maintenance of vertical height gained immediately after surgery. Primary stability was achieved with all Straumann SLA dental implants of 4.1 mm diameter and 10 or 12 mm length. The implants appeared to be osseointegrated well after a 3-month healing period. Histological investigation showed no signs of inflammation. Cranial from the native alveolar bone, newly formed mineralized tissue was observed. Also, osteoid islands as well as connective tissue were seen around the BCP particles, cranial from the front of newly formed mineralized tissue. Close bone-to-substitute contact was observed. Histomorphometric analysis showed an average bone volume/total volume (BV/TV) of 27.3% [standard deviation (SD) 4.9], bone surface/total volume (BS/TV) 4.5 mm(2)/mm(3) (SD 1.1), trabecula-thickness (TbTh) 132.1 mum (SD 38.4), osteoid-volume/bone volume (OV/BV) 7.5% (SD 4.3), osteoid surface/bone surface (OS/BS) 41.3% (SD 28.5), osteoid thickness (O.Th) 13.3 mum (SD 4.7) and number of osteoclasts/total area (N.Oc/Tar) 4.4 1/mm (SD 5.7). Although a small number of patients were treated, this study provides radiological and histological evidence in humans confirming the suitability of this new BCP for vertical augmentation of the atrophied maxilla by means of a maxillary sinus floor elevation procedure allowing subsequent dental implant placement after a 6-month healing period. The newly formed bone had a trabecular structure and was in intimate contact with the substitute material, outlining the osteoconductive properties of the BCP material. Bone maturation was evident by the presence of lamellar bone.

A Comparative Evaluation of Biphasic Calcium Phosphate Material and Bioglass in the Treatment of Periodontal Osseous Defects: A Clinical and Radiological Study

The aim of this study was to evaluate the efficacy of biphasic calcium phosphate (ossifi) and bioactive glass in the treatment of periodontal osseous defects clinically and radiographically and compare them with open-flap debridement. A total of 45 sites in two test groups (test 1, ossifi; test 2, bioactive glass) and a control group (open-flap debridement), in 35 patients, were selected in this study conducted at the department of Periodontics and Oral Implantology, D.A.V.(C) Dental College, Yamuna Nagar, India. Clinical parameters like plaque index, gingival index, pocket depth, and clinical attachment level were recorded at the baseline and at three months and six months postoperatively. Radiological parameters like the amount of defect resolution and the percentage of defect resolution were recorded at the baseline and at three months and six months postoperatively. Statistically significant difference in mean values of the plaque index, gingival index, pocket depth reduction, clinical attachment level, gain amount of defect resolution, and percentage of defect resolution were observed in all the groups at subsequent time periods. Both test groups showed significant improvement over the control in both the clinical and radiological parameters. A greater percentage of defect resolution was noticed in test 1 as compared to test 2, followed by the control.

Subcutaneous‐induced membranes have no osteoinductive effect on macroporous HA‐TCP in vivo

Induced Membranes Technique was first described to enhance bone reconstruction of large osseous defects. Previous in vitro studies established their osteoinductive potential, due to the presence of opteoblasts precursors and to high amounts of growth factors contained within. The purpose of this study was to test in vivo the osteoinductive properties of induced membranes on a macroporous HA-TCP in a nonosseous subcutaneous site. Subcutaneous-induced membranes were obtained in 21 rabbits; 1 month later, the membranes were filled with a biphasic calcium phosphate material composed of 75% hydroxyapatite (HA) and 25% beta-tricalcium phosphate associated or not with autograft. Histological and immunohistochemical studies were performed on membrane biopsies. Undecalcified and decalcified sections were qualitatively and quantitatively analyzed. (45)Ca uptake was observed and quantified on the sections using microimager analysis. Dense vascularity was found in the induced membranes. New bone formation was detected in the HA-TCP + autograft samples and increased significantly from 3 to 6 months (p < 0.05). No bone was detected in the biomaterial graft alone in the induced membranes at any time. This study showed that induced membranes placed in a nonosseous site have no osteoinductive properties on a macroporous biphasic calcium phosphate biomaterial.

Preparation and characterization of biphasic calcium phosphate ceramics of desired composition

A modified processing route for fabricating dense and porous biphasic calcium phosphate (BCP) ceramics of desired and reproducible phase composition (hydroxyapatite (HA)/beta-tricalcium phosphate (beta-TCP) ratio) has been developed. The principal idea of the route was combining a precipitation and a solid phase methods. First, a nonstoichiometric (slightly carbonated calcium-deficient) HA (CdHA) precipitate was synthesized by mixing a calcium carbonate (CaCO(3)) water suspension with an orthophosphoric acid (H(3)PO(4)) solution in abundance (related to the amount resulting in a stoichiometric HA) under definite conditions, and a powder of the precipitate was prepared and calcinated in air (860 degrees C, 1.5 h). In the second stage, a BCP ceramics of the composition determined by the calcium-deficiency in a calcinated powder (the acid abundance in a mixture) was processed by sintering powder compacts with or without a porosizer under appropriate conditions (1,200 degrees C, 2h). A calibrating dependence of the HA/beta-TCP ratio in the ceramics on the acid abundance has been plotted which enabled a controlled preparation of BCP ceramics. A correlation based on unresolved bands in nu(4)-PO (4) (3-) domain in IR-spectra of nanostructured BCP materials was found. Using the correlation, the process of CdHA --> beta-TCP transformation could be easily monitored. The density and microhardness of the BCP ceramics neglectly depended on the composition, however, the compressive strength did: the lower the HA/beta-TCP ratio, the higher the strength in the dense materials.

Macroporous biphasic calcium phosphate scaffold with high permeability/porosity ratio.

Macroporous biphasic calcium phosphate (BCP) with channel-shaped pores was produced by a novel dual-phase mixing method. The processing route includes mixing water-based BCP slurry and polymethylmethacrylate resin; shaping in a mold; and polymerization, drying, pyrolyzing, and sintering. After comparison with two other commercial macroporous BCP materials, which were produced along different routes, it was found that conventional parameters such as porosity and pore size cannot describe a macroporous structure precisely enough for the application as tissue-engineering scaffold. Instead, permeability can be seen as an intrinsic and quantitative parameter to describe the macroporous structure of various scaffolds, because it is independent of sample size and fluid used in the test. Another parameter, the permeability/porosity ratio, provides an indication of the percolative efficiency per unit porous volume of a scaffold. Structural characterizations and permeability studies of other macroporous scaffold materials were also performed, and it was found that permeability could reflect a combination of five important parameters for scaffold: (1) porosity, (2) pore size and distribution, (3) interconnectivity, (4) fenestration size and distribution, and (5) orientation of pores. Finally, the implications of relating permeability with biological performances are also discussed.