Biphasic Calcium Phosphate Particles Research Articles

Bone formation of block and particulated biphasic calcium phosphate lyophilized with Escherichia coli–derived recombinant human bone morphogenetic protein 2 in rat calvarial defects

The objective of this study was to evaluate bone formation in rat calvarial defects after surgical implantation of block or particulated biphasic calcium phosphate (BCP) lyophilized with Escherichia coli-derived recombinant human bone morphogenetic protein 2 (ErhBMP-2). Critical-size calvarial osteotomy defects were created in 5 groups of Sprague-Dawley rats. Each group received one of the following: 1) sham surgery control; 2) biphasic calcium phosphate particles (CPP); 3) biphasic calcium phosphate block (CPB); 4) ErhBMP-2-coated CPP; or 5) ErhBMP-2-coated CPB. ErhBMP was coated on BCP by a stepwise lyophilizing protocol. The new bone formation was significantly greater in ErhBMP-2-treated groups compared with the untreated group. In particular, the ErhBMP-2/CPB group showed stability of augmented areas during the period of healing, due to relevant space-providing capacity. Thus, it can be concluded that CPP and CPB lyophilized with ErhBMP-2 enhance the formation of new bone, and CPB appears to be a suitable carrier for ErhBMP-2 in which a 3-dimensional structural integrity is an important consideration factor.

Back‐scattered electron imaging and elemental analysis of retrieved bone tissue following sinus augmentation with deproteinized bovine bone or biphasic calcium phosphate

To compare resorption of a synthetic biphasic calcium phosphate (BCP) bone-graft substitute with deproteinized bovine bone (DBB) used for human maxillary sinus augmentation. Eleven patients underwent bilateral maxillary sinus floor augmentation with DBB in one side and a BCP (40%beta-tricalcium phosphate (beta-TCP) and 60% hydroxyapatite) in the contralateral side. Simultaneously, with the augmentation on each side a microimplant was placed vertically from the top of the alveolar crest penetrating the residual bone and the grafting material. Eight months after initial surgery the microimplants were retrieved with a surrounding bone core. The composition of residual graft material and surrounding bone was analysed by scanning electron microscopy and energy dispersive X-ray spectroscopy. Residual graft material of both types was present as 10-500 mum particles in direct contact with, or completely surrounded by, newly formed bone; smaller particles were also present in non-mineralized tissue. In the case of BCP the bone-graft substitute interface showed evidence of superficial disintegration of particles into individual grains. Median Ca/P ratios (at.%), determined from >200 discreet sites within residual graft particles and adjacent bone, were: DBB: 1.61 (confidence interval [CI] 1.59-1.64); BCP: 1.5 (CI 1.45-1.52); DBB-augmented bone: 1.62 (CI 1.59-1.66); BCP-augmented bone: 1.52 (CI 1.47-1.55); P=0.028 for DBB vs. BCP and DBB- vs. BCP-augmented bone. The reduction in Ca/P ratio for BCP over the healing period is consistent with the dissolution of beta-TCP and reprecipitation on the surface of calcium-deficient hydroxyapatite. The beta-TCP component of BCP may be gradually substituted by calcium-deficient hydroxyapatite over the healing period. This process and superficial degranulation of BCP particles may influence the progress of resorption and healing.

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.

Human Primary Osteocyte Differentiation in a 3D Culture System

Studies on primary osteocytes, which compose >90-95% of bone cells, embedded throughout the mineralized matrix, are a major challenge because of their difficult accessibility and the very rare models available in vitro. We engineered a 3D culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for 4 wk in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times, as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Histological analysis showed, after only 1 wk, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real-time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells, and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the cross-talk of osteocytes with tumor cells in bone metastasis, because it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could also be a very efficient tool for drug testing and bone tissue engineering applications.

The effect of citric acid on the sintering of calcium phosphate bioceramics

Biphasic calcium phosphate particles were prepared using a rapid increase of the pH value by adding an amount of concentrated ammonia solution, into a well-mixed solution containing Ca(H 2PO 4) 2·H 2O and CaCl 2 with Ca/P = 1.667 molar ratio; with or without the presence of citric acid (CiA). The precipitation of Calcium Phosphates took place at 97 °C using high-speed dispersing equipment. The samples were characterized using XRD, FT-IR, BET and SEM techniques. The thermal behaviour was studied by TG, DTG and DTA techniques. The modified precipitation method leads to the formation of biphasic needle-like particles consisted of crystalline hydroxyapatite (HA) and β-tricalcium phosphate (β-TCP). The presence of CiA in the initial solution leads to the formation of aggregated nonporous small spheroidal particles consisted of low crystallinity phases of HA and octacalcium phosphate (OCP). The later facilitates the sintering process. The calcined samples at 900 °C were consisted of calcium pyrophosphate (CPP) and TCP. α-TCP is formed in the presence of a large amount of calcium citrate complexes. The main sintering process took place at the temperature range between 750 and 1150 °C, which were strongly depended on the initial amount of the CiA in the precipitation process. These results indicated that the citrate presence in the initial solution have a strong influence on the nucleation and growth processes by complexation with calcium ions and incorporation into the solid structure during particle growth.

3D environment on human mesenchymal stem cells differentiation for bone tissue engineering

In this work a novel method was developed to create a three dimensional environment at a cellular level for bone tissue engineering. Biphasic calcium phosphate (BCP) particles of 140-200 microm were used in association with human mesenchymal stem cells (hMSCs). The cells seeded on these particles adhered and proliferated more rapidly in the first day of culture compared to culture on plastic. Analyses of hMSCs cultured without osteogenic factors on BCP particles revealed an abundant extracellular matrix production forming 3-dimensional (3D) hMSCs/BCP particles constructs after few days. Bone morphogenetic 2 (BMP-2), bone sialoprotein (BSP) and ALP gene expression using real time quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) confirmed that expression profiles were modified by the culture substrate while the addition of osteogenic medium enhanced bone markers expression. These results indicate that BCP particles alone are able to induce an osteoblastic differentiation of hMSCs that might be of interest for bone tissue engineering.

Effect of phase composition and microstructure of calcium phosphate ceramic particles on protein adsorption

The biological performance of biomaterials is strongly influenced by their protein adsorption characteristics, which are related to the structures and properties of both the biomaterial and the protein. In the present study two groups of hydroxyapatite (HA) and biphasic calcium phosphate (BCP) ceramic powders were fabricated by different drying processes. The roles of the phase composition and microstructure of the powders in the adsorption of various model proteins were evaluated. The experimental results showed that BCP always had a higher ability to adsorb fibrinogen, insulin or type I collagen (Col-I) than HA. The microporosity and micropore size of the CaP particles also had a strong impact on their protein adsorption characteristics. HA and BCP particles with higher microporosities and/or more micropores >20 nm in diameter could adsorb more fibrinogen or insulin. However, amounts of adsorbed Col-I were largely unaffected by the microstructure of HA and BCP particles.

Surface-modified biphasic calcium phosphate/poly (L-lactide) biocomposite

Biphasic calcium phosphate (BCP) powders were prepared by hydrolyzation process and surface-modified by directly grafted L-lactide (LLA) onto the surface of BCP through a chemical linkage. The grafting ratio of organic groups was 9 wt%. After surface modification, the surface of BCP powders was covered by the lamella-shaped crystal. Poly (L-lactide) was mixed with BCP to form the BCP/PLLA biocomposite. Modified BCP (mBCP) particles could be uniformly dispersed in PLLA matrix. The compressive strength of the mBCP/PLLA composite is 115 MPa, 28% higher than that of unmodified-BCP/PLLA composite. The improved mechanical strength is attributed to the enhanced adhesion between the inorganic BCP filler and the organic PLLA matrix.

<i>In Vivo</i> Performance of an Injectable Biphasic Calcium Phosphate Bone Filler

An hydrated putty was prepared by mixing submicron particles, rounded particles and granules of Biphasic Calcium Phosphate (BCP) ceramics composed of HA and β-TCP phases. The material filled entirely critical sized defects in the femoral epiphysis of NZW rabbits. After 3, 6 and 12 weeks, histology revealed that submicron particles were rapidly degraded by multinucleated TRAP-positive cells. This osteoclastic resorption stimulated bone ingrowth while the large BCP particles served as scaffold supporting bone healing by osteoconduction.

Impact of pore size on the vascularization and osseointegration of ceramic bone substitutes in vivo

The repair of bone defects with biomaterials depends on a sufficient vascularization of the implantation site. We analyzed the effect of pore size on the vascularization and osseointegration of biphasic calcium phosphate particles, which were implanted into critical-sized cranial defects in Balb/c mice. Dense particles and particles with pore sizes in the ranges 40-70, 70-140, 140-210, and 210-280 microm were tested (n = 6 animals per group). Angiogenesis, vascularization, and leukocyte-endothelium interactions were monitored for 28 days by intravital microscopy. The formation of new bone and the bone-interface contact (BIC) were determined histomorphometrically. Twenty-eight days after implantation, the functional capillary density was significantly higher with ceramic particles whose pore sizes exceeded 140 microm [140-210 microm: 6.6 (+/-0.8) mm/mm(2); 210-280 microm: 7.3 (+/-0.6) mm/mm(2)] than with those whose pore sizes were lesser than 140 microm [40-70 microm: 5.3 (+/-0.4) mm/mm(2); 70-140 microm: 5.6 (+/-0.3) mm/mm(2)] or with dense particles [5.7 (+/-0.8) mm/mm(2)]. The volume of newly-formed bone deposited within the implants increased as the pore size increased [40-70 microm: 0.07 (+/-0.02) mm(3); 70-140 microm: 0.10 (+/-0.06) mm(3); 140-210 microm: 0.13 (+/-0.05) mm(3); 210-280 microm: 0.15 (+/-0.06) mm(3)]. Similar results were observed for the BIC. The data demonstrates pore size to be a critical parameter governing the dynamic processes of vascularization and osseointegration of bone substitutes.

Competitive adsorption of bovine serum albumin and lysozyme on characterized calcium phosphates by polyacrylamide gel electrophoresis method

Characterizations of hydroxyapatite (HA), biphasic calcium phosphate (BCP) and beta tricalcium phosphate (beta-TCP) ceramic particles were carried out using X-ray diffusion (XRD), Scanning electron micrograph (SEM), Particle Sizer and Zeta potential analyzer. Competitive adsorption of bovine serum albumin (BSA) and lysozyme (LSZ) on the three calcium phosphates were investigated by polyacrylamide gel electrophoresis (PAGE) method. The results showed that HA, BCP and beta-TCP ceramic particles with irregular shapes and similar size distributions all had negative surface net charges in pH7.4 phosphate buffered saline (PBS) solution and exhibited alike behaviors of BSA and LSZ adsorption. LSZ had higher affinity for calcium phosphate ceramics than BSA and its adsorption on them didn't be almost influenced by the increasing of BSA concentration in the solution. Electrostatic interaction played an important role on the competitive adsorption of BSA and LSZ on the surface of calcium phosphate ceramic particles.

Bone repair using a new injectable self-crosslinkable bone substitute

A new injectable and self-crosslinkable bone substitute (IBS2) was developed for filling bone defects. The IBS2 consisted of a chemically modified polymer solution mixed with biphasic calcium phosphate (BCP) ceramic particles. The polymer hydroxypropylmethyl cellulose was functionalized with silanol groups (Si-HPMC) and formed a viscous solution (3 wt %) in alkaline medium. With a decrease in pH, self-hardening occurred due to the formation of intermolecular -Si-O- bonds. During setting, BCP particles, 40 to 80 microm in diameter, were added to the polymer solution at a weight ratio of 50/50. The resulting injectable material was bilaterally implanted into critically sized bone defects at the distal femoral epiphyses of nine New Zealand White rabbits. The IBS2 filled the bone defects entirely and remained in place. After 8 weeks, bone had grown centripetally and progressed towards the center of the defects. Newly formed bone, ceramic, and nonmineralized tissue ratios were 24.6% +/- 5.6%, 21.6% +/- 5.8%, and 53.7% +/- 0.1%, respectively. Mineralized and mature bone was observed between and in contact with the BCP particles. The bone/ceramic apposition was 73.4% +/- 10.6%. The yield strength for the IBS2-filled defects was 16.4 +/- 7.2 MPa, significantly higher than for the host trabecular bone tissue (2.7 +/- 0.4 MPa). This study showed that BCP particles supported the bone healing process by osteoconduction while the Si-HPMC hydrogel created intergranular space for bone ingrowth. This new injectable and self-crosslinkable bone substitute could be used conveniently in orthopedic surgery for filling critical-size bone defects.

In vivo bone regeneration with injectable calcium phosphate biomaterial: A three-dimensional micro-computed tomographic, biomechanical and SEM study

This in vivo study investigated the efficiency of an injectable calcium phosphate bone substitute (IBS) for bone regenerative procedures through non-destructive three-dimensional (3D) micro-tomographic ( μCT) imaging, biomechanical testing with a non-destructive micro-indentation technique and 2D scanning electron microscopy (SEM) analysis. The injectable biomaterial was obtained by mixing a biphasic calcium phosphate (BCP) ceramic mineral phase and a cellulosic polymer. The BCP particles were 200–500 μm or 80–200 μm in diameter. The injectable material was implanted for 6 weeks into critical-sized bone defects at the distal end of rabbit femurs. Extensive new bone apposition was noted with both 2D and 3D techniques. Micro-CT showed that newly formed bone was in perfect continuity with the trabecular host bone structure and demonstrated the high interconnectivity of the restored bone network. For both IBS formulations, SEM and μCT gave very close measurements. The only detected significant difference concerned the amount of newly formed bone obtained with IBS 80-200 that appeared significantly higher with μCT analysis than with SEM ( p=0.00007). Student t-tests did not show any significant difference in the amount of newly formed bone and remaining ceramic obtained from μCT analysis or SEM. Regression analysis showed satisfactory correlation between both the amount of newly formed bone and remaining ceramic obtained from μCT or SEM. For IBS 200-500, the newly formed bone rate inside the defect was 28.0±5.2% with SEM and yield strength of the samples was 18.8±5.4 MPa. For IBS 80-200, the newly formed bone rate inside the defect was 31.7±5.1% with SEM and yield strength of the samples was 26.8±4.5 MPa. Yield strength appeared well correlated with the amount of newly formed bone, specially observed with μCT. This study showed the ability of non-destructive techniques to investigate biological and mechanical aspects of bone replacement with injectable biomaterials.

Preparation and biocompatibility of poly (methyl methacrylate) reinforced with bioactive particles

Calcium phosphates and bioactive glasses have been used in many biomedical applications for more than 30 years due basically to their bioactive behavior. However, ceramics are too brittle for applications that require high levels of toughness and easy processability. In this work, a biphasic calcium phosphate (BCP) and a bioactive glass composition (BG) were combined with polymers to produce composites with tailorable properties and processability. The BCP particles were synthesized by a precipitation technique. The BG particles were produced by sol-gel processing. The BCP particles were treated with a silane agent to improve the compatibility between particles and the polymer matrix. Dense samples were produced by hot pressing (200 °C) a mixture of 30 wt.% of particles in poly (methyl methacrylate). The samples produced were characterized by X-ray diffraction, infrared spectroscopy and scanning electron microscopy. Mechanical properties were evaluated by a three point bending test. Samples were also submitted to in vitro bioactivity test and in vivo toxicity test. Results showed that the production of the composites was successfully achieved, yielding materials with particles well dispersed within the matrices. Evaluation of the in vivo inflammatory response showed low activity levels for all composites although composites with silane treated BCP particles led to milder inflammatory responses than composites with non-treated particles.

Long-term bone response to particulate injectable ceramic.

Biphasic calcium phosphate particles (BCP), alone or combined with a cellulosic ether vehicle in an injectable composite material (COMP), were implanted in femurs of rabbits. The long-term follow-up (up to 78 weeks) indicated: (1) BCP and COMP induced a foreign-body inflammatory reaction but without fibrous encapsulation. Phagocytosis was mediated by mononucleated macrophages (MC) and giant multinucleated cells (GMNC). Phagocytosis was stronger with COMP and required the recruitment of GMNC while it primarily involved MC in the case of BCP. (2) There appeared to be no significant difference between the bone ingrowth in the defects packed with BCP (dBCP) and in those filled with COMP (dCOMP). Bone reconstruction mostly was achieved after 4 weeks in dBCP but took more time to reach the center of dCOMP. High bone remodeling was observed at the last evaluation times, especially in the case of COMP. (3) Degradation of the materials occurred mainly during the first 4 weeks and was more severe for COMP, which probably was related to the smaller granulometry of its mineral phase. Cell-mediated degradation went on for the 78 weeks and followed two processes: phagocytosis and/or extracellular dissolution of the calcium phosphate particles.

Long‐term bone response to particulate injectable ceramic

Biphasic calcium phosphate particles (BCP), alone or combined with a cellulosic ether vehicle in an injectable composite material (COMP), were implanted in femurs of rabbits. The long-term follow-up (up to 78 weeks) indicated: (1) BCP and COMP induced a foreign-body inflammatory reaction but without fibrous encapsulation. Phagocytosis was mediated by mononucleated macrophages (MC) and giant multinucleated cells (GMNC). Phagocytosis was stronger with COMP and required the recruitment of GMNC while it primarily involved MC in the case of BCP. (2) There appeared to be no significant difference between the bone ingrowth in the defects packed with BCP (dBCP) and in those filled with COMP (dCOMP). Bone reconstruction mostly was achieved after 4 weeks in dBCP but took more time to reach the center of dCOMP. High bone remodeling was observed at the last evaluation times, especially in the case of COMP. (3) Degradation of the materials occurred mainly during the first 4 weeks and was more severe for COMP, which probably was related to the smaller granulometry of its mineral phase. Cell-mediated degradation went on for the 78 weeks and followed two processes: phagocytosis and/or extracellular dissolution of the calcium phosphate particles. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 42, 368–375, 1998.