Phenotypic expression of bone-related genes in osteoblasts grown on calcium phosphate ceramics with different phase compositions

Calcium phosphate ceramics with different hydroxyapatite (HA) and tricalcium phosphate (TCP) ratios have different chemical properties. Does the difference in phase composition affect osteoblast behavior? In this study, osteoblasts were cultured on 4 kinds of calcium phosphate ceramics, i.e. pure (HA), HT1 (HA/TCP, 70/30), HT2 (HA/TCP, 35/65), and pure TCP. Cell proliferation of SaOS-2 cells together with bone-related genes' mRNA expression and protein production in osteoblasts cultured on different calcium phosphate ceramics were detected at different time points. Data suggested that cell proliferation rate on TCP ceramics was lower than that on the other substrates tested. Generally, mRNA expressions for osteonectin and osteocalcin were similar among the four kinds of ceramics in most circumstances, whereas at six days, alkaline phosphatase mRNA expression was higher on HA and HT1 surfaces than on the other two materials. Collagen I mRNA expression was also affected by the phase composition of substrates. Osteocalcin and bone sialoprotein production in SaOS-2 cells was very similar no matter which ceramic surface the cells were grown upon. This study revealed that calcium phosphate ceramics substrate could support osteoblast growth and bone-related gene expression and its gene expression pattern explained the basis of the biocompatibility and bioactivity for calcium phosphate ceramics.     

Improved bone anchorage of hydroxypatite coated implants compared with tricalcium-phosphate coated implants in trabecular bone in dogs

Tricalcium phosphate (TCP) and hydroxyapatite (HA) ceramic coatings are bioactive coatings that have been shown to stimulate bone apposition onto ceramic-coated implants. TCP and HA ceramics have well-documented differences in physical properties, but both types of ceramics are used for stimulation of bone ongrowth to cementless endo-prosthetic components clinically. However, little is known about the difference in osteoconductive properties between these coatings when inserted into trabecular bone in a controlled experimental situation. Unloaded cylindrical gritblasted titanium (Ti-6A1-4V) implants (6 x 10 mm) coated with either hydroxyapatite (HA) or tricalcium phosphate (TCP) ceramic were inserted into the proximal humerus of 20 skeletally mature dogs. The implants were initially surrounded by a 2 mm gap. Each animal received one HA-coated implant and one TCP-coated implant. All dogs were sacrificed 6 weeks after surgery. Results were evaluated by histomorphometry and mechanical push-out test. Push-out tests demonstrated that HA-coated implants were 10-fold stronger fixated in comparison to TCP-coated implant. Bone ongrowth was significantly higher for HA-coated implants compared to TCP-coated implants. Bone volume in the gap showed a tendency to less bone volume around HA-coated implants compared to TCP-coated implants but this difference was insignificant. As expected almost all of the TCP coating were resorbed after 6 weeks and almost none of the HA coating. HA-coated implants with a grit-blasted surface provide a favorable early mechanical implant anchorage most likely due to superior ceramic stability compared to TCP-coated implants.     

Material-dependent bone induction by calcium phosphate ceramics: a 2.5-year study in dog

Bone induction by different calcium phosphate biomaterials has been reported previously. With regard to (1) whether the induced bone would disappear with time due to the absence of mechanical stresses and (2) whether this heterotopically formed bone would give rise to uncontrolled growth, a long-time investigation of porous hydroxyapatite ceramic (HA), porous biphasic calcium phosphate ceramic (TCP/HA, BCP), porous alpha-tricalcium phosphate ceramic (alpha-TCP) and porous beta-tricalcium phosphate ceramic (beta-TCP) was performed in dorsal muscles of dog, for 2.5 years. Histological observation, backscattered scanning electron microscopy observation and histomorphometric analysis were made on thin un-decalcified sections of retrieved samples. Normal compact bone with bone marrow was found in all HA implants (n = 4) and in all BCP implants (n = 4), 48 +/- 4% pore area was filled with bone in HA implants and 41 +/- 2% in BCP implants. Bone-like tissue, which was a mineralised bone matrix with osteocytes but lacked osteoblasts and bone marrow, was found in all beta-TCP implants (n = 4) and in one of the four alpha-TCP implants. Both normal bone and bone-like tissues were confined inside the pores of the implants. The results show that calcium phosphate ceramics are osteoinductive in muscles of dogs. Although the quality and quantity varied among different ceramics, the induced bone in both HA and BCP ceramics did neither disappear nor grow uncontrollably during the period as long as 2.5 years.     

Marrow cell induced osteogenesis in porous hydroxyapatite and tricalcium phosphate: a comparative histomorphometric study of ectopic bone formation

To investigate the bone formation ability of porous hydroxyapatite (HA) and tricalcium phosphate (TCP), ceramic discs were implanted with or without rat marrow cells into subcutaneous sites in syngeneic rats. The discs of HA and TCP had identical microstructures: pore size was 190-230 microns, porosity was 50-60%, and they were fully interconnected. Implants without marrow cells (discs themselves) did not show bone formation, whereas implants with marrow cells showed bone formation in the pores of the ceramics. The bone formation of both HA and TCP occurred initially on the surface of the ceramic and progressed towards the center of the pore. The de novo bone was quantitated from decalcified serial sections of the implants. One month after implantation with marrow cells, the percentage fractions of the pore area filled with bone for implanted HA and TCP were 16.9 and 15.1, respectively. At 2 months after implantation with marrow cells, the fractions of bone were 34.3 and 30.9, respectively. These results indicate that both HA and TCP ceramics can show comparable osteogenic ability in the presence of marrow cells.     

Nanoindentation study of interfaces between calcium phosphate and bone in an animal spinal fusion model.

Intertransverse process spinal fusion is a common surgical procedure for the treatment of spinal disorders. In the present study, a porous hydroxyapatite (HA)/beta-tricalcium phosphate (beta-TCP) ceramic was tested as graft material using a rabbit lumbar transverse process (L5-L6) fusion model. The porous ceramic blocks were implanted onto the dorsal decorticated surface of the lumbar transverse processes. The specimens were harvested at the seventh week after implantation. Histomorphological observation revealed that the integration of HA/beta-TCP with the host bone of the transverse process occurred by both cancellous bone formation and cartilage formation. Scanning electron microscopy-wavelength dispersive X-ray spectrometry examinations showed significant differences in calcium, phosphorus, and sulfur contents in the newly formed tissues and the porous HA/TCP implants. Nanoindentations were used to evaluate the intrinsic mechanical properties of the implants and the newly formed tissues. The Young's moduli of the newly formed cartilage, new cancellous bone, and HA/TCP, were 0.66 +/- 0.02 GPa, 2.36 +/- 0.50 GPa, and 10.2 +/- 1.21 GPa, respectively. Nanoindentation results revealed degradation of the porous ceramics and incomplete calcification of the new cancellous bone at the seventh week after implantation. Nanoindentation appeared to be a useful technique for assessing the mechanical status of spinal fusion in animal models.     

Expression of core binding factor 1 and osteoblastic markers in C2C12 cells induced by calcium phosphate ceramics in vitro

The in vivo osteoinductive capacity of porous calcium phosphate ceramics (Ca/P ceramics) with special structure and phase composition had been found for almost decades. The mechanism of the osteoinductivity of porous calcium phosphate is studied by C2C12 cells culture in this paper. C2C12 cells were cocultured with four kinds of porous Ca/P ceramics for 2 and 5 days, without adding other growth factors. The four kinds of Ca/P ceramics were pure HA sintered at 1250 degrees C and HA/TCP with a ratio of 60/40 sintered at 1100, 1200, and 1250 degrees C respectively. RT-PCR analysis found that the Ca/P ceramics induced the expression of Cbfa1, collagen type I, bone sialoprotein, and osteocalcin in C2C12 cells, while they did not induce mRNA expression of Indian hedgehog (IHH) that regulate chondrocyte differentiation. Our results showed that Ca/P ceramics alone were sufficient to induce C2C12 cells differentiation. The induction of bone-related markers expression by Ca/P ceramics in osteoprogenitor cells suggested that the osteogenesis induced by the ceramics was intramembranous and the osteoinductivity was their intrinsic property.     

In vivo study of novel biodegradable and osteoconductive CaO-SiO2-B2O3 glass-ceramics

To evaluate the possibility of novel CaO-SiO2-B2O3 glass-ceramics (CS10B) as a new bone replacement material, we compared the biodegradation and osteoconduction properties of CS10B, hydroxyapatite (HA), and tricalcium phosphate (TCP). Porous CS10B implants were prepared by the polymer sponge method. L5-6 single-level posterolateral spinal fusions were performed on 30 New Zealand white male rabbits. The animals were divided into three groups by implant material: CS10B, HA, and TCP. Radiographs were performed every 2 weeks. All animals were euthanized 12 weeks after surgery. The ratio of the area occupied by the ceramics by final and initial radiographs was calculated using radiomorphometric analysis. Uniaxial tensile strength was determined from seven cases in each group. The ratio of the area occupied by HA (88.7%+/-16.1%) was significantly higher than the others (p<0.005), and the ratio of the area occupied by CS10B (28.2%+/-9.3%) was significantly lower than those of HA and TCP (37%+/-9.6%, p<0.05). The mean values of the tensile strengths of the CS10B (182.7+/-19.9 N) and HA (191.4+/-33.5 N) were significantly higher (p<0.05) than that of TCP (141.1+/-28.2 N). CS10B had a fusion mass tensile strength similar to that of HA. Histological analysis confirmed that CS10B was well incorporated into the fusion mass. These findings suggest that CS10B is a possible bone replacement material.     

In vitro and in vivo study of microporous ceramics using MC3T3 cells,CAM assay and a pig animal model

Bone tissue engineering combines biomaterials with biologically active factors and cells to hold promise for reconstructing craniofacial defects. In this study the biological activity of biphasic hydroxyapatite ceramics (HA; a bone substitute that is a mixture of hydroxyapatite and β-tricalcium phosphate in fixed ratios) was characterized (1) in vitro by assessing the growth of MC3T3 mouse osteoblast lineage cells, (2) in ovo by using the chick chorioallantoic membrane (CAM) assay and (3) in an in vivo pig animal model. Biocompatibility, bioactivity, bone formation and biomaterial degradation were detected microscopically and by radiology and histology. HA ceramics alone demonstrated great biocompatibility on the CAM as well as bioactivity by increased proliferation and alkaline phosphatase secretion of mouse osteoblasts. The in vivo implantation of HA ceramics with bone marrow mesenchymal stem cells (MMSCs) showed de novo intramembranous bone healing of critical-size bone defects in the right lateral side of pig mandibular bodies after 3 and 9 weeks post-implantation. Compared with the HA ceramics without MMSCs, the progress of bone formation was slower with less-developed features. This article highlights the clinical use of microporous biphasic HA ceramics despite the unusually shaped elongated micropores with a high length/width aspect ratio (up to 20) and absence of preferable macropores (>100 µm) in bone regenerative medicine.     

Review paper: behavior of ceramic biomaterials derived from tricalcium phosphate in physiological condition

Various calcium phosphates are used for bone repair. Although hydroxyapatite (HA) sintered ceramics are widely used due to their osteoconductivity, its bioresorbability is so low that HA remains in the body for a long time after implantation. In contrast, tricalcium phosphate (TCP) ceramics show resorbable characters during bone regeneration, and can be completely substituted for the bone tissue after stimulation of bone formation. Therefore, much attention is paid to TCP ceramics for scaffold materials for supporting bone regeneration. This paper reviews bioresorbable properties of calcium phosphate ceramics derived from beta-TCP and alpha-TCP.     

Formation of osteoclast-like cells on HA and TCP ceramics

An essential property of bone substitute materials is that they are integrated into the natural bone remodelling process, which involves the resorption by osteoclast cells and the formation by osteoblast cells. If monocyte cells adhere to a calcium phosphate surface (bone or bone substitute material), they can fuse together and form multinucleated osteoclast cells. In this study we show that osteoclast-like cells derived from a human leukoma monocytic lineage responded in a different way to tricalciumphosphate (TCP) than to hydroxyapatite (HA) ceramics. Both ceramics were degraded by resorbing cells; however, HA enhanced the formation of giant cells. The osteoclast-like cells on HA formed a more pronounced actin ring, and larger lacunas could be observed. TCP ceramics are medically used as bone substitute materials because of their high dissolution rate. On the other hand, highly soluble calcium phosphate ceramics like TCP seem to be inappropriate for osteoclast resorption because they produce a high calcium concentration in the osteoclast interface and in the environment.     

Early weight bearing of porous HA/TCP (60/40) ceramics in vivo: a longitudinal study in a segmental bone defect model of rabbit

Porous interconnected hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) (60/40) ceramics are promising materials for hard tissue repair. However, the mechanical properties of these materials have not been accurately determined under weight-bearing conditions. In this study, newly developed HA and HA/TCP (60/40) ceramics were used with intramedullary fixation in segmental bone defects of rabbits. Early radiological, histological, densitometric and biomechanical changes were evaluated. The mean radiological grade of healing and bonding to bone was higher in HA/TCP (60/40) ceramics than that of pure HA ceramics but the difference was not statistically significant. The densities of both implanted ceramics improved with time, supported by the histological evaluation of bone matrix ingrowth into ceramic pores, whereas the densities at the bone–ceramic interface decreased gradually. Flexural resonant frequencies and three-point bending strength increased, revealing an increase in mechanical stability during this early critical time interval where implant and/or bone–implant interface failures occur frequently. It can be concluded that both HA and HA/TCP (60/40) ceramics have a limited application in the treatment of load-bearing segmental bone defects but did not fail at the early stages of implantation.     

Differential osteogenic activity of osteoprogenitor cells on HA and TCP/HA scaffold of tissue engineered bone

Biomaterial, an essential component of tissue engineering, serves as a scaffold for cell attachment, proliferation, and differentiation; provides the three dimensional (3D) structure and, in some applications, the mechanical strength required for the engineered tissue. Both synthetic and naturally occurring calcium phosphate based biomaterial have been used as bone fillers or bone extenders in orthopedic and reconstructive surgeries. This study aims to evaluate two popular calcium phosphate based biomaterial i.e., hydroxyapatite (HA) and tricalcium phosphate/hydroxyapatite (TCP/HA) granules as scaffold materials in bone tissue engineering. In our strategy for constructing tissue engineered bone, human osteoprogenitor cells derived from periosteum were incorporated with human plasma-derived fibrin and seeded onto HA or TCP/HA forming 3D tissue constructs and further maintained in osteogenic medium for 4 weeks to induce osteogenic differentiation. Constructs were subsequently implanted intramuscularly in nude mice for 8 weeks after which mice were euthanized and constructs harvested for evaluation. The differential cell response to the biomaterial (HA or TCP/HA) adopted as scaffold was illustrated by the histology of undecalcified constructs and evaluation using SEM and TEM. Both HA and TCP/HA constructs showed evidence of cell proliferation, calcium deposition, and collagen bundle formation albeit lesser in the former. Our findings demonstrated that TCP/HA is superior between the two in early bone formation and hence is the scaffold material of choice in bone tissue engineering.     

Comparison of plasma-sprayed hydroxyapatite coatings and hydroxyapatite/tricalcium phosphate composite coatings: in vivo study

This study aimed to compare biological properties, including osteoconduction, osseointegration, and shear strength, between plasma-sprayed hydroxyapatite (HA) and HA/tricalcium phosphate (TCP) coatings, using a transcortical implant model in the femora of canines. After 3 and 12 weeks of implantation, the implants with surrounding bone were assessed histologically in undecalcified sections in backscattered electron images (BEIs) under a scanning electron microscope (SEM). After short-term (3 week) follow-up, both coatings conducted new bone formation and revealed direct bone-to-coating contact. The HA/TCP coating could not enhance early host-to-coating responses. At 12 weeks, serious dissolution of the HA/TCP coatings evidently occurred. By the new bone healing index (NBHI) and apposition index (AI), we found no significant difference between HA/TCP-coated implants and HA-coated implants throughout all implant periods. At 12 weeks of implantation, some particles dissociated from the HA/TCP coating were found within the remodeling canal. After push-out measurements, the shear strength and failure mode of HA/TCP-coated implants were similar to those of HA-coated implants, and no statistical differences were found between either coating. Consequently, this study indicates that HA/TCP coatings have excellent biological response and may be considered suitable bioactive ceramic coatings for short-term clinical use.     

Silicon substitution in the calcium phosphate bioceramics

Silicon (Si) substitution in the crystal structures of calcium phosphate (CaP) ceramics such as hydroxyapatite (HA) and tricalcium phosphate (TCP) generates materials with superior biological performance to stoichiometric counterparts. Si, an essential trace element required for healthy bone and connective tissues, influences the biological activity of CaP materials by modifying material properties and by direct effects on the physiological processes in skeletal tissue. The synthesis of Si substituted HA (Si-HA), Si substituted alpha-TCP (Si-alpha-TCP), and multiphase systems are reviewed. The biological performance of these Si substituted CaP materials in comparison to stoichiometric counterparts is discussed. Si substitution promotes biological activity by the transformation of the material surface to a biologically equivalent apatite by increasing the solubility of the material, by generating a more electronegative surface and by creating a finer microstructure. When Si is included in the TCP structure, recrystallization to a carbonated HA is mediated by serum proteins and osteoblast-like cells. Release of Si complexes to the extracellular media and the presence of Si at the material surface may induce additional dose-dependent stimulatory effects on cells of the bone and cartilage tissue systems.     

Personalized implant for high tibial opening wedge: combination of solid freeform fabrication with combustion synthesis process

In this work a new generation of bioceramic personalized implants were developed. This technique combines the processes of solid freeform fabrication (SFF) and combustion synthesis (CS) to create personalized bioceramic implants with tricalcium phosphate (TCP) and hydroxyapatite (HA). These porous bioceramics will be used to fill the tibial bone gap created by the opening wedge high tibial osteotomy (OWHTO). A freeform fabrication with three-dimensional printing (3DP) technique was used to fabricate a metallic mold with the same shape required to fill the gap in the opening wedge osteotomy. The mold was subsequently used in a CS process to fabricate the personalized ceramic implants with TCP and HA compositions. The mold geometry was designed on commercial 3D CAD software. The final personalized bioceramic implant was produced using a CS process. This technique was chosen because it exploits the exothermic reaction between P?O? and CaO. Also, chemical composition and distribution of pores in the implant could be controlled. To determine the chemical composition, the microstructure, and the mechanical properties of the implant, cylindrical shapes were also fabricated using different fabrication parameters. Chemical composition was performed by X-ray diffraction. Pore size and pore interconnectivity was measured and analyzed using an electronic microscope system. Mechanical properties were determined by a mechanical testing system. The porous TCP and HA obtained have an open porous structure with an average 400?μm channel size. The mechanical behavior shows great stiffness and higher load to failure for both ceramics. Finally, this personalized ceramic implant facilitated the regeneration of new bone in the gap created by OWHTO and provides additional strength to allow accelerated rehabilitation.     

A comparative study of biphasic calcium phosphate ceramics for human mesenchymal stem-cell-induced bone formation

For the repair of bone defects, a tissue engineering approach would be to combine cells capable of osteogenic (i.e. bone-forming) activity with an appropriate scaffolding material to stimulate bone regeneration and repair. Human mesenchymal stem cells (hMSCs), when combined with hydroxyapatite/beta-tricalcium phosphate (HA/TCP) ceramic scaffolds of the composition 60% HA/40% TCP (in weight %), have been shown to induce bone formation in large, long bone defects. However, full repair or function of the long bone could be limited due to the poor remodeling of the HA/TCP material. We conducted a study designed to determine the optimum ratio of HA to TCP that promoted hMSC induced bone formation yet be fully degradable. In a mouse ectopic model, by altering the composition of HA/TCP to 20% HA/80% TCP, hMSC bone induction occurred at the fastest rate in vivo over the other formulations of the more stable 100% HA, HA/TCP (76/24, 63/37, 56/44), and the fully degradable, 100% TCP. In vitro studies also demonstrated that 20/80 HA/TCP stimulated the osteogenic differentiation of hMSCs as determined by the expression of osteocalcin.     

Phase conversion of tricalcium phosphate into Ca-deficient apatite during sintering of hydroxyapatite-tricalcium phosphate biphasic ceramics

In this study, we report a new observation on the phase conversion that occurs during the sintering of hydroxyapatite (HA)-tricalcium phosphate (TCP) biphasic ceramics. During the sintering of the HA-TCP mixture powders, a large amount of TCP was converted into HA, as detected by X-ray diffraction. The amount of TCP transformed into HA was approximately 10-90% of that initially added. From the electron probe microscopy analysis, the HA transformed from TCP was found to be Ca-deficient with Ca/P ratios of 1.62-1.64. The dissolution behavior and osteoblastic responses in a series of HA-TCP biphasic ceramics (10-90% TCP) were assessed. The solubility of the HA-TCP biphasic ceramics was intermediate between that of the HA and TCP pure ceramics. However, in the case of the HA-90% TCP biphasic ceramic, the solubility was even higher than that of pure TCP. The cell proliferation and alkaline phosphatase activity of the cells on the biphasic ceramics were lower than those on pure HA, but higher than those on pure TCP. However, particularly in the HA-50% TCP biphasic composition, the cellular responses were significantly higher than those on pure HA. It is considered that the Ca-deficient apatite newly formed from the TCP may affect in some way the solubility and biological properties of the HA-TCP biphasic ceramics.     

Effect of hydroxyapatite/tricalcium-phosphate coating on osseointegration of plasma-sprayed titanium alloy implants

This study determined the effects of a plasma-sprayed hydroxyapatite/tricalcium phosphate (HA/TCP) coating on osseointegration of plasma-sprayed titanium alloy implants in a lapine, distal femoral intramedullary model. The effects of the HA/TCP coating were assessed at 1, 3, and 6 months after implant placement. The HA/TCP coating significantly increased new bone apposition onto the implant surfaces at all time points. The ceramic coating also stimulated intramedullary bone formation at the middle and distal levels of the implants. Fluorescent bone labeling indicated that new bone formation occurred primarily during the first 3 months after implantation, with comparatively little activity detected in the latter stages of the study. There was no associated increase in pullout strength at either 3 or 6 months; however, post-pullout evaluation of the implants indicated that the HA/TCP coating itself was not the primary site of construct failure. Rather, failure was most commonly observed through the periprosthetic osseous struts that bridged the medullary cavity. The demonstrated osteoconductive activity of HA/TCP coating on plasma-sprayed titanium alloy implant surfaces may have considerable clinical relevance to early host-implant interactions, by accelerating the establishment of a stable prosthesis-bone interface.     

Ceramic drug delivery: a perspective

Different ceramic substances are offered in the market as bone substitute materials. These include monophasic calcium phosphate ceramics of tricalciumphosphate (TCP) or hydroxyapatite (HA), biphasic calcium phosphate ceramics and multiphasic bio-glasses synthetic calcium phosphate cements. Ceramics with appropriate three-dimensional geometry are able to bind and concentrate bone morphogenetic proteins in circulation and may become osteoinductive (capable of osteogenesis) and can be effective carriers of bioactive peptide or bone cell seeds and are therefore potentially useful in tissue engineering and drug delivery. An attempt has been made to review various drug delivery applications of ceramics.     

Evaluation of ceramics composed of different hydroxyapatite to tricalcium phosphate ratios as carriers for rhBMP-2

We have investigated pellet-shaped implants prepared from biphasic calcium phosphate (BCP) ceramics with five different ratios of hydroxyapatite (HAP) to beta tricalcium phosphate (beta-TCP). The purpose of this study was to evaluate these BCP ceramics as carriers for rhBMP-2. BCP ceramics impregnated with the different doses of recombinant human bone morphogenetic protein 2 (rhBMP-2) (1, 5 and 10g) were used for the experimental purpose and the ceramics without rhBMP-2 were used as control. The pellets were placed into subcutaneous pockets on the dorsum of 4-week-old male Wistar rats. The animals were sacrificed 2 and 4 weeks after implantation. Bone induction was estimated by alkaline phosphatase (ALP) activity measured at 2 weeks after implantation. Pellets were also examined radiologically, histologically and histomorphometrically. The results showed that all experimental pellets exhibited new bone formation whereas the control pellets produced only fibrous connective tissue. Here, 100% HAP ceramic showed most amount of bone formation, whereas 25% HAP to 75% TCP ceramic produced the bone least in amount among different BCP ceramics at the end of 4 weeks. This study indicates that formation of new bone depends on the ceramic content with high HAP-TCP ratio and high dose of rhBMP-2.