Three-dimensional printing of a β-tricalcium phosphate scaffold with dual bioactivities for bone repair

β-Tricalcium phosphate (β-TCP) had been widely used in the field of bone defect repair because of its osteoconduction and osteoinduction properties. However, for critical-sized bone defects, β-TCP scaffolds need to be functionalised to enhance osteoinduction and antibacterial activity. In this study, we proposed a protocol for mimicking a mussel adhesion mechanism to immobilise bone morphoprotein 2 mimetic peptide (BMP2-MP) and Ornithodoros savignyi (OS) on a three-dimensionally printed β-TCP scaffold. BMP2-MP and the OS polypeptides containing the YKYKY tail were converted into 3,4‐dihydroxyphenylalanine (DOPA) molecules via hydroxylase. The surface morphology and phase composition of the different scaffolds were analysed via scanning electron microscopy and X-ray diffraction. In addition, the binding activity of BMP2-MP and OS containing the DOPA tail to the scaffold were evaluated. The antibacterial activity of the different scaffolds was studied in vitro by performing bacteriostatic experiments against Escherichia coli and Staphylococcus aureus. The osteoinduction capability of the different scaffolds was evaluated by detecting osteogenesis-associated genes via quantitative polymerase chain reaction and by determining alkaline phosphatase expression levels. Our results demonstrated that introduction of the DOPA tail enhanced the binding capability of BMP2-MP and OS with the β-TCP scaffold, thereby enhancing the antibacterial and osteoinduction capabilities of the scaffold. A scaffold with strong antibacterial and osteoinduction capability will have good application prospects in the field of critical-sized bone defect repair.     

Improvements in phase stability and densification of β-tricalcium phosphate bioceramics by strontium-containing phosphate-based glass additive

β-tricalcium phosphate (β-TCP), which transforms to α-TCP at around 1125?°C, is characterized by poor sinterability. In this study, for the first time strontium-containing phosphate-based glass (SPG) was used as a sintering additive for β-TCP, which was sintered at 1250?°C. The results indicated that the SPG additive allowed for liquid-state sintering of β-TCP, thereby noticeably promoting the densification of β-TCP bioceramics. In the sintering process SPG reacted with β-TCP, and the metal ions from SPG were substituted for the calcium ions of β-TCP. The SPG additive effectively inhibited the phase transformation of β-TCP to α-TCP in the bioceramics. The compressive strength of porous β-TCP bioceramics was markedly increased by introducing 10?wt% SPG. The SPG is considered as an effective sintering additive to improve the phase stability and mechanical strength of porous β-TCP bioceramics.     

Enhancement of mechanical strength and osteogenesis,and inhibition of osteoclastic activity for β-tricalcium phosphate bioceramics by incorporating calcium silicate and magnesium-strontium phosphate

β-tricalcium phosphate bioceramics suffer from a drawback of poor mechanical strength and a scarcity of capacity to regulate biological performances. In the current study, the overall performances of β-tricalcium phosphate (TCP) bioceramics were improved by incorporating calcium silicate (CS) and magnesium-strontium phosphate (MSP). During the sintering process, the MSP stabilized the β phase of TCP, and the formation of MSP melt ensured effective liquid-sintering of TCP, thus conducing to lower porosity of TCP/MSP and TCP/CS/MSP bioceramics. In comparison with the TCP bioceramics, the TCP/CS and TCP/MSP bioceramics showed lower compressive strength, while the TCP/CS/MSP bioceramics attained noticeably higher compressive strength. Due to the sustained release of therapeutical ions, the TCP/CS bioceramics enhanced in vitro early-stage osteoblastic differentiation, but compromised cell proliferation; both the TCP/MSP and TCP/CS/MSP bioceramics enhanced cell proliferation and osteoblastic differentiation, and restrained osteoclastic activities. Collectively, the TCP/CS/MSP bioceramics with optimal overall performances are promising for efficaciously treating the defects of osteoporotic bone.     

Fabrication and characterization of porous β-tricalcium phosphate scaffolds coated with alginate

All porous materials have a common limitation which is lack of strength due to the porosity. In this study, two different methods have been used to produce porous β-tricalcium phosphate (β-TCP) scaffolds: liquid-nitrogen freeze casting and a combination of the direct-foaming and sacrificial-template methods. Among these two methods, porous β-TCP scaffolds with acceptable pore size and compressive strength and defined pore-channel interconnectivity were successfully fabricated by the combined direct-foaming and sacrificial-template method. The average pore size of the scaffolds was in the range of 100–150 µm and the porosity was around 70%. Coating with 4 wt% alginate on porous β-TCP scaffolds led to higher compressive strength and low porosity. In order to make a chemical link between the β-TCP scaffolds and the alginate coating, silane coupling agent was used. Treated β-TCP scaffold showed improvements in compressive strength of up to 38% compared to the pure β-TCP scaffold and 11% compared to coated β-TCP scaffold.     

Electrical polarization and ionic conduction properties of β-tricalcium phosphate bioceramics with controlled vacancies by sodium ion substitution

With the aim to understand electric polarization mechanisms of β-tricalcium phosphate as an advanced biomaterial, Na ion-substituted β-Ca₃(PO₄)₂ (Na-β-TCPs) ceramics with controlled lattice vacancies were synthesized and structural refinement was performed by the Rietveld method. The Rietveld analysis revealed that Ca and vacancies at Ca(4) sites in the β-TCP structure decreased with an increase in Na substitution. Electrical measurements by the complex impedance method revealed that the conductivity and the activation energy calculated from Cole-Cole plots rapidly decreased to a constant value with an increase in Na substitution and decrease in vacancies. The thermally stimulated depolarization current (TSDC) curve of the electrically polarized Na-β-TCP showed one large peak at 530–610 °C. However, the accumulated charge decreased with an increase in Na ions and decrease in vacancies up to 2.37 mol%, after which it became constant. These results are consistent with the presumed formation of a dipole moment between aligned Ca²⁺ ions and their vacancies along the direction of the external polarization field applied at high temperature. We conclude that the large amount of stored charge in β-TCP caused by electrical polarization is due to the low site occupancy of calcium ions and vacancies at Ca(4) sites in the β-TCP structure, which is not the case for hydroxyapatite (HAp), as previously reported.     

Graphene-oxide-modified β-tricalcium phosphate bioceramics stimulate in vitro and in vivo osteogenesis

Graphene oxide (GO) has attracted much interest for applications in bone tissue engineering; however, until now, the interaction between GO and stem cells, and the in vivo bone-forming ability of GO have not been explored. The aim of this study was to produce GO-modified β-tricalcium phosphate (β-TCP-GRA) bioceramics and then explore the material’s osteogenic capacity in vitro and in vivo, as well as unravel some of the molecular mechanisms behind this. β-TCP-GRA disks and scaffolds were successfully prepared by a simple GO/water suspension soaking method in combination with heat treatment. These scaffolds were found to significantly enhance the proliferation, alkaline phosphatase activity, and osteogenic gene expression of human bone marrow stromal cells (hBMSCs), when compared with β-TCP without GO modification (controls). Activation of the Wnt/β-catenin signaling pathway in hBMSCs appears to be the mechanism behind this osteogenic induction by β-TCP-GRA. β-TCP-GRA scaffolds led to an increased rate of in vivo new bone formation compared to β-TCP controls, indicative of the stimulatory effect of GO on in vivo osteogenesis, making GO modification of β-TCP a very promising method for applications in bone tissue engineering, in particular for the regeneration of large bone defects.     

Fabrication and characterization of honeycomb β-tricalcium phosphate scaffolds through an extrusion technique

In this study, for the first time honeycomb β-tricalcium phosphate (β-TCP) scaffolds were fabricated through an extrusion technique. The physicochemical properties and cell behaviors of the honeycomb β-TCP scaffolds were investigated. The results showed that scaffolds were characterized by ordered channel-like macropores and unidirectional interconnection. The pore structure and mechanical strength could be tailored by changing the parameters of extrusion molds. The pore size of scaffolds was in the range of 400–800 µm approximately, while their compressive strength parallel to the pore direction and porosity ranged from 14 to 20 MPa and 60–70%, respectively. The in vitro cell behavior demonstrated that cells could well attach on the surfaces and grow into the inner channel-like pores of thescaffolds; the scaffolds with higher porosity showed better cell proliferation but poorer cell differentiation. The honeycomb scaffolds fabricated by extrusion technique are potential candidate for bone tissue engineering.     

SLA 3D printing of high quality spine shaped β-TCP bioceramics for the hard tissue repair applications

β-TCP has excellent biodegradability and bioabsorption properties, and is regarded as an ideal hard tissue repair material. In the present study, 3D printing β-TCP green bodies was realized using the stereo lithography apparatus (SLA) technology. The effects of the KH-560 dispersant and solid loading on the slurry properties were investigated systematically. The optimized KH-560 addition amount and the solid loading of the slurry were 2.0 wt% and 48 wt%, respectively, and the corresponding slurry for the subsequent SLA 3D printing exhibited good fluidity, uniform dispersion and good stability. The sintering schedule of the printed β-TCP green bodies was optimized by the DSC-TG analysis. By sintering the green bodies at 1050 °C for 8 h, high quality β-TCP bioceramics without crack or deformation were fabricated. It was found that increasing the solid loading of the slurry would decrease the porosity while reducing the shrinkage degree of the β-TCP ceramics. However, the slurry could hardly be printed when its solid loading was higher than 50 wt%.     

Fabrication and characterization of porous biphasic β-tricalcium phosphate/carbonate apatite alginate coated scaffolds

In this research, biphasic β-tricalcium phosphate/carbonate apatite (β-TCP/CO₃Ap) scaffolds incorporated with alginate were fabricated. Sodium alginate was extracted from local brown seaweed, Sargassum polycystum via calcium alginate process. Biphasic β-TCP/CO₃Ap scaffolds were fabricated by polymer reticulate method. β-TCP slurry was infiltrated into the polyurethane foam (PU) foam, then sintered up to 1300?°C, soaked for 4?h and immediately quenched in still air to form biphasic β-TCP/α-TCP scaffold. Biphasic β-TCP/α-TCP scaffold was then transformed to biphasic β-TCP/CO₃Ap scaffold by dissolution-precipitation reaction with 1?M of NaHCO₃ at 170?°C for 1, 3 and 5 days. Biphasic β-TCP/CO₃Ap scaffold from 5 days dissolution-precipitation reaction was chosen to incorporate with 1%, 3% and 5% of sodium alginate, respectively, as it has the highest composition of CO₃Ap phase. FTIR and FESEM analysis confirmed the presence of characteristic functional groups of sodium alginate. Mechanical strength of biphasic β-TCP/CO₃Ap scaffold improved by increasing the concentration of sodium alginate. The highest mechanical strength achieved was 26.38 kPa for biphasic β-TCP/CO₃Ap scaffold with 5% sodium alginate coating and it was chosen to further study with the addition of 1%, 3% and 5% microspheres. FESEM analysis confirmed the attachment of microspheres on the surface of alginate/biphasic β-TCP/CO₃Ap scaffold was successful.     

Fabrication of cancellous-bone-mimicking β-tricalcium phosphate bioceramic scaffolds with tunable architecture and mechanical strength by stereolithography 3D printing

It is highly challenging to fabricate bioceramic scaffolds mimicking architecture and mechanical strength of cancellous bone. Gyroid structure, which is based on triply periodic minimal surface, highly resembles the architecture of cancellous bone. Herein, β-tricalcium phosphate (β-TCP) bioceramic scaffolds with gyroid structure were fabricated by stereolithography (SLA) 3D printing. The SLA 3D printing ensured high precision of ceramic part. The porosity (51–87%), pore size (250 – 2400 µm), pore wall thickness (< 300 µm) and compressive strength (0.6 – 16.8 MPa) of gyroid bioceramic scaffolds were readily adjusted to match various sites of cancellous bone. The gyroid bioceramic scaffolds were more favorable for cell proliferation than the grid-like bioceramic scaffolds. The cancellous-bone-mimicking gyroid bioceramic scaffolds with tunable architecture and mechanical strength were expected to efficiently repair the target bone defects.     

Influences of gallium substitution on the phase stability,mechanical strength and cellular response of β-tricalcium phosphate bioceramics

β-tricalcium phosphate (β-TCP), a well-accepted synthetic bone grafting biomaterial, is confronted with limitations of poor phase stability and lacking the capacity to mediate the biological functions. In the current study, gallium (Ga) was substituted for calcium in the β-TCP, and the influences of Ga substitution on the phase stability, compressive strength and cellular response of β-TCP bioceramics were investigated. The results indicated that substitution of at least 2.5 mol% Ga for calcium prevented the β-TCP from transforming into α-TCP at 1250 °C. The β-TCP bioceramics substituted with 2.5 mol% Ga attained the highest compressive strength. The β-TCP bioceramics substituted with 2.5 and 5 mol% Ga showed good cytocompatibility, and suppressed in vitro osteoclastic activity as well as osteoblastic differentiation. Considering the favorable mechanical strength and the inhibitory effect on the osteoclastic activity, the β-TCP bioceramics substituted with 2.5 mol% Ga are promising for treating the bone defect in the pathological state of excessively rapid bone resorption.     

Preparation and characterization of iron/β-tricalcium phosphate bio-cermets for load-bearing bone substitutes

Ceramic-metal composite materials, namely cermets, are provided with characteristics of both ceramic and metal. Herein, for the first time bio-cermets based on β-tricalcium phosphate (β-TCP) bioceramic with biodegradable iron being reinforcement phase, were fabricated using the powder metallurgic method. The phase composition, microstructure, mechanical properties and in vitro cell behaviors of bio-cermets were investigated. The results revealed that atomic diffusion occurred between the iron and β-TCP matrix during the sintering process. The bio-cermets attained remarkable increase in fracture toughness (1.16–1.55 MPa m^1/2) compared to the β-TCP bioceramic (0.54 MPa m^1/2). The bio-cermets with 10 vol% iron showed the highest compressive strength (640 MPa), significantly higher than that of plain β-TCP bioceramic (285 MPa). The in vitro cell behaviors test indicated that the bio-cermets did not showed any sign of toxicity; the iron ions released from bio-cermets up-regulated bone-related gene expression of bone mesenchymal stem cells. The bio-cermets developed in this study represent potential bone substitutes for application in the load-bearing bone defects.     

Gelatin-layered and multi-sized porous β-tricalcium phosphate for tissue engineering scaffold

The multi-sized porous β-tricalcium phosphate scaffolds were fabricated by freeze drying followed by slurry coating using a multi-sized porous sponge as a template. Then, gelatin was dip coated on the multi-sized porous β-tricalcium phosphate scaffolds under vacuum. The mechanical and biological properties of the fabricated scaffolds were evaluated and compared to the uniformly sized porous scaffolds and scaffolds that were not coated by gelatin. The compressive strength was tested by a universal testing machine, and the cell viability and differentiation behavior were measured using a cell counting kit and alkaline phosphatase activity using the MC3T3-E1 cells. In comparison, the gelatin-coated multi-sized porous β-tricalcium phosphate scaffold showed enhanced compressive strength. After 14 days, the multi-sized pores were shown to affect cell differentiation, and gelatin coatings were shown to affect the cell viability and differentiation. The results of this study demonstrated that the multi-sized porous β-tricalcium phosphate scaffold coated by gelatin enhanced the mechanical and biological strengths.     

Novel method for the synthesis of a β-tricalcium phosphate coating on a zirconia implant

A novel method for the synthesis of a thin β-tricalcium phosphate (β-TCP) coating on zirconia implants has been developed. The synthesis procedure involves two steps: (i) rapid wet-chemical deposition of a biomimetic CaP coating and (ii) subsequent post-deposition processing of the biomimetic CaP coating, which includes a heat treatment at 900 °C followed by a short sonication in a water bath. The obtained β-TCP coating showed a uniform and dense morphology with a thickness of ≈500 nm and displayed a roughness in the nanometre range (R_a = 28 nm). The β-TCP coating demonstrated an apatite-mineralization ability in a simulated body fluid and enhanced the adsorption of serum proteins on the zirconia. Moreover, the β-TCP coating adhered firmly to the zirconia substrate, developing a notable scratch resistance (L_c = 97 N) and tensile strength (52 MPa) and showed strong resistance towards mechanical forces present during implantation of the coated zirconia implant into the artificial bone.     

Evaluation of hydroxyapatite and β-tri calcium phosphate microplasma spray coated pin intra-medullary for bone repair in a rabbit model

Here we report a comparative study of the healing kinetics of surgically created artificial defects in the tibia of New Zealand white rabbits. Comparison of the healing kinetics was made for uncoated conventional SS316L intramedullary pins, and the same pins with microplasma sprayed (MIPS) pure hydroxyapatite (HAp) and beta-tri calcium phosphate (β-TCP) coatings. After thorough material characterizations including XRD, FTIR, SEM, etc., MIPS coated pins were implanted to such animals. Serum biochemistry, radiology and fluorochrome labelling were used to evaluate the comparative healing kinetics of these implants in vivo. In comparison to those of the uncoated pins, the pins coated with both MIPS HAp and β-TCP showed significant increment of alkaline phosphatase up to 15th postoperative day, insignificant changes in serum phosphorus and calcium with uneventful healing of bone defect. There was development of Havarsian canals and well-defined peripherally placed osteoblasts along with evidence of angiogenesis and comparatively more new bone formation in the defect site. On a comparative scale, the performance of the β-TCP coated intramedullary pins was much better than that of the pure HAp coated pins than the uncoated intramedullary pins.     

The biological properties of carbon nanofibers decorated with β-tricalcium phosphate nanoparticles

Carbon nanofibers decorated with β-tricalcium phosphate (β-TCP) nanoparticles (β-TCP/CNFs) have been prepared by sintering electrospun polyacrylonitrile fibers with calcium nitrate tetrahydrate as the calcium source and triethyl phosphate as the phosphorus source. Microstructure and phase composition analysis indicate that the resulting materials are composed of β-TCP nanoparticles and CNFs. And the long β-TCP/CNFs can be cut into organism-eliminable short CNFs gradually in hydrochloric acid solution due to the solubilization of β-TCP nanoparticles. The materials exhibit good biocompatibility, and have comparable effect on cell growth with pure CNFs, with their tuning ability in degradation.     

DLP printed β-tricalcium phosphate functionalized ceramic scaffolds promoted angiogenesis and osteogenesis in long bone defects

Nowadays, the repair of long bone defects remains a clinical challenge mainly due to poor oxygen and nutrients delivery. In this study, β-tricalcium phosphate (β-TCP) porous ceramic scaffolds were prepared by digital light processing (DLP) and gradient sintering process. The functionalization of scaffolds was achieved by loading hyaluronic acid-dopamine (HA-DA) coating or sphingosine 1-phosphate/hyaluronic acid-dopamine (S1P/HA-DA) coating, which solved the problem of oxygen and nutrients delivery to a certain extent by promoting blood vessels growth. Cytocompatibility assay, qRT-PCR, Alkaline phosphatase (ALP) staining and quantitative analysis demonstrated that the S1P/HA-DA/TCP scaffolds significantly promoted the proliferation and osteogenic differentiation of mouse bone marrow mesenchymal stem cells (mBMSCs). Long bone defects (22 mm), rarely reported in previous studies, were constructed on the radius of rabbits. Animal experiments showed excellent early angiogenesis and bone repair in HA-DA/TCP and S1P/HA-DA/TCP groups. In particular, the S1P/HA-DA/TCP scaffolds enhanced bone regeneration and osseointegration. Overall, these functionalized scaffolds had an effective repair on long bone defects that would have great potential for clinical applications.     

Fabrication and properties of porous β-tricalcium phosphate ceramics prepared using a double slip-casting method using slips with different viscosities

A new method to enhance the flexural strength of porous β-tricalcium phosphate (β-TCP) scaffolds was developed. This new method provides better control over the microstructures of the scaffolds and enhances the scaffolds’ mechanical properties. Using this technique, we were able to produce scaffolds with mechanical and structural properties that cannot be attained by either the polymer sponge or slip-casting methods alone or by simply combining the polymer sponge and slip-casting methods. The prepared scaffolds had an open, uniform, interconnected porous structure with a bimodal pore size of 100.0–300.0 μm. The flexural strength of the bimodal porous β-TCP scaffold sintered at 1200 °C was 56.2 MPa and had porosity of 61.4 vol%. The scaffolds obtained provide good mechanical support while maintaining bioactivity, and hence, these bioscaffolds hold promise for applications in hard-tissue engineering.     

Effects of Mn-doping on the structure and in vitro degradation of β-tricalcium phosphate

β-tricalcium phosphate (β-TCP) is an ideal biomaterial for the bone repair because of its biocompatibility and biodegradability. In this study, 0 mol%, 5 mol%, 15 mol% and 30%mol bivalent manganese ion (Mn²⁺) doped β-TCP (Mn-TCP) powders were synthesized by a sol-gel method. The amount of the dopants significantly influences the crystallinity and the parameters related with structure of β-TCP, such as the lattice parameters and crystallite dimensions. The particle size and the particle distribution of doped β-TCP powers were evaluated as well. Meanwhile, the as-synthesized powders were consolidated by sintering at 1000 °C in muffle furnace for 5 h to get Mn-TCP porous material and the degradation experiment was carried out in Simulated Body Fluid (SBF) solution for 28 days. Then, Mn-TCP porous material were characterized by Fourier-transform infrared spectroscopy (FTIR), X-ray diffraction (XRD), and scanning electron microscope (SEM). Significantly, there were bone-like apatite materials deposited on the surface of bone-like porous materials. With the increasing doping amount of Mn²⁺, the newly formed apatite-like materials decreased, while the crystallinity increased significantly. Besides, pH results showed that alkaline environment was more favorable for the formation of sedimentary materials.     

Preparation of porous β-tricalcium phosphate using starfish-derived calcium carbonate as a precursor

Porous β-tricalcium phosphate (β-TCP) was successfully prepared from starfish-derived calcium carbonate (sf-bone) under several hydrothermal conditions. The sf-bone, obtained from Patiria Pectinifera by bleaching to remove organic substances, was Mg-containing calcite granules with an interconnected microporous structure of approximately 10−50 µm of pore, and was hydrothermally treated with ammonium phosphate aqueous solutions at various pHs and temperatures. The sf-bone was converted to Mg-containing β-TCP with maintaining its microporous structure by the hydrothermal treatment for 1 day or longer in (NH₄)₂HPO₄ aqueous solution at 200 °C. This conversion was based on dissolution-reprecipitation process of Mg-containing calcite in the phosphate salt aqueous solution. Thus, conditions during the conversion, pH and temperature, affected the morphologies and crystal phases of sf-bone after the treatment depended upon both calcite dissolution and calcium phosphate-formation rates.