3D printed ß-TCP bone tissue engineering scaffolds: Effects of chemistry on in vivo biological properties in a rabbit tibia model.

In this study the effects of 3D printed SiO2 and ZnO doped tricalcium phosphate (TCP) scaffolds with interconnected pores were evaluated on the in vivo bone formation and healing properties of a rabbit tibial defect model. Pure and doped TCP scaffolds were fabricated by a ceramic powder-based 3D printing technique and implanted into critical sized rabbit tibial defects for up to 4 months. In vivo bone regeneration was evaluated using chronological radiological examination, histological evaluations, SEM micrographs and fluorochrome labeling studies. Radiograph results showed that Si/Zn doped samples had slower degradation kinetics than the pure TCP samples. 3D printing of TCP scaffolds improved bone formation. The addition of dopants in the TCP scaffolds improved osteogenic capabilities when compared to the pure scaffolds. In summary, our findings indicate that addition of dopants to the TCP scaffolds enhanced bone formation and in turn leading to accelerated healing.

Regulation of Osteogenic Markers at Late Stage of Osteoblast Differentiation in Silicon and Zinc Doped Porous TCP.

Calcium phosphates (CaPs) are one of the most widely used synthetic materials for bone grafting applications in the orthopedic industry. Recent trends in synthetic bone graft applications have shifted towards the incorporation of metal trace elements that extend the performance of CaPs to have osteoinductive properties. The objective of this study is to investigate the effects of silicon (Si) and zinc (Zn) dopants in highly porous tricalcium phosphate (TCP) scaffolds on late-stage osteoblast cell differentiation markers. In this study, an oil emulsion method is utilized to fabricate highly porous SiO2 doped ß-TCP (Si-TCP) and ZnO doped ß-TCP (Zn-TCP) scaffolds through the incorporation of 0.5 wt.% SiO2 and 0.25 wt.% ZnO, respectively, to the ß-TCP scaffold. Reverse transcriptase quantitative polymerase chain reaction (RT-qPCR) is utilized to analyze the mRNA expression of osteoprotegerin (OPG), receptor activator of nuclear kappa beta ligand (RANKL), bone morphogenetic protein 2 (BMP2), and runt-related transcription factor 2 (Runx2) at the later stage of osteoblast differentiation, day 21 and day 28. Results show that the addition of Si and Zn to the ß-TCP structure inhibited the ß to α-TCP phase transformation and enhance the density without affecting the dissolution properties. Normal BMP-2 and Runx2 transcriptions are observed in both Si-TCP and Zn-TCP scaffolds at the initial time point, as demonstrated by RT-qPCR. Moreover, the addition of both Si and Zn positively regulate the osteoprotegerin: receptor activator of nuclear factor k-ß ligand (OPG:RANKL) ratio at 21-days for Si-TCP and Zn-TCP scaffolds. These results demonstrate the effects of Si and Zn doped porous ß-TCP scaffolds on the upregulation of osteoblast marker gene expression including OPG, RANKL, BMP-2, and Runx2, indicating the role of trace elements on the effective regulation of late-stage osteoblast cell differentiation markers.

Effects of SiO2, SrO, MgO, and ZnO dopants in tricalcium phosphates on osteoblastic Runx2 expression.

Calcium phosphate materials share a compositional similarity to natural bone, which makes them excellent for use in orthopedic applications. Although these materials are osteoconductive, they lack strong osteoinductive capabilities and recent research has focused on the addition of biologics and pharmacologics with varying successes. In this study, trace elements that have been proven to play important roles in bone health and bone formation were incorporated into ß-tricalcium phosphate compacts in their oxide forms (SiO2, ZnO, SrO, and MgO). Cell material interactions were characterized using human fetal preosteoblastic cells. An MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) assay was used to evaluate cellular proliferation. Cellular differentiation was evaluated using an enzymatic colorimetric alkaline phosphatase assay as well as immunohistochemistry for Runt-related transcription factor 2 (Runx2) expression. Results prove ZnO and MgO to be effective mitogenic factors and SiO2, ZnO, and SrO to be capable of inducing rapid cellular differentiation. MgO was found to have little effect on the modulation of osteoblastic differentiation, likely due to more aggressive inherent cellular regulation of Mg(2+). In addition to the results from the study, a signaling mechanism is proposed as to the action of the dopants for further consideration.

SiO2 and ZnO dopants in three-dimensionally printed tricalcium phosphate bone tissue engineering scaffolds enhance osteogenesis and angiogenesis in vivo.

Calcium phosphate (CaP) scaffolds with three-dimensionally-interconnected pores play an important role in mechanical interlocking and biological fixation in bone implant applications. CaPs alone, however, are only osteoconductive (able to guide bone growth). Much attention has been given to the incorporation of biologics and pharmacologics to add osteoinductive (able to cause new bone growth) properties to CaP materials. Because biologics and pharmacologics are generally delicate compounds and also subject to increased regulatory scrutiny, there is a need to investigate alternative methods to introduce osteoinductivity to CaP materials. In this study silica (SiO2) and zinc oxide (ZnO) have been incorporated into three-dimensional printed ß-tricalcium phosphate (ß-TCP) scaffolds to investigate their potential to trigger osteoinduction in vivo. Silicon and zinc are trace elements that are common in bone and have also been shown to have many beneficial properties, from increased bone regeneration to angiogenesis. Implants were placed in bicortical femur defects introduced to a murine model for up to 16 weeks. The addition of dopants into TCP increased the capacity for new early bone formation by modulating collagen I production and osteocalcin production. Neovascularization was found to be up to three times more than the pure TCP control group. The findings from this study indicate that the combination of SiO2 and ZnO dopants in TCP may be a viable alternative to introducing osteoinductive properties to CaPs.

Understanding of dopant-induced osteogenesis and angiogenesis in calcium phosphate ceramics.

General trends in synthetic bone grafting materials are shifting towards approaches that can illicit osteoinductive properties. Pharmacologics and biologics have been used in combination with calcium phosphate (CaP) ceramics, however, they have recently become the target of scrutiny over safety. The importance of trace elements in natural bone health is well documented. Ions, for example, lithium, zinc, magnesium, manganese, silicon, strontium, etc., have been shown to increase osteogenesis and neovascularization. Incorporation of dopants (trace metal ions) into CaPs can provide a platform for safe and efficient delivery in clinical applications where increased bone healing is favorable. This review highlights the use of trace elements in CaP biomaterials, and offers an insight into the mechanisms of how metal ions can enhance both osteogenesis and angiogenesis.

Effects of Zinc and Strontium Substitution in Tricalcium Phosphate on Osteoclast Differentiation and Resorption.

Bone replacement materials must be able to regulate both osteoblastic synthesis of new bone and osteoclastic resorption process in order to maintain the balance of bone remodeling. Osteoclasts generate from differentiation of mononuclear cells. In the present study, we have studied the osteoclast-like-cells responses (differentiation from mononuclear cells and resorption) to beta tricalcium phosphate (ß-TCP) doped with zinc (Zn) and strontium (Sr). Osteoclast-like-cells differentiation and resorption was studied in vitro using osteoclast-like-cells precursor RAW 264.7 cell, supplemented with receptor activator of nuclear factor κß ligand (RANKL). Morphological and immunohistochemical analysis confirmed successful differentiation of osteoclast-like-cells on the doped and undoped ß-TCP substrates after 8 days of culture. Cells on the substrate surface expressed specific osteoclast markers such as; actin ring, multiple nucleus, tartrate-resistant acid phosphatase (TRAP) synthesis, and vitronectin receptor. However, quantitative TRAP assay indicated the inhibiting effect of Zn on osteoclast differentiation. Although, Zn doped ß-TCP restricted osteoclast-like-cells differentiation, the samples were resorbed much faster. An increased resorption pit volume was noticed on Zn doped ß-TCP samples after 28 days of culture compared to pure and Sr doped ß-TCP. In this work, we demonstrated that ß-TCP bone substitute materials can be successfully resorbed by osteoclast-like-cells, where both osteoclast-like-cells differentiation and resorption were modulated by Zn and/or Sr doping- a much needed property for successful bone remodeling.

Effects of silica and zinc oxide doping on mechanical and biological properties of 3D printed tricalcium phosphate tissue engineering scaffolds.

OBJECTIVES: To evaluate the effects of silica (SiO(2)) (0.5 wt%) and zinc oxide (ZnO) (0.25 wt%) dopants on the mechanical and biological properties of tricalcium phosphate (TCP) scaffolds with three dimensionally (3D) interconnected pores. METHODS: Scaffolds were created with a commercial 3D printer. Post sintering phase analysis was determined by X-ray diffraction. Surface morphology of the scaffolds was examined by field emission scanning electron microscopy (FESEM). Mechanical strength was evaluated with a screw driven universal testing machine. MTT assay was used for cellular proliferation characteristics and cellular morphology was examined by FESEM. RESULTS: Addition of dopants into TCP increased the average density of pure TCP from 90.8 ± 0.8% to 94.1 ± 1.6% and retarded the ß to α phase transformation at high sintering temperatures, which resulted in up to 2.5 fold increase in compressive strength. In vitro cell-materials interaction studies, carried out using hFOB cells, confirmed that the addition of SiO(2) and ZnO to the scaffolds facilitated faster cell proliferation when compared to pure TCP scaffolds. SIGNIFICANCE: Addition of SiO(2) and ZnO dopants to the TCP scaffolds showed increased mechanical strength as well as increased cellular proliferation.

Understanding bioactivity and polarizability of hydroxyapatite doped with tungsten.

This study investigates the use of hydroxyapatite (HAp) doped with hexavalent tungsten to improve its interaction with bone cells and to influence the polarizing capacity of HAp. Increases in dopant concentration increased the ß-TCP phases and decreased the HAp phases in sintered samples. Results of thermally stimulated depolarization current measurements suggested that doped HAp had stored fewer charge compared with pure HAp. However, the decrease in stored charge was related to fraction of HAp or ß-TCP phases present in sintered samples. Activation energy of dipole relaxation and stored charge was used to examine the mechanism of polarization. The charge stored in doped samples due to polarization was attributed to the migration of H(+) ions in HAp phases and O(2-) or Ca(2+) ions in ß-TCP phases. Hindrance of ion migration due to the presence of different phases appeared to lower charge storage ability in doped samples. In vitro study revealed an increase in bioactivity of doped HAp when compared with pure HAp. Polarization further improved the bioactivity of doped HAp. Results of our study provide evidence for the use of higher valent cations to improve biological performance of HAp ceramics.

Antibacterial and biological characteristics of silver containing and strontium doped plasma sprayed hydroxyapatite coatings.

Infection in primary total joint prostheses is estimated to occur in up to 3% of all surgery. As a measure to improve the antimicrobial properties of implant materials silver (Ag) was incorporated into plasma sprayed hydroxyapatite (HA) coatings. To offset potential cytotoxic effects of Ag in the coatings strontium (Sr) was also added as a binary dopant. HA powder was doped with 2.0 wt.% Ag(2)O, 1.0 wt.% SrO and was then heat treated at 800 °C. Titanium substrates were coated using a 30 kW plasma spray system equipped with a supersonic nozzle. X-ray diffraction confirmed the phase purity and high crystallinity of the coatings. Samples were evaluated for mechanical stability by adhesive bond strength testing. The results show that the addition of dopants did not affect the overall bond strength of the coatings. The antibacterial efficacies of the coatings were tested against Pseudomonas aeruginosa. Samples that contained the Ag(2)O dopant were found to be highly effective against bacterial colonization. In vitro cell-material interactions using human fetal osteoblast cells were characterized by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay for cell viability, field emission scanning electron microscopy for cell morphology and confocal imaging for the important differentiation marker alkaline phosphatase (ALP). Our results showed evidence of cytotoxic effects of the Ag-HA coatings, characterized by poor cellular morphology and cell death and nearly complete loss of functional ALP activity. The addition of SrO to the Ag-HA coatings was able to effectively offset these negative effects and improve performance compared with pure HA-coated samples.

ZnO, SiO2, and SrO doping in resorbable tricalcium phosphates: Influence on strength degradation, mechanical properties, and in vitro bone-cell material interactions.

To understand the combined effects of ZnO, SiO(2), and SrO doping on mechanical and biological properties of tricalcium phosphate (TCP) ceramics, dense ß-TCP compacts of different compositions (pure ß-TCP; 1.0 wt % SrO; 0.25 wt % ZnO; 1.0 wt % SrO + 0.5 wt % SiO(2); and 1.0 wt % SrO + 0.25 wt % ZnO) were prepared via dry pressing followed by sintering at 1250°C. X-ray diffraction of sintered compacts revealed that dopants retarded ß- to α-TCP phase transformation during sintering. Doping with SrO, SrO/SiO(2), and SrO/ZnO reduced compressive strength of the samples to 56% (173 ± 25 MPa), 57% (170 ± 15 MPa), and 47% (208 ± 72 MPa) of pure ß-TCP (396 ± 58 MPa), respectively. However, addition of ZnO resulted in only 7% (365 ± 69 MPa) strength degradation. The impact of dopants on long-term in vitro strength degradation was evaluated by soaking in simulated body fluid (SBF) for a period of 8 weeks. In all cases, excellent apatite growth was observed on doped ß-TCP samples. However, strength degradation rates were different depending on dopant chemistry and composition. Maximum degradation was observed in undoped and ZnO-doped ß-TCP samples, which degraded to 41% and 68% of the original strength before soaking in SBF. Finally, in vitro cell-materials interaction study using human fetal osteoblast cells demonstrated that addition of dopants improved cell attachment and proliferation. These results indicate that tailorable strength and strength degradation behavior can be achieved in ß-TCP via compositional modifications using small amount of dopants.

Mechanical, in vitro antimicrobial, and biological properties of plasma-sprayed silver-doped hydroxyapatite coating.

Implant-related infection is one of the key concerns in total joint hip arthroplasties. To reduce bacterial adhesion, we used silver (Ag)/silver oxide (Ag(2)O) doping in plasma sprayed hydroxyapatite (HA) coating on titanium substrate. HA powder was doped with 2.0, 4.0, and 6.0 wt % Ag, heat-treated at 800 °C and used for plasma spray coating using a 30 kW plasma spray system, equipped with supersonic nozzle. Application of supersonic plasma nozzle significantly reduced phase decomposition and amorphous phase formation in the HA coatings as evident by X-ray diffraction (XRD) study and Fourier transformed infrared spectroscopic (FTIR) analysis. Adhesive bond strength of more than 15 MPa ensured the mechanical integrity of the coatings. Resistance against bacterial adhesion of the coatings was determined by challenging them against Pseudomonas aeruginosa (PAO1). Live/dead staining of the adherent bacteria on the coating surfaces indicated a significant reduction in bacterial adhesion due to the presence of Ag. In vitro cell-material interactions and alkaline phosphatase (ALP) protein expressions were evaluated by culturing human fetal osteoblast cells (hFOB). Our results suggest that the plasma-sprayed HA coatings doped with an optimum amount of Ag can have excellent antimicrobial property without altering mechanical property of the Ag-doped HA coatings.