Regulation of Osteogenic and Angiogenic Markers in Alkali Treated Titanium for Hard Tissue Engineering Applications.

Titanium (Ti) and Ti alloys are of great interest in bone and dental tissue engineering applications due to their biocompatibility, corrosion resistance, and close mechanical properties to natural bone. However, the formation of fibrous tissue prevents osteointegration and results in implant loosening. Thus, physical and chemical methods are used to improve the surface properties of Ti. This study aimed to understand the role of alkali treatment conditions, including alkali medium concentration, temperature, rotation speed, and post-heat treatment. Our results showed that alkali treatment using 5 and 10 molar sodium hydroxide solution allows the formation of web-like microstructure. However, a higher concentration of 15 molar resulted in cracks along the surface. Interaction between the human fetal osteoblast cells (hfOBs) and Ti samples showed that heat treatment is necessary for increased cellular proliferation, which was not significantly different at later time points compared to the polished Ti. Alkali heat treatment did not induce inflammatory reactions at later time points. It showed an increase in vascular endothelial growth factor, osteoprotegerin/nuclear factor kappa-В ligand ratio, and osteocalcin expression, which is evidence for accelerated osteoblast cell maturation and bone remodeling in surface-modified samples. Together, these data show that alkali treatment using 5 or 10 molar of NaOH followed by heat treatment may have therapeutic effect and assist with bone tissue integration with Ti implant.

Beta-phase Stabilization and Increased Osteogenic Differentiation of Stem Cells by Solid-State Synthesized Magnesium Tricalcium Phosphate.

In this study, magnesium and strontium-doped ß-tricalcium phosphates were synthesized to understand dopant impact on substrate chemistry and morphology, and proliferation and osteogenic differentiation of mesenchymal stem cells. Under solid-state synthesis, magnesium doping stabilized the ß-phase in tricalcium phosphate, with 22% less α-phase content than control. Strontium doping increased α-phase formation by 17%, and also resulted in greater surface porosity, leading to greater crystal precipitation in vitro. Magnesium also significantly enhanced the proliferation of stem cells (P < 0.05) and differentiation into osteoblasts with increased alkaline phosphatase production (P < 0.05) at all time points. These results indicated that magnesium stabilizes ß-tricalcium phosphate in vitro and enhanced early and late-time-point osteoconduction and osteoinduction of mesenchymal stem cells.

Role of Iron on Physical and Mechanical Properties of Brushite Cements, and Interaction with Human Dental Pulp Stem Cells.

Improving the physical, mechanical and biological properties of brushite cements (BrC) is of a great interest for using them in bone and dental tissue engineering applications. The objective of this study was to incorporate iron (Fe) at different concentrations (0.25, 0.50, and 1.00 wt.%) to BrC and study the role of Fe on phase composition, setting time, compressive strength, and interaction with human dental pulp stem cells (hDPSCs). Results showed that increase in Fe concentration increases the ß-tricalcium phosphate (ß-TCP)/ dicalcium phosphate dihydrate (DCPD) ratio and prolongs the initial and final setting time due to effective role of Fe on stabilizing the ß-TCP crystal structure and retarding its dissolution kinetic, in a dose dependent manner where the highest setting time was recorded for 1.00 wt.% Fe-BrC sample. Addition of low concentrations of Fe (0.25 and 0.50 wt.%) did not have adverse effect on compressive strength and strength was in the range of 5.7-7.05 (±~1.4) MPa; however, presence of 1.00 wt.% Fe decreases the strength of BrC from 7.05 ± 1.57 MPa to 3.12 ± 1.06 MPa. Interaction between the BrCs and hDPSCs was evaluated by cell proliferation assay, scanning electron microscopy, and live/dead staining. Low concentrations of 0.25, and 0.50 wt.% of Fe did not have any adverse effect on cell attachment and proliferation; while significant decrease in cellular activity was evident in BrC samples doped with 1.00 wt. %. Together, these data show that low concentrations of Fe (equal or less than 0.50 wt. %) can be safely added to BrC without any adverse effect on physical, mechanical and biological properties in presence of hDPSCs.

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.

Sustained release of vitamin C from PCL coated TCP induces proliferation and differentiation of osteoblast cells and suppresses osteosarcoma cell growth.

The objective of this study is to understand the effect of sustained release of vitamin C from ß-tricalcium phosphate (ß-TCP) scaffold on proliferation, viability and differentiation of human fetal osteoblast cells (hFOB). The influence of pH, drug concentration, and presence of polymer on the sustained release of vitamin C from polycaprolactone (PCL) coated ß-TCP scaffolds are studied. Prolonged and sustained release of vitamin C, over 60 days is observed in PCL coated ß-TCP scaffolds compared to uncoated scaffolds. Presence of PCL helps to minimize the burst release of vitamin C from ß-TCP scaffolds in the initial 24 h of release. To evaluate the osteogenic potential of vitamin C incorporated ß-TCP scaffolds, osteoblast cells are cultured and cell morphology, proliferation, viability, and differentiation are assessed. Morphological characterization shows layer like osteoblast cell attachment in the presence of vitamin C compared to the control. MTT cell viability assay shows 2 folds increase in osteoblast cell density in the presence of vitamin C after 3,7 and 11 days of culture. Furthermore, increased ALP activity at 11 days of culture indicates the possible role of vitamin C on osteoblast differentiation. Additionally, a preliminary study shows vitamin C loaded scaffolds suppress osteosarcoma (MG-63) cell proliferation to 4 folds after 3 days compared to control. These results show a sustained release of vitamin C from PCL coated ß-TCP scaffolds improve proliferation, viability, and differentiation of osteoblasts cell as well as mitigate osteosarcoma cell proliferation, suggesting its potential application as synthetic bone graft substitutes in tissue engineering application.

Bone marrow stromal cells interaction with titanium; Effects of composition and surface modification.

Inflammation and implant loosening are major concerns when using titanium implants for hard tissue engineering applications. Surface modification is one of the promising tools to enhance tissue-material integration in metallic implants. Here, we used anodization technique to modify the surface of commercially pure titanium (CP-Ti) and titanium alloy (Ti-6Al-4V) samples. Our results show that electrolyte composition, anodization time and voltage dictated the formation of well-organized nanotubes. Although electrolyte containing HF in water resulted in nanotube formation on Ti, the presence of NH4F and ethylene glycol was necessary for successful nanotube formation on Ti-6Al-4V. Upon examination of the interaction of bone marrow stromal cells (BMSCs) with the modified samples, we found that Ti-6Al-4V without nanotubes induced cell proliferation and cluster of differentiation 40 ligand (CD40L) expression which facilitates B-cell activation to promote early bone healing. However, the expression of glioma associated protein 2 (GLI2), which regulates CD40L, was reduced in Ti-6Al-4V and the presence of nanotubes further reduced its expression. The inflammatory cytokine interleukin-6 (IL-6) expression was reduced by nanotube presence on Ti. These results suggest that Ti-6Al-4V with nanotubes may be suitable implants because they have no effect on BMSC growth and inflammation.

Effects of MgO, ZnO, SrO, and SiO2 in tricalcium phosphate scaffolds on in vitro gene expression and in vivo osteogenesis.

߭tricalcium phosphate (߭TCP) is a versatile bioceramic for its use in many orthopedic and dental applications due to its excellent biocompatibility and biodegradability. Recently, the addition of additives to ߭TCP has been proven to improve bone repair and regeneration, however, the underlying mechanism of enhanced bone regeneration is still unknown. In this study, strontium oxide (SrO), silica (SiO2), magnesia (MgO), and zinc oxide (ZnO) were added to ߭TCP for dense discs fabrication followed by in vitro evaluation using a preosteoblast cell line. Cell viability and gene expression were analyzed at day 3 and day 9 during the cell culture. MgO and SiO2 were found to significantly enhance and expedite osteoblastic differentiation. A potential mechanism was introduced to explain the additive induced osteoblastic differentiation. In addition, in vivo characterizations showed that porous 3D printed MgO-SiO2-TCP scaffolds significantly improved new bone formation after 16 weeks of implantation. This study shows beneficial effects of additives on osteoblastic viability and differentiation in vitro as well as osteogenesis in vivo, which is crucial towards the development of bone tissue engineering scaffolds.

Enhanced In Vivo Bone and Blood Vessel Formation by Iron Oxide and Silica Doped 3D Printed Tricalcium Phosphate Scaffolds.

Calcium phosphate (CaP) ceramics show significant promise towards bone graft applications because of the compositional similarity to inorganic materials of bone. With 3D printing, it is possible to create ceramic implants that closely mimic the geometry of human bone and can be custom-designed for unusual injuries or anatomical sites. The objective of the study was to optimize the 3D-printing parameters for the fabrication of scaffolds, with complex geometry, made from synthesized tricalcium phosphate (TCP) powder. This study was also intended to elucidate the mechanical and biological effects of the addition of Fe+3 and Si+4 in TCP implants in a rat distal femur model for 4, 8, and 12 weeks. Doped with Fe+3 and Si+4 TCP scaffolds with 3D interconnected channels were fabricated to provide channels for micronutrients delivery and improved cell-material interactions through bioactive fixation. Addition of Fe+3 into TCP enhanced early-stage new bone formation by increasing type I collagen production. Neovascularization was observed in the Si+4 doped samples after 12 weeks. These findings emphasize that the additive manufacturing of scaffolds with complex geometry from synthesized ceramic powder with modified chemistry is feasible and may serve as a potential candidate to introduce angiogenic and osteogenic properties to CaPs, leading to accelerated bone defect healing.

Lithium-doped ß-tricalcium phosphate: Effects on physical, mechanical and in vitro osteoblast cell-material interactions.

In this work, we have investigated the effects of lithium (Li) dopant at different concentrations and sintering temperatures on the physical and mechanical properties of ß-tricalcium phosphate (ß-TCP). Our results showed that Li addition at concentrations of 0.65 and 1.0 wt % inhibits the ß-TCP to α-TCP phase transformation. 0.15 wt % Li addition resulted in grain growth and extensive liquid phase was formed at higher concentrations. At 1150°C, compressive strength of ß-TCP increased from 138.7 ± 19.9 MPa to 170.9 ± 29.8 MPa with the addition of 0.15 wt % Li. Addition of higher amounts of Li decreased the compressive strength and the lowest compressive strength of 99.8 ± 13.7 MPa was found in samples containing 1.0 wt % Li. After 3 days of culture, osteoblast cells grew to confluence on samples containing 0.65 and 1.0 wt % Li. Cells grew to confluence on all doped samples after 11 days of culture and optical cell density was 4-5 folds higher on 0.15 and 1.0 wt % Li-doped TCP samples. Our results show that both Li content and sintering temperature have significant influence toward physicochemical and mechanical properties of ß-TCP which affects the osteoblast cell-materials interaction in Li-doped TCP scaffolds. © 2015 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 105B: 391-399, 2017.

Effects of Iron on Physical and Mechanical Properties, and Osteoblast Cell Interaction in ß-Tricalcium Phosphate.

Iron (Fe) is a vital element and its deficiency causes abnormal bone metabolism. We investigated the effects of Fe and its concentration in ß-tricalcium phosphate (ß-TCP) on physicomechanical properties and in vitro proliferation and differentiation of osteoblasts. Our results showed that Fe addition at concentrations of 0.5 wt.% (0.5 Fe-TCP) and 1.0 wt.% (1.0 Fe-TCP) inhibits the ß-TCP to α-TCP phase transformation at sintering temperature of 1250 °C. Addition of 0.25 wt.% Fe (0.25 Fe-TCP) increased the compressive strength of ß-TCP from 167.27 ± 16.2 to 227.10 ± 19.3 MPa. After 3 days of culture, surfaces of 0.5 Fe-TCP and 1.0 Fe-TCP samples were covered by osteoblast cells, compared to that of pure and 0.25 Fe-TCP. Cells grew to confluency on all Fe-doped samples after 7 days of culture and monolayer sheet-like cellular structure was found at 11 days. Optical cell density and alkaline phosphatase activity were significantly higher on Fe-doped samples and the highest values were found in 0.5 Fe-TCP samples. Our results show that Fe concentration had significant effect on physical and mechanical properties of TCP ceramics, and also on the in vitro osteoblast cellular interactions in TCP ceramics.

IGF-loaded silicon and zinc doped brushite cement: physico-mechanical characterization and in vivo osteogenesis evaluation.

Dopants play critical roles in controlling the physical, mechanical, degradation kinetics, and in vivo properties of calcium phosphates. The aim of the present study was to evaluate the effects of silicon (Si) and zinc (Zn) dopants on the physico-mechanical and in vivo osteogenesis properties of brushite cements (BrCs) alone and in combination with insulin like growth factor 1 (IGF-1). Addition of 0.5 wt% Si did not alter the setting time, ß-TCP content, and compressive strength of BrCs significantly; however, 0.25 wt% Zn incorporation was accompanied by a significant decrease in mechanical strength from 4.78 ± 0.21 MPa for pure BrC to 3.78 ± 0.59 MPa and 3.28 ± 0.22 MPa for Zn-BrC and Si/Zn-BrC, respectively. The in vivo bone regeneration properties of doped BrCs alone and in combination with IGF-1 were assessed and compared using chronological radiography, histology, scanning electron microscopy and fluorochrome labeling at 2 and 4 months post implantation in a rabbit femoral defect model. Based on in vivo characterization focusing on osteogenesis and vasculogenesis, Si-BrC and Si/Zn-BrC showed the best performance followed by Zn-BrC and pure BrCs. Addition of IGF-1 further improved bone regeneration. Our findings confirm that addition of Si and/or Zn alters the physico-mechanical properties of BrCs and promotes the early stage in vivo osseointegration and bone remodeling properties.

Phase stability and biological property evaluation of plasma sprayed hydroxyapatite coatings for orthopedic and dental applications.

In this work we have investigated the effects of strontium (Sr) dopant on in vitro protein release kinetics and in vivo osteogenic properties of plasma sprayed hydroxyapatite (HA) coatings, along with their dissolution behavior. Plasma sprayed HA coatings are widely used in load-bearing implants. Apart from osseointegration, the new generation of HA coating is expected to deliver biomolecules and/or drugs that can induce osteoinduction. This paper reports the preparation of crystalline and amorphous HA coatings on commercially pure titanium (Cp-Ti) using inductively coupled radio frequency (RF) plasma spray, and their stability at different solution pH. Coatings prepared at 110 mm working distance from the nozzle showed an average Ca ion release of 18 and 90 ppm in neutral and acidic environments, respectively. Decreasing the working distance to 90 mm resulted in the formation of a coating with less crystalline HA and phases with higher solubility products, and consequently higher dissolution over 32 days. A 92% release of a model protein bovine serum albumin (BSA) in phosphate buffer with pH of 7.4 was measured for Sr-doped HA (Sr-HA) coating, while only a 72% release could be measured for pure HA coating. Distortion of BSA during adsorption on coatings revealed a strong interaction between the protein and the coating, with an increase in α-helix content. Osteoid formation was found on Sr-HA implants as early as 7 weeks post implantation compared to HA coated and uncoated Ti implants. After 12 weeks post implantation, osteoid new bone was formed on HA implants; whereas, bone mineralization started on Sr-HA samples. While no osteoid was formed on bare Ti surfaces, bone was completely mineralized on HA and Sr-HA coatings after 16 weeks post implantation. Our results show that both phase stability and chemistry can have a significant influence toward in vitro and in vivo response of HA coatings on Ti implants.

Effects of Silicon on Osteoclast Cell Mediated Degradation, In Vivo Osteogenesis and Vasculogenesis of Brushite Cement.

Calcium phosphate cements (CPCs) are being widely used for treating small scale bone defects. Among the various CPCs, brushite (dicalcium phosphate dihydrate, DCPD) cement is widely used due to its superior solubility and ability to form new bone. In the present study, we have studied the physical, mechanical, osteoclast-like-cells differentiation and in vivo osteogenic and vasculogenic properties of silicon (Si) doped brushite cements. Addition of Si did not alter the phase composition of final product and regardless of Si level, all samples included ß-tricalcium phosphate (ß-TCP) and DCPD. 1.1 wt. % Si addition increased the compressive strength of undoped brushite cement from 4.78±0.21 MPa to 5.53±0.53 MPa, significantly. Cellular activity was studied using receptor activator of nuclear factor κß ligand (RANKL) supplemented osteoclast-like-cells precursor RAW 264.7 cell. Phenotypic expressions of the cells confirmed successful differentiation of RAW264.7 monocytes to osteoclast-like-cells on undoped and doped brushite cements. An increased activity of osteoclast-like cells was noticed due to Si doping in the brushite cement. An excellent new bone formation was found in all cement compositions, with significant increase in Si doped brushite samples as early as 4 weeks post implantation in rat femoral model. After 4 weeks of implantation, no significant difference was found in blood vessel formation between the undoped and doped cements, however, a significant increase in vasculgenesis was found in 0.8 and 1.1 wt. % Si doped brushite cements after 8 weeks. These results show the influence of Si dopant on physical, mechanical, in vitro osteoclastogenesis and in vivo osteogenic and vasculogenic properties of brushite cements.

Tricalcium phosphate and tricalcium phosphate/polycaprolactone particulate composite for controlled release of protein.

ß-Tricalcium phosphate (ß-TCP) with three different particle size ranges was used to study the effects of particle size and surface area on protein adsorption and release. Polycaprolactone (PCL) coating was applied on the particle systems to investigate its effect on particulate system properties from both structural and application aspects. The maximum loading of 27 mg/g was achieved for 100 nm particles. Bovine serum albumin (BSA) loading amount was controlled by varying the BSA loading solution concentration, as well as the sample powder's surface area. Increasing the surface area of the delivery powder significantly increased loading and release yield. Unlike the samples with low surface area, the lowest particle size samples showed sigmoidal release profile. This indicated that release was governed by different mechanisms for particles with different sizes. While the majority of samples showed no more than 50% release, the 550 nm particles demonstrated 100% release. PCL coating showed no significant ability to attenuate burst release in PBS. However, it led to a steadier release profile as compared to the bare TCP particles. FTIR analysis also proved that the secondary structure of BSA did not change significantly during the adsorption; however, minor denaturation was found during the release. The same results were found when PCL coating was applied on the TCP particles. We envision potential use of TCP and TCP+PCL systems in bone growth factor or orthopedic drug delivery applications in future bone tissue engineering application.