EFFECTS OF DOPANTS ON PHYSICAL, MECHANICAL, IN VITRO AND IN VIVO OSTEOBLASTOGENIC AND OSTEOCLASTOGENIC PROPERTIES OF CALCIUM PHOSPHATE CERAMICS FOR BONE TISSUE ENGINEERING AND VITAMIN C DELIVERY

The objective of this research is to understand the effects of different dopants such as lithium (Li), iron (Fe), silicon (Si), strontium (Sr), and magnesium (Mg) in calcium phosphate based ceramics (CaP) on their physical and mechanical properties, as well as in vitro and in vivo bone formation. The release behavior of vitamin C as a natural biomolecule from tricalcium phosphate (TCP) samples was also investigated. The hypothesis of this research is that the presence of different trace elements in CaPs influences their phase stability, microstructure, mechanical strength, and both in vitro and in vivo bioactivity. We also hypothesize that the gradual release of vitamin C from TCP can be controlled by polycaprolactone (PCL) coating and alters the interaction between TCP and bone forming cells.Presence of Li and Fe stabilized the β-TCP structure and increased its compressive strength. While addition of 0.15 wt. % Li increased the osteoblast cell proliferation, Fe addition enhanced the proliferation and differentiation of osteoblast cells in dose dependent manner. Addition of Si to brushite cement (BrC) activated the osteoclastogenesis and induced osteogenesis and vasculogenesis in vivo.Initial burst release of vitamin C from TCP samples was found at both pH of 5.0 and 7.4 and applying PCL coating decreased the cumulative release of vitamin C. Gradual release of vitamin C increased the proliferation of osteoblast cells in vitamin C and/or PCL loaded samples regardless of vitamin C concentration. However, the differentiation of cells was only induced in samples loaded with 2 µg of vitamin C.Addition of Sr and Mg to TCP and hydroxyapatite (HA) activated the signaling pathways related to enhanced osteoblastogenesis. In addition, the bone remodeling was induced in presence of Sr and Mg in osteoblast-osteoclast co-culture system. In dense TCP structures, addition of Sr enhanced the proliferation and differentiation of mesenchymal stem cells at early time points while Mg addition increased the cellular activity gradually over the course of 21 days. Our results show that presence of dopants has significant effect on the physical, mechanical, in vitro and in vivo osteogenesis CaPs for bone tissue engineering applications

Effects of Iron on Physical and Mechanical Properties, and Osteoblast Cell Interaction in β-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 β-tricalcium phosphate (β-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 β-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 β-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

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

β-Tricalcium phosphate (β-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 27mg/g was achieved for 100nm 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 550nm 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. •BSA adsorption and release kinetics is dependent on particle size and initial protein concentration.•BSA release mechanism is governed by particle size.•PCL coating cannot retard burst release effect.•Insignificant protein denaturation occurs using TCP/TCP+PCL systems

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 a new bone. In the present study, we have studied the physical, mechanical, osteoclast-like-cell differentiation and in vivo osteogenic and vasculogenic properties of silicon (Si) doped brushite cements. The addition of Si did not alter the phase composition of the final product and regardless of the Si level, all samples included β-tricalcium phosphate (β-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 a receptor activator of nuclear factor κβ ligand (RANKL) supplemented osteoclast-like-cell precursor RAW 264.7 cells. Phenotypic expressions of the cells confirmed successful differentiation of RAW 264.7 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 a 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 vasculogenesis was found in 0.8 and 1.1 wt% Si doped brushite cements after 8 weeks. These results show the influence of the Si dopant on the physical, mechanical, in vitro osteoclastogenesis and in vivo osteogenic and vasculogenic properties of brushite cements

Three-dimensional printing of biomaterials and soft materials

During the past two decades, numerous biomaterials and soft materials, including ceramics, polymers, and their composites, have been fabricated for various biomedical devices and applications in tissue engineering using three-dimensional (3D) printing. This article offers a brief overview of some of the biomaterials and soft materials fabricated using some notable 3D printing techniques and related applications. A brief perspective regarding future directions of this field is also provided

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

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 β-TCP (Si-TCP) and ZnO doped β-TCP (Zn-TCP) scaffolds through the incorporation of 0.5 wt.% SiO2 and 0.25 wt.% ZnO, respectively, to the β-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 β-TCP structure inhibited the β 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-β 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 β-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

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 β-tricalcium phosphate (β-TCP)/dicalcium phosphate dihydrate (DCPD) ratio and prolongs the initial and final setting time due to effective role of Fe on stabilizing the β-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

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 physico-mechanical, and in vivo osteogenesis properties of brushite cements (BrCs) alone and in combination with insulin like growth factor 1(IGF-1). Although addition of 0.5 wt.% Si did not alter the setting time, β-TCP content, and compressive strength of BrCs significantly, 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 after 2 and 4 months post implantation in rabbit femoral defect model. Based on different in vivo characterization, better osteogenesis and vasculogenesis was observed for Si-BrC and Si/Zn-BrC, whereas moderate bone regeneration was found in Zn-BrC as compared to pure BrCs. Excellent bone regeneration was observed when doped BrCs were combined with IGF-1. Our findings signify 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. Moreover, addition of IGF-1 further improved the performance of BrCs in terms of bone regeneration in animal model