Zirconium Ions Up-Regulate the BMP/SMAD Signaling Pathway and Promote the Proliferation and Differentiation of Human Osteoblasts

Zirconium (Zr) is an element commonly used in dental and orthopedic implants either as zirconia (ZrO2) or in metal alloys. It can also be incorporated into calcium silicate-based ceramics. However, the effects of in vitro culture of human osteoblasts (HOBs) with soluble ionic forms of Zr have not been determined. In this study, primary culture of human osteoblasts was conducted in the presence of medium containing either ZrCl4 or Zirconium (IV) oxynitrate (ZrO(NO3)2) at concentrations of 0, 5, 50 and 500 μM, and osteoblast proliferation, differentiation and calcium deposition were assessed. Incubation of human osteoblast cultures with Zr ions increased the proliferation of human osteoblasts and also gene expression of genetic markers of osteoblast differentiation. In 21 and 28 day cultures, Zr ions at concentrations of 50 and 500 μM increased the deposition of calcium phosphate. In addition, the gene expression of BMP2 and BMP receptors was increased in response to culture with Zr ions and this was associated with increased phosphorylation of SMAD1/5. Moreover, Noggin suppressed osteogenic gene expression in HOBs co-treated with Zr ions. In conclusion, Zr ions appear able to induce both the proliferation and the differentiation of primary human osteoblasts. This is associated with up-regulation of BMP2 expression and activation of BMP signaling suggesting this action is, at least in part, mediated by BMP signaling.NHMRC Project grant 63265

A Novel Bone Substitute with High Bioactivity, Strength, and Porosity for Repairing Large and Load-Bearing Bone Defects.

Achieving adequate healing in large or load-bearing bone defects is highly challenging even with surgical intervention. The clinical standard of repairing bone defects using autografts or allografts has many drawbacks. A bioactive ceramic scaffold, strontium-hardystonite-gahnite or "Sr-HT-Gahnite" (a multi-component, calcium silicate-based ceramic) is developed, which when 3D-printed combines high strength with outstanding bone regeneration ability. In this study, the performance of purely synthetic, 3D-printed Sr-HT-Gahnite scaffolds is assessed in repairing large and load-bearing bone defects. The scaffolds are implanted into critical-sized segmental defects in sheep tibia for 3 and 12 months, with bone autografts used for comparison. The scaffolds induce substantial bone formation and defect bridging after 12 months, as indicated by X-ray, micro-computed tomography, and histological and biomechanical analyses. Detailed analysis of the bone-scaffold interface using focused ion beam scanning electron microscopy and multiphoton microscopy shows scaffold degradation and maturation of the newly formed bone. In silico modeling of strain energy distribution in the scaffolds reveal the importance of surgical fixation and mechanical loading on long-term bone regeneration. The clinical application of 3D-printed Sr-HT-Gahnite scaffolds as a synthetic bone substitute can potentially improve the repair of challenging bone defects and overcome the limitations of bone graft transplantation

The Influence hydroxyapatite nanoparticle shape and size on the properties of biphasic calcium phosphate scaffolds coated with hydroxyapatite-PCL composites

We developed a composite biphasic calcium phosphate (BCP) scaffold by coating a nanocomposite layer, consisting of hydroxyapatite (HA) nanoparticles and polycaprolactone (PCL), over the surface of BCP. The effects of HA particle size and shape in the coating layer on the mechanical and biological properties of the BCP scaffold were examined. Micro-computerized tomography studies showed that the prepared scaffolds were highly porous (∼91%) with large pore size (400-700 μm) and an interconnected porous network of ∼100%. The HA nanoparticle (needle shape)-composite coated scaffolds displayed the highest compressive strength (2.1 ± 0.17 MPa), compared to pure HA/β-TCP (0.1 ± 0.05 MPa) and to the micron HA - composite coated scaffolds (0.29 ± 0.07 MPa). These needle shaped scaffolds also showed enhanced elasticity and similar stress-strain profile to natural bone. Needle shaped coated HA/PCL particles induced the differentiation of primary human bone derived cells, with significant upregulation of osteogenic gene expression (Runx2, collagen type I, osteocalcin and bone sialoprotein) and alkaline phosphatase activity compared to other groups. These properties are essential for enhancing bone ingrowth in load-bearing applications. The developed composite scaffolds possessed superior physical, mechanical, elastic and biological properties rendering them potentially useful for bone tissue regeneration.12 page(s

Micro-poro-elasticity of baghdadite-based bone tissue engineering scaffolds:A unifying approach based on ultrasonics, nanoindentation, and homogenization theory

\u3cp\u3eMicrostructure-elasticity relations for bone tissue engineering scaffolds are key to rational biomaterial design. As a contribution thereto, we here report comprehensive length measuring, weighing, and ultrasonic tests at 0.1 MHz frequency, on porous baghdadite (Ca\u3csub\u3e3\u3c/sub\u3eZrSi\u3csub\u3e2\u3c/sub\u3eO\u3csub\u3e9\u3c/sub\u3e) scaffolds. The resulting porosity-stiffness relations further confirm a formerly detected, micromechanically explained, general relationship for a great variety of different polycrystals, which also allows for estimating the zero-porosity case, i.e. Young modulus and Poisson ratio of pure (dense) baghdadite. These estimates were impressively confirmed by a physically and statistically independent nanoindentation campaign comprising some 1750 indents. Consequently, we can present a remarkably complete picture of porous baghdadite elasticity across a wide range of porosities, and, thanks to the micromechanical understanding, reaching out beyond classical elasticity, towards poroelastic properties, quantifying the effect of pore pressure on the material system behavior.\u3c/p\u3

Micro-elasticity of porous ceramic baghdadite : A combined acoustic-nanoindentation approach supported by homogenization theory

Bone tissue engineering aims at repairing damaged bone and restoring its functions with the help of biocompatible materials cultivated with cells and corresponding growth factors [1]. Besides being osteoconductive and osteoinductive, the bone substitute or scaffold should exhibit sufficient porosity for good vascular and tissue ingrowth, while not overly compromising the overall mechanical properties of the implant, i.e. its stiffness and strength. The design process of such scaffolds requires a multitude of in vitro and in vivo experiments and has proven to be a challenging task, thus giving rise to the wish for rational, computer-aided design of biomaterials, regarding not only biological and cell transport aspects, but also mechanics. Highly porous baghdadite (Ca3ZrSi2O9) scaffolds have shown promising biological responses when used for the repair of critical size defects in rabbit radial bones [2]. However, the mechanical properties of these scaffolds require further investigation. Therefore, by using structure-property relations derived from ultrasound and nanoindentation experiments, and on the basis of theoretical and applied micromechanics, the current research aims at applying the state-of-the-art methods in computational biomechanics and biomaterials to this new material to investigate its elastic properties

Micro-poro-elasticity of baghdadite-based bone tissue engineering scaffolds: A unifying approach based on ultrasonics, nanoindentation, and homogenization theory

Microstructure-elasticity relations for bone tissue engineering scaffolds are key to rational biomaterial design. As a contribution thereto, we here report comprehensive length measuring, weighing, and ultrasonic tests at 0.1 MHz frequency, on porous baghdadite (Ca3ZrSi2O9) scaffolds. The resulting porosity-stiffness relations further confirm a formerly detected, micromechanically explained, general relationship for a great variety of different polycrystals, which also allows for estimating the zero-porosity case, i.e. Young modulus and Poisson ratio of pure (dense) baghdadite. These estimates were impressively confirmed by a physically and statistically independent nanoindentation campaign. comprising some 1750 indents. Consequently, we can present a remarkably complete picture of porous baghdadite elasticity across a wide range of porosities, and, thanks to the micromechanical understanding, reaching out beyond classical elasticity, towards poroelastic properties, quantifying the effect of pore pressure on the material system behavior. (C) 2014 The Authors. Published by Elsevier B.V

Fracture behaviors of ceramic tissue scaffolds for load bearing applications

Healing large bone defects, especially in weight-bearing locations, remains a challenge using available synthetic ceramic scaffolds. Manufactured as a scaffold using 3D printing technology, Sr-HT-Gahnite at high porosity (66%) had demonstrated significantly improved compressive strength (53 ± 9 MPa) and toughness. Nevertheless, the main concern of ceramic scaffolds in general remains to be their inherent brittleness and low fracture strength in load bearing applications. Therefore, it is crucial to establish a robust numerical framework for predicting fracture strengths of such scaffolds. Since crack initiation and propagation plays a critical role on the fracture strength of ceramic structures, we employed extended finite element method (XFEM) to predict fracture behaviors of Sr-HT-Gahnite scaffolds. The correlation between experimental and numerical results proved the superiority of XFEM for quantifying fracture strength of scaffolds over conventional FEM. In addition to computer aided design (CAD) based modeling analyses, XFEM was conducted on micro-computed tomography (μCT) based models for fabricated scaffolds, which took into account the geometric variations induced by the fabrication process. Fracture strengths and crack paths predicted by the μCT-based XFEM analyses correlated well with relevant experimental results. The study provided an effective means for the prediction of fracture strength of porous ceramic structures, thereby facilitating design optimization of scaffolds

Primers Used for Real-Time Polymerase Chain Reaction.

<p>BSP, bone sialoprotein; Runx2, runt-related transcription factor 2; ALP, alkaline phosphatase; BMP2, bone morphogenetic protein 2; BMPR, BMP receptor</p><p>Primers Used for Real-Time Polymerase Chain Reaction.</p

Zirconium ions up-regulate gene expression of BMP2 and BMPR in HOBs.

<p>HOBs were treated with ZrO(NO₃)₂ and ZrCl₄ solutions at the concentrations of 5, 50 and 500 µM for 3 and 7 days. Real-time PCR was employed to analyze the gene expression of BMP2 (A), BMP receptors BMPR1a (B), BMPR1b (C) and BMPR2 (D). (A) At D3 and D7, BMP2 gene expression was significantly up-regulated in HOBs treated with ZrO(NO₃)₂ at 500 µM and ZrCl₄ at 50 µM and 500 µM, compared to the control. (B) At D3, BMPR1a expression was up-regulated in HOBs by ZrO(NO₃)₂ and ZrCl₄ only at 500 µM, compared to the control. ZrO(NO₃)₂ and ZrCl₄ at 50 and 500 µM significantly increased BMPR1a expression in HOBs treated for 7 days. BMPR1a gene expression was inhibited by ZrCl₄ at 5 µM at D3. (C) BMPR1b expression was significantly increased in HOBs treated with ZrO(NO₃)₂ and ZrCl₄ at 50 and 500 µM at D3 and D7. Increased BMPR1b expression was exhibited by ZrO(NO₃)₂ at 5 µM only at D7 whereas ZrCl₄ at 5 µM only at D3, respectively, compared with the control. (D) Zirconium solutions had no effect on the BMPR2 expression in HOBs.*p<0.05, **p<0.01 vs control.</p