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

Full deflection profile calculation and Young’s modulus optimisation for engineered high performance materials

New engineered materials have critical applications in different fields in medicine, engineering and technology but their enhanced mechanical performances are significantly affected by the microstructural design and the sintering process used in their manufacture. This work introduces (i) a methodology for the calculation of the full deflection profile from video recordings of bending tests, (ii) an optimisation algorithm for the characterisation of Young’s modulus, (iii) a quantification of the effects of optical distortions and (iv) a comparison with other standard tests. The results presented in this paper show the capabilities of this procedure to evaluate the Young’s modulus of highly stiff materials with greater accuracy than previously possible with bending tests, by employing all the available information from the video recording of the tests. This methodology extends to this class of materials the possibility to evaluate both the elastic modulus and the tensile strength with a single mechanical test, without the need for other experimental tools

Simulation of cortico-cancellous bone structure by 3D printing of bilayer calcium phosphate-based scaffolds

Traditional methods of fabrication for porous bone scaffolds are unable to accurately mimic the desirable cortico-cancellous morphology and the structure of the bone. In this study, 3D printing of a β-Tricalcium phosphate (TCP)-based paste was used to develop scaffolds simulating the two distinct cortical and cancellous layers of the natural bone. Laser microscope imaging showed that the pore sizes were 242.2±24.3. μm and 410.5±27.9. μm for the cortical and cancellous layers, respectively. Micro CT analysis revealed overall porosity and interconnectivity of 61.8±1.4% and 208707.5±52405, respectively. Mechanical properties were within the range of human cancellous bone with 10.0±2.4. MPa strength and 55.5±5.7. MPa young's modulus. The X-ray diffraction (XRD) analysis showed that the phase composition of the printed scaffolds was almost identical to pure TCP. Scanning electron microscopy (SEM) and cell vitality assessment indicated significant osteoblastic proliferation on the surface of the scaffolds. The gene expression analysis showed an increase in the level of Collagen I (Col I), Osteonectin (ON), Ostocalcin (OC), and Osteopontin (OPN) with a significant increase in OC and OPN at day 10. In conclusion, 3D printing can be used to develop a TCP-based scaffold with controllable and reproducible microstructures and favourable in vitro biological properties with potential to be further developed to be used for clinical bone regeneration

Production of nanoparticles from natural hydroxylapatite by laser ablation

Laser ablation of solids in liquids technique has been used to obtain colloidal nanoparticles from biological hydroxylapatite using pulsed as well as a continuous wave (CW) laser. Transmission electron microscopy (TEM) measurements revealed the formation of spherical particles with size distribution ranging from few nanometers to hundred nanometers and irregular submicronic particles. High resolution TEM showed that particles obtained by the use of pulsed laser were crystalline, while those obtained by the use of CW laser were amorphous. The shape and size of particles are consistent with the explosive ejection as formation mechanism

Human adipose derived stem cells are superior to human osteoblasts (HOB) in bone tissue engineering on a collagen-fibroin-ELR blend

The ultrastructure of the bone provides a unique mechanical strength against compressive, torsional and tensional stresses. An elastin-like recombinamer (ELR) with a nucleation sequence for hydroxyapatite was incorporated into films prepared from a collagen – silk fibroin blend carrying microchannel patterns to stimulate anisotropic osteogenesis. SEM and fluorescence microscopy showed the alignment of adipose-derived stem cells (ADSCs) and the human osteoblasts (HOBs) on the ridges and in the grooves of microchannel patterned collagen-fibroin-ELR blend films. The Young's modulus and the ultimate tensile strength (UTS) of untreated films were 0.58 ± 0.13 MPa and 0.18 ± 0.05 MPa, respectively. After 28 days of cell culture, ADSC seeded film had a Young's modulus of 1.21 ± 0.42 MPa and UTS of 0.32 ± 0.15 MPa which were about 3 fold higher than HOB seeded films. The difference in Young's modulus was statistically significant (p: 0.02). ADSCs attached, proliferated and mineralized better than the HOBs. In the light of these results, ADSCs served as a better cell source than HOBs for bone tissue engineering of collagen-fibroin-ELR based constructs used in this study. We have thus shown the enhancement in the tensile mechanical properties of the bone tissue engineered scaffolds by using ADSCs

Porous hydroxyapatite-bioactive glass hybrid scaffolds fabricated via ceramic honeycomb extrusion

The successful fabrication of hydroxyapatite‐bioactive glass scaffolds using honeycomb extrusion is presented herein. Hydroxyapatite was combined with either 10 wt% stoichiometric Bioglass® (BG1), calcium‐excess Bioglass® (BG2) or canasite (CAN). For all composite materials, glass‐induced partial phase transformation of the HA into the mechanically weaker β‐tricalcium phosphate (TCP) occurred but XRD data demonstrated that BG2 exhibited a lower volume fraction of TCP than BG1. Consequently, the maximum compressive strength observed for BG1 and BG2 were 30.3 ± 3.9 and 56.7 ± 6.9 MPa, respectively, for specimens sintered at 1300°C. CAN scaffolds, in contrast, collapsed when handled when sintered below 1300°C, and thus failed. The microstructure illustrated a morphology similar to that of BG1 sintered at 1200°C, and hence a comparable compressive strength (11.4 ± 3.1 MPa). The results highlight the great potential offered by honeycomb extrusion for fabricating high‐strength porous scaffolds. The compressive strengths exceed that of commercial scaffolds, and biological tests revealed an increase in cell viability over 7 days for all hybrid scaffolds. Thus it is expected that the incorporation of 10 wt% bioactive glass will provide the added advantage of enhanced bioactivity in concert with improved mechanical stability

3D bioactive composite scaffolds for bone tissue engineering

Bone is the second most commonly transplanted tissue worldwide, with over four million operations using bone grafts or bone substitute materials annually to treat bone defects. However, significant limitations affect current treatment options and clinical demand for bone grafts continues to rise due to conditions such as trauma, cancer, infection and arthritis. Developing bioactive three-dimensional (3D) scaffolds to support bone regeneration has therefore become a key area of focus within bone tissue engineering (BTE). A variety of materials and manufacturing methods including 3D printing have been used to create novel alternatives to traditional bone grafts. However, individual groups of materials including polymers, ceramics and hydrogels have been unable to fully replicate the properties of bone when used alone. Favourable material properties can be combined and bioactivity improved when groups of materials are used together in composite 3D scaffolds. This review will therefore consider the ideal properties of bioactive composite 3D scaffolds and examine recent use of polymers, hydrogels, metals, ceramics and bio-glasses in BTE. Scaffold fabrication methodology, mechanical performance, biocompatibility, bioactivity, and potential clinical translations will be discussed

Synergistic effect of nanomaterials and BMP-2 signalling in inducing osteogenic differentiation of adipose tissue-derived mesenchymal stem cells.

The lack of complete understanding in the signalling pathways that control the osteogenic differentiation of mesenchymal stem cells hinders their clinical application in the reconstruction of large bone defects and non-union bone fractures. The aim of this study is to gain insight into the interactions of bone morphogenetic protein-2 (BMP-2) and bone biomimetic scaffolds in directing osteogenic differentiation of adipose tissue-derived mesenchymal stem cells (ASCs) and the underlying signalling pathways involved. We demonstrated that bioactive glass nanoparticles (nBG) incorporated polycaprolactone (PCL) coating on hydroxyapatite/β-tricalcium phosphate (HA/TCP) scaffold exerted a synergistic effect with 3days of BMP-2 treatment in promoting osteogenic gene expression levels (Runx-2, collagen I, osteopontin and bone sialoprotein) and alkaline phosphatase activity in ASCs. Furthermore, we revealed that the synergistic effect was mediated through a mechanism of activating β1-integrin and induction of Wnt-3a autocrine signalling pathways by nBG incorporated scaffold