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.

Triple-Bioinspired Burying/Crosslinking Interfacial Coassembly Strategy for Layer-by-Layer Construction of Robust Functional Bioceramic Self-Coatings for Osteointegration Applications.

Coating bioceramics of inherent bioactivity onto biometallic implants is a straightforward yet promising solution to address poor osteointegration of the latter. One step further, it would be a nontrivial accomplishment to develop a mild, cheap, and universal route to firmly stabilizing, in principle, any ceramics onto any implant substrate, while imparting expectedly versatile biofunctional performances. Herein, we describe a triple-bioinspired burying/cross-linking interfacial coassembly strategy for enabling such ceramic coatings, which ingeniously fuses bioinspiration from sea rocks (burying assisted particle immobilization), marine mussels (universal adhesion and versatile chemical reactivity), and reef-building oysters (cross-linking rendered cohesion). Specifically, surface functionalized, aqueous dispersed ceramic particles were buried within an substrate-anchored organic matrix of polyelectrolyte multilayers (i.e., (poly(ether imide) (PEI)/poly(sodium-p-styrenesulfonate) (PSS)) n), through a new inorganic-organic hybrid layer-by-layer (LBL) coassembly scheme wherein mussel (oyster) inspired adhesive (cohesive) chemistries were exquisitely orchestrated. As a conceptual demonstration, bioactive baghdadite (Ca3ZrSi2O9) was synthesized as model ceramics, with which we constructed on medical titanium robust, biomimetic, and cross-linkable LBL self-assemblies harnessing the said strategy. Intimate substrate contacts and well-defined buried inorganic-organic interfaces were evidently seen, together with good structural and chemical stabilities, especially after cross-linking. Sustained bioactive ion releasing and appreciable biomineralization activity were confirmed in vitro. Subsequently, biological performances of the assemblies were systematically investigated with respect to surface hydrophilicity, protein adsorption, and osteoblast functions. Additionally, nanosilver deposition, which imparted the surfaces with added antibacterial potencies, was used to exemplify the strategy's versatility in allowing multifunctionality. What's more, the flexibility of our approach was testified through modifying clinically relevant complicated 3D porous scaffolds. Overall, our strategy basically met the design expectations, boding well for future medical adoption. This study offers the promise of an alternative broadly useful avenue to bioactive and functional surface design of bone implants. It may also provide insights into other multiple-bioinspired materials/interfaces for biological and other applications.

Effects of Sr-HT-Gahnite on osteogenesis and angiogenesis by adipose derived stem cells for critical-sized calvarial defect repair.

Tissue engineering strategies to construct vascularized bone grafts are now attracting much attention. Strontium-hardystonite-Gahnite (Sr-HT-Gahnite) is a strong, highly porous, and biocompatible calcium silicate based bio-ceramic that contains strontium and zinc ions. Adipose derived stem cells (ASCs) have been demonstrated to have the ability in promoting osteogenesis and angiogenesis. In this study, the effects of Sr-HT-Gahnite on cell morphology, cell proliferation, and osteogenic differentiation of ASCs were systematically investigated. The cell proliferation, migration and angiogenic differentiation of human umbilical vein endothelial cell (HUVECs) were studied. Beta-tricalcium phosphate/hydroxyapatite (TCP/HA) bioceramic scaffolds were set as the control biomaterial. Both bio-ceramics exhibited no adverse influence on cell viability. The Sr-HT-Gahnite scaffolds promoted cell attachment and alkaline phosphatase (ALP) activity of ASCs. The Sr-HT-Gahnite dissolution products enhanced ALP activity, matrix mineralization, and angiogenic differentiation of ASCs. They could also improve cell proliferation, migration, and angiogenic differentiation of HUVECs. Levels of in vivo bone formation with Sr-HT Gahnite were significantly higher than that for TCP/HA. The combination of Sr-HT-Gahnite and ASCs promoted both osteogenesis and angiogenesis in vivo study, compared to Sr-HT-Gahnite and TCP/HA bio-ceramics when administered alone, suggesting Sr-HT-Gahnite can act as a carrier for ASCs for construction of vascularized tissue-engineered bone.

Silk coating on a bioactive ceramic scaffold for bone regeneration: effective enhancement of mechanical and in vitro osteogenic properties towards load-bearing applications.

Bioactive ceramic scaffolds represent competitive choices for clinical bone reconstruction, but their widespread use is restricted by inherent brittleness and weak mechanical performance under load. This study reports the development of strong and tough bioactive scaffolds suitable for use in load-bearing bone reconstruction. A strong and bioactive ceramic scaffold (strontium-hardystonite-gahnite) is combined with single and multiple coating layers of silk fibroin to enhance its toughness, producing composite scaffolds which match the mechanical properties of cancellous bone and show enhanced capacity to promote in vitro osteogenesis. Also reported for the first time is a comparison of the coating effects obtained when a polymeric material is coated on ceramic scaffolds with differing microstructures, namely the strontium-hardystonite-gahnite scaffold with high-density struts as opposed to a conventional ceramic scaffold, such as biphasic calcium phosphate, with low-density struts. The results show that silk coating on a unique ceramic scaffold can lead to simple and effective enhancement of its mechanical and biological properties to suit a wider range of applications in clinical bone reconstruction, and also establish the influence of ceramic microstructure on the effectiveness of silk coating as a method of reinforcement when applied to different types of ceramic bone graft substitutes. Copyright © 2015 John Wiley & Sons, Ltd.

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.

In vitro response of macrophages to ceramic scaffolds used for bone regeneration.

Macrophages, the primary cells of the inflammatory response, are major regulators of healing, and mediate both bone fracture healing and the inflammatory response to implanted biomaterials. However, their phenotypic contributions to biomaterial-mediated bone repair are incompletely understood. Therefore, we used gene expression and protein secretion analysis to investigate the interactions in vitro between primary human monocyte-derived macrophages and ceramic scaffolds that have been shown to have varying degrees of success in promoting bone regeneration in vivo Specifically, baghdadite (Ca3ZrSi2O9) and strontium-hardystonite-gahnite (Sr-Ca2ZnSi2O7-ZnAl2O4) scaffolds were chosen as two materials that enhanced bone regeneration in vivo in large defects under load compared with clinically used tricalcium phosphate-hydroxyapatite (TCP-HA). Principal component analysis revealed that the scaffolds differentially regulated macrophage phenotype. Temporal changes in gene expression included shifts in markers of pro-inflammatory M1, anti-inflammatory M2a and pro-remodelling M2c macrophage phenotypes. Of note, TCP-HA scaffolds promoted upregulation of many M1-related genes and downregulation of many M2a- and M2c-related genes. Effects of the scaffolds on macrophages were attributed primarily to direct cell-scaffold interactions because of only minor changes observed in transwell culture. Ultimately, elucidating macrophage-biomaterial interactions will facilitate the design of immunomodulatory biomaterials for bone repair.

A bioceramic with enhanced osteogenic properties to regulate the function of osteoblastic and osteocalastic cells for bone tissue regeneration.

Bioceramics for regenerative medicine applications should have the ability to promote adhesion, proliferation and differentiation of osteoblast and osteoclast cells. Osteogenic properties of the material are essential for rapid bone regeneration and new bone formation. The aim of this study was to develop a silicate-based ceramic, gehlenite (GLN, Ca2Al2SiO7), and characterise its physiochemical, biocompatibility and osteogenic properties. A pure GLN powder was synthesised by a facile reactive sintering method and compacted to disc-shaped specimens. The sintering behaviour and degradation of the GLN discs in various buffer solutions were fully characterised. The cytotoxicity of GLN was evaluated by direct and indirect methods. In the indirect method, primary human osteoblast cells (HOBs) were exposed to diluted extracts (100, 50, 25, 12.5 and 6.25 mg ml(-1)) of fine GLN particles in culture medium. The results showed that the extracts did not cause any cytotoxic effect on the HOBs with the number of cells increasing significantly from day 1 to day 7. GLN-supported HOB attachment and proliferation, and significantly enhanced osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein) were compared with biphasic calcium phosphate groups (BCP, a mixture of hydroxyapatite (60wt.%) and ß-tricalcium phosphate(40wt.%)). We also demonstrated that in addition to supporting HOB attachment and proliferation, GLN promoted the formation of tartrate-acid resistance phosphatase (TRAP) positive multinucleated osteoclastic cells (OCs) derived from mouse bone marrow cells. Results also demonstrated the ability of GLN to support the polarisation of OCs, a prerequisite for their functional resorptive activity which is mainly influenced by the composition and degradability of biomaterials. Overall, the developed GLN is a prospective candidate to be used in bone regeneration applications due its effective osteogenic properties and biocompatibility.

Efficacy of novel synthetic bone substitutes in the reconstruction of large segmental bone defects in sheep tibiae.

The treatment of large bone defects, particularly those with segmental bone loss, remains a significant clinical challenge as current approaches involving surgery or bone grafting often do not yield satisfactory long-term outcomes. This study reports the evaluation of novel ceramic scaffolds applied as bone graft substitutes in a clinically relevant in vivo model. Baghdadite scaffolds, unmodified or modified with a polycaprolactone coating containing bioactive glass nanoparticles, were implanted into critical-sized segmental bone defects in sheep tibiae for 26 weeks. Radiographic, biomechanical, µ-CT and histological analyses showed that both unmodified and modified baghdadite scaffolds were able to withstand physiological loads at the defect site, and induced substantial bone formation in the absence of supplementation with cells or growth factors. Notably, all samples showed significant bridging of the critical-sized defect (average 80%) with evidence of bone infiltration and remodelling within the scaffold implant. The unmodified and modified baghdadite scaffolds achieved similar outcomes of defect repair, although the latter may have an initial mechanical advantage due to the nanocomposite coating. The baghdadite scaffolds evaluated in this study hold potential for use as purely synthetic bone graft substitutes in the treatment of large bone defects while circumventing the drawbacks of autografts and allografts.

Design and Fabrication of 3D printed Scaffolds with a Mechanical Strength Comparable to Cortical Bone to Repair Large Bone Defects.

A challenge in regenerating large bone defects under load is to create scaffolds with large and interconnected pores while providing a compressive strength comparable to cortical bone (100-150 MPa). Here we design a novel hexagonal architecture for a glass-ceramic scaffold to fabricate an anisotropic, highly porous three dimensional scaffolds with a compressive strength of 110 MPa. Scaffolds with hexagonal design demonstrated a high fatigue resistance (1,000,000 cycles at 1-10 MPa compressive cyclic load), failure reliability and flexural strength (30 MPa) compared with those for conventional architecture. The obtained strength is 150 times greater than values reported for polymeric and composite scaffolds and 5 times greater than reported values for ceramic and glass scaffolds at similar porosity. These scaffolds open avenues for treatment of load bearing bone defects in orthopaedic, dental and maxillofacial applications.

A biphasic scaffold based on silk and bioactive ceramic with stratified properties for osteochondral tissue regeneration.

Significant clinical challenges encountered in the effective long-term treatment of osteochondral defects have inspired advancements in scaffold-based tissue engineering techniques to aid repair and regeneration. This study reports the development of a biphasic scaffold produced via a rational combination of silk fibroin and bioactive ceramic with stratified properties to satisfy the complex and diverse regenerative requirements of osteochondral tissue. Structural examination showed that the biphasic scaffold contained two phases with different pore morphologies to match the cartilage and bone segments of osteochondral tissue, which were joined at a continuous interface. Mechanical assessment showed that the two phases of the biphasic scaffold imitated the load-bearing behaviour of native osteochondral tissue and matched its compressive properties. In vitro testing showed that different compositions in the two phases of the biphasic scaffold could direct the preferential differentiation of human mesenchymal stem cells towards the chondrogenic or osteogenic lineage. By featuring simple and reproducible fabrication and a well-integrated interface, the biphasic scaffold strategy established in this study circumvented the common problems experienced with integrated scaffold designs and could provide an effective approach for the regeneration of osteochondral tissue.

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.

Injectable radiopaque and bioactive polycaprolactone-ceramic composites for orthopedic augmentation.

The aim of this study was to develop and characterize an injectable bone void filler by incorporating baghdadite (Ca3 ZrSi2 O9 ) particles (average size of 1.7 µm) into polycaprolactone (PCL). A series of PCL composites containing different volume percentages of baghdadite [1 (PCL-1%Bag), 5 (PCL-5%Bag), 10 (PCL-10%Bag), 20 (PCL-20%Bag), and 30 (PCL-30%Bag)] were prepared, and their injectability, setting time, mechanical properties, radiopacity, degradation, and cytocompatibility were investigated. PCL, PCL-1%Bag, PCL-5%Bag, and PCL-10%Bag were able to be injected through a stainless steel syringe (Length: 9.0 mm, nozzle diameter: 2.2 mm) at 75°C at injection forces of below 1.5 kN. The core temperature of the injected material at the nozzle exit ranged between 55 and 60°C and was shown to set after 2.5-3.5 min postinjection in a 37°C environment. Injection force, melt viscosity, and radiopacity of the composites increased with increasing baghdadite content. Incorporation of 10-30 vol % baghdadite into PCL increased the compressive strength of the composites from 36 to 47.1 MPa, compared with that for pure PCL (31.4 MPa). Similar trend was found for the compressive modulus of the composites, which increased from 203.8 to 741 MPa, compared with that for pure PCL (205 MPa). Flexural strain of PCL, PCL-5%Bag, and PCL-10%Bag exceeded 30%, and PCL-10%Bag showed the highest flexural strength (29.8 MPa). Primary human osteoblasts cultured on PCL-10%Bag showed a significant upregulation of osteogenic genes compared with pure PCL. In summary, our results demonstrated that PCL-10%Bag could be a promising injectable material for orthopedic and trauma application.

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/ß-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 ß1-integrin and induction of Wnt-3a autocrine signalling pathways by nBG incorporated scaffold.

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 0.1MHz 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.

Nanomaterials: the next step in injectable bone cements.

Injectable bone cements (IBCs) are biocompatible materials that can be used as bone defect fillers in maxillofacial surgeries and in orthopedic fracture treatment in order to augment weakened bone due to osteoporosis. Current clinically available IBCs, such as polymethylmethacrylate and calcium phosphate cement, have certain advantages; however, they possess several drawbacks that prevent them from gaining universal acceptance. New gel-based injectable materials have also been developed, but these are too mechanically weak for load-bearing applications. Recent research has focused on improving various injectable materials using nanomaterials in order to render them suitable for bone tissue regeneration. This article outlines the requirements of IBCs, the advantages and limitations of currently available IBCs and the state-of-the-art developments that have demonstrated the effects of nanomaterials within injectable systems.

Mimicking bone microenvironment for directing adipose tissue-derived mesenchymal stem cells into osteogenic differentiation.

Adipose tissue-derived mesenchymal stem cells (ASCs) have become an increasingly interested cell source for the scientists in the fields of stem cell biology and regenerative medicine. ASCs have already been used in a number of clinical trials, and some successful outcomes have been reported in bone tissue regeneration. Here we describe the protocols which mimic the factors in bone healing microenvironment, including inflammation burst, osteoblasts, and bone biomimetic scaffolds to direct ASCs into osteogenic differentiation.

Fabrication of a novel triphasic and bioactive ceramic and evaluation of its in vitro and in vivo cytocompatibility and osteogenesis.

We report, for the first time, the synthesis of a novel triphasic and crystalline bioactive ceramic (MSM-10) with the ability to simultaneously release three types of bioactive ions (strontium (Sr), silicon (Si) and magnesium (Mg)) to the surrounding microenvironment. An MSM-10 powder with a nominal composition (wt%) of 54 Mg2SiO4, 36 Si3Sr5 and 10 MgO was prepared by the sol-gel method and fabricated as porous scaffolds using the foam replication method. The effects of the different amounts of the phases in the ceramics on the mechanical and physical properties of the scaffolds as well as their in vitro and in vivo behaviors were comprehensively investigated. Biphasic calcium phosphate (BCP, ß-tricalcium phosphate (60 wt%)/hydroxyapatite (40 wt%)) scaffolds were used as the control material. The attachment, morphology, proliferation and differentiation of primary human osteoblasts (HOBs) were investigated after cell culturing on the various scaffolds. In vitro cytotoxicity (ISO/EN 10993-5) results not only indicated the biocompatibility of MSM-10, but also its positive effects on inducing the proliferation of HOBs. Our results showed significant enhancement in osteogenic gene expression levels (Runx2, osteocalcin, osteopontin and bone sialoprotein), when HOBs were cultured on MSM-10, compared to those for BCP and other generated ceramic scaffolds. For the in vivo studies, the different types of the materials were seeded with cultured human mesenchymal stem cells (hMSC) and then subcutaneously transplanted into the dorsal surface of eight-week-old immunocompromised (NOD/SCID) mice. MSM-10 demonstrated a significant amount of new bone formation compared to the other groups tested with no macroscopic signs of inflammation or toxicity in the tissue surrounding the implants. The novel MSM-10 ceramic presents promising potential for bone regeneration in orthopaedic and maxillofacial applications.

Multiple silk coatings on biphasic calcium phosphate scaffolds: effect on physical and mechanical properties and in vitro osteogenic response of human mesenchymal stem cells.

Ceramic scaffolds such as biphasic calcium phosphate (BCP) have been widely studied and used for bone regeneration, but their brittleness and low mechanical strength are major drawbacks. We report the first systematic study on the effect of silk coating in improving the mechanical and biological properties of BCP scaffolds, including (1) optimization of the silk coating process by investigating multiple coatings, and (2) in vitro evaluation of the osteogenic response of human mesenchymal stem cells (hMSCs) on the coated scaffolds. Our results show that multiple silk coatings on BCP ceramic scaffolds can achieve a significant coating effect to approach the mechanical properties of native bone tissue and positively influence osteogenesis by hMSCs over an extended period. The silk coating method developed in this study represents a simple yet effective means of reinforcement that can be applied to other types of ceramic scaffolds with similar microstructure to improve osteogenic outcomes.