Theta-Gel-Reinforced Hydrogel Composites for Potential Tensile Load-Bearing Soft Tissue Repair Applications.

Engineering synthetic hydrogels for the repair and augmentation of load-bearing soft tissues with simultaneously high-water content and mechanical strength is a long-standing challenge. Prior formulations to enhance the strength have involved using chemical crosslinkers where residues remain a risk for implantation or complex processes such as freeze-casting and self-assembly, requiring specialised equipment and technical expertise to manufacture reliably. In this study, we report for the first time that the tensile strength of high-water content (>60 wt.%), biocompatible polyvinyl alcohol hydrogels can exceed 1.0 MPa through a combination of facile manufacturing strategies via physical crosslinking, mechanical drawing, post-fabrication freeze drying, and deliberate hierarchical design. It is anticipated that the findings in this paper can also be used in conjunction with other strategies to enhance the mechanical properties of hydrogel platforms in the design and construction of synthetic grafts for load-bearing soft tissues.

Low-Temperature Synthesis of Hollow ß-Tricalcium Phosphate Particles for Bone Tissue Engineering Applications.

ß-Tricalcium phosphate (ß-TCP) has been extensively used in bone tissue engineering in the form of scaffolds, granules, or as reinforcing phase in organic matrices. Solid-state reaction route at high temperatures (>1000 °C) is the most widely used method for the preparation of ß-TCP. The high-temperature synthesis, however, results in the formation of hard agglomerates and fused particles which necessitates postprocessing steps such as milling and sieving operations. This, inadvertently, could lead to introducing unwanted trace elements, promoting particle shape irregularity as well as compromising the biodegradability and bioactivity of ß-TCP because of the solid microstructure of particles. In this study, we introduce a one-pot wet-chemical method at low temperatures (between 160 and 170 °C) to synthesize hollow ß-TCP (hß-TCP) submicron particles of an average size of 300 nm with a uniform rhombohedral shape. We assessed the cytocompatibility of the hß-TCP using primary human osteoblasts (HOB), adipose-derived stem cells (ADSC), and antigen-presenting cells (APCs). We demonstrate the bioactivity of the hß-TCP when cultured with HOB, ADSC, and APCs at a range of particle concentrations (up to 1000 µg/mL) for up to 7 days. hß-TCP significantly enhances osteogenic differentiation of ADSC without the addition of osteogenic supplements. These findings offer a new type of ß-TCP particles prepared at low temperatures, which present various opportunities for developing ß-TCP based biomaterials.

Development of a bioactive and radiopaque bismuth doped baghdadite ceramic for bone tissue engineering.

Baghdadite (Ca3ZrSi2O9, BAG), is a Zr-doped calcium silicate that has outstanding bioactivity both in vitro and in vivo. Bioceramic scaffolds should be sufficiently radiopaque to be distinguishable in vivo from surrounding bone structures. To enhance the radiopacity of BAG, this study investigated the effect of incorporating bismuth ions into its crystalline structure (BixCa3-xZrSi2O9, x = 0, 0.1, 0.2, 0.5; BAG, Bi0.1-BAG, Bi0.2-BAG, Bi0.5-BAG, respectively). Monophasic baghdadite was retained after bismuth ion incorporation up to x = 0.2 at calcination temperatures of 1350 °C. When pressed and sintered, energy dispersive x-ray spectroscopy showed that BAG and Bi0.1-BAG retained crystalline homogeneity, but Bi0.2-BAG formed zirconium-rich crystalline regions. BAG, Bi0.1-BAG and Bi0.2-BAG exhibited non-degradation after 56 days of immersion in culture medium. Bi0.1-BAG exhibited the lowest change in culture medium pH (+0.0), compared to BAG (+0.7) and Bi0.2-BAG (+0.2) after 56 days of culture media immersion. Bi0.1-BAG exhibited similar strength and modulus to BAG (σ: 200-290 MPa; E: 4-5 GPa), and significantly higher compressive strength and modulus versus Bi0.2-BAG (σ: 150-200 MPa; E: 3.5-4 GPa) across 56 days of aqueous immersion. In vitro studies using primary human bone derived cells (HOBs) demonstrated a significant increase in HOBs proliferation when cultured on Bi0.1-BAG for seven days compared to BAG and Bi0.2-BAG. Importantly, Bi0.1-BAG showed increased radiopacity by ~33%, when compared to BAG, and by ~115% when compared to biphasic calcium phosphate. The properties of Bi0.1-BAG show promise for its use as a bioactive ceramic with sufficient radiopacity for treatment of bone defects.

Nature-inspired topographies on hydroxyapatite surfaces regulate stem cells behaviour.

Surface topography is one of the key factors in regulating interactions between materials and cells. While topographies presented to cells in vivo are non-symmetrical and in complex shapes, current fabrication techniques are limited to replicate these complex geometries. In this study, we developed a microcasting technique and successfully produced imprinted hydroxyapatite (HAp) surfaces with nature-inspired (honeycomb, pillars, and isolated islands) topographies. The in vitro biological performance of the developed non-symmetrical topographies was evaluated using adipose-derived stem cells (ADSCs). We demonstrated that ADSCs cultured on all HAp surfaces, except honeycomb patterns, presented well-defined stress fibers and expressed focal adhesion protein (paxillin) molecules. Isolated islands topographies significantly promoted osteogenic differentiation of ADSCs with increased alkaline phosphatase activity and upregulation of key osteogenic markers, compared to the other topographies and the control unmodified (flat) HAp surface. In contrast, honeycomb topographies hampered the ability of the ADSCs to proliferate and differentiate to the osteogenic lineage. This work presents a facile technique to imprint nature-derived topographies on the surface of bioceramics which opens up opportunities for the development of bioresponsive interfaces in tissue engineering and regenerative medicine.

Baghdadite Ceramics Prevent Senescence in Human Osteoblasts and Promote Bone Regeneration in Aged Rats.

Bone fractures and critical-sized bone defects present significant health threats for the elderly who have limited capacity for regeneration due to the presence of functionally compromised senescent cells. A wide range of synthetic materials has been developed to promote the regeneration of critical-sized bone defects, but it is largely unknown if a synthetic biomaterial (scaffold) can modulate cellular senescence and improve bone regeneration in aged scenarios. The current study investigates the interaction of Baghdadite (Ca3ZrSi2O9) ceramic scaffolds with senescent human primary osteoblast-like cells (HOBs) and its bone regeneration capacity in aged rats. A senescent HOB model was established by repeatedly passaging HOBs till passage 7 (P7). Compared to the clinically used hydroxyapatite/tricalcium phosphate (HA/TCP), Baghdadite prevented senescence induction in P7 HOBs and markedly negated the paracrine effect of P7 HOB secretomes that mediated the up-regulations of cellular senescence-associated gene expression levels in P2 HOBs. We further demonstrated that conditioned media extracted from Baghdadite corrected the dysfunctional mitochondria in P7 HOBs. In vivo, the bone regeneration capacity was enhanced when 3D printed Baghdadite scaffolds were implanted in a calvaria critical-sized bone defect model in both young and aged rats compared to HA/TCP scaffolds, but a better effect was observed in aged rats than in young rats. This study suggests that Baghdadite ceramic represents a novel and promising biomaterial approach to promote bone regeneration capacity in the elderly by providing an anti-senescent microenvironment.

High-Strength Fiber-Reinforced Composite Hydrogel Scaffolds as Biosynthetic Tendon Graft Material.

The development of suitable synthetic scaffolds for use as human tendon grafts to repair tendon ruptures remains a significant engineering challenge. Previous synthetic tendon grafts have demonstrated suboptimal tissue ingrowth and synovitis due to wear particles from fiber-to-fiber abrasion. In this study, we present a novel fiber-reinforced hydrogel (FRH) that mimics the hierarchical structure of the native human tendon for synthetic tendon graft material. Ultrahigh molecular weight polyethylene (UHMWPE) fibers were impregnated with either biosynthetic polyvinyl alcohol/gelatin hydrogel (FRH-PG) or with polyvinyl alcohol/gelatin + strontium-hardystonite (Sr-Ca2ZnSi2O7, Sr-HT) composite hydrogel (FRH-PGS). The scaffolds were fabricated and assessed to evaluate their suitability for tendon graft applications. The microstructure of both FRH-PG and FRH-PGS showed successful impregnation of the hydrogel component, and the tendon scaffolds exhibited equilibrium water content of ∼70 wt %, similar to the values reported for native human tendon, compared to ∼50 wt % water content retained in unmodified UHMWPE fibers. The tensile strength of FRH-PG and FRH-PGS (77.0-81.8 MPa) matched the range of human Achilles' tendon tensile strengths reported in the literature. In vitro culture of rat tendon stem cells showed cell and tissue infiltration into both FRH-PG and FRH-PGS after 2 weeks, and the presence of Sr-HT ceramic particles influenced the expression of tenogenic markers. On the other hand, FRH-PG supported the proliferation of murine C2C12 myoblasts, whereas FRH-PGS seemingly did not support it under static culture conditions. In vivo implantation of FRH-PG and FRH-PGS scaffolds into full-thickness rat patellar tendon defects showed good collagenous tissue ingrowth into these scaffolds after 6 weeks. This study demonstrates the potential viability for our FRH-PG and FRH-PGS scaffolds to be used for off-the-shelf biosynthetic tendon graft material.

Role of Biomaterials and Controlled Architecture on Tendon/Ligament Repair and Regeneration.

Engineering synthetic scaffolds to repair and regenerate ruptured native tendon and ligament (T/L) tissues is a significant engineering challenge due to the need to satisfy both the unique biological and biomechanical properties of these tissues. Long-term clinical outcomes of synthetic scaffolds relying solely on high uniaxial tensile strength are poor with high rates of implant rupture and synovitis. Ideal biomaterials for T/L repair and regeneration need to possess the appropriate biological and biomechanical properties necessary for the successful repair and regeneration of ruptured tendon and ligament tissues.

Effect of Baghdadite Substitution on the Physicochemical Properties of Brushite Cements.

Brushite cements have been clinically used for irregular bone defect filling applications, and various strategies have been previously reported to modify and improve their physicochemical properties such as strength and injectability. However, strategies to address other limitations of brushite cements such as low radiopacity or acidity without negatively impacting mechanical strength have not yet been reported. In this study, we report the effect of substituting the beta-tricalcium phosphate reactant in brushite cement with baghdadite (Ca3ZrSi2O9), a bioactive zirconium-doped calcium silicate ceramic, at various concentrations (0, 5, 10, 20, 30, 50, and 100 wt%) on the properties of the final brushite cement product. X-ray diffraction profiles indicate the dissolution of baghdadite during the cement reaction, without affecting the crystal structure of the precipitated brushite. EDX analysis shows that calcium is homogeneously distributed within the cement matrix, while zirconium and silicon form cluster-like aggregates with sizes ranging from few microns to more than 50 µm. X-ray images and µ-CT analysis indicate enhanced radiopacity with increased incorporation of baghdadite into brushite cement, with nearly a doubling of the aluminium equivalent thickness at 50 wt% baghdadite substitution. At the same time, compressive strength of brushite cement increased from 12.9 ± 3.1 MPa to 21.1 ± 4.1 MPa with 10 wt% baghdadite substitution. Culture medium conditioned with powdered brushite cement approached closer to physiological pH values when the cement is incorporated with increasing amounts of baghdadite (pH = 6.47 for pure brushite, pH = 7.02 for brushite with 20 wt% baghdadite substitution). Baghdadite substitution also influenced the ionic content in the culture medium, and subsequently affected the proliferative activity of primary human osteoblasts in vitro. This study indicates that baghdadite is a beneficial additive to enhance the radiopacity, mechanical performance and cytocompatibility of brushite cements.

Novel injectable strontium-hardystonite phosphate cement for cancellous bone filling applications.

Injectable bone cement (IBC) such as those based on methacrylates and hydraulic calcium phosphate and calcium sulfate-based cements have been used extensively for filling bone defects with acceptable clinical outcomes. There is a need however for novel IBC materials that can address some of the inherent limitations of currently available formulations to widen the clinical application of IBC. In this study, we characterized a novel hydraulic IBC formulation consisting of bioactive strontium-doped hardystonite (Sr-HT) ceramic microparticles and sodium dihydrogen phosphate, herein named Sr-HT phosphate cement (SPC). The resultant cement is comprised of two distinct amorphous phases with embedded partially reacted crystalline reactants. The novel SPC formulation possesses a unique combination of physicochemical properties suitable for use as an IBC, and demonstrates in vitro cytocompatibility when seeded with primary human osteoblasts. In vivo injection of SPC into rabbit sinus defects show minor new bone formation at the SPC periphery, similar to those exhibited in sinus defects filled with a clinically available calcium phosphate cement. The current SPC formulation presented in this paper shows promise as a clinically applicable IBC which can be further enhanced with additives.

Modulatory effect of simultaneously released magnesium, strontium, and silicon ions on injectable silk hydrogels for bone regeneration.

Injectable silk hydrogels are ideal carriers of therapeutic agents due to their biocompatibility and low immunogenicity. Injectable silk hydrogels for bone regeneration have been previously developed but often utilize expensive biologics. In this study, we have developed an injectable silk composite incorporated with a triphasic ceramic called MSM-10 (54 Mg2SiO4, 36 Si3Sr5 and 10 MgO (wt%)) capable of simultaneously releasing magnesium, silicon, and strontium ions into its environment. These ions have been previously reported to possess therapeutic effects for bone regeneration. MSM-10 particles were incorporated into the silk hydrogels at various weight percentages [0.1 (SMH-0.1), 0.6 (SMH-0.6), 1 (SMH-1) and 2 (SMH-2)]. The effects of the released ions on the physicochemical and biological properties of the silk hydrogel were comprehensively evaluated. Increased MSM-10 loading was found to hinder the gelation kinetics of the silk hydrogel through the reduction of beta-sheet phase formation, which in turn affected the required sonication time for gelation, compressive strength, force of injection, microstructure and in vitro degradation rate. Primary human osteoblasts seeded on SMH-0.6 demonstrated increased proliferation and early alkaline phosphatase activity, as well as enhanced osteogenic gene expression compared to pure silk hydrogel and SMH-0.1. In vivo results in subcutaneous mouse models showed both decreased fibrous capsule formation and increased number of new blood vessels around the injected SMH-0.1 and SMH-0.6 implants compared to pure silk hydrogels. The results in this study indicate that the ions released from MSM-10 is able to influence the physicochemical and biological properties of silk hydrogels, and SMH-0.6 in particular shows promising properties for bone regeneration.

Doped Calcium Silicate Ceramics: A New Class of Candidates for Synthetic Bone Substitutes.

Doped calcium silicate ceramics (DCSCs) have recently gained immense interest as a new class of candidates for the treatment of bone defects. Although calcium phosphates and bioactive glasses have remained the mainstream of ceramic bone substitutes, their clinical use is limited by suboptimal mechanical properties. DCSCs are a class of calcium silicate ceramics which are developed through the ionic substitution of calcium ions, the incorporation of metal oxides into the base binary xCaO-ySiO2 system, or a combination of both. Due to their unique compositions and ability to release bioactive ions, DCSCs exhibit enhanced mechanical and biological properties. Such characteristics offer significant advantages over existing ceramic bone substitutes, and underline the future potential of adopting DCSCs for clinical use in bone reconstruction to produce improved outcomes. This review will discuss the effects of different dopant elements and oxides on the characteristics of DCSCs for applications in bone repair, including mechanical properties, degradation and ion release characteristics, radiopacity, and biological activity (in vitro and in vivo). Recent advances in the development of DCSCs for broader clinical applications will also be discussed, including DCSC composites, coated DCSC scaffolds and DCSC-coated metal implants.

Strontium-doped calcium silicate bioceramic with enhanced in vitro osteogenic properties.

Gehlenite (GLN, Ca2SiAl2O7) is a bioceramic that has been recently shown to possess excellent mechanical strength and in vitro osteogenic properties for bone regeneration. Substitutional incorporation of strontium in place of calcium is an effective way to further enhance biological properties of calcium-based bioceramics and glasses. However, such strategy has the potential to affect other important physicochemical parameters such as strength and degradation due to differences in the ionic radius of strontium and calcium. This study is the first to investigate the effect of a range of concentrations of strontium substitution of calcium at 1, 2, 5, 10 mol% (S1-GLN, S2-GLN, S5-GLN and S10-GLN) on the physicochemical and biological properties of GLN. We showed that up to 2 mol% strontium ion substitution retains the monophasic GLN structure when sintered at 1450 °C, whereas higher concentrations resulted in presence of calcium silicate impurities. Increased strontium incorporation resulted in changes in grain morphology and reduced densification when the ceramics were sintered at 1450 °C. Porous GLN, S1-GLN and S2-GLN scaffolds (∼80% porosity) showed compressive strengths of 2.05 ± 0.46 MPa, 1.76 ± 0.79 MPa and 1.57 ± 0.52 MPa respectively. S1-GLN and S2-GLN immersed in simulated body fluid showed increased strontium ion release but reduced calcium and silicon ion release compared to GLN without affecting overall weight loss and pH over a 21 d period. The bioactivity of the S2-GLN ceramics was significantly improved as reflected in the significant upregulation of HOB proliferation and differentiation compared to GLN. Overall, these results suggest that increased incorporation of strontium presents a trade-off between bioactivity and mechanical strength for GLN bioceramics. This is an important consideration in the development of strontium-doped bioceramics.

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.

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.

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.