Trabecular bone organoids: a micron-scale 'humanised' prototype designed to study the effects of microgravity and degeneration.

Bone is a highly responsive organ, which continuously adapts to the environment it is subjected to in order to withstand metabolic demands. These events are difficult to study in this particular tissue in vivo, due to its rigid, mineralised structure and inaccessibility of the cellular component located within. This manuscript presents the development of a micron-scale bone organoid prototype, a concept that can allow the study of bone processes at the cell-tissue interface. The model is constructed with a combination of primary female osteoblastic and osteoclastic cells, seeded onto femoral head micro-trabeculae, where they recapitulate relevant phenotypes and functions. Subsequently, constructs are inserted into a simulated microgravity bioreactor (NASA-Synthecon) to model a pathological state of reduced mechanical stimulation. In these constructs, we detected osteoclastic bone resorption sites, which were different in morphology in the simulated microgravity group compared to static controls. Once encapsulated in human fibrin and exposed to analogue microgravity for 5 days, masses of bone can be observed being lost from the initial structure, allowing to simulate the bone loss process further. Constructs can function as multicellular, organotypic units. Large osteocytic projections and tubular structures develop from the initial construct into the matrix at the millimetre scale. Micron-level fragments from the initial bone structure are detected travelling along these tubules and carried to sites distant from the native structure, where new matrix formation is initiated. We believe this model allows the study of fine-level physiological processes, which can shed light into pathological bone loss and imbalances in bone remodelling.

Formulation of a reactive oxygen producing calcium sulphate cement as an anti-bacterial hard tissue scaffold.

Prophylactic antibiotic bone cements are extensively used in orthopaedics. However, the development of antimicrobial resistance to antibiotics, demonstrates a need to find alternative treatments. Herein, an antimicrobial honey (SurgihoneyRO-SHRO) has been successfully incorporated into a calcium sulphate (CS) based cement to produce a hard tissue scaffold with the ability to inhibit bacterial growth. Antimicrobial properties elicited from SHRO are predominantly owed to the water-initiated production of reactive oxygen species (ROS). As an alternative to initially loading CS cement with SHRO, in order to prevent premature activation, SHRO was added into the already developing cement matrix, locking available water into the CS crystal structure before SHRO addition. Promisingly, this methodology produced > 2.5 times (715.0 ± 147.3 µM/mL/g) more ROS over 24 h and exhibited a compressive strength (32.2 ± 5.8 MPa) comparable to trabecular bone after 3 weeks of immersion. In-vitro the SHRO loaded CS scaffolds were shown to inhibit growth of clinically relevant organisms, Staphylococcus aureus and Pseudomonas aeruginosa, with comparable potency to equivalent doses of gentamicin. Encouragingly, formulations did not inhibit wound healing or induce an inflammatory response from osteoblasts. Overall this study highlights the prophylactic potential of CS-SHRO cements as an alternative to traditional antibiotics.

Controlled self-assembly of chemical gardens enables fabrication of heterogeneous chemobrionic materials.

Chemical gardens are an example of a chemobrionic system that typically result in abiotic macro-, micro- and nano- material architectures, with formation driven by complex out-of-equilibrium reaction mechanisms. From a technological perspective, controlling chemobrionic processes may hold great promise for the creation of novel, compositionally diverse and ultimately, useful materials and devices. In this work, we engineer an innovative custom-built liquid exchange unit that enables us to control the formation of tubular chemical garden structures grown from the interface between calcium loaded hydrogel and phosphate solution. We show that systematic displacement of phosphate solution with water (H2O) can halt self-assembly, precisely control tube height and purify structures in situ. Furthermore, we demonstrate the fabrication of a heterogeneous chemobrionic composite material composed of aligned, high-aspect ratio calcium phosphate channels running through an otherwise dense matrix of poly(2-hydroxyethyl methacrylate) (pHEMA). Given that the principles we derive can be broadly applied to potentially control various chemobrionic systems, this work paves the way for fabricating multifunctional materials that may hold great potential in a variety of application areas, such as regenerative medicine, catalysis and microfluidics.

Formulation of an antimicrobial superabsorbent powder that gels in situ to produce reactive oxygen.

The enzymatic oxidation of glucose to produce reactive oxygen species (ROS) provides honey with antimicrobial efficacy. This mechanism offers an alternative to traditional antibiotics; however, topical use of honey is limited due to its adherent and highly viscous properties. This study aims to overcome these issues by engineering a powder-based system that eases delivery and offers in situ activation of ROS. Starch based drying agents were utilised to enable freeze drying of a medical honey, with methylated-ß-cyclodextrin (MCD) enabling the highest active incorporation (70%) while still producing a free-flowing powder. Addition of a superabsorbent, sodium polyacrylate (≤40%) was shown to facilitate in situ gelation of the powder, with an absorption capacity of up to 120.7 ± 4.5 mL g-1. Promisingly efficacy of the optimised superabsorbent powder was demonstrated in vitro against several clinically relevant Gram-negative and Gram-positive bacteria (Staphylococcus aureus, Escherichia coli and Pseudomonas aeruginosa). Alongside this no adverse effects were observed against human dermal fibroblasts. Application of the superabsorbent powder in an ex-vivo porcine wound model revealed capability to form a protective hydrogel barrier in less than 1 min. Overall, this novel ROS producing superabsorbent powder has potential to tackle topical infections without using traditional antibiotics.

The Quantification of Injectability by Mechanical Testing.

Injectable biomaterials are becoming increasingly popular for the minimally invasive delivery of drugs and cells. These materials are typically more viscous than traditional aqueous injections and may be semi-solid, therefore, their injectability cannot be assumed. This protocol describes a method to objectively assess the injectability of these materials using a standard mechanical tester. The syringe plunger is compressed by the crosshead at a set rate, and the force is measured. The maximum or plateau force value can then be used for comparison between samples, or to an absolute force limit. This protocol can be used with any material, and any syringe and needle size or geometry. The results obtained may be used to make decisions about formulations, syringe and needle sizes early in the translational process. Further, the effects of altering formulations on injectability may be quantified, and the optimum time to inject temporally changing materials determined. This method is also suitable as a reproducible way to examine the effects of injection on a material, to study phenomena such as self-healing and filter pressing or study the effects of injection on cells. This protocol is faster and more directly applicable to injectability than rotational rheology, and requires minimal post processing to obtain key values for direct comparisons.

Hexametaphosphate as a potential therapy for the dissolution and prevention of kidney stones.

The incidence of kidney stones is increasing worldwide, and recurrence is common (50% within 5 years). Citrate, the current gold standard therapy, which is usually given as potassium or sodium salts, is used because it raises urine pH and chelates calcium, the primary component of up to 94% of stones. In this study hexametaphosphate (HMP), a potent calcium chelator, was found to be 12 times more effective at dissolving calcium oxalate, the primary component of kidney stones, than citrate. HMP was also observed to be effective against other common kidney stone components, namely calcium phosphate and struvite (magnesium ammonium phosphate). Interestingly, HMP was capable of raising the zeta potential of calcium oxalate particles from -15.4 to -34.6 mV, which may prevent stone growth by aggregation, the most rapid growth mechanism, and thus avert occlusion. Notably, HMP was shown to be up to 16 times as effective as citrate at dissolving human kidney stones under simulated physiological conditions. It may thus be concluded that HMP is a promising potential therapy for calcium and struvite kidney stones.

Filling the Gap: A Correlation between Objective and Subjective Measures of Injectability.

Various injectable biomaterials are developed for the minimally invasive delivery of therapeutics. Typically, a mechanical tester is used to ascertain the force required to inject these biomaterials through a given syringe-needle system. However, currently there is no method to correlate the force measured in the laboratory to the perceived effort required to perform that injection by the end user. In this article, the injection force (F) for a variety of biomaterials, displaying a range of rheological properties, is compared with the effort scores from a 50 person panel study. The maximum injection force measured at crosshead speed 1 mm s-1 is a good proxy for injection effort, with an R2 of 0.89. This correlation leads to the following conclusions: participants can easily inject 5 mL of substance for F < 12 N; considerable effort is required to inject 5 mL for 12 N < F < 38 N; great effort is required and <5 mL can be injected for 38 N < F < 64 N; and materials are entirely non-injectable for F > 64 N. These values may be used by developers of injectable biomaterials to make decisions about formulations and needle sizes early in the translational process.

Chemobrionic structures in tissue engineering: self-assembling calcium phosphate tubes as cellular scaffolds.

A diverse range of complex patterns and mineralised hierarchical microstructures can be derived from chemobrionic systems, with formation driven by complex reaction-diffusion mechanisms far from thermodynamic equilibrium. In these experiments, self-assembling calcium phosphate tubes are generated using hydrogels made with 1 M calcium solutions layered with solutions of dibasic sodium phosphate over a range of concentrations between 0.2-1 M. Self-assembling structures prepared using 0.8 M dibasic sodium phosphate solutions were selected to assess cell-material interactions. Candidate chemobrionic scaffolds were characterised by micro-X-Ray fluorescence (µ-XRF) spectroscopy, Raman spectroscopy, powder X-ray diffraction (XRD), helium pycnometry and scanning electron microscopy (SEM). As prepared tubes were formed from non-stoichiometric hydroxyapatite (HA, Ca10-x(PO4)6-x(HPO4)x(OH)2-x (0 ≤x≤ 1)), which was confirmed as calcium deficient hydroxyapatite (CDHA, Ca9(PO4)5HPO4OH). Thermal treatment of tubes in air at 650 °C for 4 h converted the structures to beta tricalcium phosphate (ß-TCP, ß-Ca3(PO4)2). The potential of these scaffolds to support the attachment of bone marrow derived mesenchymal stem cells (BMSCs) was investigated for the first time, and we demonstrate cell attachment and elongation on the fabricated tubular structures.

Critical and diverse roles of phosphates in human bone formation.

Humans utilise biomineralisation in the formation of bone and teeth. Human biomineralisation processes are defined by the transformation of an amorphous phosphate-based precursor to highly organised nanocrystals. Interestingly, ionic phosphate species not only provide a fundamental building block of biological mineral, but rather exhibit several diverse roles in mediating mineral formation in the physiological milieu. In this review, we focus on elucidating the complex roles of phosphate ions and molecules within human biomineralisation pathways, primarily referring to the nucleation and crystallisation of bone mineral.

Formulation and viscoelasticity of mineralised hydrogels for use in bone-cartilage interfacial reconstruction.

Articular cartilage is a viscoelastic tissue whose structural integrity is important in maintaining joint health. To restore the functionality of osteoarthritic joints it is vital that regenerative strategies mimic the dynamic loading response of cartilage and bone. Here, a rotating simplex model was employed to optimise the composition of agarose and gellan hydrogel constructs structured with hydroxyapatite (HA) with the aim of obtaining composites mechanically comparable to human cartilage in terms of their ability to dissipate energy. Addition of ceramic particles was found to reinforce both matrices up to a critical concentration (< 3w/v%). Beyond this, larger agglomerates were formed, as evidenced by micro computed tomography data, which acted as stress risers and reduced the ability of composites to dissipate energy demonstrated by a reduction in tan δ values. A maximum compressive modulus of 450.7±24.9 kPa was achieved with a composition of 5.8w/v% agarose and 0.5w/v% HA. Interestingly, when loaded dynamically (1-20Hz) this optimised formulation did not exhibit the highest complex modulus instead a sample with a higher concentration of mineral was identified (5.8w/v% agarose and 25w/v% HA). Thus, demonstrating the importance of examining the mechanical behaviour of biomaterials under conditions representative of physiological environments. While the complex moduli of the optimised gellan (1.0 ± 0.2MPa at 1Hz) and agarose (1.7 ± 0.2MPa at 1Hz) constructs did not match the complex moduli of healthy human cartilage samples (26.3 ± 6.5MPa at 1Hz), similar tan δ values were observed between 1 and 5Hz. This is promising since these frequencies represent the typical heel strike time of the general population. In summary, this study demonstrates the importance of considering more than just the strength of biomaterials since tissues like cartilage play a more complex role.

Interfacial Mineral Fusion and Tubule Entanglement as a Means to Harden a Bone Augmentation Material.

A new bone augmenting material is reported, which is formed from calcium-loaded hydrogel-based spheres. On immersion of these spheres in a physiological medium, they become surrounded with a sheath of precipitate, which ruptures due to a build-up in osmotic pressure. This results in the formation of mineral tubes that protrude from the sphere surface. When brought into close contact with one another, these spheres become fused through the entanglement and subsequent interstitial mineralization of the mineral tubules. The tubular calcium phosphate induces the expression of osteogenic genes (runt-related transcription factor 2 (RUNX2), transcription factor SP7 (SP7), collagen type 1 alpha 1 (COL1A1), and bone gamma-carboxyglutamic acid-containing protein (BGLAP)) and promotes the formation of mineral nodules in preosteoblast cultures comparable to an apatitic calcium phosphate phase. Furthermore, alkaline phosphatase (ALP) is significantly upregulated in the presence of tubular materials after 10 d in culture compared with control groups (p < 0.001) and sintered apatite (p < 0.05). This is the first report of a bioceramic material that is formed in its entirety in situ and is therefore likely to provide a better proxy for biological mineral than other existing synthetic alternatives to bone grafts.

Characterisation of a novel poly (ether ether ketone)/calcium sulphate composite for bone augmentation.

BACKGROUND: Calcium sulphate (CS) has been used in bone grafting since the 1800s. It has not replaced autograft as the gold standard, however, since its dissolution occurs rapidly in bodily fluids, meaning that the material cannot support long-term bone growth. Here, the polymer poly (ether ether ketone) (PEEK) was used to slow dissolution in in vitro physiological environments and augment the mechanical properties of the material. METHODS: PEEK/CS specimens were fabricated by combining powders of PEEK and CS with water, resulting in a hardening paste. To enhance physical interactions between phases, cylindrical specimens were heat-treated to melt and fuse the PEEK. Following analysis of physical and chemical interactions by SEM and FT-IR respectively, dynamic ageing in PBS and compression testing was undertaken to measure how the PEEK influenced the mechanical properties of the final parts. Changes in structure and chemistry were determined using helium pycnometry, SEM and analysis of powder XRD patterns. RESULTS: Powders of PEEK and CS hemihydrate (CSH) (CaSO4.0.5H2O) were combined with PEEK at 0 wt%, 2.5 wt%, 20 wt%, 40 wt% and 80 wt% and at a P:L ratio of 0.85 g/mL. The subsequently hardened structures were heat-treated, which initiated the melting of PEEK and dehydration of CSD (CaSO4.2H2O) to the CS anhydrite (CSA) (CaSO4) phase, which changed colour and apparent volume. FT-IR and SEM analysis revealed heat treatment of PEEK/CS specimens facilitated both physical and chemical interactions between phases. Over a period of 21 days of ageing in PBS, the hydration of CS was determined by XRD and improved specimen longevity at all levels of PEEK wt% loading was measured compared with the control. Importantly, increasing PEEK wt% loading resulted in a marked increase in the mechanical properties of PEEK/CS specimens in terms of both compressive strength and modulus. CONCLUSIONS: Reinforcement of CS with PEEK significantly enhanced in vitro dissolution resistance, in addition to enhancing mechanical properties. This composite therefore has significant future potential as a bone graft replacement.

Biologically Analogous Calcium Phosphate Tubes from a Chemical Garden.

Calcium phosphate (CaPO4) tubes with features comparable to mineralized biological microstructures, such as Haversian canals, were grown from a calcium gel/phosphate solution chemical garden system. A significant difference in gel mass in response to high and low solute phosphate equivalent environments existed within 30 min of solution layering upon gel (p = 0.0067), suggesting that the nature of advective movement between gel and solution is dependent on the solution concentration. The transport of calcium cations (Ca2+) and phosphate anions (PO43-) was quantified and changes in pH were monitored to explain the preferential formation of tubes within a PO43- concentration range of 0.5-1.25 M. Ingress from the anionic solution phase into the gel followed by the liberation of Ca2+ ions from the gel was found to be essential for acquiring self-assembled tubular CaPO4 structures. Tube analysis by scanning electron microscopy (SEM), X-ray diffraction (XRD), and micro X-ray florescence (µ-XRF) revealed hydroxyapatite (HA, Ca10(PO4)6(OH)2) and dicalcium phosphate dihydrate (DCPD, CaHPO4·2H2O) phases organized in a hierarchical manner. Notably, the tubule diameters ranged from 100 to 150 µm, an ideal size for the permeation of vasculature in biological hard tissue.