Evaluating the swelling, erosion, and compaction properties of cellulose ethers.

Swelling, erosion, deformation, and consolidation properties can affect the performance of cellulose ethers, the most commonly used matrix former in hydrophilic sustained tablet formulations. The present study was designed to comparatively evaluate the swelling, erosion, compression, compaction, and relaxation properties of the cellulose ethers in a comprehensive study using standardised conditions. The interrelationship between various compressional models and the inherent deformation and consolidation properties of the polymers on the derived swelling and erosion parameters are consolidated. The impact of swelling (Kw) on erosion rates (KE) and the inter-relationship between Heckel and Kawakita plasticity constants was also investigated. It is evident from the findings that the increases in both substitution and polymer chain length led to higher Kw, but a lower KE; this was also true for all particle size fractions regardless of polymer grade. Smaller particle size and high substitution levels tend to increase the relative density of the matrix but reduce porosity, yield pressure (Py), Kawakita plasticity parameter (b-1) and elastic relaxation. Both KW versus KE (R2 = 0.949-0.980) and Py versus. b-1 correlations (R2 = 0.820-0.934) were reasonably linear with regards to increasing hydroxypropyl substitution and molecular size. Hence, it can be concluded that the combined knowledge of swelling and erosion kinetics in tandem with the in- and out-of-die compression findings can be used to select a specific polymer grade and further to develop and optimize formulations for oral controlled drug delivery applications.

Low temperature aqueous precipitation of needle-like nanophase hydroxyapatite.

The use of tissue engineered biodegradable porous scaffolds has become an important focus of the biomedical research field. The precursor materials used to form these structures play a vital role in their overall performance thus making the study and synthesis of these selected materials imperative. The authors present a comparison and characterisation of hydroxyapatite (HA), a popular calcium phosphate (CaP) biomaterial, synthesised by an aqueous precipitation (AP) method. The influence of various reaction conditions on the phase, crystallinity, particle size as well as morphology, molecular structure, potential in-vivo bioactivity and cell viability were assessed by XRD, SEM and TEM, FTIR, a simulated body fluid (SBF) test and a live/dead assay using MC3T3 osteoblast precursor cells, respectively. Naturally carbonated nanoparticles of HA with typically needle-like morphology were synthesised by the reported AP method. Initial pH was found to influence the crystallisation process and determine the CaP phase formed as well as the resultant particle and crystallite sizes. A marked change in particle morphology was also observed above pH 9. The use of toluene as a replacement solvent for water up to 60% was found to reduce the crystallinity of as-synthesised HA. This has marked influence on the effect of ethanolamine (5 wt%), which was found to improve HA crystallinity. SEM and EDS were used to confirm the growth of carbonated apatite on the surface of HA pellets immersed in SBF for up to 28 days. Cell culture results revealed viable cells on all samples where pH was controlled and maintained at 10-11 during precipitation, including those that used ethanolamine and toluene in preparation. When the initial alkali pH was not maintained non-viable cells were observed on HA substrates.

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 °C and melting temperature of ~90 °C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Surface Free Energy Dominates the Biological Interactions of Postprocessed Additively Manufactured Ti-6Al-4V.

Additive manufacturing (AM) has emerged as a disruptive technique within healthcare because of its ability to provide personalized devices; however, printed metal parts still present surface and microstructural defects, which may compromise mechanical and biological interactions. This has made physical and/or chemical postprocessing techniques essential for metal AM devices, although limited fundamental knowledge is available on how alterations in physicochemical properties influence AM biological outcomes. For this purpose, herein, powder bed fusion Ti-6Al-4V samples were postprocessed with three industrially relevant techniques: polishing, passivation, and vibratory finishing. These surfaces were thoroughly characterized in terms of roughness, chemistry, wettability, surface free energy, and surface ζ-potential. A significant increase in Staphylococcus epidermidis colonization was observed on both polished and passivated samples, which was linked to high surface free energy donor γ- values in the acid-base, γAB component. Early osteoblast attachment and proliferation (24 h) were not influenced by these properties, although increased mineralization was observed for both these samples. In contrast, osteoblast differentiation on stainless steel was driven by a combination of roughness and chemistry. Collectively, this study highlights that surface free energy is a key driver between AM surfaces and cell interactions. In particular, while low acid-base components resulted in a desired reduction in S. epidermidis colonization, this was followed by reduced mineralization. Thus, while surface free energy can be used as a guide to AM device development, optimization of bacterial and mammalian cell interactions should be attained through a combination of different postprocessing techniques.

An In Vitro Study to Determine the Feasibility of Combining Bone Marrow Concentrate with BST-CarGel as a Treatment for Cartilage Repair.

OBJECTIVE: The study aims were to determine whether BST-CarGel, a chitosan scaffold for cartilage repair, can be mixed with bone marrow aspirate concentrate (BMAC) to create a cell seeded implant with comparative properties to standard BST-CarGel mixed with blood. DESIGN: Whole blood and bone marrow were harvested from 12 patients who underwent cartilage repair surgery using BMAC after informed consent. A validated in vitro testing model was used to assess the following 6 conditions: (1) BST-CarGel mixed with whole blood (CG-WB), (2) BST-CarGel mixed with bone marrow (CG-BM), (3) BST-CarGel mixed with bone marrow concentrate (CG-BMAC), (4) whole blood (WB), (5) bone marrow (BM), and (6) bone marrow concentrate and batroxobin (BMAC-BTX). Cell retention and viability within the BST-CarGel/BMAC clots were investigated. RESULTS: In our study, BM and BMAC (processed using the Harvest, SmartPrep2 system and reactivated with batroxibin) when combined with BST-CarGel produced a product that had similar clot contraction, macroscopic properties, and histological appearance to standard BSTCarGel mixed with blood. Mononucleated cells from the BMAC were retained within the scaffold and remained viable until clot dissolution in vitro. CONCLUSIONS: By combining BST-CarGel with BMAC in the manner described, bone marrow-derived mononucleated cells can be retained within the chondral defect potentially negating the need for microfracture. Further in vivo work is required to confirm these potential benefits and determine if this combination will result in more durable cartilage repair and improved clinical outcomes.

Formulation of a Composite Nasal Spray Enabling Enhanced Surface Coverage and Prophylaxis of SARS-COV-2.

Airborne pathogens pose high risks in terms of both contraction and transmission within the respiratory pathways, particularly the nasal region. However, there is little in the way of adequate intervention that can protect an individual or prevent further spread. This study reports on a nasal formulation with the capacity to combat such challenges, focusing on severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2). Formulation of a polysaccharide-based spray, known for its mucoadhesive properties, is undertaken and it is characterized for its mechanical, spray distribution, and antiviral properties. The ability to engineer key mechanical characteristics such as dynamic yield stresses and high coverage is shown, through systematic understanding of the composite mixture containing both gellan and λ-carrageenan. Furthermore, the spray systems demonstrate highly potent capacities to prevent SARS-CoV-2 infection in Vero cells, resulting in complete inhibition when either treating, the cells, or the virus, prior to challenging for infection. From this data, a mechanism for both prophylaxis and prevention is proposed; where entrapment within a polymeric coating sterically blocks virus uptake into the cells, inactivating the virus, and allowing clearance within the viscous medium. As such, a fully preventative spray is formulated, targeted at protecting the lining of the upper respiratory pathways against SARS-CoV-2.

Formulation of inherently antimicrobial magnesium oxychloride cement and the effect of supplementation with silver phosphate.

The growing threat of bacterial resistance to antibiotics is driving an increasing need for new antimicrobial strategies. This work demonstrates the potential of magnesium oxychloride cements (MOC) to be used as inorganic antimicrobial biomaterials for bone augmentation. An injectable formulation was identified at a powder to liquid ratio of 1.4 g mL-1, with an initial setting time below 30 mins and compressive strength of 35 ± 9 MPa. Supplementation with Ag3PO4 to enhance the antimicrobial efficacy of MOC was explored, and shown via real time X-ray diffraction to retard the formation of hydrated oxychloride phases by up to 30%. The antimicrobial efficacy of MOC was demonstrated in vitro against Staphylococcus aureus and Pseudomonas aeruginosa, forming zones of inhibition and significantly reducing viability in broth culture. Enhanced efficacy was seen for silver doped formulations, with complete eradication of detectable viable colonies within 3 h, whilst retaining the cytocompatibility of MOC. Investigating the antimicrobial mode of action revealed that Mg and Ag release and elevated pH contributed to MOC efficacy. Sustained silver release was demonstrated over 14 days, suggesting the Ag3PO4 modified formulation offers two mechanisms of infection treatment, combining the inherent antimicrobial properties of MOC with controlled release of inorganic antimicrobials.

Tailoring Therapeutic Responses via Engineering Microenvironments with a Novel Synthetic Fluid Gel.

This study reports the first fully synthetic fluid gel (SyMGels) using a simple poly(ethylene glycol) polymer. Fluid gels are an interesting class of materials: structured during gelation via shear-confinement to form microparticulate suspensions, through a bottom-up approach. Structuring in this way, when compared to first forming a gel and subsequently breaking it down, results in the formation of a particulate dispersion with particles "grown" in the shear flow. Resultantly, systems form a complex microstructure, where gelled particles concentrate remaining non-gelled polymer within the continuous phase, creating an amorphous-like interstitial phase. As such, these materials demonstrate mechanical characteristics typical of colloidal glasses, presenting solid-like behaviors at rest with defined yielding; likely through intrinsic particle-particle and particle-polymer interactions. To date, fluid gels have been fabricated using polysaccharides with relatively complex chemistries, making further modifications challenging. SyMGels are easily functionalised, using simple click-chemistry. This chemical flexibility, allows the creation of microenvironments with discrete biological decoration. Cellular control is demonstrated using MSC (mesenchymal stem cells)/chondrocytes and enables the regulation of key biomarkers such as aggrecan and SOX9. These potential therapeutic platforms demonstrate an important advancement in the biomaterial field, underpinning the mechanisms which drive their mechanical properties, and providing a versatile delivery system for advanced therapeutics.

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.

Cell encapsulation using biopolymer gels for regenerative medicine.

There has been a consistent increase in the mean life expectancy of the population of the developed world over the past century. Healthy life expectancy, however, has not increased concurrently. As a result we are living a larger proportion of our lives in poor health and there is a growing demand for the replacement of diseased and damaged tissues. While traditionally tissue grafts have functioned well for this purpose, the demand for tissue grafts now exceeds the supply. For this reason, research in regenerative medicine is rapidly expanding to cope with this new demand. There is now a trend towards supplying cells with a material in order to expedite the tissue healing process. Hydrogel encapsulation provides cells with a three dimensional environment similar to that experienced in vivo and therefore may allow the maintenance of normal cellular function in order to produce tissues similar to those found in the body. In this review we discuss biopolymeric gels that have been used for the encapsulation of mammalian cells for tissue engineering applications as well as a brief overview of cell encapsulation for therapeutic protein production. This review focuses on agarose, alginate, collagen, fibrin, hyaluronic acid and gelatin since they are widely used for cell encapsulation. The literature on the regeneration of cartilage, bone, ligament, tendon, skin, blood vessels and neural tissues using these materials has been summarised.

Reducing MRI susceptibility artefacts in implants using additively manufactured porous Ti-6Al-4V structures.

Magnetic Resonance Imaging (MRI) is critical in diagnosing post-operative complications following implant surgery and imaging anatomy adjacent to implants. Increasing field strengths and use of gradient-echo sequences have highlighted difficulties from susceptibility artefacts in scan data. Artefacts manifest around metal implants, including those made from titanium alloys, making detection of complications (e.g. bleeding, infection) difficult and hindering imaging of surrounding structures such as the brain or inner ear. Existing research focusses on post-processing and unorthodox scan sequences to better capture data around these devices. This study proposes a complementary up-stream design approach using lightweight structures produced via additive manufacturing (AM). Strategic implant mass reduction presents a potential tool in managing artefacts. Uniform specimens of Ti-6Al-4V structures, including lattices, were produced using the AM process, selective laser melting, with various unit cell designs and relative densities (3.1%-96.7%). Samples, submerged in water, were imaged in a 3T MRI system using clinically relevant sequences. Artefacts were quantified by image analysis revealing a strong linear relationship (RR2 = 0.99) between severity and relative sample density. Likewise, distortion due to slice selection errors showed a squared relationship (RR2 = 0.92) with sample density. Unique artefact features were identified surrounding honeycomb samples suggesting a complex relationship exists for larger unit cells. To demonstrate clinical utility, a honeycomb design was applied to a representative cranioplasty. Analysis revealed 10% artefact reduction compared to traditional solid material illustrating the feasibility of this approach. This study provides a basis to strategically design implants to reduce MRI artefacts and improve post-operative diagnosis capability. STATEMENT OF SIGNIFICANCE: MRI susceptibility artefacts surrounding metal implants present a clinical challenge for the diagnosis of post-operative complications relating to the implant itself or underlying anatomy. In this study for the first time we demonstrate that additive manufacturing may be exploited to create lattice structures that predictably reduce MRI image artefact severity surrounding titanium alloy implants. Specifically, a direct correlation of artefact severity, both total signal loss and distortion, with the relative material density of these functionalised materials has been demonstrated within clinically relevant MRI sequences. This approach opens the door for strategic implant design, utilising this structurally functionalised material, that may improve post-operative patient outcomes and compliments existing efforts in this area which focus on data acquisition and post-processing methods.

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.

Physical Structuring of Injectable Polymeric Systems to Controllably Deliver Nanosized Extracellular Vesicles.

Extracellular vesicles (EVs) are emerging as a promising alternative approach to cell-therapies. However, to realize the potential of these nanoparticles as new regenerative tools, healthcare materials that address the current limitations of systemic administration need to be developed. Here, two technologies for controlling the structure of alginate based microgel suspensions are used to develop sustained local release of EVs, in vitro. Microparticles formed using a shearing technique are compared to those manufactured using vibrational technology, resulting in either anisotropic sheet-like or spheroid particles, respectively. EVs harvested from preosteoblasts are isolated using differential ultracentrifugation and successfully loaded into the two systems, while maintaining their structures. Promisingly, in addition to exhibiting even EV distribution and high stability, controlled release of vesicles from both structures is exhibited, in vitro, over the 12 days studied. Interestingly, a significantly greater number of EVs are released from the suspensions formed by shearing (69.9 ± 10.5%), compared to the spheroids (35.1 ± 7.6%). Ultimately, alterations to the hydrogel physical structures have shown to tailor nanoparticle release while simultaneously providing ideal material characteristics for clinical injection. Thus, the sustained release mechanisms achieved through manipulating the formation of such biomaterials provide a key to unlocking the therapeutic potential held within EVs.

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.

Structuring of Hydrogels across Multiple Length Scales for Biomedical Applications.

The development of new materials for clinical use is limited by an onerous regulatory framework, which means that taking a completely new material into the clinic can make translation economically unfeasible. One way to get around this issue is to structure materials that are already approved by the regulator, such that they exhibit very distinct physical properties and can be used in a broader range of clinical applications. Here, the focus is on the structuring of soft materials at multiple length scales by modifying processing conditions. By applying shear to newly forming materials, it is possible to trigger molecular reorganization of polymer chains, such that they aggregate to form particles and ribbon-like structures. These structures then weakly interact at zero shear forming a solid-like material. The resulting self-healing network is of particular use for a range of different biomedical applications. How these materials are used to allow the delivery of therapeutic entities (cells and proteins) and as a support for additive layer manufacturing of larger-scale tissue constructs is discussed. This technology enables the development of a range of novel materials and structures for tissue augmentation and regeneration.

Dissolution of bio-active dentine matrix components by mineral trioxide aggregate.

OBJECTIVES: To analyze the soluble components of setting and set mineral trioxide aggregate (MTA), assess the abilities of two varieties of MTA and Ca(OH)(2) solutions to solubilise dentine matrix proteins (DMPs) and determine if these extracts contain signalling molecules important to pulpal repair and regeneration. METHODS: The metallic ion composition of solutions of white and grey MTA (pH 11.7), 0.02M Ca(OH)(2) (pH 11.9) and 10% EDTA (pH 7.2) was determined using atomic absorption spectroscopy. Extracellular dentine matrix components from powdered human dentine were extracted using all solutions over 14 days. Extracts were analysed for concentrations of non-collagenous proteins (NCPs) and glycosaminoglycans (GAGs), and protein profiles were examined using 1D-polyacrylamide gel electrophoresis (1D-PAGE). ELISAs for TGF-beta1 and adrenomedullin (ADM) were also performed. RESULTS: Aluminium, calcium, potassium, and sodium ions were detected in both white and grey MTA solutions. MTA and Ca(OH)(2) solutions liberated similar amounts of GAGs and NCPs although yields were considerably lower than those obtained using the EDTA solution. 1D-PAGE analysis demonstrated differences in protein profiles solubilised from dentine for all solutions. All extracts contained TGF-beta1 and ADM, EDTA solution liberated significantly greater amounts of TGF-beta1, and Ca(OH)(2) and grey MTA solutions released more ADM. CONCLUSIONS: These data imply that when placed clinically soluble components of set and setting MTA may release dentine matrix components that potentially influence cellular events for dentine repair and regeneration.

Suspended Manufacture of Biological Structures.

A method for the production of complex cell-laden structures is reported, which allows high-levels of spatial control over mechanical and chemical properties. The potential of this method for producing complicated tissues is demonstrated by manufacturing a complex hard/soft tissue interface and demonstrating that cell phenotype can be maintained over four weeks of culture.

Antimicrobial peptide coatings for hydroxyapatite: electrostatic and covalent attachment of antimicrobial peptides to surfaces.

The interface between implanted devices and their host tissue is complex and is often optimized for maximal integration and cell adhesion. However, this also gives a surface suitable for bacterial colonization. We have developed a novel method of modifying the surface at the material-tissue interface with an antimicrobial peptide (AMP) coating to allow cell attachment while inhibiting bacterial colonization. The technology reported here is a dual AMP coating. The dual coating consists of AMPs covalently bonded to the hydroxyapatite surface, followed by deposition of electrostatically bound AMPs. The dual approach gives an efficacious coating which is stable for over 12 months and can prevent colonization of the surface by both Gram-positive and Gram-negative bacteria.

Biologically mediated resorption of brushite cement in vitro.

A new calcium phosphate cement is reported, which sets to form a matrix consisting of brushite, dicalcium pyrophosphate dihydrate and an amorphous phase following the mixture of beta-tricalcium phosphate with an aqueous pyrophosphoric acid solution. This reactant combination set within a clinically relevant time-frame (approximately 10 min) and exhibited a higher compressive strength (25 MPa) than previously reported brushite cements. The in vitro degradation of the beta-tricalcium phosphate-pyrophosphoric acid cement was tested in both phosphate buffered saline and bovine serum. The pyrophosphate ion containing cement reported here was found not to be hydrolysed to form hydroxyapatite in vitro like beta-tricalcium phosphate-orthophosphoric acid solution cements. This finding is significant since the formation of hydroxyapatite by hydrolysis is thought to retard in vivo degradation of brushite cements. When aged in bovine serum, the cement lost considerably more mass than when aged in phosphate buffered saline, indicating that proteins, most likely phosphatase enzymes played an important role in the degradation. As pyrophosphate ions are thought to be the source of orthophosphate ions during bone mineralisation, this new class of bone cement offers a route to new degradable synthetic bone grafting materials.

In vitro biodegradation of three brushite calcium phosphate cements by a macrophage cell-line.

Depending upon local conditions, brushite (CaHPO4 x 2 H2O) cements may be largely resorbed or (following hydrolysis to hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the osteoclast-like cell cultures, ultrastructural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement degradation in vitro.