Sensitivity of novel silicate and borate-based glass structures on in vitro bioactivity and degradation behaviour.

Three novel glass compositions, identified as NCL2 (SiO2-based), NCL4 (B2O3-based) and NCL7 (SiO2-based), along with apatite-wollastonite (AW) were processed to form sintered dense pellets, and subsequently evaluated for their in vitro bioactive potential, resulting physico-chemical properties and degradation rate. Microstructural analysis showed the carbonated hydroxyapatite (HCA) precipitate morphology following SBF testing to be composition-dependent. AW and the NCL7 formulation exhibited greater HCA precursor formation than the NCL2 and NCL4-derived pellets. Moreover, the NCL4 borate-based samples showed the highest biodegradation rate; with silicate-derived structures displaying the lowest weight loss after SBF immersion. The results of this study suggested that glass composition has significant influence on apatite-forming ability and also degradation rate, indicating the possibility to customise the properties of this class of materials towards the bone repair and regeneration process.

Novel bioglasses for bone tissue repair and regeneration: Effect of glass design on sintering ability, ion release and biocompatibility.

Eight novel silicate, phosphate and borate glass compositions (coded as NCLx, where x = 1 to 8), containing different oxides (i.e. MgO, MnO2, Al2O3, CaF2, Fe2O3, ZnO, CuO, Cr2O3) were designed and evaluated alongside apatite-wollastonite (used as comparison material), as potential biomaterials for bone tissue repair and regeneration. Glass frits of all the formulations were processed to have particle sizes under 53 µm, with their morphology and dimensions subsequently investigated by scanning electron microscopy (SEM). In order to establish the nature of the raw glass powders, X-ray diffraction (XRD) analysis was also performed. The sintering ability of the novel materials was determined by using hot stage microscopy (HSM). Ionic release potential was assessed by inductively coupled plasma optical emission spectroscopy (ICP-OES). Finally, the cytotoxic effect of the novel glass powders was evaluated for different glass concentrations via a colorimetric assay, on which basis three formulations are considered promising biomaterials.

Bond Strength and Adhesion Mechanisms of Novel Bone Adhesives.

Bone adhesives offer distinct advantages over the use of screws to attached internal fixation plates (IFPs). As the chemical composition of bone is similar to dentine, it is possible that the types of monomers used to make dentine adhesives could be utilised to affix IFPs to bone. The ability to attach a bio-resorbable IFP to porcine bone was assessed for the monomer 10-methacryloyloxydecyl dihydrogen phosphate (MDP), used either as a homopolymer or a copolymer with urethane dimethacrylate (MDP + U). Additionally, the addition of a priming step (MDP + U + P) was evaluated. The chemical interactions of the monomers with bone were assessed using XRD and imaged using TEM, revealing the formation of nano-layered structures with the MDP primer, something we believe has not been reported on bone. In a 6-week artificial aging study both MDP + U and MDP + U + P demonstrated adequate shear bond strength to affix bio-resorbable IFPs. The cytotoxicity profiles of the adhesive formulations were determined using indirect and direct contact with MC3T3 cells, with indirect conditions suggesting the MDP + U + P is as cytocompatible as the resorbable IFP. The findings of this study suggest our newly developed adhesive has the potential to be used as a bone adhesive to affix bioresorbable IFPs.

Reliable inkjet printing of chondrocytes and MSCs using reservoir agitation.

Drop-on-demand (DoD) inkjet printing has been explored for a range of applications, including those to selectively deposit cellular material, due to the high accuracy and scalability of such systems when compared with alternative bioprinting techniques. Despite this, there remain considerable limitations when handling cell suspensions due to the agglomeration and sedimentation of cells during printing, leading to a deterioration in jetting performance. The objective of this work was to design and assess the effectiveness of a custom agitation system to maintain cellular dispersion within the ink reservoir during printing. The cell printing performance of an inkjet printer was assessed with and without the use of a custom agitation system, with biological characterisation performed to characterise the impact of the agitator on cellular viability and function. Cell printing performance was retained over a 2 h printing period when incorporating an agitated reservoir, with a gradual reduction in performance observed under a non-agitated configuration. Cell assays indicated that the agitation process did not significantly affect the viability, metabolic activity or morphology of the mesenchymal stromal cell (MSC) or chondrocyte cell types. This study therefore provides a new methodology to increase process reliability within DoD printing platforms when jetting cellularised material.

How cell culture conditions affect the microstructure and nanomechanical properties of extracellular matrix formed by immortalized human mesenchymal stem cells: An experimental and modelling study.

This paper presents an investigation of how different culture media (i.e. basal and osteogenic media) affect the nanomechanical properties and microstructure of the mineralized matrix produced by the human mesenchymal stem cell line Y201, from both an experimental and theoretical approach. A bone nodule (i.e. mineralized matrix) cultured from basal medium shows a more anisotropic microstructure compared to its counterpart cultured from an osteogenic medium. As confirmed by finite element simulations, this anisotropic microstructure explains the bimodal distribution of the corresponding mechanical properties very well. The overall nanomechanical response of the bone nodule from the osteogenic medium is poorer compared to its counterpart from the basal medium. The bone nodules, from both basal and osteogenic media, have shown reverse aging effects in terms of mechanical properties. These are possibly due to the fact that cell proliferation outcompetes the mineralization process.

Development of custom-built bone scaffolds using mesenchymal stem cells and apatite-wollastonite glass-ceramics.

There is a clinical need for new bone replacement materials that combine long implant life with complete integration and appropriate mechanical properties. We have used human mesenchymal stem cells (MSCs) to populate porous apatite-wollastonite (A-W) glass-ceramic scaffolds produced by the layer manufacturing technique, selective laser sintering, to create custom-built bone replacements. Confocal and scanning electron microscopy were used to determine optimal seeding densities and to demonstrate that MSCs adhered and retained viability on the surface of A-W scaffolds over a culture period of 21 days. We found a significant increase in the number of MSCs growing on the scaffolds over 7 days. Using bromodeoxyuridine incorporation we demonstrated that MSCs proliferated on the scaffolds. Using real-time PCR we analyzed the expression of the osteogenic markers alkaline phosphatase, collagen type-I, Cbfa-1, osteocalcin, osteonectin, and osteopontin by MSCs cultured in the absence of osteogenic supplements. The expression of the osteogenic markers by MSCs was equivalent to or significantly greater on A-W scaffolds than on tissue culture plastic. We also identified significantly higher alkaline phosphatase activity on A-W compared to a commercial calcium phosphate scaffold. These results indicate for the first time the biocompatibility and osteo-supportive capacity of A-W scaffolds and their potential as patient-specific bone replacement materials.

Biological evaluation of an apatite-mullite glass-ceramic produced via selective laser sintering.

The biological performance of a porous apatite-mullite glass-ceramic, manufactured via a selective laser sintering (SLS) method, was evaluated to determine its potential as a bone replacement material. Direct contact and extract assays were used to assess the cytotoxicity of the material. A pilot animal study, implanting the material into rabbit tibiae for 4 weeks, was also carried out to assess in vivo bioactivity. The material produced by SLS did not show any acute cytotoxic effects by either contact or extract methods. There was no evidence of an apatite layer forming on the surface of the material when soaked in SBF for 30 days, suggesting that the material was unlikely to exhibit bioactive behaviour in vivo. It is hypothesized that the material was unable to form an apatite layer in SBF due to the fact that this glass-ceramic was highly crystalline and the fluorapatite crystal phase was relatively stable in SBF, as were the two aluminosilicate crystal phases. There was thus no release of calcium and phosphorus and no formation of silanol groups to trigger apatite deposition from solution within the test time period. Following implantation in rabbit tibiae for 4 weeks, bone was seen to have grown into the porous structure of the laser-sintered parts, and appeared to be very close to, or directly contacting, the material surface. This result may reflect the local environment in vivo compared to that artificially found with the in vitro SBF test and, furthermore, confirms previous in vivo data on these glass-ceramics.

Development of an arthroscopically compatible polymer additive layer manufacture technique.

This article describes a proof of concept study designed to evaluate the potential of an in vivo three-dimensional printing route to support minimally invasive repair of the musculoskeletal system. The study uses a photocurable material to additively manufacture in situ a model implant and demonstrates that this can be achieved effectively within a clinically relevant timescale. The approach has the potential to be applied with a wide range of light-curable materials and with development could be applied to create functionally gradient structures in vivo.

Three-dimensional printing of porous load-bearing bioceramic scaffolds.

This article reports on the use of the binder jetting three-dimensional printing process combined with sintering to process bioceramic materials to form micro- and macroporous three-dimensional structures. Three different glass-ceramic formulations, apatite-wollastonite and two silicate-based glasses, have been processed using this route to create porous structures which have Young's modulus equivalent to cortical bone and average bending strengths in the range 24-36 MPa. It is demonstrated that a range of macroporous geometries can be created with accuracies of ±0.25 mm over length scales up to 40 mm. Hot-stage microscopy is a valuable tool in the definition of processing parameters for the sintering step of the process. Overall, it is concluded that binder jetting followed by sintering offers a versatile process for the manufacture of load-bearing bioceramic components for bone replacement applications.

Temporary Single-Cell Coating for Bioprocessing with a Cationic Polymer.

Temporary single-cell coating is a useful tool for cell processing, allowing manipulation of cells to prevent cell attachment and agglomeration, before re-establishing normal cell function. In this work, a speckled coating method using a known polycation [poly(l-lysine), PLL] is described to induce cell surface electrostatic charges on three different cell types, namely, two bone cancer cell lines and fibroblasts. The morphology of the PLL speckled coating on the cell surface, internalization and metabolization of the polymer, and prevention of cellular aggregations are reported. Polymer concentration was found to be the key parameter controlling both capsule morphology and cell health. This approach allows a temporary cell coating over the course of 1-2 h, with cells exhibiting phenotypically normal behavior after ingesting and metabolizing the polymer. The process offers a fast and efficient alternative to aid single-cell manipulation for bioprocessing applications. Preliminary work on the application of PLL speckled cell coating in enabling reliable bioprinting is also presented.

Development of a methacrylate-terminated PLGA copolymer for potential use in craniomaxillofacial fracture plates.

We synthesised methacrylate-terminated PLGA (HT-PLGA, 85:15 LA:GA, 169kDa), for potential use as an adhesively attached craniomaxillofacial fracture fixation plate. The in vitro degradation of molecular weight, pH and flexural modulus were measured over 6weeks storage in PBS at 37°C, with commercially available high (225kDa, H-PLGA) and low (116kDa, L-PLGA) molecular weight 85:15 PLGAs used as comparators. Molecular weights of the materials reduced over 6weeks, HT-PLGA by 48%, H-PLGA by 23% and L-PLGA by 81%. HT-PLGA and H-PLGA exhibited a near constant pH (7.35) and had average flexural moduli in excess of 6GPa when produced, similar to that of the mandible. After 1week storage both exhibited a significant reduction in average modulus, however, from weeks 1-6 no further significant changes were observed, the average modulus never dropped significantly below 5.5GPa. In contrast, the L-PLGA caused a pH drop to below 7.3 by week 6 and an average modulus drop to 0.6 from an initial 4.6GPa. Cell culture using rat bone marrow stromal cells, revealed all materials were cytocompatible and exhibited no osteogenic potential. We conclude that our functionalised PLGA retains mechanical properties which are suitable for use in craniofacial fixation plates.

In vivo biocompatibility of custom-fabricated apatite-wollastonite-mesenchymal stromal cell constructs.

We have used the additive manufacturing technology of selective laser sintering (SLS), together with post SLS heat treatment, to produce porous three dimensional scaffolds from the glass-ceramic apatite-wollastonite (A-W). The A-W scaffolds were custom-designed to incorporate a cylindrical central channel to increase cell penetration and medium flow to the center of the scaffolds under dynamic culture conditions during in vitro testing and subsequent in vivo implantation. The scaffolds were seeded with human bone marrow mesenchymal stromal cells (MSCs) and cultured in spinner flasks. Using confocal and scanning electron microscopy, we demonstrated that MSCs formed and maintained a confluent layer of viable cells on all surfaces of the A-W scaffolds during dynamic culture. MSC-seeded, with and without osteogenic pre-differentiation, and unseeded A-W scaffolds were implanted subcutaneously in MF1 nude mice where osteoid formation and tissue in-growth were observed following histological assessment. The results demonstrate that the in vivo biocompatibility and osteo-supportive capacity of A-W scaffolds can be enhanced by SLS-custom design, without the requirement for osteogenic pre-induction, to advance their potential as patient-specific bone replacement materials.

Mass customization of foot orthoses for rheumatoid arthritis using selective laser sintering.

Rheumatoid arthritis is an inflammatory joint disease that can lead to pain, stiffness, and deformity, often with marked involvement of the small joints of the foot and ankle. Orthotic devices are commonly prescribed for this condition to lessen symptoms and improve function and mobility, and customized devices are most effective. The work reported in this paper has examined the feasibility of using an additive manufacturing-based approach to manufacture customized orthoses. In order to test feasibility, orthoses have been manufactured using the additive manufacturing technology of selective laser sintering, and have been evaluated through a small-scale patient trial (n = 7). The trial indicated that these orthoses performed as well as the patients' current prescribed customized devices in terms of the observed gait and subjective evaluation of fit and comfort. It is concluded that the feasibility of the additive manufacturing approach has been demonstrated, and further development of a mass customization system to deliver orthoses, together with exploitation of the design freedom offered by the manufacturing method, will give the overall approach significant clinical potential.