Gowsihan Poologasundarampillai

Evaluation of 3-D bioactive glass scaffolds dissolution in a perfusion flow system with X-ray microtomography

Bioactive glass has high potential for bone regeneration due to its ability to bond to bone and stimulate osteogenesis whilst dissolving in the body. Although three-dimensional (3-D) bioactive glass scaffolds with favorable pore networks can be made from the sol-gel process, compositional and structural evolutions in their porous structures during degradation in vivo, or in vitro, have not been quantified. In this study, bioactive glass scaffolds were put in a simulated body fluid flow environment through a perfusion bioreactor. X-ray microtomography (μCT) was used to non-destructively image the scaffolds at different degradation stages. A new 3-D image processing methodology was developed to quantify the scaffolds pore size, interconnect size and connectivity from μCT images. The accurate measurement of individual interconnect size was made possible by a principal component analysis-based algorithm. During 28 days of dissolution, the modal interconnect size in the scaffold was reduced from 254 to 206 μm due to the deposition of mineral phases. However, the pore size remained unchanged, with a mode of 682 μm. The data presented are important for making bioactive glass scaffolds into clinical products. The technique described for imaging and quantifying scaffold pore structures as a function of degradation time is applicable to most scaffold systems.

Cotton-wool-like bioactive glasses for bone regeneration

Inorganic sol-gel solutions were electrospun to produce the first bioactive three-dimensional (3-D) scaffolds for bone tissue regeneration with a structure like cotton-wool (or cotton candy). This flexible 3-D fibrous structure is ideal for packing into complex defects. It also has large inter-fiber spaces to promote vascularization, penetration of cells and transport of nutrients throughout the scaffold. The 3-D fibrous structure was obtained by electrospinning, where the applied electric field and the instabilities exert tremendous force on the spinning jet, which is required to be viscoelastic to prevent jet break up. Previously, polymer binding agents were used with inorganic solutions to produce electrospun composite two-dimensional fibermats, requiring calcination to remove the polymer. This study presents novel reaction and processing conditions for producing a viscoelastic inorganic sol-gel solution that results in fibers by the entanglement of the intermolecularly overlapped nanosilica species in the solution, eliminating the need for a binder. Three-dimensional cotton-wool-like structures were only produced when solutions containing calcium nitrate were used, suggesting that the charge of the Ca(2+) ions had a significant effect. The resulting bioactive silica fibers had a narrow diameter range of 0.5-2μm and were nanoporous. A hydroxycarbonate apatite layer was formed on the fibers within the first 12h of soaking in simulated body fluid. MC3T3-E1 preosteoblast cells cultured on the fibers showed no adverse cytotoxic effect and they were observed to attach to and spread in the material.

Hierarchically structured titanium foams for tissue scaffold applications.

We present a novel route for producing a new class of titanium foams for use in biomedical implant applications. These foams are hierarchically porous, with both the traditional large (>300μm) highly interconnected pores and, uniquely, wall struts also containing micron scale (0.5-5μm) interconnected porosities. The fabrication method consists of first producing a porous oxide precursor via a gel casting method, followed by electrochemical reduction to produce a metallic foam. This method offers the unique ability to tailor the porosity at several scales independently, unlike traditional space-holder techniques. Reducing the pressure during foam setting increased the macro-pore size. The intra-strut pore size (and percentage) can be controlled independently of macro-pore size by altering the ceramic loading and sintering temperature during precursor production. Typical properties for an 80% porous Ti foam were a modulus of ∼1GPa, a yield strength of 8MPa and a permeability of 350 Darcies, all of which are in the range required for biomedical implant applications. We also demonstrate that the micron scale intra-strut porosities can be exploited to allow infiltration of bioactive materials using a novel bioactive silica-polymer composite, resulting in a metal-bioactive silica-polymer composite.

Electrospun silica/PLLA hybrid materials for skeletal regeneration

This paper presents the electrospinning of new silica/poly(L-lactic acid) (PLLA) based organic/inorganic sol–gel hybrid materials for skeletal defect regeneration. Here, hybrids are defined as inorganic and organic species interacting at the molecular level such that there are no distinguishable phases above the nanoscale. In previous hybrids, calcium has not been successfully incorporated into the silica network, limiting bioactivity. Here, an alkoxide source of calcium (calcium methoxyethoxide (CME)) was successfully used with a modified dual syringe reactive electrospinning (DSRS) setup to produce a calcium and silica containing PLLA sol–gel hybrid fibermat. Fibermats with continuous and homogeneous fibers were successfully produced that showed controlled and tailorable release of soluble silica in aqueous media. The CME-derived PLLA hybrid was observed to be hydrophilic and showed indication of bone-like apatite formation within 12 h of soaking in simulated body fluid (SBF). After soaking in SBF, the entire fiber surface was coated by a homogeneous thickness of apatite that had a plate-like crystal structure. The reversal of PLLAs hydrophobicity has tremendous implications on the biological properties of the material. All the electrospun materials were easy to handle and could be easily shaped into complex shapes. The tensile strength of the hybrid fibermats was found to be enhanced by the addition of silica, where a twofold increase in the tensile strength of 20 wt% silica containing PLLA fibermats compared to that of PLLA fibermats was observed.

Poly(γ-glutamic acid)/silica hybrids with calcium incorporated in the silica network by use of a calcium alkoxide precursor

Current materials used for bone regeneration are usually bioactive ceramics or glasses. Although they bond to bone, they are brittle. There is a need for new materials that can combine bioactivity with toughness and controlled biodegradation. Sol-gel hybrids have the potential to do this through their nanoscale interpenetrating networks (IPN) of inorganic and organic components. Poly(γ-glutamic acid) (γ-PGA) was introduced into the sol-gel process to produce a hybrid of γ-PGA and bioactive silica. Calcium is an important element for bone regeneration but calcium sources that are used traditionally in the sol-gel process, such as Ca salts, do not allow Ca incorporation into the silicate network during low-temperature processing. The hypothesis for this study was that using calcium methoxyethoxide (CME) as the Ca source would allow Ca incorporation into the silicate component of the hybrid at room temperature. The produced hybrids would have improved mechanical properties and controlled degradation compared with hybrids of calcium chloride (CaCl2), in which the Ca is not incorporated into the silicate network. Class II hybrids, with covalent bonds between the inorganic and organic species, were synthesised by using organosilane. Calcium incorporation in both the organic and inorganic IPNs of the hybrid was improved when CME was used. This was clearly observed by using FTIR and solid-state NMR spectroscopy, which showed ionic cross-linking of γ-PGA by Ca and a lower degree of condensation of the Si species compared with the hybrids made with CaCl2 as the Ca source. The ionic cross-linking of γ-PGA by Ca resulted in excellent compressive strength and reduced elastic modulus as measured by compressive testing and nanoindentation, respectively. All hybrids showed bioactivity as hydroxyapatite (HA) was formed after immersion in simulated body fluid (SBF).

A comparative study of oxygen diffusion in tissue engineering scaffolds

Tissue engineering scaffolds are designed to support tissue self-healing within physiological environments by promoting the attachment, growth and differentiation of relevant cells. Newly formed tissue must be supplied with sufficient levels of oxygen to prevent necrosis. Oxygen diffusion is the major transport mechanism before vascularization is completed and oxygen is predominantly supplied via blood vessels. The present study compares different designs for scaffolds in the context of their oxygen diffusion ability. In all cases, oxygen diffusion is confined to the scaffold pores that are assumed to be completely occupied by newly formed tissue. The solid phase of the scaffolds acts as diffusion barrier that locally inhibits oxygen diffusion, i.e. no oxygen passes through the scaffold material. As a result, the oxygen diffusivity is determined by the scaffold porosity and pore architecture. Lattice Monte Carlo simulations are performed to compare the normalized oxygen diffusivities in scaffolds obtained by the foam replication (FR) method, robocasting and sol–gel foaming. Scaffolds made by the FR method were found to have the highest oxygen diffusivity due to their high porosity and interconnected pores. These structures enable the best oxygen supply for newly formed tissue among the scaffold types considered according to the present numerical predictions.

Strategies for the chemical analysis of highly porous bone scaffolds using secondary ion mass spectrometry

Understanding the distribution of critical elements (e.g. silicon and calcium) within silica-based bone scaffolds synthesized by different methods is central to the optimization of these materials. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) has been used to determine this information due to its very high surface sensitivity and its ability to map all the elements and compounds in the periodic table with high spatial resolution. The SIMS image data can also be combined with depth profiles to construct three-dimensional chemical maps. However, the scaffolds have interconnected pore networks, which are very challenging structures for the SIMS technique. To overcome this problem two experimental methodologies have been developed. The first method involved the use of the focused ion beam technique to obtain clear images of the regions of interest and subsequently mark them by introducing fiducial marks; the samples were then analysed using the ToF-SIMS technique to yield the chemical analyses of the regions of interest. The second method involved impregnating the pores using a suitable reagent so that a flat surface could be achieved, and this was followed by secondary ion mapping and 3D chemical imaging with ToF-SIMS. The samples used in this work were sol-gel 70S30C foam and electrospun fibres and calcium-containing silica/gelatin hybrid scaffolds. The results demonstrate the feasibility of both these experimental methodologies and indicate that these methods can provide an opportunity to compare various artificial bone scaffolds, which will be of help in improving scaffold synthesis and processing routes. The techniques are also transferable to many other types of porous material.

X-ray Tomographic Imaging of Tensile Deformation Modes of Electrospun Biodegradable Polyester Fibers

Electrospinning allows the production of fibrous networks for tissue engineering, drug delivery and wound healing in healthcare. It enables the production of constructs with large surface area and a fibrous morphology that closely resembles the extracellular matrix of many tissues. A fibrous structure not only promotes cell attachment and tissue formation, but could also lead to very interesting mechanical properties. Poly(3-hydroxybutyrate-co-4-hydroxybutyrate) (P(3HB-co-4HB)) is a biodegradable polyester that exhibits large (>400%) elongation before failure. In this study, synchrotron X-ray phase contrast imaging was performed during tensile deformation to failure on a non-woven fibre mat of P(3HB-co-4HB) fibres. Significant reorientation of the fibres in straining direction was observed, followed by localised necking and eventual failure. From an original average fibre diameter of 4.3 μm a bimodal distribution of fibre diameter (modal diameters of 1.9 and 3.7 μm) formed after tensile deformation. Extensive localised necking (thinning) of fibres between (thicker) fibre-fibre contacts was found to be the cause for non-uniform thinning of the fibres, a phenomenon that is expected, but has not been observed in 3D previously. The data presented here has implications not only in tissue regeneration but for fibrous materials in general.

High-Density Protein Loading on Hierarchically Porous Layered Double Hydroxide Composites with a Rational Mesostructure

Hierarchically porous biocompatible Mg-Al-Cl-type layered double hydroxide (LDH) composites containing aluminum hydroxide (Alhy) have been prepared using a phase-separation process. The sol-gel synthesis allows for the hierarchical pores of the LDH-Alhy composites to be tuned, leading to a high specific solid surface area per unit volume available for high-molecular-weight protein adsorptions. A linear relationship between the effective surface area, SEFF, and loading capacity of a model protein, bovine serum albumin (BSA), is established following successful control of the structure of the LDH-Alhy composite. The threshold of the mean pore diameter, Dpm, above which BSA is effectively adsorbed on the surface of LDH-Alhy composites, is deduced as 20 nm. In particular, LDH-Alhy composite aerogels obtained via supercritical drying exhibit an extremely high capacity for protein loading (996 mg/g) as a result of a large mean mesopore diameter (>30 nm). The protein loading on LDH-Alhy is >14 times that of a reference LDH material (70 mg/g) prepared via a standard procedure. Importantly, BSA molecules pre-adsorbed on porous composites were successfully released on soaking in ionic solutions (HPO4(2-) and Cl(-) aqueous). The superior capability of the biocompatible LDH materials for loading, encapsulation, and releasing large quantities of proteins was clearly demonstrated.

3D printing of biocomposites for osteochondral tissue engineering

Abstract 3D printing in tissue engineering and regenerative medicine has vastly expanded in recent years due to the ability to fabricate complex multimaterial biomimetic structures that resemble the in vivo tissue architecture, allowing a range of applications to be explored. This has allowed development of scaffolds for osteochondral applications which more closely resemble the native architecture and multimaterial composition of the tissue compared with conventional fabrication techniques. This chapter provides an overview of 3D printing techniques, materials, and scaffold requirements utilised currently in osteochondral tissue engineering. Future challenges and perspectives on the utilisation of 3D printing and materials within osteochondral tissue engineering are provided.