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.

Preconditioned 70S30C bioactive glass foams promote osteogenesis in vivo.

Bioactive glass scaffolds (70S30C; 70% SiO2 and 30% CaO) produced by a sol-gel foaming process are thought to be suitable matrices for bone tissue regeneration. Previous in vitro data showed bone matrix production and active remodelling in the presence of osteogenic cells. Here we report their ability to act as scaffolds for in vivo bone regeneration in a rat tibial defect model, but only when preconditioned. Pretreatment methods (dry, pre-wetted or preconditioned without blood) for the 70S30C scaffolds were compared against commercial synthetic bone grafts (NovaBone® and Actifuse®). Poor bone ingrowth was found for both dry and wetted sol-gel foams, associated with rapid increase in pH within the scaffolds. Bone ingrowth was quantified through histology and novel micro-CT image analysis. The percentage bone ingrowth into dry, wetted and preconditioned 70S30C scaffolds at 11 weeks were 10±1%, 21±2% and 39±4%, respectively. Only the preconditioned sample showed above 60% material-bone contact, which was similar to that in NovaBone and Actifuse. Unlike the commercial products, preconditioned 70S30C scaffolds degraded and were replaced with new bone. The results suggest that bioactive glass compositions should be redesigned if sol-gel scaffolds are to be used without preconditioning to avoid excess calcium release.

Bioactive glass foam scaffolds are remodelled by osteoclasts and support the formation of mineralized matrix and vascular networks in vitro.

Remodelling of scaffolds and new bone formation is critical for effective bone regeneration. Herein is reported the first demonstration of resorption pits due to osteoclast activity on the surface of sol-gel bioactive glass foam scaffolds. Bioactive glass foam scaffolds are known to have osteogenic potential and suitable pore networks for bone regeneration. Degradation of the scaffolds is known to be initially solution mediated, but for effective bone regeneration, remodelling of the scaffold by osteoclasts and vascularisation of the scaffold is necessary. The culture of C7 macrophages on a bioactive glass scaffold induces the cells to differentiate into (TRAP(+ve) ) osteoclasts. They then form distinctive resorption pits within 3 weeks, while MC3T3-E1 pre-osteoblasts deposit mineralized osteoid on their surfaces in co-culture. The scaffolds are of the 70S30C (70 mol% SiO2 , 30 mol% CaO) composition, with modal pore and interconnect diameters of 373 µm and 172 µm respectively (quantified by X-ray micro-tomography and 3D image analysis). The release of soluble silica and calcium ions from 70S30C scaffolds induces an increase in osteoblast numbers as determined via the MTT assay. Scaffolds also support growth of endothelial cells on their surface and tube formation (characteristic of functional microvasculature) following 4 days in culture. This data supports the hypothesis that 70S30C bioactive glass scaffolds promote the differentiation of the 3 main cell types involved in vascularized bone regeneration.

Protein interactions with nanoporous sol-gel derived bioactive glasses.

Sol-gel derived bioactive glasses are excellent candidates for bone regenerative implant materials as they bond with bone, stimulate bone growth and degrade in the body. Their interactions with proteins are critical to understanding their performance after implantation. This study focuses on the interactions between fibrinogen and sol-gel glass particles of the 70S30C (70 mol.% SiO(2), 30 mol.% CaO composition). Sol-gel silica and melt-derived Bioglass® were also used for comparison. Fibrinogen penetration into the nanoporous glasses was observed by live tracking the fluorescent-labelled fibrinogen with confocal microscopy. The effect of pore size on protein penetration was investigated. Nanoporous networks with modal pore diameters larger than 6 nm were accessible to fibrinogen. When the modal nanopore diameter was decreased to 2 nm or less, the penetration of fibrinogen was inhibited. The surface properties of the glasses, which can be modulated by media pH, glass composition and final stabilisation temperature in the sol-gel process, have effects on fibrinogen adsorption via long-range Coulombic forces before the adsorption and via short-range interactions such as hydrogen bonding after the adsorption.