Synchrotron X-ray microtomography for assessment of bone tissue scaffolds
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- Yue, S., Lee, P.D., Poologasundarampillai, G. et al. J Mater Sci: Mater Med (2010) 21: 847. doi:10.1007/s10856-009-3888-9
X-ray microtomography (μCT) is a popular tool for imaging scaffolds designed for tissue engineering applications. The ability of synchrotron μCT to monitor tissue response and changes in a bioactive glass scaffold ex vivo were assessed. It was possible to observe the morphology of the bone; soft tissue ingrowth and the calcium distribution within the scaffold. A second aim was to use two newly developed compression rigs, one designed for use inside a laboratory based μCT machine for continual monitoring of the pore structure and crack formation and another designed for use in the synchrotron facility. Both rigs allowed imaging of the failure mechanism while obtaining stress–strain data. Failure mechanisms of the bioactive glass scaffolds were found not to follow classical predictions for the failure of brittle foams. Compression strengths were found to be 4.5–6 MPa while maintaining an interconnected pore network suitable for tissue engineering applications.
Natural bone healing is generally only successful if the defect is small, so when a defect exceeds ~1 cm3 bone grafting is often needed. Bone tissue engineering is thought to be a promising way to regenerate lost bone where an ideal bone tissue engineering method uses a temporary template (scaffold) cultured with osteogenic cells harvested from the patient [1–3]. In this ideal scenario, the cells are expanded in culture, seeded on a scaffold in a bioreactor and once enough new tissue has been generated in vitro, the tissue/scaffold composite can then be implanted into the defect site of the patient.
The scaffold should act as both a guide and a stimulus for bone regeneration in vitro and then in vivo. The design criteria of an ideal scaffold for bone regeneration are many [1, 4]. Having a suitable interconnected porous network is important as the pore size and more importantly the interconnect size must be large enough to enable cell migration, fluid exchange and eventually bone ingrowth and vascularisation. The minimum interconnect diameter for vascularised bone ingrowth is thought to be in excess of 100 μm . The mechanical properties of the scaffold should also match that of the host tissue because the scaffold needs to have enough strength to retain its structure in a load bearing environment after implantation and without being so stiff that it shields surrounding bone from load .
Sol–gel derived bioactive glass foam scaffolds are promising candidates for bone tissue engineering because they bond to bone, degrade in the body and release soluble silica species and calcium ions which are thought to stimulate osteogenesis at the genetic level [7–9]. In addition, the bioactive glass foam has a cancellous bone-like porous structure with suitable pore and interconnects size for tissue engineering .
High resolution X-ray micro-computed tomography (μCT) is a powerful tool for scaffold characterisation. Unlike many other techniques for pore and interconnect size quantification, including scanning electron microscopy (SEM) and mercury intrusion porosimetry (MIP), μCT can non-destructively obtain three dimensional (3D) image of a scaffold . Now, when combined with 3D image analysis techniques, it can provide not only qualitative but also quantitative information of the scaffold can be obtained [10, 12].
μCT can be divided into two types in terms of X-ray sources : synchrotron radiation (SR) and laboratory based Micro/Nano focus X-ray tube heads. Synchrotron μCT usually has a higher resolution, and faster scanning process than those of laboratory based μCT. Synchrotron radiation is generated from an electron storage ring and can be monochromatic (having a specific wavelength), therefore different elements can be distinguished according to their atomic number in the reconstructed images. The speed of scanning process of synchrotron μCT is massively increased compared to laboratory based μCT by the use of a finite size source (parallel) instead of point source (pencil beam).
The aim of this work was to assess whether there is a significant benefit to using synchrotron μCT over high resolution laboratory sources when characterising scaffolds for bone tissue engineering. Bioactive glass foam scaffolds are used as example scaffolds. Their pore networks were quantified using 3D image analysis and mechanical properties and failure mechanisms assessed using load cells specially designed for scanning in μCT. Tissue ingrowth and changes in scaffolds as a function of time in vivo, were also observed.
2 Materials and methods
2.1 Foam synthesis
A bioactive glass foam of the 70S30C composition (70 mol% SiO2, 30 mol% CaO) was prepared using the sol–gel foaming method as described previously [4, 7]. The sol preparation involved mixing 0.2 mol/l nitric acid with water, followed by addition of tetraethyl orthosilicate (TEOS) and calcium nitrate (all Sigma). The molar ratio of water to TEOS (R ratio) was 12:1. Aliquots of 50 ml of the sol were foamed with vigorous agitation with the addition of 3 ml of 5 vol% HF (catalyst) and 0.35 ml of Teepol (surfactant, Thames Mead Ltd., London). As the foamed sol approached gelling point it was cast into cylindrical Teflon moulds sealed and then aged at 60°C for 72 h. The samples were then dried at 130°C, stabilised at 600°C and sintered at 800°C for 2 h.
2.2 In vivo experiment
Following general anaesthesia and surgical preparation, a cube shaped bioactive glass foam scaffold, with 1 mm dimensions, was implanted between the muscular fascia and the tibia of an 8-week-old CD1 male mouse. This study was carried out following appropriate local and national (Home Office) ethical approval. After 4 weeks, the corrosion casting of vessels was performed as detailed previously  by trans-cardiac perfusion with Mercox CL-2B (Vilene Med Co, Tokyo Japan) diluted with 20% methylmethacrylate monomers (Merck Darmstadt, Germany). Upon complete polymerization, the entire lower limbs containing the scaffolds were removed, fixed in 10% neutral buffered formalin and the surrounding tissue dissected free to allow easy placement of the sample in the chamber. The samples were imaged using monochromatic synchrotron μCT (ESRF, Grenoble, France; Beamline ID19) with a special resolution of 0.55 μm. The reconstructed image was then rendered with commercial image analysis packages (VGStudio MAX 1.2, Volume Graphics GmbH, Heidelberg, Germany).
2.3 3D image analysis and compression testing
The pore and interconnect sizes of the scaffold were quantified with algorithms which have been described previously [10, 12], including a dilation based distance transform, a 3D watershed algorithm [14, 15], and a novel in house algorithm to locate the interconnects. [10, 12]. The size of the spherical pore was represented by the diameter length of a sphere with equivalent volume. The quantification of the interconnect size has been improved by a principle component analysis (PCA) based method. The PCA based method can find the natural moment of the interconnects by calculating the eigensystem of the covariance matrix of its voxels coordinates in the image. Then the interconnect voxels can be projected to its principle plane and the area of the interconnect can be calculated by a convex hull algorithm.
2.4 In situ compression
Using an in-house developed compression rig (Fig. 1b), the compressive strength of similar scaffolds was assessed while they were in the X-ray beam of a laboratory based μCT machine. Scaffolds with sintering temperatures of 600°C and 800°C were compared. Seven samples (3 mm × 3 mm × 5 mm) of each were tested. The rig’s main components included a Harmonic drive ME-02-L rotary encoder, a Heidenhain 602E linear encoder, a Harmonic drive RH-8-3006 actuator, an ENTRAN ELFS-T3M-250N load cell, a stationary platen and a mobile platen. The rotary encoder was the driver for the moving platen while the calibrated linear encoder (0.1 μm resolution) read the displacement of the platform. The load cell was 250 N but the maximum load applied during testing was restricted to below 180 N. The shafts are isolated with air-bearings to provide sufficient lubrication to protect the system from any static and dynamic frictional forces. A NextMove NBA-100-MC01 control board was used to provide communication between the testing system and the accompanying WorkBench software in a Windows operation system. This rig enabled 2D transmission images of the scaffold to be captured in real time under continual strain, while load/compression data was collected at a strain rate of 0.001 mm s−1. Transmission images were collected every 0.4 s. Mode of failure and crack path could therefore be observed.
3 Results and discussion
3.1 Synchrotron imaging of explanted scaffolds and tissue
3.2 Bioactive glass foam failure mechanisms
Returning to the 3D image analysis of the scaffold prior to testing (Fig. 4), the results show that the pore network was suitable for tissue engineering applications with a mean pore size of 265 μm and a mean interconnect size greater than 100 μm. Figure 4a shows that the majority of pores were in the range 100–300 μm and Fig. 4b shows that 17 of the 20 interconnects had diameters in the range 50–200 μm and half the total interconnects were in excess of 100 μm in diameter. During time of implantation the interconnect size is expected to increase as dissolution occurs.
Synchrotron μCT can be used to obtain high resolution images of explanted scaffolds and bone tissue. Resolution of mouse explants is sufficient to identify porosity and morphology of the bone and mineral distribution within the scaffold pores and struts. Using the corrosion casting technique on sacrifice of the mouse allows imaging of microvessel development around and inside the scaffold. There is a clear benefit of using the synchrotron over laboratory based μCT.
Compression testing in especially designed rigs that can fit inside the μCT machines combined with image analysis techniques allow simultaneous quantification of the pore structure, imaging of crack initiation, determination of crack path and mode of failure while obtaining compression strength data.
Julian Jones is a Royal Academy of Engineering/Engineering and Physical Science Research Council (EPSRC) Research Fellow. The authors also acknowledge financial support from the Philip Leverhulme Prize and EPSRC (GR/T26344). The European Synchrotron Radiation Facility especially the team of beam line ID19, especially Elodie Boller is greatly acknowledged for the provision of synchrotron radiation facilities.