Micro-CT as a tool for nanoporosity investigation of bone engineered scaffolds

Transcription

1 Micro-CT as a tool for nanoporosity investigation of bone engineered scaffolds K. Szlazak 1, J. Jaroszewicz 1, J. Idaszek 1, B. Ostrowska 1, Christian Hellmich 2, W. Swieszkowski 1 1 Faculty of Materials Science and Engineering, Warsaw University of Technology, Warsaw, Poland 2 Institute for Mechanics of Materials and Structures, Vienna University of Technology (TU Wien), Austria Aims Nowadays medicine needs material to heal, unite and complete defects of the affected tissue. It often involves with creating three dimensional constructs. Biodegradable materials, which are currently available for bone tissue regeneration, still have limitations regarding their degradation rate. In this case there is a growing need for novel quantitative techniques for the characterization of nanoporoisty, which can appear after degradation process. One of this method is microcomputed tomography (micro-ct). It has a wide variety of applications in bioengineering varying from imaging, through 2D and 3D quantitative analysis to in situ tests. In comparison to conventional methods used for evaluation of degradation of tissue engineering products which are based on 2D visualization, the micro-ct not only allows the observation in all of orientations in non-destructive way, but also give an access to nanoporosity. The aim of this study was to show an application of micro-ct in quantitative analysis of nanoporosity in scaffolds during degradation process in vitro. Method As a novel composite for bone tissue engineering application, we consider scaffolds made of polycaprolactone (PCL, Sigma-Aldrich; USA), poly(lactic-co-glycolic) acid (PLGA, Resomer 855S; Boehringer Ingelheim; Germany) and tricalcium phosphate (TCP, Sigma-Aldrich, USA). Three components where used because of different biological, chemical and mechanical properties, Addition of PCL significantly increased initial ultimate tensile strength (UTS 0) and Young's modulus of the scaffolds. The PLGA was used as a biodegradable device. This polymer is composed of α-hydroxy acids and undergo relatively fast bulk erosion. The products of the hydrolytic degradation can be accumulated in the core of a device and released in a burst-like fashion into the surrounding environment. Due to the drop of ph, which in vivo can be as low as 2 units, a strong inflammatory reaction can be induced resulting in a local resorption of the bone. TCP is a component a human bone and is needed for tissue regeneration [1]. All of the scaffolds, with customized geometry and controlled internal architecture were fabricated using rapid prototyping method (Bioscaffolder, Syseng, Germany). Cylinder-like samples with elliptic hole (Fig. 1), diameter of 6 mm, height of 10 mm and with an average pore size of µm were obtained.

2 Figure 1. A cylindrical 3D model of scaffold with elliptic hole (a, SolidWorks 2012) and 3D reconstruction of 3D printed scaffold The degradation study was carried out in (PBS). The specimens were sterilized under UV light and subsequently incubated in 15 ml of PBS at 37 C for a period of 56 days. The PBS was replaced after 2, 4, 6, and 8 weeks. At each time point on average 8 samples were removed from PBS and rinsed with deionized water. Composite scaffolds was scanned by means of scanning electron microscopy (SEM, Phenom Pro-X, Netherlands). before (Fig. 3a, b) and after (Fig. 3c, d) degradation study. Fractures were done by pair of tweezers after 5 minutes cooling in liquid nitrogen [3]. Respective fracture (without coated) micrographs were taken at an accelerating voltage of 5 kv. These scaffolds after degradation study contain pores of two sizes: the larger ones are in the order of one to several micrometers ( micropores ), while the smaller ones have a characteristic size of less than one micrometer ( nanopores ).

3 Figure 3. Microstructure of composite scaffolds before (a surface, b fracture) and after (c surface, d fracture) eight week degradation in phosphate buffered saline (PBS). In order to characterize the degradation process in 3D, advanced microcomputed tomography image analysis was used. Scaffolds were scanned by means of SkyScan 1172 (Bruker, Belgium) before and after degradation study with the same scanning parameters. The X-ray tube voltage was 40kV and current was 250 µa. Resolution was 9.93 μm, performing a 180-degree rotation with a step size of 0.2 degrees and 10 frames were averaged for each rotation. Additionally, ring artefacts were reduced trough selection of a random movement amplitude of 50. The latest SkyScan (Bruker, Belgium) software (NRecon, DataViewer, CTAn, and CTVox) and ImageJ (1.49m, Wayne Rasband, National Institutes of Health, USA) was used to reconstruct and process images and to get virtual slices and volumes rendering reconstructions. In the reconstructed image slices, the voxel-specific X-ray attenuation coefficients µ are given in terms of gray values X, according to the linear relation (1): X = a μ + b (1)

4 with a and b as voxel-independent constants. Next, we translate such voxel-specific gray values to voxel-specific nanoporosities nano. Therefore, we consider the average rule for attenuation coefficients of composite materials [4,5,6], reading for our case as (2): μ = φ nano μ air + (1 φ nano )μ bm (2) with μ air as the X-ray attenuation coefficient of air filling the nanoporous space, and with μ bm as the X-ray attenuation coefficient of bulky material (µ bm = µ PCL = µ PLGA). Inserting (1) into (2) yields an average rule for gray values, reading as (3): X = φ nano X air + (1 φ nano )X bm (3) which can be transformed, as to provide the voxel-specific nanoporosity from gray values, in the form (4) (Dejaco et al. [7] formula): φ nano (X) = X deg X bm X air X bm (4) with X air as the gray value of an air-filled voxel, and X bm as the gray value of a voxel which is totally filled by dense PCL and PLGA, right peak, X bm=148 (Fig. 4, Ia). Xair=39 can be identified from the air peak of a gray value histogram (Fig. 4 Ia and Ib). X deg are reconstructed slices of scaffold after degradation study. PCL scaffolds had the same historgrams of gray values (Fig. 4 IIa and IIb) before and after degradation study, so in this case there is no nanoporosity in this resolution. Figure. 4. Reconstructed stack histogram of composite (I) and PCL (II) scaffolds before and after degradation Results

5 Total nanoporosity of the composite scaffold (Fig. 5) is known from the formula (4). The average value of the nanoporosity for this case is 13,4%, but for the whole of analysis samples this value is 12,41±1,11%. Distribution of nanoporosity can be also visualized in 3D (Fig. 6). The highest content of nanoporosity is on the crosses (Fig. 7) and in the inner (elliptic whole) and outer part of the scaffold. Figure. 5. Total nanoporosity of composite scaffold Figure 6. Three-dimensional visualisation of the nanoporosity distribution within the composite scaffold.

6 Figure 7. Nanoporosity distribution of the composite scaffold : 2D cross section (a) and 3D reconstruction (b). Conclusion Micro-CT ensure a careful assessment of a structure of bone scaffolds. Despite of micrometric resolution of reconstructed images, this tool gives an access to nanoscale based on gray value infomation. In addition to a microstructure observation of the entire volume of tested materials, micro-ct allows quantitative analysis of nanoporosity. Acknowledgments This work has been supported by the National Science Centre no. DEC- 2012/07/D/ST8/ References: 1. Idaszek J., Zinn M., Obarzanek-Fojt M., Zell V., Swieszkowski W., Bruinink A., Tailored degradation of biocompatible poly(3-hydroxybutyrate-co-3- hydroxyvalerate)/calcium silicate/poly(lactide-co-glycolide) ternary composites: An in vitro study, Materials Science and Engineering: C, volume 33, issue 7, october 2013, pages Hubbel J. H., Seltzer S. M., Tables of X-ray Mass Attenuation Coefficients and Mass Energy-Absorption Coefficients from 1 kev to 20 MeV for Elements Z 5 1 to 92 and 48 Additional Substances of Dosimetric Interest (National Institute of Standards and Technologies, July 2004) Zawadzak E., Ryszkowska J., Struktura i właściwości nanokompozytów poliuretanowych otrzymywanych w wyniku zmiennego sposobu wprowadzania tlenku cyrkonu modyfikowanego metalami ziem rzadkich, Czasopismo Techniczne, Wydawnictwo Politechniki Krakowskiej, 6, pages , 2006,

7 4. Crawley E., Evans W., Owen G., A theoretical analysis of the accuracy of singleenergy CT bone measurements Physics in Medicine and Biology, 33 (10) (1988), pages Scheiner S., Sinibaldi R., Pichler B., Komlev V., Renghini C., Vitale-Brovarone C., Rustichelli F., Hellmich C., Micromechanics of bone tissue-engineering scaffolds, based on resolution error-cleared computer tomography, Biomaterials, 30 (12) (2009), pages Hellmich C., Kober C., Erdmann B., Micromechanics-based conversion of CT data into anisotropic elasticity tensors, applied to FE simulations of a mandible Annals of Biomedical Engineering, 36 (2008), pages Dejaco A. et al., From micro-ct to multiscale mechanics of double-porous hydroxyapatite granules for regenerative medicine, Journal of Biomechanics, Volume 45, Issue 6, pages , 2012