Biomaterials (v.28, #15)

Imaging Techniques for Biomaterials Characterization by David G. Castner; Robert E. Guldberg (2379).

Surface plasmon resonance (SPR) sensing has long been used to study biomolecular binding events and their kinetics in a label-free way. This approach has recently been extended to SPR microscopy, which is an ideal tool for probing large microarrays of biomolecules for their binding interactions with various partners and the kinetics of such binding. Commercial SPR microscopes now make it possible to simultaneously monitor binding kinetics on >1300 spots within a protein microarray with a detection limit of ∼0.3 ng/cm2, or <50 fg per spot (<1 million protein molecules) with a time resolution of 1 s, and spot-to-spot reproducibility within a few percent. Such instruments should be capable of high-throughput kinetic studies of the binding of small (∼200 Da) ligands onto large protein microarrays. The method is label free and uses orders of magnitude less of the precious biomolecules than standard SPR sensing. It also gives the absolute bound amount and binding stoichiometry.
Keywords: Surface plasmon resonance microscopy; Surface plasmon resonance imaging; Bioaffinity; Kinetics; Protein arrays; DNA arrays;

Probing molecular recognition sites on biosurfaces using AFM by Vincent Dupres; Claire Verbelen; Yves F. Dufrêne (2393-2402).
Knowledge of the molecular forces that drive receptor–ligand interactions is a key to gain a detailed understanding of cell adhesion events and to develop novel applications in biomaterials science. Until recently, there was no tool available for analyzing and mapping these forces on complex biosurfaces like cell surfaces. During the past decade, however, single-molecule atomic force microscopy (AFM) has opened exciting new opportunities for detecting and localizing molecular recognition forces on artificial biosurfaces and on living cells. In this review, we describe the general principles of the AFM technique, present procedures commonly used to prepare samples and tips, and discuss a number of applications that are relevant to the field of biomaterials.
Keywords: Atomic force microscopy; Biosurfaces; Molecular recognition; Single molecules; Functionalized tips; Receptor–ligand interactions;

Dynamic force microscopy imaging of plasmid DNA and viral RNA by Ferry Kienberger; Lilian T. Costa; Rong Zhu; Gerald Kada; Manuela Reithmayer; Lilia Chtcheglova; Christian Rankl; Ana B.F. Pacheco; Stefan Thalhammer; Vassili Pastushenko; Wolfgang M. Heckl; Dieter Blaas; Peter Hinterdorfer (2403-2411).
Plasmid DNA and viral RNA were imaged in a liquid environment by dynamic force microscopy (DFM) and fine structures of DNA with heights of 1.82±0.66 nm were obtained in topographical images. In simultaneously acquired phase images, DNA could be imaged with better contrast at lower imaging forces. By splitting the cantilever oscillation signal into lower and upper parts, the contribution of the adhesion between tip and sample to the topographical images was eliminated, resulting in better signal-to-noise ratio. DFM of the single stranded RNA genome of a human rhinovirus showed loops protruding from a condensed RNA core, 20–50 nm in height. The mechanical rigidity of the RNA was determined by single molecule pulling experiments. From fitting RNA stretching curves to the Worm-Like-Chain (WLC) model a persistence length of 1.0±0.17 nm was obtained.
Keywords: Atomic force microscope; Dynamic force microscopy; DNA; RNA; Imaging;

Multivariate analysis strategies for processing ToF-SIMS images of biomaterials by Bonnie J. Tyler; Gaurav Rayal; David G. Castner (2412-2423).
Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a hyperspectral imaging technique. Each pixel in a two-dimensional ToF-SIMS image (or each voxel in a three-dimensional (3-D) ToF-SIMS image) contains a full mass spectrum. Thus, multivariate analysis methods are being increasingly used to process biomaterial ToF-SIMS images so the maximum amount of information can be extracted from the images. This study examines the use of principal component analysis (PCA) and maximum autocorrelation factors (MAF) on four different ToF-SIMS images. These images were selected because they represent significant challenges for biomedical ToF-SIMS image processing (topographical features, low count rates, surface contaminants, etc.). With PCA four different types of scaling methods (auto, root mean, filter, and shift variance scaling) were used. The effect of two preprocessing methods (normalization and mean centering) was also examined for both PCA and MAF. The more computational intense MAF provided the best results for all the images investigated in this study, doing the best job of reducing the number of variables required to describe the image, enhancing image contrast and recovering key spectral features. MAF was particularly good at identifying subtle features that were often lost in PCA and impossible to visualize in single peak images. However, the combination of PCA with either root mean or shift variance scaling provided similar results to MAF. Thus, these combinations offer promising alternatives to MAF for working with large data sets encountered in 3-D imaging. Also, the new method of filter scaling is promising for processing low count rate images with salt and pepper noise. Normalization proved an important tool for deconvoluting chemical effects from topographic and/or matrix effects. Mean centering aided in reducing the dimensionality of the data, but in one case resulted in a loss of information.
Keywords: Image analysis; Molecular imaging; SIMS; Surface analysis; Micro-patterning;

The current paradigm in designing biomaterials is to optimize material chemical and physical parameters based on correlations between these parameters and downstream biological responses, whether in vitro or in vivo. Extensive developments in molecular design of biomaterials have facilitated identification of several biophysical and biochemical variables (e.g. adhesion peptide density, substrate elastic modulus) as being critical to cell response. However, these empirical observations do not indicate whether different parameters elicit cell responses by modulating redundant variables of the cell–material interface (e.g. number of cell–material bonds, cell–matrix mechanics). Recently, fluorescence resonance energy transfer (FRET) has been applied to quantitatively analyze parameters of the cell–material interface for both two- and three-dimensional adhesion substrates. Tools based on FRET have been utilized to quantify several parameters of the cell–material interface relevant to cell response, including molecular changes in matrix proteins induced by interactions both with surfaces and cells, the number of bonds between integrins and their adhesion ligands, and changes in the crosslink density of hydrogel synthetic extracellular matrix analogs. As such techniques allow both dynamic and 3-D analyses they will be useful to quantitatively relate downstream cellular responses (e.g. gene expression) to the composition of this interface. Such understanding will allow bioengineers to fully exploit the potential of biomaterials engineered on the molecular scale, by optimizing material chemical and physical properties to a measurable set of interfacial parameters known to elicit a predictable response in a specific cell population. This will facilitate the rational design of complex, multi-functional biomaterials used as model systems for studying diseases or for clinical applications.
Keywords: Extracellular matrix (ECM); Integrin; Fibronectin (FN); Fluorescence; Green fluorescent protein (GFP);

Non-invasive evaluation of alginate/poly-l-lysine/alginate microcapsules by magnetic resonance microscopy by Ioannis Constantinidis; Samuel C. Grant; Susanne Celper; Isabel Gauffin-Holmberg; Kristina Agering; Jose A. Oca-Cossio; Jonathan D. Bui; Jeremy Flint; Christine Hamaty; Nicholas E. Simpson; Stephen J. Blackband (2438-2445).
In this report, we present data to demonstrate the utility of 1H MR microscopy to non-invasively examine alginate/poly-l-lysine/alginate (APA) microcapsules. Specifically, high-resolution images were used to visualize and quantify the poly-l-lysine (PLL) layer, and monitor temporal changes in the alginate gel microstructure during a month long in vitro culture. The thickness of the alginate/PLL layer was quantified to be 40.6±6.2 μm regardless of the alginate composition used to generate the beads or the time of alginate/PLL interaction (2, 6, or 20 min). However, there was a notable difference in the contrast of the PLL layer that depended upon the guluronic content of the alginate and the alginate/PLL interaction time. The T 2 relaxation time and the apparent diffusion coefficient (ADC) of the alginate matrix were measured periodically throughout the month long culture period. Alginate beads generated with a high guluronic content alginate demonstrated a temporal decrease in T 2 over the duration of the experiment, while ADC was unaffected. This decrease in T 2 is attributed to a reorganization of the alginate microstructure due to periodic media exchanges that mimicked a regular feeding regiment for cultured cells. In beads coated with a PLL layer, this temporal decrease in T 2 was less pronounced suggesting that the PLL layer helped maintain the integrity of the initial alginate microstructure. Conversely, alginate beads generated with a high mannuronic content alginate (with or without a PLL layer) did not display temporal changes in either T 2 or ADC. This observation suggests that the microstructure of high mannuronic content alginate beads is less susceptible to culture conditions.
Keywords: MR imaging; Alginate encapsulation; Poly-l-lysine;

Advances in nanotechnology, in particular the development of novel types of nanoparticles, will result in advanced tools for biomedical research and clinical practice. One exciting aspect of future nanomaterial research will be the possibility to combine therapy and imaging in multifunctional nanoparticle designs. In this context, anisotropic particles with subcellular dimensions may offer so far unattainable capabilities, because they could provide access to directional information with respect to nanoparticle–cell interactions. We have recently developed an electrified jetting process, which can produce water-stable polymer particles with two distinct phases.To address the first critical hurdle towards the application of these biphasic nanocolloids as imaging probes, short-term biocompatibility was evaluated using model cell culture systems. Exposure of human endothelial cells and murine fibroblasts to biphasic nanocolloids made of 0.5% polyacrylic acid and 4.5% poly(acrylamide-co-acrylic acid) did not affect cell proliferation as determined by a colorimetric proliferation assay. Moreover, double staining with Annexin V and propidium iodide and subsequent flow cytometric analysis indicated high cell viability, although slightly decreased viability was observed at the highest dose tested (1 mg particles/106 seeded cells). Particle internalization as well as surface binding occurred simultaneously for both cell types, as evidenced by confocal laser scanning microscopy. Taken together, these results suggest excellent short-term biocompatibility in physiological systems for wide concentration ranges of the biphasic nanocolloids and open possibilities for future work investigating receptor- or surface marker-mediated targeting.
Keywords: Nanocolloids; Biocompatibility; Janus particles; Polymers;

Particles currently used in arterial embolization therapy have several disadvantages, most importantly their radiolucency. This means the radiologist cannot precisely asses the fate of embolization particles. Microspheres that combine two additional features have been designed. By incorporating an iodine containing monomer, radiopaque microspheres were obtained that display good visibility under standard X-ray conditions. Incorporation of methacrylic acid makes the surface of the spheres suitable for surface functionalization. Here, thrombin was covalently attached to the surface of the radiopaque microspheres. By induction of a thrombus, improved anchoring of the embolization spheres in the blood vessel can be obtained. The immobilized thrombin induced a biphasic response of the blood namely: (1) fast deposition of fibrin on the surface resulting in sphere aggregation and (2) additional thrombin generation in the surrounding blood and a subsequent local thrombus formation. These microspheres with both intrinsic X-ray visibility and a biofunctionalized surface can potentially improve embolization therapies.
Keywords: Microsphere; Radiopacity; Embolization; Thrombin; Coagulation;

FT-IR imaging of native and tissue-engineered bone and cartilage by Adele Boskey; Nancy Pleshko Camacho (2465-2478).
Fourier transform infrared (FT-IR) imaging and microspectroscopy have been extensively applied to the analyses of tissues in health and disease. Spatially resolved mid-IR data has provided insights into molecular changes that occur in diseases of connective or collagen-based tissues, including, osteoporosis, osteogenesis imperfecta, osteopetrosis and pathologic calcifications. These techniques have also been used to probe chemical changes associated with load, disuse, and micro-damage in bone, and with degradation and repair in cartilage. This review summarizes the applications of FT-IR microscopy and imaging for analyses of bone and cartilage in healthy and diseased tissues, and illustrates the application of these techniques for the characterization of tissue-engineered bone and cartilage.
Keywords: Bone tissue engineering; Cartilage tissue engineering; Bone; Cartilage vibrational spectroscopy; Review;

Nondestructive micro-computed tomography for biological imaging and quantification of scaffold–bone interaction in vivo by G. Harry van Lenthe; Henri Hagenmüller; Marc Bohner; Scott J. Hollister; Lorenz Meinel; Ralph Müller (2479-2490).
Scaffolds, also called bioscaffolds, are needed in all tissue engineering applications as carriers for cells and biochemical factors, as constructs providing appropriate mechanical conditions, or as a combination of the two. The aim of this paper is to present recent developments in micro-computed tomography (μCT) analyses of scaffolds. The focus will be on imaging and quantification aspects in bone research, and will deal with the assessment of scaffold architecture and how it interacts with bone tissue. We show that micro-architectural imaging is a nondestructive and noninvasive procedure that allows a precise three-dimensional (3D) measurement of scaffold architecture. Direct μCT-based image analysis allows to accurately quantify scaffold porosity, surface area, and 3D measures such as pore size, pore distribution, and strut thickness; furthermore, it allows for a precise measurement of bone growth into the scaffold and onto its surface. This methodology is useful for quality control of scaffold fabrication processes, to assess scaffold degradation kinetics, and to assess bone tissue response. Even more so, in combination with bioreactors or in vivo animal models, μCT allows to qualitatively and quantitatively assess the spatial and temporal mineralization of bone tissue formation in scaffolds; such longitudinal studies improve the assessment of bone response due to scaffold architecture. Computational models will be helpful in further analyses of these data in order to improve our understanding of mechanical and biochemical stimuli on bone formation, and are likely to provide valuable knowledge to optimize scaffold design.
Keywords: Scaffold; Micro-computed tomography; Micro-structure; Bone tissue engineering; Bioreactor;

Assessment of bone ingrowth into porous biomaterials using MICRO-CT by Anthony C. Jones; Christoph H. Arns; Adrian P. Sheppard; Dietmar W. Hutmacher; Bruce K. Milthorpe; Mark A. Knackstedt (2491-2504).
The three-dimensional (3D) structure and architecture of biomaterial scaffolds play a critical role in bone formation as they affect the functionality of the tissue-engineered constructs. Assessment techniques for scaffold design and their efficacy in bone ingrowth studies require an ability to accurately quantify the 3D structure of the scaffold and an ability to visualize the bone regenerative processes within the scaffold structure. In this paper, a 3D micro-CT imaging and analysis study of bone ingrowth into tissue-engineered scaffold materials is described. Seven specimens are studied in this paper; a set of three specimens with a cellular structure, varying pore size and implant material, and a set of four scaffolds with two different scaffold designs investigated at early (4 weeks) and late (12 weeks) explantation times. The difficulty in accurately phase separating the multiple phases within a scaffold undergoing bone regeneration is first highlighted. A sophisticated three-phase segmentation approach is implemented to develop high-quality phase separation with minimal artifacts. A number of structural characteristics and bone ingrowth characteristics of the scaffolds are quantitatively measured on the phase separated images. Porosity, pore size distributions, pore constriction sizes, and pore topology are measured on the original pore phase of the scaffold volumes. The distribution of bone ingrowth into the scaffold pore volume is also measured. For early explanted specimens we observe that bone ingrowth occurs primarily at the periphery of the scaffold with a constant decrease in bone mineralization into the scaffold volume. Pore size distributions defined by both the local pore geometry and by the largest accessible pore show distinctly different behavior. The accessible pore size is strongly correlated to bone ingrowth. In the specimens studied a strong enhancement of bone ingrowth is observed for pore diameters>100 μm. Little difference in bone ingrowth is measured with different scaffold design. This result illustrates the benefits of microtomography for analyzing the 3D structure of scaffolds and the resultant bone ingrowth.
Keywords: Micro-CT; Bone ingrowth; Hydroxyapatite; Scaffolds; Pore structure;

Bulk and interface investigations of scaffolds and tissue-engineered bones by X-ray microtomography and X-ray microdiffraction by R. Cancedda; A. Cedola; A. Giuliani; V. Komlev; S. Lagomarsino; M. Mastrogiacomo; F. Peyrin; F. Rustichelli (2505-2524).
This review is presented of recent investigations concerning the structure of ceramic scaffolds and tissue-engineered bones and focused on two techniques based on X-ray radiation, namely microtomography (microCT) and microdiffraction. Bulk 3D information, with micro-resolution, is mainly obtained by microCT, whereas microdiffraction provides useful information on interfaces to the atomic scale, i.e. of the order of the nanometer. Since most of the reported results were obtained using synchrotron radiation, a brief description of the European Synchrotron Radiation Facility (ESRF) is presented, followed by a description of the two techniques. Then examples of microstructural investigations of scaffolds are reported together with studies on bone architecture. Finally, studies on ex vivo tissue-engineered bone and on bone microstructure in vivo are presented.
Keywords: Bone tissue engineering; Bone marrow; Scaffold; Calcium phosphate; Image analysis; XRD (X-ray diffraction);

Noninvasive image analysis of 3D construct mineralization in a perfusion bioreactor by Blaise D. Porter; Angela S.P. Lin; Alexandra Peister; Dietmar Hutmacher; Robert E. Guldberg (2525-2533).
Although the beneficial effects of perfusion on cell-mediated mineralization have been demonstrated in several studies, the size of the mineralized constructs produced has been limited. The ability to quantify mineralized matrix formation non-invasively within 3D constructs would benefit efforts to optimize bioreactor conditions for scaling-up constructs to clinically relevant dimensions. In this study, we report a micro-CT imaging-based technique to monitor 3D mineralization over time in a perfusion bioreactor and specifically assess mechanisms of construct mineralization by quantifying the number, size, and distribution of mineralized particle formation within constructs varying in thickness from 3 to 9 mm. As expected, mineralized matrix volume and particle number increased with construct thickness. Analyzing multiple concentric volumes inside each construct indicated that a greater proportion of the mineral volume was found within the interior of the perfused constructs. Interestingly, intermediate-sized 6 mm thick constructs were found to have the highest core mineral volume fraction and the largest mineralized particles. Two complementary mechanisms of increasing total mineral volume were observed in the 6 and 9 mm constructs: increasing particle size and increasing the number of mineralized particles, respectively. The rate of mineralized matrix formation in the perfused constructs increased from 0.69 mm3/week during the first 3 weeks of culture to 1.03 mm3/week over the final 2 weeks. In contrast, the rate of mineral deposition in the static controls was 0.01 mm3/week during the first 3 weeks of culture and 0.16 mm3/week from week 3 to week 5. The ability to monitor overall construct mineralization non-invasively coupled with quantitative analysis of mineralized particle size, number, and distribution offers a powerful tool for elucidating how mineral growth mechanisms are affected by cell type, scaffold material and architecture, or bioreactor flow conditions.
Keywords: Bioreactor; Bone tissue engineering; Image analysis;