Interactions between tissues, cells, and biomaterials: an advanced evaluation by synchrotron radiation-based high-resolution tomography

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Interactions between tissues, cells, and biomaterials: an advanced evaluation by synchrotron radiation-based highresolution tomography

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Alessandra Giuliani1, Serena Mazzoni1, Adrian Manescu2 and Giuliana Tromba3 1

Department of Clinical Sciences, Universita` Politecnica delle Marche, Ancona, Italy 2 “Victor Babes” University of Medicine and Pharmacy, Timisoara, Romania 3 Sincrotrone Trieste S.C.p.A, Trieste, Italy

1.1 CONDUCTION, INDUCTION, AND CELL TRANSPLANTATION IN TISSUE ENGINEERING: THE LIMITATIONS OF CROSS-TALK STUDIES BY CONVENTIONAL TECHNIQUES Tissue loss or damage due to congenital defects, disease, and injury are major clinical problems. The majority of anatomical districts are comprised of several tissues for which the preferred method of replacement is through autologous grafting. For instance, autografts are standard for bone grafts because of their biocompatibility, immunogenic characteristics, and because they offer all the essential properties required: they achieve osteoinduction through bone morphogenetic proteins (BMPs) and other growth factors (GFs); osteogenesis, by means of osteoprogenitor cells; and osteoconduction, because autografts are implanted in the shape of 3D porous matrixes (Amini et al., 2012). However, there is often insufficient host tissue for a complete repair of a defect; or for specific diseases, sites, and tissues, the replacement is clinically forbidden. Furthermore, autologous transplants entail extremely expensive procedures, often inducing significant donor site injury and morbidity (Alsberg et al., 2001; Iezzi et al., 2016).

Materials for Biomedical Engineering: Hydrogels and Polymer-based Scaffolds. DOI: https://doi.org/10.1016/B978-0-12-816901-8.00001-8 © 2019 Elsevier Inc. All rights reserved.

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In this context, tissue engineering aims to restore function to or replace damaged or diseased tissues through the application of biological and engineering rationales (Alsberg et al., 2001). The approaches normally adopted, alone or in combination, include: conduction, induction, and cell (normally stem cell) transplantation (Langer and Vacanti, 1993; Putnam and Mooney, 1996). The choice of the correct approach depends on several factors, including the site and the size of the defect, the availability of cells in surrounding areas, cell migration kinetics, and the presence or absence of sufficient vascularization. In conductive approaches (Fig. 1.1, panels A and B), the biomaterial acts as a 3D matrix for endogenous cells migration, grafting, proliferation, and differentiation. These cells form the new tissue that, hopefully, will be integrated with the host tissue and with the grafted biomaterial that, depending on its composition, may or may not degrade over time. Sometimes, however, endogenous cell migration, differentiation of these cells, and the following tissue formation are processes that need to be controlled. In this case, an inductive approach to tissue engineering appears to be the best solution (Fig. 1.1, panels C and D). Bioactive scaffolds, GFs, drug, or plasmid DNA delivery are used to induce cell migration and control cellular behavior. Conductive and inductive methods are usually used in cases of limited defects, as in most maxillary or mandibular bone sites, when the presence of optimized biomaterials promote cell migration from the host tissue into the scaffolds. However, the repair of large defects often also requires the direct transplantation of cells. This approach (Fig. 1.1, panels E and F) is also required when there are not enough available cells in the tissue surrounding the defect, or when endogenous cell migration to the damaged site would require too long a time (Alsberg et al., 2001). In these cases, a biopsy is usually taken from a donor source, cells are isolated and expanded in vitro, and then seeded on a duly prepared scaffold to proliferate and form new tissue. In turn, this tissue is normally implanted into the damaged area. Autologous cells give insignificant immune response, but have the disadvantage of requiring a long time to expand to the needed quantities. Allogeneic cells, which are genetically different cells from the same species as the patient and even more so xenogeneic cells derived from a different species than the patient, present the opposite problems: they are ready available but strongly increase the possibility of immunological reactions. This is the case, for example, when trying to repair muscle damage in Duchenne muscular dystrophy (DMD) by transplanting myogenic progenitors directly into the muscles. In fact, this procedure has shown to suffer from the problems of limited cell survival and reduced migration of these cells in the muscles (Farini et al., 2012). In particular, cell therapies consist of the use of stem cell populations that have been previously manipulated and cultured in vitro, with the objective of repairing and regenerating damaged tissues. In this context, the presence in the heart of primitive cells, able to generate the different structures of the myocardium, has been recently documented (Giuliani, 2012).

1.1 Conduction, Induction, and Cell Transplantation

FIGURE 1.1 Description of the three approaches to tissue engineering. (A and B) Conduction—A scaffold controls and selects cells infiltrating the defect site from the outside in order to repair it. The scaffold may be resorbed in time or surgically removed. (C and D) Induction—Bioactive molecules bind with selected host cells (with receptors for the molecules) that migrate to the defect site forming a new extracellular matrix. (E and F) Cell transplantation—Cells are transplanted from a donor source to a scaffold. Afterwards, the cell/scaffold construct is grafted into the defect site to achieve tissue regeneration in conjunction with host cells that migrate to the defect site.

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The monitoring of the longitudinal outcomes of conduction, induction, and cell therapy requires the use of nondestructive methods that are capable of identifying the location, amount, and extent of cellular survival and fate, as well as of evaluating, qualitatively and quantitatively, the tissue growth under different conditions. This type of study has been frequently performed in the recent literature, for instance to engineer bone (Appel et al., 2013; Giuliani et al., 2011, 2014a; Olubamiji et al., 2014), cartilage (Olubamiji et al., 2014; Zehbe et al., 2010a), and tendon (Gigante et al., 2013). In this context, imaging techniques are assuming an increasingly important role, not only for a rigorous characterization of the properties and functions of biomaterials, but also to investigate the kinetics of their biological behavior in conduction, induction, and cell transplantation processes. Several advanced 2D imaging technologies are available to complement histological evaluation and to study complex biological events occurring at the interface between tissues and biomaterials (Appel et al., 2013; Nam et al., 2015). However, 2D imaging technologies present several limitations. In fact (Zehbe et al., 2010a), the current methods of optical microscopy require a sectioning of serial sections that takes a long time and they are severely limited in their ability to analyze opaque 3D biological structures. Furthermore, although electron microscopy (EM) methods offer good 3D topographic representation (by EM scanning), highresolution imaging is restricted to extremely thin samples (by transmission EM). Moreover, for the previously mentioned techniques, sample preparation sometimes causes significant damage to the specimen, practically inhibiting the visualization and quantification of the cells and tissues spatial distribution, present and formed within porous biomaterials in vitro and in vivo conditions. Furthermore, for regeneration of vascularized tissues, such as bone or muscle, there is a need for imaging techniques able to quantify 3D vascular ingrowth, particularly for recent innovative studies focused on exploring the potential to enhance regeneration via therapeutic angiogenesis strategies (Langer et al., 2009; Appel et al., 2013; Giuliani et al., 2017). The imaging modality most extensively applied for this purpose, especially for bone tissue engineering studies (Giuliani et al., 2013; Manescu et al., 2016a; Komlev et al., 2009; Belicchi et al., 2009), is high-resolution X-ray computed tomography (microCT). In particular, the use of synchrotron-produced X-rays has several advantages with respect to X-rays produced by laboratory or industrial sources. In this chapter, several major experiences applying synchrotron radiation (SR)-based microCT to explore regeneration in bone, tendons, cartilage, and skeletal and cardiac muscle sites were reviewed. Conductive, inductive, and cell transplantation approaches were overviewed, although different emphasis was given to each strategy depending on the specified engineered tissue. In addition, innovative protocols were explored to track donor cells after transplantation in order to clarify their role in tissue regeneration.

1.2 X-Ray Computed Microtomography: A Challenging Diagnostic Tool

1.2 X-RAY COMPUTED MICROTOMOGRAPHY: A CHALLENGING DIAGNOSTIC TOOL A new concept of diagnostic by imaging was developed at the beginning of the 1970s, when the first equipment for X-rays computed tomography (CT) was produced. The CT technique overcame several limitations of conventional X-ray radiology, many of which were mainly due to the 2D nature of the radiological images that had been used up to that moment in medical diagnostics. Indeed, conventional and digital X-ray radiology are imaging methods with constraints linked to their two-dimensionality: radiographs provide a 2D image of a 3D object, not accurately replicating the anatomy that is being assessed. Anatomical structures may superimpose causing misleading signals for radiograph interpretation. Indeed, 2D radiographs usually show minor structure damages compared to those actually present and do not reveal the interactions between soft and hard tissues (Manescu et al., 2016b). For these reasons, the diagnostic contribution provided by the CT was pioneering, allowing for the visualization of internal details of the sample with unprecedented precision, in a nondestructive way and achieving a contrast up to 1000 times better than conventional radiography (Claesson, 2001). Tomography data are a collection of cross-sectional images obtained from either transmission or reflection configurations and collected by illuminating the sample from many different directions (Kak and Slaney, 1988). The first applications were in diagnostic medicine, but today CT is a characterization technique also used in several nonmedical imaging applications. X-ray microtomography (microCT) exploits the same physical principles of conventional CT usually used in medical diagnostics, but, unlike this, reaches a spatial resolution of up to 0.1 μm (Weitkamp et al., 2010), that is, about three orders of magnitude higher. The number of projections and of data points per projection define the spatial resolution: thus, large datasets contain more information (i.e., more pixels in a smaller object is equivalent to better spatial resolutions). Indeed, the choice of spatial resolution versus overall sample size is a crucial issue in microCT. Both 3D conventional CT and microCT employ X-rays to virtually reconstruct the samples of interest based on their attenuation coefficient. In an ideal setup, the sample must absorb a value close to and at most equal to 90% of the incident photons: in this configuration, it is expected to achieve the best signal-to-noise ratio. Tomographic images are reconstructed starting from its projections, that is, the radiographies acquired over 180
sample rotation (or reciprocal rotation of the detection system around a fixed object). Hundreds of 2D projection radiographs are taken at several different angles: each of them is a projection of absorption density distribution, in the direction of the photons, onto the plane perpendicular

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to the direction of the beam. This means that, if the sample is imaged in different orientations, 3D (i.e., volumetric) information on the sample structure can be obtained in a second phase using computer algorithms. This second phase, referred to as “tomographic image reconstruction,” is based on solving an inverse problem, estimating an image from its line integrals on different directions, and is theoretically equivalent to the inversion of the Radon Transform of the image. Indeed, in 1917 Radon solved the problem to reconstruct a function from radiographic projections: this finding was exploited as Hounsfield’s invention of the X-ray computed tomographic scanner for which the same Hounsfield received the Nobel Prize in 1972. Nowadays, there are two types of algorithms with different approaches (Penczek, 2010) for the reconstruction phase: transform-based methods which exploit analytic inversion formulae; and series expansion methods based on linear algebra. The traditional reconstruction algorithm used in most practical applications of microCT is the filtered back projection (FBP), a Fourier-based technique. FBP is derived from the Fourier slice theorem, described in detail elsewhere (Kak and Slaney, 1988). The advantages of FBP are that its implementation is straightforward and execution is relatively fast. Another approach is based on iterative reconstruction algorithms: these give an initial guess of the attenuation coefficients (Webb, 2003) and compare such estimations with those actually acquired. The correction of the initial matrix is made in an iterative way for each projection in a first step and for the whole dataset in a second step until the residual error between the measured data and estimated matrix falls below a predesignated value. Iterative schemes are scarcely used in standard CT. 3D renderings of the data obtained after the reconstruction are easily obtained by stacking up the slices and may be sectioned in arbitrary ways to better locate and quantify the details. Indeed, if the 2D slices and the 3D reconstructions are fascinating tools to perform qualitative observations of the internal structure of biomaterials, cells and tissues, the real benefit is the quantitative information that can be extracted from 3D datasets (Ohgushi et al., 1989). Different methods may be applied to extract quantitative architectural parameters from tomographic images. For instance, in the field of bone research, different protocols have been proposed for bone microarchitecture quantification. The 3D mean intercept length method provides a good estimation of trabecular thickness and spacing based on structural geometry assumptions, for example, parallel plate model (Hildebrand and Ruegsegger, 1997a). However, using 3D images such assumptions can be avoided, allowing for the achievement of new model-independent quantitative parameters (Hildebrand and Ruegsegger, 1997b).

1.3 HI-RES Tomography

1.3 INNOVATIVE APPROACHES TO HIGH-RESOLUTION TOMOGRAPHY BY SYNCHROTRON RADIATION By properly selecting the photon energy, the interaction between X-rays and biological structures provides semitransparency of tissues, allowing penetration of even large specimens. Based on the algorithms described in the previous paragraph, angular projections can be used for tomographic imaging. The imaging of cells inside biological tissues is challenging with conventional microCT devices, because of several experimental conditions linked to the limited spatial and structural resolution. A major problem is the low difference in density and absorption contrast between cells and surrounding tissue. Therefore, a monochromatic X-ray beam, sufficiently high photon flux, and coherent beam properties are key requirements, currently only achieved with synchrotron light (Zehbe et al., 2010a). SR is an electromagnetic light created when charged particles (for instance electrons) are emitted by an electron gun and are then linearly accelerated by an electric field. Next, the particles are further accelerated to near the speed of light in two connected rings (the first, named the “booster ring,” and the second the “storage ring”). In the storage ring, as the electrons travel round the ring, they pass through different types of magnets and, in the process, they produce X-rays. Indeed, these magnets cause the electrons to change direction: this results in a change in their velocity vector and, consequently, in the emission of SR. In particular, when the electron is moving fast enough, the emitted energy is at X-ray wavelength (Giuliani et al., 2014b; Olubamiji et al., 2014; http://www.esrf.eu/ about/synchrotron-science/synchrotron). The properties of SR significantly improve contrast sensitivity in X-ray imaging systems (Cancedda et al., 2007; Giuliani et al., 2010), as specifically discussed in detail in the next paragraphs for the different anatomical districts. X-rays produced by synchrotron facilities have several advantages compared to those delivered by desktop laboratory sources. In particular, they offer several possibilities: 1. to get a high photon flux, achieving measurements with high signal-to-noise ratio as well as high spatial resolutions; 2. to tune the X-ray source, allowing measurements at different energies; 3. to set a specific monochromatic X-ray radiation, eliminating the beam hardening effects; 4. to perform parallel beam acquisitions, allowing the use of specific tomographic reconstruction algorithms. Due to the listed advantages offered by synchrotron light sources, several 3D imaging techniques have been developed in order to serve and support biomedical research.

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In a pure absorption experimental setup, the sample is as close as possible to the detector, with a minimization of image blurring. At synchrotron facilities, parallel photon beams are made available from a wide cross-sectional source; they pass through the sample and are collected by a 2D detector. Being the X-ray beam parallel, the projection of each slice of the object on the detector is not dependent of all the other slices (Stock, 2009). Absorption tomography (Fig. 1.2, panel A) is based on the Beer-Lambert law, which describes that the intensity of monochromatic X-rays that transmit the specimen decreases exponentially as a function of the line integral of the linear attenuation coefficients along the X-ray path:  ðN  I 5 I0 3 exp 2 μðxÞdx

(1.1)

2N

where μ is the linear absorption coefficient at position x along a particular beam direction. Tomography solves the problem of assigning the correct values of μ to each position along the photon path, knowing only the values of the line integral but for a large number of paths through the sample (i.e., rays sent through the sample from different angles). As they pass through the different phases of the sample, the photons interact in the electron shells of the atoms they pass. Absorption tomography exploits that the attenuation of X-ray beams of a given energy varies with the atomic electron density of the imaged material and its bulk density. The differences in the X-ray attenuation rate within the samples are represented by different peaks in the gray-scale histogram, corresponding to the different phases. However, by modulating the sample-to-detector distance, contrast is also generated by phase differences among the scattered X-ray waves (Fig. 1.2, panel B). In particular, this phase-contrast (PhC) effect puts into evidence the interface and edges between two materials, and it is particularly useful when media with similar absorption coefficients should be discriminated (Fiori et al., 2012). Indeed, differently from conventional X-ray tomography, in the PhC approach, the image contrast is not based solely on attenuation of the beam. In this case, the effect of an X-ray beam penetrating the sample is described by the refractive index, n 5 1 2 δ 1 iβ

(1.2)

where δ is the refractive index decrement and β is the attenuation index. δ is actually proportional to the mean electron density of the specific phase, which in turn is nearly proportional to its mass density. Moreover, the δ value is much larger than the imaginary part β in nonmineralized tissues, suggesting that the phase approach provides greater sensitivity than the absorption approach when studying these tissues. The methods used for the reconstruction of the refractive index n are typically based on a two-step approach: the phase projections are extracted in the first step while the refractive index decrement δ is reconstructed by applying a conventional FBP or similar algorithms in the second step (Giuliani et al., 2014a,b).

1.3 HI-RES Tomography

FIGURE 1.2 (A) Standard (absorption-based) tomography—The sample is mounted on a translationrotation stage (standard SR-microCT setup). The detector (made up of a scintillator, light microscope optics, and a CCD) is mounted on a translation stage. Projections are acquired with the detector close to the sample and, afterwards, are processed with the filtered back projection algorithm for the reconstruction of the 3D absorption index. (B) Propagation-based phase-contrast imaging—The imaging source consists, also in this case, of monochromatized SR X-rays. The long source to sample distance yields a high degree of spatial coherence. The detector, mounted on a translation stage at a long distance from the sample, allows free space propagation of the beam after the sample. In this case, the application of the FBP algorithm produces an edge-enhancement effect, which is proportional to the Laplacian of the refractive index. (C) In-line phase tomography (holotomography), from Ref. (Giuliani et al., 2013)—The tomographic acquisition is performed with the detector distances (D1, D2,. . ., Dn) from the sample. For each rotation angle, a phase retrieval algorithm is applied to projections acquired at each distance, providing the phase maps, in turn, are then processed with the FBP algorithm to recover the 3D refractive index decrement.

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For the first phase (Langer et al., 2010), two main classes of algorithms can be identified in literature: they are alternatively based on a linearization with respect to the propagation distance, which yields what is known as the transport of intensity equation (TIE) (Teague, 1982a,b), or on a linearization with respect to the object, which yields the contrast transfer function (Cloetens et al., 1999). Furthermore (Manescu et al., 2016a,b), phase retrieval usually implies the reconstruction of the two different real-valued 3D distributions, δ and β; such reconstruction generally requires the acquisition of 2D projections, at least at two different sample-detector distances at each view angle (Fig. 1.2, panel C). However, in some cases, it can be shown a priori that the real and imaginary parts of the refractive index are proportional to each other, that is: β5ε δ

(1.3)

where the proportionality constant ε does not depend on the spatial coordinates. This assumption is possible only for special classes of objects, such as pure phase (i.e., weakly absorbing) objects, or homogeneous objects, such as objects consisting predominantly of a single material (possibly with a spatially varying density) (Gureyev et al., 2004, 2006, 2009). Moreover (Giuliani et al., 2014a,b), Bronnikov suggested an algorithm providing a direct reconstruction of the refractive index and avoiding the first phase retrieval step. It established a fundamental relation between the 3D Radon transform of the object function and the 2D Radon transform of the phase-contrast projection (Bronnikov, 2000). Thus, a reconstruction algorithm is derived in the form of a FBP. In recent years, there has been increasing interest in the mentioned approaches to evaluate different biomaterials’ performance by means of SR-microCT. Tissue regeneration derived from hosting sites grafting with different types of biomaterials (with or without stem cells seeding), was recently explored using SRmicroCT (Cancedda et al., 2007; Giuliani et al., 2014a, 2016; Rominu et al., 2014; Gigante et al., 2013). Evaluation of newly formed tissue is usually based on histology, by observation of one or more sections; however, conventional histological evaluation and corresponding histomorphometric measurements provide only 2D information with the consequent risk that the selected sections do not properly represent the entire biopsy specimen (Giuliani, 2016). Furthermore, if the involvement of neighboring tissues with different morphology (bone, unmineralized extracellular matrix, regenerated vessels, etc.) on the regeneration process of defects or tissues is unknown or still not clearly verified, 3D analyzing methods, including high-resolution SR-microCT, are indicated to explore the dynamic and spatial distribution of regenerative phenomena in these anatomic structures. Traditionally, absorption imaging with SRmicroCT in medical applications was performed with almost no distance between sample and detector, obtaining significant information on morphometric distribution of the bioengineered structures (Cancedda et al., 2007; Giuliani et al., 2014a, 2016; Renghini et al., 2013; Pozdnyakova et al., 2010). However, homogeneous materials,

1.4 Skeletal Tissue Engineering

with a low attenuation coefficient (like collagen, polymers, thermoset and thermoplastic matrices, unmineralized extracellular matrix, vessels, nerves, etc.), or heterogeneous materials, with a narrow range of attenuation coefficients (like the case of heterologous bone scaffolds or graded mineralized bone), produce insufficient contrast for absorption imaging. For such materials, the imaging quality can be enhanced through the use of PhC-microCT, with an increased distance between sample and detector (Manescu et al., 2016a,b; Giuliani et al., 2014b; Albertini et al., 2009). In addition, whereas PhC-microCT can be based on a single distance between the detector and the sample (for the special classes of objects previously mentioned), holotomography (HT) involves PhC imaging at several distances (Fig. 1.2, panel C), then combining the phase shift information to generate 3D reconstructions. HT is helpful when the material of interest has extremely small variations in attenuation coefficients, which lead to unsatisfactory imaging results even with phase-contrast techniques on a single-distance and the phase retrieval algorithms previously described (Giuliani et al., 2013; Giuliani, 2016).

1.4 SKELETAL TISSUE ENGINEERING 1.4.1 BONE Bone structures have fundamental functions in the body. When congenital defects, trauma, or diseases are present, there is a significant need for bone replacement (Alsberg et al., 2001). The combination of living cells, biologically active molecules, and a structural scaffold to form a construct able to promote the repair and regeneration of bone is the fundamental concept underlying bone engineering. The scaffold plays a crucial role, being expected to support cell colonization, migration, growth, and differentiation. In parallel with bone formation, the scaffold may undergo degradation, releasing products that have to be biocompatible or that are easily excreted or subjected to metabolism (Hutmacher et al., 2007). In this context, imaging techniques, including SR-PhC-microCT investigations, were extensively applied to investigate the properties of several biomaterials proposed to act as scaffolds. Since each bone site performs multiple functional roles, it is unlikely that a single scaffold would serve as a universal support for the regeneration of bone tissue. The considerations for scaffold design are, hence, complex and include material composition, architecture, structural mechanics, surface properties, degradation properties and products, together with the composition of any added biological component and, of course, the changes in all of these factors with time (Hutmacher et al., 2007). Allowing an accurate 3D examination of samples, SR-based microCT was not only employed to reconstruct, at high resolution, the complex architecture of bone

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tissue at different scales (Langer et al., 2012; Peyrin et al., 2014; Giuliani et al., 2018a) and in different genetic and environmental conditions (Tavella et al., 2012; Costa et al., 2013; Canciani et al., 2015; Giuliani et al., 2018b), but it is also increasingly becoming a powerful tool for engineered bone characterization in different skeletal sites. In this scenario, interesting microCT studies have been performed on different biomaterials that have previously been indicated as bone-substitute (Olubamiji et al., 2014). SR-microCT was exploited by Yue (Yue et al., 2010) to characterize the scaffold morphology, the mineral distribution within scaffold pores, and the tissue ingrowth in 4-week-old explants of a bioactive glass foam scaffold implanted between the muscle and tibia of a mouse. Similarly, SR-microCT was used to successfully identify scaffold architecture and bone ingrowth into cell-loaded hydroxyapatite scaffolds implanted in immunodeficient mice for 8 weeks (Mastrogiacomo et al., 2004). The bone ingrowth was estimated in terms of total volume fraction, distribution, and thickness in the pores of the implant and the scaffold architecture was analyzed in terms of the porosity and spatial distribution of walls. The same group explored the ability of SR-microCT to examine the progressive resorption of and bone ingrowth into scaffolds implanted in immunodeficient mice for repair times of 8, 16, or 24 weeks (Cancedda et al., 2007; Papadimitropoulos et al., 2007; Komlev et al., 2006). When using a hydroxyapatite scaffold (Engipore), a single peak was observed in the X-ray absorption histogram before implantation, corresponding to the biomaterial used for the manufacturing of the scaffold itself (Fig. 1.3, panel A). After implantation, an additional peak was observed at lower X-ray absorption values, corresponding to the newly formed bone (Fig. 1.3, panel B). It is possible to observe that the newly formed bone peak shifted to higher values of linear attenuation coefficient with the increasing of the implantation time: this is explained by the progressive mineralization of the bone. In dental districts, successful bone regeneration using biphasic calcium phosphate materials was reported in some clinical applications for maxillary sinus elevation (Mangano et al., 2013; Ohayon, 2014). Special morphologies of 3D scaffolds, in the shape of granules or structured blocks, were shown to realize promising scaffolds to be used either in an acellular strategy (pure scaffold grafting and its colonization by endogenous cells) (Mangano et al., 2013) or combining the biomaterial with cells in vitro (Barboni et al., 2013). While previously reported studies were usually based on single time points, the long-term kinetics of bone regeneration being not fully investigated, a recent clinical study (Giuliani et al., 2016) reported a quantitative kinetics evaluation of blocks versus granules in biphasic calcium phosphate scaffolds carried out by SR microCT. Twenty-four bilateral sinus augmentations were performed and grafted with HA/β-TCP 30/70, 12 with granules and 12 with blocks. The samples were retrieved at 3, 5/6 and 9 months from grafting and were evaluated for bone regeneration, graft resorption, neovascularization, and morphometric parameters. Big

1.4 Skeletal Tissue Engineering (A)

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FIGURE 1.3 Hydroxyapatite scaffold (Engipore) before (A) and after 8 (empty dots), 16 (full dots), and 24 (triangles) weeks of implant (B). Profiles of gray levels for the whole sample. The peaks on the right (xD6.5 cm21) correspond to the hydroxyapatite scaffold while in (B), the central peak (xD2.5 cm21), corresponds to the new bone. This peak shifts to the right after 24 weeks of implantation, due to an increase in mineral content. From Cancedda et al. (2007).

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quantities of newly formed bone were detected in the retrieved biopsies, together with a good rate of biomaterial resorption and the formation of new vessels (Fig. 1.4). While the morphometric parameters were comparable up to 5/6 months from grafting, 9 months after grafting microCT revealed that the sites grafted with blocks (Fig. 1.4, panels E and F) mimicked the healthy native bone of the maxillary site slightly better than those grafted with particulates (Fig. 1.4, panels B and C). Other significant studies in literature (Cancedda et al., 2007; Komlev et al., 2009) have demonstrated, by SR-microCT, the bioactive role of TCP or of TCP in combination with HA in bone regeneration even if, in these cases, the regeneration was limited to ectopic sites of animal models. Naturally produced bioceramics are an interesting alternative to biphasic calcium phosphate materials. For instance, coral has been used for a long time as a scaffold for bone tissue engineering because of its porous and interconnected

FIGURE 1.4 MicroCT 3D reconstruction of (A) granule-based TCP/HA scaffold before grafting, (B and C) granule-based TCP/HA scaffold 9 months after grafting, (D) block-based TCP/HA scaffold before grafting and (E and F) block-based TCP/HA scaffold 9 months after grafting. (BE) Residual scaffold 9 months after grafting. Legend for panels C and F— dark gray: regenerated vessels; light phase: newly formed bone, and white phase: scaffold. From Giuliani, A., Manescu, A., Mohammadi, S., Mazzoni, S., Piattelli, A., Mangano, F., et al., 2016. Quantitative kinetics evaluation of blocks versus granules of biphasic calcium phosphate scaffolds (HA/β-TCP 30/70) by synchrotron radiation X-ray microtomography: a human study. Implant. Dent. 25 (1), 615.

1.4 Skeletal Tissue Engineering

architecture, its high compressive breaking stress, and high biocompatibility and resorption properties. All these characteristics make coral a promising candidate for use as delivery vehicles for cells. (Piattelli et al., 2016; Iezzi et al., 2016). The calcium-carbonate coral-derived material, named Biocoral, is a naturally produced ceramic biomaterial, such as animal and plant skeletons (hydroxyapatite or calcium carbonate). SR-microCT revealed in 3D that 6 months after grafting, a huge amount of newly formed bone was present in the retrieved Biocoral-based samples, coupled with a good rate of biomaterial resorption and the formation of a homogeneous and rich net of new vessels. The morphometric parameters were comparable to those obtained in the biphasic calcium phosphate-based samples after the same amount of time from grafting, with the exception of the connectivity index for which biphasic calcium phosphate-based samples exhibited the most well-connected structure (Giuliani et al., 2014a). The previously reported clinical cases demonstrate that mineralized-scaffold grafting is a major strategy for the repair and reconstruction of bone defects and that the SR-microCT technique plays a fundamental role in advanced characterization of such bone tissue-engineered constructs. However, the reconstruction of the mandible is still a challenge for oral and maxillofacial surgeons, at least until now. Indeed, the various methods that have been used, including insertion of bone grafts and allogeneic materials, are still not completely satisfactory: alloplastic materials carry the risk of bacterial infection and can perforate skin or oral mucosa (Engstrand, 2012), the harvesting of bone grafts is associated with morbidity and possible functional impairment at the donor site (Pieske et al., 2009), and the products used in some procedures can have costly, time-consuming manufacturing processes and controversial ethical issues (Sukumar and Drı´zhal, 2008). In this context, some researchers (Giuliani et al., 2013) sought to study the bone regeneration process in injured human mandibles repaired with autologous dental pulp stem cells (DPSCs)/collagen sponge biocomplex implants. They reported that stem cells unexpectedly regenerated a compact rather than a spongy bone type. This was assessed by SR-based HT, fully exploiting the sensitivity of phase-contrast imaging as discussed in Section 1.3. SR-HT reconstructed 3D images and analyzed tissues at resolutions comparable to histology. However, with respect to morphometric analyses conducted through histology, SR-HT generated additional and more reliable quantitative information because the entire 3D samples, rather than only selected 2D sections, were assessed.

1.4.2 CARTILAGE Degenerative, rheumatic, or traumatic processes are the predominant causes of articular cartilage damage. Biologic agents can block such deterioration, but the tissue has only limited regenerative potential and drugs are still unable to rebuild cartilage. Such limited ability of articular cartilage to regenerate often makes joint arthroplasty unavoidable (Makris et al., 2015). Consequently, cartilage tissue

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engineering was attempted by autologous cells conduction, sometimes filling the defect area with cell-scaffold constructs, or enhancing the regeneration through biochemical targeting mechanisms (Zehbe et al., 2010a). Zehbe et al. (2009) showed the efficiency of SR-microCT to image volumetric cartilage morphology, evaluating the spatial distribution of single cells inside the tissue and their quantification, and comparing their findings to conventional histological techniques. Recently, a series of acellular and cellular regenerative products and techniques were tested in order to promote the development of functional articular cartilage which could revolutionize joint care over the next decade. Acellular scaffolds are usually made of collagen or hyaluronic-acid-based materials, while cellular strategies use either primary cells or stem cells with or without the support of a scaffold. In their research on scaffolds for cartilage tissue engineering, Zehbe et al. (2009) used a directional freezing process to structure water-based solutions of gelatin. The electrolysis of water prior to freezing resulted in the introduction of gas bubbles inside the gelatin solution before its directional freezing. The addition of other components, like salts, acids, ceramics, or polymer particles, allowed for the synthesis of composite scaffolds and for achieving different pore morphologies. The scaffolds, after seeding with porcine chondrocytes, were stained with a combined Au/Ag stain to enhance the absorption contrast in SR-microCT. While only some cells showed enhanced absorption contrast, most cells did not show any difference in contrast to the surrounding scaffold and were consequently not detectable using conventional greyscale threshold methods. Therefore, an imagebased 3D segmentation tool was used on the tomographic data, revealing a multitude of nonstained cells. However, although a lot of studies referred to cartilage engineering concentrate on in vitro examinations, SR-PhC-microCT is currently used for the visualization of tissue-engineered repair in exercised tissues. In fact, providing the phase-contrast X-ray imaging a substantially enhanced contrast resolution for soft tissues compared to conventional absorption techniques, some works (Bravin, 2003; Coan et al., 2005, 2010) demonstrated that SRPhC-microCT is able to show structural properties of the cartilage matrix in excised tissues. The level of detail within the tomographic images is sufficiently consistent to allow for the differentiation between osteoarthritic and healthy cartilage (Coan et al., 2010). Indeed, the edges and interfaces between cartilage and calcified cartilage/subchondral bone are clearly discernible because of the beam refraction occurring at the edges between tissues with different refraction indexes. The osteoarthritic samples showed significantly lower chondrocyte distribution homogeneity, less chondrocyte alignment, lower height of tangential, transitional, and radial zones, and a higher prevalence of superficial cartilage damage (Coan et al., 2010). Practically, if in the future phase-contrast imaging will be translated into the clinical scenario, then it could become an important tool for the evaluation of

1.4 Skeletal Tissue Engineering

osteoarthritis (especially for early detection and monitoring of the disease) and, in general, a valuable noninvasive alternative to routine histological examination of tissue-engineered cartilages.

1.4.3 TENDONS Research on tissue engineering recently involved some attempts at tendon repair. This field is currently facing a significant challenge because of the urgency for developing strategies that will lead to a clinically effective and commercially successful product (Shearn et al., 2011). Indeed, this challenge is primarily motivated by the fact that injured tendons have limited repair ability after full-thickness lesions. Treatments normally involve surgical repair but usually the damaged tendon is unable over time to bear the mechanical stress of daily activities, with a consequent high risk of refailure. Autografts, allografts, xenografts, and synthetic prostheses have been tested for tendon augmentation. Although autografts have always been considered the best solution, the substantial morbidity of the donor site has pushed research toward alternative solutions (Longo et al., 2010). Silk, carbon fibers, Gore-Tex, and LARS1 have been tested as alternatives to autografts, but several cases have highlighted endurance and durability problems related to frictional problems (Gigante et al., 2013; Thomas et al., 2011) and side effects ranging from immunogenic reactions to necrotic phenomena (McKibbin, 1984). Better results were achieved in the early 1990s with the introduction of collagen membranes. In this context, some authors (Gigante et al., 2013) performed an ex vivo study, assessing the tendon regeneration ability of a new oriented collagen-I membrane in an experimental animal model, using conventional histological analysis and SR-microCT examination. Ten New Zealand White rabbits were sectioned in the central third of the patellar tendon that was grafted with a membrane obtained from purified equine Achilles tendon. The contralateral patellar tendons were cut longitudinally and evaluated as sham-operated control. Histological and SR-microCT findings showed satisfactory graft integration with native tendon without adverse side effects. SR-microCT experiments were performed in absorption configuration at the ID19 beamline of the European Synchrotron Radiation Facility (ESRF, Grenoble, France) and in phase-contrast setup at the SYRMEP beamline of the ELETTRA Synchrotron Radiation Facility (Trieste, Italy). Morphometric data and fiber thickness distribution analysis, obtained by the ESRF experiments, showed a more connected fiber stacking in the treated tendon than in the lesion site of the control tendon. A more uniform distribution of fiber thickness was found in treated compared to control tendons, suggesting possible better biomechanical performances, after healing, of the treated tendons. Furthermore, the microCT data allowed to evaluate also the mass density of treated tendons in terms of density. The implant site of treated tendons and the lesion area of control tendons were not significantly different, confirming good

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FIGURE 1.5 New Zealand White rabbit tendon grafted with collagen-I membrane. SR-PhC-microCT 3D reconstruction. Micrometric fiber distribution 6 months after surgery.

integration of the oriented collagen-I membrane in the treated site. A 3D reconstruction of a treated tendon is shown in Fig. 1.5. However, tendon tissue engineering also needs to employ more clinically relevant models of tendon injury, such as degenerative tendons. There is an urgent need to translate the previously described successes from small to larger animal models, with the objective of beginning to explore the clinical implications of these treatments (Shearn et al., 2011). In this direction, the use of SR-microCT was demonstrated to be, without a doubt, a fundamental tool because of its unique ability to quantitatively provide, at 3D level, the longitudinal outcome of a particular treatment based on the use of innovative biomaterials and the interaction of these with the host tissues.

1.5 MUSCLE TISSUE ENGINEERING 1.5.1 SKELETAL MUSCLES Skeletal muscles usually have good regeneration capacities, but, in cases of severe trauma or diseases, the loss of muscle functionality is inevitable (Qazi et al., 2015).

1.5 Muscle Tissue Engineering

In the past years several mechanisms of skeletal muscle repair have been explained thanks to research in the field of skeletal muscle tissue engineering. In this direction, various types of cells and bioactive factors, playing an important role during regeneration, were identified (Edmunds and Gargiulo, 2015). For instance, the delivery of myogenic stem cells to the sites of muscle lesions through systemic circulation is a promising approach to treating DMD. This is considered a valid alternative to the attempts to repair muscle damages by transplanting myogenic progenitors directly into muscles, this solution being inhibited by limited cell survival and limited migration of donor cells in the muscles (Belicchi et al., 2009). In the study reported in Torrente et al. (2006), the authors used SR-microCT for 3D visualization of stem cells, previously labeled with FeO (Endorem) nanoparticles, transplanted via intra-arterial infusion. SR-microCT showed the distribution of intra-arterially delivered stem cells within muscle biopsies, providing biological insights into the early processes of muscle stem cell homing. As shown in Torrente et al. (2006), labeled CD133 1 stem cells were distributed around the vessels of muscle tissues within 24 h of their intra-arterial transplantation. The same authors repeated the previous SR-microCT experiment on live animals (Farini et al., 2012), scanning and visualizing the leg injected with labeled CD133 1 stem cells at different times from cell administration. It was found that the majority of intra-arterially injected stem cells accessed the muscle tissues after several rounds of recirculation, but within the first 2 h of the injection. The originality of this study lies in the fact that it was the first investigation of the kinetics of the distribution of intra-arterially injected human stem cells into the capillary system of downstream dystrophic muscles. The efficient transplantation of stem cells into the muscle of dystrophin-deficient mice further supported the approach of intra-arterial delivery of cells for cell-based clinical therapies of neuromuscular diseases, such as DMD (Farini et al., 2012). However, delivering stem cells through systemic routes also presents some obstacles; indeed, the injected cells may become trapped intravenously in other organs, like the liver, spleen, or lungs, so that only a small portion enters the muscle microvasculature and migrates to the dystrophic muscles (Belicchi et al., 2009). Therefore, in order to improve the therapeutic effects of cells and GFs, several biomaterials have been tested on animal models in order to find a suitable biomaterial to serve as a template for guided tissue reorganization. This biomaterial should be a matrix that provides optimum microenvironmental conditions to cells, a delivering system capable of releasing bioactive factors in a controlled manner, and be able to act as local niches for in situ tissue regeneration (Qazi et al., 2015). Several materials have been tested as scaffolds for muscle tissue engineering (Albertini et al., 2009): natural materials (like collagen and alginate), cellular tissue matrices (like bladder submucosa and small intestinal submucosa), and

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synthetic polymers [like polyglycolic acid (PGA), polylactic acid (PLLA), and polylactic-co-glycolic acid (PLGA)]. In this context, SR-microCT is a promising imaging modality for the assessment of such engineered scaffolds and their morphometric parameters, such as porosity, pore size, and interconnectivity. The study of cellular integration and interaction with engineered scaffolds is achieved through microCT, as images can be acquired sequentially over time with minimal negative affects to cells from X-ray dose. Based on data from the literature, and considering that synthetic polymers have the advantages of degrading by nonenzymatic hydrolysis and producing nontoxic molecules, some researchers (Albertini et al., 2009) selected PGA/PLLA (a mixture of 50% PGA and 50% PLLA) as a bioscaffold to test cell viability after their loading. They used SR-PhC-microCT to visualize in 3D the ECM organization, after in vitro seeding on PGA/PLLA of bone marrowderived human and murine mesenchymal stem cells induced to myogenic differentiation and previously labeled with iron oxide nanoparticles. X-ray microCT allowed for the detection, with high spatial resolution, of the 3D structural organization of ECM within the bioscaffold, clarifying how the presence of cells modified the construct arrangement. Furthermore, species-specific differences between the matrix produced by human and murine cells were observed. However, in order to achieve sufficient X-ray contrast, muscle tissues and the previously listed biomaterials often need to be freeze-dried or kept under dry conditions instead of standard culture conditions, sometimes significantly affecting certain cell or tissue types (Edmunds and Gargiulo, 2015). For this reason, the study by microCT of skeletal muscle engineered biopsies is still challenging and requires an optimization of experimental phase-contrast setups.

1.5.2 HEART The engineering of heart myocardium has experienced exciting progress in the past 10 years. Advances in stem cell biology, tissue engineering, and knowledge of biomaterials suggest that cardiac tissue engineering techniques will be strongly used in preclinical research and drug development in the coming years. In order to reach these objectives, several steps need to be taken, including the standardization of myocyte production methods, the establishment of simple and efficient protocols for the vascularization of biomaterials (patches), systems for maturation of myocytes, and, finally, thorough selection of predictive diagnostic techniques and tools for preclinical investigations (Hirt et al., 2014). Traditionally, the engineering of 3D cardiac tissue was motivated by the need to produce in vitro tissue surrogates for cardiac repair, but also by the fascinating experience of observing a heart muscle beating in a dish. Unfortunately, despite the numerous papers that are published on the subject every year, cardiac tissue engineering has not yet entered clinical practice and has still not found wide application in preclinical drug development (Hirt et al., 2014).

1.5 Muscle Tissue Engineering

Published reports have contributed to identifying possible cellular therapy approaches to generate new myocardium, involving transcoronary and intramyocardial injection of progenitor cells (Giuliani et al., 2011). While light fluorescence scanning and transmission EM techniques attempt to visualize the tissue-rebuilding process but are limited to 2D local information, in vivo imaging methods, like MRI, PET, and conventional CT, could play a major role in achieving the quantification of the rebuilding process, including longitudinal cell tracking. However, these 3D techniques present intrinsic limitations to identifying the localization and fate of the injected cells in both clinical and experimental settings, as exhaustively described by Terrovitis et al. (2010). In this context, some researchers (Giuliani et al., 2011) explored the use of microCT in absorption and phase-contrast setups as experimental techniques with high spatial resolution for the detection of rat cardiac progenitor cells (CPCs) previously labeled with iron oxide nanoparticles inside infarcted rat hearts, 1 week after injection and in ex vivo conditions. Through microCT, they were able to observe, in 3D, the presence of these cells after migration to the damaged cardiac tissue, with important structural details that are difficult to visualize using conventional bidimensional imaging techniques. Indeed, 3D visualization of the spatial distribution of the grafted cells with respect to the myocardium and vascular system was obtained. In particular, the X-ray absorption of the labeled cells was higher than that of host tissues, allowing their visualization as bright spots in the 3D images (Fig. 1.6). One week after injection, labeled cells were distributed mostly in proximity and toward the damaged infarcted area (Fig. 1.6F), demonstrating migration of CPCs from the injection site (Fig. 1.6D), that is, around the coronary binding. It was also possible to identify finger-like cell structures in the inner part of the left ventricular wall (Fig. 1.6F). This is another example of useful information not detectable by conventional histological methods. Together with these particular structures, attributable to cell clusters, single smaller units were also observed in all areas of the heart, such as in the atria, the large vessels (image not shown), and in the right ventricle (Fig. 1.6D). These are important new data; in particular, they constitute a confirmation that these cells can migrate through the myocardium through a biological mechanism which is still unknown. This new 3D imaging approach, combining absorption and phase-contrast data with the fusion method (Stokking et al., 2003), appears to be an important way of investigating cellular events involved in cardiac regeneration and represents a promising tool for future clinical applications. However, cardiac tissue engineering techniques have yet to reveal their full potential prior to the introduction of stem cells for drug screening and modeling of a patient’s specific disease. Indeed, the limited success of cardiac cell therapy studies highlights the requirement for further research focused on the objective of finding improved cell delivery methods (Hirt et al., 2014). SR-based microCT techniques are able to give unevaluable support in this direction. Indeed, when imaging soft tissues with hard X-rays, like for an engineered heart, phase contrast is often preferred over conventional attenuation

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FIGURE 1.6 MicroCT reconstruction of an infarcted rat heart injected with 5 3 105 rat CPCs. (A) Upper part of the heart (base). (B) Same volume as in (A), with all the phases but the rare (Continued)

1.5 Muscle Tissue Engineering

L

contrast due to its superior sensitivity. However, it is unclear which of the numerous phase tomography methods yields the optimum results for given experimental conditions. Therefore, some authors (Lang et al., 2014) compared three phase tomography methods implemented at the beamline ID19 of the European Synchrotron Radiation Facility: X-ray grating interferometry (XGI), propagationbased phase tomography, that is, single-distance phase retrieval (SD-PhC), and HT, using the entire heart of a rat. They showed that the spatial resolution available to detect morphological features is about a factor of two better for HT and SD-PhC compared to XGI, whereas the XGI data generally exhibited much better contrast-to-noise ratios for anatomical features and in density measurements. In this direction, as a crucial goal of cardiac tissue engineering is the development of implantable constructs, such as cardiac patches, which provide physical and biochemical supports for myocardial regeneration, a key problem is the quantitative monitoring in situ, in a nondestructive way, of the success of these constructs. This is fundamental for longitudinal assessments necessary to translate studies from ex vivo to in vivo, in animal models and human patients. Izadifar (2016) produced experimental nanoparticles with the aim of temporally modulating the GF release in cardiac patches. In the same study, SR-PhC methods for visualization and quantitative assessment of 3D-printed cardiac patches implanted in a rat myocardial infarction model were explored. The results showed that polymer and external aqueous phase concentrations are the most significant process parameters affecting nanoparticle physical and release characteristics. The experimental nanoparticles, produced in order to overcome the limitations of PLGA nanoparticles, were made of a protein-encapsulating PLGA core and a poly(L-lactide) (PLLA)-rate regulating shell, thus allowing for low burst effect, protein structural integrity, and time-delayed release patterns. Izadifar (2016) also created patches from alginate strands using a 3D printing technique which were surgically implanted on rat hearts for their assessment by SR-PhC-microCT. Phase-retrieved images depicted visible and quantifiable structural details of the patch at a low radiation dose. The microstructural features of fibrin and alginate, which were low-density ( . 97% water) constituents of the patch, were clearly visualized and quantitatively characterized from the phaseretrieved PhC-microCT slices of the implanted patch. The MicroCT technique

labeled cells virtually made transparent. CPCs migrated here from the site of injection. (C) Middle part of the heart (equatorial portion). CPCs were injected here. (D) Same volume as in (C), with all the phases but the labeled cells virtually made transparent. (E) Bottom part of the heart (apex). This portion was largely involved by infarction, as shown by the thinning of the wall. (F) Same volume as in (E), with all the phases but the labeled cells virtually made transparent. From Giuliani, A., Frati, C., Rossini, A., Komlev, V.S., Lagrasta, C., Savi, M., et al., 2011. High-resolution Xray microtomography for three-dimensional imaging of cardiac progenitor cell homing in infarcted rat hearts. J. Tissue. Eng. Regen. Med. 5 (8), e168e178.

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provided anatomical details including the microvessels surrounding the implanted patch. Notably, these microstructural and anatomical features of the patch and heart were achieved without the usage of any contrast agent, suggesting that SRPhC-microCT is of great potential for noninvasive assessment of patch structural changes, myocardium regeneration, and vascularization in live animal studies for cardiac tissue engineering.

1.6 NEW FRONTIERS 1.6.1 CENTRAL AND PERIPHERAL NERVOUS SYSTEM Conventional therapy approaches to neurological diseases present several problems, mainly linked to the restricted intrinsic regeneration capacity of neurons. Therefore, currently, many researchers in various fields, including surgery, internal medicine, pharmacology, medical device technology, chemistry, and cell biology, are actively attempting to find solutions and to establish new therapies for curing neurological diseases. Recently, cell-based regenerative medicine has appeared as one of the most promising approaches for treating such diseases. Regenerative therapy by the direct injection of dissociated cells has been performed in clinical settings, but several published works (Ibarretxe et al., 2012; Lu et al., 2004; Bertani et al., 2005) confirmed only modest therapeutic benefits. Regarding the delivery of drugs into the central nervous system (CNS), the main obstacle is the presence of the bloodbrain barrier (BBB), since it forms a barrier, hindering the delivery of therapeutic agents from the bloodstream. To circumvent it, various drug carrier systems were developed. Carriers of drugs proven to be more efficient as systemic delivery systems include liposomes, polymeric nuclei, polymer micelles, NPs, and ceramic dendrimers (Oliveira et al., 2010). Of these, only liposomes and polymeric NPs have been widely exploited in the administration of brain drugs. A promising strategy to increase therapeutic efficacy while reducing systemic side effects is based on the local administration of therapeutic agents through a polymeric and biocompatible delivering system implanted at the target site (Jain et al., 2006). This approach offers many advantages: in addition to bypassing the BBB, it avoids systemic side effects and toxicity, inactivation of the peripheral drug, and the need to modify the surface of the vehicle. There are also disadvantages of local administration: the dosage cannot be adjusted after implantation, the rate of drug release usually decreases over time, and it may be necessary to repeat the implant for long-term release, requiring invasive surgery. Restoring peripheral nerve damage is another crucial issue in tissue engineering. One promising approach is based on nerve conduits, consisting of biodegradable polymers attempting to realize a preformed structure, serving as guidance for axonal growth in vivo.

1.6 New Frontiers

Some authors (Zehbe et al., 2010b) proposed a rather simple methodology to control cell growth by applying an electrical potential. They demonstrated that SR-microCT can be used to confirm the efficacy from a 3D morphological point of view. In fact, as cells usually grow in vitro in a nonordered way, they developed a technique to print microelectrodes on polymer sheets via a method named “inverse inkjet printing.” The method uses a sputter coater to establish a gold metallization, allowing a coded position of vital cells with the protein fibrin on the anode part of the electrodes to be obtained (Zehbe et al., 2010a). The electrodes presented parallel-aligned rows of thin gold made by the sputter coating process. All rows were contacted anodically allowing for the deposition of cells and fibrin. Afterwards, several microelectrodes, with the top cells, were stacked, fixated using glutaraldehyde, rinsed in distilled water, and freeze-dried for further SR-microCT investigations. The microCT results were rather interesting and unexpected. Being that the experiments were performed in absorption setup, the authors expected to observe strongly absorbing deposited gold structures and a weekly absorbing polymer substrate. Further, as the deposited cells were not stained with any metal stain, they did not expect to observe the cells themselves, as they have a low density and are thin compared to the polymer substrate. Nevertheless, interestingly and unexpectedly, cells were imaged well and in good accordance with the fluorescence microscopic images acquired previously. Another fundamental challenge in neuronal tissue engineering is the imaging of the human brain at cell level and in 3D. In vivo methods lack spatial resolution, and optical microscopy has a limited penetration depth. Research (Hieber et al., 2016) showed that PhC-microCT was able to visualize a volume of up to 43 mm3 of human post mortem or biopsy brain samples. The method was demonstrated on the cerebellum. They automatically identified 5000 Purkinje cells with an error of less than 5% at their layer and determined the local surface density to 165 cells per mm2 on average. Moreover, they highlighted that microCT allowed, by segmentation, the discrimination of subcellular structures, including the dendritic tree and Purkinje cell nucleoli, without the need of a staining process. Once again, SR-PhC-microCT was shown to be successful in the 3D analysis of soft tissues, achieving automatic cell feature quantification, with isotropic resolution and in a label-free manner. This result is expected to open, in the near future, new possibilities in the study of engineered tissues through different types of biomaterials in the CNS and peripheral nervous system districts.

1.6.2 VASCULARIZATION Satisfactory vascularization is a required condition for a clinical outcome of tissue-engineered sites. Thus, it is no wonder that several imaging modalities have been tested for the visualization of new vessels in tissue engineering. Currently, vascular imaging modalities are classified into three major groups (Upputuri et al., 2015): nonoptical methods (X-ray, magnetic resonance,

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ultrasound, and positron emission imaging), optical methods (optical coherence, fluorescence, multiphoton, and laser speckle imaging), and hybrid methods (photoacoustic imaging). However, referring to the first group, the observation of blood vessels using absorption-based X-ray imaging is challenging. To image vessel network structures, contrast agents, or corrosion casts, generating absorption contrast can be introduced and imaged using conventional X-ray techniques. In these cases, radiopaque contrast agents, such as barium sulfate or Microfil, are injected into the vasculature of the animal prior to sacrifice to allow for ex vivo imaging of the samples (Nam et al., 2015). Some researchers (Arkudas et al., 2010) created an automatic observer-independent quantitative method to analyze vascularization using microCT. An arteriovenous loop was created in the medial thigh of 30 rats and was placed in a particulated porous hydroxyapatite and β-TCP matrix, filled with fibrin with or without the application of fibrin-gelimmobilized angiogenetic GFs, vascular endothelial GF, and basic fibroblast GF. In both groups, microCT showed that the arteriovenous loop had led to the generation of dense vascularized connective tissue with differentiated and functional vessels inside the matrix. Quantitative analysis of microCT data also allowed for the assessment of several complex vascularization parameters within the 3D constructs, demonstrating an early improvement of vascularization through the application of exogenous GFs. However, in agreement with several papers, the authors commented that the proposed method presented two limitations, namely the limited resolution of microCT and the incomplete filling of the vessels by the contrast agent. Due to these inherent limitations, complete reliance on absorption contrast leads to significant challenges when attempting to simultaneously identify multiple tissue features in a single sample (e.g., microvascular and calcified tissue structure). Depending on vessel properties and conditions, phase-contrast techniques can provide details on vascular structure in the absence of exogenous contrast agents (Appel et al., 2011). Interferometric, analyzer-based and propagation-based tomography allowed for the detection of the vascular structure within the liver, the first method capable of resolving vessels as small as 50 μm in diameter. Instead, in-line holography micrographs were capable to visualize blood vessels 20 μm thick in diameter in the auricle region of live mice. An in-line technique, named pseudo-holotomography, allowed for the observation, in 3D, of the neovascularization of a porous ceramic scaffold, subcutaneously implanted in mice (Komlev et al., 2009). Silicon-stabilized TCP/MSC composites were implanted subcutaneously on the back of immunodeficient mice. The mice were sacrificed 24 weeks after implantation and the extracted constructs were investigated by absorption-based microCT and HT. MicroCT 3D images were reconstructed from a series of 2D projections using a 3D FBP algorithm, while the “holographic” acquisitions were treated, at each sample-to-detector distance, with a phase retrieval procedure based on TIE with a successive

1.6 New Frontiers

reconstruction of the 3D images using the FBP algorithm. To combine both δ and β maps, an optimized alignment-and-matching procedure for one tomographic and three HT volumes was used (pseudo-HT process). Using this method, the hard and soft tissues and the vascular network were simultaneously imaged and quantified. Another work (Giuliani et al., 2013) assessed the quality of the regenerated vessel network, with in-line HT, in mandible grafts (made of DPSCs seeded on collagen-I scaffolds) 3 years after the grafting intervention. In this study, an innovative method for phase retrieval, experimented by Langer (Langer et al., 2010) and described in Section 1.3, was applied. HT allowed not only to acquire qualitative and quantitative information on hard tissue, but also assessed the presence and the distribution of the blood vessels within the investigated specimens, as shown in Fig. 1.7. Besides the specific results previously described, phase-contrast methods are expected to be extremely useful when considering the vascularization of tissue engineered constructs (Giuliani et al., 2017). In particular, the progress associated to these X-ray imaging techniques could be extrapolated to angiogenesis and microvasculogenesis studies in pathologies characterized by inflammation and tissue damage, such as diabetes and osteoporosis (Giuliani et al., 2013).

FIGURE 1.7 Synchrotron X-ray HT. Portion of a 3D reconstruction for a human mandible revealing the vascularization net. From Giuliani, A., Manescu, A., Mohammadi, S., Mazzoni, S., Piattelli, A., Mangano, F., et al., 2016. Quantitative kinetics evaluation of blocks versus granules of biphasic calcium phosphate scaffolds (HA/β-TCP 30/70) by synchrotron radiation X-ray microtomography: a human study. Implant. Dent. 25 (1), 615.

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1.7 CONCLUSIONS The previously reported studies have applied SR-microCT with great success at imaging different engineered tissue and for the characterization of the chosen scaffolds. They show unequivocally that SR-microCT is a promising methodology to investigate tissues or biomaterial-cell engineered constructs, due to the superior photon quality generated in modern synchrotron facilities and to the multiple methods of preparation of the experimental setup (not always feasible with conventional X-ray tube microCT devices). However, the majority of these examinations focused on tissue samples, sometimes from humans (dental districts) and often from animal models or else they are in vitro studies (Olubamiji et al., 2014). This is mainly due to the high radiation dose combined to the large exposure times to achieve extremely high resolution. In this context, it is important to consider that phase contrast imaging was also effective using low radiation doses; thus, these procedures were fully compatible with preclinical and clinical standards (Hagen et al., 2014). By decreasing CT scan time and ROI, the effective dose could be further reduced in the near future, possibly without significant changes in the image quality. This means that PhCmicroCT has the potential to ultimately become a fundamental diagnostic tool for testing the effectiveness of regenerative medicine therapies, also achieving the monitoring of biomaterial behavior and functionality in vivo after transplantation into live organisms.

REFERENCES Albertini, G., Giuliani, A., Komlev, V., Moroncini, F., Pugnaloni, A., Pennesi, G., et al., 2009. Organization of extracellular matrix fibers within polyglycolic acid-polylactic acid scaffolds analyzed using X-ray synchrotron-radiation phase-contrast micro computed tomography. Tissue Eng., Part C Methods 15 (3), 403411. Alsberg, E., Hill, E.E., Mooney, D.J., 2001. Craniofacial tissue engineering. Crit. Rev. Oral Biol. Med. 12 (1), 6475. Amini, A.R., Laurencin, C.T., Nukavarapu, S.P., 2012. Bone tissue engineering: recent advances and challenges. Crit. Rev. Biomed. Eng. 40 (5), 363408. Appel, A., Anastasio, M.A., Brey, E.M., 2011. Potential for imaging engineered tissues with X-ray phase contrast. Tissue Eng. Part B Rev. 17 (5), 321330. Appel, A.A., Anastasio, M.A., Larson, J.C., Brey, E.M., 2013. Imaging challenges in biomaterials and tissue engineering. Biomaterials 34 (28), 66156630. Arkudas, A., Beier, J.P., Pryymachuk, G., Hoereth, T., Bleiziffer, O., Polykandriotis, E., et al., 2010. Automatic quantitative microcomputed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct. Tissue Eng. Part C Methods 16, 15031514. Barboni, B., Mangano, C., Valbonetti, C.L., Marruchella, G., Berardinelli, P., Martelli, A., et al., 2013. Synthetic Bone Substitute engineered with amniotic epithelial cells enhances bone regeneration after maxillary sinus augmentation. PLoS One 8 (5), e63256.

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FURTHER READING Garcia-Bennett, A.E., Kozhevnikova, M., Ko¨nig, N., Zhou, C., Leao, R., Kno¨pfel, T., et al., 2013. Delivery of differentiation factors by mesoporous silica particles assists advanced differentiation of transplanted murine embryonic stem cells. Stem Cells Transl. Med. 2 (11), 906915.