Biological tissues consist of cells and the extracellular matrix (ECM) [2]. The ECM consists of various proteins such as collagen, fibronectin, and laminin, as well as proteoglycans. Therefore, the ECM is regarded as a multicomponent material. Almost all the cells in our body adhere to the ECM, and they do so mainly by means of the interaction between integrin and ECM proteins. Integrin is a transmembrane protein present on the surface of cells that binds to ECM proteins such as collagen, fibronectin, and laminin [3]. The interaction between integrin and ECM proteins translates cues from the ECM into intracellular signal transduction. This adhesion-mediated signal transduction depends on the combinations of integrin and ECM proteins, demonstrating that cells can sense the compositions of the ECM. Therefore, cells behave distinctly depending on the compositions of the ECM. Moreover, cells sense the physical properties of the ECM, such as structural and mechanical properties, through adhesion-mediated signal transduction, and cells respond to these physical properties of the ECM [4–10]. The cell-ECM interaction affects various cellular behaviors such as proliferation, morphology change, adhesion, migration, and differentiation, and thus, the interaction directs the morphogenetic process. Therefore, designing the physical properties of cell scaffolds is crucial for constructing functional engineered tissues.

Highly ordered structures are often observed in the ECM of connective tissues such as bone, tendon, cornea, and blood vessel. For example, oriented collagen fiber arrays are present in the tendon tissue [11]. The highly ordered structures of the ECM affect not only mechanical properties but also various cellular behaviors in the connective tissues. Pioneering studies on the effects of the ordered structures of ECMs on cell behaviors were conducted by Weiss [4, 12], who showed that cells aligned parallel to the orientation of fibers in blood clots and fish scales (Fig. 12.2). This phenomenon is known as “contact guidance.” Weiss and Taylor also reproduced contact guidance in vitro using glass that had been scored using a MicroLathe [12]: cells elongated along an orientation axis of the microgrooves of the scored glass. Since these seminal studies, the mechanism of contact guidance has been intensively investigated. Although contact guidance has been widely suggested to occur as a result of anisotropic force generation by lamellipodia and filopodia [13, 14], the mechanism of contact guidance remains unresolved. Conversely, contact guidance has been widely reported to induce directional cell migration [15–17]. Directional cell migration is well known to play a key role in tissue morphogenesis [2]. Therefore, the control of contact guidance is crucial when constructing engineered tissues that feature biomimetic hierarchical structures, and hence, methods are required to produce cell scaffolds exhibiting anisotropic structures (anisotropic cell scaffolds) and to control the anisotropic structures of the scaffolds. As introduced in Sect. 12.4, various techniques have been developed to prepare anisotropic cell scaffolds and these techniques have been applied in the field of tissue engineering.

At a macroscopic scale, cell density and morphology and the structure, properties, and compositions of the ECM of native tissues are heterogeneously distributed. The heterogeneities observed in native tissues are spatially regulated and are not completely random. For example, the bone density and the hierarchical structure present at the peripheral parts of long bones are considerably distinct from those at inner parts. Long bones contain cortical bone at the periphery and cancellous bone in the interior. The cortical bone exhibits high bone density and a highly ordered hierarchical structure, whereas the cancellous bone exhibits low bone density and a spongelike structure composed of trabecular bones. The macroscopic inhomogeneity contributes the mechanical properties of long bones [18–20]. By contrast, the confined compression modulus of joint cartilage increases with increasing distance from the articular surface [21]. The compression modulus gradient contributes the functions of cartilage, such as lubrication and shock absorption. Furthermore, the vascular networks present in native tissues consist of vessels of various diameters and lengths and feature self-similar structures [22, 23]. The hierarchical structure of vascular networks results in the efficient transportation of oxygen and nutrients. Thus, mimicking the heterogeneities of tissues is critical for the construction of highly functional engineered tissues.

Tissue morphogenetic processes are controlled through the spatial regulation of cellular functions. Therefore, spatially regulating cellular behavior is crucial for obtaining engineered tissues that mimic the heterogeneities of native tissues. As mentioned in Sect. 12.2.1, cellular behaviors depend on local compositions, structures, and mechanical properties of the ECM. Therefore, controlling the spatial distributions of the compositions, structures, and mechanical properties of cell scaffolds can facilitate the spatial regulation of cellular behavior. For example, culturing cells on a cell scaffold exhibiting an elastic modulus gradient generates a cell density gradient (Fig. 12.3) [24–26]. The formation of a cell density gradient can be attributed to the directional migration of cells induced by the physical cue provided by the elastic modulus gradient of the cell scaffold. This type of directional migration is known as “durotaxis.”

In recent years, various methodologies have been developed to control the spatial distribution of compositions, structures, and mechanical properties of cell scaffolds. In Sect. 12.5, several examples of the methods used to regulate the spatial distributions of composition, structure, and mechanical properties of cell scaffolds are presented and the applications of these methods in the field of tissue engineering are described.

Polymers featuring rigid or semiflexible structures exhibit isotropic-to-anisotropic phase transition. Several biopolymers such as collagen and DNA are regarded as main-chain liquid crystal polymers because these biopolymers possess rigid helical structures. Concentrated solutions of liquid crystal biopolymers exhibit diverse liquid crystalline textures [48–52]. For example, slow evaporation of concentrated DNA solutions generates a liquid crystalline phase that presents a columnar hexagonal structure. These liquid crystalline textures depend on the concentration of DNA molecules and the molecular weight of DNA and on the types of salts added. Similarly, highly concentrated Type I collagen solutions exhibit liquid crystalline phases that mimic the ordered structures observed in the collagen matrix of connective tissues (Fig. 12.5) [51–54]. The morphologies of liquid crystal phases change from a spherulite phase to a dense cholesteric phase when the concentration of collagen is increased [53, 54]. Giraud-Guille and coworkers obtained a dense collagen matrix displaying highly ordered structures by neutralizing the pH of highly concentrated collagen solutions [55] and then used the dense collagen matrix to investigate various properties of fibroblast, such as migration, density, and expression of matrix metalloproteinases, on collagen hydrogels [56]. The fibroblasts seeded on the surface of collagen hydrogels infiltrated the hydrogels to reach deep parts; however, the fibroblasts infiltrated the dense collagen hydrogel considerably more slowly than they infiltrated dilute collagen hydrogels. These results indicate that the dense collagen matrix reduces the infiltration of cells from the surface of the gel to the interior of the gel. By contrast, another group reported that a cholesteric collagen film cast from a dense collagen solution can induce the contact guidance of human fibroblasts [57].

External fields such as flow and magnetic fields are tools that can be used to manipulate molecular alignment in scaffolds. When flow fields such as shear flow field and stretched flow field are applied to polymer solutions, polymer chains are deformed and oriented along the direction of the flow field [58–62]. If we apply the flow fields to polymer solutions during the gelation processes, we can obtain hydrogels featuring anisotropic structures. Elsdale and Bard prepared hydrated collagen lattices (HCLs) featuring anisotropic structures by applying a unidirectional drainage flow field to neutralized collagen solutions at semi-dilute concentrations (approximately 0.1 wt%) during the gelation processes [5]. In the anisotropic HCLs, collagen fibers aligned parallel to the drainage direction. Using anisotropic HCLs, the investigators examined the morphology, motility, adhesion, and proliferation of human embryonic lung fibroblasts under 2D and 3D conditions and observed that the fibroblasts aligned parallel to the orientation of the collagen fibers in the HCLs; the orientation of the fibroblasts here could be attributed to contact guidance as described in the preceding paragraph. Dunn and Ebendal prepared anisotropic HCL by using same method and found that chick-heart fibroblasts cultured on wet anisotropic HCL aligned parallel to the orientation of collagen fibers, whereas fibroblasts cultured on air-dried anisotropic HCL did not exhibit contact guidance [6]. These results suggest a key role of 3D culture conditions in the contact guidance of cells. Recently, Lanfer et al. developed a method to finely control the orientation and density of collagen fibrils by combining a microfluidic system and flow field-induced orientation of collagen fibrils [63]; these investigators showed that the degree of collagen fibril orientation and the density of collagen fibrils adsorbed on glass substrates can be controlled by regulating collagen concentration and flow rate and the surface properties of glass substrates, which can be modified by coating with copolymers. Using this anisotropic collagen substrate, Lanfer and coworkers investigated contact guidance and differentiation of human bone marrow-derived mesenchymal stem and progenitor cells (hMSCs) and C2C12 cells (a mouse myoblast cell line) [64] and determined that the myotube assembly of C2C12 cells was regulated by the orientation of collagen fibrils adsorbed on the glass substrate. However, the osteogenic and adipogenic differentiation behaviors of hMSCs cultured on collagen substrates that did or did not feature the anisotropic structure were not markedly different, although contact guidance of the hMSCs was induced on the collagen substrate featuring the anisotropic structure.

Magnetic field can also be used to manipulate the molecular alignment in cell scaffolds. Torbet et al. prepared anisotropic cell scaffolds composed of various fibrous proteins by using a strong magnetic field [65–67]. These investigators showed that the birefringence of the fibrous protein scaffolds, which is related to the degree of fibril orientation, can be controlled by the magnitude of the applied magnetic field. For example, anisotropic collagen hydrogels can be prepared by applying magnetic fields stronger than 1.9 tesla [66]. The contact guidance behaviors of various cells embedded in magnetically aligned collagen scaffolds have been investigated. Guido and Tranquillo described a method to systematically and quantitatively investigate contact guidance behaviors in anisotropic collagen gels prepared by applying magnetic fields [68] and demonstrated that a cell orientation parameter that can be used to quantify cell orientation distribution was linearly proportional to the birefringence intensity of the anisotropic collagen gels. A modified magnetic molecular alignment method was used to prepare an engineered corneal stroma composed of the multilayered collagen gel, in which the orientation of the collagen fibrils changed orthogonally in each layer [69]; keratocytes seeded on the engineered corneal stroma infiltrated the scaffold and the cells aligned parallel to the orientation of the collagen fibrils in each layer.

Anisotropic cell scaffolds prepared using isotropic-to-anisotropic phase transitions exhibit highly ordered microscopic structures that may mimic the microscopic structure of the ECM in connective tissues. Furthermore, because external fields can be applied to finely control molecular alignment in cell scaffolds, the cell scaffolds prepared using flow fields feature well-defined anisotropic structures. These anisotropic cell scaffolds can be used for investigating cellular behaviors under highly physiological conditions and can be used as the template for reconstructing the local anisotropic structures of native tissues. However, because many of the anisotropic cell scaffolds introduced above are homogeneous at a macroscopic scale, they cannot serve as templates that mimic the macroscopic structure of native tissues. Therefore, controlling the macroscopic heterogeneities of cell scaffolds is also crucial.

As mentioned in Sect. 12.2.2, directional cell migration plays a key role in tissue morphogenesis. The mechanisms that control the direction of cell migration have been intensively investigated in the field of developmental biology. Many of the directional cell migrations observed during development have been widely reported to be regulated by concentration gradients of ligand molecules such as fibroblast growth factors (FGFs), bone morphogenetic proteins, and stromal cell-derived factors. These ligands are known as “chemokines,” and the directional cell migration induced by a concentration gradient of chemokines is called “chemotaxis.” If a concentration gradient of chemokines can be generated in vitro, the morphogenetic processes that occur in vivo could be reproduced. Therefore, controlling chemotaxis in vitro can be considered a critical part of the methodology used to construct engineered tissues featuring biomimetic hierarchical structures. However, because several chemokines are water-soluble biomolecules, maintaining a concentration gradient of chemokines long term is challenging. Hence, techniques are required to immobilize chemokines on cell scaffolds. A polyethylene glycol (PEG) hydrogel featuring a concentration gradient of covalently immobilized FGF was developed using a gradient maker and photopolymerization [70]. On the PEG hydrogel featuring the FGF concentration gradient, vascular smooth muscle cells (VSMCs) aligned parallel to the direction of the concentration gradient and migrated from a region of low FGF concentration to a region of high FGF concentration. Therefore, PEG hydrogels can be used for investigating chemotaxis in vitro, and the technique of immobilizing FGF to generate the concentration gradient can be used to control chemotaxis and cell orientation.

Cells not only sense chemical stimuli by means of specific ligand-receptor interactions but also detect physical stimuli through the adhesive interactions that occur between cells and the ECM. As mentioned in Sect. 12.2.3, cells seeded on cell scaffolds featuring an elastic modulus gradient migrate from soft regions to rigid regions, and this directional cell migration is called “durotaxis.” Therefore, by regulating the spatial distribution of various properties of cell scaffolds, directional cell migration is controlled to construct engineered tissues that exhibit highly biomimetic structures. Numerous cell scaffolds featuring gradients of mechanical properties have been developed to investigate cellular behaviors under physiological conditions in vitro and to test the potential utility of such cell scaffolds in the field of tissue engineering. Microfluidic devices have been frequently used to fabricate cell scaffolds that exhibit gradients of mechanical properties [25, 26].Collagen-coated polyacrylamide hydrogels that exhibit an elastic modulus gradient have been prepared by photopolymerizing precursor solutions under a cross-linker gradient generated using a gradient generator, which was a microfluidic device fabricated using poly(dimethylsiloxane) [71]. VMSCs seeded on this gradient polyacrylamide hydrogel migrated from the soft side to the rigid side. The durotaxis of VMSCs was evaluated using the tactic index developed for a biased persistent random walk, and the durotaxis was shown to increase when the magnitude of the elastic modulus gradient was increased. Moreover, the degree of cell orientation was also reported to increase when the magnitude of the elastic modulus gradient was increased. Thus, cell scaffolds featuring an elastic modulus gradient can control not only the spatial distribution of cells but also cell morphology. Gradient biomaterials composed of ECM proteins are useful for investigating the effects of gradient properties on cellular behaviors under physiological conditions. A 3D collagen gel featuring an elastic modulus gradient was developed using an H-shaped microfluidic device [72] and used to investigate the effect of the elastic modulus gradient on neurite growth; neurite growth was directed and enhanced by the elastic modulus gradient, demonstrating that the 3D collage gel can be used as a substrate to guide the directional regrowth of axons in various nervous systems.