Regulation of Cell/Tissue Responses by Architecture of Scaffolds

Scaffolds for tissue engineering should be porous and permeable to permit the ingress of cells, nutrients and oxygen diffusion, as well as waste removal. Porosity and pore size of scaffolds play critical roles in tissue formation, which have been extensively discussed elsewhere [74, 101-104]. Generally, the scaffolds should possess certain characteristic including high porosity with an interconnected pore structures for efficient cell ingrowth. In addition, a minimum pore size is required, which is correlated with cell size. For example, the minimum pore size is considered to be about 100 pm for the migration of osteogenetic cells into the pores [102], whereas pore sizes greater than 500 pm are beneficial for rapid vascularization and survival of transplanted cells in fibrovascular tissues [82]. Satisfaction of the minimum pore size together with mechanical integrity requires sufficient mechanical properties of the scaffolds.

As mentioned in Sect., the high mechanical properties of the reinforced scaffolds may augment the porosity and thus provide microstructure with interconnected pores and extremely large surface-area-to-volume ratio. This is advantageous to cell ingrowth, proliferation, and tissue regeneration [98, 105-107]. Moreover, the high mechanical properties may balance the mechanical function with cell ingrowth, thus providing a sequential transition in which the regenerated tissue assumes function as the scaffold degrades [103].

In addition to the pore structure, the nanoscale characteristics of the incorporated nanofibers or nanotubes positively contribute to the cell adhesion and proliferation [108]. Several studies have revealed that the incorporation of nanofibers or nanotubes into scaffolds facilitates cell attachment and cell proliferation [94, 106, 109]. For instance, Woo et al. [110] found that the nano-fibrous architecture built in threedimensional scaffolds increased cell adhesion by two-fold. It is widely accepted that macro-, micro- and nano-sized topographical factors stimulate behavioral changes in both cells and tissues [78]. However, the nanostructures elicit a higher degree of biological plasticity compared with micro- or macro- structures. For example, osteoblasts cultured on a nanograined Al2O3 and TiO2 substrate (grain sizes less than 100 nm) were found with obvious improvement in adhesion, proliferation and mineralization compared with those cultured on a micrograined surface [111, 112]. The fate of hMSCs could be controlled only by the nanotopography of culture substrates [113]. Specifically, small TiO2 nanotubes («30 nm diameter) promoted hMSC adhesion without noticeable differentiation, whereas larger ( « 70 to 100 nm diameter) nanotubes elicited a dramatic stem cell elongation ( « 10-fold increased), which induced cytoskeletal stress and selective differentiation into osteoblast-like cells. One important factor responsible for the increased adhesion, growth and maturation of cells in the reinforced scaffolds is the surface-area-to-volume ratio. Incorporation of nanofibers/nanotubes may increase the surface-to-volume ratio of scaffolds [45], which provides more room for cell adhesion and survival. Similarly, more room is provided for protein adsorption and ECM deposition, resulting in

Scanning electron micrographs demonstrating the morphological similarities between

Fig. 5.11 Scanning electron micrographs demonstrating the morphological similarities between (a) nitrogen-doped-MWNTs and (b) collagen, both structures are long and rod-like with a periodic structure along their long axis, scale bars: 200 nm [37]

more ligands for cell adhesion (see Sect. 5.3). In addition, the organization of endo- geneous ECM protein may be affected. Lee et al. [114] once found that the endogenous ECM produced by human ACL fibroblast organized along the fibers. Another factor for the improved cell adhesion and growth is the morphological similarities of nanofibers and nanotubes to the natural ECM, thus providing cells a more in vivo- like environment [115]. For instance, CNTs can be synthesized with dimensions comparable to the proteins of ECM (Fig. 5.11), forming a fibrillar/ECM-protein mimicking morphology, thus stimulating and strengthening integrin mediated adhesions between CNTs and cells [37]. The embedded fibers in nanofiber-reinforced hydrogel may form ECM-like microstructures and thus provide more binding sites for cell adhesion and proliferation, which was observed to significantly accelerate the proliferation of hMSCs [106].

In addition to facilitating cell adhesion and proliferation, nanofibers/nanotubes were observed to regulate cell morphology and orientation [116]. For example, fibroblast cells on gelatin/PCL nanofiber composites containing unidirectional nanofibers clearly orientated along the direction of the aligned nanofibers; on the other hand, cells on composites containing bidirectional nanofibers took an orientation in the criss-crossed pattern of the fibers (Fig. 5.12) [117]. The same phenomenon was observed on the case of Keratinocytes grown for 48 h in the presence of parallel or rectangularly arranged polyvinyl alcohol (PVA) nanofibers [74]. Previous study has also found that cell elongation resulted from the nanofibers in scaffolds could force hBMSCs into an osteogenic morphology [73]. Specially, hBMSCs cultured on PCL nanofibers had smaller cell area, higher aspect ratio, lower roundness and more branching than hBMSCs on PCL films. This spindly, branched morphology was similar to hBMSCs cultured on PCL films in the presence of osteogenic supplements, suggesting that the nanofibers could drive osteogenic differentiation of hBMSCs via controlling cell shape (Fig. 5.13). Linear cell orientation on nanofibers should be contributed to contact-cell guidance. According to Albuschies’s

T3 fibroblasts grown on gelatin/PCL nanofiber composites with (a) bi-directional and (b) unidirectional fiber orientations (white arrows show orientation of cells) [117]

Fig. 5.12 3 T3 fibroblasts grown on gelatin/PCL nanofiber composites with (a) bi-directional and (b) unidirectional fiber orientations (white arrows show orientation of cells) [117]

hBMSC morphology

Fig. 5.13 hBMSC morphology (400x) after 1d culture for (a) PCL _BNF (PCL nanofibers), (b) PCL_SC (PCL films) and (c) PCL_SC + OS (PCL films in the presence of osteogenic supplements). Actin is red (AlexaFluor 546 phalloidin) and projections of confocal z-stacks are shown [73]

research, when cells cultured on flat versus nanofibrillar interfaces, transient filopo- dia are initially (t < 1 min) in contact with both flat surfaces and nanowires; however, as time passes (t « 5 min), the transient filopodia adhere and align with individual nanowires and quickly direct cell spreading towards nanowire adhesion sites, while most filopodia retract from the flat glass surfaces (Fig. 5.14) [118]. The retraction of filopodia on flat surface and the formation of aligned filopodia on nanowires indicate that the interaction between cells and substrates is influenced by nanostructures on substrates. The aligned filopodia formation can explain why cells adopt an orientated morphology when they grow on aligned nanofibers. It should be noted that cell alignment due to fiber orientation is dependent on the fiber diameter and cell type. For example, Liu et al. [119] found that a critical minimum diameter of 0.97 pm exists for cell orientation to occur. At lower fiber diameter, no big difference in aspect ratio was observed relative to the control samples on film. On the contrary, for endothelial cells, aligned fiber diameter over 1.2 pm seems to have less influence on cell orientation. Using electrospun aligned polycaprolactone (PCL)/ collagen fibers with different fiber diameters (100 nm, 300 nm and 1200 nm), Whited and Rylander [120] found that the fiber-directed cell orientation was signifi-

Schematics of the topography recognizing function of filopodia on flat surface and nanowires

Fig. 5.14 Schematics of the topography recognizing function of filopodia on flat surface and nanowires (NMs). (a) filopodia initiate the very first substrate contacts to both, NWs and flat surfaces at a time where no ruffles and no lamellipodia were present; (b-c) as time progresses, most of the initial filopodia peeled off from flat surfaces, while the filopodia were able to pull the NWs into alignment thereby increasing the NW-filopodia contact areas. [118]

cantly greater for primary human umbilical vein endothelial cells (HUVEC) on 100- and 300-nm scaffolds as compared to 1200 nm scaffolds.

Not only cell orientation but also cell elongation was observed on nanofibers, especially on aligned fibers. For example, the length of keratinocytes grown on a flat glass surface is around 30 mm, but in the case of aligned nanofibers the cells reach 80 mm or more in length. Besides the whole cell morphology, the cell nuclei also adopted a distinctly elongated shape [74]. Similar linear cell orientation and elongation guided by nanofibers were observed in the case of stem cells [73]. The morphological similarity between nanofibers and ECM, as well as the organized ECM along the fibers should be responsible for cell elongation on the aligned nanofibers. The phenomena of cell orientation and elongation along nanofibers indicate the potential applications of nanofibers reinforced scaffolds in promoting the formation of various artificial tissues or organs, such as blood vessel, tendon, nerve, etc., in which cell orientation and elongation are crucial for their performance.

As indicated in Sect. 5.4.2, nanofibers and nanotubes reinforced cell proliferation and growth are better than their unreinforced counterparts, one contributor of which is the enhanced mechanical properties. Beside, the elongated cell morphology on nanofibers might be another contributor. Cell elongation is accompanied by a high degree of cytoskeleton organization parallel to the direction of fiber alignment, which results in greater cytoskeletal stiffness [85, 120, 121]. Thereby, the cell-matrix interface force (ligand-receptor binding) increases, which further promotes the assembly of focal adhesions (FAs). It has been found for a long time that FAs function as a node on which a complex signaling network is assembled and regulated [122]. Specifically, FA assembly and signaling result in the phosphorylation of FA kinase (FAK). FAK appears to be a key component of the mechanotrans- duction apparatus, and both FAK and its interacting proteins are candidate transducers (Integrins in mechanotransduction), which can activate pathways known to promote cell proliferation, such as Shc pathway and MAPK pathway [123, 124]. It should be reminded that FAs and cytoskeletal stiffness are the mechanotransduc- tion mechanics by which the external mechanical loadings are transmitted to cells and the mechanical properties of the scaffolds are perceived by cells (Fig. 5.15) (see Sect. 5.4). Besides, the mechanical properties of the scaffolds are influenced by the scaffold architecture as well. Therefore, the observed cell/tissue responses on nano- fibers/nanotubes reinforced scaffolds are more often the combinational or syner-

Intergrin-mediated force transmission

Fig. 5.15 Intergrin-mediated force transmission. Integrins (blue) are heterodimers of a and в subunits that form bonds with the extracellular matrix on the outside of the cell. In response to force, several proteins are recruited to the integrin cytoplasmic в domain. Critical proteins among these are talin and filamin. Talin, in turn, recruits vinculin, a prominent mechanotransducing protein [125]

getic results from the mechanical and architectural and even chemical properties of the scaffolds. Understanding of their interplays helps to take advantage of their positive contributes to cell/tissue responses and further to design desirable scaffolds for specific application.

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