Hydrogel-Based Biomimetic Environment for In Vitro Cell and Tissue Manipulation
A biomimetic environment fabricated with synthetic material would be an effective tool for reproducing the tissue-developmental process and even for achieving tissue engineering in vitro. A hydrogel material is one candidate for this tool, because a hydrogel normally shows harmless properties in regard to cells and tissue, and it can be tuned chemically and physically to obtain the desired form. Accordingly, fibrin gel was utilized to reproduce the 3D cellular orientations found in muscle tissue, fabricate tendon-like mineralized tissue, and regulate vascular formation. In this context, cell and tissue manipulations within the gel were led by in vitro physical and chemical stimulations. In this chapter, the approach used for manipulating cells and tissues using the designed hydrogel is discussed.
KeywordsBiomimetic environment Cell manipulation Hydrogel In vitro tissue engineering
Thanks to recent advances in cell and molecular biology, researchers have gradually started to understand how molecules are concerned with the expression of cellular functions. They have also started to understand how the surrounding molecules guide cellular behavior. Engineers and chemists have also started to participate in this in-vitro cellular guidance, develop methods for so-called “in-vitro cellular guidance,” since they can design and construct an environment that is suitable for manipulating cell functions. For example, Chen et al. used a microprinting system that can control the cell-adhesion shape by applying the patterned coating of fibronectin on two-dimensional tissue-culture substrates. They indicated that the mesenchymal stem cells (MSCs) shape regulates the switch in lineage commitment by modulating endogenous RhoA activity. Expressing dominant-negative RhoA committed MSCs to become adipocytes, while constitutively active RhoA caused osteogenesis . Discher and Mooney indicated that hydrogel with different mechanical stiffnesses regulate cell proliferation, cell differentiation, and even the uptakes of non-viral vectors [2, 3, 4]. Since the environment surrounding a cell can be easily tuned by materials and devices according to our favorable design, a newly developed research methodology (integrative biology) is now recognized as a newcomer to find something new that cannot be found by conventional biological methods [5, 6]. Here, we consider that reproducing the tissue-development process in vitro is investigated as a robust tool for understanding cellular behavior in the tissue-developmental stage so that in vitro tissue engineering becomes possible.
13.2 Cell and Matrix Patterning Using Hydrogel with Static Mechanical Stimulation
13.3 Three-Dimensional Patterning of Mineralized Cell Groups in Hydrogel
The cell dynamics within the strained fibrin gel were investigated. The fibrinogen solution containing myoblast (C2C12) was used to form a gel, which was continuously subjected to 25 % strain. The cells in the fibrin gel display a specific alignment, that is, parallel to the strain direction. Interestingly, the direction of cell proliferation was identical to that of cell alignment (Fig. 13.1d). A single seeded cell therefore divided multiple times, and the oriented cells subsequently formed linear groups aligned parallel to the strain direction (Fig. 13.1e, f), in a similar manner to the cellular organization found in a longitudinal section of native skeletal muscle tissue. It is assumed that the positions of the cells in the fibrin gel are restricted to the spaces between the fibrin bundles such that they align and proliferate parallel to the strain direction. This assumption is supported by a typical SEM image (Fig. 13.1g), which shows cells positioned in the spaces between the bundle-like structures of the fibrin gel.
To investigate the alternations of cellular functions in strained fibrin gel, the cells were cultured in gels with different strain rates. At day eight, the gel subjected to a higher strain rate had enhanced cell proliferation compared to the gel subjected to a lower strain rate. The mRNA expressions of Opn and Oc, namely, osteogenic differentiation markers, were investigated at day four. Both Opn and Oc expressions decreased with the increasing strain rate from up to 50 %. These results suggest that cell functions in the strained fibrin gel are regulated by the alteration of strain rate. To confirm this suggestion, cell-derived mineralization in the gel (subjected to varied strain rate during the culture period) was investigated. Mineralization caused by cell differentiation was detected only in the sample that was subjected to strain rate decreased from 50 to 0 % at day 21 (III). In contrast, mineral deposition was not detected in the gel that was subjected to strain rate reduced from 50 to 0 % at day 28 (II) or in the gel that was subjected to 50 % strain maintained for 50 days (I) (Fig. 13.2e) .
13.4 Microvessel Patterning Using Fibrin Gel with Dynamic Mechanical Stimulation
As mentioned here, the hydrogel system can be physically tuned by applying mechanics. The cultured cells used in the physical stimulations show different behavior according to the surrounding architectures or stimulation conditions. Conventionally, the cell behavior was regulated only to confirm the effect of soluble factors that were newly-cloned. However, recent studies aiming to modulate cells and tissue to fabricate cell-based functional materials, or even to achieve biological tissue synthesis in vitro, have been performed [16, 17, 18, 19, 20, 21]. In the present study, as well as chemical stimulation, physical stimulation is also considered as a promising candidate to modulate cell and tissue functions. Moreover, the trials on these cell and tissue manipulations would be valuable to help understanding of the biological unknown during the tissue-developmental process.
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