Katsuko Furukawa

The effect of substrate microtopography on focal adhesion maturation and actin organization via the RhoA/ROCK pathway

Recently, a growing number of reports have reported that micro- or nanoscale topography enhances cellular functions such as cell adhesion and stem cell differentiation, but the mechanisms responsible for this topography-mediated cell behavior are not fully understood. In this study, we examine the underlying processes and mechanisms behind specific topography-mediated cellular functions. Formation of focal adhesions (FA) was studied by culturing cells on different kinds of topographies, including a flat surface and surfaces with a micropatterned topography (2 μm lattice pattern with 3 μm intervals). We found that the formation and maturation of focal adhesions were highly dependent on the topography of the substrate although the shape, morphology and spreading of cells on the different substrates were not significantly affected. Focal adhesion maturation and actin polymerization were also promoted in cells cultured on the micropatterned substrate. These differences in cell adhesion led us to focus on the Rho GTPases, RhoA and downstream pathways since a number of reports have demonstrated that RhoA-activated cells have highly enhanced focal adhesions and actin activation such as polymerization. By inhibiting the Rho-associated kinase (ROCK) and downstream myosin II, we found that the FA formation, actin organization, and FAK phosphorylation were dramatically decreased. The topographical dependency of FA formation was also highly decreased. These results show that the FA formation and actin cytoskeleton organization of cells on the microtopography is regulated by the RhoA/ROCK pathway.

Three-dimensional seeding of chondrocytes encapsulated in collagen gel into PLLA scaffolds.

Tissue engineering approaches have been clinically tried to repair damaged articular cartilages. It is an essential step to uniformly seed chondrocytes into 3D scaffolds in order to reconstruct tissue-engineered cartilages in vitro, but the tissue engineering could not have been provided with efficient cell seeding methods. Type I collagen is clinically used and known as a cytocompatible material, having recognition sites for integrins. Collagen gel encapsulating chondrocytes has been tried for making regenerated cartilages, but it is found difficult to have the gel keep its original shape after long-term culture, because of shrinking. On the other hand, 3D scaffolds, either of a nonwoven structure or a sponge-like structure, involve difficulty in that chondrocytes could not be uniformly seeded, although they have adequate initial mechanical properties. In this study, by combining collagen gelation with a nonwoven PLLA scaffold, we achieved uniform cell seeding into the 3D scaffold. Bovine articular chondrocytes were mixed with type I collagen solution, and the solution was poured into the nonwoven PLLA scaffold (1.5 mm thick, f 15 mm). The collagen–chondrocyte mixture was made into gel at 37°C for 1 h. The 0.39% collagen mixture was viscous enough to prevent cells from precipitating during gelation. Almost all chondrocytes were able to be incorporated into the PLLA scaffolds by mixing with collagen solution and subsequently making into gel, while 30–40% of the chondrocytes seeded as a cell suspension were not trapped into the PLLA scaffolds. The method presented, where chondrocytes were mixed with collagen solution, and the mixture was incorporated into a 3D scaffold, then made into gel in the scaffold, could serve as an alternative for in vitro cartilage regeneration, also simultaneously having the advantages of both materials.

3D Culture of Osteoblast-Like Cells by Unidirectional or Oscillatory Flow for Bone Tissue Engineering

A medium perfusion system is expected to be beneficial for three‐dimensional (3D) culture of engineered bone, not only by chemotransport enhancement but also by mechanical stimulation. In this study, perfusion systems with either unidirectional or oscillatory medium flow were developed, and the effects of the different flow profiles on 3D culturing of engineered bone were studied. Mouse osteoblast‐like MC 3T3‐E1 cells were 3D‐cultured with porous ceramic scaffolds in vitro for 6 days under static and hydrodynamic conditions with either a unidirectional or oscillatory flow. We found that, in the static culture, the cells proliferated only on the scaffold surfaces. In perfusion culture with the unidirectional flow, the proliferation was significantly higher than in the other groups but was very inhomogeneous, which made the construct unsuitable for transplantation. Only the oscillatory flow allowed osteogenic cells to proliferate uniformly throughout the scaffolds, and also increased the activity of alkaline phosphatase (ALP). These results suggested that oscillatory flow might be better than unidirectional flow for 3D construction of cell‐seeded artificial bone. The oscillatory perfusion system could be a compact, safe, and efficient bioreactor for bone tissue engineering. Biotechnol. Bioeng. 2009;102: 1670–1678.

Rapid and large-scale formation of chondrocyte aggregates by rotational culture

Chondrocytes in articular cartilage synthesize collagen type II and large sulfated proteoglycans, whereas the same cells cultured in monolayer (2D) dedifferentiate into fibroblastic cells and express collagen type I and small proteoglycans. On the other hand, a pellet culture system was developed as a method for preventing the phenotypic modulation of chondrocytes and promoting the redifferentiation of dedifferentiated ones. Because the pellet culture system forms only one cell aggregate each tube by a centrifugator, the pellet could not be applied to produce a tissue-engineered cartilage. Therefore, we tried to form chondrocyte aggregates by a rotational culture, expecting to form a large number of aggregates at once. In order to increase cell–cell interactions and decrease chondrocyte–material interaction, dishes with low retention of protein adsorption and cell adhesiveness were used. In addition, rotational shaking of the dish including cells was attempted to increase the cell–cell interaction. The shaking speed was set at 80 rpm, so the cells would be distributed in the center of the dish to augment the frequency of cell–cell contact. Under these conditions, bovine articular chondrocytes started aggregating in a few hours. At 24–36 h of rotational culture, aggregates with smooth surfaces were observed. Parameters such as increase of culture time and addition of TGF-β controlled diameters of the aggregates. There were many fusiform cells at the periphery of the aggregates, where the cells tended to form a multilayered zone in cross sections. In addition, lacune-like structure, which was almost the same as pellet culture, was observed. It was found that the internal structure of the aggregates was similar to that of pellets reported previously. Therefore, the aggregates formed by a rotational culture could become an essential component to make tissue-engineered artificial cartilage.

The switching of focal adhesion maturation sites and actin filament activation for MSCs by topography of well-defined micropatterned surfaces.

Securing robust cell adhesion between cells and biomaterials is one of key considerations for tissue engineering. However, the cell adhesion investigation by the biophysical effects such as topography or rigidity of substrates has only been recently reported. In this study, we examined the spatial property of focal adhesions by changing the height of micropatterns in two kinds of microtopography (grid and post) and the stiffness of the substrates. We found that the focal adhesion localization is highly regulated by topographical variation (height) of gird micropattens but not the rigidity of substrates or the function of actin cytoskeleton, although the latters strongly influence the focal adhesion size or area. In detail, the change of the height of the grid micropatterns results in the switching of focal adhesion sites; as the height increases, the localization of focal adhesion is switched from top to bottom areas. This study demonstrates that the localization of focal adhesion on well-defined micropatterned substrates is critically determined by the topographical variation in the micropatterns.

Formation of human fibroblast aggregates (spheroids) by rotational culture.

In the current study, we attempted to form aggregates of fibroblasts by rotationally shaking, declining fibroblast–material interactions, and augmenting cell–cell interactions. In addition, to promote cell–cell interactions, the medium was supplemented with insulin, dexamethasone, and basic fibroblast growth. Under such improved culture conditions, normal neonatal human dermal fibroblasts formed spheroidal aggregates within 1 day of rotation on a rotational shaker. The aggregates that formed had irregular shapes and were composed from only several cells after 12 h. However, they became nearly spheroidal after 24 h of shaking. The aggregates were approximately 240 μm in diameter. After 36 h of shaking, their shape became more rounded and their surfaces became smoother. No evidence of necrosis in the center of the aggregates was observed, although a small number of dead cells was scattered throughout the aggregates. After 24–36 h, aggregates of normal human fibroblasts were collected and reinoculated onto a scaffold composed of polyglycolic acid, which is used commercially as a scaffold for artificial skin, coated with collagen. The aggregates were successfully trapped to the mesh of polyglycolic acid and became attached within 24 h. Therefore, the aggregates could provide an alternative method for seeding fibroblasts to scaffold for an artificial skin, such as a mesh of polyglycolic acid.

Optimization of allograft implantation using scaffold-free chondrocyte plates.

If a tissue-engineered cartilage transplant is to succeed, it needs to integrate with the host tissue, to endure physiological loading, and to acquire the phenotype of the articular cartilage. Although there are many reported treatments for osteochondral defects of articular cartilage, problems remain with the use of artificial matrices (scaffolds) and the stage of implantation. We constructed scaffold-free three-dimensional tissue-engineered cartilage allografts using a rotational culture system and investigated the optimal stage of implantation and repair of the remodeling site. We evaluated the amounts of extracellular matrix and gene expression levels in scaffold-free constructs and transplanted the constructs for osteochondral defects using a rabbit model. Allografted 2-week constructs expressed high levels of proteoglycan and collagen per DNA content, integrated with the host cartilage successfully, and were able to counter physiological loads, and the chondrocyte plate contributed reparative mesenchymal stem cells to the final phenotype of the articular cartilage.

Hybrid of gel-cultured smooth muscle cells with PLLA sponge as a scaffold towards blood vessel regeneration.

Although rapid formation of a smooth inner surface is important in constructing an artificial vascular graft, a conventional model that uses a biodegradable polymer such as polyglycolic acid needs long-term culture to form it. In another model, which uses collagen gel, it is reported that prompt formation of the smooth inner surface was achieved. But the mechanical properties were not suitable, resulting in rupture under high pressure at the arterial level. Therefore, we propose a new artificial vascular graft model made of biodegradable polymer, gel, and cells. At first we manufactured an artificial vascular graft (i.d. 5 mm, o.d.7 mm) consisting of poly-l-lactic acid (PLLA) with open pore structures by using gas-forming methods. After mixing human normal aortic smooth muscle cells (SMCs) with type I collagen solution, pores of the PLLA scaffold were filled with the mixture. The collagen mixture was made into gel in the pores of the PLLA scaffold, incubating at 37°C. WET-SEM analysis showed that the prompt formation of a smooth inner surface was achieved in the new model. The ratio of incorporation of SMCs into the artificial vascular graft became approximately 100% by using the cell–collagen mixture, whereas only 40% of SMCs were trapped in the conventional model where SMCs were inoculated as a cell–medium suspension. Therefore, it was suggested that the new artificial vascular graft model was superior in smooth inner surface formation and cell inoculation, compared with conventional models using either biodegradable polymer or gel.

Tissue-engineered skin using aggregates of normal human skin fibroblasts and biodegradable material

Higher-density inoculation of fibroblasts into a three-dimensional scaffold should accelerate wound healing after skin implantation. This study attempted to develop tissue-engineered skin with a higher density of fibroblasts. We first attempted to fabricate three-dimensional high-cell-density aggregates (spheroids) of normal human fibroblasts for application to tissue-engineered skin. Our method consisted of rotational shaking with nontreated dishes, decreasing fibroblast-meterial interactions, and augmenting cell-cell interaction. To prompt aggregate formation, the medium was supplemented with insulin, dexamethasone, ascorbic acid, and basic fibroblast growth factors that potentiate secretion of extracellular matrices. Under such improved conditions, fibroblasts were able to form spheroidal aggregates within 24 to 36h of rotational culture. Although the formed aggregates were irregular in shape and were composed of only several cells after 12h, they became almost spheroidal after 24h. The aggregates grew even more round after 36h, and their surfaces became smooth. After 36h of rotational culture, the fibroblast aggregates were collected and reinoculated onto a biodegradable mesh composed of polyglycolic acid coated with collagen. The aggregates were trapped in the material and became attached after 24h. Finally, because transforming growth factor-β3 (TGF-β3) is known to accelerate wound healing, we conducted a semiquantitative analysis of TGF-β3 mRNA in both the fibroblast monolayers (two-dimensional culture) and the aggregates (three-dimensional culture). Analysis of TGF-β3 mRNA expression showed that mRNA expression was greater in the fibroblasts of aggregates than in a monolayer. Therefore, our newly developed dermal graft is expected to accelerate wound healing faster than conventional grafts.

Recent technological advancements related to articular cartilage regeneration

Some treatments for full thickness defects of the articular cartilage, such as the transplantation of cultured chondrocytes have already been performed. However, in order to overcome osteoarthritis, we must further study the partial thickness defects of articular cartilage. It is much more difficult to repair a partial thickness defect because few repair cells can address such injured sites. We herein show that bioengineered and layered chondrocyte sheets using temperature-responsive culture dishes may be a potentially useful treatment for the repair of partial thickness defects. We also show that a chondrocyte-plate using a rotational culture system without the use of a scaffold may also be useful as a core cartilage of an articular cartilageous defect. We evaluated the properties of these sheets and plates using histological findings, scanning electrical microscopy, and photoacoustic measurement methods, which we developed to evaluate the biomechanical properties of tissue-engineered cartilage. In conclusion, the layered chondrocyte sheets and chondrocyte-plates were able to maintain the cartilageous phenotype, thus suggesting that they could be a new and potentially effective therapeutic product when attached to the sites of cartilage defects.