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Three-dimensional porous scaffolds play an important role for tissue engineering. The recent developments of porous scaffolds and their preparation methods, especially those developed by our group, are summarized in this review. A method for the preparation of biodegradable porous scaffolds has been developed by using pre-prepared ice particulates as porogen material. A kind of composite biodegradable porous scaffolds has been developed by forming collagen microsponges in the pores or interstices of a synthetic polymer sponge or mesh. A composite sponge of synthetic polymer, collagen and hydroxyapatite has been developed for hard tissue engineering. Bovine articular cartilage-like tissue has been engineered by culturing chondrocytes in the PLGA-collagen scaffolds.

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.

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 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.

To enhance the liver-specific functions of rat hepatocyte aggregates without the addition of exogenous growth factors, polylactic acid-polyglycolic acid (PLGA)/gelatin microcapsules that release insulin, dexamethasone, epidermal growth factors, and glucagon were prepared and incorporated into the hepatocyte aggregates in suspension culture. Precoating the capsules with fibronectin enhanced the incorporation of the microcapsules into the hepatocyte aggregates. In a growth factor- and hormone-free culture medium, these microcapsule-containing aggregates showed a sustained cell number and an ammonium detoxification capacity compared with two types of control culture. One was the culture of microcapsule-free aggregates with albumin-containing control capsules and the other was the culture of capsule-free aggregates that were supplied with the same factors and hormones from the culture medium at concentration levels expected from the release kinetics of the microcapsules. Our new methodology demonstrates that the performance and duration of bioartificial liver systems can be enhanced due to a more efficient maintenance of cell number by using such growth factor- and hormone-releasing microcapsules.

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.

The effects of hardystonite (Ca2ZnSi2O7, CSZn) and tricalcium phosphate (β-TCP) on the proliferation and osteogenic differentiation of human bone marrow-derived mesenchymal stem cells (MSCs) were compared by directly culturing MSCs on ceramic disks (contact mode) or separately culturing cells with ceramic disks (non-contact mode). In non-contact mode, the CSZn ceramic supported MSC proliferation more strongly than did the β-TCP ceramic. However, in contact mode, the MSCs proliferated more quickly on the β-TCP ceramic than they did on the CSZn ceramic. Alkaline phosphatase (ALP) staining and osteogenic gene expression analysis showed that the CSZn and β-TCP ceramics had significant effects on the promotion of the osteogenic differentiation of MSCs in both non-contact and contact mode. Furthermore, in contact mode, the CSZn disk promoted the osteogenic differentiation of MSCs more strongly than did the β-TCP disks. Even without the induction of dexamethasone and β-glycerophosphate, CSZn stimulated the osteogenic differentiation of MSCs. These results suggest that CSZn ceramic would be a useful candidate material for bone regeneration and hard tissue engineering. [ABSTRACT FROM AUTHOR]

Three-dimensional cultures of pancreatic islet cells in porous scaffolds or hydrogels have been constructed as a biohybrid artificial pancreas. A thin mesh of a PLGA-collagen hybrid was used to culture rat RIN-5F cells. The hybrid mesh was coated with laminin, fibronectin, vitronectin, type IV collagen, and poly(L-lysine) were evaluated and mesh without coating was used as a control. Cell adhered and proliferated on all of the coated and uncoated meshes. The cells formed spheroids in the uncoated, poly(L-lysine)-, fibronectin-, vitronectin-, and type IV collagen-coated hybrid meshes, while forming a layered structure in the laminin-coated hybrid mesh. Cell adhesion on the coated PLGA-collagen hybrid meshes was higher than that for the uncoated hybrid mesh. The laminin-coated hybrid mesh showed the greatest level of adhesion. The insulin secretion capacity of RIN-5F cells was at the same level for all coated and uncoated PLGA-collagen hybrid meshes and higher than that of cells cultured on cell culture plates. The 3D cultured PLGA-collagen hybrid meshes promoted insulin production capacity. Gene expression analysis showed that genes encoding insulin I, insulin II, and the pancreatic transcription factor PDX-1 (pancreas/duodenum homeobox 1) was expressed. These results indicate that the PLGA-collagen hybrid meshes support adhesion, proliferation, and differentiation of RIN-5F cells that allows culturing pancreatic islet cells on 3D constructs. [ABSTRACT FROM AUTHOR]

Rat and human bone marrow cells (BMCs) were cultured on a composite ceramic of zinc-containing β-tricalcium phosphate and hydroxyapatite (ZnTCP/HAP) with a (Ca+Zn)/P molar ratio of 1.60 and varying zinc contents. After a 2-week culture of the BMCs in the presence of β-glycerophosphate and dexamethasone, many macroscopic mineralized areas with high alkaline phosphatase (ALP) activity were seen on the ZnTCP/HAP ceramic disks. The ALP activity increased with increasing zinc content in the ceramics. The highest ALP activity was observed when the BMCs were cultured on the ceramics with 1.26 wt % zinc, and the ceramics released zinc ions at concentrations from 2.2 to 7.2 μg/mL into the culture medium. Zinc ions were incorporated into mineralized areas produced by BMCs. BMCs also were directly cultured onto the culture dish surface, and the addition of 100 μM of free ZnCl2 (6.5 μg/mL) to the culture medium significantly increased the ALP activity of the BMCs relative to the culture medium without the ZnCl2addition. The maximum zinc concentration required to enhance mineralization was higher in human BMCs than in rat BMCs. The present study demonstrates the superiority of ZnTCP/HAP ceramics over TCP/HAP in supporting the osteogenic differentiation of BMCs, and thus these ceramics are safe and useful in clinical settings, such as for bone reconstructive surgery. © 2003 Wiley Periodicals, Inc. J Biomed Mater Res 67A: 1115–1122, 2003 [ABSTRACT FROM AUTHOR]