Recently, regenerative medicine is considered one of the most promising treatments for defective or damaged tissues. Cell-based therapies have been clinically performed for some tissues. And more recently, advanced therapies have shifted towards tissue engineering using biodegradable scaffolds. Cooperation between life science and engineering has led to rapid progress in tissue engineering. On the other hand, we have developed novel technology “cell sheet engineering”, which can avoid the limitations of single cell suspension injection or tissue reconstruction using biodegradable scaffolds. We aim to reconstruct various tissues using cell sheet engineering and apply the technology to regenerative medicine. Clinical trials have been performed for some tissues and significant improvement can be observed.
There are still two major problems that have not been overcome in artificial liver support systems, although they have been studied for long years. One of the problems is about the source of cells, which can proliferate and differentiate as we desired. The other is construction of blood vessels or micro-channels to exchange oxygen and nutrients. Recently, proliferative fetal hepatocytes are gradually recognized as remarkable cell source, because energetic studies in the field of molecular biology have enabled differentiation of proliferative fetal hepatocytes in vitro. In addition, transplantation of bioartificial liver tissue constructed with hepatocytes and biodegradable polymers has been also established as another methods to support liver functions. In this review, we introduce studies about liver support system using such cell source and methods.
Surface modification of poly(ethylene-co-vinyl alcohol)(EVA) was carried out by ozone oxidation in order to introduce carboxyl group. And the calcium phosphate crystaline was deposited on the surface through alternate soaking in CaCl2 and Na2HPO4 solution. It was confirmed that the crystalline was hydroxyapatite with low degree of crystallinity having carbonate apatite by X ray diffraction and IR spectra.
The purposes of this study were to investigate the differentiations and proliferations of epithelial cells of the periodontal ligament (PDL) after three-dimensional culture using collagen gel. The cells were obtained from PDL of porcine primary teeth. The collagen gels contained fibroblast were added the epithelial cells on the surface and incubated under the air/medium interface for 1, 2, 3 and 4 weeks. The paraffin sections were stained with H-E and the cryo-sections were immunostained for keratin. The histological specimens showed that the proliferated epithelial cells had a stratified structure after 1 week, but there was no keratinized layer. The dispersing tendency of the cells in the epithelial layer was seen after 2-week incubation. The immunochemical findings showed that the epithelium of the cultured tissue were expressed keratin. As the conclusion, this culture will be a useful tissue for the research of proliferation of epithelial cells related with apical periodontitis.
Periodontal ligament cells cultured on hydroxyapatite coated poly(ethylene-co-vinyl alcohol) expressed higher alkaline phosphatase activity and osteocalcin secretion but lower proliferation than on carboxyl group introduced EVA, collagen immobilized EVA and tissue culture dish. It suggested that the cells might differentiate to osteoblast and cementoblast like cells in response to the hydroxyapatite surface
We made experimental defect of maxilla bone of beagles and tried bone augmentation using implants applied Platelet-rich Plasma (PRP) and autologous bone. The newly- formed bone was labeled with calcein and alizarin red and was observed by confocal laser scanning microscopic (CLSM) with time. As a result, an ossification was seen in postoperative eight weeks, and a portion revealed caleification to implant circumference. In addition, an increase of formation and remodeling of bone were found at implant circumference in postoperative 12 weeks. From these results, it was thought that an implants applied PRP and autologous bone augmented bone at bone defects. In addition, it was suggested that CLSM was useful to observe the newly-formed bone at implant circumference.
The aim of this study was to investigate the morphology, growth and type I collagen expression of human pulp derived cells on the poly(ethylene-co-vinyl alcohol) (EVA) coated with collagen (+C) film. Cell morphology and growth curve for EVA+C group was similar to that for control group, which was cultured on the normal cell culture dish. However, for EVA group cell shape showed no extension and the growth was down-regulated. There was no difference in expression of type I collagen in the cultured cells among the three groups. These results suggested that the immobilization of collagen to EVA facilitate an attachment and extension of human pulp derived cells on the EVA film without down regulation of type I collagen.
The initial calcification of enamel was observed by the high resolution electron microscope. With gultalaldehyde fixation, the granular or amorphous structure at 20-50 nm in diameter and rod like structure of 20-50 nm in width having high density fibers in the center, were observed near the Tomes process of the ameloblasts. The grainy materials at 20-50 nm in diameter secreted by the ameloblasts were observed, when the animals were fixed with tannic acid. The hollow structures located to a distance of the several 100 nm from the cell membrane. And also the tubular structures had high density fibrils in the center. The diameter of these two structures was same as about 20-50 nm. The central fibers formed as twisted threads, strands or beads were less than 1 nm in diameter, were similar size of the atoms composed of apatite crystal. These phenomena showed the pattern of initial enamel crystals. The amorphous stippled materials fused and formed the tubular structures, which were enamel protein secreted by ameloblast. The hydrophobic small part of the enamelin captured the molecules or atoms of apatite crystal elements. Atoms and molecules accumulated in the tubule and arranged irregularly to form the strand structure in the center. As the result, the initial precursor of crystal formed in the tubular structures. It is to say the precursor hardly packed by the amorphous organic materials of enamel protein. It is the reason that the structure was insoluble by the acid attack and stained with lead-acetate. It is concluded that this amorphous structure is the precursor of initial enamel crystal. The c-axis of the crystal growth is decided by the arrangement of these tubular structures of enamelin, which was the crystal developing in the place. The tubules arranged almost perpendicular to the cell membrane of Tomes process, because the C-terminal of the enamelin had an affinity at the cell membrane of the secreting face of Tomes process, the N-terminal attached to the sheathlin in the cross striation of enamel prism. When atoms accumulated and saturated in the tubular structures, the amorphous precursor crystal, so called as strands or crystal nuclei, changed suddenly to the apatite crystal, having several lattices of atomic images, by the thermal energy potential. This initial crystal was recognized ribbon-shaped crystal of enamel. Next, the atoms adhered on the side of these crystals and grew to the plate forms. These plate-crystals increase the thickness, and finally changed to a hexagonal cylinder shape with the epitaxial growth. New crystallization of the precursor of crystals always occurs between crystals, and new crystals fused matured hexagonal crystals. Finally these crystals formed the irregular polygonal crystals. These processes take place in the spaces of dissolved amelogenin. In other words, the crystal size and form were depended on the shape and size of the space of the amelogenin, because the spaces appeared by the dissolve of amelogenin.