A bi-phasic scaffold consisting of a columnar formaldehyde-acetalized polyvinyl alcohol (PVF) sponge and a cylindrical porous hydroxyapatite (HA) with a hollow center was devised. Rat bone marrow cells (rBMCs) were seeded into the sponge placed in the hollow center of the cylindrical porous HA. The bi-phasic scaffold, a cylindrical porous HA and a PVF sponge separated from a bi-phasic scaffold after rBMC seeding, and a PVF sponge without rBMCs as a negative control, were implanted for 6 weeks into rat dorsal subcutaneous tissue. In each construct, bone formation was examined histologically and osteocalcin was measured immunochemically. Bone formation was observed in the bi-phasic scaffold and also in the cylindrical porous HA isolated from the bi-phasic scaffold. A significant difference in the quantity of osteocalcin was observed between the bi-phasic scaffold and the isolated cylindrical porous HA. No bone formation was found in the isolated PVF sponge. The bi-phasic scaffold as an outer layer of the scaffold seemed to inhibit the outflow of rBMCs from the PVF sponge. This type of bi-phasic scaffold may have two specific characteristics: Attachment of cells both in PVF sponge and cylindrical porous HA.

To regenerate teeth and bones, a scaffold is essential. Hydroxyapatite has been used in many studies, but scaffolds made of hydrogel or sponge are also effective. The hardness of hydroxyapatite is a disadvantageous property for shaping. A sponge is suitable as a scaffold because the shape of the scaffold can be matched to the defect. Sodium alginate (AL) has excellent biocompatibility and a sponge can be made from this gel by lyophilization. The purpose of this study was to promote hard tissue formation with a sponge made of AL gel or AL gel and chondroitin sulfate (Chs). Sponges were made from AL gel, which were then used as a scaffold to investigate their effectiveness for the formation of hard tissue or bone. Hard tissue formation in the pores of these AL sponges was estimated in vitro and in vivo. In the sponge made from AL gel, the concentration of AL and the addition of Chs affected bone formation. Concentration of AL would affect the shape and size of the pores. ALP activity in the sponges was also enhanced by Chs. The amount of osteocalcin (OC) produced in the sponge by rat bone marrow cells increased depending on the AL and Chs concentrations in the gel. The level of OC amount in the sponges made from AL gel containing Chs was notable in vivo. Bone formation in the sponge in vivo was affected by the addition of Chs in AL gel. The quantity of OC and the bone formation in AL sponges in subcutaneous tissue in vivo suggested that AL sponges can be useful as a scaffold. 2.2. Preparation of AL Sponge Scaffolds and Microstructural Observation by SEM Each gel was poured into a ring made of stainless steel with a 6-mm inner diameter, 8-mm outer

Because of the three-dimensional structure of bone or hard tissue such as a tooth, a scaffold is necessary for its regeneration by cellular engineering. Commonly, for in vivo examination, hydroxyapatite (HA) has been used as such a scaffold. Cylindrical HA with a hollow center, which included a columnar formalin-treated polyvinyl alcohol sponge, was used in this examination as a scaffold. The sponge had been coated with L-tryptophan or L-lysine before insertion into the hollow center of the HA. Rat bone marrow cells (rBMCs) derived from the femur were seeded in the sponge before insertion into the hollow center of HA. The number of rBMCs seeded in each sponge was 1.5 × 10 6 . These scaffolds were implanted subcutaneously into the backs of Fischer 344 rats for 6 weeks. In the amino-acid-coated sponge in HA, osteogenesis was found histologically. An osteocalcin level of approximately 10 µg was measured in the scaffolds with L-tryptophan-coated formalized polyvinyl alcohol sponge containing rBMCs, 4 µg on average in the scaffolds with L-lysine-coated sponge containing the cells and about 2 µg in each scaffold with uncoated sponge containing the cells. The structure of the scaffolds used in this study was thought to be suitable for osteogenesis by rBMCs. It was concluded that tryptophan, as a factor for bone formation by stem cells, functioned by promoting cell adhesion and the differentiation of stem cells into osteoblasts.

One great advantage of bone marrow is that a large number of stem cells can be obtained.

Introduction: The purpose of this study was to assess the significant proliferation of dental pulp-derived stem cells in vitro from rats with the systemic administration of immunosuppressant in subcutis. There must be a sufficient number of stem cells for tooth regeneration. However, number of mesenchymal stem cells in the dental pulp tissue is a small. Then, the proliferation of stem cells must be accelerated for hard tissue formation. The subcutaneous injection of the immunosuppressant would enhance the hard tissue forming ability of dental pulp cells of rat. It was hypothesized in this study that differentiation of stem cells into blasts would be effectively promoted by suppression of the systemic immune response. Materials and methods: The dental pulp cells of rats with immunosuppressant injection subcutaneously were cultured with or without addition of the immunosuppressant in the medium containing dexamethasone for calcified nodule formation. Ca2+ by decalcification of calcified nodules were quantitatively analysed. Statistical comparisons between the quantities of Ca2+ were performed using two-way unrepeated ANOVA followed by post hoc analysis with Tukey-Kramer’s test. Differences of p < 0.01 were considered significant. Results: The proliferation and differentiation of stem cells among dental pulp cells was inhibited by the presence of immunosuppressive agents in the culture medium. However, stem cells obtained from rats after systemic administration of an immunosuppressive agent exhibited a high ability to form calcified nodules. Conclusions: To promote proliferation and differentiation of stem cells, systemic administration of an immunosuppressant to individuals prior to harvesting stem cells would be recommended.

Some chemicals can promote the differentiation of stem cells and the formation of hard tissues. In this study, the effects of transferrin on calcified nodule formation were evaluated using bone marrow cells obtained from the femora of Fischer 344 rats. Transferrin was added to the culture medium to promote the proliferation and differentiation of stem cells among the bone marrow cells into osteoblasts or chondroblasts. Calcified nodule formation was confirmed macroscopically in the culture medium and evaluated quantitatively by measuring Ca 2+ in the solution after demineralization using formic acid. The cells were cultured in 2 ml of minimum essential medium with 20 μl of solution containing 100, 200 or 400 ng of transferrin. Dexamethasone was also added to the medium at 10 nmol. Subculturing was performed for 2 weeks. The concentration of Ca 2+ after decalcification of the calcified nodule formed in the bone marrow cell culture with dexamethasone was approximately 10 g/ml. The addition of 100~200 ng of transferrin to the bone marrow cell culture resulted in the maximum concentration of Ca 2+ and size of the formed calcified nodule in the medium. For calcified nodule formation by bone marrow cells, the optimal concentration of transferrin in the culture medium was suggested to be 200 ng in 2 ml. This study suggested that transferrin is beneficial together with dexamethasone. It may play an important role in bone formation in vivo.

No abstract