Papers by Author: Yoshinori Takakura

Abstract: Marrow mesenchymal cells contain stem cells and can regenerate tissue. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However there is room for improvement in the culture methods; and here, we examined the optimal frequency of medium changing. Marrow cells were collected from the femur of a Fisher 7 week-old male rat. At 2 weeks after primary culture in a standard medium (MEM containing 15% bovine fetal serum), the cultures were trypsinized to prepare a cell suspension and divided into two groups, with or without addition of dexamethasone (Dex) to the osteogenic medium. To investigate optimal frequency, we further divided into 5 sub-groups; no changing (M0), 1 time/week (M1), 2/week (M2), 3/week (M3), and everyday (M7). After 2 weeks of subculture, the tissue was harvested and then ALP activity and calcium and DNA contents measured. In both of the Dex groups, there was significantly high ALP activity in the higher frequency group; but there was no significant DNA content. Also, in the Dex(+)group, there was a significant increase of calcium content in only the M3 and M7 sub-groups. Thus, we showed that the osteogenic potential of cultured bone is cultivated by increasing the frequency of medium changing.

Abstract: Osseous tissue can be formed by culturing marrow cells with compounds such as dexamethasone and that a bone matrix cultured in this manner possesses BMP activity. We have reported that artificial bones with a high level of osteogenic potential can be prepared by culturing artificial bone materials with cultured osseous tissue. Here, in an attempt to develop activated cultured bone constructs with even greater osteogenic potential, the effects of the female hormone estriol on osteogenesis were investigated. Bone marrow cells were collected from the femur shafts of 7-week-old male Fischer rats, and subjected to primary and secondary cultures. During secondary culture with or without dexamethasone (Dx), 10-5, 10-6, 10-7, 10-8 or 10-9 M of estriol was added to a standard culture medium containing ascorbic acid and β-glycerophosphosphate. The alkaline phosphatase(ALP) activity and Ca levels were measured and statistically analyzed. There was a significant difference in ALP activity between the control group and the estriol groups, and ALP activity was the highest in the 10-7 and 10-8 M groups, being about 2.5 times higher than in the control group. Similar results were seen for Ca levels. Furthermore, in vivo study showed10-7M-estriol-treated-cultured bone/ceramic construct has significant high osteogenic potential when it is grafted into in vivo. Estriol has been reported to increase bone mass, and the results of the present study suggest that the osteogenic potential of cultured bone constructs can be more than doubled by adjusting the concentration of estriol in bone marrow cell culture. Therefore, the use of estriol may be able to facilitate osteogenesis in bone regeneration therapy.

Abstract: Alumina ceramics have excellent mechanical and biocompatible properties, but are bioinert and hence have no bone-bonding properties. We took a tissue engineering approach in an attempt to modify the ceramic surface and so provide an osteogenic/osteoconductive milieu. We used fresh human bone marrow cells obtained from the iliac crest by needle aspiration for culture expansion of mesenchymal stem cells (MSC) followed by in vitro osteogenic differentiation on both tissue culture polystyrene (TCPS) and alumina ceramics. We have succeeded in expanding the number of MSC from all 35 cases and compared the differentiation capability of selected MSC on alumina ceramics to that on TCPS. The cells on both substrata showed extensive alkaline phosphatase staining and mineralization as evidenced by calcein uptake. Biochemical analyses revealed high levels of alkaline phosphatase activity, osteocalcin expression, and calcium content. These data indicate that an alumina ceramic surface can support a differentiation cascade of MSC resulting in osteoblastic phenotype expression of the cells. Based on these results, we have performed clinical applications of tissue engineered total joint replacements for osteoarthritic patients.