Norimasa Tsuji

In Vivo Osteogenesis in Porous Hydroxyapatite Scaffold Processed in Hyaluronic Acid Solution

Porous hydroxyapatite (HA) scaffolds were processed in hyaluronic acid solution. Bone marrow cells obtained from the bone shaft of femurs of Fischer 344 rats at 1×106/ml concentration were seeded in pores of the scaffolds. The scaffolds were implanted in the dorsal subcutaneous tissue of rats for 2, 4, 6 or 8 weeks. Removed HA scaffolds at 2 and 4 week after dorsal subcutaneous implantation were histologically examined. At all experimental periods, osteocalcin in the scaffold was immunochemically measured for the quantitative analysis of osteogenesis by bone marrow cells in the porous HA scaffolds. Moreover, value of alkaline phosphatase (ALP) activity in the scaffolds was measured. Osteocalcin measured in scaffolds without bone marrow cells was 1.3 ng in an average and the ALP activity was 62.2 μmol at 4 week. In hyaluronic acid processed scaffold with bone marrow cells, quantity of osteocalcin increased from 1.6 ng at 2 week to 2.2 ng at 4 week after implantation of the scaffold. Histologically, many pores containing bone in the scaffolds immersed in hyaluronic acid solution were detected. Significant difference of the quantity of osteocalcin was recognized between 2 and 4 week implantation. There was no significant difference in the quantity of osteocalcin between the scaffolds implanted for 4 and 8 weeks. Value of ALP activity of the scaffold implanted for 4 weeks showed significant difference comparing with that implanted for 6 and 8 weeks. From the results of this study, quantitative increase of the bone formation in the pores of HA scaffolds would be able to observe from 6 to 8 weeks after implantation on the scaffolds by immersion in hyaluronic acid solution

Comparison of Hard Tissue Formation in Two Porous Hydroxyapatite Scaffolds Treated with Hyaluronic Acid Sodium Salt

The purpose of this study was to estimate hard tissue formation in two types of porous columnar hydroxyapatite (HA) in order to use as a scaffold for regeneration of dentine-pulp complex. Hard tissue formation in the columnar HA scaffold with a hollow center was compared to that in the columnar HA scaffold without a hollow center. The scaffolds were immersed in hyaluronic acid sodium salt solution and were soaked in bone marrow cell suspension. They were respectively implanted into dorsal subcutis of rats for 4 weeks. Serially sectioned paraffin specimens were made and observed histologically. The scaffolds with a hollow center showed new hard tissue formation in many pores between the superficies and the wall of hollow. On the other hand, in the scaffolds without a hollow center, hard tissue formation was observed in only a few pores in the area near the external superficies. The results of this study suggested that the supply of nutrition and bioactive substance from the surrounding tissue were indispensable for differentiation of bone marrow cells and formation of new hard tissue in scaffold. A large contact area of a scaffold to the surrounding tissue may contribute to nutrition supply into the pores.

Osteogenic Influence of Lysine in Porous Hydroxyapatite Scaffold

The purpose of this study was to estimate influence of lysine for osteogenesis in the porous hydroxyapatite (HA) scaffolds with bone marrow cells. The HA scaffolds were soaked in 100mM concentration of lysine solution. They were kept in bone marrow cell suspension at 1×106 cells/ml density. Another HA scaffolds without immersion in lysine solution were kept in the cell suspension at 1×106 or 1×107 cells/ml density. They were respectively implanted into dorsal subcutis of rats for 4 weeks. Serially sectioned paraffin specimens were made and observed histologically. In several sections, total pores and ones with bone were counted. Many pores containing bone were found in1×107 cells/ml concentration group. The significant difference was between 1×107 cells/ml group, the lysine group, and 1×106 cells/ml group. Although more bone formation was seen in lysine group than in 1×106 cells/ml group. There was no significant difference between the groups. Concentration of lysine to add in culture medium or scaffold should be improved respectively.

Hard Tissue Formation by Bone Marrow Stem Cells in Sponge Scaffold with Dextran Coating

A scaffold is necessary for bone regeneration, because the structure of hard tissue is three-dimensional. For application in dentistry, it is desirable for the scaffold to be modified the geometry easily according to the defect. Therefore it is thought that a sponge is suitable as a scaffold. A sponge (Polyvinyl formal®: PVF) was purchased for this study. The sponge was made from polyvinyl alcohol (PVA) with formalin preparation. In this study, the PVF sponges were coated with dextran, because dextran may affect cell adhesion. The effect of dextran for hard tissue formation by bone marrow stem cells in the PVF scaffold was examined in vivo. PVF scaffolds (5x5x5 mm) were immersed in each dextran solution of 10 kDa (2 g/dl) and 500 kDa (4 g/dl) and air dried. The coating for the PVF scaffolds by dextran was confirmed by SEM. Rat femur bone marrow cells of 1.107 were seeded in PVF scaffolds. They were implanted for four weeks in rat dorsal subcutaneous tissue. In removed scaffolds, hard tissue formation was examined histologically. Bone formation was conspicuously found in the scaffold coated with 10 kDa of dextran. Value of ALP activity measured biochemically in the scaffold was 58.5 mM/scaffold. By immunochemical examination, it was measured that quantity of osteocalcin in the scaffold was 25.9 ng/scaffold and that of Ca was 129.2 ig/scaffold. These values showed a significantly higher in comparison with those of the scaffold with 500 kDa dextran coating and without coating. It was suggested in this study that coating of the scaffold with dextran induce increase of quantity of hard tissue formation in PVF scaffold in vivo. It is concluded that 10 kDa dextran coating of PVF scaffolds effectively contribute to adhesion of bone marrow cells in the scaffold.