Masataka Yoshikawa

Histopathological study of a newly developed root canal sealer containing tetracalcium-dicalcium phosphates and 1.0% chondroitin sulfate*

We studied the possibility of the clinical use of a calcium phosphate-type newly developed sealer composed of tetracalcium phosphate, dicalcium phosphate dihydrate, and a modified McIlvains buffer solution (TDM). Another sealer using the buffer solution, to which 2.5% chondroitin sulfate was added to promote wound healing (TDM-S), was also studied. TDM and TDM-S were histopathologically compared with another type of calcium phosphate sealer (ARS), which is commercially available in Japan, in the dorsal subcutaneous tissue and in the periapical tissue of rats. TDM and TDM-S caused no inflammatory reactions in the subcutaneous tissue. The periapical tissue reacted mildly to them. ARS caused severe inflammatory reactions in both the subcutaneous and the periapical tissue. These results indicate that TDM-S has excellent histocompatibility and potential as a root canal sealer.

Reconstruction of alveolar bone defect by calcium phosphate compounds.

Osteoconductivity of newly developed calcium phosphate cements (CP-1, CP-2) was estimated in mandibular periapical alveolar bone of rats. The powder phase of CP-1 was an equimolar mixture of tetracalcium phosphate and dicalcium phosphate dihydrate, and that of CP-2 was alpha-tricalcium phosphate. The liquid phase of CP-1 and CP-2 was a solution containing tannic acid and citric acid. CP-1, CP-2, an apatitic sealer (ARS), or a zinc oxide eugenol sealer (ZOE) was respectively applied in the mechanically injured periapical regions through the root canals of both mandibular first molars of 15 rats. A further 15 rats were used as controls with no material in the region. The results of histopathological examination at 1, 3, and 5 weeks after operation were analyzed by the chi(2) test (95% confidence level). At 1 week, foreign-body giant cells were observed around CP-1 and CP-2, but not around ARS or ZOE. Congestion of small blood vessels was seen in bone defect areas of the controls. After 5 weeks, statistically significant bone reconstruction was induced by application of CP-1 (80.0%), CP-2 (90.0%) compared with ARS (33.3%). Fibrous scaring was seen in the controls. It is concluded that resolvability of CP-1 and CP-2 should contribute to osseous healing.

A maxillary central incisor having two root canals geminated with a supernumerary tooth.

This article reports the treatment of a maxillary left central incisor geminated with a supernumerary tooth. A patient having labial swelling around the maxillary incisors was referred to this hospital for diagnosis and endodontic treatment of the maxillary left central incisor. Clinical and radiographic examinations revealed a tooth having two roots and three canals. Examination of a rubber base impression of the cleaned canals showed there was neither perforation of, nor communication with, the radicular groove in the tooth.

Osteocompatibility and biocompatibility of tetracalcium phosphate cement containing cellulose

ABSTRACT We performed in vitro and in vivo experiments on the cytotoxicity and histotoxicity of the cement prepared by kneading an equimolar mixture of tetracalcium phosphate and dicalcium phosphate dihydrate with modified McIlvains buffer solution containing cellulose. The colony formation of cells (Balb/3T3) was not inhibited. The cement showed desirable biocompatibility in the dorsal subcutaneous tissue of rats and in the femoral condyles of rabbits. The cement placed in rat molar canals caused apical closure with a cementum-like calcified product. These observations suggest that the cement is useful for application to bone defects and as a dental cement.

In vivo estimation of calcium phosphate cements containing chondroitin sulfate in subcutis

Calcium phosphate cements using an equimolar mixture of tetracalcium phosphate and dicalcium phosphate dihydrate (TeDCPD) for the powder phase were experimentally developed for use in endodontic treatment. The fundamental cement is comprised of TeDCPD kneaded with modified McIlvains buffer solution containing calboxymethyl cellulose sodium salt (CEM-1). In the liquid phase of the modified one (CEM-2), chondroitin sulfate (CS) was added in place of the salt. The final concentration of CS in CEM-2 is 1%. Another one (CEM-3) contained 2% CS finally in place of the salt. X-ray diffract meter (XRD) was used to examine the crystalline phases of the cements. The tissue compatibility of the cements was examined histologically in the subcutaneous tissue using rats. The XRD results showed no dibasic calcium phosphate phase to be traced in CS containing two cements after 1 day of kneading. There were more multinucleated giant cells appearing around CEM-1 than around CEM-2 or CEM-3 after 4 weeks. Fibroblasts, collagen fibers and small vessels infiltrated into the internal porous structure of CEM-3. Excluding CEM-3, two cements were encapsulated with a dense fibrous connective tissue layer. We conclude that CS, in the experimentally developed cement, contributed to biocompatibility and bioactivity of the cement.

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.

α-Tricalcium Phosphate Cements and the Granules to Dental Pulp and Periapical Tissue

c-tricalcium phosphate (c-TCP) granules and c-TCP cements kneaded with high or low concentration of organic acid solution were respectively placed on the dental pulp surface after pulp extirpation to estimate pulp tissue responses and hard tissue formation in rat mandibular first molar canals. The tissue responses in the residual pulp in the middle and apical portion of the root canals and periapical area were histopathologically estimated 1, 3 and 5 weeks postoperatively. c-TCP granules and c-TCP cement kneaded with a low concentration of citric acid solution induced acute inflammation in the pulp tissue. c-TCP cements kneaded with high concentration of citric acid solution induce hard tissue deposition at the apical portion of root canal. It is concluded that fine granules dispersed from the material in the tissue, even though it is a biocompatible material, caused inflammatory responses.