Papers by Author: Noriko Kotobuki

Abstract: Hydroxyapatite (HA) ceramics together with various kinds of osteogenic cells have been used in bone tissue engineering. It is well known that the ceramics structure and composition affect cell proliferation / differentiation. In this study, three different types of HA ceramics were used to investigate initial cell attachment followed by osteoblastic differentiation of human mesenchymal stromal cells (MSCs). The results indicated that micro-pore affected the cell attachment and porosity (pore diameter and inter-pore connection) was the key to allow spacious distribution of the viable cells in the ceramics. This study also confirmed that surface pore areas of HA ceramics support the differentiation of human MSCs and thus the ceramics have the capability to regenerate damaged bone tissue.

Abstract: Since 2001, we have clinically utilized human bone marrow-derived mesenchymal stem cells (MSCs) for bone regeneration. The osteogenic ability of MSCs has been assessed by measurement of alkaline phosphatase activity and calcium deposition. As for the detection of in vitro calcium deposition, we have established the method using calcein, which is a calcium-binding fluorescence material. Using this fluorescence material, we could observe the calcium deposition and then estimate the value of calcium deposition. In this report, we cultured rat MSCs on culture plate as well as transparent β-TCP, and calcium deposition was visualized and quantitated using an image analyzer. After 2 weeks differentiation of rat MSCs to osteoblasts, calcium deposition on β-TCP was observed as a signal of calcium-binding fluorescence. This fluorescence signal was also quantitated with an image analyzer.

Abstract: Since 2001, we have started tissue engineered approach for hard tissue repair using mesenchymal stromal cells (MSCs) derived from patient’s bone marrow. MSCs were culture expanded on culture dish, then applied on various ceramics including hydroxyapatite (HA) ceramics. The MSCs on the ceramics were further cultured in osteogenic media to induce osteognenic differentiation. The differentiation resulted in appearance of bone forming osteoblasts as well as bone matrix on the ceramics, thus we could fabricate the tissue engineered bone. We have reported that the tissue engineered bone is effective for treatment of large bone defect, which is difficult to repair only with artificial materials such as HA ceramics. The present study focused on osteogenic capability of cryopreserved human MSCs derived from patients who already were treated by the tissue engineered bone. The MSCs showed high alkaline phosphatase activity together with abundant bone matrix formation when cultured in osteogenic media. The MSCs also showed in vivo new bone formation when implanted at subcutaneous sites of athymic nude rats. Based on the results, we concluded that the tissue engineering approach is a reliable method to be used in hard tissue regeneration.

Abstract: This study focused on in vivo osteogenic capability of bone marrow mesenchymal stem cells (MSCs) seeded on ceramic scaffold. Human MSCs from a single donor were seeded on hydroxyapatite porous ceramic (HAP) and were induced to the osteogenic lineage during in vitro culture condition, then the MSCs/HAP composites were implanted subcutaneously into immunodeficient rats. The cellular activities of the composites were assayed in order to evaluate the distribution and differentiation capability of seeded MSCs before and after implantation. These results showed that the new bone, after implantation, was derived from the donor MSCs, which adhered to the surface of the ceramics pore areas during in vitro culture. Therefore, the engrafted donor cells proliferated and showed continuous osteogenic differentiation within the recipients. Consequently, our study demonstrates the usefulness of MSCs/HAP composites for clinical applications.

Abstract: After culture expansion of mesenchymal stem cells (MSCs) from a few milliliter of fresh patient’s bone marrow, we applied the MSCs on alumina ceramic ankle prosthesis and further cultured in an osteogenic medium for 2 weeks. After the culture, the MSCs differentiated into osteoblasts, which fabricated bone matrix on the surface of ceramic prosthesis. The expansion of MSCs followed by osteogenic differentiation was done using the commercially available medium with some chemicals and patient’s own serum. The MSCs well proliferated and differentiated into osteoblasts, even the MSCs were from old aged (more than 70 years old) patients. The tissue engineered ceramic prostheses were implanted into osteoarthritic patients. Typical X-ray findings showed that radiodense areas began to appear around the cell-seeded areas on the prosthesis about 2 to 3 months after the operation. These findings confirmed the importance of tissue engineering approach for early bone fixation and the approach can be done using small number of bone marrow cells and patient’s own serum without adding animal-derived products.

Abstract: The objective of this study was to evaluate cell adhesion and proliferation on the hydroxyapatite (HAp)-coated silk fibroin (SF) fabric. Nano-scaled sintered HAp particles were covalently coated on SF chemically modified by graft polymerization. After the fabrication of the HAp/SF composite, mesenchymal cells (MCs) derived from EGFP-expressing transgenic rat bone marrow were seeded on the composite and cultured for 10 days. Fluorescence and scanning electron microscopy (SEM) revealed that the cells adhered and actively proliferated on the composites comparable to those on tissue culture polystyrene (TCPS) dishes. The results suggest that the composites are suitable for mesenchymal cell culture scaffolds and useful materials for regenerative medicine.

Abstract: Ceramics of hydroxyapatite (Ca10(PO4)6(OH)2: HA) and β-tricalcium phosphate (β-Ca3(PO4)2: β-TCP), were prepared by spark plasma sintering (SPS) at the temperatures from 800 °C to 1000 °C for 10 min with a heating rate of 25 °C·min-1. The HA ceramics prepared at 900 °C and 1000 °C showed transparency. On the other hands, transparent β-TCP ceramics was obtained by SPS at 1000 °C. In analysis of the densification behavior during sintering of HA and β-TCP by SPS, dominant sintering mechanism was plastic flow in the early stage of densification. Transparent ceramics should be the most suitable materilas to investigate the interface between human cells and ceramics.

Abstract: We have cultured mesenchymal cells (MSC) on various types of ceramic disks and used these tissue-engineered ceramics for hard tissue regeneration. In this approach, observation of cultured cell morphology is important even if culture substrata are calcium phosphate ceramics, which usually show bioactive nature. However, due to the opaque nature of the ceramics, cells observation is very difficult. Here, we demonstrate light microscopic observation of rat MSC cultured on transparent β-tricalcium phosphate ceramics (β-TCP). The culture was performed in osteogenic medium. Thus, the cell differentiated into bone-forming osteoblasts, which fabricated a mineralized matrix on the ceramic disks. Microscopic observation revealed that the cascade of osteogenic differentiation after attachment/proliferation of MSC on the ceramic disks was similar to that on a culture grade polystyrene dish. These results confirmed the excellent property of β-TCP for MSC culture leading to hard tissue regeneration.

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.