Papers by Author: Motohiro Hirose

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: In the field of tissue engineering for bone regeneration, there have been many studies that examined in the bone forming ability of the porous biomaterials with mesenchymal stem cells (MSCs). To promote the tissue engineering approach in clinical situation, there is a need for the establishment and standardization of evaluation methods for measuring the in vivo bone forming ability. In this study, we examined the seeding process using rat MSCs to ascertain whether it is a valid protocol for various materials. Our results showed that the cell seeding process for the fabrication method of MSCs/materials composite influenced the number and distribution of the MSCs in the materials, therefore the process is a key to show new bone formation which derived from the seeded MSCs. Here, we describe the detailed process which can show consistent new bone formation in pores of the materials.

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: Cultured osteoblasts with mineralized matrix (regenerative cultured bone; RCB) have been used for patients having osseous defects. The RCB can be fabricated on various ceramic substrata using patient’s mesenchymal stem cells (MSCs) at our cell processing center (CPC). Since we have to transport the RCB for hospitals outside of our town, the RCB should maintain the cell viability for a long time. To determine a suitable condition for transportation of the RCB, stability of the RCB was analyzed by biochemical assays. Even outside CO2 incubator, the RCB kept high level of viability until 24 hours at 25°C and also showed low level of cytotoxicity for 24 hours at 37°C and 25°C. On the other hand, the RCB incubated for 24 hours at 4°C outside CO2 incubator resulted in extremely low level of viability with obvious cytotoxicity. These data indicated that stability of the RCB can be maintained for 24 hours at 37°C and 25°C, but not at 4°C. Therefore, the RCB derived from patient’s MSCs can be transported and utilized for the patients at hospitals far away from the CPC.

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.