Papers by Author: Yoshinori Takakura

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: 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: HA has a high affinity for bone as well as various tissues. In the present study, we investigated an affinity for abdominal organs. Coralline hydroxyapatite ceramic (HA, cubic structure 4x4x4mm, Interpore 500) was used in this experiment. We made two incisions in the lower back of a 5-week-old male nude mouse, and implanted HA blocks. One was placed around the liver at the right side and another one was placed around the kidney at the left side. The organ fibrous capsule was not removed. At 6 weeks after implantation, mice were sacrificed under overanesthesia and HA blocks were retrieved and prepared for histological analysis. In the HE stain of HA blocks around liver, liver tissue is invaded into the HA pore areas. Hepatocyte proliferation in trabecular pattern was seen in contact with the surfaces of many HA pores. Within some pores, hepatic lobular pattern, Glisson sheath or central vein could be detected. In the HA around kidney, renal tissue was observed in many pores. The pore areas of HA were fullfilled with grumerulus and urinary tube tissues. In contact with the surfaces of some HA blocks, the tissue invasion of pancreas and spleen tissue were recognized. These results indicate that porous HA has a high affinity for the celiac organs, and has a stimulatory effect on celiac organ regeneration. Especially, concerning the regeneration of kidney, it has not been reported yet, so this report is very interesting. HA is also very useful as a scaffold of the organ regeneration.

Abstract: Posterolumbar fusion, which involves placing a bone graft in the posterolateral portion of the spine, has been applied to patients with lumbar instability due to structural defects or regressive degeneration. However, harvesting cancellous bone from the ilium is associated with severe postoperative pain, and patients experience more pain at the harvest site than at the graft site, thus resulting in poor patient satisfaction. If a tissue engineering approach was used to produce autogenous bone ex vivo with culture techniques, spinal fusion could be performed without damaging normal tissues. In all patients, 10 to 20 mL of bone marrow fluid was collected from the ilium and cultured in MEM containing autologous serum or fetal bovine serum and an antibiotic. After two weeks in primary culture, the marrow mesenchymal cells were seeded onto porous beta-TCP block, and tissue engineered bone were fabricated as we reported previously. Decompressive laminectomy and posterolateral lumbar fusion with use of the tissue engineered bone thus obtained were then done. In all patients, the implanted artificial bone survived and bone regeneration was detected radiographically, and the clinical symptoms were improved. Short term follow-up has shown that the bone implants were effective in all of the patients. There were no adverse reactions related to implantation. The use of this tissue engineered bone makes it possible to perform osteogenetic treatment without harvesting autogenous bone, thus avoiding pain and pelvic deformity at the site of bone collection and reducing the burden on the patient.

Abstract: Introduction: Marrow mesenchymal cells contain stem cells and can regenerate tissues. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However, in cases with low numbers of active cells, culture is often unsatisfactory. If frozen marrow cells retain their osteogenic potential, we could clinically use them in regeneration therapy as alternatives to high active cells obtained from youngsters. Here, we examined osteogenic potential of frozen human mesenchymal stem cells in combination with recombinant human bone morphogenetic protein (rhBMP) using biochemical and histological analyses. Method: Marrow fluid was aspirated from the human iliac bone of a 46-year-old man with lumbar canal stenosis during surgery. Two weeks after primary culture in standard medium, bone marrow mesenchymal stem cells (BMSCs) were trypsinized for the preparation of a cell suspension, and cells were concentrated to 106 cells/ml by centrifugation. Cells were kept at – 80 °C until use. To impregnate porous hydroxyapatite (HA) with rhBMP, 1 3g rhBMP/20 3l 0.1 % trifluoroacetic acid was applied on HA, and then desiccated under vacuum. In the present study, we used 4 subgroups: BMSC/rhBMP/HA, BMSC/HA, rhBMP/HA, and HA only. HA constructs from the 4 subgroups were implanted at subcutaneous sites on the back of 5-week-old nude mice (BALB/cA Jcl-nu). Eight weeks after implantation, implanted HA constructs were harvested, and biochemical and histological analyses were performed. Alkaline phosphatase activity (ALP) and human osteocalcin (hOs) levels were measured. Results and Discussion: ALP activity and hOs in the BMSC/BMP/HA subgroup were 2 or 3 times that in the BMSC/HA subgroup. Histological analysis showed that significant bone formation was observed in these two subgroups, and supported biochemical data. However, in the BMP/HA and HA only subgroups, significant bone formation could not be detected histologically nor biochemically. These results indicated that a combination of rhBMP and BMSCs, and only with a minimal amount of 1 3g rhBMP, allowed successful generation of human bone. In the human body, rhBMP in the order of milligrams is necessary for bone formation. However, by combining BMSCs, HA and rhBMP, only a small amount of rhBMP was needed to dramatically enhance osteogenic potential. As we reported here, cryopreserved BMSCs also showed high osteoblastic activity. In conclusion, this study provided histological and biochemical evidence that combination of cryopreserved BMSCs, BMP, and porous HA could enhance osteogenic potential.

Abstract: Availability, storage and transportation of engineered bone tissue fabricated in vitro are major practical problems associated with adequate use of bone replacement grafts for the treatment of bone diseases. The ability to maintain viable engineered bone tissue would facilitate future clinical applications. In the present study, we investigated time required for transportation of engineered bone removed from cool storage, from the culture room to the operating room; and examined effects of cool storage on survival of engineered bone tissue. Bone marrowcells were obtained from the iliac bone of a 60-year-old male affected with lumbar spondylosis, and then incubated in standard medium. After two weeks in primary culture, cultured cells were trypsinized, and a concentrated cell suspension was incubated with a porous beta-TCP block. After 3 weeks of subculture with the osteogenic medium containing dexamethasone etc., engineered bone tissue was collected, stored for 0, 6, 12, 24 hours at 4 °C, and was subcutaneously implanted into the back of nude mice. Six weeks after implantation, implants were harvested. Before and after implantation, significant activity could be detected in all animals. In in vitro and in vivo situations, osteogenic activity of engineered bone tissue could be maintained even after 24 hours. These results provided information on appropriate storage conditions for engineered bone tissue.

Abstract: Introduction: Osteogenesis occurs in porous hydroxyapatite (HA) when HA blocks combined with marrow mesenchymal cells are grafted in vivo. In vitro bone formation occurs in HA pores when HA combined with marrow cells is cultured in osteogenic medium containing dexamethasone. Cultured bone/HA constructs possess higher osteogenic ability when they are grafted in vivo. Marrow mesenchymal cells (MSCs) contain many stem cells which can generate many tissue types. In the present study, we investigated osteogenic potential of cultured bone/HA combined with MSCs. Materials and Methods: Marrow cells were obtained from the femoral bone shaft of male Fischer 344 rats (7 weeks old), and were cultured in T-75 flasks. Primary cultured cells were trypsinized and combined with porous HA (5x5x5 mm, Interpore 500). The composites were subcultured in osteogenic medium containing dexamethasone. One tenth of primary cells were transferred into new T-75 flasks containing standard medium. After 2 weeks, MSCs were trypsinized, combined with cultured-bone/HA constructs, and prepared for implantation. MSC/cultured-bone/HA constructs, cultured bone/HA constructs, and HA alone were subcutaneously implanted into syngeneic rats. These implants were harvested at 2 or 4 weeks post-implantation, and prepared for histological and biochemical analyses. Results: Alkaline phosphatase activity and osteocalcin content of MSC /cultured bone/HA constructs were much higher than those of cultured bone/HA constructs at 2 and 4 weeks post-implantation. Histological examination supported these findings. Discussion and Conclusion: MSCs show high ability of cell proliferation. In addition, MSCs can generate new blood vessels which would support regeneration of bone tissue. Here, we suggested that MSCs could promote osteogenesis. We also showed that excellent engineered bone tissue could be fabricated by combining MSCs and cultured bone derived from dexamethasone-treated MSC culture.

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: Subjects were graft patients with pseudoarthrosis (average age, 60.3 years; range, 17-85 years). Pseudoarthrosis affected the thoracolumbar spine, the femur, the clavicle, the humerus and the metatarsal. From the ilium (tibia in one patient), 10-20 ml of bone marrow fluid was collected, and then, it was immediately transferred to the culture room and incubated in a flask containing MEM with 15% autologous or fetal bovine serum, etc.. After 2 weeks in primary culture, cells were released by trypsin treatment and were subsequently incubated with porous beta-TCP in order to prepare tissue-engineered artificial bone, according to the previously reported modified culturing technique. Tissue-engineered artificial bone was grafted around the non-union site of each affected long bone, while tissue-engineered artificial bone was grafted via the pedicle of each affected vertebral body. In all patients, favorable bone formation was seen at three months after surgery. In the patients with pseudoarthrosis of the spine, CT and MRI confirmed favorable vertebral body formation. In the patients with pseudoarthrosis of a long bone, the artificial bone was remodeled and favorable bone union was confirmed. In 2 patients in whom bone biopsy was performed during pin removal, bone regeneration was confirmed histologically. With present type of tissue-engineered artificial bone, an artificial material with a high bone regeneration capacity can be prepared by aspiration, which is minimally invasive, and thus when compared to iliac bone grafts, it is possible to radically reduce postoperative pain without damage of autologous bone.

Abstract: The effect of genistein, a soybean isoflavone, on new bone formation by bone marrow cells from mature humans was examined. After informed consent was obtained from a 55-year-old woman with osteoporosis and lumbar spondylosis deformans，bone marrow cells were collected from her ilium, cultured in the standard medium of MEM containing fetal calf serum and then cultured with or without the addition of genistein to the bone-forming medium containing dexamethasone etc.． In humans, when genistein was added to the bone-forming medium, genistein (10-7 M and 10-8 M)caused a significant increase in the levels of alkaline phosphatase avtivity and DNA content compared with cells not cultured in genistein． In conclusion，genistein was found to promote bone formation at lower concentrations，and thus may be useful as a bone formation-promoting factor.