Papers by Author: Jin Iida

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: 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.

Abstract: Marrow mesenchymal cells contain stem cells and can regenerate tissue. We previously reported the clinical application of autologous cultured bone to regeneration therapy. However there is room for improvement in the culture methods; and here, we examined the optimal frequency of medium changing. Marrow cells were collected from the femur of a Fisher 7 week-old male rat. At 2 weeks after primary culture in a standard medium (MEM containing 15% bovine fetal serum), the cultures were trypsinized to prepare a cell suspension and divided into two groups, with or without addition of dexamethasone (Dex) to the osteogenic medium. To investigate optimal frequency, we further divided into 5 sub-groups; no changing (M0), 1 time/week (M1), 2/week (M2), 3/week (M3), and everyday (M7). After 2 weeks of subculture, the tissue was harvested and then ALP activity and calcium and DNA contents measured. In both of the Dex groups, there was significantly high ALP activity in the higher frequency group; but there was no significant DNA content. Also, in the Dex(+)group, there was a significant increase of calcium content in only the M3 and M7 sub-groups. Thus, we showed that the osteogenic potential of cultured bone is cultivated by increasing the frequency of medium changing.

Abstract: Osseous tissue can be formed by culturing marrow cells with compounds such as dexamethasone and that a bone matrix cultured in this manner possesses BMP activity. We have reported that artificial bones with a high level of osteogenic potential can be prepared by culturing artificial bone materials with cultured osseous tissue. Here, in an attempt to develop activated cultured bone constructs with even greater osteogenic potential, the effects of the female hormone estriol on osteogenesis were investigated. Bone marrow cells were collected from the femur shafts of 7-week-old male Fischer rats, and subjected to primary and secondary cultures. During secondary culture with or without dexamethasone (Dx), 10-5, 10-6, 10-7, 10-8 or 10-9 M of estriol was added to a standard culture medium containing ascorbic acid and β-glycerophosphosphate. The alkaline phosphatase(ALP) activity and Ca levels were measured and statistically analyzed. There was a significant difference in ALP activity between the control group and the estriol groups, and ALP activity was the highest in the 10-7 and 10-8 M groups, being about 2.5 times higher than in the control group. Similar results were seen for Ca levels. Furthermore, in vivo study showed10-7M-estriol-treated-cultured bone/ceramic construct has significant high osteogenic potential when it is grafted into in vivo. Estriol has been reported to increase bone mass, and the results of the present study suggest that the osteogenic potential of cultured bone constructs can be more than doubled by adjusting the concentration of estriol in bone marrow cell culture. Therefore, the use of estriol may be able to facilitate osteogenesis in bone regeneration therapy.