Nobuhisa Satoh

Raloxifene: its ossification-promoting effect on female mesenchymal stem cells

BackgroundRaloxifene acts like estrogen in preventing bone loss in postmenopausal women, but it selectively activates biological responses in bone tissue. It has a direct effect on osteoblasts’ differentiation and bone formation in bone marrow culture. However, the point at which raloxifene has an effect on bone marrow-derived mesenchymal stem cells (MSCs), regardless of sex difference, is not known. The purpose of this study was to examine the osteogenic effect of raloxifene on MSCs derived from female and male rats and to assess the sex difference of raloxifene with or without osteogenic supplements (OSs) in the regulation of bone formation.MethodsFemale and male rat bone marrow cells were cultured with or without OSs. In each experimental group, 10-6 M or 10-8 M raloxifene was added. As a control, cells were cultured without raloxifene. Histologically, mineralization was assessed by alizarin red S staining. Biochemically, alkaline phosphatase (ALP) activity, calcium content, and osteocalcin content were assessed.ResultsOn histological analysis, mineralized nodules were seen on alizarin red S staining in the groups treated with OS. On the biochemical analysis, OS increased ALP activity, calcium content, and osteocalcin content. Among female groups with OSs, 10-6 M raloxifene significantly increased ALP activity, calcium content, and osteocalcin content compared with the controls. Among male groups, raloxifene had negligible effects.Conclusions10-6 M Raloxifene had no ossification-inducing effect on female MSCs, but it had an ossification-promoting effect; it had no osteogenic effect on male MSCs. Therefore, raloxifene has a sex difference with regard to its osteogenic effect on MSCs. Moreover, combined treatment with raloxifene plus OS has an effect on female MSCs. These results provide a useful insight into the possible influence of raloxifene after MSC transplantation in clinical practice.

Posterior Fixation of a Cervical Fracture Using the RRS Loop Spine System and Polyethylene Tape in an Elderly Ankylosing Spondylitis Patient: A Case Report

An 80-year-old woman presented with neck pain and paraparesis of Frankel C in her upper and lower extremities after falling. Imaging revealed an ankylosing cervical spine and a fracture line running obliquely from the anterior C3-4 to the posterior C4-5 level. Posterior fixation from the occi pit to T3 was performed using the RRS Loop Spine System and concomitant polyethylene tape fixation. This system is characterized by the uniqueness of how it screws to the occi pit and its use of a fixation rod with a larger diameter than in other instrumentation devices for use in the cervical region. Sublaminar banding using polyethylene tape was used to secure fixation. Her postoperative course was unremarkable, and her neck pain was relieved, although neurological improvement was minor. To our knowledge, this is the first report of an application of the RRS Loop Spine System to an ankylosing spondylitis patient with a cervical fracture.

Temporal changes in the tensile strength of ultra-high-molecular-weight polyethylene cable embedded in muscle tissue

BACKGROUND Wires and cables have been used extensively for spinal sublaminar wiring, but damages to the spinal cord due to compression by metal wires have been reported. We have used more flexible ultra-high-molecular-weight polyethylene cable (Tekmilon tape) instead of metal wires since 1999 and have obtained good clinical outcomes. Although the initial strength of Tekmilon tape is equivalent to metal wires, the temporal changes in the strength of Tekmilon tape in the body should be investigated to show that sufficient strength is maintained over time until bone union is complete. METHODS Tekmilon tape was embedded into the paravertebral muscle of 10-week-old male Japanese white rabbits. Samples were embedded for 0, 1, 3, 6 or 12 months. At the end of each period, sequential straight tensile strength and sequential knot-pull tensile strength were measured. FINDINGS The initial strength of Tekmilon tape in muscle tissue was maintained over time, with 92% straight tensile strength and 104% knot-pull tensile strength at 6months, and values of 77% and 100% at 12 months, respectively. Since single knot is clinically relevant, it is very important that the knot-pull tensile strength did not decrease over a 12-month period. This suggests that temporal changes in the tensile strength of Tekmilon tape are negligible at 1 year. INTERPRETATION Tekmilon tape maintains sufficient strength in vivo until bone union has occurred. It is useful for sublaminar wiring instead of metal materials due to its flexibility and strength and may reduce the risk of neurological damage.

Posterolateral Lumbar Fusion by Tissue Engineered Bone

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.

Osteogenetic Effect on Cortical Bone of Cultured Bone/Ceramics Implants

Hydroxyapatite(HA) has been reported to have a good affinity for cancellous bone with high osteoblastic activity, binding directly to such bone. However, little work has been done concerning the strength of binding to cortical bone with a low cellular activity. Bio-artificial bone with a high osteogenetic capacity can be produced by combining cultured bone tissue with an artificial bone material. We examined this bio-artificial bone for its osteogenetic capacity around the bone tissue to evaluate whether the bone is applicable to osteogenetic treatment. Bone marrow cells were collected from the femurs of 7-week-old male Fischer rats, placed into two T75 flasks, and incubated in standard medium. After 2 weeks in primary culture, the cells were seeded on a porous hydroxyapatite block, and incubated in an osteogenic medium prepared by adding 10 nM dexamethasone, ascorbic acid and b-glycerophosphate. to the standard medium. Two weeks later, cultured HA constructs were implanted onto the cortical bone of the femur. At four or eight weeks after implantation, the femur was removed and radiographically and histologically examined for osteogenesis. The bio-artificial bone of HA impregnated with cultured marrow cells bound firmly to the femoral cortex both radiographically and histologically, and calcification indicating new bone formation was observed around the HA. The use of this bio-artificial bone makes it possible to perform osteogenetic treatment without damage to autogenous bone, avoiding pain at the site of bone collection and deformity of the pelvis, and reducing the burden on the patient. Introduction Many studies reported that when marrow cells were cultured in a medium containing compounds such as dexamethasone bone-like tissue formed, and that this cultured bone tissue possessed a calcified collagen matrix, contained osteocalcin (a bone specific protein) and exhibited bone morphogenetic protein activity [1-4]. The results of biochemical and gene expression analyses showed that the osteoblast activity of cultured cells was high. We have reported that it is possible to prepare artificial bone with a potent osteogenic potential by culturing artificial bone materials with functionally active bone tissue [5-10]. We have also documented that the osteoblast activity of such cultured bone constructs is similar to that of cancellous bone [11,12]. Autologous cancellous bone is used for spinal fixation and treatment of pseudoarthrosis due to its high osteogenic potential. Cancellous bone is often harvested from the ilium, but this process can result in pelvic deformation, and there are limitations on the amount of bone that can be harvested. Furthermore, patients often experience uncomfortable levels of pain at the site of bone harvest. On the other hand, cultured bone constructs, with properties comparable to cancellous bone, can be produced by harvesting marrow cells using bone marrow aspiration, which is a minimally invasive procedure. Key Engineering Materials Online: 2003-12-15 ISSN: 1662-9795, Vols. 254-256, pp 745-748 doi:10.4028/www.scientific.net/KEM.254-256.745

Osteogenic Potential of Tissue Engineered Bone by Combination of Marrow Mesenchymal Cells and Cultured Bone/Ceramic Constructs

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.

Bone Regeneration from Frozen Marrow Mesenchymal Cells/Recombinant Human Bone Morphogenetic Protein/Hydroxyapatite Transplantation

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.

Cool Storage of Human Tissue Engineered Bone for Bone Regeneration Therapy

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.