Key Engineering Materials Vols. 309-311

Abstract: Carbonate apatite (CO3Ap) was synthesized at 60+1°C and pH 7.4+0.2, to develop a new biodegradable scaffold biomaterial. The synthetic CO3Ap was mixed with a neutralized collagen gel and the CO3Ap-collagen mixtures with different kinds of CO3Ap contents and porosity were frozen and dried in lyophilized into the sponges. CO3Ap-collagen mixtures were also lyophilized into sponges in a HAp frame ring with 0.5 mm pores. To examine the degree of cell invasion, mouse MC3T3-E1 cells were grown in αMEM with 10% heat-inactivated FBS in 96-well plates containing the CO3Ap-collagen sponges at 37°C in a 5% humidified atmosphere. Under pentobarbital anesthesia, samples of UV-irradiated CO3Ap-collagen sponges with frames were surgically implanted beneath the periosteum cranii of rats. SEM observation of CO3Ap-collagen sponges showed favorable pores for cell invasion. Approximately 50~300 µm size pores seemed to continue into the deep bottom. X-ray high-resolution microtomography revealed a clear image of 3D structure of the sponges. 70 wt% CO3Ap-collagen sponge seemed to be most favorable biomaterial from the viewpoint of the natural bone properties. Then, to avoid the shrinkage of the sponges, we successfully made a hybridized CO3Ap-collagen sponge with a frame. When these sponge-frame complexes were implanted beneath the periosteum cranii of rats, newly created bone was observed toward the inner core of the complex from the surface of the periosteum cranii.

Abstract: In the field of bone tissue engineering using cells combined with scaffolds, it is important to efficiently load cells into porous scaffolds. We devised a novel cell-loading method
into porous beta-tricalcium phosphate (β-TCP) blocks. In this study, we compared this method with two conventional cell-loading methods in terms of cell-loading efficiency and in vivo bone formation capability. Bone marrow stromal cells (BMSCs) were obtained from the femurs of Fisher rats. After about 10 days of culture, BMSCs were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using three loading methods: a soaking method, a post low-pressure method and a pre low-pressure method (the novel method). These β-TCP cubes were cross-sectioned and stained with toluidine blue and cell-counted. Cell-loading efficiency was significantly higher when using the novel methods. For the study of in vivo bone formation capability, nearly confluent BMSCs were exposed in an osteogenic medium supplemented with 10-7 M dexamethasone, 50µg/ml L-ascorbic acid phosphate and 10mM β-glycerophosphate for 4 days. These osteogenic cells were harvested and suspended in the plasma of the Fisher rats at a concentration of 2×106 cells/ml. This cell suspension was loaded into porous β-TCP cubes (5×5×5mm) by using the three cell-loading methods. Immediately, these β-TCP cubes were implanted at subcutaneous sites in the backs of 7-week-old male Fisher rats and harvested at postoperative 3 and 6 weeks. After cross-sectioning, these sections were stained with hematoxylin and eosin, and the new bone formation area was quantified. Consistent with cell-loading efficiency, in vivo bone formation capability was significantly higher in the novel method at postoperative 6 weeks. We showed the usefulness of the novel cell-loading method in bone tissue engineering.

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: 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: 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: 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: 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: In this study, porous bioceramics (titanium foam with diamond-like carbon coatings, glass foam and zirconium oxide foam) were produced using expansion in vacuum. The porosity, the pore size and pore morphology can be adjusted in agreement with the application. The different 3D structures were obtained by varying the parameters of the process. The microstructure and morphology of the porous materials were observed by scanning electron microscopy (SEM) and optical microscopy. The foam exhibit an open-cell structure with interconnected macropores, which provide the potential for tissue ingrowths and the transport of the body fluids.

Abstract: Various works have been done to produce a cellular form of bioactive ceramics for a scaffold. However, the most of these cellular implants have low compressive strength. In this study, therefore, glass-infiltrated cellular alumina with compressive strength of 7.3MPa was first prepared. Bioactive glass was then coated on the cellular alumina. When the specimen was reacted in simulate body fluid, hydroxyapatite developed on the bioactive glass coat in 18 hours.