Takafumi Yoshikawa

Tissue-engineered bone using mesenchymal stem cells and a biodegradable scaffold.

Bone marrow has been shown to contain a population of rare cells capable of differentiating to the cells that form various tissues. These cells, referred to as mesenchymal stem cells (MSCs), are capable of forming bone when implanted ectopically in an appropriate scaffold. The aim of this study was to investigate the potential of a new &bgr;-tricalcium phosphate (&bgr;-TCP) as a scaffold and to compare the osteogenic potential between &bgr;-TCP and hydroxyapatite (HA). The &bgr;-TCP and HA loaded with MSCs were implanted in subcutaneous sites and harvested at 1, 2, 4, and 8 weeks after implantation for biochemical and histological analysis. Biochemically, in both &bgr;-TCP and HA composites, the alkaline phosphatase activity in the composites could be detected and was maintained at a high level for 8 weeks. In the histological analysis, active bone formation could be found in both the &bgr;-TCP and HA composites. These findings suggest that &bgr;-TCP could play a role as a scaffold as well as HA. The fabricated synthetic bone using biodegradable &bgr;-TCP as a scaffold in vivo is useful for reconstructing bone, because the scaffold material is absorbed several months after implantation.

Enhancement of the in vivo osteogenic potential of marrow/hydroxyapatite composites by bovine bone morphogenetic protein

A composite of marrow mesenchymal stem cells and porous hydroxyapatite (HA) has in vivo osteogenic potential. To investigate factors enhancing the osteogenic potential of marrow/HA composites, we prepared a bone morphogenetic protein (BMP) fraction from the 4M guanidine extract of bovine bone by heparin-sepharose affinity chromatography. Marrow/HA composites or composites containing marrow mesenchymal stem cells, BMP, and HA (marrow/BMP/HA composites) were implanted subcutaneously in 7-week-old male Fischer rats. BMP/HA composites and HA alone were also implanted. The implants were harvested after 2, 4, or 8 weeks and were prepared for histological and biochemical studies. Histological examination showed obvious de novo bone formation together with active osteoblasts at 2 weeks, as well as more extensive bone formation at 4 and 8 weeks in many pores of the marrow/BMP/HA composites. The marrow/HA composites did not induce bone formation at 2 weeks, but there was moderate bone formation at 4 weeks. At 2 weeks, only marrow/BMP/HA composites resulted in intensive osteogenic activity, judging from alkaline phosphatase and osteocalcin expression at both the protein and gene levels. These results indicate that the combination of marrow mesenchymal stem cells, porous HA, and BMP synergistically enhances osteogenic potential, and may provide a rational basis for their clinical application, although further in vivo experiment is needed.

In vivo osteogenic capability of cultured allogeneic bone in porous hydroxyapatite: immunosuppressive and osteogenic potential of FK506 in vivo.

Fischer or ACI rat marrow cells were obtained from femoral shafts and were cultured to confluence in Eagles minimal essential medium (EMEM) supplemented with 15% fetal bovine serum. After trypsinization, the cells were subcultured on porous hydroxyapatite (HA; Interpore 500) blocks in the presence of β‐glycerophosphate and 10 nM dexamethasone (Dex). After 2 weeks of subculture, a mineralized bone matrix with osteogenic cells developed on the HA pore surfaces. ACI or Fischer cultured bone tissue/HA constructs were implanted subcutaneously into the backs of Fischer rats and the immunosuppressant FK506 was given to the rats for 4 weeks. Implants were harvested 4 weeks and 8 weeks after insertion. At 4 weeks, the ACI constructs (allografts) showed high levels of osteogenic parameters (alkaline phosphatase [ALP] activity and osteocalcin content) and bone formation was observed together with active osteoblasts without obvious accumulation of inflammatory cells. At 8 weeks, active osteoblasts and progressive bone formation were still observed, while osteogenic parameters remained high and osteocalcin messenger RNA (mRNA) was detected. Without FK506 administration, the allografts showed neither bone formation nor osteocalcin mRNA and there were only trace levels of the osteogenic parameters. In the case of Fischer constructs (isografts), extensive bone formation was detected and all the osteogenic parameters were higher with FK506 than without FK506 at both 4 weeks and 8 weeks. These results indicate that cultured bone tissue/HA constructs possess a high osteogenic potential, even as allografts, and that FK506 not only has an immunosuppressive action, but also promotes bone formation.

Bone reconstruction by cultured bone graft

Abstract Grafting of the patients own bone is ideal from the perspective of osteogenic potential, and also produces the best results clinically. If a tissue engineering approach is used to produce autogenous bone ex vivo with culture techniques, extensive bone defects could be repaired without any damage to the normal tissues. Maniatopoulos et al. reported the formation of calcified bone-like tissue when bone marrow cells were cultured with dexamethasone and beta-glycerophosphate. Combining this cultured bone tissue with synthetic bone material has allowed the production of synthetic bone containing proliferating bone marrow cells with increased osteogenic potential. The in vitro biochemical and morphological study showed that the synthetic bone with a matrix similar to the normal bone and a high level of osteoblastic activity, can be produced by culture in just over 2 weeks. The histological and biochemical analyses of the synthetic bone after subcutaneous implantation in vivo, revealed that bone formation commenced 1 week after implantation and continued for 8 weeks. Expression of alkaline phosphatase and osteocalcin mRNA was confirmed at 1–2 weeks after the implantation of synthetic bone, and was comparable to the expression level in the normal cancellous bone. These findings also demonstrated that this synthetic bone exhibits a high osteogenic potential in vivo. When human bone marrow cells are cultured using the method of Maniatopoulos et al., bone tissue forms in vitro. Cultured synthetic bone, which has been confirmed by animal experiments to possess high osteogenic potential, can also be produced using human bone marrow cells. The method of creating synthetic bone described here allows the grafting of autogenous bone while preserving normal tissue. In addition, culture can be used to increase the number of bone marrow cells, thereby allowing the repair of extensive bony defects.

Bone and soft tissue regeneration by bone marrow mesenchymal cells

Culture of bone marrow blood produces bone marrow mesenchymal cells that adhere to the bottom of the culture dish. These cells are known to include cells that can differentiate in various directions. Culturing these marrow mesenchymal cells with differentiation factors and biomaterials can achieve regeneration of bone and soft tissue. The present study showed that culture of marrow mesenchymal cells with porous artificial bone material in an osteogenic culture medium containing dexamethasone could produce new bone tissue on the artificial material. This bio-artificial bone graft substitute could effectively regenerate bone tissue when it was implanted in vivo. Porous artificial bone material treated with a small amount of bone morphogenetic protein (BMP) and perfused with marrow mesenchymal cells also markedly stimulated bone regeneration by the interaction between the cells and BMP. Similarly, when an artificial dermis made of collagen sponge was perfused with marrow mesenchymal cells and used to cover a skin defect, the regeneration of blood vessels was stimulated. Since the present results demonstrated the ability of marrow mesenchymal cells to regenerate bone and soft tissue, these cells are expected to be useful for tissue regeneration therapy.

Prospects for bone fixation—development of new cerclage fixation techniques

Abstract The principles for the fixation and treatment of bone fractures have been well established based on the multidisciplinary studies performed by the Arbeitsgemeinschaft fur Osteosynthesefragen/Association for the Study of Internal Fixation (AO/ASIF group). As the basis for modern internal fixation of fractures, these principles are presently supported by the majority of orthopedic surgeons. However, with recent advances in bioengineering and biomaterials, it has become necessary to reconsider these principles. We focused our attention on cerclage fixation, which is not considered so important by the AO group, and developed two new cerclage materials. One is a nylon strap that is in wide industrial use. In the field of orthopedics, Partridge first employed the nylon strap for internal fixation and demonstrated its efficacy in 1976. However, its use was thereafter discontinued because of various complications. We developed a nylon strap for temporary fracture fixation and demonstrated its efficacy in clinical studies. We also developed a bioabsorbable thread for the tight tying of bones. Bone fixation with a thread is sometimes used clinically, but adequate fixation often cannot be obtained because of loosening of the knot. Therefore, we prepared a blended thread by mixing poly- l -lactic acid (PLLA) and e-polycaprolactone (PCL) fiber, so that it could be fixed tightly by melting. The fatigue strength of this thread was higher than that of a stainless-steel wire with the same cross-sectional area. We are aiming to establish more effective and convenient methods of treatment by developing new bone fixation materials and examining their efficacy. In the future treatment of fractures, it will be important to develop new instruments and bone fixation materials for the purpose of standardizing the procedures used by surgeons.