Mika Tadokoro

Cell sheet transplantation of cultured mesenchymal stem cells enhances bone formation in a rat nonunion model

Orthopedic surgeons have long been troubled by cases involving nonunion of fractured bones. This study aimed to enhance bone union by cell sheet transplantation of mesenchymal stem cells. A nonunion model was made in rat femur, and rat bone marrow cells were cultured in medium containing dexamethasone and ascorbic acid phosphate to create a cell sheet that could be scraped off as a single sheet. Cell sheets were transplanted onto fractured femurs without a scaffold in the model. X-ray and histological analysis were performed at 2, 4 and 8 weeks. Ultrasonography and biomechanical analysis were performed at 8 weeks. X-ray photographs and histological sections showed callus formation around the fracture site in the cell sheet-transplanted group (sheet group). Bone union was obtained in the sheet group at 8 weeks. By contrast, the control group (without sheet transplantation) showed nonunion of the femur. The results of pullout evaluation in the vertical direction of the femur in the sheet group were significantly better than that of the control group. Analysis of the origin of de novo formed bone using the Sry gene, which was used as a marker for donor cells, showed that transplanted cells without scaffolds could survive and differentiate into osteogenic lineage cells in vivo. These results showed that the femoral fracture in our model was completely cured by the transplantation of a cell sheet created by tissue engineering techniques. Thus, we think that cell sheet transplantation can contribute to hard tissue reconstruction in cases involving nonunion, bone defects and osteonecrosis.

Induction of Pluripotent Stem Cells from Human Third Molar Mesenchymal Stromal Cells

The expression of four transcription factors (OCT3/4, SOX2, KLF4, and MYC) can reprogram mouse as well as human somatic cells to induced pluripotent stem (iPS) cells. We generated iPS cells from mesenchymal stromal cells (MSCs) derived from human third molars (wisdom teeth) by retroviral transduction of OCT3/4, SOX2, and KLF4 without MYC, which is considered as oncogene. Interestingly, some of the clonally expanded MSCs could be used for iPS cell generation with 30–100-fold higher efficiency when compared with that of other clonally expanded MSCs and human dermal fibroblasts. Global gene expression profiles demonstrated some up-regulated genes regarding DNA repair/histone conformational change in the efficient clones, suggesting that the processes of chromatin remodeling have important roles in the cascade of iPS cells generation. The generated iPS cells resembled human embryonic stem (ES) cells in many aspects, including morphology, ES marker expression, global gene expression, epigenetic states, and the ability to differentiate into the three germ layers in vitro and in vivo. Because human third molars are discarded as clinical waste, our data indicate that clonally expanded MSCs derived from human third molars are a valuable cell source for the generation of iPS cells.

The osteogenic differentiation of rat bone marrow stromal cells cultured with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles

There is an increasing interest in developing novel macromolecular vehicles for the intracellular and controlled delivery of bioactive molecules, since they can allow modulation of the cellular functions in a more effective manner ex vivo, and maintain the cellular phenotype in vivo upon re-implantation. The present study was designed to investigate the effect of combining novel dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer (Dex-loaded CMCht/PAMAM) nanoparticles and, both HA and SPCL scaffolds (3D system) on the proliferation and osteogenic differentiation of rat bone marrow stromal cells (RBMSCs) in vitro. A luminescent cell viability assay using RBMSCs was performed for screening cytotoxicity of the developed HA and SPCL scaffolds. Results corroborated previous ones which have demonstrated in vitro, the superior performance of the HA and SPCL scaffolds on supporting cells adhesion and proliferation. Furthermore, this work showed that RBMSCs seeded onto the surface of both HA and SPCL scaffolds differentiate into osteoblasts when cultured in the presence of 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles. In addition, results demonstrated that Dex-loaded CMCht/PAMAM dendrimer nanoparticles combined with the HA enhance osteogenesis by increasing ALP activity and mineralization of the extra-cellular matrix. The pre-incubation of stem cells with these kinds of nanoparticles allows the delivery of Dex inside the cells and directly influences their cellular fate, being a promising new tool to be used in cells and tissue engineering strategies.

In Vivo Osteogenic Capability of Human Mesenchymal Cells Cultured on Hydroxyapatite and on β-Tricalcium Phosphate

The aim of the current study was to examine in vitro osteogenic capability and in vivo bone formation of mesenchymal stromal cells (MSCs) on two kinds of calcium phosphate ceramics. MSCs derived from human bone marrow were seeded on either hydroxyapatite (HA) ceramic or beta-tricalcium phosphate (beta-TCP) ceramic and then cultured in a medium supplemented with a donors serum, vitamin C, beta-glycerophosphate, and dexamethasone. The culture revealed the expression of alkaline phosphatase activity, indicating the osteogenic differentiation of the MSCs on the ceramics (fabrication of tissue-engineered construct). The constructs were then implanted subcutaneously into nude rats for 8 weeks. New bone formation was observed in both types of ceramics, and human-specific Alu sequence was detected by in situ hybridization analysis. Quantitative microcomputed tomography showed that the volume of the new bone in the HA ceramic was greater than that in the beta-TCP ceramic in six of seven cases. These results suggest that human MSCs cultured on ceramics could retain their osteogenic capability even after ectopic implantation and provide a rationale for the use of tissue-engineered constructs derived from a patients MSCs and calcium phosphate ceramics in bone tissue regeneration.

Human mesenchymal stem cells as a stable source of VEGF-producing cells.

Vascular endothelial growth factor (VEGF) is a positive regulator and plays a crucial role in angiogenesis. We demonstrate that VEGF was highly expressed in cultures of human bone marrow‐derived mesenchymal stem cells (hMSCs) and the high expression level was maintained during prolonged culture periods (checked up to passage 10). We also confirmed that in vivo hMSCs engrafted into immunodeficient mice could survive and secreted human VEGF. These findings suggest that implantation of hMSCs is a practical means as a source of VEGF production and might be effective in neoangiogenesis. Copyright

Ex vivo culturing of stromal cells with dexamethasone-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles promotes ectopic bone formation.

Recently, our group has proposed a combinatorial strategy in tissue engineering principles employing carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles (CMCht/PAMAM) towards the intracellular release and regimented supply of dexamethasone (Dex) aimed at controlling stem cell osteogenic differentiation in the absence of typical osteogenic inducers, in vivo. In this work, we have investigated if the Dex-loaded CMCht/PAMAM dendrimer nanoparticles could play a crucial role in the regulation of osteogenesis, in vivo. Macroporous hydroxyapatite (HA) scaffolds were seeded with rat bone marrow stromal cells (RBMSCs), whose cells were expanded in MEM medium supplemented with 0.01 mg ml(-1) Dex-loaded CMCht/PAMAM dendrimer nanoparticles and implanted subcutaneously on the back of rats for 2 and 4 weeks. HA porous ceramics without RBMSCs and RBMSCs/HA scaffold constructs seeded with cells expanded in the presence and absence of 10(-8) M Dex were used as controls. The effect of initial cell number seeded in the HA scaffolds on the bone-forming ability of the constructs was also investigated. Qualitative and quantitative new bone formation was evaluated in a non-destructive manner using micro-computed tomography analyses of the explants. Haematoxylin and Eosin stained implant sections were also used for the histomorphometrical analysis. Toluidine blue staining was carried out to investigate the synthesis of proteoglycan extracellular matrix. In addition, alkaline phosphatase and osteocalcin levels in the explants were also quantified, since these markers denote osteogenic differentiation. At 4 weeks post-implantation results have shown that the novel Dex-loaded carboxymethylchitosan/poly(amidoamine) dendrimer nanoparticles may be beneficial as an intracellular nanocarrier, supplying Dex in a regimented manner and promoting superior ectopic de novo bone formation.

In vivo survival and osteogenic differentiation of allogeneic rat bone marrow mesenchymal stem cells (MSCs).

Marrow mesenchymal stem cells (MSCs) are multipotent progenitor cells and reported to be immunoprivileged as well as immunosuppressive. Hence, MSCs might be ideal candidates for allogeneic transplantation to induce regeneration of damaged tissues/organs. To confirm the differentiation capability of allogeneic MSCs in vivo is important for the acceleration of regenerative medicine. Consequently, we have established an in vivo rat model using subcutaneous implantation of a hydroxyapatite (HA) ceramic/MSCs composite. Osteogenic differentiation was used as an indicator of differentiation. When syngeneic MSCs were implanted, MSCs showed osteogenic differentiation as evidenced by new bone formation as well as high alkaline phosphatase (ALP) activity. When allogeneic MSCs were implanted, none of the allografts survived or showed osteogenic differentiation. However, when the recipient rats were treated with FK506 immunosuppressant, allogeneic MSCs showed osteogenic differentiation. Although this finding might not be adequate for the acceleration of regenerative medicine, these results did confirm that MSCs are not intrinsically immunoprivileged but that under appropriate immunosuppressant treatment, allogeneic MSCs can survive and show differentiation capability in vivo.

New Bone Formation by Allogeneic Mesenchymal Stem Cell Transplantation in a Patient with Perinatal Hypophosphatasia

Mesenchymal stem cells (MSCs) can show osteogenic differentiation capability when implanted in vivo, as well as cultured in vitro; therefore we attempted to use allogeneic MSCs for an 8-month-old patient with hypophosphatasia. MSCs were obtained by culture expansion of fresh marrow from the patients father. Some of the MSCs were further cultured under osteogenic conditions on a culture dish or porous hydroxyapatite ceramics, resulting in cultured osteoblasts and osteogenic constructs, respectively. The MSCs and osteoblasts were injected into the patient, and the constructs were implanted locally. After traditional bone marrow transplantation, the MSCs, osteoblasts, and osteogenic constructs were used for treatment and to improve the patients respiratory condition and skeletal abnormality. The condition worsened again, and an MSC booster shot was administered. At the same time, the construct was retrieved. The respiratory condition improved, and the retrieved construct showed de novo bone derived from both donor and patient cells. We demonstrated the importance of allogeneic MSC transplantation for hypophosphatasia and the constructs as an alternative to bone fragments that provided further osteogenic capability in the patient.

Hard tissue-forming potential of stem/progenitor cells in human dental follicle and dental papilla

OBJECTIVE The existence of stem/progenitor cells in dental tissue has been suggested but their characterization in the human tooth germ remains elusive. The purpose of this study was to investigate these cells in human dental follicles and dental papillae at the crown-forming stage and compare their potential for hard tissue formation. DESIGN We used dental follicle cells (DFCs) and dental papilla cells (DPCs) derived from dental follicles and dental papillae at the crown-forming stage and compared their proliferative capacity, cell surface antigens and ability to form hard tissue in vitro and in vivo. RESULTS Both DFCs and DPCs had extensive proliferation ability, expressed similar cell surface antigens and were capable of forming hard tissue in vivo as well as in vitro. However, there were two differences between DFCs and DPCs. First, DPCs had a significantly higher calcium accumulation than that in DFCs. Second, DFCs expressed a cementoblast marker, whereas DPCs expressed an odontoblast marker. CONCLUSIONS We propose that dental follicles and dental papillae at the crown-forming stage contain different types of stem/progenitor cells and may have hard tissue-forming ability in a possibly origin-specific lineage direction.

Scaffold-free cell sheet injection results in bone formation.

We previously reported a new cell transplantation method in which mesenchymal stem cells (MSCs) were cultured as cell sheets. The cultured MSC sheets showed high alkaline phosphatase (ALP) activities and osteocalcin (OC) contents. In the present study, we transplanted such sheets by injection to assess whether the injectable MSC sheets could form bone tissue at subcutaneous sites. At 4 weeks after the subcutaneous injection, the injected areas showed hard mass formation. Each mass consisted of newly formed bone, as evaluated by radiographic, histological and gene expression analyses as well as three‐dimensional computed tomography (3D‐CT). Histological analyses revealed extracellular bone matrix together with osteocytes and active osteoblasts. Real‐time PCR analyses showed high ALP and OC mRNA expressions. We also injected the cell sheets into dead bone to determine whether the lost osteogenic potential could be rescued, and histological analyses revealed that the injected cell sheets supplied osteogenic potential to the dead bone. The present study clearly indicates that osteogenic MSC sheets can be transplanted via injection through a needle and that bone formation results in the injected areas. Owing to its usage of a needle for fabrication of in vivo bone tissue, this injection method can be applied as a minimally invasive approach for hard tissue reconstruction. Copyright