Hidemi Nakata

Osteogenic Potential of Mouse Adipose-Derived Stem Cells Sorted for CD90 and CD105 In Vitro.

Adipose tissue-derived stromal cells, termed ASCs, play an important role in regenerative applications. They resemble mesenchymal stem cells owing to their inexhaustibility, general differentiation potential, and plasticity and display a series of cell-specific and cluster-of-differentiation (CD) marker profiles similar to those of other somatic stem cells. Variations in phenotypes or differentiation are intimately associated with CD markers. The purpose of our study was to exhibit distinct populations of ASCs with differing characteristics for osteogenic differentiation. The primary cell batch of murine-derived ASCs was extracted from subcutaneous adipose tissue and the cells were sorted for the expression of the surface protein molecules CD90 and CD105 using flow cytometry. Each cell population sorted for CD90 and CD105 was analyzed for osteogenic potency after cell culture. The results suggested that ASCs exhibit distinct populations with differing characteristics for osteogenic differentiation: unsorted ASCs stimulated comparable mineralized nodule formation as bone marrow stromal cells (BMSCs) in osteogenic medium and viral transfection for BMP2 accelerated the formation of mineralized nodules in CD90 and/or CD105 positive ASCs with observation of decrease in CD105 expression after 14 days. Future studies assessing different immunophenotypes of ASCs should be undertaken to develop cell-based tissue engineering.

Low-Intensity Pulsed Ultrasound Stimulation Enhances Heat-Shock Protein 90 and Mineralized Nodule Formation in Mouse Calvaria-Derived Osteoblasts.

Low-intensity pulsed ultrasound (LIPUS) has demonstrated its positive effects on osteogenic differentiation of mesenchymal stem cells and the proliferation and differentiation of osteoblasts, negative effects on osteoclast growth, and promotion of angiogenesis, leading to improvement of the tissue perfusion. Heat-shock proteins (HSPs) are initially identified as molecules encouraged and expressed by heat stress or chemical stress to cells and involved in the balance between differentiation and apoptosis of osteoblasts. However, it remains unclear if the effect of LIPUS on osteoblast differentiation could involve HSP expression and contribution. In this study, mouse calvarial osteoblasts were exposed to LIPUS at a frequency of 3.0 MHz by 30 mW/cm(2) for 15 min or to 42°C heat shock for 20 min at day 3 of cell culture and examined for osteogenesis with pursuing induction of HSP27, HSP70, and HSP90. LIPUS as well as heat shock initially upregulated HSP90 and phosphorylation of Smad1 and Smad5, encouraging cell viability and proliferation at 24 h, enhancing mineralized nodule formation stronger by LIPUS after 10 days. However, HSP27, associated with BMP2-stimulated p38 mitogen-activated protein kinase during osteoblast differentiation, was downregulated by both stimulations at this early time point. Notably, these two stimuli maintained Smad1 phosphorylation with mineralized nodule formation even under BMP2 signal blockage. Therefore, LIPUS might be a novel inducer of osteoblastic differentiation through a noncanonical signal pathway. In conclusion, LIPUS stimulation enhanced cell viability and proliferation as early as 24 h after treatment, and HSP90 was upregulated, leading to dense mineralization in the osteoblast cell culture after 10 days.

Osteogenic Potential of Mouse Periosteum-Derived Cells Sorted for CD90 In Vitro and In Vivo

The treatment of bone defects still presents complex problems, although various techniques have been developed. The periosteum is considered a good source of osteogenic precursor cells for new bone formation. It can be collected easily in the clinical setting and is less invasive to the donor site. However, the murine skull periosteum has a poor cellular component, and growth is very slow, making it important to identify a culture method for efficient growth. In the present study, we used three‐dimensional cell migration with atelocollagen and gelatin media and found that both were effective for promoting the proliferation of periosteum‐derived cells. Moreover, atelocollagen medium is expected to provide an added benefit as a scaffold structure in the ambient temperature of the human body. The selection of a proper surface marker for osteogenesis is imperative for bone regeneration. CD90 is a mesenchymal stem cell marker. Periosteum‐derived cells sorted with CD90 showed higher proliferative capacity and osteogenic potential than that of unsorted periosteum‐derived cells in vivo and in vitro. Thus, periosteum‐derived cells sorted with CD90 are expected to be a good source for bone regeneration.

Aesthetical Reconstruction of an Anterior Tooth in an Alveolar Cleft Site: A Case Report and 3-Year Follow-up Findings

Abstract Abstract An alveolar cleft often displays the furrowed shape of the buccal site because of the lack of alveolar bone and gingival contracts. A 35-year-old woman presented with a repaired cleft lip and a non-grafted alveolar cleft associated with a missing lateral incisor on the left side of the maxilla. The central incisor had been an abutment tooth of a bridge for 15 years; however, it fractured and was extracted. Orthodontic treatment was antecedent to implant reconstruction because the edentulous space was not adequate for a single tooth replacement. As the palate was closed by the mucosa, bone grafting was not performed at the cleft site beforehand. Dental implant placement was performed within the narrowed, beamlike bone, and hydroxyapatite particles were used to augment the ridge. A relaxing incision and expansion of the invaginated mucosa improved the buccal shape of the gum. The usage of a narrow-type implant and bone substitutes facilitated the minimization of surgical stress, even though she did not undergo autologous bone-grafting surgery. Additionally, the aesthetical reconstruction of the anterior region significantly improved her quality of life at the 3-year follow-up.

Indirect osteoblast differentiation by liposomal clodronate

Bisphosphonates impair function of osteoclasts and prevent bone resorption, the mechanism of which has been studied extensively. However, the possible effects of bisphosphonates on chondroblast differentiation and calcium deposition by osteoblasts have only been demonstrated recently. Moreover, cells from monocytic lineage are capable of stimulating osteoblast proliferation. Hence, susceptibility of osteoblasts to various factors requires further investigation. A primary culture of bone marrow‐derived stromal cells was treated with liposomal clodronate (0.1, 0.5, or 1.0 mg/ml) or conditioned medium from liposomal clodronate. Liposomal clodronate (0.25 mg) was injected into mouse femur for in vivo experiments. The effects of liposomal clodronate were examined by alkaline phosphatase staining and/or activity assay, and real‐time RT‐PCR was used for studying the effect on osteogenic gene expression. Administration of liposomal clodronate to bone marrow‐derived mesenchymal stromal cell culture enhanced alkaline phosphatase activity and mRNA levels of Runx2 and Dlx5. In addition, conditioned medium from liposomal clodronate also stimulated osteogenic characteristics similar to those of observed in vitro, and the number of exosomes in the conditioned medium was highest when pre‐treated with liposomal clodronate. Western blot analysis revealed the presence of RANK proteins in exosomes collected from conditioned medium of liposomal clodronate. Identical observations were obtained in vivo, as liposomal clodronate‐injected mouse femur showed increased alkaline phosphatase activity and Runx2 and Dlx5 mRNA expressions, even though the numbers of monocytes and macrophages were reduced. In conclusion, osteoblast differentiation was promoted via soluble RANK‐containing exosomes in response to clodronates.

Osteogenic Potential of Adipose-Derived Macrospheroids Cocultured with CD11b+ Monocytes

PURPOSE Among potential cell-based therapies, adipose-derived stem cells (ASCs) have been proposed as a promising source of stem cells for tissue regeneration. Although many recent clinical trials have investigated the use of adipose tissue or ASCs in transplantation, analysis of the microstructures of outgrowing macrosized spheroids (macrospheroids) or three-dimensional coculture of ASC spheroids and monocyte/macrophage lineages has not been performed. The aim of this study was to analyze the microstructures of murine-derived ASC macrospheroids and the growth and osteogenic potential of these macrospheroids in a three-dimensional environment and after calcification induction by coculture with monocytes. MATERIALS AND METHODS The histologic structures of murine-derived ASC macrospheroids and the expression of marker genes for multipotency within these macrospheroids were analyzed by hematoxylin and eosin staining and in situ hybridization. ASC macrospheroid microstructures were observed by transmission electron microscopy, and cell proliferation in the spheroids was analyzed. Additionally, the growth and osteogenic potential of these macrospheroids were assessed in two-dimensional and three-dimensional environments and after calcification induction by coculture with monocytes. RESULTS The expression of Oct3/4, Nanog, and Sox2 was detected even in the deep zone of spheroids, although higher expression was observed at the surface. Cell proliferation was detected within the spheroid centers. Observation of spheroid microstructure revealed extracellular matrix production within the spheroid architecture. Transplantation of a spheroid on the hydroxyapatite disc resulted in three-dimensional cell growth, filling the disc. Coculture of the spheroids with monocytes led to the formation of many osteoclast-like, multinucleated cells, and calcification was observed after 3 weeks of coculture. CONCLUSION ASC spheroids exhibited high capacity for dynamic three-dimensional growth and osteogenic differentiation. Furthermore, ASC spheroids promoted monocyte differentiation into osteoclast-like cells, which may enhance the osteogenic potential of ASC spheroids.

Clinical Outcomes of Esthetic and Functional Rehabilitation with Dental Implants in Patients with Alveolar Cleft

Background and aim: Alveolar cleft is notable congenital deformity in the oral and maxillofacial region. And missing incisor is often associated with the cleft site. Owing to recent advances in bone grafting techniques, dental implant treatment has become an appreciable method of restoring the edentulous space at the cleft site in addition to conventional methods using bridges or dentures. In this study, we investigated the risk factors related to esthetic outcomes of implant treatment at alveolar cleft sites. Materials and methods: A total of 13 patients treated with dental implants for missing teeth associated with an alveolar cleft were examined. The patients’ gender, cleft type, and number of dental implants were recorded. The ages of patients at bone grafting for cleft closure, additional bone grafting prior to dental implant surgery, and dental implant placement were also investigated. Seven risk factors were assessed to evaluate the relation to esthetic outcomes. Results: Five to thirteen years after the final prostheses were delivered, neither marginal bone resorption nor loss of dental implants were found. The esthetic outcomes differed among patients according to several risk factors. Discussion and conclusion: Oral rehabilitation with dental implants represented a promising treatment for alveolar cleft sites when bone graft was appropriate. However, the esthetic outcomes of this treatment depend upon multiple risk factors. Type of Clefts r-s l-s Bilateral Total Male 1 3 1 5 Female 0 6 2 8 Total 1 9 3 13 r-s: right side l-s: left side Table 1: Description of the 13 patients evaluated in the present study. Journal of Tissue Science & Engineering J o u r n a l o f T iss ue S cience &ngine e r i n g

Prosthetic recovery utilizing residual dental implants: A case report involving a unilateral milled-bar-magnet attachment combination

Few options for functional recovery using residual dental implants are available after removal of disintegrated implants. This report describes the application of a maxillary implant-supported removable partial denture (RPD) retained by a unilateral milled-bar-magnetic attachment combination fixed to the remaining two implants as an alternative for functional recovery without surgery. A custom milled-bar with keepers was fixed to the two remaining unparallel unilateral implants in a 78-year-old woman who visited our department after dental implant treatment. Although 2 of the 4 initial implants were lost, additional surgery was unfeasible; thus, a magnetic RPD with a clasp on the ipsilateral second molar was placed. Peri-implant infection and bone resorption did not occur in the 4 years after implant-supported RPD placement. In the present case, a custom milled-bar-magnetic attachment and a clasp combination have been effective for retaining an implant-supported RPD even with an inadequate unilateral design.