Kazumasa Nakao

Evaluation of pluripotency in human dental pulp cells.

PURPOSEnPostnatal stem cells have been isolated from various tissues, including bone marrow, neural tissue, skin, retina, and dental epithelium. Recently, adult stem cells have been isolated from human dental pulp. Postnatal stem cells have been isolated from a variety of tissues. Previously, it was generally accepted that the differentiation potential of postnatal stem cells was lineage restricted.nnnMATERIALS AND METHODSnNormal impacted third molars were collected from adults and normal exfoliated deciduous teeth (SHED; stem cells from human exfoliated deciduous teeth) by single-colony selection and magnetic activated cell sorting.nnnRESULTSnBMP-2 treatment groups produced alkaline phosphatase in the cells and also produced and secreted osteocalcin in the culture medium, and were capable of inducing an upregulated expression of Osteocalcin or Sox9, Col 2, and Col X by reverse transcriptase polymerase chain reaction (RT-PCR). For adipogenic differentiation, there is potential for SHED and dental pulp stem cells (DPSC) to express 2 adipocyte-specific transcripts, PPARgamma2 and LPL, in vitro, as do bone marrow mesenchymal stem cells by RT-PCR.nnnCONCLUSIONnThis study demonstrated that pluripotential cells isolated from the pulp of human teeth expanded in vitro and differentiated into osteoblasts, chondrocytes, and adipocytes. DPSC and SHED are not only derived from a very accessible tissue resource but also capable of providing enough cells for potential clinical applications.

Human Induced Pluripotent Stem Cells Differentiated into Chondrogenic Lineage Via Generation of Mesenchymal Progenitor Cells

Human induced pluripotent stem cells (hiPSCs) exhibit pluripotency, proliferation capability, and gene expression similar to those of human embryonic stem cells (hESCs). hESCs readily form cartilaginous tissues in teratomas in vivo; despite extensive effort, however, to date no efficient method for inducing mature chondrocytes in vitro has been established. hiPSCs can also differentiate into cartilage in vivo by teratoma formation, but as with hESCs, no reliable system for in vitro chondrogenic differentiation of hiPSCs has yet been reported. Here, we examined the chondrogenic differentiation capability of hiPSCs using a multistep culture method consisting of embryoid body (EB) formation, cell outgrowth from EBs, monolayer culture of sprouted cells from EBs, and 3-dimensional pellet culture. In this culture process, the cell density of monolayer culture was critical for cell viability and subsequent differentiation capability. Monolayer-cultured cells exhibited fibroblast-like morphology and expressed markers for mesenchymal stem cells. After 2-3 weeks of pellet culture, cells in pellets exhibited a spherical morphology typical of chondrocytes and were surrounded by extracellular matrix that contained acidic proteoglycans. The expression of type II collagen and aggrecan in pellets progressively increased. Histological analysis revealed that over 70% of hiPSC-derived pellets successfully underwent chondrogenic differentiation. Using the same culture method, hESCs showed similar histological changes and gene expression, but differentiated slightly faster and more efficiently than hiPSCs. Our study demonstrates that hiPSCs can be efficiently differentiated into the chondrogenic lineage in vitro via generation of mesenchymal progenitor cells, using a simplified, multistep culture method.

Osteoinduction by microbubble-enhanced transcutaneous sonoporation of human bone morphogenetic protein-2.

Bone morphogenetic protein‐2 (BMP‐2) is believed to participate in bone healing and regeneration. Previous studies using BMP‐2 in clinical applications have encountered difficulties that include the lack of an efficient, safe and simple delivery system, and expensive proteins and matrices. The gene transfer approach is a promising option for utilizing BMP‐2. Viral vector‐mediated gene transfer is efficient, but safety concerns prevent its clinical application for common diseases. Sonoporation is a simple and inexpensive method that only requires a plasmid and a sonoporation device.

The Local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth

Recent studies revealed C-type natriuretic peptide (CNP) and its receptor, guanylyl cyclase-B (GC-B) are potent stimulators of endochondral bone growth. As they exist ubiquitously in body, we investigated the physiological role of the local CNP/GC-B in the growth plate on bone growth using cartilage-specific knockout mice. Bones were severely shorter in cartilage-specific CNP or GC-B knockout mice and the extent was almost the same as that in respective systemic knockout mice. Cartilage-specific GC-B knockout mice were shorter than cartilage-specific CNP knockout mice. Hypertrophic chondrocyte layer of the growth plate was drastically reduced and proliferative chondrocyte layer, along with the proliferation of chondrocytes there, was moderately reduced in either cartilage-specific knockout mice. The survival rate of cartilage-specific CNP knockout mice was comparable to that of systemic CNP knockout mice. The local CNP/GC-B system in growth plate is responsible for physiological endochondral bone growth and might further affect mortality via unknown mechanisms.

Pluripotency of mesenchymal cells derived from synovial fluid in patients with temporomandibular joint disorder

AIMSnMesenchymal stem cells are an interesting source of material for regenerative medicine. The present study aimed at characterizing the phenotype and differentiation potential of adherent synovial fluid-derived cells from temporomandibular joint (TMJ) disorder patients.nnnMAIN METHODSnSynovial fluid collection takes place during TMJ cavity irrigation arthrocentesis under local anesthesia. The synovial fluid-derived adherent cells were fibroblast-like and spindle-shaped. Ex vivo-expanded synovial fluid-derived cells were shown to express STRO-1 and CD146, previously found to be present in bone marrow mesenchymal stem cells. Further, they were identified as being capable of differentiating into a variety of cell types including osteoblasts, chondrocytes, adipocytes, and neurons.nnnKEY FINDINGSnThe present study demonstrates that human pluripotent cells can be isolated from synovial fluid. These synovial fluid-derived cells cannot only be derived from a very accessible resource, but are also capable of providing sufficient cells for potential clinical applications.nnnSIGNIFICANCEnThese cells may play a role in the regenerative response during arthritic diseases and are promising candidates for developing novel cell-based therapeutic approaches for postnatal skeletal tissue repair.

Osteoinduction by repeat plasmid injection of human bone morphogenetic protein-2.

Bone morphogenetic protein‐2 (BMP‐2) is an osteoinductive protein and is considered useful for the treatment of skeletal disorders. Previous studies using BMP‐2 in clinical applications have encountered difficulties, including the lack of an efficient, safe, inexpensive and simple delivery system. The gene transfer approach is a promising option for utilizing BMP‐2. Although viral vector‐mediated gene transfer is efficient, safety concerns prevent its clinical application for common diseases. On the other hand, plasmid‐based gene transfer is a safe method and can be harnessed for practical applications.

Impact of different surgery modalities to correct class III jaw deformities on the pharyngeal airway space.

Objective: The objective of the study was to compare the outcome of different modalities of orthognathic surgery to correct class III jaw deformities concerning the pharyngeal airway space, especially in patients with other predisposing factors for the development of obstructive sleep apnea. Methods: Lateral cephalograms of 30 Japanese patients (12 males and 18 females, 24.4 [SD, 6.8] years), who underwent surgical-orthodontic treatment for class III jaw deformities, were obtained. Patients were divided into 3 groups: Group A included patients who underwent bilateral sagittal split ramus osteotomy; group B patients underwent bimaxillary surgery, and group C patients underwent intraoral vertical ramus osteotomy. Lateral cephalograms were assessed before surgery and around 3 months and 1 year after surgery. The paired t-test was used to compare the groups, and P < 0.05 was considered significant. Results: In groups A and C who underwent sagittal split ramus osteotomy and intraoral vertical ramus osteotomy, respectively, the pharyngeal airway was constricted significantly at the 3 levels of the pharyngeal airway space on short- and long-term follow-up, whereas in group B, who underwent bimaxillary surgery, no significant changes were noted on long-term follow-up. Conclusions: Bimaxillary surgery rather than only mandibular setback surgery is preferable to correct class III jaw deformity to prevent narrowing of the pharyngeal airway, which might be a predisposing factor in the development of obstructive sleep apnea syndrome.

Increased Bone Turnover and Possible Accelerated Fracture Healing in a Murine Model With an Increased Circulating C-Type Natriuretic Peptide

Recent studies have revealed that C-type natriuretic peptide (CNP) is a potent stimulator of endochondral bone growth. Nevertheless, the effect of CNP on bone turnover has not yet been well studied. To elucidate this issue, we investigated the bone phenotype of a mouse model with elevated plasma CNP concentrations (SAP-CNP-Tg mice) in the present study. Microcomputed tomography (CT) analysis revealed less bone in femurs, but not in lumber vertebrae, of young adult SAP-CNP-Tg mice than that of wild-type mice. Bone histomorphometry of the tibiae from 8-week-old SAP-CNP-Tg mice showed enhanced osteoblastic and osteoclastic activities, in accordance with elevated serum levels of osteocalcin and tartrate-resistant acid phosphatase-5b, respectively. Next we performed an open and stabilized femoral fracture using 8-week-old SAP-CNP-Tg mice and compared the healing process with age-matched wild-type mice. An immunohistochemical study revealed that CNP and its receptors, natriuretic peptide receptor-B and natriuretic peptide clearance receptor, are expressed in hard calluses of wild-type mice, suggesting a possible role of CNP/natriuretic peptide receptor-B signaling in fracture repair, especially in bone remodeling stage. On micro-CT analysis, a rapid decrease in callus volume was observed in SAP-CNP-Tg mice, followed by a generation of significantly higher new bone volume with a tendency of increased bone strength. In addition, a micro-CT analysis also showed that bone remodeling was accelerated in SAP-CNP-Tg mice, which was also evident from increased serum osteocalcin and tartrate-resistant acid phosphatase-5b levels in SAP-CNP-Tg mice at the remodeling stage of fracture repair. These results indicate that CNP activates bone turnover and remodeling in vivo and possibly accelerates fracture healing in our mouse model.

The Effects of C-type Natriuretic Peptide on Craniofacial Skeletogenesis

C-type natriuretic peptide (CNP) is a potent stimulator of long bone and vertebral development via endochondral ossification. In the present study, we investigated the effects of CNP on craniofacial skeletogenesis, which consists of both endochondral and membranous ossification. Morphometric analyses of crania from CNP knockout and transgenic mice revealed that CNP stimulates longitudinal growth along the cranial length, but does not regulate cranial width. CNP markedly increased the length of spheno-occipital synchondrosis in fetal murine organ cultures, and the thickness of cultured murine chondrocytes from the spheno-occipital synchondrosis or nasal septum, resulting in the stimulation of longitudinal cranial growth. Mandibular growth includes endochondral and membranous ossification; although CNP stimulated endochondral bone growth of condylar cartilage in cultured fetal murine mandibles, differences in the lengths of the lower jaw between CNP knockout or transgenic mice and wild-type mice were smaller than those observed for the lengths of the upper jaw. These results indicate that CNP primarily stimulates endochondral ossification in the craniofacial region and is crucial for midfacial skeletogenesis.

Experimental study of osteoinduction using a new material as a carrier for bone morphogenetic protein-2

We evaluated the usefulness of artificial collagen as a new carrier for recombinant human bone morphogenetic protein-2 (rhBMP-2) by comparing it with that of atelopeptide collagen, which is derived from porcine skin, and which we have previously shown to be useful for the induction of bone. rhBMP-2 5μg with either atelopeptide collagen 3mg or artificial collagen 3mg was implanted into the calf muscle of 10-week-old Wistar rats (n=3 in each group). Three rats were given artificial collagen alone and acted as controls (n=3). Radiographic evaluation, histological analysis, and biochemical examinations were made on day 21 after implantation. Soft radiographs (wavelength 10-0.10nm) showed opaque shadows in both groups. Histological analysis showed that new bone had formed in both experimental groups. Endochondral ossification was found at the outermost edge of the implanted collagen in the atelopeptide group. However, there was less ossification in the implanted collagen in the artificial collagen group. On biochemical examination, alkaline phosphatase activity and calcium concentrations in both experimental groups were higher than in the control group, and were higher in the atelopeptide group than in the artificial collagen group. Our results suggest that artificial collagen is useful as a carrier for rhBMP-2 designed to promote the formation of new bone.