In vitro differentiation of human dental follicle cells with dexamethasone and insulin

The dental follicle is an ectomesenchymally derived connective tissue harboring precursor cells for the tooth supporting apparatus. In this study, we examined gene expression of freshly isolated human dental follicle cells during osteogenic differentiation in vitro. These plastic adherent fibroblastic cells express Notch-1, nestin and vimentin. We differentiated dental follicle cells with dexamethasone or insulin-based protocols into membrane-like structures containing mineralizing foci. An analysis of mineralized tissue with atomic force microscopy illustrated a bone and cementum-like structure. A real-time RT-PCR analysis was developed to investigate expression of typical osteoblast or cementoblast related genes during differentiation. Gene expressions of osteocalcin (OCN), bone morphogenic protein (BMP)-2 and nestin were increased during the both differentiation approaches. Our work demonstrates differentiation of dental follicle cells with an insulin-based protocol for the first time.     

TGF-beta3 induces ectopic mineralization in fetal mouse dental pulp during tooth germ development

Several members of the transforming growth factor (TGF)-beta superfamily are expressed in developing teeth from the initiation stage through adulthood. Of those, TGF-beta1 regulates odontoblast differentiation and dentin extracellular matrix synthesis. However, the molecular mechanism of TGF-beta3 in dental pulp cells is not clearly understood. In the present study, beads soaked with human recombinant TGF-beta3 induced ectopic mineralization in dental pulp from fetal mouse tooth germ samples, which increased in a dose-dependent manner. Further, TGF-beta3 promoted mRNA expression, and increased protein levels of osteocalcin (OCN) and type I collagen (COL I) in dental pulp cells. We also observed that the expression of dentin sialophosphoprotein and dentin matrix protein 1 was induced by TGF-beta3 in primary cultured dental pulp cells, however, not in calvaria osteoblasts, whereas OCN, osteopontin and osteonectin expression was increased after treatment with TGF-beta3 in both dental pulp cells and calvaria osteoblasts. Dentin sialoprotein was also partially detected in the vicinity of TGF-beta3 soaked beads in vivo. These results indicate for the first time that TGF-beta3 induces ectopic mineralization through upregulation of OCN and COL I expression in dental pulp cells, and may regulate the differentiation of dental pulp stem cells to odontoblasts.     

Type I collagen regulated dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of rat dental pulp cells

In this study, we investigated the effect of type I collagen on dentin matrix protein-1 (Dmp-1) and osteocalcin (OCN) gene expression of dental pulp cells. The mRNA level of Dmp-1 gene was down-regulated; however, OCN gene expression was up-regulated by the culture of dental pulp cells with type I collagen. These findings imply that type I collagen regulates mRNA level of Dmp-1 and OCN gene that are predominantly expressed in active odontoblasts. The change of gene expression by type I collagen was suppressed by the blocking of collagen-integrin interaction. We could conclude that the effect of type I collagen was mediated via binding of collagen to integrin receptors.     

Ectopic osteogenesis and chondrogenesis of bone marrow stromal stem cells in alginate system

In orthopedics, the regeneration and repair of cartilage or bone defects after trauma, cancer, or metabolic disorders is still a major clinical challenge. Through developmental plasticity, bone marrow mesenchymal stem cells (BMSSCs) are important seed cells for the musculoskeletal tissue engineering approach. The present study sought to determine the ectopic osteogenic and chondrogenic ability of BMSSCs in combination with a scaffolding material made from alginate gel. After isolation from the bone marrow of BALB/C mice, BMSSCs were expanded in vitro and induced to chondrogenesis or osteogenesis for 14 days, respectively. Subsequently, these induced cells were seeded into alginate gel, and the constructs implanted into BALB/C nude mice subcutaneously for up to 8 weeks. In the histological analysis, the transmission electron microscopy of the retrieved specimens at various intervals showed obvious trends of ectopic cartilage or bone formation along with the alteration of the cellular phenotype. Simultaneously, the results of the immunohistochemical staining and RT-PCR both confirmed the expression of specific extracellular matrix (ECM) markers for cartilaginous tissue, such as collagen type II (Col-II), SOX9, and aggrecan, or alternatively, markers for osteoid tissue, such as osteopontin (OPN), osteocalcin (OCN), and collagen type I (Col-I). During subcutaneous implantation, the elevating production of ECM and the initiation of the characteristic structure were closely correlated with the increase of time. In contrast, there was an apparent degradation and resorption of the scaffolding material in blank controls, but with no newly formed tissues. Finally, the constructs that were made of non-induced BMSSCs nearly disappeared during the 8 weeks after implantation. Therefore, it is suggested that alginate gel, which is combined with BMSSCs undergoing differentiation into skeletal lineages, may represent a useful strategy for the clinical reconstruction of bone and cartilage defects.     

Induction of osteoblast differentiation indices by statins in MC3T3-E1 cells

Statins inhibit 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase, which catalyzes conversion of HMG-CoA to mevalonate, a rate-limiting step in cholesterol synthesis. The present study was undertaken to understand the events of osteoblast differentiation induced by statins. Simvastatin at 10(-7) M markedly increased mRNA expression for bone morphogenetic protein-2 (BMP-2), vascular endothelial growth factor (VEGF), alkaline phosphatase, type I collagen, bone sialoprotein, and osteocalcin (OCN) in nontransformed osteoblastic cells (MC3T3-E1), while suppressing gene expression for collagenase-1, and collagenase-3. Extracellular accumulation of proteins such as VEGF, OCN, collagenase-digestive proteins, and noncollagenous proteins was increased in the cells treated with 10(-7) M simvastatin, or 10(-8) M cerivastatin. In the culture of MC3T3-E1 cells, statins stimulated mineralization; pretreating MC3T3-E1 cells with mevalonate, or geranylgeranyl pyrophosphate (a mevalonate metabolite) abolished statin-induced mineralization. Statins stimulate osteoblast differentiation in vitro, and may hold promise drugs for the treatment of osteoporosis in the future.     

TGF-β1-induced collagen promotes chicken ovarian follicle development via an intercellular cooperative pattern

Follicle development is a complex process under strict regulation of diverse hormones and cytokines including transforming growth factor β (TGF-β) superfamily members. TGF-β is pivotal for the regulation of ovarian functions under physiological and pathological conditions. In this study, effect of TGF-β1 on chicken follicle development was examined through investigating the accumulation and action of collagen, an indispensable member of the extracellular matrix (ECM) involved in this process. The granulosa cells (GCs) and theca cells (TCs) were separated from growing follicles of the laying chicken for treatment of TGF-β1 and analysis of expression of ECM components and key proteins in intracellular signaling pathways. Results showed that collagen was mainly distributed in the follicular theca layer and was produced with the formation of the granulosa layer during ovarian development. Collagen accumulation increased with follicle growth and treatment of GCs with TGF-β1 elicited an increased expression of collagen. After production from GCs, collagen was transferred to the neighboring TCs to promote cell proliferation and inhibit apoptosis. Treatment of collagen remarkably increased expression of p-ERK, mitogen-activated protein kinase (MAPK), and p-MAPK, but treatment with hydroxylase inhibitor (to break collagen structure) reversed these alterations. In conclusion, during follicle growth collagen was secreted by GCs under TGF-β1 stimulation and was subsequently collaboratively transferred to neighboring TCs to increase cell proliferation and thus to promote follicle development via an intercellular cooperative pattern during development of chicken growing follicles.     

Histochemical examination of systemic administration of eldecalcitol combined with guided bone regeneration for bone defect restoration in rats

The aim of this experiment was to elucidate the histological alterations after systemic administration of eldecalcitol (ELD) combined with guided bone regeneration during the restoration of bone defect healing in rats. The femurs of 8-week-old Wister rats were used to generate bone defect models. The defect was covered with a collagen membrane, and ELD group was administrated with eldecalcitol (50 ng/kg body weight) intragastrically once every other day. Femora were harvested at 1, 2, 4 and 8 weeks post-surgery. Decalcify tissue slices were made and used for histological and immunohistochemical examination. Bone biomarkers of RANKL, OPG and osteocalcin (OCN) were detected by western blot. The results revealed that the system administration of ELD could improve new bone formation demonstrated by the increased bone volume/tissue volume ratio and accelerated mineralization. ELD suppressed osteoclastic bone resorption by reducing the number of osteoclasts, decreasing the expression of cathepsin-K and the ratio of RANKL/OPG at the early stage of bone defect restoration (1 and 2 weeks) and upregulating OCN expression at the later stage of bone defect healing (4 and 8 weeks). These data suggested that systemic administration of eldecalcitol accelerated bone formation and promoted bone maturation by decreasing bone resorption and promoting bone mineralization during bone defect restoration.     

Treatment of diabetic wounds with fetal murine mesenchymal stromal cells enhances wound closure

The dental follicle is a mesenchymal tissue that surrounds the developing tooth germ. During tooth root formation, periodontal components, viz., cementum, periodontal ligament (PDL), and alveolar bone, are created by dental follicle progenitors. Here, we report the presence of PDL progenitors in mouse dental follicle (MDF) cells. MDF cells were obtained from mouse incisor tooth germs and immortalized by the expression of a mutant human papilloma virus type 16 E6 gene lacking the PDZ-domain-binding motif. MDF cells expressing the mutant E6 gene (MDF ^E6-EGFP cells) had an extended life span, beyond 150 population doublings (PD). In contrast, normal MDF cells failed to proliferate beyond 10 PD. MDF ^E6-EGFP cells expressed tendon/ligament phenotype-related genes such as Scleraxis (Scx), growth and differentiation factor-5, EphA4, Six-1, and type I collagen. In addition, the expression of periostin was observed. To elucidate the differentiation capacity of MDF ^E6-EGFP cells in vivo, the cells were transplanted into severe combined immunodeficiency mice. At 4 weeks, MDF ^E6-EGFP cell transplants had the capacity to generate a PDL-like tissue that expressed periostin, Scx, and type XII collagen and the fibrillar assembly of type I collagen. Our findings suggest that MDF ^E6-EGFP cells can act as PDL progenitors, and that these cells may be a useful research tool for studying PDL formation and for developing regeneration therapies. This work was supported by a Grant-in Aid for the High-Tech Research Center Project from the Ministry of Education, Culture, Sports, Science, and Technology of Japan, the AGU High-Tech Research Center Project, the 2003-Multidisciplinary Research Project from MEXT, and grants from the Ministry of Education, Culture, Sports, Science, and Technology of Japan.     

Adipose tissue-deprived stem cells acquire cementoblast features treated with dental follicle cell conditioned medium containing dentin non-collagenous proteins in vitro

Adipose tissue-derived stem cells (ADSCs), which are easily harvested and show excellent pluripotency potential, have generated considerable interest in regenerative medicine. In this study, the differentiation of ADSCs was assessed after treatment with dental follicle cell conditioned medium (DFCCM) containing dentin non-collagenous proteins (dNCPs). ADSCs exhibited a fibroblast-like morphology and high proliferative capacity. However, after treatment with dNCPs/DFCCM, ADSCs changed from a fibroblast-like to cementoblast-like morphology and significantly lost their proliferative capacity. Alkaline phosphatase activity and in vitro mineralization behaviour of ADSCs were significantly enhanced. Mineralization-related markers including cementum attachment protein, bone sialoprotein, osteocalcin, osteopontin and osteonectin were detected at mRNA or protein levels, whereas dentin sialophosphoprotein and dentin sialoprotein were not detected, implying a cementoblast-like phenotype. These results demonstrate that ADSCs acquired cementoblast features in vitro with dNCPs/DFCCM treatment and could be a potential source of cementogenic cells for periodontal regeneration.