Role of reactive oxygen species (ROS) in the regulation of adipogenic differentiation of human maxillary/mandibular bone marrow-derived mesenchymal stem cells.

BACKGROUND: Maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs) exhibit a unique property of lower adipogenic potential than other bone marrow-derived MSCs. However, the molecular mechanisms regulating the adipogenesis of MBMSCs remain unclear. This study aimed to explore the roles of mitochondrial function and reactive oxygen species (ROS) in regulating the adipogenesis of MBMSCs. METHODS AND RESULTS: MBMSCs exhibited significantly lower lipid droplet formation than iliac BMSCs (IBMSCs). Moreover, the expression levels of CCAAT/enhancer-binding protein ß (C/EBPß), C/EBPδ, and early B cell factor 1 (Ebf-1), which are early adipogenic transcription factors, and those of peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which are late adipogenic transcription factors, were downregulated in MBMSCs compared to those in IBMSCs. Adipogenic induction increased the mitochondrial membrane potential and mitochondrial biogenesis in MBMSCs and IBMSCs, with no significant difference between the two cell types; however, intracellular ROS production was significantly enhanced only in IBMSCs. Furthermore, NAD(P)H oxidase 4 (NOX4) expression was significantly lower in MBMSCs than in IBMSCs. Increased ROS production in MBMSCs by NOX4 overexpression or treatment with menadione promoted the expression of early adipogenic transcription factors but did not induce that of late adipogenic transcription factors or lipid droplet accumulation. CONCLUSIONS: These results suggest that ROS may be partially involved in the process of MBMSC adipogenic differentiation from undifferentiated cells to immature adipocytes. This study provides important insights into the tissue-specific properties of MBMSCs.

Elucidation of adipogenic differentiation regulatory mechanism in human maxillary/mandibular bone marrow-derived stem cells.

OBJECTIVE: This study aims to investigate the underlying molecular mechanisms that regulate the adipogenic differentiation of maxillary/mandibular bone marrow-derived mesenchymal stem cells (MBMSCs). DESIGN: MBMSCs and iliac bone marrow-derived MSCs (IBMSCs) were compared for osteogenic, chondrogenic, and adipogenic differentiation. Cell surface antigen expression was examined using flow cytometry, and stem cell marker expression was assessed using real-time polymerase chain reaction (PCR). Various adipogenic regulatory factors' expression was evaluated using real-time PCR and western blotting. RESULTS: No significant differences in cell surface antigen profiles or stem cell marker expression in MBMSCs and IBMSCs were observed. MBMSCs and IBMSCs displayed similar osteogenic and chondrogenic potentials, whereas MBMSCs showed significantly lower adipogenic potentials than those shown by IBMSCs. Expression of CCAAT/enhancer binding protein ß (C/EBPß), C/EBPδ, early B-cell factor 1 (Ebf-1), and Krüppel-like factor 5 (KLF5), which are early adipogenic differentiation factors, was suppressed in MBMSCs compared to that in IBMSCs. Peroxisome proliferator-activated receptor-γ (PPARγ) and C/EBPα, which play important roles in the terminal differentiation of adipocytes, was lower in MBMSCs than that in IBMSCs. Furthermore, the level of zinc finger protein 423 (Zfp423), which is involved in the commitment of undifferentiated MSCs to the adipocyte lineage, was significantly lower in MBMSCs than that in IBMSCs. CONCLUSIONS: MBMSCs are negatively regulated in the commitment of undifferentiated MSCs to the adipocyte lineage (preadipocytes) as well as in the terminal differentiation of preadipocytes into mature adipocytes. These results may elucidate the site-specific characteristics of MBMSCs.

Efficacy of bone grafting materials in preserving the alveolar ridge in a canine model.

Preservation of the alveolar ridge after tooth extraction is an essential component for ideal implant positioning. Furthermore, preservation of bone around the implant after implant placement is an essential component for implant treatment. We aimed to evaluate the efficacy of bone grafting materials in preserving the alveolar ridge after implant placement. Implants were placed in regenerated bone without grafting material or with beta-tricalcium phosphate, bovine bone substitute, or carbonate apatite transplantation. In all groups, the bone healed and the implants were successfully placed within the bone. No significant differences in insertion torque and implant stability quotient values were found. The amount of bone around the implant 5 weeks after implant placement was significantly reduced in the bovine bone substitute group; however, implants placed in regenerated bone achieved sufficient initial fixation and osseointegration.

Efficacy of chitinase-3-like protein 1 as an in vivo bone formation predictable marker of maxillary/mandibular bone marrow stromal cells.

INTRODUCTION: Maxillary/mandibular bone marrow stromal cells (MBMSCs) are a useful cell source for bone regeneration in the oral and maxillofacial region. To further ensure the clinical application of MBMSCs in bone regenerative therapy, it is important to determine the bone formation capacity of MBMSCs before transplantation. The aim of this study is to identify the molecular marker that determines the in vivo bone formation capacity of MBMSCs. METHODS: The cell growth, cell surface antigens, in vitro and in vivo bone formation capacity of MBMSCs were examined. The amount of chitinase-3-like protein 1 (CHI3L1) secreted into the conditioned medium was quantified. The effects of CHI3L1 on the cell growth and osteogenic differentiation potential of MBMSCs and on the cell growth and migration of vascular endothelial cells and fibroblasts were examined. RESULTS: The cell growth, and in vitro and in vivo bone formation capacity of the cells treated with different conditions were observed. MBMSCs that secreted a large amount of CHI3L1 into the conditioned medium tended to have low in vivo bone formation capacity, whereas MBMSCs that secreted a small amount of CHI3L1 had greater in vivo bone formation capacity. CHI3L1 promoted the migration of vascular endothelial cells, and the cell growth and migration of fibroblasts. CONCLUSION: Our study indicates that the in vitro osteogenic differentiation capacity of MBMSCs and the in vivo bone formation capacities of MBMSCs were not necessarily correlated. The transplantation of high CHI3L1 secretory MBMSCs may suppress bone formation by inducing fibrosis at the site. These results suggest that the CHI3L1 secretion levels from MBMSCs may be used as a predictable marker of bone formation capacity in vivo.

Effects of denture adhesives and mouth moisturizers to human oral fibroblast and human keratinocyte cells using direct and indirect cell culture systems.

The purpose of this study was to evaluate the effects of commercialized denture adhesives and mouth moisturizers using direct and indirect cell cultures for in vitro examinations with human fibroblast and epithelial cells. Denture adhesives (Faston, Poligrip Powder, New Poligrip Free, Tafugurippu Kurimu, Polident Adhesive, Tafugurippu Tomei) and mouth moisturizers (Concool Mouth Gel, Biotene Oral Balance Gel) were subjected to live and dead detection and pH level determination. The mouth moisturizers showed higher cytotoxicity effects comparing with control on every cell cultures and cells, and pH level did not show any significant differences. However, there was no correlation of type of denture adhesive or mouth moisturizer with cytotoxicity. We concluded that cytotoxicity affects human cells regardless of type of material, though some dependence was noted.

Vascular endothelial growth factor-C induces osteogenic differentiation of human mesenchymal stem cells through the ERK and RUNX2 pathway.

Vascular endothelial cell growth factor C (VEGF-C) is a member of the VEGF family and plays a role in a variety of biological activities including lymphangiogenesis, angiogenesis, and neurogenesis through VEGF receptor 2 (VEGFR2) and 3 (VEGFR3). However, it has not been elucidated whether VEGF-C promotes osteogenic differentiation. Herein, we investigated the effects of VEGF-C on osteogenic differentiation in human mesenchymal stem cells (MSCs) and evaluated the underlying molecular mechanisms. VEGF-C treatment significantly increased RUNX2 expression, and led to the promotion of osteogenic marker gene expression and mineralization of MSCs. VEGF-C treatment induced the phosphorylation of VEGFR2 and VEGFR3 in MSCs. Treatment with the VEGFR3-specific ligand VEGF-C156S also promoted MSC mineralization. Furthermore, co-treatment with VEGFR2 and VEGFR3 kinase inhibitors blocked VEGF-C-induced MSC mineralization. VEGF-C treatment activated ERK signaling in MSCs, and inhibition of ERK signaling effectively suppressed VEGF-C-induced RUNX2 expression and mineralization. These results indicate that VEGF-C-induced MSC osteogenesis is mediated through VEGFR2 and VEGFR3, and followed the activation of the ERK/RUNX2 signaling pathway.

Low-serum culture with novel medium promotes maxillary/mandibular bone marrow stromal cell proliferation and osteogenic differentiation ability.

OBJECTIVES: The purpose of this study was to evaluate the effect of low-serum STK2 medium on the isolation and osteogenic differentiation of human maxillary/mandibular bone marrow stromal cells (MBMSCs). MATERIALS AND METHODS: Human MBMSCs were obtained from patients undergoing dental implant treatment. These cells were cultured in serum-free medium or STK2 medium containing 1  % fetal bovine serum (low-serum) or α-MEM containing 10  % fetal bovine serum (control). Proliferation on the culture surface, cell surface antigen expression, and mRNA levels of neural crest and osteogenic markers were examined. Alkaline phosphatase assay and Alizarin red staining were used to assess osteogenic differentiation potential. Immunoblotting analysis was performed to detect ERK phosphorylation. RESULTS: Low-serum and control MBMSCs were positive for CD73, CD90, and CD105 and negative for CD14, CD34, CD45, CD271, and HLA-DR. CD140a was absent in low-serum cells but present in control cells. Low-serum MBMSCs proliferated more than control MBMSCs. After induction of osteogenic differentiation, alkaline phosphatase activity and osteocalcin mRNA levels were higher in low-serum MBMSCs than in control cells, and Alizarin red staining was stronger in low-serum MBMSCs than in control cells. Low-serum culture promoted ERK phosphorylation. CONCLUSIONS: MBMSCs precultured in low-serum medium exhibited a greater cumulative cell number and a higher osteogenic differentiation capacity than those cultured in control medium. CLINICAL RELEVANCE: These findings indicate that low-serum STK2 culture might be useful to promote MBMSC proliferation and osteogenic differentiation. This method requires less autologous blood collection for cell expansion than conventional methods, thus reducing the burden on patients.

Regulation and Biological Significance of Formation of Osteoclasts and Foreign Body Giant Cells in an Extraskeletal Implantation Model.

The implantation of biomaterials induces a granulomatous reaction accompanied by foreign body giant cells (FBGCs). The characterization of multinucleated giant cells (MNGCs) around bone substitutes implanted in bone defects is more complicated because of healing with bone admixed with residual bone substitutes and their hybrid, and the appearance of two kinds of MNGCs, osteoclasts and FBGCs. Furthermore, the clinical significance of osteoclasts and FBGCs in the healing of implanted regions remains unclear. The aim of the present study was to characterize MNGCs around bone substitutes using an extraskeletal implantation model and evaluate the clinical significance of osteoclasts and FBGCs. Beta-tricalcium phosphate (ß-TCP) granules were implanted into rat subcutaneous tissue with or without bone marrow mesenchymal cells (BMMCs), which include osteogenic progenitor cells. We also compared the biological significance of plasma and purified fibrin, which were used as binders for implants. Twelve weeks after implantation, osteogenesis was only detected in specimens implanted with BMMCs. The expression of two typical osteoclast markers, tartrate-resistant acid phosphatase (TRAP) and cathepsin-K (CTSK), was analyzed, and TRAP-positive and CTSK-positive osteoclasts were only detected beside bone. In contrast, most of the MNGCs in specimens without the implantation of BMMCs were FBGCs that were negative for TRAP, whereas the degradation of ß-TCP was detected. In the region implanted with ß-TCP granules with plasma, FBGCs tested positive for CTSK, and when ß-TCP granules were implanted with purified fibrin, FBGCs tested negative for CTSK. These results showed that osteogenesis was essential to osteoclastogenesis, two kinds of FBGCs, CTSK-positive and CTSK-negative, were induced, and the expression of CTSK was plasma-dependent. In addition, the implantation of BMMCs was suggested to contribute to osteogenesis and the replacement of implanted ß-TCP granules to bone.

Diversity of multinucleated giant cells by microstructures of hydroxyapatite and plasma components in extraskeletal implantation model.

UNLABELLED: Foreign body giant cells (FBGCs) and osteoclasts are multinucleated giant cells (MNGCs), both of which are formed by the fusion of macrophage-derived mononuclear cells. Osteoclasts are distinct from FBGCs due to their bone resorption ability; however, not only morphological, but also functional similarities may exist between these cells. The characterization and diversity of FBGCs that appear in an in vivo foreign body reaction currently remain incomplete. In the present study, we investigated an in vivo foreign body reaction using an extraskeletal implantation model of hydroxyapatite (HA) with different microstructures. The implantation of HA granules in rat subcutaneous tissue induced a foreign body reaction that was accompanied by various MNGCs. HA granules composed of rod-shaped particles predominantly induced cathepsin K (CTSK)-positive FBGCs, whereas HA granules composed of globular-shaped particles predominantly induced CTSK-negative FBGCs. Plasma, which was used as the binder of ceramic granules, stimulated the induction of CTSK-positive FBGCs more strongly than purified fibrin. Furthermore, the implantation of HA composed of rod-shaped particles with plasma induced tartrate-resistant acid phosphatase (TRAP)-positive MNGCs in contrast to HA composed of globular-shaped particles with purified fibrin, which predominantly induced CTSK-negative and TRAP-negative typical FBGCs. These results suggest that CTSK-positive, TRAP-positive, and CTSK- and TRAP-negative MNGCs are induced in this subcutaneous implantation model in a manner that is dependent on the microstructure of HA and presence or absence of plasma. STATEMENT OF SIGNIFICANCE: We attempted to elucidate the mechanisms responsible for the foreign body reaction induced by the implantation of hydroxyapatite granules with different microstructures in rat subcutaneous tissue with or without plasma components as the binder of ceramic granules. By analyzing the expression of two reliable osteoclast markers, we detected tartrate-resistant acid phosphatase-positive multinucleated giant cells, cathepsin K-positive multinucleated giant cells, and tartrate-resistant acid phosphatase- and cathepsin K-negative multinucleated giant cells. The induction of tartrate-resistant acid phosphatase-positive multinucleated giant cells was plasma component-dependent while the induction of cathepsin K-positive multinucleated giant cells was influenced by the microstructure of hydroxyapatite. This is the first study to show the conditions dividing the three kinds of multinucleated giant cells in the foreign body reaction.

Candidates cell sources to regenerate alveolar bone from oral tissue.

Most of the cases of dental implant surgery, especially the bone defect extensively, are essential for alveolar ridge augmentation. As known as cell therapy exerts valuable effects on bone regeneration, numerous reports using various cells from body to regenerate bone have been published, including clinical reports. Mesenchymal cells that have osteogenic activity and have potential to be harvested from intra oral site might be a candidate cells to regenerate alveolar bone, even dentists have not been harvested the cells outside of mouth. This paper presents a summary of somatic cells in edentulous tissues which could subserve alveolar bone regeneration. The candidate tissues that might have differentiation potential as mesenchymal cells for bone regeneration are alveolar bone chip, bone marrow from alveolar bone, periosteal tissue, and gingival tissue. Understanding their phenotype consecutively will provide a rational approach for alveolar ridge augmentation.

Evaluation of trabecular bone formation in a canine model surrounding a dental implant fixture immobilized with an antimicrobial peptide derived from histatin.

JH8194 induces osteoblast differentiation, although it was originally designed to improve antifungal activity. This suggests that JH8194 is useful for implant treatment. Therefore, the aim of this study was to evaluate the osseointegration capacity of JH8194-modified titanium dental implant fixtures (JH8194-Fi). The implants were randomly implanted into the edentulous ridge of dog mandibles. Healing abutments were inserted immediately after implant placement. Three weeks later, peri-implant bone levels, the first bone-to-implant contact points, and trabecular bone formation surrounding the implants were assessed by histological and digital image analyses based on microcomputed tomography (microCT). The histological analysis revealed an enhancement of mature trabecular bone around the JH8194-Fi compared with untreated fixtures (control-Fi). Similarly, microCT combined with analysis by Zed View™ also showed increased trabecular bone formation surrounding the JH8194-Fi compared with the control-Fi (Student's t-test, P < 0.05). JH8194 may offer an alternative biological modification of titanium surfaces to enhance trabecular bone formation around dental implants, which may contribute to the transient acquirement of osseointegration and the long-term success of implant therapy.

Impact of zinc fingers and homeoboxes 3 on the regulation of mesenchymal stem cell osteogenic differentiation.

We propose zinc fingers and homeoboxes 3 (ZHX3) as new osteogenic markers for mesenchymal stem cells (MSC). ZHX3 mRNA expression was upregulated within 1-6 h after incubation of MSCs in the osteogenic induction medium, and reached maximum levels after 24 h of incubation. Two to 4 days later, ZHX3 mRNA levels had decreased sharply. Maximal mRNA levels were 3- to 5-fold higher than those in the undifferentiated state. In contrast, Runt-related transcription factor2 (RUNX2) mRNA expression was downregulated at 2-4 h after incubation, and levels were only enhanced 1.4-fold after 12 and 24 h of incubation. Further, Osterix mRNA levels increased only 1.6-fold after 4 and 24 h of incubation. Thus, ZHX3 expression may be a better marker of MSC osteogenic differentiation than RUNX2 or Osterix expression at the initial stage of differentiation. Knockdown of ZHX3 using 2 distinct small interfering RNA (siRNA) oligonucleotides had little effect on cell morphology or on MSC proliferation, regardless of the differentiation state of the cells. However, ZHX3 siRNAs suppressed Osterix, but not RUNX2 mRNA expression, within 1 h of osteogenic differentiation, and this suppression was sustained for at least 24 h. The 2 ZHX3 siRNAs also suppressed alkaline phosphatase induction and matrix mineralization (assessed using alizarin red staining), and, further, suppressed the calcium content of the cultures at a later stage of differentiation (days 6-21). The effects of ZHX3 siRNAs on the osteogenic differentiation were comparable to those of RUNX2 and Osterix siRNAs. These findings suggest that ZHX3 is involved in the switch from the undifferentiated state of MSC to an osteogenic program, and that ZHX3 may be useful as an early osteogenic differentiation marker.

Comprehensive analysis of chemotactic factors for bone marrow mesenchymal stem cells.

To understand which growth factors/cytokines can affect migration of mesenchymal stem cells (MSCs) to injured tissues, we compared the effects of many (26) growth factors/cytokines on the migration activity of rabbit and human MSCs using a microchemotaxis chamber. Among them, platelet-derived growth factor (PDGF)-BB, PDGF-AB, epidermal growth factor (EGF), HB-EGF, transforming growth factor (TGF-alpha), insulin growth factor (IGF-I), hepatocyte growth factor (HGF), fibroblast growth factor (FGF-2), and thrombin consistently enhanced the migration of rabbit and human MSCs at appropriate concentrations. PDGF-BB showed the greatest effect on migration. Various combinations of these factors further enhanced the migration of MSCs, whereas combinations of factors that shared common cell-surface receptors did not induce the additive stimulation. On the other hand, some combinations, including that of FGF-2 or thrombin with PDGF-BB, suppressed the migration activity of MSCs. These findings suggest that combinations of growth factors are important to eliciting the maximal chemotactic effect. The factors that induced the migration of MSCs also enhanced their proliferation, suggesting that migration and proliferation can take place simultaneously. The above factors were also effective in stimulating the migration of fibroblasts, but thrombin alone selectively enhanced the migration of MSCs, suggesting that thrombin is useful to stimulate migration of MSCs without migration of fibroblasts.