Metformin facilitates the proliferation,migration, and osteogenic differentiation of periodontal ligament stem cells in vitro

Periodontitis is one of the main causes of tooth loss and has been confirmed as the sixth complication of diabetes. Metformin promotes the osteogenic differentiation of stem cells. Periodontal ligament stem cells (PDLSCs) are the best candidate stem cells for periodontal tissue regeneration. Herein, we aimed to identify the effects of metformin on the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro. PDLSCs were isolated by limiting dilution, and their characteristics were assessed by colony formation assay and flow cytometry. Cell counting and migration assays were used to investigate the effects of metformin on proliferation and migration. The osteogenic differentiation ability of PDLSCs was detected by alkaline phosphatase (ALP) activity and Alizarin Red S staining. Gene and protein levels of osteogenesis‐related markers were determined by quantitative real‐time polymerase chain reaction (qRT‐PCR) and western blot analysis, respectively. Metformin treatment at 10 μM did not affect PDLSC proliferation, while at 50 and 100 μM, metformin time‐dependently enhanced PDLSC proliferation and significantly increased cell numbers after 5 and 7 days of stimulation (P < 0.05). In addition, 50 μM metformin exhibited a maximal effect on migration, ALP activity, and mineral deposition (P < 0.05). Furthermore, 50 μM metformin significantly upregulated the gene expression levels of ALP, BSP, OPN, OCN, and Runx2 and the protein expression of ALP and Runx2 (P < 0.05). In summary, our study confirms that metformin facilitates the proliferation, migration, and osteogenic differentiation of PDLSCs in vitro and could be used as a new strategy for periodontal tissue regeneration.     

The effects of platelet-rich plasma derived from human umbilical cord blood on the osteogenic differentiation of human dental stem cells

Platelet-rich plasma (PRP) is an emerging therapeutic application because PRP contains various growth factors that have beneficial effects on tissue regeneration and engineering. Mesenchymal stem cells and PRP derived from peripheral blood have been well studied. In this study, we investigated the effects of PRP derived from human umbilical cord blood (UCB-PRP) on proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHEDs), dental pulp stem cells (DPSCs), and periodontal ligament stem cells (PDLSCs). Three types of dental stem cells were primarily isolated and characterized by flow cytometric analysis. Dental stem cells were exposed to various concentrations of UCB-PRP, which resulted in the proliferation of dental stem cells. Treatment with 2% UCB-PRP resulted in the highest level of proliferation. The ALP activity of DPSCs and PDLSCs increased following treatment with UCB-PRP in a dose-dependent manner up to a concentration of 2%. ALP activity decreased with higher concentration of UCB-PRP. The effects of UCB-PRP on calcium deposition were similar to those on proliferation and ALP activity. Treatment with 2% UCB-PRP resulted in the highest calcium depositions in DPSCs and PDLSCs; however, treatment with 1% UCB-PRP resulted in the highest calcium deposition in SHEDs. The concentrations of platelet-derived growth factor-AB and transforming growth factor-β1 in UCB-PRP were investigated and found to be comparable to the amounts in peripheral blood. Overall, UCB-PRP had beneficial effects on the proliferation and osteogenic differentiation of dental stem cells. Determination of the optimal concentration of UCB-PRP requires further investigation for clinical applications.     

Canonical Wnt signaling differently modulates osteogenic differentiation of mesenchymal stem cells derived from bone marrow and from periodontal ligament under inflammatory conditions

Background

Cellular plasticity and complex functional requirements of the periodontal ligament (PDL) assume a local stem cell (SC) niche to maintain tissue homeostasis and repair. Here, pathological alterations caused by inflammatory insults might impact the regenerative capacities of these cells. As bone homeostasis is fundamentally controlled by Wnt-mediated signals, it was the aim of this study to characterize the SC-like capacities of cells derived from PDL and to investigate their involvement in bone pathophysiology especially regarding the canonical Wnt pathway.

Methods

PDLSCs were investigated for their SC characteristics via analysis of cell surface marker expression, colony forming unit efficiency, proliferation, osteogenic differentiation and adipogenic differentiation, and compared to bone marrow derived mesenchymal SCs (BMMSCs). To determine the impact of both inflammation and the canonical Wnt pathway on osteogenic differentiation, cells were challenged with TNF-α, maintained with or without Wnt3a or DKK-1 under osteogenic induction conditions and investigated for p-IκBα, p-NF-κB, p-Akt, β-catenin, p-GSK-3β, ALP and Runx2.

Results

PDLSCs exhibit weaker adipogenic and osteogenic differentiation capacities compared to BMMSCs. TNF-α inhibited osteogenic differentiation of PDLSCs more than BMMSCs mainly through regulating canonical Wnt pathway. Blocking the canonical Wnt pathway by DKK-1 reconstituted osteogenic differentiation of PDLSCs under inflammatory conditions, whereas activation by Wnt3a increased osteogenic differentiation of BMMSCs.

Conclusions

Our results suggest a diverse regulation of the inhibitory effect of TNF-α in BMMSCs and PDLSCs via canonical Wnt pathway modulation.

General significance

These findings provide novel insights on PDLSC SC-like capacities and their involvement in bone pathophysiology under the impact of the canonical Wnt pathway.     

Rho‐kinase inhibitor Y‐27632 facilitates the proliferation,migration and pluripotency of human periodontal ligament stem cells

The selective in vitro expansion and differentiation of multipotent stem cells are critical steps in cell‐based regenerative therapies, while technical challenges have limited cell yield and thus affected the success of these potential treatments. The Rho GTPases and downstream Rho kinases are central regulators of cytoskeletal dynamics during cell cycle and determine the balance between stem cells self‐renewal, lineage commitment and apoptosis. Trans‐4‐[(1R)‐aminoethyl]‐N‐(4‐pyridinyl)cylohexanecarboxamidedihydrochloride (Y‐27632), Rho‐associated kinase (ROCK) inhibitor, involves various cellular functions that include actin cytoskeleton organization, cell adhesion, cell motility and anti‐apoptosis. Here, human periodontal ligament stem cells (PDLSCs) were isolated by limiting dilution method. Cell counting kit‐8 (CCK8), 5‐ethynyl‐2′‐deoxyuridine (EdU) labelling assay, cell apoptosis assay, cell migration assay, wound‐healing assay, alkaline phosphatase (ALP) activity assay, Alizarin Red S staining, Oil Red O staining, quantitative real‐time polymerase chain reaction (qRT‐PCR) were used to determine the effects of Y‐27632 on the proliferation, apoptosis, migration, stemness, osteogenic and adipogenic differentiation of PDLSCs. Afterwards, Western blot analysis was performed to elucidate the mechanism of cell proliferation. The results indicated that Y‐27632 significantly promoted cell proliferation, chemotaxis, wound healing, fat droplets formation and pluripotency, while inhibited ALP activity and mineral deposition. Furthermore, Y‐27632 induced PDLSCs proliferation through extracellular‐signal‐regulated kinase (ERK) signalling cascade. Therefore, control of Rho‐kinase activity may enhance the efficiency of stem cell‐based treatments for periodontal diseases and the strategy may have the potential to promote periodontal tissue regeneration by facilitating the chemotaxis of PDLSCs to the injured site, and then enhancing the proliferation of these cells and maintaining their pluripotency.     

Dental Follicle Cells Rescue the Regenerative Capacity of Periodontal Ligament Stem Cells in an Inflammatory Microenvironment

Aims

Periodontal ligament stem cells (PDLSCs) are one of the best candidates for periodontal regeneration. Their function could be impaired in periodontitis microenvironment. Dental follicle cells (DFCs), serving as precursor cells and mesenchymal stem cells, have intimate connection with PDLSCs. However, it is still unknown whether DFCs could provide a favorable microenvironment to improve the proliferation and differentiation capacity of PDLSCs from healthy subjects (HPDLSCs) and patients diagnosed with periodontitis (PPDLSCs).

Methods

HPDLSCs, PPDLSCs and DFCs were harvested and identified using microscopic and flow cytometric analysis. Then, the coculture systems of DFCs/HPDLSCs and DFCs/PPDLSCs were established with 0.4 µm transwell, in which all the detection indexs were obtained from HPDLSCs and PPDLSCs. The expression of stemness-associated genes was detected by real-time PCR, and the proliferation ability was assessed using colony formation and cell cycle assays. The osteogenic differentiation capacity was evaluated by real-time PCR, western blot, ALP activity, Alizarin Red S staining and calcium level analysis, while the adipogenic differentiation capacity was determined by real-time PCR and Oil Red O staining. The cell sheet formation in vitro was observed by HE staining and SEM, and the implantation effect in vivo was evaluated using HE staining and Masson’s trichrome staining.

Results

PPDLSCs had a greater proliferation capability but lower osteogenic and adipogenic potential than HPDLSCs. DFCs enhanced the proliferation and osteogenic/adipogenic differentiation of HPDLSCs and PPDLSCs to different degrees. Moreover, coculture with DFCs increased cell layers and extracellular matrix of HPDLSCs/PPDLSCs cell sheets in vitro and improved periodontal regeneration by HPDLSCs/PPDLSCs in vivo.

Conclusions

Our data suggest that the function of PPDLSCs could be damaged in the periodontitis microenvironment. DFCs appear to enhance the self-renewal and multi-differentiation capacity of both HPDLSCs and PPDLSCs, which indicates that DFCs could provide a beneficial microenvironment for periodontal regeneration using PDLSCs.     

CTHRC1 promotes osteogenic differentiation of periodontal ligament stem cells by regulating TAZ

Collagen triple helix repeat containing 1 (CTHRC1) is associated with bone metabolism. Alveolar bone has an ability to rapidly remodel itself to adapt its biomechanical environment and function. However, whether CTHRC1 is expressed in alveolar bone tissue and the role of CTHRC1 in alveolar bone remodeling remain unclear. We used orthodontic tooth movement (OTM) rat model to study the effects of CHTRC1 in alveolar bone remodeling in vivo. We found that CTHRC1 was expressed in normal physiological condition of osteocytes, bone matrix, and periodontal ligament cells in rat. During the OTM, the expression of CTHRC1, Runx2 and TAZ were increased. We further studied the effects of CTHRC1 on osteogenic differentiation of human periodontal ligament stem cells in vitro. CTHRC1 can positively regulate the expression of TAZ and osteogenic differentiation markers like Col1, ALP, Runx2 and OCN. Overexpression of CHTRC1 increased osteogenic differentiation of PDLSCs, which could be abolished by TAZ siRNA. Our results suggest that CTHRC1 plays an important role in alveolar bone remodeling and osteogenic differentiation of PDLSCs.     

Mitochondria transfer reverses the inhibitory effects of low stiffness on osteogenic differentiation of human mesenchymal stem cells

Microenvironment biophysical factors such as matrix stiffness can noticeably affect the differentiation of mesenchymal stem cells (MSCs). In this mechanobiology transduction process, mitochondria are shown to be an active participant. The present study aims to systematically elucidate the phenotypic and functional changes of mitochondria during the stiffness-mediated osteogenic differentiation. Additionally, the effect of mitochondria transfer on the osteogenesis of impaired MSCs caused by stiffness was investigated. Human periodontal ligament stem cells (PDLSCs) were used as model cells in the current study. Low stiffness restrained the cell spreading and significantly inhibited the proliferation and osteogenic differentiation of PDLSCs. Mitochondria of PDLSCs cultured on low stiffness exhibited shorter length, rounded shape, fusion/fission imbalance, ROS and mitophagy level increase, and ATP production reduction. The inhibited mitochondria function and osteogenic differentiation capacity were recovered to near-normal levels after transferring the mitochondria of PDLSCs cultured on the high stiffness. This study indicated that low matrix stiffness altered the mitochondrial morphology and induced systematical mitochondrial dysfunction during the osteogenic differentiation of MSCs. Mitochondria transfer was proved to be a feasible technique for maintaining MSCs function in vitro by reversing the osteogenesis ability.     

Effect of heparin and alendronate coating on titanium surfaces on inhibition of osteoclast and enhancement of osteoblast function

The failure of orthopedic and dental implants has been attributed mainly to loosening of the implant from host bone, which may be due to weak bonding of the implant material to bone tissue. Titanium (Ti) is used in the field of orthopedic and dental implants because of its excellent biocompatibility and outstanding mechanical properties. Therefore, in the field of materials science and tissue engineering, there has been extensive research to immobilize bioactive molecules on the surface of implant materials in order to provide the implants with improved adhesion to the host bone tissue.In this study, chemically active functional groups were introduced on the surface of Ti by a grafting reaction with heparin and then the Ti was functionalized by immobilizing alendronate onto the heparin-grafted surface. In the MC3T3-E1 cell osteogenic differentiation study, the alendronate-immobilized Ti substrates significantly enhanced alkaline phosphatase activity (ALP) and calcium content. Additionally, nuclear factor kappa B ligand (RANKL)-induced osteoclast differentiation of RAW264.7 cells was inhibited with the alendronate-immobilized Ti as confirmed by TRAP analysis. Real time PCR analysis showed that mRNA expressions of osteocalcin and osteopontin, which are markers for osteogenesis, were upregulated in MC3T3-E1 cells cultured on alendronate-immobilized Ti. The mRNA expressions of TRAP and Cathepsin K, markers for osteoclastogenesis, in RAW264.7 cells cultured on alendronate-immobilized Ti were down-regulated. Our study suggests that alendronate-immobilized Ti may be a bioactive implant with dual functions to enhance osteoblast differentiation and to inhibit osteoclast differentiation simultaneously.     

Immunomodulatory properties of human periodontal ligament stem cells

Tissue engineering utilizing periodontal ligament stem cells (PDLSCs) has recently been proposed for the development of new periodontal regenerative therapies. Although the use of autologous PDLSC transplantation eliminates the potential of a significant host immune response against the donor cells, it is often difficult to generate enough PDLSCs from one donor source due to the variation of stem cell potential between donors and disease state of each patient. In this study, we examined the immunomodulatory properties of PDLSCs as candidates for new allogeneic stem cell‐based therapies. Human PDLSCs displayed cell surface marker characteristics and differentiation potential similar to bone marrow stromal stem cells (BMSSCs) and dental pulp stem cells (DPSCs). PDLSCs, BMSSCs, and DPSCs inhibited peripheral blood mononuclear cell (PBMNC) proliferation stimulated with mitogen or in an allogeneic mixed lymphocyte reaction (MLR). Interestingly, gingival fibroblasts (GFs) also suppressed allogeneic PBMNC proliferation under both assay conditions. PDLSCs, BMSSCs, DPSCs, and GFs exhibited non‐cell contact dependent suppression of PBMNC proliferation in co‐cultures using transwells. Furthermore, conditioned media (CM) derived from each cell type pretreated with IFN‐γ partially suppressed PBMNC proliferation when compared to CMs without IFN‐γ stimulation. In all of these mesenchymal cell types cultured with activated PBMNCs, the expression of TGF‐β1, hepatocyte growth factor (HGF) and indoleamine 2, 3‐dioxygenase (IDO) was upregulated while IDO expression was upregulated following stimulation with IFN‐γ. These results suggest that PDLSCs, BMSSCs, DPSCs, and GFs possess immunosuppressive properties mediated, in part, by soluble factors, produced by activated PBMNCs. J. Cell. Physiol. 219: 667–676, 2009. © 2009 Wiley‐Liss, Inc.     

牙源性干细胞复合微渠多孔羟基磷灰石支架成骨性能研究*

目的: 探讨牙源性干细胞复合微渠多孔羟基磷灰石支架(grooved porous hydroxyapatite scaffolds, HAG支架)的成骨性能,为骨缺损修复治疗提供新手段。方法: 从健康成人第三磨牙中提取牙周膜干细胞(periodontal ligament stem cells, PDLSCs)及牙髓干细胞(dental pulp stem cells, DPSCs)分别接种于HAG支架上,进行多向分化鉴定及碱性磷酸酶(alkaline phosphatase,ALP)活性测定;并通过CCK-8检测细胞增殖能力;逆转录聚合酶链反应(qRT-PCR)检测骨形态发生蛋白2(bone morphogenetic protein 2, BMP-2)、骨钙素(osteocalcin, OCN)和骨桥蛋白(osteopontin, OPN)等成骨相关基因的表达。体内研究中将搭载PDLSCs和DPSCs的HAG支架移植到裸鼠的背部皮下,8周后取材,组织切片后采用苏木精-伊红(HE)染色观察新骨形成,提取组织蛋白采用Western blot检测ALP、OCN等成骨相关蛋白的表达。结果: 体外研究中DPSCs复合HAG支架组的细胞增殖能力、ALP活性,以及成骨相关基因ALP、BMP2、OCN等的表达均高于PDLSCs复合HAG支架组。体内研究中HE染色显示,PDLSCs复合HAG支架组及DPSCs复合HAG支架组均较空白HAG支架组有更多细胞生长区、纤维细胞增生及骨基质形成,且DPSCs复合HAG支架组的骨基质面积更大,成纤维细胞数量更多;PDLSCs复合HAG支架组及DPSCs复合HAG支架组成骨相关蛋白的表达量均高于空白HAG组,且DPSCs复合HAG支架组中ALP蛋白表达量显著高于PDLSCs复合HAG支架组。结论: PDLSCs、DPSCs复合HAG支架在体内外均表现出良好的成骨性能,其中DPSCs复合HAG支架的成骨性能更为优异。     

两种Wnt 通路抑制剂对牙周膜干细胞作用的比较研究

目的:本实验主要探讨Wnt通路抑制剂XAV-939相比DKK1在牙周膜干细胞增殖及矿化中的作用差异。方法:酶消化法培养牙周膜干细胞,鉴定后,用CCK8试剂盒检测XAV-939和DKK1对牙周膜干细胞增殖能力的影响,茜素红染色及定量检测XAV-939和DKK1对牙周膜干细胞成骨分化能力的影响,q RT-PCR检测DKK1和XAV-939对牙周膜干细胞Wnt通路相关基因GSK-3β和β-catenin及成骨分化相关基因ALP,DSPP,BSP,OCN,RUNX-2的影响。结果:XAV-939和DKK1都可以通过抑制Wnt通路来抑制牙周膜干细胞的增殖及成骨分化。当没有外源性Wnt蛋白刺激时,XAV-939作为Wnt通路抑制剂的抑制作用要强于DKK1,而加入外源性Wnt蛋白后,XAV-939与DKK1的作用效果相当。结论:XAV-939对比DKK1,具有更为广泛而稳定的抑制效果。XAV-939可以作为高效的Wnt通路抑制剂应用于未来关于牙周膜干细胞和Wnt通路相关实验研究中。