Xingmei Feng

TNF-α triggers osteogenic differentiation of human dental pulp stem cells via the NF-κB signalling pathway.

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells (MSCs) characterised by self‐renewal and multi‐lineage differentiation, including chondrocytes, adipocytes, neural cells and osteoblasts, which make it an attractive choice for tissue engineering purposes. Tumour necrosis factor α (TNF‐α) had the positive effect on the mineralisation of bone marrow MSCs and stromal cells derived from human adipose tissue. However, the effect of TNF‐α on DPSCs is unclear. We found that TNF‐α activated the NF‐κB pathway during the osteogenic differentiation of DPSCs. TNF‐α also increased mineralisation and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP), runt‐related transcription factor 2 (RUNX2) and collagen type I (COL I) during this process. PDTC, an NF‐κB inhibitor, blocked the osteogenic differentiation induced by TNF‐α. No effect of TNF‐α on proliferation of DPSCs or cell cycle was detected. In summary, TNF‐α promotes mineralisation and mineralisation‐related gene expression through the NF‐κB signalling pathway in DPSCs, which may provide a foundation for autologous transplantation of DPSCs.

Insulin‐like growth factor 1 can promote proliferation and osteogenic differentiation of human dental pulp stem cells via mTOR pathway

Insulin‐like growth factor 1 (IGF‐1) is a multifunctional peptide that can enhance osteogenic differentiation of bone marrow mesenchymal stem cells (BMMSCs). However, it remains unclear whether IGF‐1 can promote osteogenic differentiation of human dental pulp stem cells (DPSCs). In our study, DPSCs were isolated from the impacted third molars, and treated with IGF‐1. Osteogenic differentiation abilities were investigated. We found that IGF‐1 activated the mTOR signaling pathway during osteogenic differentiation of DPSCs. IGF‐1 also increased the expression of runt‐related transcription factor 2 (RUNX2), osteocalcin (OCN), osterix (OSX) and collagen type I (COL I) during this process. Rapamycin, an mTOR inhibitor, blocked osteogenic differentiation induced by IGF‐1. Meanwhile, CCK‐8 assay and flow cytometry results demonstrated that 10–200 ng/mL IGF‐1 could enhance proliferation ability of DPSCs and 100 ng/mL was the optimal concentration. In summary, IGF‐1 could promote proliferation and osteogenic differentiation of DPSCs via mTOR pathways, which might have clinical implications for osteoporosis.

Repeated lipopolysaccharide stimulation promotes cellular senescence in human dental pulp stem cells (DPSCs)

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cell (MSC) characterized by multi-lineage differentiation making it an attractive choice for tissue regeneration. However, before DPSCs can be used for cell-based therapy, we have to understand their biological properties in response to intrinsic and extrinsic stimuli such as lipopolysaccharide (LPS). DPSCs were therefore stimulated with LPS and senescence was evaluated by senescence-associated β-galactosidase (SA-β-gal) staining, with cell number and cell-cycle arrest being examined by BrdU assay and flow cytometry, respectively. The morphology of DPSCs was characterized by their flat shape, increased size and increased SA-β-gal activity after repeated stimulation (3 or 6 times) with LPS. Reactive oxygen species (ROS) staining showed that the number of ROS-stained cells and the DCFH fluorescent level were higher in the LPS-treated DPSCs compared with those in the untreated DPSCs. Protein and mRNA expression levels of γ-H2A.X and p16INK4A were also increased in DPSCs with repeated LPS stimulation. We found that the LPS bound with Toll-like receptor 4 (TLR4) and that TLR4 signaling accounted for p16INK4A expression. Further results indicated that the senescence of DPSCs stimulated repeatedly with LPS was reversed by p16INK4A short interfering RNA. The DNA damage response and p16INK4A pathways might be the main mediators of DPSC senescence induced by repeated LPS stimulation. Thus, DPSCs tend to undergo senescence after repeated activation, implying that DPSC senescence starts after many inflammatory challenges. Ultimately, these findings should lead to a better understanding of DPSC-based clinical therapy.

p16INK4A mediates age-related changes in mesenchymal stem cells derived from human dental pulp through the DNA damage and stress response

Mesenchymal stem cells derived from human dental pulp (DP-MSCs) are characterized by self-renewal and multi-lineage differentiation, which play important roles in regenerative medicine. Autologous transfers, as non-immunogenic, constitute the safest approach in cellular transplantations. However, their use may be limited by age-related changes. In the study, we compared DP-MSCs isolated from human in five age groups: 5-12 y, 12-20 y, 20-35 y, 35-50 y, and >50 y. We tested the effect of age on proliferation, differentiation, senescence-associated β-galactosidase (SA-β-gal), cell cycle and programmed cell death. DP-MSCs showed characteristics of senescence as a function of age. Meanwhile, the expression of p16(INK4A) and γ-H2A.X significantly increased with age, whereas heat shock protein 60 (HSP60) was decreased in the senescent DP-MSCs. Reactive oxygen species (ROS) staining showed the number of ROS-stained cells and the DCFH fluorescent level were higher in the aged group. Further we examined the senescence of DP-MSCs after modulating p16(INK4A) signaling. The results indicated the dysfunction of DP-MSCs was reversed by p16(INK4A) siRNA. In summary, our study indicated p16(INK4A) pathway may play a critical role in DP-MSCs age-related changes and the DNA damage response (DDR) and stress response may be the main mediators of DP-MSCs senescence induced by excessive activation of p16(INK4A) signaling.

JAB1 accelerates odontogenic differentiation of dental pulp stem cells

Jun activation domain-binding protein 1 (JAB1) is a multifunctional protein that participates in the control of cell proliferation and the stability of multiple proteins. JAB1 regulates several key proteins, and thereby produces varied effects on cell cycle progression, genome stability and cell survival. Some studies have shown that the loss of JAB1 in osteochondral progenitor cells severely impairs embryonic limb development in mice. However, the biological significance of JAB1 activity in the odontogenic differentiation of dental pulp stem cells (DPSCs) remains unclear. This study aimed to determine the role of JAB1, a key player in tooth development, in reparative dentin formation, especially odontogenic differentiation. We found that increased expression of JAB1 promoted odontogenic differentiation of DPSCs via Wnt/β-catenin signaling. The role of JAB1 in the odontogenic differentiation of DPSCs was further confirmed by knocking down JAB1. Our findings provide novel insights on odontogenic differentiation of DPSCs.

AGS3 is involved in TNF-α medicated osteogenic differentiation of human dental pulp stem cells.

Dental pulp stem cells (DPSCs) are multipotent adult stem cells capable of differentiating along the osteoblast, adipocyte, and chondrocyte lineages. Regulating differentiation of DPSCs may be a useful tool for regenerative medicine and cell-based therapy in oral diseases. Multisignaling pathways are involved in osteogenic differentiation of DPSCs. Recent studies show that cAMP/PKA/CREB signaling could stimulate the expression of genes such as bone morphogenic proteins 2 (BMP2), inhibitor of DNA binding 2 (ID2), bone sialoprotein, osteocalcin, and type XXIV collagen, which have been implicated in osteogenesis and bone formation. Activator of G-protein signaling 3 (AGS3, gene name G-protein signaling modulator-1, Gpsm1), an accessory protein for G-protein signaling, plays an important role in regulating the phosphorylation of cyclic AMP response element-binding protein (p-CREB). However, the involvement of AGS3 in osteogenic differentiation of DPSCs has not been explored. Our data indicated that increased expression of AGS3 would inhibit osteogenic differentiation of DPSCs exposed to inflammatory cytokine tumor necrosis factor α (TNF-α) via cAMP/PKA/CREB signaling. The negative role of AGS3 in osteogenic differentiation was further confirmed by knocking down and over expression of AGS3. Our findings may provide clinical implications for osteoporosis.

Runx2 modified dental pulp stem cells (DPSCs) enhance new bone formation during rapid distraction osteogenesis (DO).

Distraction osteogenesis (DO) remains a major challenge in orthopedic and craniofacial surgery. The transplantion of mesenchymal stem cells (MSCs) could reduce the treatment period and the associated complications by increasing new bone formation during long-bone DO. Runt-related transcription factor 2 (Runx2) encodes a nuclear protein that is a pivotal regulator of osteoblast differentiation. It significantly stimulates calcium accumulation and alkaline phosphatase (ALP) activity in dental pulp stem cells (DPSCs). In this study, we investigated the effects of gene therapy using Runx2 on new bone formation during tibia DO of rabbits. The distraction gap of the rabbits was injected with adenovirus (Adv)-Runx2-green fluorescent protein (GFP)-transfected DPSCs (overexpression group, Group OE) or Adv-GFP-transfected DPSCs (negative control group, Group NC). Rabbits in the control group (Groups CON) were injected with physiologic saline. The generation of new bone tissue in the distraction gap was studied by radiographic examination, micro-computed tomography (CT) evaluation, histological analyze, and Mechanical testing at weeks 8 in the consolidation period. Excellent bone formation in the distracted callus was observed in Group OE and Group NC. Moreover, the OE group showed better bone formation and the highest bone mineral density (BMD) and bone mineral content (BMC). Group CON animals showed inadequate bone formation in the distracted callus compared to the other groups. The results suggest that gene therapy using Runx2-modified DPSCs was more effective during bone deposition and new bone formation in tibia DO.

RAC1 regulate tumor necrosis factor-α-mediated impaired osteogenic differentiation of dental pulp stem cells.

Human dental pulp contains a rapidly proliferative subpopulation of precursor cells termed dental pulp stem cells (DPSCs) that show self‐renewal and multilineage differentiation, including neurogenic, chondrogenic, osteogenic and adipogenic. We previously reported that tomuor necrosis factor‐α (TNF‐α) (10 ng/mL) triggered osteogenic differentiation of human DPSCs via the nuclear factor‐κB (NF‐κB) signaling pathway. While previous studies showed that cells treated with TNF‐α at higher concentrations showed decreased osteogenic differentiation capability. In this study we analyze the function of TNF‐α (100 ng/mL) on osteogenic differentiation of human DPSCs for the first time and identify the underlying molecule mechanisms. Our data revealed that TNF‐α with higher concentration significantly reduced mineralization and the expression of bone morphogenetic protein 2 (BMP2), alkaline phosphatase (ALP) and runt‐related transcription factor 2 (RUNX2). Further, we revealed that TNF‐α could suppress the osteogenic differentiation of DPSCs via increasing the expression of RAC1, which could activate the Wnt/β‐catenin signaling pathway and liberate β‐catenin to translocate into the nucleus. Genetic silencing of RAC1 expression using siRNA restored osteogenic differentiation of DPSCs. Our findings may provide a potential approach to bone regeneration in inflammatory microenvironments.

SIRT1 was involved in TNF-α-promoted osteogenic differentiation of human DPSCs through Wnt/β-catenin signal

Dental pulp stem cells (DPSCs), as one type of mesenchymal stem cells (MSCs), have the capability of self-renewal and differentiating along the various directions, including osteogenic, chondrogenic, neurogenic, and adipogenic. We previously study and found that tumor necrosis factor-α (TNF-α) promoted osteogenic differentiation of human DPSCs via the Wnt/β-catenin signaling pathway in low concentration while inhibited that in high concentration. In the abovementioned process, we found that sirtuin-1 (SIRT1) had the same change compared with the characteristic protein of bone formation, such as bone morphogenetic protein 2 (BMP2), runt-related transcription factor 2 (Runx2), and collagen I (COL1). We asked whether SIRT1 could regulate osteogenesis of DPSCs. In inflammation microenvironment constructed by TNF-α, we tested the expression changing of SIRT1 and analyzed the function of SIRT1 on osteogenic differentiation of DPSCs. SIRT1 deacetylated β-catenin, and then promote its accumulation in the nucleus. Accumulated β-catenin can lead to transcription of osteogenic characteristic genes. Using the activator of SIRT1, resveratrol, could promote the above-mentioned process of osteogenic differentiation. SIRT1 could regulate osteogenesis of DPSCs through Wnt/β-catenin signal. SIRT1, as a regulator of differentiation of DPSCs, may be a new target for cell-based therapy in oral diseases and other regenerative medicine.

The role of oncostatin M regulates osteoblastic differentiation of dental pulp stem cells through STAT3 pathway

Dental pulp stem cells (DPSCs) are a type of mesenchymal stem cells, which have the self-renewal and multi-lineage differentiation potential, including chondrocytes, adipocytes, neural cells and osteoblasts. So they play a significant role in pulp repair and bone regeneration. Oncostatin M (OSM), one of the IL-6 family cytokines, inhibits adipogenic differentiation and stimulates osteogenic differentiation of human bone marrow mesenchymal stem cells. However, the effect of OSM on DPSCs is unclear. We found that OSM induced osteogenic differentiation of DPSCs, promoting matrix mineralization as measured by Alizarin Red S staining. OSM also increased expression of osteogenesis-associated gene products Alkaline phosphatase, Bone morphogenetic protein 2 (BMP2), Runt-related transcription factor 2 and Osteocalcin (OCN) as assessed by immunoblotting. We also found that OSM activated the Signal Transducer And Activator Of Transcription 3 (STAT3) pathway during the osteogenic differentiation of DPSCs. Blocking the osteogenic differentiation by silencing of STAT3 can significantly inhibit OSM-induced osteogenic differentiation of DPSCs and the expression of related genes, furthermore matrix mineralization was also suppressed. In summary, OSM promotes osteoblastic differentiation of DPSCs and osteogenesis-related genes expression through the JAK3/STAT3 signaling pathway which may be useful for the autologous transplantation of DPSCs.