Wenjia Liu

High levels of β-catenin signaling reduce osteogenic differentiation of stem cells in inflammatory microenvironments through inhibition of the noncanonical Wnt pathway.

Periodontal ligament stem cells (PDLSCs), a new population of mesenchymal stem cells (MSCs), have been isolated from the periodontal ligament (PDL). The capacity of multipotency and self‐renewal makes them an excellent cell source for bone regeneration and repair. However, their bone‐regeneration ability could be awakened in inflammatory microenvironments, which may be the result of changes in their differentiation potential. Recently, genetic evidences has shown that the Wnt pathway plays an important role in bone homeostasis. In this study we have determined the specific role of β‐catenin in osteogenic differentiation of PDLSCs obtained from inflammatory microenvironments (P‐PDLSCs). The inflammatory microenvironment, while inhibiting osteogenic differentiation potential, promotes proliferation of MSCs. A higher the level of β‐catenin in P‐PDLSCs than in H‐PDLSCs (PDLSCs obtained from a healthy microenvironment) resulted in the same disparity in canonical Wnt signaling pathway activation between each cell type. Here we show that activation of β‐catenin suppresses the noncanonical Wnt/Ca2+ pathway, leading to increased proliferation but reduced osteogenic differentiation of P‐PDLSCs. Downregulation of the levels of β‐catenin by treatment with dickkopf‐1 (DKK‐1) leads to activation of the noncanonical Wnt/Ca2+ pathway, which, in turn, results in the promotion of osteogenic differentiation in P‐PDLSCs. Interestingly, β‐catenin can affect both the canonical Wnt/β‐catenin pathway and the noncanonical Wnt/Ca2+ pathway. Our data indicate that β‐catenin plays a central role in regulating osteogenic differentiation of MSCs in inflammatory microenvironments. Given the important role of Wnt signaling in osteogenic differentiation, it is possible that agents that can modify this pathway may be of value in bone regeneration by MSCs in chronic inflammatory microenvironments.

MiR-17 modulates osteogenic differentiation through a coherent feed-forward loop in mesenchymal stem cells isolated from periodontal ligaments of patients with periodontitis.

Chronic inflammatory diseases, such as rheumatoid arthritis and periodontitis, are the most common causes of bone tissue destruction. Recently, human periodontal ligament tissue‐derived mesenchymal stem cells (PDLSCs), a population of multipotent stem cells, have been used to reconstruct tissues destroyed by chronic inflammation. However, the impact of the local inflammatory microenvironment on tissue‐specific stem cells and the mechanisms controlling the effects of the local inflammatory environment remain poorly understood. In this study, we found that the multidifferentiation potential of mesenchymal stem cells (MSCs) isolated from periodontitis‐affected periodontal ligament tissue (P‐PDLSCs) was significantly lower than that of MSCs isolated from healthy human periodontal ligament tissue (H‐PDLSCs). Inflammation in the microenvironment resulted in an inhibition of miR‐17 levels, and a perturbation in the expression of miR‐17 partly reversed the differentiation potential of PDLSCs in this microenvironment. Furthermore, inflammation in the microenvironment promoted the expression of Smad ubiquitin regulatory factor one (Smurf1), an important negative regulator of MSC osteogenic differentiation. Western blotting and 3′ untranslated regions (3′‐UTR) reporter assays confirmed that Smurf1 is a direct target of miR‐17 in PDLSCs. Our data demonstrate that excessive inflammatory cytokine levels, miR‐17, and Smurf1 were all involved in a coherent feed‐forward loop. In this circuit, inflammatory cytokines led to direct activation of Smurf1 and downregulation of miR‐17, thereby increasing degradation of Smurf1‐mediated osteoblast‐specific factors. The elucidation of the molecular mechanisms governing MSC osteogenic differentiation in a chronic inflammatory microenvironment could provide us with a better knowledge of chronic inflammatory disorder and improve stem cell‐mediated inflammatory bone disease therapy. STEM CELLS 2011;29:1804–1816

Integration of a calcined bovine bone and BMSC-sheet 3D scaffold and the promotion of bone regeneration in large defects

Reconstruction of large area bone defect with mechanical integrity to the skeleton is important for patients rehabilitation. However with the limitation of scaffold material and suitable seed cell sources, the best treating strategy remains to be identified though various tissue engineering methods were reported. In this study, we investigated the feasibility of applying calcined bovine bone (CBB) which was coated by allograft bone marrow mesenchymal stem cells (BMSC)-sheet as a 3D scaffold material in bone repairing tissue engineering. The new scaffold material was implanted into osteoporosis rat cranial bone defects and repairing critical size bone defects (8 mm diameter). Data showed that CBB-BMSC-sheet combination had a stronger potential in osteogenic differentiation and mineralized formation both in vitro and in vivo than CBB-BMSC combination. In in vitro study BMSC-sheet had a more feasible characteristic upon bone repairing including richer ECM, larger mineralized area and stronger ALP activity in addition with a significant higher mRNA expression of osteogenic maker such as BMP-2, b-FGF, Col 1a1, OSX and Runx-2 than the control group. In in vivo study 3D reconstruction of micro CT, HE staining and bone strength results showed that newly formed bone in CBB-BMSC-sheet group was significant higher than that in CBB-BMSC group at 4, 8 and 12 weeks after transplantation in the aspect of area and volume. What was more, results indicated that allograft BMSC-sheet had survivaled in the scaffold material and participated in the newly formed bone which had the same thickness with surrounding autologous bone tissues after transplantation. Results of our study demonstrated that CBB-BMSC-sheet combination was a promising strategy in healing of large area bone defect in osteoporosis.

TCF3, a novel positive regulator of osteogenesis, plays a crucial role in miR-17 modulating the diverse effect of canonical Wnt signaling in different microenvironments

Wnt signaling pathways are a highly conserved pathway, which plays an important role from the embryonic development to bone formation. The effect of Wnt pathway on osteogenesis relies on their cellular environment and the expression of target genes. However, the molecular mechanism of that remains unclear. On the basis of the preliminary results, we observed the contrary effect of canonical Wnt signaling on osteogenic differentiation of periodontal ligament stem cells (PDLSCs) in the different culture environment. Furthermore, we found that the expression level of miR-17 was also varied with the change in the culture environment. Therefore, we hypothesized that miR-17 and canonical Wnt signaling may have potential interactions, particularly the inner regulation relationship in different microenvironments. In this paper, we observed that canonical Wnt signaling promoted osteogenesis of PDLSCs in the fully culture medium, while inhibited it in the osteogenic differentiation medium. Interestingly, alteration in the expression level of endogenous miR-17 could partially reverse the different effect of canonical Wnt signaling. Furthermore, the role of miR-17 was because of its target gene TCF3 (transcription factor 3), a key transcription factor of canonical Wnt pathway. Overexpression of TCF3 attenuated the effect of miR-17 on modulating canonical Wnt signaling. Finally, we elucidated that TCF3 enhanced osteogenesis both in vitro and in vivo. In brief, the different level of miR-17 was the main cause of the different effect of canonical Wnt signaling, and TCF3 was the crucial node of miR-17–canonial Wnt signaling regulation loop. This understanding of microRNAs regulating signaling pathways in different microenvironments may pave the way for fine-tuning the process of osteogenesis in bone-related disorders.

Mechanical stress regulates osteogenic differentiation and RANKL/OPG ratio in periodontal ligament stem cells by the Wnt/β-catenin pathway.

BACKGROUND The balance between osteoblastic and osteoclastic activity is critical in orthodontic tooth movement (OTM). Mesenchymal stem cells (MSCs) play an important role in maintaining bone homeostasis, and periodontal ligament stem cells (PDLSCs) are tissue-specific MSCs in the periodontal ligament. However, whether PDLSCs are required for periodontal tissue remodeling during OTM is not fully understood. METHODS Here, we used PDGFRα and Nestin to trace PDLSCs during OTM in rats. We treat human PDLSCs with 100kpa static pressure for 1h or 12h in vitro, and examined the phenotypic changes and expression of RANKL and OPG in these cells. RESULTS In vivo, we found that positive signals of PDGFRα and Nestin in the PDL gradually increased and then decreased on the pressure side to which pressure was applied. In vitro, the osteogenic differentiation of PDLSCs was significantly increased after force treatment for 1h relative to 12h. In contrast, the expression ratio of RANKL/OPG was reduced at 1h and significantly increased at 12h. Furthermore, we found that the Wnt/β-catenin pathway was dynamically activated in the PDL and in PDLSCs after mechanical stimulation. Importantly, the canonical Wnt pathway inhibitor DKK1 blocked the osteogenesis effect and rescued the ratio of RANKL/OPG in PDLSCs under force treatment for 1h. CONCLUSIONS Our findings reveal that PDLSCs participate in OTM and that the Wnt/β-catenin pathway maintains bone homeostasis during tooth movement by regulating the balance between osteoblastic and osteoclastic activity. GENERAL SIGNIFICANCE We describe a novel potential mechanism related to tooth movement.

Licochalcone A up-regulates of FasL in mesenchymal stem cells to strengthen bone formation and increase bone mass

The role of bone marrow-derived mesenchymal stem cells(BMSCs)in the pathogenesis and therapy of osteoporosis has drawn increasing attention in recent years. In the development of osteoporosis, it has been demonstrated that many changes occurred in the behavior of BMSCs. For example, the biological system of FasL pathways mediated differentiation of ERK and GSK-3β-catenin pathway was damaged. Here we found that 0.35 mg/L Licochalcone A (L-A) had a strong effect in increasing the osteogenic differentiation and mineralization of BMSCs both in vivo and in vitro by up-regulating FasL and further playing a role in regulating the ERK and GSK-3β-catenin systems. It has also demonstrated that the administration of L-A could restore the biological function of the damaged BMSCs differentiation by recovering or protecting bone mass in a disease state through activating the endosteal bone formation and partially inhibiting bone resorption in acute estrogen deficiency model. Results of our study suggested that careful titration of MSC was response to L-A and up-regulated FasL pathways mediating differentiation of ERK and GSK-3β-catenin biological systems under disease state in vivo, restore the impaired function, is one of the ways of L-A relieve or treatment osteoporosis.

Autophagy promotes MSC-mediated vascularization in cutaneous wound healing via regulation of VEGF secretion

Vascularization deficiency caused a lot of diseases, such as diabetes ulcer and myocardial infarction. Mesenchymal stem cells (MSCs), with the self-renewal and multipotent differentiation capacities, have been used for many diseases treatment through regulation microenvironment. Numerous studies reported that MSCs transplantation could largely improve cutaneous wound healing via paracrine secretion of growth factors. However, whether MSCs take part in the angiogenesis process directly remains elusive. Previous study proved that autophagy inhibited immunosuppressive function of MSCs and prevented the degradation of MSCs function in inflammatory and senescent microenvironment. Here, we proved that autophagy determines the therapeutic effect of MSCs in cutaneous wound healing through promoting endothelial cells angiogenesis and demonstrated that the paracrine of vascular endothelial growth factor (VEGF) in MSCs was required in wound site. We further revealed that autophagy enhanced the VEGF secretion from MSCs through ERK phosphorylation directly. Collectively, we put forward that autophagy mediated paracrine of VEGF plays a central role in MSCs cured cutaneous wound healing and may provide a new therapeutic method for angiogenesis-related diseases.

Autophagy controls mesenchymal stem cell properties and senescence during bone aging

Bone marrow‐derived mesenchymal stem cells (BMMSCs) exhibit degenerative changes, including imbalanced differentiation and reduced proliferation during aging, that contribute to age‐related bone loss. We demonstrate here that autophagy is significantly reduced in aged BMMSCs compared with young BMMSCs. The autophagy inhibitor 3‐methyladenine (3‐MA) could turn young BMMSCs into a relatively aged state by reducing their osteogenic differentiation and proliferation capacity and enhancing their adipogenic differentiation capacity. Accordingly, the autophagy activator rapamycin could restore the biological properties of aged BMMSCs by increasing osteogenic differentiation and proliferation capacity and decreasing adipogenic differentiation capacity. Possible underlying mechanisms were explored, and the analysis revealed that autophagy could affect reactive oxygen species and p53 levels, thus regulating biological properties of BMMSCs. In an in vivo study, we found that activation of autophagy restored bone loss in aged mice. In conclusion, our results suggest that autophagy plays a pivotal role in the aging of BMMSCs, and activation of autophagy could partially reverse this aging and may represent a potential therapeutic avenue to clinically treat age‐related bone loss.

Manganese superoxide dismutase is required to maintain osteoclast differentiation and function under static force

Bone homeostasis is maintained by the balance of osteoblasts (OBs) and osteoclasts (OCs). Increased activity of OCs not only contributes to pathological bone resorption, such as osteoporosis and periodontitis, but also is responsible for physiological conditions like orthodontic tooth movement (OTM). However, the detailed mechanism by which orthodontic force promotes the formation of OCs is still poorly understood. In this study, we confirmed that static force promoted the differentiation of human cord monocytes (HMNCs) into OCs depending on loading time and magnitude. Protein expression profiles among HMNCs, HMNCs subjected to static force and mature OCs were established via 2-DE and MALDI-TOF-MS analyses. Total respective protein spot numbers of 549 ± 13, 612 ± 19 and 634 ± 16 were detected in each of the gels by image analysis. The five proteins identified were plasminogen activator inhibitor 2 (PAI-2, Spot 1), peroxiredoxin-6 (PRD-6, Spot 3), manganese superoxide dismutase (SOD2, Spot 6), Rho GDP-dissociation inhibitor 2 (Rho-GDI2, Spot 11) and L-lactate dehydrogenase B chain (L-LDH, Spot 15). More importantly, we revealed that SOD2 was required to maintain monocyte differentiation into functional OCs and may become a potential target for regulating the efficiency of OTM in the future.

Jawbone microenvironment promotes periodontium regeneration by regulating the function of periodontal ligament stem cells

During tooth development, the jawbone interacts with dental germ and provides the development microenvironment. Jawbone-derived mesenchymal stem cells (JBMSCs) maintain this microenvironment for root and periodontium development. However, the effect of the jawbone microenvironment on periodontium tissue regeneration is largely elusive. Our previous study showed that cell aggregates (CAs) of bone marrow mesenchymal stem cells promoted periodontium regeneration on the treated dentin scaffold. Here, we found that JBMSCs enhanced not only the osteogenic differentiation of periodontal ligament stem cells (PDLSCs) but also their adhesion to titanium (Ti) material surface. Importantly, the compound CAs of PDLSCs and JBMSCs regenerated periodontal ligament-like fibers and mineralized matrix on the Ti scaffold surface, both in nude mice ectopic and minipig orthotopic transplantations. Our data revealed that an effective regenerative microenvironment, reconstructed by JBMSCs, promoted periodontium regeneration by regulating PDLSCs function on the Ti material.