ADAM10 is essential for cranial neural crest-derived maxillofacial bone development.

Growth disorders of the craniofacial bones may lead to craniofacial deformities. The majority of maxillofacial bones are derived from cranial neural crest cells via intramembranous bone formation. Any interruption of the craniofacial skeleton development process might lead to craniofacial malformation. A disintegrin and metalloprotease (ADAM)10 plays an essential role in organ development and tissue integrity in different organs. However, little is known about its function in craniofacial bone formation. Therefore, we investigated the role of ADAM10 in the developing craniofacial skeleton, particularly during typical mandibular bone development. First, we showed that ADAM10 was expressed in a specific area of the craniofacial bone and that the expression pattern dynamically changed during normal mouse craniofacial development. Then, we crossed wnt1-cre transgenic mice with adam10-flox mice to generate ADAM10 conditional knockout mice. The stereomicroscopic, radiographic, and von Kossa staining results showed that conditional knockout of ADAM10 in cranial neural crest cells led to embryonic death, craniofacial dysmorphia and bone defects. Furthermore, we demonstrated that impaired mineralization could be triggered by decreased osteoblast differentiation, increased cell death. Overall, these findings show that ADAM10 plays an essential role in craniofacial bone development.

Chemokine CCL2 up-regulated in the medullary dorsal horn astrocytes contributes to nocifensive behaviors induced by experimental tooth movement.

To test the hypothesis that the astrocytic chemokine (C-C motif) ligand 2 (CCL2) plays an important role in nocifensive behaviors after experimental tooth movement (ETM), the expression and cellular localization of CCL2 and astrocyte activation in the medullary dorsal horn (MDH) were determined by immunohistochemistry in rats. The dose-dependent effects of intrathecal C-C chemokine receptor type 2 (CCR2) antagonists on these changes in nocifensive behaviors were evaluated. Exogenous CCL2 was added to medullary dorsal horn slices to evaluate its contributory role in the induction of extracellular signal-regulated kinase (ERK) activation ex vivo. We found a significant increase in the expression of CCL2 and glial fibrillary acidic protein (GFAP), corresponding well to the nocifensive behaviors after ETM. In addition, application of recombinant CCL2 led to ERK activation, which could be attenuated effectively by pretreatment with CCL2-neutralizing antibody ex vivo. The magnitude of the nocifensive behavior could be reduced by medullary CCR2 antagonists in a dose-dependent manner. Therefore, the astrocytic CCL2 is actively involved in the development and maintenance of tooth-movement pain and thus may be a potential target for analgesics in orthodontic nocifensive responses control.

Effect of mechanical stretch on the proliferation and differentiation of BMSCs from ovariectomized rats.

Osteoporosis is characterized by a broken balance between bone formation and bone resorption. Mechanical stress has been considered to be an important factor in bone modeling and remodeling. However, biological responses of stromal cells in osteoporosis to mechanical stimuli remain unknown. To explore the correlation between mechanical stress and osteoblastic differentiation of bone mesenchymal stem cells (BMSCs) in osteoporosis, we built an osteoporosis model in ovariectomized (OVX) rats, and then investigated proliferation, alkaline phosphatase (ALP) activity, and the expression of osteoblastic genes in BMSCs under mechanical stress of 5 and 10% elongation, using the Flexercell Strain system. The proliferation of BMSCs was detected using alamarBlue. The expression of osteoblastic genes was analyzed by real-time quantitative polymerase chain reaction. Protein expression was examined by Western blotting. BMSCs (OVX) and BMSCs (Sham-operated, Sham in short) proliferations were inhibited at 5 and 10% elongation at day 3, compared with the un-stretched group, while BMSCs (OVX) proliferation was slower than BMSCs (Sham). ALP activity increased significantly at 10% elongation in both cells, but it was less active in BMSCs (OVX) than BMSCs (Sham). At days 3 and 7, the mRNA expression of osteoblastic genes was unregulated by mechanical stretch (5 and 10 % elongation); however, osteoblastic gene expression in BMSCs (OVX) was less than that in BMSCs (Sham). The mRNA and protein expression of Runx2 showed similar trends in BMSCs (OVX) under mechanical stretch. These results indicate that the mechanical stretch stimulates osteoblastic differentiation of BMSCs (OVX); however, this differentiation was weaker than that of BMSCs (Sham).

Leptin-Induced Angiogenesis of EA.Hy926 Endothelial Cells via the Akt and Wnt Signaling Pathways In Vitro and In Vivo.

Angiogenesis involves the activation of endothelial cells followed by capillary formation. Leptin, the protein product of the ob gene, can induce the angiogenic potential of endothelial cells. However, the underlying cellular mechanism still remains to be elicited. We firstly evaluated the in vitro effects of leptin on proliferation and angiogenic differentiation of endothelial cell line EA.hy926. Leptin was found to potently induced cell proliferation, expression of angiogenic gene, migration and tube formation. Then we investigated the roles of the Akt and Wnt signaling pathways in the aforementioned processes. It showed that Akt and Wnt signaling pathways could be activated by leptin, while inhibition of the Akt and Wnt signaling pathways by siRNAs effectively blocked the leptin-induced angiogenesis. Finally, we used electrospinning to fabricated leptin-immobilized linear poly(L-lactide-co-caprolactone) (PLCL)-leptin. The in vivo vessel formation of PLCL-leptin was evaluated using subcutaneous implants in Sprague-Dawley rats. The histological and immunofluorescence revealed that cell infiltration with PLCL-leptin was much more significant than that with the control PLCL group. More importantly, the number of laminin+ vessels and CD31+ cells in PLCL-leptin grafts was significantly higher than in control grafts. The study demonstrated that it is via Akt and Wnt signaling pathways that leptin promotes the proliferation and angiogenic differentiation of endothelial cells and the capacity of endogenous tissue regeneration makes the novel leptin-conjugated PLCL promising materials for grafts.

The Effects of Leptin on the Proliferation and Differentiation of Primary Chondrocytes in Vitro and Cartilage Regeneration in Vivo.

Patients with cartilage damage have various discomforts, including pain, clicks, deformities, and dysfunction. Chondrocytes are a crucial component of cartilage restoration; however, their limited proliferative ability and degenerative specificity dramatically reduce their effectiveness. In the present study, the effects of leptin on chondrocyte proliferation, chondrogenic and secretion marker gene expression, and chondrocyte cartilage matrix component secretion were evaluated in vitro. The roles of the mitogen-activated protein kinase (MAPK) and protein kinase B (AKT) signaling pathways in these processes were also investigated. More importantly, a leptin sustained release system was developed using a hydrogel with calcium alginate microspheres and was transplanted into cartilage defects in rabbit femurs to analyze the effect of leptin on promoting cartilage restoration. The results showed that leptin promoted cell proliferation and chondrocyte gene expression in a dose-dependent manner, and a concentration of 100 ng/mL leptin had the greatest effect. The activation of the P38 and AKT signaling pathways might be responsible for these effects. An improved in vivo restoration outcome was observed in the leptin sustained release group compared with the control group. These results suggest that leptin could be used as a suitable drug for cartilage restoration.

Lithium-containing biomaterials stimulate bone marrow stromal cell-derived exosomal miR-130a secretion to promote angiogenesis.

The chemical signals of biomaterials could influence bone marrow stromal cells (BMSCs)-endothelial cells (ECs) communication during vascularized bone regeneration. However, the underlying mechanisms still remain unknown. Exosomes, a series of extracellular vesicles, have recently emerged as potential paracrine mediators in cell-cell communication. However, whether exosomes and exosomal microRNAs (miRNAs) are involved in the chemical signals of biomaterials-modulated BMSCs-ECs communication are unknown. Hence, in the present study, a model Li-incorporated bioactive glass ceramic (Li-BGC) was applied to explore the chemical signals of biomaterials mediated cell-cell communication between BMSCs and ECs. Our results showed that Li-BGC directly promoted the pro-angiogenic capability of HUVECs in vitro and new blood vessel ingrowth in vivo. Moreover, Li-BGC activated Wnt/ß-catenin, AKT and NF-κB signaling pathways, while AKT signaling pathway might function as the upstream of Wnt/ß-catenin and NF-κB signaling pathways. More importantly, Li-BGC further facilitated the pro-angiogenic capacity of HUVECs by eliciting the expression of exosomal pro-angiogenic miR-130a in BMSCs-derived exosomes, which subsequently leading to the downregulation of PTEN protein and activation of AKT pathway, ultimately resulting in the elevated proliferation, migration and tube formation of endothelial cells, as well as the upregulated expression of pro-angiogenic genes. Our findings may provide new insights into the regulatory roles of the chemical signals of biomaterials in BMSCs-ECs communication via stimulating exosomal miR-130a secretion and PTEN/AKT signaling pathway in the angiogenic process of bone remodelling.

Mechanical strain promotes osteogenic differentiation of bone mesenchymal stem cells from ovariectomized rats via the phosphoinositide 3­kinase/Akt signaling pathway.

Osteoporosis has become an overwhelming public health problem worldwide. As an elementary physiological factor to regulate bone formation and regeneration, mechanical strain may be used as a non­invasive intervention in osteoporosis prevention and treatment. However, little is known regarding the underlying mechanism. The aim of the current study was to investigate the effect of continuous mechanical strain (CMS) on osteogenic differentiation of bone mesenchymal stem cells (BMSCs) from ovariectomized rats (OVX BMSCs). In addition, involvement of the phosphatidylinositol 3­kinase (PI3K)/Akt signaling pathway in biomechanical signal transduction and its function were evaluated. The results demonstrated that OVX BMSCs subjected to CMS exhibited higher alkaline phosphatase (ALP) activity and deeper staining at 24 and 48 h. In addition, CMS upregulated the mRNA expression levels of ALP, collagen type I, runt related transcription factor 2 (Runx2), as well as the protein expression level of Runx2 in a time­dependent manner. The PI3K/Akt signaling pathway was rapidly activated by CMS, with its phosphorylation level reaching its maximum in a short duration and a large quantity of phosphorylated­Akt remaining in the nucleus. Pre­treatment with a selective blocker significantly blocked the strain­induced activation of PI3K/Akt and reduced the commitment of OVX BMSCs into osteoblasts, demonstrating a dominated regulative effect of PI3K/Akt signaling in strain­induced osteogenesis. These results indicated that CMS induced the early differentiation of OVX BMSCs towards an osteogenic phenotype by activating the PI3K/Akt signaling pathway.

ADAM10 modulates SOX9 expression via N1ICD during chondrogenesis at the cranial base.

The cranial base is the foundation of the craniofacial structure, and any interruption of the cranial base can lead to facial deformity. The cranial base develops from two synchondroses via endochondral ossification. Chondrogenesis is an important step in endochondral ossification. A disintegrin and metalloprotease (ADAM) 10 participates in the Notch1 signalling pathway, which has been reported to regulate chondrogenesis via a SOX9-dependent mechanism. However, little is known about the function of ADAM10 in chondrogenesis. In this study, adam10-conditional-knockout (cKO) mice exhibited sharper naso-labial angles and flatter skulls than wild-type (WT) mice. In the sagittal plane, SOX9 was more widespread in the cranial base in Adam10-cKO mice than in WT mice. For in vitro experiments, we used the ATDC5 cell line as a model to investigate the role of ADAM10 in chondrogenesis. Plasmid 129 was designed to decrease the expression of Adam10; the resulting downregulation of Adam10 reduced the production of N1ICD. Plasmid 129 increased the expression of SOX9 under chondrogenic induction, and this increase could be inhibited by transfection with exogenous N1ICD. Collectively, these results show that ADAM10 participates in chondrogenesis by negatively regulating SOX9 expression in an N1ICD-dependent manner during cranial base development.

[CCL2/CCR2 signaling activation contributes to tooth movement pain].

OBJECTIVE: To test the hypothesis that the CCL2/CCR2 signaling pathway plays an important role in pain induced by experimental tooth movement. METHODS: Male Sprague- Dawley rats weighing between 200 and 300 g were used in this study. Expression of CCL2/CCR2 in the trigeminal ganglion (TG) was determined by Western blotting 0 h, 4 h, 1 d, 3 d, 5 d, 7 d after tooth movement. Localization of the CCL2 was revealed by immunohistochemistry. Changes in body weight, nocifensive behaviors, and the effects of CCL2/CCR2 antagonists on these changes in pain behaviors were evaluated. Exogenous CCL2 was injected into periodontal tissues and added to TG neurons in culture and the resulting c-fos expression and pain responses were detected. In addition, the expression and cellular localization of CCL2 in the medullary dorsal horn (MDH) was determined by immunohistochemistry 3 d and 14 d after tooth movement. RESULTS: Experimental tooth movement led to a statistically significant increase in CCL2/CCR2 expression at the protein level from day 3 to 7 after application of force initiating tooth movement.When compared with control group (1.000 ± 0.000), CCL2 increased to (2.620 ± 0.128), (3.300 ± 0.197) and (1.740 ± 1.290) at day 3, 5 and 7 respectively, which were statistically significant (P < 0.05). CCR2 expression levels were (1.636 ± 0.061) and (1.766 ± 0.126) compared with that in control group (1.000 ± 0.000) at day 3 and 5 respectively with statistical significance (P < 0.05). Both of them peaked on day 5 (3.3 and 1.8 time compared to control group). Application of recombinant CCL2 led to the up-regulation of c-fos expression in vivo and in vitro, and triggered a corresponding nocifensive behavior in rats. The magnitude of the nocifensive behavior could be reduced by a CCR2 antagonist, and by CCL2 neutralizing antibody. Furthermore, we found a significant increase in the expression of CCL2, corresponding well to the up-regulation of the time spent on nocifensive behaviors after ETM. In addition, CCL2 was up-regulated in TG neurons and astrocytes in Vc. CONCLUSIONS: The CCL2/CCR2 axis was modulated by experimental tooth movement and involved in the development of tooth movement pain, and thus palyed an important role in orthodontic pain mechanism.

Chemokine ligand 2 in the trigeminal ganglion regulates pain induced by experimental tooth movement.

OBJECTIVE: To test the hypothesis that the chemokine ligand 2/chemokine receptor 2 (CCL2/CCR2) signaling pathway plays an important role in pain induced by experimental tooth movement. MATERIALS AND METHODS: Expression of CCL2/CCR2 in the trigeminal ganglion (TG) was determined by Western blotting 0 hours, 4 hours, 1 day, 3 days, 5 days, and 7 days after tooth movement. CCL2 localization and cell size distribution were revealed by immunohistochemistry. The effects of increasing force on CCL2 expression and behavioral changes were investigated. Furthermore, the effects of CCL2/CCR2 antagonists on these changes in pain behaviors were all evaluated. Exogenous CCL2 was injected into periodontal tissues and cultured TG neurons with different concentrations, and then the pain responses or c-fos expression were assessed. RESULTS: Experimental tooth movement led to a statistically significant increase in CCL2/CCR2 expression from day 3 to day 7, especially in small to medium-sized TG neurons. It also triggered an increase in the time spent on directed face-grooming behaviors in a force magnitude-dependent and CCL2 dose-dependent manner. Pain induced by experimental tooth movement was effectively blocked by a CCR2 antagonist and by CCL2 neutralizing antibody. Also, exogenous CCL2 led to an increase in c-fos expression in cultured TG neurons, which was blocked by CCL2 neutralizing antibody. CONCLUSIONS: The peripheral CCL2/CCR2 axis is modulated by experimental tooth movement and involved in the development of tooth movement pain.

[Spatiotemporal expression of ADAM10 during cranial base suture development].

PURPOSE: To explore the expression of a disintegrin and metalloproteinase 10 (ADAM10) during mouse's development of cranial base synchondrosis. METHODS: Histological and biochemical method including alcian blue-alizarin red staining, IHC, ICC and Western blot were applied to observe and analyze the expression of ADAM10 in mouse cranial base. RESULTS: The spheno-occipital synchondrosis and spheno-ethmoid synchondrosis were of similar histological structure, including resting zone, proliferative zone and hypertrophic zone. ADAM10 was expressed widely in synchondrosis,especially in the hypertrophic zone. During development of cranial base, ADAM10 was highly expressed from embryo period to infantile period, while there was a huge decrease when it came to adulthood. As an excellent cell line to mimic chondrogenesis, ATDC5 expressed ADAM10 in cytoplasm, particularly around the nucleus. CONCLUSIONS: The expression of ADAM10 during mouse's development of cranial base synchondrosis is spatiotemprally different. Supported by National Natural Science Foundation of China (10972142), National Science Foundation for Distinguished Young Scholars of China (81000451),Science Foundation for Young Scholars of Shanghai Municipal Public Health Bureau (2010y142), Foundation for Distinguished Youth Teachers of Shanghai Municipal Commission of Education [(2010)83], Key Research Project of Science and Technology Committee of Shanghai Municipality (12JC1405700) and Innovation Team Project of Shanghai Municipal Commission of Education.

Osteoclastogenesis accompanying early osteoblastic differentiation of BMSCs promoted by mechanical stretch.

Mechanical stress plays a crucial role in bone formation and absorption. In previous studies, we verified the osteoblastogenesis of bone mesenchymal stem cells (BMSCs) affected by intermittent traction stretch. However, little is known about the osteoclastogenesis process under mechanical stimulation and its underlying association with osteoblastogenesis. In the present study, we investigated the osteoclastogenesis of BMSCs under this special mechanical stress. BMSCs were subjected to 10% elongation for 1-7 days using a Flexcell Strain Unit and then the mRNA levels of osteoclastic genes were examined. The results indicated time-dependent varying of mRNA levels of the receptor activator of nuclear factor-κB ligand (RANKL) and osteoprotegerin (OPG) in BMSCs at different stretching time points. The ratio of RANKL/OPG increased at the early stage of mechanical stimulation (5 days) and decreased to a low level at a later stage (7 days). Findings of this study may help to understand the correlations between osteoblastogenesis and osteoclasteogenesis when mechanical stretch induces the osteoblastic differentiation of BMSCs.