Neutrophils are involved in early bone formation during midpalatal expansion.

OBJECTIVE: Midpalatal expansion (MPE) is routinely employed to treat transverse maxillary arch deficiency. Neutrophils are indispensable for recruiting bone marrow stromal cells (BMSCs) at the initial stage of bone regeneration. This study aimed to explore whether neutrophils participate in MPE and how they function during bone formation under mechanical stretching. MATERIALS AND METHODS: The presence and phenotype of neutrophils in the midpalatal suture during expansion were detected by flow cytometry and immunofluorescence staining. The possible mechanism of neutrophil recruitment and polarization was explored in vitro by exposing vascular endothelial cells (VECs) to cyclic tensile strain. RESULTS: The number of neutrophils in the distracted suture peaked on Day 3, and N2-type neutrophils significantly increased on Day 5 after force application. The depletion of circulatory neutrophils reduced bone volume by 43.6% after 7-day expansion. The stretched VECs recruited neutrophils via a CXCR2 mechanism in vitro, which then promoted BMSC osteogenic differentiation through the VEGFA/VEGFR2 axis. Consistently, these neutrophils showed higher expression of canonical N2 phenotype genes, including CD206 and Arg1. CONCLUSIONS: These results suggested that neutrophils participated in early bone formation during MPE. Based on these findings, we propose that stretched VECs recruited and polarized neutrophils, which, in turn, induced BMSC osteogenic differentiation.

Matrix mechanics regulate the polarization state of bone marrow-derived neutrophils through the JAK1/STAT3 signaling pathway.

Matrix mechanics regulate essential cell behaviors through mechanotransduction, and as one of its most important elements, substrate stiffness was reported to regulate cell functions such as viability, communication, migration, and differentiation. Neutrophils (Neus) predominate the early inflammatory response and initiate regeneration. The activation of Neus can be regulated by physical cues; however, the functional alterations of Neus by substrate stiffness remain unknown, which is critical in determining the outcomes of engineered tissue mimics. Herein, a three-dimensional (3D) culture system made of hydrogels was developed to explore the effects of varying stiffnesses (1.5, 2.6, and 5.7 kPa) on the states of Neus. Neus showed better cell integrity and viability in the 3D system. Moreover, it was shown that the stiffer matrix tended to induce Neus toward an anti-inflammatory phenotype (N2) with less adhesion molecule expression, less reactive oxygen species (ROS) production, and more anti-inflammatory cytokine secretion. Additionally, the aortic ring assay indicated that Neus cultured in a stiffer matrix significantly increased vascular sprouting. RNA sequencing showed that a stiffer matrix could significantly activate JAK1/STAT3 signaling in Neus and the inhibition of JAK1 ablated the stiffness-dependent increase in the expression of CD182 (an N2 marker). Taken together, these results demonstrate that a stiffer matrix promotes Neus to shift to the N2 phenotype, which was regulated by JAK1/STAT3 pathway. This study lays the groundwork for further research on fabricating engineered tissue mimics, which may provide more treatment options for ischemic diseases and bone defects. STATEMENT OF SIGNIFICANCE.

[The effect of Foxc2 overexpression on the osteogenic properties of C3H10T1/2 cells].

PURPOSE: To investigate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation of C3H10T1/2 cells. METHODS: C3H10T1/2 cells were transfected with plenti-Foxc2 and selected with puromycin for stable clones. The expression of Foxc2 was determined by real-time PCR and Western blot. Cell proliferation was detected by CCK-8 kit. Cell cycle and apoptosis were detected by flow cytometry. The level of osteogenic biomarkers Runx2, OPN, OCN and adipogenic biomarker PPARγ were quantified by real-time PCR and Western blot. Alkaline phosphatase (ALP) staining and oil red staining were conducted to evaluate the effect of Foxc2 overexpression on osteogenic and adipogenic differentiation. Statistical analysis was performed using SPSS 17.0 software package. RESULTS: C3H10T1/2-Foxc2 cell line was successfully constructed and verified by direct sequencing and Foxc2 overexpression in vitro. Cell proliferation was reduced and cell cycle was blocked in G1/G0 phase. Enhanced ALP staining and reduced oil red staining were observed in C3H10T1/2-Foxc2 cells as compared with the control. Foxc2 overexpression up-regulated Runx2, OPN, OCN during osteogenic differentiation and down-regulated PPARγduring adipogenic differentiation. CONCLUSIONS: C3H10T1/2 cell line stably expressing Foxc2 gene was successfully established, cell proliferation was reduced, osteogenesis biomarkers were up-regulated during the osteogenesis by overexpression Foxc2, PPARγwas down-regulated during adipogenesis.

[The effect of estrogen on adipogenic differentiation of rat bone mesenchymal stem cells].

PURPOSE: In this study, 10⁻9 mol/L 17 ß-estradiol (E2) was applied in the adipogenic differentiation of rat bone mesenchymal stem cells (rBMSCs) and the effect of E2 was explored. METHODS: Rat BMSCs were obtained from the femurs and tibias of SD rats. 10⁻9 mol/L E2 was involved in the adipogenic differentiation of rBMSCs. Oil red staining, real time PCR and Western blot were carried out to detect the effect of 10⁻9 mol/L E2 on adipogenic differentiation of rBMSCs. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The use of 10⁻9 mol/L E2 decreased the amount of lipid droplets in rBMSCs and weakened the expression of adipogenic related genes and proteins like C/EBP α, C/EBP ß, PPAR γ, aP2, and ARDP, which were significantly lower than the adipogenic induced group. CONCLUSIONS: The use of 10⁻9 mol/L E2 inhibited adipogenic differentiation of rBMSCs significantly in vitro.

[The effect of estrogen on proliferation and osteogenic differentiation of rat bone marrow mesenchymal stem cells].

PURPOSE: Different concentrations of 17 ß-estradiol (E2) were applied in the osteogenic differentiation of rat bone mesenchymal stem cells (BMSCs), and the proliferation and apoptosis of BMSCs were explored. METHODS: BMSCs were obtained from the femurs and tibias of SD rats. The proliferation curve was conducted to rBMSCs in culture medium containing 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol by CCK-8 for 7 days. Annexin V and PI for flow cytometry were applied to detect the impact of E2 on apoptosis of rBMSCs. After 1, 7, 11 and 14 days of osteogenic induction, the activity of alkaline phosphatase (ALP) was assayed; ALP staining was performed on day 7 and day 14; Alizarin red staining for calcium deposits was carried out on day 21. Concentrations of 0, 10(-9), and 10(-7) mol/L 17 ß-estradiol were administrated to rBMSCs for real-time PCR of osteogenic related genes on day 1, 3, 5, 7, 14, and day 21. The data was statistically analyzed using SPSS 19.0 software package. RESULTS: The effect of 17 ß-estradiol on proliferation and apoptosis of rBMSCs was not obvious. However, after osteogenic induction, the ALP activity and Alizarin red staining were significantly stronger in the groups containing 17 ß-estradiol. Especially, the use of 17 ß-estradiol with the concentration of 10(-9) mol/L enhanced the expression of osteogenic related genes like RUNX2, ALP, COL I, and OCN, which was significantly higher than other groups. CONCLUSIONS: 17 ß-estradiol promotes osteogenic differentiation of BMSCs in a dose-dependent pattern in vitro.

[Changes of the microarchitecture of alveolar bone due to different duration of ovariectomy: a Micro-CT study in rats].

PURPOSE: To study the changes of microarchitecture of alveolar bone due to different duration of ovariectomy in rats. METHODS: Twenty-four virgin Sprague-Dawley rats were randomly assigned to ovariectomy group (OVX) or sham-ovariectomy group (sham). OVX rats were subjected to bilateral ovariectomy and sham-ovariectomy was conducted in sham rats. Six rats of each group were sacrificed respectively 3 months and 6 months after surgery. The right semi-maxilla of all rats were scanned by Micro-CT, and the inter-radicular alveolar bone of the maxillary first molar was analyzed. Statistical analysis was carried out with SPSS 16.0 software package. RESULTS: Two-dimensional and three-dimensional reconstructed images of the alveolar bone showed porotic changes in rats both 3 months and 6 months after ovariectomy, including thinner, looser trabeculae and expanded bone marrow. When compared with corresponding sham rats, BMD, BV/TV and Tb.Th of alveolar bone significantly decreased in OVX rats both 3 months and 6 months after ovariectomy (P<0.05). Tb.N and Tb.Sp significantly increased in both OVX groups (P<0.05). When compared with 3 months after ovariectomy, the rats 6 months after ovaroectomy shared deceased BMD, BV/TV and Tb.Th (P<0.05) and increased Tb.N (P<0.05). CONCLUSIONS: Bone loss and deterioration of trabeculae of alveolar bone aggravates with the extended duration of ovariectomy in OVX rats.