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.

Cytochalasin D Promotes Osteogenic Differentiation of MC3T3-E1 Cells via p38-MAPK Signaling Pathway.

BACKGROUND: Bone defect caused by trauma, tumor resection, infection or congenital malformation is a common clinical disease. Bone tissue engineering is regarded as a promising way of bone defect reconstruction. Thus, agents that can promote osteogenesis have received great attention. Cytochalasin D (Cyto D), a metabolite derived from molds, proves to be able to modify actin, reorganize cytoskeleton, and then promote the osteogenic differentiation. OBJECTIVE: The purpose of this study was to explore the effect and mechanism of Cyto D on osteogenic differentiation of mouse pre-osteoblast MC3T3-E1 cells. METHODS: The optimum concentration of Cyto D was explored. The osteogenic differentiation of MC3T3-E1 cells induced by Cyto D was assessed by alkaline phosphatase (ALP) staining, Alizarin Red S (ARS) staining, western blotting and quantitative real-time polymerase chain reaction (RT-qPCR). In addition, a specific pathway inhibitor was utilized to explore whether MAPK pathways were involved in this process. RESULTS: The results showed that the optimized concentration of action was 10-2µg/ml. The expression of Runx2, OCN and OSX was up-regulated by the supplement of Cyto D. ALP activity, calcium deposition, and phosphorylation level of p38 protein were also improved. Inhibition of the pathway significantly reduced the activation of p38, and the expression of osteogenic-related genes. CONCLUSION: Cyto D can promote the osteogenic differentiation of MC3T3 cells via the p38-MAPK signaling pathway, but not the ERK1/2 or JNK, and it is a potential agent to improve the osteogenesis of MC3T3 cells.

Development process of traumatic heterotopic ossification of the temporomandibular joint in mice.

PURPOSE: The exact development process underlying traumatic heterotopic ossification of the temporomandibular joint (THO-TMJ) is largely unclear. In this study, we try to explore the histological development process of THO-TMJ. MATERIALS AND METHODS: Condylar cartilage of one-month-old male mice was partially removed from the left joint with small scissors to induce THO-TMJ. The phenotypes were observed using gross observation, microcomputed tomography (micro-CT) scans and histological examination from one month to six months after surgery. RESULTS: The micro-CT examination results showed that the injured condyle integrated with ectopic bone tissue to form an osteophyte and that the volume and density of the osteophyte grew exponentially with time. Hematoxylin and eosin (H&E), safranin O and fast green staining of the THO-TMJ specimens revealed that the ectopic bone tissue was mainly nonmineralized fibrous tissue 1 month after surgery. This tissue gradually transformed into cartilage 3 months after surgery. Finally, the tissues transformed into mature bone tissue 6 months after surgery. Immunofluorescence staining showed VEGF-α expression in the heterotopic tissue 1 month after surgery, and the expression of Sox9 in the heterotopic tissue was obvious 3 months after surgery. Furthermore, OCN expression was evident in most of the heterotopic tissue 6 months after surgery. The results also showed clear hypoxia-inducible factor 1-alpha (Hif-1α) expression in the injured chondrocytes of the condyle, especially in the articular proliferative zone and fibrocartilaginous zone. CONCLUSIONS: The THO-TMJ imaging characteristics indicated an exponential change with time. Histologically, the development process of THO-TMJ is an endochondral ossification process and includes three stages, fibroproliferative, chondrogenic and osteogenic stage. In addition, Hif-1α, which was expressed in some of the injured chondrocytes, may play an essential role in the initial THO-TMJ.

Effects of a single condylar neck fracture without condylar cartilage injury on traumatic heterotopic ossification around the temporomandibular joint in mice.

OBJECTIVES: In this study, we tried to explore the effects of a single condylar neck fracture without condylar cartilage injury during the pathogenesis process of traumatic heterotopic ossification around the temporomandibular joint (THO-TMJ). STUDY DESIGN: One-month-old C57 BL/6 J male mice were divided into 2 groups. In group 1, condylar cartilage was partially removed in the right joint to induce THO. In group 2, a single fracture on the condylar neck was created using small scissors. The condylar head was repositioned to its original place if any displacement occurred. The phenotypes were observed using gross observation, micro-computed tomography, and histologic examination. RESULTS: The results showed obvious hyperplasia in the right condyle in group 1, with ectopic bones and cartilage in the periarticular region. In group 2, the surface of condyle was smooth, but the size of the right condylar head became smaller. CONCLUSIONS: Taking these findings together, we concluded that it is condylar cartilage injury, and not a single condylar neck fracture without condylar cartilage injury, that contributes to the development of THO-TMJ.

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.

Subcutaneous C Shape Muscular Flap for Correcting the Depression of Alar Base in Affected Side in Patients With Unilateral Complete Cleft Lip/Palate During Primary Surgery.

PURPOSE: The depression of alar base in affected side in patients with unilateral complete cleft lip/palate (CL/P) is one of common clinical features. In this study, the authors try to explore the effect of subcutaneous C shape muscular flap for correcting the depression of alar base in affected side in patients with unilateral complete CL/P during primary surgery. METHODS: A total of 30 patients with unilateral complete CL/P who received primary correction of the lip nose deformity were included in this study. The C flap was used to drop and lengthen the height of upper lip in unaffected side, and the subcutaneous muscular flap was dissected from the C flap and positioned at the alar base in the affected side of upper lip to correct the depression. Then the surgical effect was evaluated based on clinical examination during follow-up. RESULTS: Alar base symmetry was obtained in 26 patients of this case series, and 4 patients showed slight improvement in alar base symmetry. No major complications such as flap necrosis, infection, or hypertrophic scars were observed during surgery and follow-up. No additional incisions and operative time were necessary. CONCLUSION: The subcutaneous C shape muscular flap described in this article could offer enough muscular support and markedly correct the depression of nostril and alar base in affected side in primary lip nose repair with no additional incisions and operative time for patients with unilateral complete CL/P.

[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.

Injured condylar cartilage leads to traumatic temporomandibular joint ankylosis.

PURPOSE: The exact mechanisms of traumatic temporomandibular joint ankylosis (TTMJA) are largely unknown. In this study, we explore the role of injured condylar cartilage in the development of TTMJA. MATERIAL AND METHODS: One-month-old male mice were divided into two groups. In group 1, condylar cartilage was partially removed in the right joint using a small scissors to induce ankylosis. In group 2, condylar cartilage was completely removed in the same right joint using a small scissors to induce ankylosis. The phenotypes were observed using gross observation, micro-computed tomography scans and histological examination. RESULTS: The results revealed a great deal of ectopic cartilage and bone formation in the right periarticular region in all mice in group 1, whereas there was only a small amount of ectopic cartilage present in 26.7% of the mice in group 2. Additionally, there was stronger expression of FGF9 and weaker expression of OPN in the right temporomandibular joint region in group 2 at 7 days after surgery. CONCLUSIONS: These results suggest that the injured cartilage, not the injured bone, plays a crucial role in the development of TTMJA. In addition, it offers a useful TTMJA animal model to study the molecular mechanisms of TTMJA based on the gene manipulation technology, such as gene knock-out and knock-in as well as transgenic or gene mutation.

[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.

[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.

[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.