Effect of Mdivi-1 treatment on the inflammatory response induced by TNF-α.

<p>The cells were treated with 10 μM Mdivi-1 for 24 h. (A) Representative images of Mitotracker Red staining. Lower panels show magnified images corresponding to the indicated images. (B) Quantification of average mitochondrial length. (C) Representative images of Mitotracker Red staining. Lower panels show magnified images corresponding to the indicated images. (D) Quantification of average mitochondrial length. (E) Representative images of TMRM staining. (F) Level of mitochondrial membrane potential. (G) Level of ATP production. (H-K) Gene expression levels of osteogenic markers, ALP activity, ALP expression level and cellular activity, respectively. Image intensity was quantified using NIH Image J software. (Scale bar = 10 μm). N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group and NAC-added group. (L) Working hypothesis: ROS-induced oxidative stress resulting from TNF-α exposure triggers an increase in Drp1 expression leading to mitochondrial dynamic imbalance, resulting in mitochondrial dysfunction and subsequent osteogenic dysfunction.</p

Primers for the analysis of target genes by qRT-PCR.

<p>Primers for the analysis of target genes by qRT-PCR.</p

Alterations in osteogenic function of MC3T3-E1 cells during TNF-α-triggered inflammation.

<p>(A-E) Gene expression levels of Runx2, ALP, OPG and RANKL, as well as ALP activity, respectively, were determined in cell lysates. (F) Expression of ALP was directly examined in a 24-well plate. (G) Cellular activity was determined by the CCK-8 method. N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group.</p

Changes in mitochondrial function and morphology of MC3T3-E1 cells during inflammation.

<p>(A) Representative images of Mitosox red staining. (B) Level of mitochondrial ROS was assessed by Mitosox red staining intensity. (C) Representative images of TMRM staining. (D) Level of mitochondrial membrane potential was assessed by TMRM staining intensity. (E) ATP production was detected by an ATP assay kit. (F) Representative images of Mitotracker Red staining. Middle panels show magnified images corresponding to the indicated bilateral images. (G) Quantification of average mitochondrial length. (H) Quantification of immunoreactive bands of Drp1. Representative immunoblots are shown in the lower panel. Image intensity was quantified using NIH Image J software. (Scale bar = 10 μm). N = 5–7 cell lines/group. P< 0.05 versus the vehicle-treated group.</p

Preparation, <i>in vitro</i> degradability, cytotoxicity, and <i>in vivo</i> biocompatibility of porous hydroxyapatite whisker-reinforced poly(L-lactide) biocomposite scaffolds

<p>Biodegradable and bioactive scaffolds with interconnected macroporous structures, suitable biodegradability, adequate mechanical property, and excellent biocompatibility have drawn increasing attention in bone tissue engineering. Hence, in this work, porous hydroxyapatite whisker-reinforced poly(L-lactide) (HA-w/PLLA) composite scaffolds with different ratios of HA and PLLA were successfully developed through compression molding and particle leaching. The microstructure, <i>in vitro</i> mineralization, cytocompatibility, hemocompatibility, and <i>in vivo</i> biocompatibility of the porous HA-w/PLLA were investigated for the first time. The SEM results revealed that these HA-w/PLLA scaffolds possessed interconnected pore structures. Compared with porous HA powder-reinforced PLLA (HA-p/PLLA) scaffolds, HA-w/PLLA scaffolds exhibited better mechanical property and <i>in vitro</i> bioactivity, as more formation of bone-like apatite layers were induced on these scaffolds after mineralization in SBF. Importantly, <i>in vitro</i> cytotoxicity displayed that porous HA-w/PLLA scaffold with HA/PLLA ratio of 1:1 (HA-w₁/PLLA₁) produced no deleterious effect on human mesenchymal stem cells (hMSCs), and cells performed elevated cell proliferation, indicating a good cytocompatibility. Simultaneously, well-behaved hemocompatibility and favorable <i>in vivo</i> biocompatibility determined from acute toxicity test and histological evaluation were also found in the porous HA-w₁/PLLA₁ scaffold. These findings may provide new prospects for utilizing the porous HA whisker-based biodegradable scaffolds in bone repair, replacement, and augmentation applications.</p

Activation of p38 signaling pathway in Dex-induced oxidative damage in gingival tissue.

<p>Quantification of immunoreactive bands of p-p38 normalized to t-p38 in vehicle group, Dex group and after treatment with NAC and CsA as designed. Representative immunoblots are shown in lower panels. Image intensity was quantified using NIH Image J software. N = 12 mice/group.</p

Changes in oxidative stress and CypD expression in Dex-treated gingival tissue.

<p>(A) Level of mitochondrial ROS was assessed by Mitosox red staining intensity. (B) Representative images of Mitosox red staining. (C) Level of total ROS was assessed by HO-1staining intensity. (D) Representative images of HO-1 staining. (E) Level of total ROS was assessed by EPR values. The peak height in the spectrum represents the level of total ROS. Representative EPR spectra are shown in the lower panel. (F) Cco activity was determined in tissue homogenate. (G) Quantification of immunoreactive bands of CypD. Representative immunoblots are shown in the lower panel. Image intensity was quantified using NIH Image J software. N = 12 mice/group.</p

Effect of CypD blockade on the Dex-induced oxidative damage in HGF-1 cells.

<p>HGF-1 cells were treated with siRNA for ppif or control siRNA as designed and then performed the following measurements. (A) Immunoblotting for CypD in cells transfected with siRNA-ppif or control siRNA. (B) Representative images of Mitosox red staining. (C) Level of mitochondrial ROS was assessed by Mitosox red staining intensity. (D) Cco activity was determined in cell lysate. (E) Apoptosis was detected by flow cytometry. (F) Values represent means ± SD of three experiments. Image intensity was quantified using NIH Image J software. (Scale bar = 10μm). N = 5–7 cell lines/group.</p

Effect of NAC and CsA treatment on the Dex-induced oxidative damage in gingival tissue.

<p>Mice were treated with designated concentrational NAC (150 mM) and CsA (100 mM) for 21 days and then performed the following measurements. (A) Level of mitochondrial ROS was assessed by Mitosox red staining intensity. (B) Representative images of Mitosox red staining. (C) Level of total ROS was assessed by HO-1staining intensity. (D) Representative images of HO-1 staining. (E) Level of total ROS was assessed by EPR values. The peak height in the spectrum represents the level of total ROS. (F) Representative EPR spectra. (G) Cco activity was determined in tissue homogenate. Image intensity was quantified using NIH Image J software. N = 12 mice/group.</p