Igf 1

Cytokines retained in the DTSs and tECM-DTSs, including transforming growth factor beta 1 (TGF-β1), vascular endothelial growth factor (VEGF), insulin-like growth factor 1 (IGF-1) and SDF-1, were measured using ELISA. Soluble molecules were extracted from the DTSs and tECM-DTSs specimens using Tissue Extraction Reagent I (FNN0071, Thermo Fisher Scientific, USA) with a protease inhibitor (1% phenylmethane sulfonyl fluoride, 1% PMSF, Sigma) at 4°C for 24 h. The extracted lysates were homogenized, and centrifuged at 10 000 rpm for 10 min at 4°C, and then the supernatants were collected. ELISA measurements of the extracted lysates were performed (n = 6 for each group) according to the manufacturer’s instructions (TGF-β1 and VEGF, NeoBioscience, China; IGF-1, RayBiotech, USA; SDF-1, DL-develop, China).

The maxillae of each mouse collected at the endpoint were defleshed and soaked in toluidine blue solution. Stained maxillae were photographed using a dissection microscope (Nikon), and total alveolar bone loss was calculated by measuring the cemento-enamel junction (CEJ) to the alveolar bone crest decalcification (ABC) distances on the buccal side of each root [41 (link)]. Some maxillae were fixed in 10% formaldehyde and then 10% EDTA solution at 4 °C for 3 to 4 weeks. The decalcified maxillae samples were then embedded in paraffin and sectioned (7-μm thickness) for TRAP and hematoxylin and eosin (H&E) staining. For immunofluorescence staining, the decalcified maxillae samples were embedded in OCT compound (Fisher Scientific) and sectioned (6 μm thickness) using a cryostat. The expression of Sema4D was monitored using biotin-conjugated anti-Sema4D-mAb, followed by avidin conjugated to AlexaFluor 488. Gingival tissue homogenates were obtained from the gingival tissue of each mouse at the endpoint, and Sema4D, TNF-α, RANKL, OPG, IL-10 and IGF-1 production in the homogenate was analyzed using an ELISA kit purchased from the following sources: Sema4D: RayBiotech; TNF-α, RANKL, OPG, IL-10 and IGF-1: R&D Systems.

Growth hormone (GH; Cloud-Clone Corp. Wuhan, China), insulin-like growth factor 1 (IGF-1; RayBio, Norcross, GA, USA), insulin-like growth factor binding protein-3 (IGFBP-3; RayBio, Norcross, GA, USA) and insulin receptor substrate-1 (IRS-1; Cloud-Clone Corp. Wuhan, China) were determined in serum of controls and treated animals by ELISA following the manufacturer’s instructions.