Alizarin red stain

PCL was purchased from Beijing Heyanling Pharmaceutical Development Co. Ltd. (Beijing, China) and had been identified as the Genuine Medicinal Herb by Professor Lei Yan in Shandong First Medical University and Shandong Academy of Medical Sciences. A BCIP/NBT Alkaline Phosphatase Color Development Kit, DAPI Staining Solution, Blocking Buffer for Immunol Staining, Antifade Mounting Medium, a bicinchoninic acid (BCA) protein assay kit, and penicillin-streptomycin were purchased from Beyotime (Shanghai, China). A ReverTra Ace® Qpcr RT Kit and Taq SYBR® Green Qpcr Premix were purchased from TOYOBO (Shanghai, China). β-Glycerophosphate, P-nitrophenyl phosphate, and ascorbic acid-2-phosphate were purchased from Sigma-Aldrich (St. Louis, MO, USA). TRIzol was obtained from Invitrogen (Carlsbad, CA, USA). The anti-GAPDH, anti-PPARγ, anti-AhR, and CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H + L) antibodies were obtained from Proteintech Group (Wuhan, China). Alizarin red stain was obtained from Cyagen Biosciences (Guangzhou, China). The primers were synthesized by BGI (Shenzhen, China).

Sodium tetraborate (Na₂B₄O₇.10H₂O) was purchased from Beijing Chemical Works (Beijing, China). PVA (≈99% hydrolyzed, Mw ≈130,000) and TEOS were obtained from Sigma-Aldrich. The rabbit BMSCs were supplied by American Type Culture Collection (ATCC, MD, United States). Low-glucose Dulbecco’s modified Eagle’s medium (LG-DMEM), fetal bovine serum (FBS), and streptomycin–penicillin were obtained from Gibco^® Life Technologies (CA, United States). The Cell Counting Kit-8 (CCK-8) kit and Calcein-AM/PI staining kit were supplied by Beyotime Biotechnology (Shanghai, China). The mediums for osteogenic differentiation of BMSCs and Alizarin Red stain were obtained from Cyagen Biosciences (CA, United States). Phosphate buffer (PBS) and 4% paraformaldehyde were provided by Solarbio (Beijing, China). The Eastep Super Total RNA Extraction Kit was purchased from Promega (Shanghai, China), and the Perfect Real-Time RT reagent kit was supplied by Takara Bio (Dalian, China). Hematoxylin–eosin stain was obtained from Thermo Fisher Scientific, MA, United States. Primary antibodies, runt-related transcription factor-2 (Runx-2), type I collagen (Col-1), osteocalcin (OCN), and osteopontin (OPN) were purchased from Abcam (Cambridge, United Kingdom), and secondary antibodies were obtained from Jackson ImmunoResearch Laboratories (West Grove, PA, United States).