Masson trichrome

This study was approved by the Animal Ethical and Welfare Committee of Central South University. The hUC‐MSCs transfected with pCDH‐linc02349 or pCDH‐control vector were induced in osteogenic medium at 37°C for 7 days. Then, the cells were harvested, and 2 × 10⁶ cells were incubated with Bio‐Oss Collagen scaffolds (5 × 5 × 1.75 mm³) (Geistlich) scaffolds for 1 hour, which were transplanted subcutaneously in right back of BALB/c nude mice(5 weeks old, male, n = 5 per group). After 8 weeks, the Bio‐Oss Collagen were obtained and fixed in 4% paraformaldehyde. The implants were analysed via Quantum GX micro‐CT Imaging System (PerkinElmer), with the Quantum Supporting Xcapture Software (PerkinElmer) used to analyse the BV/TV. After that, implants were decalcified in 10% EDTA for 14 days, embedded with paraffin and sliced into sections (4 μm thickness). The sections were stained with H&E (Boster) and Masson Trichrome (Solarbio). In addition, IHC staining was performed with anti‐OPN. The images were taken using CKX41 optical microscope (Olympus).

All animal experiments were conducted under the ethical committee guidelines of the Affiliated Stomatological Hospital of Chongqing Medical University. Cells from each group (approximately 3×10⁶ cells per group) were loaded on β-tricalcium phosphate (β-TCP) porcelain granules (Bio-lu Biomaterials, Shanghai, China) and subcutaneously implanted the mixture into the flanks of 6-week-old BALB/c nude mice. 8 weeks later, these implants were obtained, fixed with paraformaldehyde (4%), decalcified in EDTA decalcification solution (Servicebio, Wuhan, China), and embedded in paraffin. Tissues embedded were serially sliced (5 μm) and processed for hematoxylin and eosin (H&E; Solarbio, Beijing, China) staining and Masson trichrome (Solarbio, Beijing, China) staining. Furthermore, the immunohistochemistry (IHC) staining was also carried out with anti-OCN antibodies (Abcam, Cambridge, UK) as previously described (33 (link)). The images were acquired by digital section scanner VS200 (Olympus, Japan).

To assess cell and nuclear clearance as well as preservation of collagen, hematoxylin & eosin and Masson Trichrome staining (Solarbio, China) were performed after fixation in 10% formalin, paraffin embedding, and sectioning.

Hearts were harvested and fixed in 4% paraformaldehyde. Sections were stained with Masson Trichrome (Solarbio, China) to evaluate the myocardial fibrosis area. The density of capillaries was determined by von Willebrand Factor (vWF) (Cell Signaling Technology, USA) staining.

Following μCT analysis, the samples were decalcified with 10% EDTA for 4 weeks. Sections of 4.5 μm were prepared using a conventional method [31 (link)]. H&E and Masson trichrome staining (Solarbio, Beijing, China) were performed as the manufacturer’s instruction. The stained sections were observed using an inverted microscope (IX81, Olympus).

After 8 weeks of osteogenic differentiation in vivo, all nude mice were sacrificed, and the Bio-Oss Collagen was stripped from the subcutaneous skin and fixed in 4% PFA. The specimens were analyzed using Quantum GX microCT Imaging System (PerkinElmer, CA, USA). BV/TV and BMD were measured using Quantum Supporting Xcapture Software (PerkinElmer, CA, USA). Then the specimens were decalcified in 10% EDTA solution for 14 days, followed by embedding with paraffin and sectioning. The sections (4.0 μm) were stained with H and E (Boster, USA) and Masson Trichrome (Solarbio, Beijing, China). In addition, IHC staining was performed with anti-OCN and anti-OSX to evaluate the expression of osteogenic markers. The images were taken using CKX41 Optical microscope (Olympus, Japan).