Ha tcp

BMSCs were cultured to construct cell sheets. A sandwich structure of three layers of BMSCs sheets composited with two layers of HA/TCP (20 mg, HA/TCP = 6/4, 50–200 nm) (Sigma-Aldrich) were packaged for subcutaneous grafting. Under anesthesia with 30 mg/kg pentobarbital sodium, the implants were transplanted on the back of 8-week-old NOD/SCID mice subcutaneously. Twelve weeks after transplantation, the implants were collected for fixing, decalcification and H&E staining. The areas of new bone formation were photographed and were estimated by Image-Pro Plus 6.0 (Media Cybernetics).

The 6-week-old NOD/SCID mice (Fourth Military Medical University, Xi'an, China) were randomly divided into 12 groups (grouping situation can be found in Supplementary Table S3). Each group included three mice and six transplant complexes (each mouse was transplanted with two complexes in the dorsal region). For a single transplant complex, ~5 × 10⁶ cells were mixed with 20 mg HA-TCP (Sigma, St Louis, MO, USA) and subcutaneously implanted into pockets of NOD/SCID mice in the dorsal region. General anesthesia was administered by intramuscular injection of pentobarbital sodium (0.1 ml/100 g) for all surgical procedures. All animal procedures were approved by the Animal Care Committee of the Fourth Military Medical University (license number: 2015, kq-012). After 4 weeks, the mice were killed by cervical dislocation under general anesthesia. The implants were then removed, fixed with 4% paraformaldehyde and decalcified in 10% EDTA (pH 6.0) for 7 days. For histological analyses, the implants were embedded in paraffin, sectioned and stained with H&E or Masson's Trichrome stain (BaSO Diagnostic Inc., Guangdong, China) according to the manufacturer's instructions.

The NOD/SCID mice around 4‐week old purchased from Provincial Animal Center (Guangdong, China) were randomly divided into four groups with five mice per group. For the transplantation, around 5 × 10⁶ cells were loaded onto 20 mg hydroxyapatite‐tricalcium phosphate scaffold (HA‐TCP; Sigma, St. Louis, MO) and subcutaneously implanted into the dorsal region of NOD/SCID mice. General anesthesia was performed by intraperitoneal injection of pentobarbital sodium (0.1 ml/100 g) for all surgical procedures. All animal procedures were approved by the Animal Care Committee of Southern Medical University. After 4 weeks, the mice were killed by cervical dislocation under general anesthesia. The xenografts were removed, fixed with 4% paraformaldehyde and decalcified in 10% ethylenediaminetetraacetic acid (pH 6.0) for 7 days. For histological analyses, the xenografts were then embedded in paraffin, sectioned and stained with hematoxylin and eosin (H&E; Beyotime Institute of Biotechnology, Shanghai, China), or Masson's Trichrome stain (Sigma‐Aldrich; Merck KGaA), according to the protocols of manufacturer.