Histochemical examination of adipose derived stem cells combined with β-TCP for bone defects restoration under systemic administration of 1α,25(OH)2D3

• More details about the alizarin red stain were added to the Materials and methods section.
• The preparation of the sections and the statistical analysis method were explained more explicitly.
• Low magnitude views of HE staining were added to provide a gross impression of the bone defects restoration.
• High magnifications of TRAP staining were provided for the easier identification of TRAP-positive osteoclasts.

The purpose of this study was to evaluate the effects of osteogenic differentiated adipose-derived stem cell (ADSC) loaded beta-tricalcium phosphate (β-TCP) in the restoration of bone defects under intraperitoneal administration of 1α,25-dihydroxyvitamin D3(1α,25(OH)2D3). ADSCs were isolated from the fat tissue of 8 week old Wister rats and co-cultured with β-TCP for 21 days under osteogenic induction. Then the ADSC-β-TCP complexes were implanted into bone defects in the femora of rats. 1α,25(OH)2D3 (VD) or normal saline (NS) was administrated intraperitoneally every other day after the surgery. Femora were harvested at day 7, day 14 and day 28 post-surgery. There were 4 groups for all specimens: β-TCP-NS group; β-TCP-ADSC-NS group; β-TCP-VD group and β-TCP-ADSC-VD group. Alkaline phosphatase (ALP) was up-regulated obviously in ADSC groups compared with non-ADSC groups at day 7, day 14 and day 28, although high expression of runt-related transcription factor 2 (RUNX2) was only seen at day 7. Furthermore, the number of TRAP-positive osteoclasts and the expression of cathepsin K (CK) were significantly decreased in VD groups compared with non-VD groups at day 7 and day 14. As a most significant finding, the β-TCP-ADSC-VD group showed the highest BV/TV ratio compared with the other three groups at day 28. Taken together, ADSC-loaded β-TCP under the administration of 1α,25(OH)2D3 made a promising therapy for bone defects restoration.

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