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A A B B

C D
C D

G

E F

H

I J K

Figure 4. The potential of trabeculae-like biomimetic bone-filling material (TBM) as an organoid. (A) Schematic diagram of TBM embedding mouse
adipose-derived mesenchymal stem cells (ADSCs) in vitro. (B) Cell viability of ADSCs embedded in HAMA hydrogel or TBM cultured in vitro, as
detected by Cell Counting Kit-8. (C and D) Cell proliferation and viability of ADSCs embedded in HAMA hydrogel or TBM cultured in vitro, as
detected by 5-ethynyl-2’-deoxyuridine (EdU) staining and Calcein AM/PI staining (C: scale bar: 50 μm; magnification: ×23). Red arrows indicate live
and dead cells in the first and second rows, respectively. (E and F) Distribution of ADSCs in TBM, as detected by alizarin red-alcian blue staining (E;
Scale bar: 50μm; Magnification: ×17 and ×126 for left images and right images, respectively) and safranin O staining (F; scale bar: 50μm; magnification:
×126). Red and yellow arrows indicate the ADSCs in the hydrogel layer or upon the porous skeletal structure. (G) Micro-computed tomography
reconstructed images of the ADSCs embedded in TBM and cultured in vitro for 1 week (up) and 4 weeks (below) (scale bar: 750 μm; magnification: ×12).
(H and I) Protein expression of alkaline phosphatase (ALP) and runt-related transcription factor 2 (RUNX2) in ADSCs cultured in TBM, as detected by
immunofluorescence staining (H) and western blotting (I). Red arrows indicate ALP and RUNX2 expression in cells. (J and K) Expression levels of Alp
and Runx2 in ADSCs cultured in TBM, as detected by transcription-polymerase chain reaction (data represented as mean ± SD, n = 3).
Notes: TBM: Blank TBM; Ber: TBM loaded with bergamottin; MSA: TBM loaded with empty recombinant tRNA; anti138: TBM loaded with the
recombinant miR-138-5p antagonist; *p<0.05; **p<0.01.

effects in bone defect mouse models. Tibial and calvarial were humanely sacrificed and the TBM’s therapeutic effects
bone defect mouse models were constructed, and the TBM on bone defects were evaluated (Figures 6A and 7A). The
loaded with either bergamottin or miR-138-5p antagonist TBM exhibited no hepatorenal toxicity in mice and did
was implanted into the defect regions. After 3 weeks, mice not affect the morphology of tissues in several organs,

Volume 1 Issue 2 (2025) 10 doi: 10.36922/OR025040003