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2.16. Western blotting reconstructed and calibrated at an isotropic voxel size
of 10.5 mm (70 kVp, 114 mA, 200 ms integration time,
For western blotting, TBM-embedded ADSCs were lysed 3
using a cell lysis buffer (P0013, Beyotime, China). The 260 thresholds, and 1200 mg HA/cm ). Using SCANCO
lysates were electrophoresed, and proteins were transferred software, regions of interest within the bone defect
onto nitrocellulose membranes (66485, Pall, USA). The areas were manually contoured for three-dimensional
membranes were blocked with 5% skimmed milk and reconstruction (sigma = 1.2, supports = 2, and threshold
incubated overnight at 4°C with primary antibodies. After = 200). Parameters such as bone mineral density (BMD),
washing, the membranes were incubated with horseradish bone volume to tissue volume (BV/TV), trabecular number
peroxidase (HRP)-conjugated secondary antibodies. Protein (Tb.N), and bone mineral content (BMC) were calculated
bands were visualized using enhanced chemiluminescence to evaluate bone microarchitecture. 31
(BL520B, Biosharp, China) and exposed using an imager 2.20. Immunohistochemistry
(Fusion FX, Vilber Lourmat, France). The following
antibodies were supplied by HUABIO (China) and used All antibodies were purchased from Santa Cruz Biotech
at a dilution of 1:2000: ALP Rabbit pAb (ET1601-21), (USA). Inflammatory marker protein levels were evaluated
RUNX2 Rabbit pAb (ET1612-47), and GAPDH Rabbit using CD3 mouse monoclonal antibody (mAb) (1:100;
pAb (ET1601-4, RRID: AB_3069615). HRP-conjugated sc-20047, RRID: AB_627014) and CD68 mouse mAb
anti-immunoglobulin G antibodies (1:2000; CW0102- (1:100; sc-20060, RRID: AB_627158). Bone-specific
RRID-AB_2814710 and CW0103-RRID-AB_2814709, markers were osteocalcin (OCN) mouse mAb (1:200;
CWBIO, China) were used for detection. sc-73464, RRID: AB_1126894) and RUNX2 mouse mAb
(1:200; sc-390351, RRID: AB_2892645). The incubation
2.17. Bone defect and fracture modeling with primary antibodies was at 4°C overnight, and for
A total of 120 2-month-old C57BL/6 male mice (25 ± secondary antibodies was at room temperature for 1.5 h. 28
0.5 g) were used in bone defect and fracture models. For 2.21. Statistical analyzes
the bone defect model, 1.2 mm boreholes were created in
the tibiae or calvariae using a dentist’s drill, followed by All experiments were independently repeated at least
27
the insertion of TBM. Mice were humanely euthanized at 3 times, with each experiment performed in triplicate.
3 weeks post-surgery using CO . For the fracture model, The statistical analyzes were conducted using GraphPad
2
tibial fractures were induced, and TBM was implanted Prism version 10.1.2 software (GraphPad Software, USA;
into the fracture sites. Mice were humanely euthanized at www.graphpad.com). For comparison involving three or
4 weeks post-surgery. more groups, ordinary one-way analysis of variance with
Bonferroni post hoc analysis was used to assess variance.
2.18. In vivo hydrogel biocompatibility Differences between the two groups were evaluated using
Blood samples were collected, and supernatants Student’s t-test. Data are presented as mean ± standard
were isolated for alanine transaminase, aspartate deviation. A p<0.05 was considered statistically significant
aminotransferase, blood urea nitrogen, and creatinine for all comparisons.
blood analyzes using the ADVIA 2400 Chemistry System
(Siemens Healthcare Diagnostics, USA). In addition, tissue 3. Results
samples from the liver, spleen, lung, kidney, and heart were 3.1. Construction and characterization of the TBM
collected, fixed, and embedded in paraffin. Sections were
prepared and stained with hematoxylin and eosin (HE) for The TBM was prepared using a combination of silk protein,
histological evaluation under optical microscopy. chitosan, matrigel, BCP, and HAMA. An organic collagen-
like network was first formed by mixing silk protein,
2.19. Bone histomorphometry chitosan, and matrigel. BCP was added to the organic
The effects of the TBM on bone repair were evaluated using network, mixed thoroughly, and immediately lyophilized
double calcein labeling and micro-computed tomography to form the Core, which provides mechanical support.
(micro-CT) imaging (v.6.5, Viva CT40, SCANCO Medical, The core was then coated with the organic network-BCP
Switzerland), as previously described. 28,29 To assess bone mixture. The resulting construct underwent rapid freezing
regeneration and bone resorption, paraffin sections were and lyophilization to generate the Porous. Finally, the
prepared for Masson’s trichrome staining, Goldner’s structure was coated with a HAMA hydrogel to form the
trichrome staining, and tartrate-resistant acid phosphatase TBM (Figure 1A).
(TRAP) staining. 30,31 The TBM exhibited a microstructure resembling
Bone microstructures were analyzed using micro-CT, trabecular bone, with a relatively uniform pore size range
with a focus on the bone defect regions. Images were of 50 – 250 μm. The water content of the TBM was 40.81%

Volume 1 Issue 2 (2025) 5 doi: 10.36922/OR025040003