2.5 In vivo experiments
2.5.1 Rat model of cranial defect
Sprague–Dawley rats (male,
weighing 280–320 g) were kept in
a specific pathogen-free facility with a controlled temperature of
22±2°C, humidity of 55±5%, a light/dark cycle of 12/12 h, and free
access to water and food. To create the critical-sized defect models,
the rats were anesthetized with isoflurane gas, and a skin incision was
made to expose the cranium. Then 5-mm-diameter defects were created
bilaterally. Then the defects
were covered with COL membrane, DP, or left empty (blank control group),
and the wound was subsequently closed.
All rats were housed and fed
routinely until being euthanized at the designated time points.
2.5.2 Micro-computed tomography (μ-CT) analysis
Cranial samples harvested at 8 weeks post-surgery were fixed in 4%
paraformaldehyde. The fixed samples were scanned using µ-CT system
(Scanco Medical, Bassersdorf, Switzerland) with 70 kV voltage, 114 mA
electric current, and 700-ms integration time. The obtained images were
analyzed with the software of the μ‐CT 80 system for 3D construction.
Cylinders with 5 mm diameter and 1 mm height were chosen as the volume
of interest (VOI). Bone mineral density (BMD) and bone volume/tissue
volume (BV/TV) were calculated for quantitative analysis of bone
regeneration within each VOI.
2.5.3 Histological evaluation
Immediately after μ-CT analysis, samples were decalcified and embedded
in paraffin. Sections were prepared and subjected to Masson’s trichrome
staining. The sections were observed under a
stereoscopic microscope
(Eclipse E600, Nikon, Tokyo, Japan), and the proportion of newly
regenerated bone was evaluated using Image-Pro Plus software (Media
Cybernetics).
2.5.4 Immunohistochemical staining
At 1 week post-surgery, collected tissues were prepared into 10-μm-thick
sections and stained using primary antibodies targeting the macrophage
pan marker CD68 (ab125212, Abcam, USA), M1 phenotype marker iNOS
(ab15323, Abcam, USA), and M2 phenotype marker CD206 (ab64693, Abcam,
USA). Briefly, dewaxed sections were incubated with primary antibodies
overnight and then incubated with secondary antibody for 30 min. The DAB
kit (Proteintech, USA) was used to detect immunoreactions, and stained
sections were viewed under an optical microscope (Leica DMI 6000B
Microsystems, Germany). The proportion of positive cells was calculated
under 40× magnification.