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.