Strictly following the guidelines of the Institutional Animal Care and Use Committee at The Chinese University of Hong Kong, 12-week-old male Sprague-Dawley rats were randomly divided into four groups, shaved, and prepped for aseptic surgery. A midline skin flap was raised over the parietal bones and reflected caudally to expose the midsagittal and transverse sutures. The periosteum was incised along the midsagittal suture and the right or left transverse suture and removed to expose the parietal bone. A 5-mm-diameter defect was created using a trephine with normal saline irrigation during processing, and a section of the bone was removed to expose the dura mater. 3D porous peptide–functionalized HA hydrogels (n = 3 per group) 5 mm in diameter and 1 mm thick were seeded with 1 million rMSCs with trilineage differentiation potential (46). After 7 days of in vitro osteogenic induction in the incubator, the hydrogels were implanted into the calvarial defects. All experimental animals were maintained until 8 weeks from the day of defect creation. After the rats were euthanized, the parietal bones were harvested and decalcified with 10% EDTA solution. The subsequent H&E staining and histological analysis were performed as described in previous publications (9).

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