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However, the current loading method has focused on physical mixing, which has limited release control. Therefore, alternative conjugation of bioactive agents with hydrogels is highly recommended. Direct chemical conjugation of synthetic peptides containing a functional moiety with a hydrogel would be ideal. Here, we synthesized a bioactive calcium accumulating peptide (CAP) containing a collagen binding motif, which can induce osteogenic differentiation. A tyrosine residue in CAP was used to directly chemically conjugate the peptide with a gelatin\u2010based enzymatically crosslinked hydroxyphenyl propionic acid hydrogel under H<jats:sub>2<\/jats:sub>O<jats:sub>2<\/jats:sub>\/Horse radish peroxidase conditions. To test the acceleration of bone formation, human periodontal ligament stem cells (PDLSCs) were loaded into a chemically conjugated CAP hydrogel. The CAP hydrogel induced bone mineralization around the PDLSCs and increased osteogenic marker expressions <jats:italic>in vitro<\/jats:italic>. It also recovered a bone layer in a calvarial defect 4 weeks postimplantation. In summary, an injectable CAP hydrogel scaffold system was developed as a potentially useful engineered microenvironment to enhance bone restoration, and it could be utilized as a vehicle for bioactive delivery of stem cells in tissue regenerative therapy. \u00a9 2017 Wiley Periodicals, Inc. 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