Craniosynostosis occurs in one of 2500 live human births and may manifest as craniofacial disfiguration, seizure, and blindness. Craniotomy is performed to reshape skull bones and resect synostosed cranial sutures. We demonstrate for the first time that autologous mesenchymal stem cells (MSCs) and controlled-released TGFβ3 reduced surgical trauma to localized osteotomy and minimized osteogenesis in a rat craniosynostosis model. Approximately 0.5mL tibial marrow content was aspirated to isolate mononucleated and adherent cells that were characterized as MSCs. Upon resecting the synostosed suture, autologous MSCs in collagen carriers with microencapsulated TGFβ3 (1ng/mL) generated cranial suture analogs characterized as bone–soft tissue–bone interface by quantitative histomorphometric and μCT analyses. Thus, surgical trauma in craniosynostosis can be minimized by a biologically viable implant. We speculate that proportionally larger amounts of human marrow aspirates participate in the healing of craniosynostosis defects in patients. The engineered soft tissue–bone interface may have implications in the repair of tendons, ligaments, periosteum and periodontal ligament.