The purpose of this study was to investigate a capability of PuraMatrix (PM), which is a self-assembling peptide nanomaterial, as a scaffold for bone regeneration in combination with dog mesenchymal stem cells (dMSCs) and/or platelet-rich plasma (PRP) using tissue engineering and regenerative technology. Initially, teeth were extracted from an adult hybrid dog's mandible region. After 4 weeks, bone defects were prepared on both sides of the mandible with a trephine bar. The following graft materials were implanted into these defects: (1) control (defect only), (2) PM, (3) PM/PRP, (4) PM/dMSCs, and (5) PM/dMSCs/PRP. From scanning electron microscope images, PM had a three-dimensional nanostructure, and dMSCs attached on the surface of PM. At 2, 4, and 8 weeks after implantation, each sample was collected from the graft area with a trephine bar and assessed by histologic and histomorphometric analyses. It was observed that the bone regenerated by PM/dMSCs/PRP was of excellent quality, and mature bone had been formed. Histometrically, at 8 weeks, newly formed bone areas comprised 12.39 +/- 1.29% (control), 25.28 +/- 3.92% (PM), 27.72 +/- 3.15% (PM/PRP), 50.07 +/- 3.97% (PM/dMSCs), and 58.43 +/- 5.06% (PM/dMSCs/PRP). The PM/dMSCs and PM/dMSCs/PRP groups showed a significant increase at all weeks compared with the control, PM, or PM/PRP (P < 0.05 at 2, 4, and 8 weeks, analysis of variance). These results showed that MSCs might keep their own potential and promote new bone regeneration in the three-dimensional structure by PM scaffolds. Taken together, it is suggested that PM might be useful as a scaffold of bone regeneration in cell therapy, and these results might lead to an effective treatment method for bone defects.