## 4 Discussion
The absence of hydroxyapatite can be attributed to the freeze drying of TCP and gelatin scaffolds under reduced pressure immediately after mixing, which reduced the contact time with moisture. Moreover, the FTIR analysis did not reveal any significant change between \(\alpha\)-TCP alone and the composite. The peak for GS (1631 cm\({}^{-1}\)) slightly shifted to a higher wave number (1640 cm\({}^{-1}\)) in the \(\alpha\)-TCP/GS, due to the electrostatic interaction between calcium ions in \(\alpha\)-TCP and carboxyl groups in gelatin [25; 26]. In the present study, seeding was performed via droplet seeding; DFATs displayed a rounded appearance, and were evenly dispersed within the pores of the scaffolds 24 h post-seeding. The seeding of droplets and the subsequent static culture is associated with the concentration of cells around the scaffold, as air becomes trapped in the center of the porous scaffold [27; 28]. In this experiment, the cell suspension volume was set at 50 \(\upmu\)L. When the volume of the cell suspension was 50 \(\upmu\)L, the cell numbers of GS and \(\alpha\)-TCP/GS were 4.4 \(\times\) 10\({}^{6}\) and 3.4 \(\times\) 10\({}^{6}\), respectively, 24 h after DFAT seeding. Undifferentiated pluripotent progenitor cells for bone regeneration therapy are commonly used in vitro to differentiate osteoblasts using bone-forming medium, prior to cell transplantation. [PERSON] et al. [29] demonstrated that no significant difference was noted in the BMD of three-dimensional spheroids DFAT transplantation with or without osteogenic stimulation into rat calvarial defects. In this study, DFATs were transplanted in vitro without osteoblast induction, considering the cost issues of future clinical applications. However, we identified remaining \(\alpha\)-TCP and new bone surrounding the \(\alpha\)-TCP on the CT and HE staining images of the \(\alpha\)-TCP/DFATs group. Bone regeneration occurred upon DFAT transplantation into bone defects; [PERSON] et al. [30] demonstrated that the therapeutic angiogenic effects of DFAT cells occurred due to secretion of angiogenic factors and pericyte differentiation by transforming growth factor \(\beta\)1 signaling, via Smad2/3. The transplanted cells release a variety of factors that accelerate healing, thereby accelerating the healing of the bone defect, and causing bone formation. Angiogenesis during bone healing can be confirmed by immunohistochemistry staining for vWF, platelet endothelial cell adhesion molecule (CD31), and the pericyte marker alpha-smooth muscle actin (\(\alpha\)-SMA) [31]. Newly formed vessels stained by factor VIII, which forms a complex with vWF, were prominent around the surface of \(\alpha\)-TCP particles. In conclusion, this study demonstrated that \(\alpha\)-TCP granules and gelatin sponges possessed adequate mechanical strength. \(\alpha\)-TCP did not convert to hydroxyapatite upon contact with water, and a stable \(\alpha\)-TCP/GS composite was formed by electrostatic inter actions, which were verified based on their infrared peak shifts. Bone regeneration was promoted in the DFAT /\(\alpha\)-TCP/GS group by increasing the bone volume, as evidenced by \(\mu\)-CT analyses. The transplantation of the \(\alpha\)-TCP/GS comprising DFAT cells enhanced bone regeneration and vascularization, demonstrating its potential for healing critical-size bone defects. Among the groups studied in a rat calvarial defect model in this study, the group transplanted with \(\alpha\)-TCP-GS with DFAT cells demonstrated greatest bone regeneration after 4 weeks of transplantation. [PERSON], H.K. and [PERSON]. (Yoshitomo [PERSON]) conceived and designed the experiments; [PERSON], [PERSON] performed the experiments; [PERSON] analyzed the data; [PERSON] contributed reagents/materials/analysis tools; [PERSON], H.K., [PERSON] ([PERSON]) and [PERSON] wrote the paper. All authors have read and agreed to the published version of the manuscript. This work was supported in part by JSPS KAKENHI grant nos.17K11864 and 20K10195.