## 4 Discussion  
The cells that form the regenerating tissue, including mature, progenitor, and stem cells, play an important role in the success of tissue regeneration. Numerous studies have evaluated MSCs combined with scaffolds for inducing in vitro or in vivo tissue regeneration. Gelatin is a natural polymer derived from partially hydrolyzed collagen [20; 21]. Our previous study using gelatin sponge for bone regeneration indicated the value of using rat DFATs in conjunction with a platelet-rich plasma [19]. In addition, gelatin chemically modified with epigallocatechin gallate in combination with DFATs can facilitate the induction of bone regeneration [22]. However, the application of gelatin to hard tissue regeneration scaffolds is limited, due to the low mechanical strength of the latter. We studied the in vivo bone healing potential of DFAT-loaded \(\alpha\)-TCP/gelatin sponge, transplanted into an athymic critical-sized rat calvarial defect model. In our previous study [19], GS was fabricated using a 1% gelatin solution. The combination of \(\alpha\)-TCP particles with 1% gelatin solution precipitated \(\alpha\)-TCP particles at the bottom of the solution; this may be attributed to their sol state at 4 \({}^{\circ}\)C. Therefore, we used a 2% gelatin solution in the gel state. Although the strength of the composite sponge is likely to increase with the number of \(\alpha\)-TCP granules added to the gelatin, adding excess \(\alpha\)-TCP granules makes it fragile and impairs the ability to form a composite sponge. The 120 mg \(\alpha\)-TCP/GS composite displayed a mechanical strength similar to that of 60 mg  
Figure 16: Tissue specimen of immunostaining (Factor VIII). (**A**) \(\alpha\)-TCP/GS group and (**B**) \(\alpha\)-TCP/GS + DFAT group. \(\alpha\)-TCP/GS, composite gelatin sponge; GS, gelatin scaffolds; and DFAT, dedifferentiated fat cell. Arrows indicate blood vessels. The scale bar represents a length of 100 \(\upmu\)m.  
Figure 17: Results of the analysis of the number of new blood vessels; Student’s \(t\)-test; \({}^{*}p<0.05\); n = 4. \(\alpha\)-TCP/GS; however, it collapsed when grasped with tweezers because of the numerous \(\alpha\)-TCP granules. In addition to sufficient mechanical strength, the operability for grafting is an important requirement for bone defect graft materials. The high-temperature peak of the GS sponge in the TGA profile was higher than that in our previous study [19], because of a change in the gelatin concentration from 1% to 2%. In the TGA profile of the \(\alpha\)-TCP/GS sponge, there was no significant weight loss at high temperatures (above 600 \({}^{\circ}\)C); this could be explained by use of 60 mg \(\alpha\)-TCP/GS (96.2 wt% of \(\alpha\)-TCP content). [PERSON] et al. [23] revealed that there is no significant change in the weight of hydroxyapatite even at high temperatures. Our findings indicated that \(\alpha\)-TCP maintained its thermal stability. We prepared a composite sponge by mixing gelatin and porous \(\alpha\)-TCP. The ceramic body comprised a continuous pore structure with a pore diameter of approximately 10-50 \(\upmu\)m, and a sponge-like scaffold with a grain size of 500 \(\upmu\)m. The porous body of TCP can gradually dissolve away from the bone defect during bone repair, and can be replaced by the regenerated bone. This microporous biosensorable ceramic is useful as a bone filler for medical applications; this is because its porous structure facilitates cell invasion [24]. However, the solubility of porous \(\alpha\)-TCP may be higher than that of the original \(\alpha\)-TCP; thus, it is more likely to be completely absorbed by the body before bone regeneration can repair the defect [2]. In this study, a mixture of porous \(\alpha\)-TCP and gelatin was effective for controlling the degradation rate of the implanted \(\alpha\)-TCP. In addition, there was no significant difference between the XRD patterns of \(\alpha\)-TCP/GS and \(\alpha\)-TCP.