## 4 Discussion  
Sericin has demonstrated the ability to enhance bone regeneration in a regular animal model [1]. Silk mat, which is the combination of sericin and fibroin, has increased bone regeneration in clinical trials [22]. In this study, a diabetic animal model was used to determine whether it had a similar effect on a surgically created defect. According to microCT and histological analysis, group S showed more new bone formation compared to group G and group N (Tables 1 and 2). Studies of sericin have increased in biomedical programs. Stable sericin biomaterials are manufactured by precipitation with ethanol, cross-linking, or incorporation with other polymers [23]. In vitro, mineralized sericin provides better cellular adhesion and spreading than sericin alone [24]. By combining sericin with CaCl\({}_{2}\) and Na\({}_{2}\)CO\({}_{3}\), spherical particles are formed and mineralized to hydroxyapatite microspheres [25]. Investigation of enhancing osseointegration by coating sericin on titanium surfaces has also been conducted [26]. Sericin has been found to promote the growth and proliferation of mouse osteoblasts [26]. In this study, sericin was extracted by sonication and centrifuging with a filter. Since there was no cross-linking, precipitation with ethanol, or blending with another polymer, the manufacturing procedure was simple. \begin{table}
\begin{tabular}{c c c c} \hline
**Group** & **G** & **N** & **S** \\ \hline Bone volume (mm\({}^{3}\)) & 1.24 \(\pm\) 1.05 & 1.71 \(\pm\) 1.65 & 3.87 \(\pm\) 2.51 * \\ \hline \end{tabular}
\end{table} 
Table 1: Bone volume in region of interest measured by microCT. Figure 1: Micro computerized tomogram (MicroCT) analysis. Scanned images were used for three-dimensional reconstructions. The size of the primary surgical defect was referenced as the region of interest (G: gelatin only group, N: unfilled control group, S: sericin combined with gelatin group). Several studies have provided evidence of sericin's healing properties, particularly in diabetic models, primarily promoting wound healing. Sericin is used for the treatment of corneal lesions in the type 2 DM model [27]. When 2.4 g/kg of sericin was given to a diabetic animal orally, the treated animals showed reduced blood glucose level and increased neurofilament protein expression in the sciatic nerve [28]. Sericin has a therapeutic effect on dysfunctions of the growth hormone/insulin-like growth factor I axis, which is caused by DM [29]. In this study, a high molecular fraction of sericin was grafted using a gelatin sponge, which had a role of a scaffold for sericin. Compared to the gelatin sponge-only group (G), the sericin combined with gelatin sponge (S) group showed significantly higher new bone formation (Figure 2). Bone healing after trauma requires neovascularization and an adequate blood supply [30; 31]. Impaired angiogenesis could be the primary cause of delayed fracture healing. Macrophages are essential cells for angiogenesis [32]. Macrophages orchestrate the healing process by phagocytosis and secretion of numerous kinds of cytokines. Macrophage-originated cytokines control wound healing from the inflammatory phase to the remodeling phase [32]. Macrophages can be classified grossly as M1 and M2. M1-type macrophages are predominant in the inflammatory phase, whereas M2-type macrophages constitute the main population in the remodeling phase [33]. Vascular endothelial growth factors (VEGFs) are a key cytokine for angiogenesis and are secreted by M1-type macrophages [33; 34]. Sericin is an M1-type macrophage polarizing material and increases VEGFs via the hypoxia-inducible factor-1\(\alpha\) mediated pathway [35]. Therefore, the sericin application might help wound healing in diabetic animals via VEGF elevation and subsequent angiogenesis. Since this study was an observational study of new bone formation, the underlying mechanism of sericin-induced bone formation was under investigation.