## 3 Results  
### Experiment 1: Analgesic Effect of BO  
Improvement of locomotive function through BO administration was evaluated using the rotarod test. In the acute phase (on days 1 and 3), the latencies to fall off the rotarod apparatus (walking time) became significantly lower in the DMM group than in the control group (\(p\) < 0.01). However, this decrease was inhibited in the DMM + BO group (\(p\) < 0.05). In the chronic phase (on day 14 and later), the walking time on the rotarod apparatus in the DMM group was significantly lower than that in the control and sham groups (\(p\) < 0.01). These decreases were significantly inhibited in the DMM + BO group (on days 14 and 21; \(p\) < 0.01, on day 28; \(p\) < 0.05). Moreover, the walking time on day 28 in the DMM + BO group showed no significant difference compared with that in the control group (Figure 3). Suppressive Effect of BO on ERK1/2 Phosphorylation in the Dorsal Horn of the Spinal Cord in the Acute Phase after DMM Surgery  
The spinal cord was harvested 1 day after DMM surgery in order to analyze the degree of ERK1/2 phosphorylation. In the DMM group, immunofluorescence staining showed a significant increase in the number of pERK1/2-positive cells in the dorsal horn of the spinal cord compared to that in the control group (7.7 \(\pm\) 2.5 vs. 3.3 \(\pm\) 0.8, \(p\) \(<\) 0.05). After the oral administration of BO, a significantly lower number of pERK1/2-positive cells was noted in the dorsal horn of the spinal cord (7.7 \(\pm\) 2.5 vs. 3.3 \(\pm\) 0.8, \(p\) \(<\) 0.05) (Figure 4A,B). Furthermore, the results of Western blotting revealed an increased pERK1/2 expression, which was significantly blocked by BO administration (\(p\) \(<\) 0.05) (Figure 4C,D). Suppressive Effect of BO on ERK1/2 Phosphorylation in the Dorsal Horn of the Spinal Cord in the Chronic Phase after DMM Surgery  
The spinal cord was also harvested 28 days after the DMM surgery. In the DMM group, immunofluorescence staining revealed a significant increase in the number of pERK1/2-positive cells in the dorsal horn of the spinal cord compared to that in the control group (9.3 \(\pm\) 2.7 vs. 2.5 \(\pm\) 1.1, \(p\) \(<\) 0.05). Oral administration of BO resulted in a significantly lower number of pERK1/2-positive cells in the dorsal horn of the spinal cord than that in the DMM group (9.3 \(\pm\) 2.7 vs. 5.6 \(\pm\) 1.6, \(p\) \(<\) 0.05) (Figure 5A,B). The results of Western blotting also showed significantly increased pERK1/2 expression, which was blocked by BO administration (\(p\) \(<\) 0.05) (Figure 5C,D). Figure 3: Rotarod test. All data are presented as mean \(\pm\) standard deviation (\(n\) = 5 per group). \(\dagger\)\(p\)\(<\) 0.05, \(\dagger\)\(p\)\(<\) 0.01 vs. Control, \({}^{*}\)\(p\)\(<\) 0.05, \({}^{**}\)\(p\)\(<\) 0.01 vs. DMM. DMM, destabilization of the medial meniscus; BO, bioigito. Figure 4: Expression of pERK1/2 in the right dorsal horn of the spinal cord in the acute phase (1 day after DMM surgery). **(A)** Representative images of immunofluorescence staining in each group (red; pERK1/2, blue; nucleus). Upper images; magnification: \(\times\)200, Scale bar = 150 \(\upmu\)m. Lower images; magnification: \(\times\)600, Scale bar = 50 \(\upmu\)m. White arrow (\(\
earrow\square\)) indicates a pERK1/2-positive cell. **(B)** The numbers of pERK1/2-positive cells. Each \(n\) = 5, \(\upmu\)t \(p\) \(<\) 0.01 vs. Control, * \(p\) \(<\) 0.05 vs. DMM. **(C)** Immunoblot images of pERK1/2 and total ERK1/2 in the right dorsal horn of the spinal cord. **(D)** The quantified data of the immunoblot for pERK1/2 normalized to total ERK1/2. Each \(n\) = 5, \(\upmu\)p \(<\) 0.05 vs. control, * \(p\) \(<\) 0.05 vs. DMM. All data are presented as mean \(\pm\) standard deviation. pERK, phosphorylated extracellular signal-regulated kinase; DMM, destabilization of the medial meniscus; BO, bioogito. Figure 5: Expression of pERK1/2 in the right dorsal horn of the spinal cord in the chronic phase (28 days after DMM surgery).