Fig. 3

In vivo activity of sicXer-iaf and boXer-iaf in 2.5 mm drill-hole defects in healthy rats four weeks after surgery. A1 Overview of movat pentachrome stained sections of (from left to right) empty defect, sicXer-iaf, boXer-100-iaf, boXer-500-iaf, and boXer-2500-iaf. Sections reveal enhanced new bone formation, with boXer-100-iaf exhibiting superior osteoid integration and trabecular continuity compared to controls, indicative of structural recovery similar to healthy bone. Histological analysis of A2 alkaline phosphatase (ALP) staining showed increased ALP expression for boXer-100-iaf, consistent with active bone formation, while A3 tartrate-resistant acid phosphatase (TRAP) demonstrated significantly fewer TRAP-positive cells in boXer-2500-iaf-treated samples (P = 0.002), suggesting a decrease in bone resorption. B1 Histomorphometrical analysis of new bone formation (bone volume [BV]/tissue volume [TV]) in the initially created defect zone. Mineralized tissue was significantly higher in boXer-100-iaf treated animals when compared to empty defect (P = 0.038), and significantly lower for boXer-2500-iaf as compared to empty defect (P ≤ 0.001), sicXer-iaf (P = 0.002), boXer-100-iaf (P ≤ 0.001), and boXer-500-iaf (P = 0.002), respectively. Mann-Whitney test; *P < 0.05, **P < 0.01, and ***P ≤ 0.001, respectively. Grey-colored significance bars indicate comparison of boXer-2500-iaf to all other materials, black-colored in comparison to the empty control. Comparative histomorphometric analysis of B2 number of osteoblasts (No. Ob) / trabecular area (TAr) based on ALP staining in the initially created defect zone and B3 number of osteoclasts (No. Oc) /TAr based on TRAP staining. ALP staining showed increased expression in boXer-100-iaf. A significant decrease (P = 0.002) in TRAP positive cells was observed in boXer-2500-iaf treated samples. B4 Immunohistomorphometry showed a significant increase the OPG/RANKL levels in boXer-100 when compared to all the other groups (P ≤ 0.001). C1 An increase in the size of the inflammation zone as compared to the empty defect (no inflammation) was seen for sicXer, boXer-100-iaf, boXer-500-iaf, and boXer-2500-iaf (all P ≤ 0.001). boXer-2500-iaf showed significantly higher inflammation compared to sicXer (P = 0.002), boXer-100-iaf (P ≤ 0.001), and boXer-500-iaf (P = 0.004). Grey-colored significance bars indicate comparison of boXer-2500-iaf to all other materials, black-colored in comparison to the empty control. C2 A simultaneous decrease of ED1 counts was seen in boXer-100-iaf treated animals. Corresponding immunohistochemical stainings are shown in Supplementary Fig. S3. Mann-Whitney test; *P < 0.05, **P < 0.01, and ***P ≤ 0.001, respectively. D. Mass spectrometric images of bone sections. D1-D3 Mass images of the Ca⁺ and Si⁺ distribution given as overlay (Ca⁺ in red color, Si⁺ in green color) of empty defect (control), sicXer-iaf, and boXer-2500-iaf. D4 and D5 mass images of the B⁺ distribution. Pixels brightness correlates with count rate of B⁺. Orange line in D5 gives the summed counts of B⁺ seen in the mass image summed up along the y-axis. D6 Si⁺ image of sicXer-iaf. E. In vivo bortezomib concentrations in E1 the drill hole and surrounding bone/tissue environment as well as E2 the percentual concentration gradient from the drill hole (100%) to the condyle massive (< 5%), the distal (< 2.5%) and proximal shaft (~ 1%)