*3.5. Histological Analysis*

H&E stained materials are demonstrated in Figure 4. A progressive bone matrix deposition was observed during the experimental period. Presence of a fibrillary material, similar to FBP structure, inside the defect 3 days after surgery on the group treated only with FBP evidences that it has adhered to injury site. Bone fragments probably from the surgical procedure were also observed adhered to the fibers. There was a significant increase in cellularity associated to the biomaterial. On the group treated with FBP + MSCs the presence of newly formed trabecular bone on the defect margins was evident after 21 days as well as in the FBP + MSC-D treated group. At day 42, from histological perspective, all defects were partially repaired, although in the FBP + MSC-D treated group newly formed bone was more abundant and its structure more similar to normal mature bone tissue.

**Figure 4.** Histological analysis of bone regeneration tissue in femur of rats at 3 (D03), 21 (D21) and 42 (D42) days post injury stained with H&E. FBP (fibrin biopolymer only); FBP + MSCs (fibrin biopolymer + mesenchymal stem cells); FBP + MSC-D (fibrin biopolymer + differentiated mesenchymal stem cells); Control show a non-injured bone for comparison. Arrows: fibrillary material; dashed circle: bone fragments.

Figure 5 shows collagen fibers formation through picrosirius staining under polarized light. Yellow-reddish staining represents mature thick fibers, as demonstrated by the FBP group, while green staining shows recent synthesized and immature fibers. On the first 3 days there was no evidence of collagen formation in all groups. After 21 days, there were observed thin immature green fibers within thick yellow and red mature collagen showing an increase in collagen synthesis in all three groups. In addition, there were also a high number of cells adhered to the scaffold claiming that MSCs injected with the FBP remained at injury site and have differentiated for matrix synthesis. Our results show that even after 21 days cells were associated with the fibrin structure strengthening the use of FBP as a scaffold for cell delivery. The collagen synthesis pattern was similar in FBP and FBP + MSC groups at day 42, but it was higher on group FBP + MSC-D.

**Figure 5.** Picrosirius staining under polarized light showing collagen formation in injury filling in femur of rats at 3, 21 and 42 days post injury. FBP (fibrin biopolymer only); FBP + MSCs (fibrin biopolymer + mesenchymal stem cells); FBP + MSC-D (fibrin biopolymer + differentiated mesenchymal stem cells). Control shows a non-injured bone for comparison.
