A modo de conclusión: ideas para un enfoque alternativo de

In all groups, bone regeneration started at the proximal and distal bone adjacent to the defect and progressed towards the central area of the defect (Fig. 4). The defect was almost completely bridged (defined as a remaining gap <0.5 mm) in seven rats of the BMP-2/bFGF group, seven rats of the BMP-2 group, five rats of the bFGF group and one rat of the unloaded group. The average remaining gap distance was 0.33±0.34 mm for the BMP-2/bFGF group, 0.51±0.74 mm for the BMP-2 group, 0.83±0.53 mm for the bFGF group and 1.20±0.56 mm for the unloaded group. The 2D images of cross-sections along the vertical axis of the femurs also confirmed

throughout the interconnected porosity of the scaffolds as confirmed by micro-CT (Fig. 1C and D). Scanning electron micrographs revealed the porous structure made of fused titanium particles as well as the surface morphology of titanium (Fig. 3A and B). The porous structure of the scaffolds was fully filled with nanostructured colloidal gelatin gels (Fig. 3C). As a consequence, original surface morphology of titanium was replaced by nanoparticulate morphology of colloidal gelatin gels consisting of nanoparticles ranging between 100-200 nm in diameter (Fig. 3D)[18]_.

Figure 3. Microscopic images of the porous titanium scaffolds before (A, B) and after (C, D) incorporation of the colloidal gelatin gels. Low (A, C) and high (B, D) magnification.

3.2 In vivo evaluation 3.2.1. Clinical evaluation

All rats were able to tolerate weight-bearing activities immediately after surgery, and all implantation sites healed without complications. All animals remained healthy during the entire study period, except for one rat that received the BMP-2 group, which died due to anesthesia-related complications during the in vivo micro-CT scan made at four weeks.

Figure 4. Representative 3D micro-CT images of bone bridging the porous titanium scaffolds containing the unloaded gels (A), bFGF gels (B), BMP-2 gels (C) and BMP-2/bFGF gels (D) after twelve weeks implantation. Porous titanium scaffolds are shown in grey whereas bone appears in brown.

Figure 5. Representative transversal micro-CT images of the porous titanium scaffolds containing the unloaded (A), bFGF (B), BMP-2 (C) and BMP-2/bFGF (D) gels after 12 weeks implantation. Porous titanium scaffolds and fixation screws appear in black whereas bone appears in dark grey. Scale bar = 1 mm.

3.2.2. In vivo and ex vivo micro-CT evaluation

In all groups, bone regeneration started at the proximal and distal bone adjacent to the defect and progressed towards the central area of the defect (Fig. 4). The defect was almost completely bridged (defined as a remaining gap <0.5 mm) in seven rats of the BMP-2/bFGF group, seven rats of the BMP-2 group, five rats of the bFGF group and one rat of the unloaded group. The average remaining gap distance was 0.33±0.34 mm for the BMP-2/bFGF group, 0.51±0.74 mm for the BMP-2 group, 0.83±0.53 mm for the bFGF group and 1.20±0.56 mm for the unloaded group. The 2D images of cross-sections along the vertical axis of the femurs also confirmed

substantial bone ingrowth into pores and the inner medullary space originating from proximal and distal ends (Fig. 5).

Figure 6. A) In vivo micro-CT quantification of total bone volume (BV) at four, eight twelve weeks after implantation. B) Ex vivo micro-CT quantification of total BV after twelve weeks of implantation. Total BV was defined as total volume of bone formed within the 6 mm defect area. Horizontal and vertical bars indicate statistically significant differences (p<0.05) between groups.

Bone regeneration was quantified using in vivo micro-CT by measuring bone inside the defect (total BV) at different time points following implantation. All groups showed an increase at each time point, but there was significantly more bone regenerated in the defects that received the GF-loaded gels than those that received the unloaded gels after twelve weeks (Fig. 6). BMP-2 gels strongly enhanced bone formation during the first four weeks, and total BV was significantly more than unloaded gels at four weeks (Fig. 6A). Total BV continued to increase between four and eight weeks, and reached a plateau phase during the last four weeks (8-12 weeks). bFGF gels did not show a significant increase in total BV after four weeks, but between four and eight weeks total BV increased rapidly. Finally, bFGF gels resulted in 40.0±7.6 mm3

total BV, which was comparable to BMP-2 gels (42.9±18.9 mm3_{). BMP-2/bFGF gels}

continuously enhanced bone formation during the entire follow-up period, resulting in the highest average total BV at twelve weeks (49.6±8.6 mm3_{) (Fig. 6B).}

Bone formation was most profoundly enhanced outside the porous titanium scaffolds (outer BV) Porous titanium scaffolds with BMP-2/bFGF gels resulted in significantly more bone than those with BMP-2 gels, or bFGF gels (Fig. 7A). Bone ingrowth into the porous titanium scaffold (porous PV), however, was not significantly enhanced by the GF-loaded gels (Fig. 7B). Porous BV was 13.6±7.2 mm3 _{with BMP-2/bFGF gels,}

15.3±7.6 mm3 _{with BMP-2 gels, 14.4±4.2 mm}3 _{with bFGF gels and 10.4±5.3 mm}3

with unloaded gels. bFGF-gels resulted in significantly more bone inside the porous titanium scaffold (inner BV) than BMP-2 gels. Inner BV was 6.1±0.8 mm3 _{with bFGF}

gels and 4.0±1.6 mm3 _{(p=0.033) with BMP-2 gels (Fig. 7C), respectively. The latter}

was comparable to BMP-2/bFGF gels (4.3±2.1 mm3_{) and unloaded gels (4.2±1.4}

mm3_).

Figure 7. Ex vivo micro-CT quantification of outer BV (A), porous BV (B) and inner BV (C) at twelve after weeks implantation. Outer BV was defined as bone formed outside the porous titanium scaffolds; porous BV: bone formed inside the porous space of the titanium scaffolds; and inner BV: bone formed in the medullary canal of the scaffold. Horizontal bars indicate statistical significant differences (p<0.05).