Evaluation of Bone Remodeling with Fluorescent Markers in Rats after Implant

GOIA TS; VIOLIN KB; PORTIANSKY EL; BRESSIANI AH.

Brasil

Congreso; International Microscopy Congress 17; 2010

The understanding of the growth pattern of bone tissue after implantation may help the development of implants with specific features. Moreover, it may establish the recovery time necessary for each step in bone repair. This study aimed to identify the different stages of bone remodeling during repair after surgical implantation of porous metallic implants with the aid of fluorescent markers in rats. Implants of Ti-13Nb-13Zr alloy with mean porosity of 30% and cylinder shaped were surgically positioned in femurs of 10 weeks old male Wistar rats. The used fluorescent markers for bone tissue were calcein, alizarin and tetracycline at concentrations of 8 mg/kg, 20 mg/kg and 10 mg/kg, and administered on day 7, 14 and 21 after surgery, respectively. Animals were euthanized on day 28 and bone-implant samples were fixed with formalin solution. The blocks of calcified tissue samples were prepared using Technovit® 9100 NEU resin and the histological slides from the blocks were performed using Isomet® cutter. The slides without staining were evaluated under fluorescent microscopy using the filter UV-2E/C for calcein, filter B-2E/C for alizarin and no filter for tetracycline from Nikon® E-1000. Monochrome images were taken, digitally pseudocolored and merged using the ImagePro Plus® 6.3 analysis image software. Green pseudocolor was used for calcein, red for alizarin and blue for tetracycline. Calcein (green color) was seen in the bone after implantation followed by the next stage of remodeling where alizarin (red color) was observed. Tetracycline (blue color) was also observed in both stages. When calcein and alizarin colocalized a yellow color was detected. The same was true for the colocalization of alizarin and tetracycline (magenta color). From the analysis of the data it appears that calcein can be seen during the second week of bone repair while alizarin appears on the third week. The magenta/blue areas were observed mostly during the fourth and last week of repair. Inside the implant the internalization of the bone ingrowths takes place mostly during the third week of repair (red color) surrounded by green areas (second week) and few blue dots (FIG. 1).