Bone regeneration in surgically created defects filled with autogenous bone: an epifluorescence microscopy analysis in rats

Abstract

Although the search for the ideal bone substitute has been the focus of a large number of studies, autogenous bone is still the gold standard for the filling of defects caused by pathologies and traumas, and mainly, for alveolar ridge reconstruction, allowing the titanium implants installation.

Objectives: The aim of this study was to evaluate the dynamics of autogenous bone graft incorporation process to surgically created defects in rat calvaria, using epifluorescence microscopy.

Material and methods: Five adult male rats weighing 200-300 g were used. The animals received two 5-mm-diameter bone defects bilaterally in each parietal bone with a trephine bur under general anesthesia. Two groups of defects were formed: a control group (n=5), in which the defects were filled with blood clot, and a graft group (n=5), in which the defects were filled with autogenous bone block, removed from the contralateral defect. The fluorochromes calcein and alizarin were applied at the 7th and 30th postoperative days, respectively. The animals were killed at 35 days.

Results: The mineralization process was more intense in the graft group (32.09%) and occurred mainly between 7 and 30 days, the period labeled by calcein (24.66%).

Conclusions: The fluorochromes showed to be appropriate to label mineralization areas. The interfacial areas between fluorochrome labels are important sources of information about the bone regeneration dynamics.

Figure 1

Rat calvaria scheme in an upper view. The 5-mmdiameter surgical bone defects were localized bilaterally at the parietal bones and were filled as illustrated

Figure 2

Surgical procedure. A) Surgical access. A linear incision in an anterior-posterior direction was made in the median region of calvaria and the dermoperiostic was detached. B) Trephine bur used to prepare the bone defects. C) The bone block removed from the left side was positioned in the right side defect. D) An approximate view of the defects to be filled

Figure 3

Schematic presentation of the application of calcein (7th postoperative day) and Alizarin (30th postoperative day) application (20 mg/kg body weight, IM)

Figure 4

Preparation of the 150-μm-thick sections. The arrows indicate the wear direction to the center of the defect

Figure 5

Standardization of the analyzed area. The square lines delimit the center of the defect, selected to be the analyzed area. The yellow arrow show the block graft and the white arrows show the boards of the defect

Figure 6

Image superposition scheme. The calcein and the alizarin images were superimposed in a computer program to obtain the final studied images

Figure 7

Total area (TA) standardization. A) The dashed lines delimit the area; B) Imagelab 2000® program calculated the area in pixels

Figure 8

Calcein (A) and Alizarin (B) labeled areas delimited by dashed lines. (Imagelab 2000®)

Figure 9

Fluorochrome-labeled areas in the Control Group. Three of five animals did not show labeled areas (green = calcein ; red = alizarin)

Figure 10

Fluorochrome-labeled areas in the Graft Group. The five animals showed strong labeled areas (green = calcein; red = alizarin)

Figure 11

Percentage reached by calcein and alizarin in relation to the total area (TA). (G = Graft Group; C = Control Group). Results are expressed as mean ± standard deviation *Differs significantly from the Control group labeled by calcein (P = 0.008) (Mann-Whitney Test) **Differs significantly from the Graft Group labeled by calcein. (P = 0.008) (Mann-Whitney Test) *** Differs significantly from the Graft Group labeled by alizarin. (P = <0.001) (T Test)

Figure 12

Sum of the areas labeled by calcein and alizarin in the Graft (G) and Control (C) groups. Results are expressed as mean ± standard deviation * Differs significantly from the Control Group. (P < 0.001) (T Test)