Automatic quantitative micro-computed tomography evaluation of angiogenesis in an axially vascularized tissue-engineered bone construct.

INTRODUCTION

We invented an automatic observer-independent quantitative method to analyze vascularization using micro-computed tomography (CT) along with three-dimensional (3D) reconstruction in a tissue engineering model.

MATERIALS AND METHODS

An arteriovenous loop was created in the medial thigh of 30 rats and was placed in a particulated porous hydroxyapatite and beta-tricalcium phosphate matrix, filled with fibrin (10 mg/mL fibrinogen and 2 IU/mL thrombin) without (group A) or with (group B) application of fibrin-gel-immobilized angiogenetic growth factors vascular endothelial growth factor (VEGF¹⁶⁵) and basic fibroblast growth factor (bFGF). The explantation intervals were 2, 4, and 8 weeks. Specimens were investigated by means of micro-CT followed by an automatic 3D analysis, which was correlated to histomorphometrical findings.

RESULTS

In both groups, the arteriovenous loop led to generation of dense vascularized connective tissue with differentiated and functional vessels inside the matrix. Quantitative analysis of vascularization using micro-CT showed to be superior to histological analysis. The micro-CT analysis also allows the assessment of different other, more complex vascularization parameters within 3D constructs, demonstrating an early improvement of vascularization by application of fibrin-gel-immobilized VEGF¹⁶⁵ and bFGF.

CONCLUSIONS

In this study quantitative analysis of vascularization using micro-CT along with 3D reconstruction and automatic analysis exhibit to be a powerful method superior to histological evaluation of cross sections.