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

Abstract

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.