Evaluation of angiogenesis of bioactive glass in the arteriovenous loop model

Abstract

In this study, the angiogenetic effect of sintered 45S5 Bioglass® was quantitatively assessed for the first time in the arteriovenous loop (AVL) model. An AVL was created by interposition of a venous graft from the contralateral side between the femoral artery and vein in the medial thigh of eight rats. The loop was placed in a Teflon isolation chamber and was embedded in a sintered 45S5 Bioglass® granula matrix filled with fibrin gel. Specimens were investigated 3 weeks postoperatively by means of microcomputed tomography, histological, and morphometrical techniques. All animals tolerated the operations well. At 3 weeks, both microcomputed tomography and histology demonstrated a dense network of newly formed vessels originating from the AVL. All constructs were filled with cell-rich, highly vascularized connective tissue around the vascular axis. Analysis of vessel diameter revealed constant small vessel diameters, indicating immature new vessel sprouts. This study shows for the first time axial vascularization of a sintered 45S5 Bioglass® granula matrix. After 3 weeks, the newly generated vascular network already interfused most parts of the scaffolds and showed signs of immaturity. The intrinsic type of vascularization allows transplantation of the entire construct using the AVL pedicle.

FIG. 1.

The arteriovenous loop was placed in the Teflon isolation chamber that was filled with the sintered 45S5 Bioglass^®-granulated matrix and fibrin gel with a fibrinogen concentration of 10 mg/mL and a thrombin concentration of 2 I.U./mL. (A) arteriovenous (AV) loop placed on the first half of the matrix; (B) Chamber filled with the complex matrix. Color images available online at www.liebertpub.com/tec

FIG. 2.

Macroscopic appearance upon explantation: After removing the Teflon isolation chamber, Bioglass^® granules showed no signs of degradation or resorption. Vascularization of matrices and patency of the arteriovenous loops were visible because of yellow filling of functional vessels with Microfil^®. Color images available online at www.liebertpub.com/tec

FIG. 3.

Hematoxylin and eosin stainings: The newly formed tissue was localized around the vascular pedicle (arrows) with a reduction in the central part and the periphery of the arteriovenous loop. Functional vessels were observed by black filling of lumina with Microfil in the connective tissue. Magnification ×25. Color images available online at www.liebertpub.com/tec

FIG. 4.

Immunhistochemical stainings: Endothelial cells could be detected using Lectin stainings (A). Alpha-smooth muscle actin immunohistochemical stainings showed smooth muscle cells inside the vessel walls and the connective tissue (B). Using ED1 stainings, some macrophages could be detected without showing multinucleated giant cells (C). Magnification ×100. Color images available online at www.liebertpub.com/tec

FIG. 5.

MicroCT analysis strategy of accumulated vessel length with regard to vessel diameter: Measured raw data sinograms (A) were reconstructed with tomographic reconstruction software. The three-dimensional visual images and STL models were generated using imaging software after labeling with a global threshold and using a 26-side growing algorithm on all layers (B). After binarization, additional skeletonization using thinning algorithms was performed to determine the network structure of the vessels. The vessel network was divided into segments with individual length and mean diameter (C). Color images available online at www.liebertpub.com/tec

FIG. 6.

Morphometrical analysis of blood vessel distribution of cross sections. At 3-week explantation time point, there was an equal number of blood vessels in the arterial and venous parts of the constructs with a decrease in the central part of the specimens detectable.

FIG. 7.

Microcomputed tomography (μCT) analysis of accumulated vessel length with regard to the vessel radius. The histogram displays a peak at a vessel radius of 10 μm with a rapid decrease in the vessel length in larger vessels. The μCT had a resolution of 6.9 to 8 μm/voxel.