Automatic quantification of angiogenesis in 2D sections: a precise and timesaving approach

Autor: Johannes G. Hilgert, Nico Leibig, Jennifer Covi, Ulrich Kneser, Christian Weis, Raymund E. Horch, Andreas Arkudas, Volker J. Schmidt
Rok vydání: 2015
Předmět:
Zdroj: Journal of Microscopy. 259:185-196
ISSN: 0022-2720
Popis: Summary Introduction The standardized characterization of angiogenesis is crucial in the field of tissue engineering as sufficient blood supply is the limiting factor of mass transfer. However, reliable algorithms that provide a straight forward and observer-independent assessment of new vessel formation are still lacking. We propose an automatic observer-independent quantitative method (including downloadable source code) to analyze vascularization using two-dimensional microscopic images of histological cross-sections and advanced postprocessing, based on a ‘positive- and negative-experts’ model and a (corrected) nearest neighbour classification, in a vascularized tissue engineering model. Materials and Methods An established angioinductive rat arteriovenous loop model was used to compare the new automatic analysis with a common 2D method and a μCT algorithm. Angiogenesis was observed at three different time points (5, 10 and 15 days). Results In line with previous results, formation of functional new vessels that arose from the venous graft was evident within the three-dimensional construct and a significant (p < 0.05) increase in vessel count and area was observed over time. The proposed automatic analysis obtained precise values for vessel count and vessel area that were similar to the manually gained data. The algorithm further provided vectorized parameterization of the newly formed vessels for advanced statistical analysis. Compared to the μCT-based three-dimensional analyses, the presented two-dimensional algorithm was superior in terms of small vessel detection as well as cost and time efficiency. Conclusions The quantitative evaluation method, using microscopic images of stained histological sections, ‘positive- and negative-experts’-based vessel segmentation, and nearest neighbour classification, provides a user-independent and precise but also time- and cost-effective tool for the analysis of vascularized constructs. Our algorithm, which is freely available to the public, outperforms previous approaches especially in terms of unambiguous vessel classification and statistical analyses. Lay Description Blood supply is the crucial limiting factor in Tissue Engineering. While small constructs can be nourished by diffusion up to 200 μm larger three-dimensional construct require an extrinsic or intrinsic blood supply with its own vascular network. The intrinsic blood supply has major advantages for Tissue Engineering compared to the extrinsic vascularization. It provides immediately blood to the center of a three-dimensional construct, instead of a period of ingrowths form periphery into the core of the construct. Additionally, it is possible to transfer an intrinsic or axially vascularized construct as free tissue transplantation. Though, a reliable vascular network within the newly formed construct is required. Therefore reliable evaluation of the vascularization in tissue engineered constructs is critical for their preclinical assessment. To investigate the various attempts of angiogenesis, a reproducible tool for quantification of the vessel number is necessary to achieve an objective evaluation for this matter. The manuscript describes a new method to evaluate automatically vascularization in histological 2D sections in a standardized and high quality manner. The algorithm enables to segment vessels unambiguously und user-independently. It is straight forward because users only have to define "experts" as representative examples for vessels at the beginning of analyses and the process of quantification is performed automatically. The vectorized parameterization, which is the equivalent of a coordinate system transformation, enables on the one hand to calculate a whole new set of parameters (e.g. cumulative amount of vessels vs. distance to main vessels) and allows on the other hand a better comparison between the samples (even of different sizes). The used nearest neighbour classification is ideally suited to analyze vascularized constructs, because hereby relationship to the defined reference point (e.g. main vessel, center of the construct) is measured and not spatial orientation within the histological specimen. With the described method whole histological sections are analysed, not only Region of Interests (ROI), which makes it more accurate regarding quantification of newly formed vessels.
Databáze: OpenAIRE
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