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

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.

Rho-inhibition by local application of c3-toxin for enhancement of axonal sprouting in a rat end-to-side nerve repair model.

INTRODUCTION: The aim of the presented study was to investigate nerve regeneration after application of C3-Toxin, a Rho-GTPase inhibitor and to correlate morphometry, neurophysiology, and function in an end-to-side peroneal/tibial nerve repair model of the rat. MATERIALS AND METHODS: Twenty rats with a peroneal to tibial end-to-side neurorrhaphy were divided into two groups: 1) control group, 2) C3 fusion toxin group with intrafascicular application of 1 µg/100 µl C3 fusion toxin. Survival time was 8 weeks. Nerve conduction velocities and motor function were analyzed and histomorphological evaluation consisting of measurement of intraneural collagen level, axon count, total nerve area, myelination index, and N-ratio followed. RESULTS: Evaluation of motor function and nerve conduction did not show any statistical differences. Histological analysis revealed higher axon count, thicker myelin sheaths, and higher myelination index in the C3 fusion toxin group (P < 0.001). Comparison of N-ratio and intraneural collagen level were without statistical significance. CONCLUSION: The current study shows that application of C3 fusion toxin leads to higher myelination and increases axonal sprouting.

Retrospective evaluation of diagnostic accuracy of free flap monitoring with the Cook-Swartz-Doppler probe in head and neck reconstruction.

The Cook-Swartz-Doppler probe is an easy to handle and reliable tool for free flap monitoring. In the head and neck region different confounders can affect the read out. We therefore analyzed the use of the Doppler probe regarding these potential difficulties and to compare the diagnostic accuracy in arterial or venous monitoring of free flaps in the head and neck region. A retrospective study was performed in which all patients were included who underwent free flap surgery in the head and neck region in the Department of Plastic Surgery and the Department of Maxillofacial Surgery of our institution between 2010 and 2018 and were monitored with an implanted Doppler probe. 147 free tissue transfers were included. No significance was found for arterial and venous placement of the Doppler probe for sensitivity (artery 83.3%; vein 84.6%; p = 0.87), specificity (artery 89.2%; vein 96.1%; p = 0.17) and negative predictive value (artery 96.7%; vein 94.2%; p = 0.55). A better positive predictive value for placing the Doppler probe around the artery (82.7%) than the vein (61.1%) was found in our study (p = 0.056). The better positive predictive value in arterial monitoring suggests that this is the more reliable measuring method to assess flap perfusion in the head and neck region.

The Neuro-spheroid--A novel 3D in vitro model for peripheral nerve regeneration.

BACKGROUND: In order to reduce in vivo animal experiments in peripheral nerve regeneration research, in vitro models are desirable. Common two dimensional (2D) co-culture models lack the complex interactions of three dimensional (3D) physiological structures. The aim of the study was to establish a neuronal 3D spheroidal sprouting assay for peripheral nerve regeneration. NEW METHOD: Spheroids consisting of Schwann cells (SC, 500 cells/spheroid) and NG108-15 cells (NG, 50 cells/spheroid), a hybrid cell line, were formed in hanging drops and were embedded in a 3D collagen matrix. Spheroid sprout lengths were compared to those of the neurites of NG in a 2D co-culture with SC. Lengths were measured using phase contrast images taken every day over 10 days. Additionally we took fluorescence images to visualize the PKH26-labeled NG in both culture systems. RESULTS: Initially thin neurites grew out in both co-cultures, over time the sprouts' diameter in the 3D culture increased. The direct comparison of the sprout length revealed significantly longer neurites in the 3D co-culture from day 7 until day 10 (p<0.001). COMPARISON WITH EXISTING METHODS: Other co-culture models either display processes in 2D or need complex matrices to create 3D structures. Our spheroidal model is easy to establish, highly flexible and nevertheless 3D. CONCLUSIONS: The 3D-Schwann cell-neuron spheroid model shows that by simply transferring a 2D into a 3D co-culture with multiplication of cell-cell contacts, a significant increase of neurite length can be achieved. The model is a relatively simple method for the investigation of neurite development in vitro.