Ultrastructural changes in esophageal tissue undergoing stretch tests with possible impact on tissue engineering and long gap esophageal repairs performed under tension

Preparation of the specimens and stretch tests

Ovine esophagi from 1-month-old lambs were obtained from the local abattoir and transported on ice to the laboratory for the commencement of the investigations within 4–6 h. All experiments were conducted under the guidelines of the Committee for Animal Research, Federal Ministry of Science and Research, Vienna, Austria.

The methodology of the stretch tests remained similar to our previously published research (Fig. 1)¹⁶. The esophagi were divided into 4 groups (4 esophagi/group): control, Group 1 (G1), Group 2 (G2), Group 3 (G3) stretched to 20{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, 30{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} and 40{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} of their original length respectively. On arrival in the laboratory, the esophagus was rinsed with PBS to remove any contents within the lumen. The middle 70–80 mm portion of the esophagus was prepared and utilized for the investigations (Fig. 1a). The ends of the esophagus were secured with yarns to the plastic screws of the strain instrument. Gage markers for the length measurements via the video extensometer were glued to the esophagus 1 cm apart in the mid portion of the investigated segment. The uniaxial stretch test experiments were performed on a computer-controlled screw-driven high precision tensile/compressive testing machine adapted for small biological specimens (μ-Strain Instrument ME 30–1, Messphysik) (Fig. 1b).

Experimental setup. (a) Mid-sections of the esophagus was investigated with gage markers glued for video extensometer detection. (b) The ends were secured with yarns to plastic screws for insertion in the equipment. The esophagus was immersed in a Perspex container filled with PBS at body temperature.

The specimens were investigated in a Perspex container filled with PBS solution (pH 7.3 containing 100 mg/l EDTA) maintained at 37 ± 0.1 °C by a heater-circulation unit (type Ecoline E 200, Lauda; Lauda-Königshofen, Germany) The tensile force was measured with a 25 N class 1 strain gage-load cell (model F1/25 N, AEP converter)¹⁷. The upper and lower crossheads of the testing machine are moving in opposite directions to enable the gage region of the samples in the constant field of view. A crosshead stroke resolution of 0.04 μm and a minimum load resolution of 1 mN of the 25 N load cell were employed. Digital control of the electric drive of the machine as well as data acquisition of the crosshead position and the applied load was done by an external digital controller (EDC 5/90 W, DOLI; Munich, Germany). For automatic gage mark and edge recognition a PC-based (CPU 586) video extensometer (model ME 46–350, Messphysik) was used^{17, 18}. The experiments were conducted at 10 mm/min with samples subjected to 5 cycles each to achieve the desired stretch per group.

Electron microscopic investigation

After completion of the stretch experiments, all esophagi from all groups were sampled multiple times and locations. The harvested 1–2 mm³ muscular layer samples 4 groups were fixed in 2.45{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} glutaraldehyde and 2.45{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} paraformaldehyde in a 0.1 M sodium cacodylate buffer (pH = 7.4) at room temperature for 4 h and at 4 °C for 16 h. The samples were washed in a 0.1 M sodium cacodylate buffer (pH = 7.4) at room temperature for 3 h and then post-fixed with 2{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} osmium tetra oxide at room temperature for 2 h. Afterwards the tissue was dehydrated in graded series of ethanol (50{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, 70{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, 90{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, 96{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, and 100{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847}, each for 30 min at room temperature) and embedded in TAAB epoxy resin (TAAB Laboratories Equipment Ltd., UK). To determine the range of interest of the embedded samples, semi-thin Sects. (500 nm) of the embedded tissue was stained with 0.5{64d42ef84185fe650eef13e078a399812999bbd8b8ee84343ab535e62a252847} toluidine blue (FLUKA, Lot: BCBL 8513 V) in an aqueous solution and analyzed with light microscopy (Nikon Eclipse E800, equipped with a Nikon DN100 camera). For transmission electron microscopy (TEM), ultra-thin sections were prepared (70 nm) with ultramikrotom (LEICA, UC6, Wetzlar, Germany) and transferred on to copper grids. Additionally, they were stained with uranyl acetate (TED PELLA Inc., Lot: CA-1206) and lead citrate (DELTA MICROSCOPIES, Lot: REY 2618). For transmission electron microscopy FEI Tecnai G2 20 (Hillsboro, Oregon, United States) was used, equipped with CCD camera (Gatan US1000, Gatan, Pleasanton, USA) to obtain pictures. For image post-processing and measurements i-Team V10.1 (Olympus Soft Imaging Solutions GmbH, Technologiepark, Münster, Germany) was used.

Quantification analysis

To compare and quantify the ultrastructural changes in the tissues the following measurements were done in all 4 groups. To objectively determine the severity of intracellular damage, correlations between stretch levels and the diameter of the muscle cells as well as distance between myofibrils in the muscle cells were analyzed. To quantify the effect of mechanical stress on to extracellular matrix and connections between muscle cells the distance between cell membranes lying next each other was measured (Fig. 2). Previous three morphological parameters were measured in all groups as many times as possible on every sample (average number of measurements done per groups: Distance between muscle cells N = 299, Diameter of the muscle cells N = 213, Distance between myofibrils N = 970).

TEM measurements method. Electron microscopic image showing measured distance between cells (lines) and myofibrils (double arrows); distance of phospholipid membranes measured in 1–2 μm gradients and at the level of Z lines of sarcomeres.

For statistical analysis IBM SPSS Statistics 27.0.1 (Armonk, New York, USA) was used. Continuous data were tested for normality and presented in means and standard deviations. Outliers were interpreted as measurement errors and excluded. Independent-Samples Kruskal–Wallis test was used to compare groups. The null hypothesis was that the distribution of measured distances is the same across the 4 Groups. A p < 0.05 was considered to be significant. Significance values have been adjusted by the Bonferroni correction for multiple tests.