Study of the Mechanical and Structural Properties of Porcine-Derived Small Intestine Submucosa Decellularized with Triton X-100 at Different Concentrations

Research Article

Austin J Biomed Eng. 2020; 5(1): 1041.

Study of the Mechanical and Structural Properties of Porcine-Derived Small Intestine Submucosa Decellularized with Triton X-100 at Different Concentrations

Sordelli A1*, Jaunarena JH1, Loresi M1, D’adamo M1, Albite R1, Costa L1, Leonardi L2, Londoño Calderón CL2, Froimowicz P2, DeBadiola F3, Moldes JM3, Giúdice C4, Güeglio G4 and Villoldo GM1,4

1Institute of Translational Medicine and Biomedical Engineering, Hospital Italiano de Buenos Aires, Argentina

2School of Engineering, Institute of Technology in Polymers and Nanotechnology, Argentina

3Pediatric Urology Department, Hospital Italiano de Buenos Aires, Argentina

4Urology Department, Hospital Italiano de Buenos Aires, Argentina

*Corresponding author: Sordelli A, Institute of Translational Medicine and Biomedical Engineering, Hospital Italiano de Buenos Aires, Buenos Aires, Argentina, CONICET, Potosí St 4240, Buenos Aires, C1199ACL, Argentina

Received: September 25, 2020; Accepted: October 20, 2020; Published: October 27, 2020


Introduction: Porcine-derived small intestine submucosa has been widely used in tissue engineering. Cell components must be removed to preclude transplant rejection in animal models. Known as decellularization, this type of process can damage collagenous fibers and thus affect the integrity of the extracellular matrix. Purpose: Determine whether structural and physical changes occur in SIS when applying increased concentrations of a non-ionic detergent, Triton X-100, together with fixed concentrations of 0.1% sodium azide. Materials and Methods: SIS was collected, sectioned and placed in distilled water for 24 h before different concentrations of Triton X-100 were applied for 48 h. Samples were removed and stained with hematoxylin and eosin, Masson’s trichrome and Hoechst. Physical characterization was carried out via traction assays; we calculated the rupture strain, ultimate tensile strength and Young’s modulus, and we performed infrared spectroscopy and thermogravimetric analyses. We determined the concentration of remaining DNA.

Results: Regardless of the concentration of detergent used, no cells were revealed at 48 h post-treatment. The patterns of rupture strain, ultimate tensile strength and Young’s modulus, as well as the infrared spectra, exhibited no significant differences between the use of high and low detergent concentrations. A higher detergent concentration results in a lower residual DNA concentration.

Conclusions: It can be concluded that, irrespective of the concentration of Triton X-100 used, at 48 hours post-treatment, decellularization is efficient and does not alter the structural or the physical characteristics of the material. Residual DNA amounts decrease with higher doses and longer treatment times.

Keywords: SIS (Porcine-derived small intestine submucosa); Decellularization; Tissue engineering; Regenerative medicine; Characterization


SIS: Porcine-derived Small Intestine Submucosa; ECM: Extracellular Matrix; FTIR: Performed Infrared Spectroscopy TGA: Thermogravimetric Analyses; PFA: Paraformaldehyde; H&E: Hematoxylin and Eosin; CEPI: Research Ethics Committee; GAGs: Glucosaninoglycans


Matrices derived from natural tissue, such as porcine-derived Small Intestine Submucosa (SIS), have been successfully used both in lab and clinical settings [1-6]. SIS can be easily harvested, processed and handled [2], is biocompatible, degrades easily and resorbs rapidly into the receptor tissue [7,8]. When SIS is transplanted into animals different processes are triggered, such as neovascularization, infiltration with fibroblasts, release of growth factors and other biologically active substances required for restoration processes [9,10].

Natural collagenous matrices need to be decellularized to be used as scaffolds [11]. Several kinds of detergents have been employed at different concentrations for SIS decellularization, but ideal concentrations and exposure times for maximum performance and minimum damage remain unclear. We aimed to analyze the effectiveness of the SIS-decellularization process and find variations in the physical and mechanical properties of SIS under increased concentrations of Triton X-100 combined with fixed concentrations of sodium azide.

Materials and Methods

This study was approved by the Research Ethics Committee of our institution.

SIS collection

SIS was harvested from two male pigs weighing approximately 15kg each. Under aseptic conditions and general anesthesia at 10 minutes post-euthanasia, a median supra-infraumbilical incision was made to obtain a 20cm of jejunum. Mesenteric vessels were bound together at the intestinal margin and the remaining intestine was resealed. The specimen was placed in saline solution and the enteric contents were removed.

SIS processing

Slightly-modified SIS was prepared following Badylack et al.’s protocol [2]. The specimen was placed in saline solution, was cut open at the mesenteric border and the seromuscular layer was removed by careful dissection, moistened gauze was used to mechanically remove the mucosal layer. A 1mm-thick transparent membrane was obtained, sectioned into 5x4 cm rectangles and placed on an orbital shaker (Thermo Scientific) with distilled water for continuous agitation at 200rpm and 37oC for 24 hours. These were then immersed in 250ml screw-cap glass containers (Schott) containing 0.5, 1, 1.5 and 2% Triton X-100 and 0.1% sodium azide solutions and shaken continuously. Samples were collected after 24 and 48 hours.

Histology and morphologic analysis

The matrices were fixed in 4% Paraformaldehyde (PFA), processed and embedded in paraffin. Then they were stored in increased alcohol concentrations (70-100%) and finally cleared with xylene. After this, the paraffin-embedded matrices were sectioned to 4μm thickness.

Routine staining was performed, including Hematoxylin and Eosin (H&E) to validate that decellularization had been satisfactory; Masson’s trichrome to determine the structure of the ECM, and Hoechst to visualize eventually remaining cell nuclei. A Nikon Eclipse E400 microscope was used.

DNA quantification

DNA was extracted from frozen SIS exposed to different detergent concentrations. Each membrane was sectioned into 1g small chunks, which were then placed in a tube. The DNA extraction kit (QIAamp DSP DNA Blood Mini QIAGEN) was filled with a softer tissue lysis buffer (Buffer ATL) and an enzyme (Proteinase K) for SIS molecular disintegration. After incubation at 56 and 37oC respectively, a stronger lysis buffer (Buffer AL) was added to the tube for a 10-minute reincubation at 70oC. Then 100% ethanol was added in a QIAamp Mini spin column of the extraction kit.

After successive spin steps and buffer (AW and AW2) addition, the column was transferred to a tube and DNA was extracted with AE buffer. The DNA in each SIS sample was quantified using the GENESYS 10S spectrophotometer and the ADN.mfx method (which measures wavelengths using a factor) and the result (average after five measurements) was expressed in μg. A curve was plotted with the untreated, water-treated and detergent-treated SIS values, the latter including different detergent concentrations and exposure times.

Hydration time measurement

SIS rectangles were placed on an Explorer Ohaus balance with draft shield to determine their dry weight and were subsequently hydrated in a beaker with saline solution at 25oC. These were then weighed at 5-minute intervals until the matrices’ weight remained unchanged in three consecutive readings and reached a stable weight. The “optimal hydration time” was defined as the first stable reading was obtained.

Mechanical characterization

Uniaxial tensile tests were performed on rectangular decellularized and hydrated SIS samples of 50.8mm in nominal width and varying lengths, i.e. lengths depending on the intestine they had been longitudinally sectioned from. All tests were carried out at room temperature in an Instron 5982 dynamometer with a 1kN load cell at a crosshead speed of 10mm/min until complete fracture. All this data was analyzed using INSTRON’s Bluehill 3® software; stress-strain curves were obtained and calculations were performed to determine the properties of the material. Load-elongation curves were obtained and converted into stress-strain curves using the following: Stress s=F/Ao, where F is the force applied to N and AO is the initial crosssection of each test specimen calculated as its width times its thickness. The thickness was measured using a micrometer (accuracy=0.001 mm) once the test tubes were subjected to a preload of 0.5 N.

Strain ε=Δl/lo, where Δl is the elongation equal to l– lo, and l and lo, the instantaneous and reference length between clamps, respectively.

Young’s modulus was obtained as the initial slope of the stressstrain curve for linear elastic behavior, i.e. where the material deforms but remains recoverable. The ultimate tensile strength (sUTS) was measured as the maximum stress withstood before material fracture, and the strain at break (eb) as the maximum strain experimented by the material. At least six measurements were performed for each group. The average values and their corresponding standard deviations were reported.

SIS lyophilization for Fourier-Transform Infrared (FT-IR) spectroscopy

In order to perform FT-IR spectroscopy and Thermogravimetric Analysis (TGA), samples were first lyophilized in a LABCONCO Freezone 2.5 lyophilizer, model 7670530, for 24-48 hours at room temperature.

FT-IR spectroscopy

Fourier-transform infrared spectra were recorded on a Shimadzu IR Affinity-1 spectrophotometer on net samples. For each spectrum 40 scans were averaged, spectral window was from 450 to 4000 cm-1, at a resolution of 4cm-1. Each sample was measured 6 times in different regions, thus generating a spectrum reflecting the average of all six.

Thermogravimetric analysis

Thermograms for TGA were recorded using a Shimadzu TGA- 50 thermogravimetric analyzer. Sample temperatures were increased once, from room temperature to 600oC. Nitrogen atmosphere (30mL/ min) was used and the heating ramp rate was set at 10oC/min in all cases. Thermal decomposition temperature was determined as the onset on the curve of the first derivative of each thermogram.


Data are presented as absolute values or percentages as appropriate. Statistical line, dot and bar charts were used, with 95% confidence intervals represented by the whiskers. A one-way ANOVA was used for group comparison while pair comparison (post hoc) was performed with the Bonferroni correction. For this purpose, IBM SPSS v19.0 (SPSS Inc., Chicago, IL, USA) software was used. Values of p<0.05 were regarded as statistically significant.


SIS collection

At a macroscopic level, a 1 mm-thick, elastic and resistant off white translucent membrane was observed.

Microstructural features

Collagenous fiber structures were preserved after the detergentbased decellularization protocol. Evaluation revealed that decellularization caused variable results (typical of biological samples) and that Triton X-100 treatment causes reduced thickness in some ECM areas. In most cases, however, the structure was preserved, and presented only some regional variations in terms of fiber alignment but no significant differences (i.e., the ultrastructure was preserved). No further damage to the collagenous fibers was observed after increasing the detergent concentration.

Hoechst staining revealed a slight variation in the count of cells attached to SIS (Figure 1). In order to estimate the nuclei count across samples, these were sorted out into groups of n=5 samples per treatment; five points were chosen randomly in each Hoechststained section (with 40x magnification) and averaged. A high nuclei count was observed in the native (untreated) SIS sample, but after placing the SIS in distilled water and applying continuous agitation for 24 hours, a significantly decreased nuclear content was observed although the nuclei count remained high. Histological results revealed a small number or absence of remaining nuclei after the 24-hour protocol (Figure 1C), and a complete absence of nuclear material in the samples following the 48-hour protocol (Figure 1D). The difference observed was statistically significant (p<0.05) when comparing native SIS with each of the groups after 48 hours.