Tissue harvest and storage
Nine pairs of human cadaveric legs (age = 82 ± 10 years old, 5 male, 4 female) were obtained from the Radboud University Medical Center (Department of Medical Imaging, Anatomy, Radboudumc, Nijmegen, The Netherlands). All procedures regarding the use of human tissue were strictly obeyed Article 67–69, of the ‘Wet op de Lijkbezorging’. None of the cadaveric legs had previous operative treatments of the knee joint. Specimens were wrapped in saline solution-soaked gauze and stored in a −20 °C freezer upon arrival. Legs were thawed at room temperature for 24 hours before tissue procurement. Menisci were procured by an incision perpendicular to the meniscal horn attachments and human remains were transported back (after harvesting of other tissues, e.g. tendons, for other research purposes) to the Radboud University Medical Center for anonymous cremation. After procurement, menisci were washed in phosphate buffered saline (PBS) for 1 hour at room temperature to remove excess blood and stored at −20 °C until further processing. Only macroscopically intact menisci without fibrillations or ossifications were used. In total, 14 medial (width = 46.8 ± 6.3 mm, thickness = 5.2 ± 0.7 mm) and 14 lateral menisci (width = 42.0 ± 2.7 mm, thickness = 6.7 ± 1.0 mm) were included in this study.
Decellularization
Decellularization was carried out based on a developed protocol previously developed for tendons and ligaments [5, 37]. In short, frozen menisci tissue was freeze-thawed five times in liquid nitrogen and 37 °C distilled water, washed in distilled water at room temperature overnight, and soaked in 1% Triton X-100 (Merck) in a Tris-EDTA buffer (pH 7.6; Merck, Sigma-Aldrich) for 24 hours. Then, samples were shortly washed in distilled water before soaking them for 24 hours in Benzonase (25 U/ml; Sigma-Aldrich) in a Tris buffer containing magnesium chloride hexahydrate (37 °C, pH 7.6; Sigma-Aldrich). Samples were then subjected to three washes of one hour in PBS + 2.7 mM EDTA (pH 7.6) at 37 °C and overnight incubation in distilled water at 4 °C. Next, samples were soaked in 1% Triton X-100 for 72 hours, shortly washed in distilled water, soaked in Benzonase for 48 hours at 37 °C, and washed three times 1 hour each in PBS + 2.7 mM EDTA at 37 °C. Finally, samples were washed in distilled water for 2 days. Distilled water was replaced every day. All steps were performed at room temperature on a rotator shaker unless stated otherwise. After the decellularization procedure, samples were stored at −80 °C until further usage.
Sterilization
Sterilization was performed in a Supercritical Fluid Extractor system (Waters Corporation) using supercritical carbon dioxide and peracetic acid (HCM Medical, Nijmegen). The sterilization procedure consisted of three steps: 1) A static process for 15 min with a flow of 20 g/min, a vessel temperature of 25 °C, and a pressure of 60 bar. 2) A second static process for 240 min with a flow of 20 g/min, a vessel temperature of 37 °C, and a pressure of 160 bar. 3) A dynamic flushing step, at a flow of 30 g/min, a vessel temperature of 37 °C, and a pressure of 160 bar. Samples were stored at −20 °C until further usage.
Sample digestion
To measure DNA, GAGs, and hydroxyproline (HYP), samples were digested, according to Kim et al. [19]. Briefly, a small piece (~ 3 mm × 3 mm) from the middle portion of each sample was taken and lyophilized for 48 hours. Three pieces of 2–3 mg of freeze-dried tissue were used as technical replicate from each meniscus and digested in 500 μL Papain digestion buffer [130 μg Papain (Sigma-Aldrich) per ml in 5 mM L-cysteine hydrochloride (Sigma-Aldrich) + 5 mM Na2EDTA (VWR)] for 24 hours at 60 °C in an Eppendorf ThermoMixer® at 300 rpm.
Determination of DNA content
The double-stranded DNA (dsDNA) content was quantified in decellularized (N = 8), decellularized + sterilized (N = 8), and native (2x N = 8) samples using the Qubit dsDNA High Sensitivity assay (Thermo Fisher Scientific) according to the manufacturers’ protocol. Readings were taken using the Qubit® 2.0 Fluorometer and data was normalized to tissue dry weight, obtained before digestion.
Visualization of cell nuclei
Preparation of tissue sections
Tissue sections of native (N = 3), decellularized (N = 3), and decellularized + sterilized (N = 3) samples were embedded in OCT Compound (Tissue-Tek®) and frozen on dry ice. Embedded samples were sectioned longitudinally at 6 μm and were fixed in 3.7% formaldehyde (Merck) for 20 min.
Hematoxylin and eosin stain
To visualize the remaining nuclei after decellularization and sterilization, tissue sections were stained with Hematoxylin and Eosin (H&E). Sections were incubated in Mayer’s Hematoxylin solution (Sigma-Aldrich) for 10 min, washed in distilled and tap water, incubated in aqueous Eosin Y solution (Sigma-Aldrich) for 1 min, and washed again. Finally, tissue sections were dehydrated in increasing series of ethanol and mounted with Entellan (Merck).
DAPI stain
To identify remaining cell nuclei after decellularization and sterilization, tissue sections were stained with 4′,6-diamidino-2-phenylindole dihydrochloride (DAPI; Sigma-Aldrich). Tissue sections were incubated in 1 μg/mL DAPI solution for 30 min and mounted with Mowiol (Merck).
Mechanical testing
Mechanical properties of medial menisci samples of five donors were determined using an unconfined stress-relaxation ball indentation test. Samples were prepared according to a previously described protocol by Maier et al. [24]. In short, one cylinder (diameter = 6 mm) per meniscus sample was punched out of the central portion using a biopsy punch (kai Europe GmbH). The superior and inferior surface of the cylindrical samples were cut parallel, resulting in cylinders of about 3 mm in height. During testing, the samples were placed in a custom-designed mold with a circular cavity (diameter = 6 mm, depth = 0.3 mm) to prevent lateral movement of the samples during testing. The custom-made indenter and tip consisted of a steel ball (diameter = 6 mm). Mechanical testing was performed on a mechanical tester (Criterion Model 42, MTS, Eden Prairie, Minnesota) with a 50 N load cell. A test cycle consisted of the following four phases: 1) Preloading at 0.1 N; 2) Dynamic compression from pre-load to 7 N at a velocity of 5 mm/min; 3) Stress relaxation at 7 N for 1 min (the indenter position was fixed at 7 N), and 4) Unloading to 0.15 N at a velocity of 1 mm/min. After an interval of 1 min, the next test cycle started, with a total of five repetitions. Load, indenter position, and time were recorded during testing. A graphical overview of one test cycle can be found in Fig. 1.
From the data, relative compression, as an indicator for tissue viscosity, was calculated from the indenter position at the end of dynamic compression in the 5th test cycle. The Young’s modulus was determined during the 2nd step of each test cycle, calculated from the linear slope of the engineering stress-strain curve. And finally, the residual force, a measure of the tissue’s viscoelastic behavior, was defined as the recorded load at the end of the 3rd step (static compression) of each test cycle.
Determination of hydroxyproline and glycosaminoglycan content
The total HYP content of decellularized (N = 8), decellularized + sterilized (N = 8), and native (2x N = 8) meniscus was determined, as a measure of collagen, according to Huszar et al. [16]. Total sulfated GAG content was quantified using a dimethyl methylene blue (DMMB) assay according to Farndale et al. [8]. A more detailed description of these assays can be found in the Supplements.
Structural properties
Picro Sirius red stain
The effect of decellularization and/or sterilization on collagen fibers was visualized using the Picro Sirius Red Stain Kit (Connective Tissue Stain; Abcam). Hydrated tissue sections were incubated in Picro Sirius Red solution for 60 min and rinsed with 3% acetic acid and absolute ethanol. Then, tissue sections were dehydrated in absolute ethanol and mounted with Entellan.
Alcian blue stain
Alcian Blue was used to visualize the effect of decellularization and/or sterilization on glycosaminoglycans. In short, tissue sections were incubated in 3% acetic acid solution (Abcam) for 3 min, incubated in 1% Alcian Blue solution (pH 2.5; Alcian Blue-8GX (Sigma Aldrich) dissolved in 3% Acetic Acid) for 30 min, and washed in 3% acetic acid, tap water, and distilled water. Subsequently, tissue sections were incubated in Nuclear Fast Red solution (Abcam) for 5 min, and washed again. Tissue sections were dehydrated in increasing series of ethanol and mounted with Entellan.
Determination of cell cytotoxicity
Tissue sections of native (N = 3), decellularized (N = 3), and decellularized + sterilized (N = 3) samples were embedded in OCT Compound and frozen on dry ice. Then, samples were longitudinally sectioned at 300 μm thickness. Meniscus sections were freeze-dried for 48 hours, punched to a diameter of 4 mm, in order to fit the well, and sterilized in serial dilutions of ethanol, 70%, 80%, 90%, and absolute, for 20 min each, followed by UV sterilization for 15 min. Once sterilized, the sections were washed in sterile PBS three times 20 min each and once in culture media (DMEM [4.5 g/mL glucose, 4.0 mM L-glutamine, 25 mM HEPES], 10% FBS, 1% P/S, 1% nonessential amino acids) for 20 min. Finally, tissue sections were incubated in culture media overnight at 37 °C and 5% CO2.
Human dermal fibroblasts (NHDF-Ad, Lonza CC-2511) at passage 7 were seeded on the meniscus tissue sections (2000 cells per tissue) and submerged in 100 μL culture media and incubated at 37 °C and 5% CO2. To determine cell cytotoxicity, an assay measuring Lactate Dehydrogenase (LDH) release was performed using the CyQUANT Cytotoxicity Assay Kit (Invitrogen) according to manufacturer’s protocol. Experiments were performed on day 1, 4, and 7. Cells cultured on a flat polystyrene surface were used as a negative control (0% cytotoxicity) and the maximum LDH release as a positive control (100% cytotoxicity).
Statistical analysis
Since there are structural differences between the medial and lateral meniscus, the meniscus from left or right knee, and within the meniscus itself [25], it was chosen to perform the statistical analysis in an unpaired fashion. All statistical analyses were performed in GraphPad Prism version 9.1 (GraphPad Software, Inc., San Diego, CA). One-way ANOVA or Kruskal-Wallis tests with, respectively, Šídák or Dunn’s post hoc multiple comparison tests were performed with significance level set at p < 0.05. Results are expressed as mean ± standard deviation.