Tissue sample preparation

Human tissue was temporarily stored after surgical resection in serum-free Dulbecco’s modified Eagle’s medium (Gibco, Life Technologies, Darmstadt, Germany) with 2% (v/v) penicillin-streptomycin and 1.2% (v/v) amphotericin B at room temperature. Further processing and measurements were carried out within 24 h of surgery, and the samples were fully immersed in DMEM to reduce the possibility of artifacts caused by dehydration and drying (Erisken et al., 2010). Due to the surgical procedure, the samples were received as fragments (<1 cm × 1 cm). From these tissue fragments, only areas with a clearly identifiable anulus fibrosus collagen architecture were selected (Figure 2). Only one sample per human patient was analysed. Samples were embedded in water-soluble embedding medium (Tissue-Tek O.C.T. Compound, Sakura Finetek, Alphen aan den Rijn, Netherlands) and sectioned with 35 µm thickness (Leica cryotome type CM3050S, Leica Biosystems, Wetzlar, Germany). The slices were rinsed with phosphate-buffered saline to remove the water-soluble embedding medium and they were then either prepared for later immunohistochemical analyses or AFM.

Selection of anulus fibrosus from intraoperatively obtained tissue. Lamellar structure of the anulus fibrosus. The black arrows point at the lamellar structure of the anulus fibrosus in bovine (A) and human IVD samples (B). This lamellar structure can already easily be recognised by the naked eye.

The bovine discs were harvested from the animals within 24 h after death and kept frozen until further processing. To evaluate the variability of the data acquired with the technique used, five independent tissue sample were resected from the same bovine disc at different locations in the bovine anulus fibrosus at a similar radial position. To evaluate how much results depend on the location of the tissue origin with the disc, five samples were measured along the radius of the disc from the inner nucleus to the outer anulus in each of five different bovine IVDs. The rest of the tissue preparation was carried out as in human tissue.