Cartilage labelling for mechanical testing in T-peel configuration

Abstract

Purpose: The purpose of this study was to find a suitable method of labelling cartilage samples for the measurement of distraction distances in biomechanical testing.

Methods: Samples of bovine cartilage were labelled using five different methods: hydroquinone and silver nitrate (AgNO3), potassium permanganate (KMnO4) with sodium thiosulphate (Na2S2O3), India ink, heat, and laser energy. After the labelling, we analysed the cartilage samples with regard to cytotoxity by histochemical staining with ethidiumbromide homodimer (EthD-1) and calcein AM. Furthermore, we tested cartilages labelled with India ink and heat in a T-peel test configuration to analyse possible changes in the mechanical behaviour between marked and unlabelled samples.

Results: Only the labelling methods with Indian ink or a heated needle showed acceptable results in the cytotoxity test with regard to labelling persistence, accuracy, and the influence on consistency and viability of the chondrocytes. In the biomechanical T-peel configuration, heat-labelled samples collapsed significantly earlier than unlabelled samples.

Conclusion: Labelling bovine cartilage samples with Indian ink in biomechanical testing is a reliable, accurate, inexpensive, and easy-to-perform method. This labelling method influenced neither the biomechanical behaviour nor the viability of the tissue compared to untreated bovine cartilage.

Fig. 1

a and b The pictures and illustrations show the three steps of cartilage block preparation. Osteochondral blocks harvested from the patellofemoral groove of bovine hind legs were cut with a scalpel in horizontal and perpendicular directions. The prepared cartilage region is further dissected by a sledge microtome, which finishes the cartilage tissue for T-peel testing by partially incising the block from one side. c The exact geometrical parameters of the prepared cartilage block are given. Parts of the block can be adjusted by clamps, so that the left part of the block can be peeled and measured with a biomechanical testing device

Fig. 2

a The picture illustrates the custom-made clamps for mounting the samples into a mechanical testing machine. The clamps are surrounded by a sterile plexiglas bowl to keep the samples irrigated during the T-peel test. b Mechanical testing machine for measuring the applied force for T-peeling

Fig. 3

The upper pictures illustrate the T-peel progress of the cartilage block. The numbers 1 to 6 on pictures a and c refer to the markers on the cartilage sample. It is obvious that the distance between the markers increase during the peeling process. The diagram below shows a typical stress–strain curve depicting the initial bending of the sample until rupture of the cartilage block starts. Only after the rupture, the force needed to peel the cartilage tissue remains the same

Fig. 4

Live/Dead®-viability test: red staining indicates dead cells, and green staining viable cells. The area of labeling application is marked by arrows. a Heat labelled cartilage with large areas of necrotic cartilage shown in red colour. b Ink labelled cartilage with hardly any necrotic cartilage shown in green colour; the black spot visible within the green area is ink

Fig. 5

Overview of the investigations depending on the labelling methods used