Non-destructive monitoring of viability in an ex vivo organ culture model of osteochondral tissue

Abstract

Organ culture is an increasingly important tool in research, with advantages over monolayer cell culture due to the inherent natural environment of tissues. Successful organ cultures must retain cell viability. The aim of this study was to produce viable and non-viable osteochondral organ cultures, to assess the accumulation of soluble markers in the conditioned medium for predicting tissue viability. Porcine femoral osteochondral plugs were cultured for 20 days, with the addition of Triton X-100 on day 6 (to induce necrosis), camptothecin (to induce apoptosis) or no toxic additives. Tissue viability was assessed by the tissue destructive XTT (2,3-bis[2-methoxy-4-nitro-5-sulfophenyl]-2H-tetrazolium-5-carboxyanilide tetrazolium salt) assay method and LIVE/DEAD ® staining of the cartilage at days 0, 6 and 20. Tissue structure was assessed by histological evaluation using haematoxylin & eosin and safranin O. Conditioned medium was assessed every 3-4 days for glucose depletion, and levels of lactate dehydrogenase (LDH), alkaline phosphatase (AP), glycosaminoglycans (GAGs), and matrix metalloproteinase (MMP)-2 and MMP-9. Necrotic cultures immediately showed a reduction in glucose consumption, and an immediate increase in LDH, GAG, MMP-2 and MMP-9 levels. Apoptotic cultures showed a delayed reduction in glucose consumption and delayed increase in LDH, a small rise in MMP-2 and MMP-9, but no significant effect on GAGs released into the conditioned medium. The data showed that tissue viability could be monitored by assessing the conditioned medium for the aforementioned markers, negating the need for tissue destructive assays. Physiologically relevant whole-or part-joint organ culture models, necessary for research and pre-clinical assessment of therapies, could be monitored this way, reducing the need to sacrifice tissues to determine viability, and hence reducing the sample numbers necessary.

Ex vivo model unravelling cell distribution effect in hydrogels for cartilage repair

“…Changes in the cartilage during culture observed for the equine plugs were similar to those reported for other species (Chen et al, 1997;de Vries-van Melle et al, 2012;Elson et al, 2015;Schwab et al, 2016). The native cartilage swelled and GAGs leached into the medium, possibly due to the damaged collagen network at the surface of the plugs and the defect edges (Chen et al, 1997;de Vries-van Melle et al, 2012;Elson et al, 2015;Schwab et al, 2016). However, in the current study, both the water and GAG content were stable between days 29 and 57, even though GAGs leached continuously into the medium, indicating that GAGs were produced and that a new stable situation was reached.…”

Ex vivo model unravelling cell distribution effect in hydrogels for cartilage repair_suppl

“…The current study focused on equine OC plugs, as the horse is a widely used pre-clinical model for cartilage repair strategies (Malda et al, 2012). Changes in the cartilage during culture observed for the equine plugs were similar to those reported for other species (Chen et al, 1997;de Vries-van Melle et al, 2012;Elson et al, 2015;Schwab et al, 2016). The native cartilage swelled and GAGs leached into the medium, possibly due to the damaged collagen network at the surface of the plugs and the defect edges (Chen et al, 1997;de Vries-van Melle et al, 2012;Elson et al, 2015;Schwab et al, 2016).…”

Effect of osteochondral graft orientation in a biotribological test system

“…The release of sulfated glycosaminoglycans (sGAGs) into the culture medium in organ cultures with full‐depth articular cartilage is used to detect or quantify tissue degradation . In our study, the release of the sGAGs was used as a parameter to determine the breakdown of the cartilage concerning different orientations of the OC grafts.…”

“…Subsequently, the osteochondral samples were cultivated in a growth medium (GIBCO DMEM/F12 GlutaMAX‐I, Invitrogen, LifeTech Austria, Vienna, Austria) with antibiotics (penicillin 200 U/ml; streptomycin 0.2 mg/ml) and Amphotericin B 2.5 μg/ml (Sigma–Aldrich Chemie GmbH) for 7 days. This step was introduced to overcome a decrease in metabolic activity and equilibrate the ex vivo environment after the harvesting procedure . After seven days of incubation, tribological tests for each animal were performed within the next two days at 39°C.…”

“…Among them, currently used techniques include bone marrow stimulating methods (e.g., microfracture, drilling, abrasion arthroplasty, or spongialization), chondrocyte implantation directly (autologous chondrocyte implantation [ACI]) or in combination with biomaterials (matrix‐associated chondrocyte implantation [MACI]). Also, intra‐articular injection of lubricants (e.g., hyaluronan), as well as the implantation of single or multiple autologous grafts (mosaicplasty), are used as treatment options …”