Imaging Cell Viability on Non-transparent Scaffolds — Using the Example of a Novel Knitted Titanium Implant

Abstract

Intervertebral disc degeneration and disc herniation is one of the major causes of lower back pain. Depletion of extracellular matrix, culminating in nucleus pulposus (NP) extrusion leads to intervertebral disc destruction. Currently available surgical treatments reduce the pain but do not restore the mechanical functionality of the spine. In order to preserve mechanical features of the spine, total disc or nucleus replacement thus became a wide interest. However, this arthroplasty era is still in an immature state, since none of the existing products have been clinically evaluated. This study intends to test the biocompatibility of a novel nucleus implant made of knitted titanium wires. Despite all mechanical advantages, the material has its limits for conventional optical analysis as the resulting implant is non-transparent. Here we present a strategy that describes in vitro visualization, tracking and viability testing of osteochondro-progenitor cells on the scaffold. This protocol can be used to visualize the efficiency of the cleaning protocol as well as to investigate the biocompatibility of these and other non-transparent scaffolds. Furthermore, this protocol can be used to show adherence pattern of cells as well as cell viability and proliferation rates on/in the scaffold. This in vitro biocompatibility testing assay provides a propitious tool to analyze cell-material interaction in non-transparent and opaque scaffolds.

Feasibility of Cell Lines for In Vitro Co-Cultures Models for Bone Metabolism

“…But not only alterations in liquid volume but also the carrier itself may represent a challenge. When being non-transparent, the carrier limits microscopic analyses, a factor that might be partly circumvented by using fluorophores [128]. However, penetration depth and possible autofluorescence of the carrier material remain limiting factors.…”

Use of in vitro bone models to screen for altered bone metabolism, osteopathies, and fracture healing: challenges of complex models

“…An increase in culture/reaction volume would dilute factors secreted by cells in the culture medium and thus effectively lowers the sensitivity of established analytical methods. However, the use of scaffolds bares also other challenges: The scaffold limits microscopic analyses—the use of fluorophores may circumvent some of these limitations (Tendulkar et al 2016 ); however, penetration depth and possible autofluorescence of the material remain limiting factors. Assay substrates and reaction products require time for perfusion.…”

“…The scaffold limits microscopic analyses—the use of fluorophores may circumvent some of these limitations (Tendulkar et al 2016 ); however, penetration depth and possible autofluorescence of the material remain limiting factors.…”

“…Similar to all fluorescent tracers or markers, the method works very well in 2D cultures, but quickly reaches its limits in 3D cultures with non-transparent scaffolds (Fig. 2 b) (Tendulkar et al 2016 ). To make things worse, our transfected SaOS-2 cells showed altered mitochondrial and alkaline phosphatase (ALP) activity, which in turn affected the formation of mineralized matrix by these cells (Fig.…”

Exogenous Delivery of Link N mRNA into Chondrocytes and MSCs—The Potential Role in Increasing Anabolic Response

“…Therefore, synthetic mRNA encoding Link N was generated and the transfection efficiency was analyzed in both cell types. Functional Link N mRNA delivery augmented the anabolic response in primary human chondrocytes and SCP1 cells, promoting extracellular matrix related gene expression in 2D cell cultures as well as in 3D cultures after seeding cells on knitted titanium scaffolds [22]. Thus, the delivery of synthetic Link N mRNA into chondrocytes and MSCs presents a new therapeutic strategy for augmenting the anabolic response of cells for improving cartilage and/or IVDs regeneration.…”