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Bioengineering

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

Published: September 7th, 2016

DOI:

10.3791/54537

1Siegfried Weller Institute for Trauma Research at the BG Trauma Center, Eberhard Karls Universität Tübingen, 2Department of Orthopaedics, BG Trauma-Center, 3Buck GmbH and Co.KG

Here we present a fluorophore based imaging technique to detect cell viability on a non-transparent titanium scaffold as well as to detect glimpses of the scaffold impurities. This protocol troubleshoots the drawback of imaging cell-cell or cell-metal interactions on non-transparent scaffolds.

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.

Chronic back pain is a multifactorial disease. The interest in a minimally invasive treatment option for the degenerative disc disease has grown since the 1950s. Until today, multi-segmental fusion of the spinal column is the most widely used treatment. Since, this method often leads to limitations in the mobility of the affected segment1,2, exploration of the arthroplasty era became a wide interest. Significant advancements in total disc replacement and nucleus replacement has become a good alternative to treat chronic back pain1. Despite the huge progress, none of the methods has been clinically evaluated. The less rigid nucleus implants repres....

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NOTE: Immortalized human mesenchymal stromal precursor cells (SCP-1 cells) were used for the experiments. SCP-1 cells were provided by Prof. Matthias Schieker12.

1. Expansion of SCP-1 Cells

  1. Prior to working with the SCP-1 cells, properly clean the working area (designated biosafety cabinet I) with 70% ethanol (v/v) wearing gloves.
  2. In the cleaned biosafety cabinet prepare an appropriate volume of cell culture medium by mixing the required components as indicated.......

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Preliminary results showed that the described novel nucleus implant not only has good damping features but also is biocompatible with SCP-1 cells. During the production process of the implant, it comes in contact with strong corrosive and toxic substances (lubricant, mordant, electro-polishing solution). With the help of indirect fluorescent staining techniques we were able to visualize remaining impurities and consequently optimize a cleaning protocol showing significant reduction in sub.......

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The scaffold surface plays an important role in its interaction with surrounding tissue in vivo thereby determining implants functional durability. Thus, the bio-compatibility of the scaffold is studied by in vitro assays using cells (SCP1 cell line), when plated on the scaffolds.

Microscopy techniques that function well with thin and optically transparent scaffolds are poorly suited for non-transparent scaffolds to study the biocompatibility. This is mainly because the non-t.......

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Project is partially funded by Zentrales Innovationsprogramm Mittelstand (ZIM) des Bundesministeriums für Wirtschaft und Energie -KF3010902AJ4. The publication fee has been covered by the BG trauma hospital Tübingen, Germany.

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Name Company Catalog Number Comments
6/24/48 well plates, T25/ T75 culture flask Greiner Bio-One GmbH *
* 24 well plates Greiner Bio-One GmbH CELLSTAR 662 160
* 48 well plates Corning Incorporated USA 3548
* 6 well plates Falcon 353046
* T25 Greiner Bio-One GmbH 690 175
* T75 Greiner Bio-One GmbH 658 175
Acetic acid, purum ≥ 99,0 % Carl Roth 3738.4
Acetone Carl Roth 5025.1
Axioplan-2  Carl Zeiss, Germany
Biological safety cabinets Thermo Scientific safe 2020
Calcein acetoxymethyl ester (calcein AM) Sigma 17783
Cell Culture Incubtator Binder, Tuttlingen, Germany 9040-0078
Filter unit (0.22µm) Millipore, IRL SLGP033RS
Centrifuges 5810 R And 5417 R Thermo Fisher Scientific, NY Megafuge 40R
Dimethylsulfoxid (DMSO) Carl Roth 4720.2
Dulbecco’s PBS without Ca & Mg Sigma H15-002
Ethanol 99 %  SAV liquid prod. GmBH 475956
Ethidium homodimer Sigma 46043
EVOS Fluorescence imaging system Life technologies AMF4300
Fetal Bovine Serum (FCS) Gibco 10270-106
Hemocytometer Hausser Scientific, PA, USA
Hoechst 33342 Sigma 14533-100MG
Knitted titanium nucleus implant Buck co & KG,Germany
MEM Alpha Modification with Glutamine w/o nucleoside Sigma E15-832
Omega microplate Reader BMG Labtech,Germany FLUOstar Omega
Penicillin/Streptomycin Sigma P11-010
Resazurin sodium salt Sigma 199303-1G
Sulforhodamine B sodium salt Sigma S1402-1G
Test tube rotator Labinco B.V.,The Netherlands Model LD-76
TRIS (hydroxymethyl) aminomethan Carl Roth AE15.1
Triton Carl Roth 3051.2
Trypan Blue 0.5 % Carl Roth CN76.1
Trypsin/EDTA Sigma L11-004

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