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

Summary

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.

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.

Introduction

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 segment^1,2, 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....

Protocol

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 in Table 1. In order to maintain sterility of the basal medium, add supplements by passing through sterile filters with a pore size ....

Results

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.......

Discussion

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.......

Materials

List of materials used in this article

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 (0.22 µm)	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