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 represent a promising alternative to total disc replacement, provided that the annulus fibrosus is intact3,4. However, the currently present nucleus implants on the market are often associated with complications like changes in vertebral body, dislocation, vertical height loss of the disc and the lack of necessary associated mechanical rigidity5. In order to overcome the current drawbacks, a novel nucleus implant made of knitted titanium wires has been successfully developed6. Due to the unique knitted structure, this newly developed scaffold has shown distinguished biomechanical characteristics, e.g., damping feature, pore size, loading capacity and reliability7. Aiming to test the biocompatibility of this novel nucleus implant, depicted severe limitations in the (optical) analysis techniques attributed to the non-transparent nature of the implant.
In order to test the biocompatibility, cell-metal interaction plays a prominent role8-10. An interaction between the cells and the scaffold is necessary for the stabilization and hence for the better implant integration within the host system. However, an increasing ingrowth depth might alter the mechanical properties of the scaffold. Aiming to investigate whether the scaffold surface provides a base for cell attachment, proliferation and differentiation or whether the metal affects cell viability, it is important to troubleshoot the common well-known problem of imaging cells on/in non-transparent and opaque scaffolds. In order to overcome this limitation several fluorescent based techniques were explored. Companies provide a large range of fluorophores to visualize living cells, cellular compartments, or even specific cellular states11. Fluorophores for this experiment were chosen with the help of the online tool spectral viewer in order to best fit our fluorescent microscope.
The developed strategy for the analysis of the adherent cells behavior on/in the non-transparent knitted titanium scaffold involves the following: 1) fluorescent (green fluorescent protein/GFP) labeling of the osteochondro-progenitor cells to allow tracking of the cells on the scaffold, 2) measuring the viability (mitochondrial activity) of the cells, and 3) visualizing cell-cell and cell-material interactions within the scaffold. The procedure has the advantage that it can be easily transferred to other adherent cells and other non-transparent or opaque scaffold. Furthermore, viability and ingrowth pattern can be monitored over several days, thus it can be used with limited amounts of scaffold material or cells.
The present study demonstrates the successful use of our current protocol to measure the cell viability and visualize in-growth pattern of osteochondro-progenitor cells on/in the non-transparent knitted titanium scaffold. Furthermore, the developed protocols might be used in order to determine the scaffold impurities and to check cleaning protocols.
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
2. Counting of SCP-1 Cells
3. GFP Transfection of SCP-1 Cells
NOTE: In order to observe SCP-1 cell growth on and into the knitted titanium scaffold over a certain culture period we marked the cells with green fluorescent protein (GFP). Overexpression of GFP is achieved by infection with adenovirus particles coding for GFP. Replication incompetent (-E1/-E3) adenovirus particles coding for green fluorescent protein (GFP) were used to infect SCP-1 cells. The virus particles were obtained from Prof. Steven Dooley13 by collecting culture supernatant of recombinant adenovirus (Ad5-GFP) transfected HEK293T cells (Biosafety lab II). Three repeated freeze (-80 °C) and thaw (37 °C in the water bath) cycles ensured that no HEK293T cells remain viable to produce new virus particles. Using this adenovirus seed stock can efficiently infect the SCP-1 cells without producing new virus particles. Thus, the infected cells can be handled in a Biosafety Lab I.
4. Cleaning of Knitted Titanium Scaffolds
5. Imaging Scaffold Structures by Indirect Fluorescence
NOTE: The present protocol describes the imaging of scaffold structures by indirect fluorescence using the fluorophore sulforhodamine B which gives a bright red fluorescence at an ex/em wavelength of 565/586 nm. However, the fluorophore can be changed to better fit for given microscope settings or possible auto-fluorescence of the scaffold.
6. In Vitro Biocompatibility Assay
Figure 5: Timeline of in vitro assay. (A) Experimental setup for plating cells. (B) Illustration of procedure, emphasizing importance of beginning protocol and cell functionality validation till day 7. Please click here to view a larger version of this figure.
7. Resazurin Conversion measurement
NOTE: Resazurin conversion assay is used for measuring the mitochondrial activity and thus indirectly cell proliferation. Resazurin reduction to resorufin generates a fluorescent signal, which is based on the mitochondrial activity associated with viable cell numbers (Figure 7A).
8. Live-dead Staining
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 substance load on the scaffold. Figure 3 shows the efficiency of established cleaning protocol.
The success of implants used for arthroplasty treatment is determined by events that takes place at the cell-material interface. Figure 4 shows the cells attached on the scaffold after 24 hr of plating, as described in the protocol section 6. A significant transfection efficiency of SCP-1 cells was observed as we could image the growth pattern of mesenchymal stromal precursor cells on the scaffold (refer Figure 2). Direct visualization confirms the biocompatibility of the scaffold and also depicts the adherence pattern on the scaffold surface (Figure 4). Fluorescence staining can be done further to examine cell interaction with and spreading on the scaffold surface.
Fluorophores were successfully applied in order to examine cell death and proliferation over a period of time on the scaffold. Live-dead-staining images exemplify how staining can successfully be done on the scaffold to confirm the percent viability of cells over a period of time. Figure 6 shows blue nuclear staining (Hoechst 3342) in all cells, red fluorescently labelled (ethidium homodimer) dead cells, and green labelling for incorporation of calcein-AM as viability marker. Calcein AM is converted to calcein which exhibits a bright green fluorescence in the presence of calcium ions in the cytoplasm of the cells. Hoechst 33342 is cell wall permeable and intercalates into the cellular DNA. This way all cells will show blue nuclei (refer Figure 6). Ethidium homodimer is not cell wall permeable, thus it will only intercalate into the DNA of dead cells. This way, dead cells will show red nuclei. Furthermore, cell viability and fold increase in cell number on scaffold over a week was quantified by resazurin conversion assay and represented graphically (Figure 7).
Figure 1: Cell counting with a Hemocytometer. (A) Setup of a chamber assembly. (B) Illustration of counting chambers; 4 x 4 counting chamber is used for a cell count. Please click here to view a larger version of this figure.
Figure 2: GFP transfection efficiency. SCP1 cells exhibit a strong green fluorescence indicating positive ad-GFP- transfection efficiency. Scale bar = 1,000 µm, 4X magnification. Please click here to view a larger version of this figure.
Figure 3: Sulforhodamine B staining negative images capture. (A) Scaffold before cleaning. Arrow indicates the presence of toxic/corrosive substances on scaffold. (B) Scaffold after the cleaning protocol. Scale bar = 1,000 µm, 4X magnification. Please click here to view a larger version of this figure.
Figure 4: Adherence pattern of SCP1 cells on scaffold. GFP signal indicates cell adherence and growth pattern on the surface of the knitted titanium scaffold. Scale bar = 1,000 µm, 4X magnification. Please click here to view a larger version of this figure.
Figure 6: Co-fluorescence staining of cells on scaffold. (A) The Hoechst nuclear staining (blue) and (B) the Calcein-AM cytoplasmic staining (green). (C) Arrow indicates the presence of dead cell due to uptake of ethidium homodimer-1 stain (red). (D) shows the merged image. Scale bar = 1,000 µm, 4X magnification. Please click here to view a larger version of this figure.
Figure 7: Resazurin conversion assay. (A) Biochemical reduction reaction of redox dye (resazurin) into an end product (resorufin) which emits fluorescence and undergoes colorimetric changes. (B) Mitochondrial activity was measured when cells were plated on 0.75 mg/cm³ density scaffold. Data was collected using fluorescence based measuring instrument. Fluorescence intensity at 590 nm (y-axis) at defined time points (x-axis) is depicted as a result of quantitative cell viability measurement (Ex = 540 nm, Em = 590 nm). The statistical significance was determined using Two way ANOVA and standard error of the mean (SEM) is shown as an error bars. Considering the scaffold physical properties, e.g., pore size and mechanical properties (e.g., damping feature) together, 0.75 mg/cm³ density scaffold was used for biocompatibility characterization. Please click here to view a larger version of this figure.
Media components | Concentration |
Basal αMEM media (%) | 90 |
Serum (%) | 10 |
Pen/Strep (%) | 1 |
Table 1: Cell culture (αMEM) Media composition.
Problem | Cause | Solution |
Cell viability imaging on Non-transparent scaffold | Scaffold prevents light from penetrating without distortion | Use fluorophore based imaging technique for cell assessment. |
Interference of fluorescence intensity | Auto fluorescence, background signal | Pay attention to the fluorophores and use appropriate based on particular scaffold properties. |
Cell viability assessment on scaffold over a culture period | Repeated measurements over a long time | Transfect the cells with ad-GFP-virus particles. |
Cell functionality assessment on non-transparent scaffold | Fluorescence imaging technique allows only cell spreading pattern analysis. | Perform resazurin conversion assay (quantitative cell viability measurement) in a combination with imaging technique. |
Table 2: Summary table: troubleshooting the cell viability imaging on non-transparent scaffold.
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-transparent scaffolds prevent light from penetrating without significant distortion15. To partly overcome these problems we herewith establish a method for cell assessment on/in knitted titanium made scaffolds using various fluorophores.
In order to enable knitting followed by folding of the titanium wires, the material comes into contact with strong corrosive and toxic substances (lubricant, mordant, electro-polishing solution), which might alter the biocompatibility of the scaffold if traces remain in/on the scaffold. With the help of a developed indirect-fluorescence protocol (Protocol 5) we could visualize the scaffold structure. Furthermore, scaffold characteristics, e.g., the material thickness, the individual pore size and shape, or the connective density, were analyzed using ImageJ. A higher magnified epifluorescence microscopic image allowed visualizing impurities in the scaffold as well as on the scaffold surface. Figure 3b thus represents the confirmatory result of the successfully developed cleaning protocol. The principle of this indirect staining protocol can be easily reproduced to other non-transparent scaffolds, taking into consideration the individual scaffold properties, e.g., pore size and corresponding diffusion which affects the incubation time. Also, auto-fluorescence of the scaffold and microscopic settings affects the choice of fluorophores used. The online tool fluorescence spectral viewer can help to choose the adequate fluorophores.
Cell-metal interaction has been examined indirectly by analyzing the adherence pattern of cells on the scaffold. Protocol 6 describes the methodology of how cells can be monitored in vitro if plated on non-transparent scaffolds in culture systems by using a GFP transfection strategy. Based upon preliminary results of in vitro assays, it has been predicted that the surface properties such as composition, micro-topography and roughness16 might play an important role in establishing adherence and spreading of target cells. Titanium being a biomaterial thus might be acting as a substrate template to provide base for cell attachment (refer Figure 4).
Implant surface topography has been reported to influence cell behavior16. In the present study, we analyzed the cell growth/spreading and viability using fluorescence staining techniques in combination with quantitative viability measurements. Analysis revealed subtle variation in cell percent viability and spreading depending on the scaffold material. However, cell functionality assessed quantitatively by resazurin conversion assay (mitochondrial activity), was not significantly affected by the scaffold material. The measurement of the mitochondrial activity by resazurin conversion has the advantage that it is not cell toxic and thus can be performed repeatedly over a long culture period. Special care has to be taken when washing off residual resazurin working solution, so to not accumulate background signal (false positive results). Despite these advantages, the resazurin conversion assay will not give any information on the cell spreading on the scaffold. The GFP infected cells hence can be tracked over a long culture period (GFP signal remained constant for over 14 days), thereby enabling to visualize specific growth pattern on the scaffold surface. Visualization of cells deeper in the scaffold is still limited by the scaffold material and thus will require dissection of the implant. The combination of these two techniques has the major advantage that it can be easily transferred to other cell types, considering that incubation time might have to be adapted to the cell type of interest. However, care has to be taken when transferring this method to other non-transparent scaffolds, e.g., collagen based scaffolds which generally exhibit a strong green auto-fluorescence17. In this case other fluorescent tags might be used. The combination of above stated two methods therefore has several advantages (see Table 2).
Scaffold characteristics, e.g., pore size might trap cells inside the scaffold. If not enough nutrients are supplied, these cells might die and secrete proteases that affect cell viability of the surrounding cells/tissue. We were able to adapt a fluorescent based staining protocol that is able to visualize live and dead cells in and on the knitted titanium scaffold. Similar to the indirect fluorescence staining protocol, the principle of this staining protocol can be easily transferred to other non-transparent scaffolds. While doing so, the individual scaffold properties, e.g., pore size and corresponding diffusion as well as possible auto-fluorescence, microscopic setting have to be taken into consideration as they might affect the incubation time and the choice of fluorophores used. Here, again the online tool fluorescence spectral viewer can help to choose the adequate fluorophores.
In summary, in vitro results indicate that the proposed knitted titanium nucleus implant model has a biological profile. Initial attachment of mesenchymal stromal precursor cells (SCP-1 cells) on this material suggests that this titanium alloy implant material is biocompatible. Although we have not analyzed association of different topographical parameters, scaffold surface modification might enhance cell adherence proliferation as well as differentiation18. Optimum compatibility between scaffold and cells raise the probability of better implant integration into the surrounding tissue and so improving in vivo longevity after the treatment19. Using the above stated test setup opens up the possibility to measure and visualize improvements in the biological performance of the scaffold induced by surface modifications. The preference of scaffolds with bioactive surface over unmodified implant designs suggests the better performance20 in terms of osteo-chondrogenic integration. This study is further enhanced by other reports on the knitted titanium implant where its mechanical property and bioactivity have been reported 6,7.
The authors have nothing to disclose.
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.
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 |