Tissue engineering is characterized by seeding cells on a scaffold ex vivo in order to repair damaged tissue of human body in a regenerative way [10, 14]. To achieve this goal, there is the requirement that biomaterials must be compatible with human tissue, adhesive to human cells and biodegradable at a rate commensurate with the production of a new cell matrix. An implant material with a biomimetic structure combined with an incorporation of compatible cells has a successful precursor for regeneration . In that regard, seeding of viable cells precisely into scaffolds can be a significant hurdle in tissue engineering. As previously shown, inkjet printing technology could be a potent solution [4, 9, 10]. Especially when there is the requirement to seed different types of cells in anatomically exact locations of one scaffold, to attain biological function .
There are two different mechanisms of inkjet printing: continuous inkjet printing (CIJ) and drop-on-demand inkjet printing (DOD) . In CIJ a stream of fluid is passed through a small orifice and breaks up into small droplets due to Rayliegh instability . To control the ejection direction, the droplets are electrically charged and steered by an electrostatic or magnetic field. Unused droplets are fed back to the stream of fluid. In contrast to CIJ, DOD ejects droplets when required only. Mostly the droplets are formed by using heat or mechanical compression . In thermal DOD fluid is vaporised in the printhead by heating. The expansion and collapse of small gas bubbles cause the ejection of the droplets. In piezoelectric DOD, as performed here, the mechanical compression of the fluid chamber is realised by a rapid change in the shape of a piezo ceramic.
CIJ printers operate faster than DOD printers. But there is the need of an electrically conducting fluid and there is the risk of contamination of fluid by fed back process. Due to that, CIJ is limited for an application in cell printing . In comparison, thermal as well as piezoelectric DOD printers have been reported in the literature as suitable for printing viable cells [3, 11, 14].
In this work, piezoelectric DOD inkjet printing is applied to human dental follicle stem cells (hDFSC). In general, periodontal tissues include diverse cell types, including stem cells [7, 12]. It is well known, that stem cells are able of self-renewal and they have the potential to differentiate in dependence to their division . HDFSCs are easily accessible in dental practice through commonly extracted teeth . For that reason, the hDFSCs can provide a source for experimental and future clinical applications in periodontal tissue or bone regeneration approaches [2, 5, 8].
2 Material and methods
2.1 Cells and culture conditions
Human dental follicle stem cells (hDFSC) were isolated as described by . Wisdom teeth from young volunteers were extracted by the Department of Oral and Maxillo-facial Plastic Surgery, University of Rostock After extraction the dental follicles were washed immediately several times with a cold PBS solution containing increasing concentration of 2-10 % penicillin/streptomycin (GIBCO, Carlsbad, USA). The dental follicles were dissected and digested in serum-free DMEM F-12 (Invitogen, Germany) supplemented with dispase II 1 mg/ml (Sigma-Aldrich Chemie, Germany) for 2 hours at 37 °C. The enzyme mixture was removed and tissue was cultured in DMEM F-12 medium containing 10 % fetal calf serum (PAA Laboratories, Germany) at 37 °C, 5 % CO2.
For the inkjet printing investigations a cell suspension with concentration of 6.6 × 106 cells ml−1 in DMEM with 10 % FCS was prepared. Cellular count and viability of hDFSC pre-printing were determined by Trypan Blue dye enumeration (see chapter 2.3). HDFSCs were stained with Trypan Blue, viable cells were counted using a Neubauer counting chamber and an inverted light microscope Olympus CKX41 (Olympus, Germany).
2.2 Printing process
Cell size and shape of hDFSCs was determined with light microscope Olympus BX51 equipped with digital camera UC 30 (Olympus, Germany) pre-printing to verify acceptable size referring the nozzle diameter of the printhead. For inkjet printing experiments a Nanoplotter 2.1 (Gesim, Germany) was equipped with the printhead NanoTip HV (Gesim, Germany). The Nanoplotter enables non-contact printing of sub-nanoliter volumes with high density and high precision droplet positioning. The printhead has a nozzle diameter of 53 μm and consists of a glass capillary bonded to an annular piezoelectric actuator. It follows the piezoelectric DOD principle, i.e. a drop is released exactly when the piezo ceramic actuator is triggered. The nominal droplet volume is 400 pl (droplet ~90 μm in diameter, depending on utilized fluid). Fluids with a viscosity up to 20 mPas can be printed. The printhead contains no valves and cannot actively aspirate samples into its pump chamber, which is therefore instead done by an implemented dilutor. The tip of the printhead is shown in Figure 1.
Before and after the dosage, the correct function of the printhead is checked with a stroboscope. The printhead is illuminated by a LED that flashes with the frequency of the printhead (500 Hz). The resulting image is analyzed for droplet volume and droplet positioning estimation. The droplet volume estimation is calculated on the basis of rotational body. To assess correct droplet positioning, there is a check of trajectory of the droplet.
Printing the hDFSC suspension, the printhead was set to a voltage of 75 V and a pulse of 50 μs. The drops were dosed with 50.000 droplets over a time of 100 s. The print-head was filled from a 96-well plate and ejected the suspension onto a microscope slide. The printing process was performed at room temperature (21 °C).
2.3 Cell viability post-printing
The printing process was immediately followed by a Trypan Blue dye exclusion test in order to determine viable hDFSCs. The test is based on the principle that viable cells have an intact cell membrane, which excludes Trypan Blue dye. In contrast, dead cells lose the integrity of the membrane and absorb the dye. HDFSC suspension was mixed with Trypan Blue dye in a ratio of 1:1. The cells were visualized with the light microscope Olympus BX51. Dead hDFSCs take up dye and get stained. Viable cells exclude the dye and remain unstained .
3 Results and discussion
3.1 Printing process
Before starting the printing process hDFSCs were checked in light microscope. They show a spherical shape with a diameter around 15 μm to 20 μm.
Figure 2 and 3 show the stroboscope tests before and after printing. Both figures show a formation of a single droplet from the printhead. Any satellites outside the trajectory of the main drop could be seen. The trajectories of droplets show an insignificant deviation to vertical line, referred to as angle failure of 2.06°. The angle failure increases insignificantly by 0.21° up to 2.27°. In contrast, there is a significant decrease of droplet volume by 35 % from 347 pl to 224 pl, whereas the droplet volume should remain stable. Due to the diameter of cells in proportion to nozzle diameter of the printhead (53 μm), sedimentations or agglomerations of the cells may affect the printing performance.
3.2 Cell viability post-printing
As seen in Figure 4 the bulk of the printed hDFSCs remains unstained by Trypan Blue dye. The integrity of their membrane is intact. In result, they did not absorb dye and are bright colored. It is assumed that these cells are viable. In contrast, dead cells absorb the dye and are in a dark blue color. Concluding DOD inkjet printing via piezoelectric printhead causes less negative effect on viability of hDFSCs. Nethertheless, the processed Trypan Blue dye exception test as described in 2.3 is limited.
In conclusion, piezoelectric DOD inkjet printing by using Nanoplotter 2.1 equipped with the printhead NanoTip HV, can be applied for the seeding of viable hDFSCs. Limitation factors in printing performance may be sedimentation or agglomeration of the cells near the nozzle in the printhead during printing process. The performed Trypan Blue dye exception test post printing shows promising results. No significant influence on cell viablility has been observed so far. The performance of printing has to be optimized, regarding stable volume and trajectory of the dosage droplets over several minutes.
Piezoelectric DOD inkjet printing offers some control in droplet formation through variable driving parameters, in particular voltage, pulse width and frequency of triggering Especially exakt cell count and viability of hDFSCs post printing needs to be validated by additional methods. Furthermore, the re-cultivation on plastic surfaces and metabolic activity of cells will be determined.
Similar results to this work were obtained in [3, 11, 14]. The authors used other DOD inkjet printing devices and other types of viable cells. Therefore it can be said that DOD inkjet printing can be used for the seeding of viable cells. This provides further ambition to unite the capability of inkjet printing regarding potentials of hDFSCs in tissue enginieering and future potential dental stem cell therapies.
We wish to acknowledge the support of the Department of Oral and Maxillofacial Plastic Surgery, University of Rostock which provided our research with hDFSCs.
We also would like to thank the Minis-terium für Wirtschaft, Bau und Tourismus MecklenburgVorpommern which provided funding of the Gesim Nanoplotter 2.1 with financial resources by the Europäischer Fonds für regionale Entwicklung EFRE (European Regional Development Fund ERDF).
Conflict of interest: Authors state no conflict of interest. Material and Methods: Informed consent: Informed consent has been obtained from all individuals included in this study. Ethical approval: The research related to human use has been complied with all the relevant national regulations, institutional policies and in accordance the tenets of the Helsinki Declaration, and has been approved by the authors’ institutional review board or equivalent committee.
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