Time-lapse microscopy of wound healing: the effects of histatin variants in vitro

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Time-lapse microscopy of wound healing:

the effects of histatin variants in vitro

Master thesis dentistry

Daniëlle Spies

Academic Centre for Dentistry Amsterdam

Department of Oral Biochemistry

F.J. Bikker, PhD

J.G.M. Bolscher, PhD

Department of Preventive Dentistry

B.P. Krom, PhD

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Abstract

Wound healing in the oral cavity proceeds faster than in skin. Saliva is shown to play an important role in this process. It has been found that histatins are the major wound-closure stimulating factors in human saliva. The main focus of this research was to determine the influence of several variants of histatin-1 on cell spreading and cell migration in oral epithelial cells in vitro as an

indication of wound healing capacity. The peptides used for this purpose are: histatin-1, cyclic histatin-1, serine phosphorylated histatin-1, tyrosine sulfated (3,5,9,12,29) histatin-1, and scrambled histatin-1. Cell-spreading was described by calculating the relative number of spread cells. To determine cell

migration, a scratch-assay was conducted. The dynamics of cell spreading and cell migration were recorded with a time-lapse microscope. It can be concluded that histatin-1, cyclic histain-1 and serine phosphorylated histatin-1 all significantly stimulated the speed and extent of cell migration in vitro. Histatin-1 and its variants did not influence cell spreading. This study showed a dynamic view in cell spreading and cell migration as influenced by histatin-1 and its variants supporting the role of histatin-1 as a wound healing factor.

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Introduction

Histatins are antimicrobial peptides present in human saliva. They are

secreted by the parotid, submandibular and sublingual glands, and the Von Ebner glands on the tongue (Piludu, et al., 2006). Histatins exert antifungal and antibacterial properties through different working mechanisms. The histatins exert their antimicrobial effect by targeting the fungal mitochondria

(Kavanagh & Dowd, 2004), whilst in bacteria they inhibit the bacterial proteases (Mackay, Denepitiya, Iacono, Krost, & Pollock, 1984) and cause membrane defects (den Hertog, et al., 2005). In the acquired salivary pellicle on dental enamel histatin-1 is also found to reduce the adhesion of

Streptococcus mutans (Shimotoyodome, Kobayashi, Tokimitsu, & Matsukubo,

2006). When adsorbed onto dental enamel, histatin is likely to be more

resistant to proteolytic degradation (McDonald, Goldberg, Tabbara, Mendes, & Siqueira, 2011).

Wound healing in the oral cavity proceeds faster than in skin. Also, scar formation is reduced in mucosal wounds compared to lesions in skin (Wong, et al., 2009). Patients suffering from hyposalivation are thereby more

susceptible to oral infections and delayed wound healing, suggesting that saliva promotes wound healing. Oudhoff and co-authors have hence concluded that histatins are the major wound-closure stimulating factors in human saliva by stimulating cell spreading and migration (Oudhoff, et al., 2008) (Oudhoff, et al., 2009) (Van Dijk, Nazmi, Bolscher, Veerman, & Stap, 2015). Interestingly, cyclization of histatin-1 enhanced its molar activity by a 1000-fold indicating that the effect of histatin-1 may be structure dependent (Oudhoff, et al., 2009). The activation of histatin-1 in epithelial cells shows similarities with the activation of growth factors. A stereospecific interaction between histatin-1 and the receptor on the membrane is suggested in which the uptake of histatin-1 occurs via active energy-dependent mechanisms (Oudhoff, et al., 2008).

The concentration of histatin in saliva varies throughout the day. Gusman and co-authors found that overall histatin-1 concentrations are up to three times higher in stimulated secretions from the submandibular and sublingual glands than from the parotid glands (2,8-12,2 and 0,7-2,8 mg% respectively)

(Gusman, et al., 2004). Tyrosine sulfated histatins are produced in the submandibular and sublingual glands. The sulfation occurs when sulfate is transferred to the hydroxyl group of tyrosine residues. It is also stated that the sulfated histatins are the result of an incomplete removal of the sulfate prior to granule storage (Cabras, et al., 2007). The sulfated histatins make up to 10% of the total amount of histatin-1 (Messana, et al., 2015). Phosphorylation of

histatin-1 enhances the ability to bind to hydroxyapatite significantly (Driscoll, Zuo, Choi, Troxler, & Oppenheim, 1995). Phosphorylation of histatin-1 occurs at the serine residue; Messana and others have calculated that up to 80% of histatin-1 occurs phosphorylated (Messana, et al., 2015).

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Human saliva is shown to play an important role in wound healing. This research is conducted to observe the activity of histatin-1 on cell spreading and cell migration in vitro using time-lapse microscopy. In previous research on cell spreading and migration, images were obtained manually at large time intervals creating a static profile. For better understanding of the process of wound healing, a more dynamic view is needed to observe the action of cell spreading and migration continuously throughout time: hence the use of time-lapse microscopy. The phosphorylated and sulfated variants of histatin-1 (also present in human saliva) had not yet been explored on promoting wound healing. The main focus of this research was to determine the

influence of several variants of histatin-1 on cell spreading and cell migration in oral epithelial cells in vitro as an indication of wound healing capacity.

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Materials and methods

Cell culture

The cells used for this research are HO-1-N-1 buccal epithelial carcinoma cells obtained from the Japanese Collection of Research Bioresources (Osaka, Japan). They were cultured at 37o_{C with a relative humidity of 90% and 5%}

CO2. Dulbecco’s modified Eagle medium (DMEM/F-12) (Life Technologies,

Carlsbad, USA) was used for culture, supplemented with 10% fetal calf serum (HyClone, South Logan, USA) and 2% antibiotics (100µg/ml penicillin +

100µm/ml streptomycin + 250ng/ml amfotericin) (Sigma Aldrich, St. Louis, USA). The cells were cultured in flasks and multi-well plates (Greiner Bio-One, Alphen aan den Rijn, Netherlands). Three times a week the cells (about 4·106

per flask) were washed with Dulbecco’s Phosphate Buffered Saline and trypsinized with 0,25% trypsin-EDTA (Life Technologies, Carlsbad, USA) to be dispersed into three new flasks each containing 15 ml of medium for further culturing. A total of three flasks was continuously present for this stusy: one was saved to continue culturing and two were used for the experiments. After each experiment the cells from the leftover flask were allocated to constitute a new set of three culture bottles. A Neubauer chamber was used for cell counting (Marienfeld, Lauda-Königshofen, Germany).

Peptides

The peptides used for this research are: histatin-1, cyclic histatin-1, serine

phosphorylated histatin-1, tyrosine sulfated histatin-1 (sulfation at location 3, 5, 9, 12, 29 in the sequence), and scrambled histatin-1 (sequences are found in table 1). The compounds used as controls were serum-free medium (negative control) and Epidermal Growth Factor (EGF) diluted in serum-free medium (positive control). The histatins were synthesized and purified as described by Van Dijk and co-authors (Van Dijk, Nazmi, Bolscher, Veerman, & Stap, 2015). The EGF was acquired externally (Biosource, Invitrogen, ThermoFisher

Scientific, USA). The final concentrations of the peptides used for the experiment are found in table 2.

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Table 1: Histatin variants used for the experiments

Peptide Sequence Molecular

mass (Da)1

Histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4848.2 ± 0.5}

Histatin-1-scramble SDHSRHEEFKPRFHYHGGDYYRGRSKNFYHLEYKDHNH _{4848.2 ± 0.5}

Cyclic histatin-1 GGDSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDNLPET~2 _5356.0_{± 0.5} Serine phosphorylated histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.2 ± 0.5} Tyrosine sulfated (3) histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.0 ± 0.5} Tyrosine sulfated (5) histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.0 ± 0.5} Tyrosine sulfated (9) histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.0 ± 0.5} Tyrosine sulfated (12) histatin-1 DSHEKRHHGYR RKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.0 ± 0.5} Tyrosine sulfated (29) histatin-1 DSHEKRHHGYRRKFHEKHHSHREFPFYGDYGSNYLYDN _{4928.0 ± 0.5}

1_{Data obtained from UniProt (National Institute of Health)}

2_{The amino acids GG and LPET were needed for cyclization by the enzyme sortase} Table 2: Final concentrations used for the experiments. The compounds were diluted in serum-free medium.

Compound Final concentration in well

Negative control -

EGF 10ng/ml

Histatin-1

Histatin-1-scramble Cyclic histatin-1

Serine phosphorylated histatin-1 Tyrosine sulfated (3) histatin-1 Tyrosine sulfated (5) histatin-1 Tyrosine sulfated (9) histatin-1 Tyrosine sulfated (12) histatin-1 Tyrosine sulfated (29) histatin-1

10µM

Assays

For the cell spreading experiment 12 wells were used. From their flask, the cells were trypsinized and centrifuged for isolation and then put in serum-free medium to be distributed among the wells. Each of the wells contained 2·104

cells in 0,5 ml serum-free medium and was advanced with 0,5 ml of one of the compounds as listed in table 2. Each variant was repeated three times

randomly. The plate was then transported into the Bioflux-system and time-lapse recording started.

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To determine cell migration, a scratch-assay was conducted. Two days before onset of the experiment, the cells were washed and trypsinized from their culture bottles. The cells were then set about 3·105_{per well (for a total of}

12 wells) containing 1,0 ml medium with fetal calf serum and antibiotics. About six hours prior to recording, the cells were starved by replacing the medium present in the wells with serum-free medium. After starvation, in each cell layer a scratch was made using the tip of a pipette. Debris was removed by rinsing with PBS and 0,5 ml serum-free medium was added to each well and the wells were advanced with 0,5 ml of the various compounds as listed above. The plate was then transported into the Bioflux-system and time-lapse recording started.

A total of six experiments were conducted, of which two contained cyclic histatin-1 and the phosphorylated and sulphated histatins. All experiments contained the positive and negative control. One experiment was discarded for the results deviated significantly from the others (results of reliability test are found in appendix 2). The amount of wells for each compound used for

analysis, is listed in table 3.

Table 3: Amount of wells for each compound.

Compound Total wells

cell spreading Total wells scratch-assay

Negative control 9 12

EGF 9 12

Histatin-1 9 9

Histatin-1-scramble 9 9

Cyclic histatin-1 6

Serine phosphorylated histatin-1 6 Tyrosine sulfated (3) histatin-1 6 Tyrosine sulfated (5) histatin-1 6 Tyrosine sulfated (9) histatin-1 6 Tyrosine sulfated (12) histatin-1 6 Tyrosine sulfated (29) histatin-1 6

Both cell spreading and cell migration were examined with a time-lapse microscope. The plate was placed in a CO2 incubator (Tokai Hit, Japan) and

mounted on the stage of an Axio Observer Z1 inverted microscope (Zeiss, Jena, Germany). Real-time images were acquired using the Bioflux Meta Imaging Series Software Version 7.8.1 (Molecular Devices, Downington, PA, USA) according to the following settings; 5x magnification, brightfield, 50ms exposure. For 24 hours, every 10 minutes a picture was taken of each of the wells. After image acquisition cell spreading and migration were calculated and plotted using ImageJ software Version 1.48V (rsb.info.nih.gov/ij).

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Prior to conducting the assays with the time-lapse microscope, an experiment using a regular cell imaging microscope served as a pilot study to determine the distribution of cells per well needed for the scratch-assay using time-lapse microscopy (images of the pilot study are found in appendix 1).

Picture analysis

Pictures acquired with the Bioflux software were saved in .tiff extension and image stacks were saved in .avi to visualize the dynamics. For cell spreading, the upper right quadrant of the acquired images was used to count the number of cells spread per 100 cells present. The scratches were traced manually using a point-sensitive touchpad after which the perimeter was calculated and converted from pixels to µm.

The cell-spreading was described by the relative number of spread cells at t= 24 hr compared to t= 0 hr. The outcome regarding the scratch-assays was determined to the speed and extent of cell migration as measured by the decreasing surface of the scratch. The extent of migration per hour was forth calculated as the surface that got covered by cells divided by the surface of the wound at t= 0 hr. The speed of wound closure per hour was calculated by the surface that got covered with cells divided by the duration of the

experiment. Statistics were conducted with IBM SPSS Statistics V21 (SPSS Inc., Chicago, USA) using multiple one-way ANOVA tests at a significance of p<0,05.

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Results

Cell spreading

To determine the amount of cell spreading, wells containing 2·104_{cells were}

advanced with either histatin-1, histatin-1-scramble, EGF, or serum-free medium (negative control) and images were obtained using time-lapse microscopy. In figure 1 a typical example of cell spreading is shown. Cell-spreading was described by calculating the relative number of spread cells as shown in figure 2 (total cell spreading at the end of the experiment).There are no significant differences in cell spreading for histatin-1compared with the negative control; only the wells containing EGF showed more cells spread than the wells advanced with histatin-1-scramble.

Figure 1: A typical example of cell spreading;

time points shown are at onset and after 8, 16, 24 hours.

t= 0 hr

t= 8 hr

t= 16 hr

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Figure 2: Cell spreading expressed as the relative number of spread cells (%) at the end of the experiment. *Significant difference compared with histatin-1-scramble.

Scratch-assay

To determine the speed and extent of cell migration, a scratch was made in each cell layer and the dynamics were recorded with the time-lapse

microscope. The surface of the scratch was measured and speed and extent of the migration were calculated. Figure 3 shows images from the time-lapse recording of a well containing histatin-1 and the negative control. The cells migrated towards each other and as time progressed the rate of migration lessened. The fastest rate of migration for histatin-1 and serine phosphorylated histatin-1 appeared in the first 5-6 hours (figure 4).

Histatin-1, cyclic histatin-1 and the serine phosphorylated histatin-1

significantly stimulated both the speed and extent of cell migration when compared to the negative control (figure 5 and 6). The effectiveness of

histatin-1 and the serine phosphorylated histatin-1 is equal to that of EGF. Also, histatin-1 and the serine phosphorylated histatin-1 work significantly better than all sulfated variants and scrambled histatin-1.

Dynamics

By stacking the images acquired from the time-lapse microscope, videos were created showing the process of cell spreading and cell migration as influenced by the variants of histatin-1. An example of cell spreading, and the videos concerning cell migration can be viewed online via this link:

http://tinyurl.com/histatin 0 5 10 15 20 25 30 35 40 45 50 Compounds Rela tive num b er of sp rea d cells ( % )

Cell spreading

EGF Histatin-1 Negative control Histatin-1-scramble *

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12 Histatin-1 t= 0 hr Negative control t= 0 hr t= 8 hr t= 8 hr t= 16 hr t= 16 hr t= 24 hr t= 24 hr

Figure 3: Images acquired from the scratch-assay containing Histatin-1 (left column) and the negative control (right column); time points shown are at onset and after 8,16,24 hours. The numbers correspond to the width of the scratch (µm).

669 850 907 1026 991 1017 1033 1041

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Figure 4: The decrease in wound surface for each of the compounds (all sulfated histatins are shown as one). The averages of all wells as noted in table 3 for the scratch-assay are shown. The acquired images were assembled to one hour time intervals for calculating the outcomes.

Figure 5: The average speed of cell migration for the various compounds. The

averages of all wells as noted in table 3 for the scratch-assay are shown. *Significant difference compared with the negative control. For comparison in between groups, see appendix 3. Sp= Serine phosphorylated, Ts= tyrosine sulfated.

0 50000 100000 150000 200000 250000 1 3 5 7 9 11 13 15 17 19 21 23 C ell mig rat ion (decr eas e of w oun d sur face in pix els ) Time (hours) EGF Negative control Histatin-1 Cyclic histatin-1 Serine phosphorylated histatin-1

All sulfated histatins

0 0,5 1 1,5 2 2,5 3 3,5 A ver ag e speed of cell mig rat ion (µm/min ) _* * * *

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Figure 6: The extent of wound closure for the various compounds as percentage of the surface that got covered by cells at the end of the experiment. The averages of all wells as noted in table 3 for the scratch-assay are shown. *Significant difference compared with the negative control. For comparison in between groups, see appendix 3. Sp= Serine phosphorylated, Ts= tyrosine sulfated.

0 5 10 15 20 25 Ex tent of w oun d clos ur e (% ) * * * *

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Discussion

The main focus of this research was to determine the influence of several variants of histatin-1 on cell spreading and cell migration in oral epithelial cells in vitro as an indication of wound healing capacity. Cell-spreading was

described by calculating the relative number of spread cells. To determine cell migration, a scratch-assay was conducted. The surface of the scratch was measured and the speed and extent of the migration were calculated. The dynamics of cell spreading and cell migration were recorded with a time-lapse microscope.

In previous research, large time intervals were set between the measurements and this resulted in a static view of the ongoing process of wound healing. It was unclear how the progress was developing between the given time points, especially concerning the speed of cell migration. The time-lapse microscope automatically obtained images every ten minutes. Stacking all the images presented a dynamic view on cell spreading and cell migration. The fastest rate of cell migration for the wells advanced with histatin-1 and serine phosphorylated histatin-1 appeared in the first 5-6 hours. Such

differences in speed can only be examined accurately in a dynamic view of the process.

From the current research it can be concluded that histatin-1, cyclic histatin-1 and the serine phosphorylated histatin-1 stimulated both the speed and extent of cell migration when compared to the negative control. Due to the limited number of experiments though, caution is advised when interpreting the results. In correspondence to the conclusions of Oudhoff and co-authors, histatin-1 and cyclic histatin-1 stimulate cell migration (Oudhoff, et al., 2008) (Oudhoff, et al., 2009). Their experiments ended after 16 and 18 hours

respectively, but the current study shows that the process of cell migration is still ongoing at 24 hours and may even last for many hours more since the scratch had not been fully covered at the end of the experiment.

Epidermal growth factor was used as a positive control and served with the negative control as proof that the cells responded to the presence of certain compounds in the wells. EGF often showed different results than the variants of histatin-1 and this may be explained by the ability of EGF to stimulate cell proliferation on top of cell spreading and migration.

Histatin-1 and the variants used in this experiment were added to each well with a final concentration of 10µM. Various concentrations may affect cell spreading and cell migration in distinctive ways. It is possible that the

threshold for histatin-1, phosphorylated histatin-1 and cyclic histatin-1 was reached at 10µM, but that the sulfated histatin variants require a higher concentration to exert a similar stimulating effect on cell migration. At the same time, perhaps a lower concentration of the promoting histatin variants may result in the same amount of stimulation.

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In this research, histatin-1 had no influence on cell spreading, whereas previous research concluded histatin-1 to have a stimulating effect with reference to this process too (Oudhoff, et al., 2009). Oudhoff and co-authors used the same epithelial cells and similar methods as in this current research to conduct cell spreading, but they added histatin-1(12-38): a truncated variant of histatin-1. Apparently, this truncated variant promotes cell spreading to a higher extent than histatin-1 as a whole.

Oudhoff and co-authors (Oudhoff, et al., 2009) discussed that the

effectiveness of histatin-1 may not only be sequence-, but also structure dependent, and this might elucidate the found results with the

phosphorylated and sulfated histatin variants. Serine phosphorylated histatin-1 and tyrosine sulfated histatin-1 naturally occur alongside histatin-1 in human saliva. None of the sulfated histatin variants promoted cell migration, whilst the phosphorylated histatin-1 did exert a positive effect. The submandibular and sublingual glands are responsible for most of the histatin-1 produced and are the only glands to produce the sulfated histatin-1. It was said that the sulfated histatins are the result of an incomplete removal of the sulfate prior to granule storage (Cabras, et al., 2007), therefore the activity of the sulfated histatin-1 may not be primarily targeted at stimulating cell migration. Even though it is only found in the highest histatin-producing glands, sulfated histatin-1 makes up for only 10% of the total amount of histatin-1 in human saliva, making it unlikely to play a big role in wound healing as well. In this research, histatin-1 was sulfated on merely one location in the sequence. It is unknown how polysulfation of histatin-1 will affect cell migration, but it is possible that due to a different structure it may show better results.

Wong and co-authors found that wound healing in the oral cavity proceeds faster than in skin (Wong, et al., 2009). Also, hyposalivation causes delayed healing of mucosal lesions. Saliva plays therefore an important role in tissue recovery. Previous research presented a static view on the process of wound healing as stimulated by histatin-1. The current study shows a dynamic view in cell spreading and cell migration as influenced by histatin-1 and its variants supporting the role of histatin-1 as a wound healing factor. Histatin-1 may serve as an important component in tissue recovery and wound healing medications used in dentistry as well as in general medicine. Although more assays are advised to be performed (especially investigating multiple

concentrations of the histatin variants used), it can be concluded that histatin-1, cyclic histatin-1 and serine phosphorylated histatin-1 significantly increase the speed and extent of cell migration in vitro presenting themselves as stimulating factors in wound healing.

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Acknowledgements

“Shoot for the moon. Even if you miss, you’ll land among the stars” - Les Brown

I would like to thank Jan Bolscher, Floris Bikker and Bastiaan Krom for their guidance in this project and supporting my plans in this assay. Many thanks to Jan in particular for teaching me to culture and nurture the cells. I also want to thank Kamran Nazmi, for providing me with all the supplies and peptides needed for this research. A big thank you to Michel Hoogenkamp as well for setting up the Bioflux-system every week and teaching me to work with the ImageJ software.

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References

Cabras, T., Fanali, C., Monteiro, J., Amado, F., Inzitari, R., Desiderio, C., et al. (2007). Tyrosine Polysulfation of Human Salivary Histatin 1. A

Post-Translational Modification Specific of the Submandibular Gland. Journal

of Proteome Research, 6(7): 2472-2480.

Den Hertog, A., van Marle, J., van Veen, H., Van't Hof, W., Bolscher, J., Veerman, E., et al. (2005). Candidacidal effects of two antimicrobial peptides: histatin 5 causes small membrane defects, but LL-37 causes massive disruption of the cell membrane. The Biochemical journal, 388(Pt2): 689-695.

Driscoll, J., Zuo, Y., Choi, J., Troxler, R., & Oppenheim, F. (1995). Functional Comparison of Native and Recombinant Human Salivary Histatin 1.

Journal of Dental Research, 74(12): 1837-1844.

Gusman, H., Leone, C., Helmerhorst, E., Nunn, M., Flora, B., Troxler, R., et al. (2004). Human salivary gland-specific daily variations in histatin concentrations determined by a novel quantitation technique.

Archives of Oral Biology, 49: 11-22.

Kavanagh, K., & Dowd, S. (2004). Histatins: antimicrobial peptides with

therapeutic potential. Journal of Pharmacy and Pharmacology, 56(3): 285-289.

Mackay, B., Denepitiya, L., Iacono, V., Krost, S., & Pollock, J. (1984). Growth-inhibitory and bactericidal effects of human parotid salivary histidine-rich polypeptides on Streptococcus mutans. Infection and Immunity, 44(3): 692-701.

McDonald, E., Goldberg, H., Tabbara, N., Mendes, F., & Siqueira, W. (2011). Histatin 1 Resists Proteolytic Degradation when adsorbed to

Hydroxyapatite. Journal of Dental Research, 90(2): 268-272.

Messana, I., Cabras, T., Iavarone, F., Manconi, B., Huang, L., Martelli, C., et al. (2015). Chrono-proteomics of human saliva: variations of the salivary proteome during human development. Journal of proteome research, 14(4): 1666-1677.

National Institute of Health. (n.d.). Universal Protein Resource. Retrieved

november 1, 2015, from Uniprot: http://www.uniprot.org/uniprot/P15515 Oudhoff, M., Bolscher, J., Nazmi, K., Kalay, H., Van 't Hof, W., Nieuw

Amerongen, A., et al. (2008). Histatins are the major wound-closure stimulating factors in human saliva as identified in a cell culture assay.

The FASEB Journal, 22(11): 3805-3812.

Oudhoff, M., Kroeze, K., Nazmi, K., Van den Keijbus, P., Van 't Hof, W.,

Fernandex-Borja, M., et al. (2009). Structure-activity analysis of histatin, a potent wound healing peptide from human saliva: cyclization of histatin potentiates molar activity 1000-fold. The FASEB Journal, 23(11): 3928-3935.

Piludu, M., Lantini, M., Cossu, M., Piras, M., Oppenheim, F., Helmerhorst, E., et al. (2006). Salivary histatins in human deep posterior lingual glands (of Von Ebner). Archives of Oral Biology, 51(11): 967-973.

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Shimotoyodome, A., Kobayashi, H., Tokimitsu, I., & Matsukubo, T. Y. (2006). Statherin and Histatin 1 Reduce Parotid Saliva-Promoted Streptococcus mutans Strain MT8148 Adhesion to Hydroxyapatite Surfaces. Caries

Research, 40(5): 403-411.

Van Dijk, I., Nazmi, K., Bolscher, J., Veerman, E., & Stap, J. (2015). Histatin-1, a histidine-rich peptide in human saliva, promotes substrate and cell-cell adhesion. The FASEB Journal, 29(8): 3124-3132.

Wong, J., Gallant-Behm, C., Wiebe, C., Mak, K., Hart, D., Larjava, H., et al. (2009). Wound healing in oral mucosa results in reduced scar formation as compared with skin: Evidence from the red Duroc pig model and humans. Wound repair and regeneration, 17(5): 717-729.

Full-link videos:

https://www.youtube.com/watch?v=DwzsaBNgOh4&list=PL1CuOLtydayFIcIh GpEREWLKq3KAvXqyY

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Appendix

Appendix 1: images scratch-assay EVOS

The experiment using the EVOS microscope served as a pilot study prior to using the time-lapse microscope. The purpose of this experiment was to determine the duration of the recording, width of the scratch and the

influence of EGF on the cells to create the best surroundings when recording with the time-lapse microscope.

Pictures were taken using the EVOS FL Cell Imaging system at 4x

magnification (Life Technologies, Carlsbad, USA). In a range of 27 hours, 3 photos were taken (figure 7). At the given time points the cells were removed from the incubator and an image was obtained, after which the cells

returned to the incubator. For the images obtained with the EVOS, each well contained a black vertical marker to adjust the plate for each picture easily. Alongside this vertical line the width of the scratch was measured in pixels and then converted to µm. Figure 7 demonstrates that histatin-1 show to stimulate cell migration to a larger extent than histatine-1-scramble and the negative control. Condition t= 0 hr t=23 hr t=27 hr Negative control EGF Histatin-1 Histatin-1-scramble

Figure 7: Images obtained from the EVOS microscope. Scale: 634 pixels = 1000 µm. The blue line shows the extent of wound healing with the numbers corresponding to the wound diameter (µm).

948 1137 763 883 1012 1047 891 1055 1198 634 679 836

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Appendix 2: results ANOVA reliability scratch-assays

The reliability of the scratch-assays was determined using an ANOVA-test to compare the assays mutually on significant differences. Scratch-assay

experiment 2 (blue circled) showed significant mean differences with all other assays and had therefore been discarded from analysis.

Multiple Comparisons Dependent Variable: VAR00002

LSD

(I) VAR00001 (J) VAR00001

Mean

Difference (I-J) Std. Error Sig.

95% Confidence Interval Lower Bound Upper Bound

1,00 2,00 -2,13130* ,32109 ,000 -2,8717 -1,3909 3,00 -,60849 ,32109 ,095 -1,3489 ,1320 4,00 -,57210 ,32109 ,113 -1,3125 ,1683 2,00 1,00 2,13130* ,32109 ,000 1,3909 2,8717 3,00 1,52282* ,32109 ,001 ,7824 2,2633 4,00 1,55921* ,32109 ,001 ,8188 2,2997 3,00 1,00 ,60849 ,32109 ,095 -,1320 1,3489 2,00 -1,52282* ,32109 ,001 -2,2633 -,7824 4,00 ,03639 ,32109 ,913 -,7041 ,7768 4,00 1,00 ,57210 ,32109 ,113 -,1683 1,3125 2,00 -1,55921* ,32109 ,001 -2,2997 -,8188 3,00 -,03639 ,32109 ,913 -,7768 ,7041

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22 Appendix 3: results ANOVA scratch-assays Speed of cell migration

All groups as listed below have been compared mutually on the speed of cell migration. The blue circles show significant differences between the groups (duplicate results not included).

Group 1: negative control Group 2: EGF

Group 3: cyclic histatin-1

Group 4: serine phosphorylated histatin-1 Group 5: tyrosine sulfated (3) histatin-1 Group 6: tyrosine sulfated (5) histatin-1 Group 7: tyrosine sulfated (9) histatin-1 Group 8: tyrosine sulfated (12) histatin-1 Group 9: tyrosine sulfated (29) histatin-1 Group 10: histatin-1

Group 11: histatin-1-scramble

LSD

(I) VAR00001 (J) VAR00001

Mean

1,00 2,00 -1,98108* ,34743 ,000 -2,6735 -1,2887 3,00 -1,11230* ,42551 ,011 -1,9603 -,2643 4,00 -1,77032* ,42551 ,000 -2,6184 -,9223 5,00 -,37922 ,42551 ,376 -1,2273 ,4688 6,00 -,37828 ,42551 ,377 -1,2263 ,4698 7,00 -,41318 ,42551 ,335 -1,2612 ,4349 8,00 -,54633 ,42551 ,203 -1,3944 ,3017 9,00 -,56803 ,42551 ,186 -1,4161 ,2800 10,00 -1,58969* ,37526 ,000 -2,3376 -,8418 11,00 ,05854 ,37526 ,876 -,6894 ,8064 2,00 1,00 1,98108* ,34743 ,000 1,2887 2,6735 3,00 ,86878* ,42551 ,045 ,0207 1,7168 4,00 ,21077 ,42551 ,622 -,6373 1,0588 5,00 1,60186* ,42551 ,000 ,7538 2,4499 6,00 1,60280* ,42551 ,000 ,7548 2,4508 7,00 1,56790* ,42551 ,000 ,7199 2,4159 8,00 1,43475* ,42551 ,001 ,5867 2,2828 9,00 1,41305* ,42551 ,001 ,5650 2,2611 10,00 ,39139 ,37526 ,300 -,3565 1,1393 11,00 2,03962* ,37526 ,000 1,2917 2,7875

(23)

23 3,00 1,00 1,11230* ,42551 ,011 ,2643 1,9603 2,00 -,86878* ,42551 ,045 -1,7168 -,0207 4,00 -,65802 ,49134 ,185 -1,6372 ,3212 5,00 ,73308 ,49134 ,140 -,2462 1,7123 6,00 ,73402 ,49134 ,140 -,2452 1,7133 7,00 ,69912 ,49134 ,159 -,2801 1,6784 8,00 ,56597 ,49134 ,253 -,4133 1,5452 9,00 ,54427 ,49134 ,272 -,4350 1,5235 10,00 -,47740 ,44853 ,291 -1,3713 ,4165 11,00 1,17084* ,44853 ,011 ,2769 2,0648 4,00 1,00 1,77032* ,42551 ,000 ,9223 2,6184 2,00 -,21077 ,42551 ,622 -1,0588 ,6373 3,00 ,65802 ,49134 ,185 -,3212 1,6372 5,00 1,39110* ,49134 ,006 ,4119 2,3703 6,00 1,39204* ,49134 ,006 ,4128 2,3713 7,00 1,35714* ,49134 ,007 ,3779 2,3364 8,00 1,22398* ,49134 ,015 ,2448 2,2032 9,00 1,20229* ,49134 ,017 ,2231 2,1815 10,00 ,18062 ,44853 ,688 -,7133 1,0745 11,00 1,82886* ,44853 ,000 ,9349 2,7228 5,00 1,00 ,37922 ,42551 ,376 -,4688 1,2273 2,00 -1,60186* ,42551 ,000 -2,4499 -,7538 3,00 -,73308 ,49134 ,140 -1,7123 ,2462 4,00 -1,39110* ,49134 ,006 -2,3703 -,4119 6,00 ,00094 ,49134 ,998 -,9783 ,9802 7,00 -,03396 ,49134 ,945 -1,0132 ,9453 8,00 -,16711 ,49134 ,735 -1,1463 ,8121 9,00 -,18881 ,49134 ,702 -1,1680 ,7904 10,00 -1,21047* ,44853 ,009 -2,1044 -,3166 11,00 ,43776 ,44853 ,332 -,4561 1,3317 6,00 1,00 ,37828 ,42551 ,377 -,4698 1,2263 2,00 -1,60280* ,42551 ,000 -2,4508 -,7548 3,00 -,73402 ,49134 ,140 -1,7133 ,2452 4,00 -1,39204* ,49134 ,006 -2,3713 -,4128 5,00 -,00094 ,49134 ,998 -,9802 ,9783 7,00 -,03490 ,49134 ,944 -1,0141 ,9443 8,00 -,16805 ,49134 ,733 -1,1473 ,8112 9,00 -,18975 ,49134 ,700 -1,1690 ,7895 10,00 -1,21142* ,44853 ,009 -2,1053 -,3175 11,00 ,43682 ,44853 ,333 -,4571 1,3307 7,00 1,00 ,41318 ,42551 ,335 -,4349 1,2612 2,00 -1,56790* ,42551 ,000 -2,4159 -,7199 3,00 -,69912 ,49134 ,159 -1,6784 ,2801

(24)

24 4,00 -1,35714* ,49134 ,007 -2,3364 -,3779 5,00 ,03396 ,49134 ,945 -,9453 1,0132 6,00 ,03490 ,49134 ,944 -,9443 1,0141 8,00 -,13315 ,49134 ,787 -1,1124 ,8461 9,00 -,15485 ,49134 ,754 -1,1341 ,8244 10,00 -1,17652* ,44853 ,011 -2,0704 -,2826 11,00 ,47172 ,44853 ,296 -,4222 1,3656 8,00 1,00 ,54633 ,42551 ,203 -,3017 1,3944 2,00 -1,43475* ,42551 ,001 -2,2828 -,5867 3,00 -,56597 ,49134 ,253 -1,5452 ,4133 4,00 -1,22398* ,49134 ,015 -2,2032 -,2448 5,00 ,16711 ,49134 ,735 -,8121 1,1463 6,00 ,16805 ,49134 ,733 -,8112 1,1473 7,00 ,13315 ,49134 ,787 -,8461 1,1124 9,00 -,02170 ,49134 ,965 -1,0009 ,9575 10,00 -1,04336* ,44853 ,023 -1,9373 -,1494 11,00 ,60488 ,44853 ,182 -,2890 1,4988 9,00 1,00 ,56803 ,42551 ,186 -,2800 1,4161 2,00 -1,41305* ,42551 ,001 -2,2611 -,5650 3,00 -,54427 ,49134 ,272 -1,5235 ,4350 4,00 -1,20229* ,49134 ,017 -2,1815 -,2231 5,00 ,18881 ,49134 ,702 -,7904 1,1680 6,00 ,18975 ,49134 ,700 -,7895 1,1690 7,00 ,15485 ,49134 ,754 -,8244 1,1341 8,00 ,02170 ,49134 ,965 -,9575 1,0009 10,00 -1,02167* ,44853 ,026 -1,9156 -,1278 11,00 ,62657 ,44853 ,167 -,2673 1,5205 10,00 1,00 1,58969* ,37526 ,000 ,8418 2,3376 2,00 -,39139 ,37526 ,300 -1,1393 ,3565 3,00 ,47740 ,44853 ,291 -,4165 1,3713 4,00 -,18062 ,44853 ,688 -1,0745 ,7133 5,00 1,21047* ,44853 ,009 ,3166 2,1044 6,00 1,21142* ,44853 ,009 ,3175 2,1053 7,00 1,17652* ,44853 ,011 ,2826 2,0704 8,00 1,04336* ,44853 ,023 ,1494 1,9373 9,00 1,02167* ,44853 ,026 ,1278 1,9156 11,00 1,64824* ,40117 ,000 ,8487 2,4478 11,00 1,00 -,05854 ,37526 ,876 -,8064 ,6894 2,00 -2,03962* ,37526 ,000 -2,7875 -1,2917 3,00 -1,17084* ,44853 ,011 -2,0648 -,2769 4,00 -1,82886* ,44853 ,000 -2,7228 -,9349 5,00 -,43776 ,44853 ,332 -1,3317 ,4561 6,00 -,43682 ,44853 ,333 -1,3307 ,4571

(25)

25

7,00 -,47172 ,44853 ,296 -1,3656 ,4222

8,00 -,60488 ,44853 ,182 -1,4988 ,2890

9,00 -,62657 ,44853 ,167 -1,5205 ,2673

10,00 -1,64824* ,40117 ,000 -2,4478 -,8487

*. The mean difference is significant at the 0.05 level. Extent of cell migration

All groups as listed below have been compared mutually on the extent of cell migration. The blue circles show significant differences between the groups (duplicate results not included).

Group 1: negative control Group 2: EGF

Group 3: cyclic histatin-1

Group 4: serine phosphorylated histatin-1 Group 5: tyrosine sulfated (3) histatin-1 Group 6: tyrosine sulfated (5) histatin-1 Group 7: tyrosine sulfated (9) histatin-1 Group 8: tyrosine sulfated (12) histatin-1 Group 9: tyrosine sulfated (29) histatin-1 Group 10: histatin-1

Group 11: histatin-1-scramble

LSD

(I) VAR00001 (J) VAR00001

Mean

1,00 2,00 -15,63552* 2,65771 ,000 -20,9323 -10,3387 3,00 -7,95591* 3,25502 ,017 -14,4431 -1,4687 4,00 -12,91396* 3,25502 ,000 -19,4012 -6,4267 5,00 -2,90259 3,25502 ,375 -9,3898 3,5846 6,00 -2, 75422 3,25502 ,400 -9,2415 3,7330 7,00 -2,86564 3,25502 ,382 -9,3529 3,6216 8,00 -3,87651 3,25502 ,238 -10,3637 2,6107 9,00 -3,83073 3,25502 ,243 -10,3180 2,6565 10,00 -12,81275* 2,87065 ,000 -18,5340 -7,0915 11,00 ,25311 2,87065 ,930 -5,4681 5,9743 2,00 1,00 15,63552* 2,65771 ,000 10,3387 20,9323 3,00 7,67961* 3,25502 ,021 1,1924 14,1669 4,00 2,72157 3,25502 ,406 -3,7657 9,2088 5,00 12,73293* 3,25502 ,000 6,2457 19,2202 6,00 12,88131* 3,25502 ,000 6,3941 19,3685

(26)

26 7,00 12,76989* 3,25502 ,000 6,2826 19,2571 8,00 11,75902* 3,25502 ,001 5,2718 18,2463 9,00 11,80479* 3,25502 ,001 5,3176 18,2920 10,00 2,82277 2,87065 ,329 -2,8984 8,5440 11,00 15,88863* 2,87065 ,000 10,1674 21,6098 3,00 1,00 7,95591* 3,25502 ,017 1,4687 14,4431 2,00 -7,67961* 3,25502 ,021 -14,1669 -1,1924 4,00 -4,95805 3,75857 ,191 -12,4489 2,5328 5,00 5,05332 3,75857 ,183 -2,4375 12,5441 6,00 5,20169 3,75857 ,171 -2,2891 12,6925 7,00 5,09027 3,75857 ,180 -2,4005 12,5811 8,00 4,07940 3,75857 ,281 -3,4114 11,5702 9,00 4,12518 3,75857 ,276 -3,3656 11,6160 10,00 -4,85684 3,43109 ,161 -11,6950 1,9813 11,00 8,20902* 3,43109 ,019 1,3709 15,0472 4,00 1,00 12,91396* 3,25502 ,000 6,4267 19,4012 2,00 -2,72157 3,25502 ,406 -9,2088 3,7657 3,00 4,95805 3,75857 ,191 -2,5328 12,4489 5,00 10,01137* 3,75857 ,010 2,5205 17,5022 6,00 10,15974* 3,75857 ,009 2,6689 17,6506 7,00 10,04832* 3,75857 ,009 2,5575 17,5391 8,00 9,03745* 3,75857 ,019 1,5466 16,5283 9,00 9,08322* 3,75857 ,018 1,5924 16,5740 10,00 ,10121 3,43109 ,977 -6,7369 6,9394 11,00 13,16707* 3,43109 ,000 6,3289 20,0052 5,00 1,00 2,90259 3,25502 ,375 -3,5846 9,3898 2,00 -12,73293* 3,25502 ,000 -19,2202 -6,2457 3,00 -5,05332 3,75857 ,183 -12,5441 2,4375 4,00 -10,01137* 3,75857 ,010 -17,5022 -2,5205 6,00 ,14838 3,75857 ,969 -7,3424 7,6392 7,00 ,03696 3,75857 ,992 -7,4539 7,5278 8,00 -,97391 3,75857 ,796 -8,4647 6,5169 9,00 -,92814 3,75857 ,806 -8,4190 6,5627 10,00 -9,91016* 3,43109 ,005 -16,7483 -3,0720 11,00 3,15570 3,43109 ,361 -3,6824 9,9939 6,00 1,00 2,75422 3,25502 ,400 -3,7330 9,2415 2,00 -12,88131* 3,25502 ,000 -19,3685 -6,3941 3,00 -5,20169 3,75857 ,171 -12,6925 2,2891 4,00 -10,15974* 3,75857 ,009 -17,6506 -2,6689 5,00 -,14838 3,75857 ,969 -7,6392 7,3424 7,00 -,11142 3,75857 ,976 -7,6022 7,3794 8,00 -1,12229 3,75857 ,766 -8,6131 6,3685 9,00 -1,07652 3,75857 ,775 -8,5673 6,4143

(27)

27 10,00 -10,05853* 3,43109 ,005 -16,8967 -3,2204 11,00 3,00733 3,43109 ,384 -3,8308 9,8455 7,00 1,00 2,86564 3,25502 ,382 -3,6216 9,3529 2,00 -12,76989* 3,25502 ,000 -19,2571 -6,2826 3,00 -5,09027 3,75857 ,180 -12,5811 2,4005 4,00 -10,04832* 3,75857 ,009 -17,5391 -2,5575 5,00 -,03696 3,75857 ,992 -7,5278 7,4539 6,00 ,11142 3,75857 ,976 -7,3794 7,6022 8,00 -1,01087 3,75857 ,789 -8,5017 6,4799 9,00 -,96510 3,75857 ,798 -8,4559 6,5257 10,00 -9,94711* 3,43109 ,005 -16,7853 -3,1090 11,00 3,11875 3,43109 ,366 -3,7194 9,9569 8,00 1,00 3,87651 3,25502 ,238 -2,6107 10,3637 2,00 -11,75902* 3,25502 ,001 -18,2463 -5,2718 3,00 -4,07940 3,75857 ,281 -11,5702 3,4114 4,00 -9,03745* 3,75857 ,019 -16,5283 -1,5466 5,00 ,97391 3,75857 ,796 -6,5169 8,4647 6,00 1,12229 3,75857 ,766 -6,3685 8,6131 7,00 1,01087 3,75857 ,789 -6,4799 8,5017 9,00 ,04577 3,75857 ,990 -7,4450 7,5366 10,00 -8,93624* 3,43109 ,011 -15,7744 -2,0981 11,00 4,12962 3,43109 ,233 -2,7085 10,9678 9,00 1,00 3,83073 3,25502 ,243 -2,6565 10,3180 2,00 -11,80479* 3,25502 ,001 -18,2920 -5,3176 3,00 -4,12518 3,75857 ,276 -11,6160 3,3656 4,00 -9,08322* 3,75857 ,018 -16,5740 -1,5924 5,00 ,92814 3,75857 ,806 -6,5627 8,4190 6,00 1,07652 3,75857 ,775 -6,4143 8,5673 7,00 ,96510 3,75857 ,798 -6,5257 8,4559 8,00 -,04577 3,75857 ,990 -7,5366 7,4450 10,00 -8,98202* 3,43109 ,011 -15,8202 -2,1439 11,00 4,08384 3,43109 ,238 -2,7543 10,9220 10,00 1,00 12,81275* 2,87065 ,000 7,0915 18,5340 2,00 -2,82277 2,87065 ,329 -8,5440 2,8984 3,00 4,85684 3,43109 ,161 -1,9813 11,6950 4,00 -,10121 3,43109 ,977 -6,9394 6,7369 5,00 9,91016* 3,43109 ,005 3,0720 16,7483 6,00 10,05853* 3,43109 ,005 3,2204 16,8967 7,00 9,94711* 3,43109 ,005 3,1090 16,7853 8,00 8,93624* 3,43109 ,011 2,0981 15,7744 9,00 8,98202* 3,43109 ,011 2,1439 15,8202 11,00 13,06586* 3,06886 ,000 6,9496 19,1821 11,00 1,00 -,25311 2,87065 ,930 -5,9743 5,4681

(28)

28 2,00 -15,88863* 2,87065 ,000 -21,6098 -10,1674 3,00 -8,20902* 3,43109 ,019 -15,0472 -1,3709 4,00 -13,16707* 3,43109 ,000 -20,0052 -6,3289 5,00 -3,15570 3,43109 ,361 -9,9939 3,6824 6,00 -3,00733 3,43109 ,384 -9,8455 3,8308 7,00 -3,11875 3,43109 ,366 -9,9569 3,7194 8,00 -4,12962 3,43109 ,233 -10,9678 2,7085 9,00 -4,08384 3,43109 ,238 -10,9220 2,7543 10,00 -13,06586* 3,06886 ,000 -19,1821 -6,9496 *. The mean difference is significant at the 0.05 level.

Time-lapse microscopy of wound healing: the effects of histatin variants in vitro