University of Groningen
Salivary lubrication and xerostomia
Vinke, Jeroen
DOI:
10.33612/diss.133408765
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Publication date: 2020
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Vinke, J. (2020). Salivary lubrication and xerostomia. University of Groningen. https://doi.org/10.33612/diss.133408765
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Chapter 6
General discussion
CHAPTER 6
General discussion
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General discussion
Salivary lubrication is important for oral functions like mastication, swallowing and talking as well as to protect oral hard and soft tissues against wear and abrasion. Changes in salivary composition and amount can influence the lubrication properties and thereby the functionality of saliva. Xerostomia, the subjective feeling of dry mouth, can be a result of a decreased lubrication1.
For the relief of symptoms related to oral dryness, several dry mouth relieving agents have been used by patients. The past decades, the number of commercially available products has exploded, prompted by commercial interests. Saliva substitutes are fluids or gels composed of food thickeners such as starch, glycerine, glycols and polysaccharides like celluloses, xanthan gum and Aloe vera. Porcine gastric mucin is
also used. Sometimes antimicrobials and food colourants or taste enhancers are added to these substitutes.
The efficacy of saliva substitutes to relieve dry mouth sensation and comparison of different types have been assessed through clinical tests, in which patients were asked to use products and score their dry mouth sensation, usually by completing xerostomia questionnaires or visual analogue scales.
Although lubrication is one of the main functions in the oral cavity provided by saliva, it has been mostly neglected in xerostomia research. More focus than on lubrication was laid on flow rates, pH and viscosity of saliva as well as on stringiness of saliva of people experiencing a dry mouth2,3. When lubricating
properties of saliva were studied, these properties were tested on artificial surfaces4,5. The last decade,
tribology has gained more interest in biomedical and food technology research6–8, because it has been
shown that tribology matches better with oral perception than rheology9. Tribology is the science of
friction, wear and lubrication between interacting surfaces in relative motion and the properties of those surfaces. The focus of the research described in this thesis was to create and test an ex vivo testing system
for quantifying intra-oral lubrication as well as to use this system to explore the lubricating properties of saliva of healthy volunteers, saliva of patients and saliva substitutes. Additionally, a lubrication enhancing strategy was tested.
Ex vivo measurements of lubrication
As mentioned above, there is a need to develop an ex vivo test system. In Chapter 2, two systems were
described and compared for measuring lubricating properties in terms of friction. In both systems, the friction between two opposing surfaces was measured. The opposing surfaces differed in the systems. In one system, biological tissue was used and in the other system polydimethylsiloxane. Biological tissue had been used before to study the lubricating properties of saliva, but the novelty of the system described in
Chapter 2 was the introduction of the lubrication parameters ‘Relief’ and ‘Relief period’ as indicators for
dry mouth. Using these parameters, measures of oral relief as felt by patients after applying symptom-relieving agents in the mouth (Relief) and the duration of that relief (Relief period) were created. The Relief was calculated by dividing the coefficient of friction on dry tissue (to mimic dry mouth conditions) by the coefficient of friction in a lubricated situation. In both systems, the lubricating properties of water, two saliva substitutes (Saliva Orthana and Dentaid Xeros) and several types of saliva (stimulated and unstimulated whole saliva, reconstituted whole saliva, glandular sublingual/submandibular saliva, parotid saliva) were measured. The tongue-enamel friction system better differentiated between lubricating agents than the polydimethylsiloxane system and was therefore found to be more sensitive.
Relief period was found to be very variable. The standard deviations were often so large that no statistical differences could be found. We considered biological variation in the stiffness and presence of ridges on the tongues as a possible cause for this large variation in Relief period. These biological variations remained despite a strict selection criterion (surface roughness, tissue stiffness, damage, size and direction
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of ridges) for the tongues. Controlling variation in tongue surface characteristics was difficult to minimize though and only a much larger size of experiments would have lowered the standard deviations. Ridges could function as drainage or storage channels leading to less dispersion of saliva on the track, but also could lead to a lower evaporation rate leading to the restoration of the salivary conditioning film (SCF) and longer Relief periods. Controlling the laboratory’s environmental conditions like temperature and humidity could also reduce the variation between Relief periods between separate experiments. To minimize differences, the hydration level of all tongues were brought to a similar state by incubating the tissue in a buffered solution. Best would be testing in intraoral air humidity, however, no solution was found to adequately protect the machinery electronics against moisture. Using the system despite these flaws in the system characteristics can lead to variances in measuring Relief period. Still, it has been shown that the parameter ’Relief period‘ can be used to show major improvements in salivary lubricating properties using smart strategies to enhance the duration of the Relief period, i.e., by using muco-adhesives to recruit native occurring mucins10. The parameter ‘Relief’ is not dependent as much as the
‘Relief period’ on changing laboratory conditions since this measure is taken immediately after lubricating the surface. Relief is predominantly determined on the lubricant and surface properties.
The extent to which we can compare the Relief with truly sensed relief in the oral cavity remains speculative since dry mouth is a subjective sensation. Actual salivary flow rates insufficiently predict whether a person might experience oral dryness11. Differences in mouthfeel were linked to differences in
intraoral friction12,13. We found that changes in mouthfeel related better with the differences in Relief as
measured in the tongue-enamel system than with that in the fully artificial system. The artificial system was insufficiently sensitive to measure differences in Relief. Therefore, the Relief from the tongue-enamel friction system is easier translatable to real mouthfeel than Relief measured with the artificial friction system.
We performed experiments in which Relief correlated positively with perceived mouthfeel in healthy volunteers. These experiments were performed by sipping different kinds of drinks (water (neutral), pear juice (soft) and red wine (astringent)) and scoring sensed roughness of oral surfaces and dry mouth sensation. The same drinks were mixed with saliva and subjected to the tongue-enamel friction system. These experiments could be more elaborated to find more robust correlations. For instance, a broader spectrum of foods and drinks mixed with saliva could be subjected to the system and a mouthfeel score for the same substances could get achieved by using a panel of volunteers. If the correlations appear more robust, the system as proposed in this thesis has the potential to gain impact in the saliva (substitute) and food research.
Measurement of lubrication of saliva from healthy controls and xerostomia patients
Once the ex vivo friction system was established, this system was used to assess the lubricating properties
of saliva from healthy controls and patients with xerostomia (Chapter 3). It was assessed how the
lubricating properties and factors like whole salivary flow rate, and concentrations and output of protein and the MUC5b glycoprotein may explain dry mouth sensation.
The concentrations of mucin in saliva of healthy controls and patients experiencing xerostomia did not differ. This confirms the findings of others14,15 who proposed that not the concentration but functional
integrity of salivary mucins is impaired. Altered mucin glycosylation15, mucin hyposulphation16, and
decreased mucin sialylation17 all may result in a decrease in mucin water retaining capability, and thus in
reduced mucosal hydration. The loss of hydration potentially explains the loss in salivary lubricating properties15 since the lubrication is facilitated by electrostatic repulsion of hydrated residues18.
Impaired mucin integrity leads to decreased salivary film thickness. The latter was found evident in dry mouth patients. The decreased salivary film thickness apparently leads to dry mouth sensation even when
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mucin levels were unchanged and total protein levels were increased15. The residual salivary film thickness
is maintained by adsorbed salivary proteins that retain water and serve as a lubrication layer19. The film
thickness varies between different sites in the oral cavity. For instance, the salivary film thickness in healthy subjects varies from 17 µm on the palatal mucosa to 77 µm on the anterior tongue. A salivary film thickness of <10 µm on the palatal mucosa is accompanied by a dry mouth sensation when at the same time the salivary film thickness on the anterior tongue was reduced to 30 µm19. When saliva flow
rate remains above the critical value to compensate for evaporation, absorption through the oral mucosa, and clearance down the throat, mucosal wetness will continue to be sufficient and dry mouth sensation will not develop19–21.
Hyposalivation would lead to an inhomogeneous spread of saliva throughout the oral cavity but in our research, we worked with the same volume of saliva (20 µl) that was always dispersed over the same area on the tongue (2.33 cm2). Equal coverage of the testing area with 20 µl of saliva results in a film thickness
of about 80 µm. Such a thick layer is higher than the critical value (<10 µm) and the equivalent value on the tongue (30 µm) for dry mouth sensation. The salivary film was sufficiently thick and could have been the cause that differences in lubricating properties of saliva could not have been measured, despite the possible impairment of mucin structural integrity and water-retaining properties. What we did see, however, when working with lower volumes of saliva, was that the measured Relief was lower. A volume of 10 µl of saliva provides a film thickness of about 40 µm on the tongue-enamel interface. This could be the cause of a lowered Relief. Based on these preliminary results we could conclude that insufficient replenishment of the residual salivary film thickness might be a reason for dry mouth sensation. Whether partly disfunctionalized mucins might play a role was not clear from these experiments. In research concerning lubricating properties of xerostomia patients’ saliva should incorporate all of the previously mentioned factors.
The functionality of saliva substitutes
Saliva substitutes are exogenous and their functioning should ideally be independent of the salivary flow rates of patients. So, in Chapter 4 we used the tongue-enamel friction system to systematically and
objectively quantify the lubricating properties of saliva substitutes. Quartz crystal microbalance with dissipation (QCM-D) was used to assess the adsorption of saliva substitutes on existing SCFs and the ability to interact with it. Only for a couple of saliva substitutes (Saliva Orthana, Aldiamed, Saliva Natura, and Glandosane sprays and Biotène and Gum Hydral gels), we found either interaction with saliva-coated surfaces and lubrication enhancement.
Evaluation of saliva substitutes was done in great numbers before, often in clinical studies where dry mouth patients participated and used saliva substitutes. Subjective dry mouth questionnaires were frequently completed and salivary flow rates were often assessed in addition. Such studies were summarized and reviewed by, amongst others, Hahnel et al.22 and Furness et al.23. These authors found
no clear indications for saliva substitutes to function. Survey results were frequently contradictory and altogether, there was no saliva substitute that was considerably better than other products (control, placebo’s, natural sialagogues, water or other saliva substitutes). In some studies, rheological tests were performed between several saliva substitutes in order to test whether viscosities were similar compared with saliva. Only in high shear rates (>10 s-1) the viscosity of saliva was sometimes matched by saliva
substitutes due to the non-Newtonian characteristics that saliva displays as a fluid24–27. However, a saliva
substitute that matches the viscosity of saliva can still display far inferior lubricating properties28. This
illustrates how rheology and tribology are different fields of science as also recognized by others7,29.
Rheology focuses on the properties of the fluid under certain conditions and tribology focuses on the interaction between fluids or lubricants between sliding surfaces under motion.
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The tongue-enamel friction system enables us to compare the lubricating properties of saliva substitutes, and in less extent also the capability to keep the tongue surface lubricated for a longer period. The results of the tongue-enamel lubrication studies provide an objective understanding of the mechanism of how saliva substitutes perform ex vivo. The results confirm why many saliva substitutes in vivo lack functionality.
Many saliva substitutes lack ingredients that truly enhance lubricating properties.
QCM-D was used in adsorption studies of polymers on hydroxyapatite and silica showing that interaction and adsorption of exogenous polymers on biological tissues can be studied30. QCM-D can also be used
to study the ability of substances, i.e., saliva substitutes, to perturb or interact with a pre-adsorbed SCF. Mass adsorption and the film structural softness were measured before31. In that perspective, using
QCM-D for assessing saliva substitutes in wet conditions in vitro is a promising technique. The QCM-D
experiments already provided an understanding of the interaction of saliva substitutes to pre-adsorbed SCFs and the order in which they adhere under fluid shear. The study described in chapter 4 provided the data (on Relief and interaction to a precoated SCF) that subscribes the thoughts of researchers that saliva substitutes do not have the required composition to provide relief to xerostomia patients22,23. Most
saliva substitutes tested did not perform better than water concerning Relief or a long-term Relief period which is in line with the conclusions of the reviews performed by, amongst others, Hahnel et al.22 and
Furness et al.23.
A novel strategy for saliva substitutes
Since regular saliva substitutes did not properly work in relieving dry mouth sensation ex vivo32, and there
is a lack of proof of functionality in vivo22,23 we felt the urge to introduce a new strategy to fight dry mouth
symptoms. We hypothesized that when saliva substitutes contain components that strongly interact with the existing SCF on oral surfaces, the integrity and thickness of the SCF will be further enhanced. This might also improve the hydration of the oral cavity. Therefore in the study described in Chapter 5, we
tried to restore salivary lubrication using the native salivary mucins that are still sufficiently present in patients’ saliva15. Recombinant cationic supercharged polypeptides (SUPs) can interact electrostatically
with salivary components and strengthen the SCFs on the oral surface. Sessile SUPs recruit mucins from saliva and create a composite double-layer on the oral surfaces. This composite double-layer is composed of a compacted rigid base layer and a very soft top layer composed predominantly of mucins. In chapter 5, the presence of this double-layer formation was proven by QCM-D and X-ray photoelectron spectroscopy data and surface topography was assessed by using atomic force microscopy. The SUP-induced salivary double-layer was shown to keep the tongue surface hydrated for a longer period in the model system. This observation is considered important since saliva provides hydration lubrication in the boundary lubrication regime18. Interestingly, in Chapter 3 no differences in lubrication (Relief) between
patient and healthy saliva was found, which recurred in chapter 5, where also no differences in Relief were found between healthy volunteers and xerostomia patients. Even when improving the hydration state of the saliva using SUPs, the lubricating properties of saliva from healthy volunteers and patients remained equal at macro-scale lubrication (using the tongue-enamel friction system and the tribochemist). These findings suggest that the lubricating ability of saliva remains intact, at least when sufficient saliva is present.
Duration of lubrication or Relief period was observed to be prolonged by 7 times for both healthy and patients’ saliva after SUP treatment. The SCFs from patient and healthy saliva did not retain water for a long time (5-10 min). Water probably evaporated very quickly due to the low ambient humidity. Addition of SUPs, and especially K108cys, extended the Relief period of patients’ saliva to over 20 min
in ambient conditions. This implies that K108cys has the potential to improve the naturally occurring water retaining properties and lubrication, taking a protective role from the soft and hard tissues that are
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insufficiently wetted. In vivo, this period would probably be even higher because the humidity in the oral
cavity is higher. Furthermore, once a robust SCF is acquired with the help of the SUPs the SCF can be
rehydrated with just water without the need of re-administration of SUPs. We did not test this ability of rehydration of SUP treated SCF in our work because of time constraints. Only a few saliva substitutes that we tested in Chapter 4, provided a similar long Relief period, however of which some were easily washed away with buffer. Using SUPs for providing a long-term Relief would be beneficial since SUPs can recruit freshly secreted mucins through electrostatic interactions and hydrogen bonds. SUPs could fulfil an important role as being the active ingredient in a to be developed saliva substitute whether or not in combination with remineralizing and antimicrobial agents. Additions could enhance protection of the oral cavity against antimicrobials or erosion or provide taste. Food thickeners would be abundant since the lubricating properties would be provided by the native occurring saliva.
Future perspectives
A physical testing system that can quantify the lubricating properties of saliva and saliva substitutes was developed. We expect that the developed system will be a great aid in saliva substitute manufacturing. This system can be used as an ex vivo testing setup in the development of future saliva substitutes for
relieving dry mouth sensation before going for clinical tests. After favourable ex vivo testing, a saliva
substitute can be subjected to clinical trials, to test whether improved lubrication properties relieve dry mouth sensation in vivo, and, not of least importance, to comply to patient’s wishes and expectations.
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References
1. Von Bültzingslöwen, I. et al. Salivary dysfunction associated with systemic diseases: systematic review and clinical
management recommendations. Oral Surg. Oral Med. Oral Pathol. Oral Radiol. Endod. 103 Suppl, S57.e1-15 (2007).
2. Vissink, A. et al. A clinical comparison between commercially available mucin- and CMC-containing saliva
substitutes. Int. J. Oral Surg. 12, 232–238 (1983).
3. Van der Reijden, W. A., Veerman, E. C. I. & Van Nieuw Amerongen, A. Rheological properties of commercially available polysaccharides with potential use in saliva substitutes. tID Pergamon Biorheol. 31, 631–42 (1994).
4. Bongaerts, J. H. H., Rossetti, D. & Stokes, J. R. The lubricating properties of human whole saliva. Tribol. Lett. 27,
277–287 (2007).
5. Stokes, J. R., Boehm, M. W. & Baier, S. K. Oral processing, texture and mouthfeel: From rheology to tribology and beyond. Current Opinion in Colloid and Interface Science 18, 349–359 (2013).
6. Kho, H.-S. Understanding of xerostomia and strategies for the development of artificial saliva. Chinese J. Dent. Res. Off. J. Sci. Sect. Chinese Stomatol. Assoc. 17, 75–83 (2014).
7. Laguna, L., Farrell, G., Bryant, M., Morina, A. & Sarkar, A. Relating rheology and tribology of commercial dairy colloids to sensory perception. Food Funct. 8, 563–573 (2017).
8. Sarkar, A., Kanti, F., Gulotta, A., Murray, B. S. & Zhang, S. Aqueous lubrication, structure and rheological properties of whey protein microgel Particles. Langmuir 33, 14699–14708 (2017).
9. Krop, E. M., Hetherington, M. M., Holmes, M., Miquel, S. & Sarkar, A. On relating rheology and oral tribology to sensory properties in hydrogels. Food Hydrocoll. 88, 101–113 (2019).
10. Wan, H., Vissink, A. & Sharma, P. K. Enhancement in xerostomia patient salivary lubrication using a mucoadhesive.
J. Dent. Res. (2020). doi:10.1177/0022034520917675
11. Van der Putten, G. J., Brand, H. S., Schols, J. M. G. A. & De Baat, C. The diagnostic suitability of a xerostomia questionnaire and the association between xerostomia, hyposalivation and medication use in a group of nursing home residents. Clin. Oral Investig. 15, 185–192 (2011).
12. Camacho, S., Liu, K., van der Linden, A., Stieger, M. & van de Velde, F. Formation, clearance and mouthfeel perception of oral coatings formed by emulsion-filled gels. J. Texture Stud. 46, 399–410 (2015).
13. Pradal, C. & Stokes, J. R. Oral tribology: Bridging the gap between physical measurements and sensory experience.
Curr. Opin. Food Sci. 9, 34–41 (2016).
14. Pramanik, R., Osailan, S. M., Challacombe, S. J., Urquhart, D. & Proctor, G. B. Protein and mucin retention on oral mucosal surfaces in dry mouth patients. Eur. J. Oral Sci. 118, 245–253 (2010).
15. Chaudhury, N. M. A., Shirlaw, P., Pramanik, R., Carpenter, G. H. & Proctor, G. B. Changes in saliva rheological properties and mucin glycosylation in dry mouth. J. Dent. Res. 94, 1660–1667 (2015).
16. Castro, I. et al. Decreased salivary sulphotransferase activity correlated with inflammation and autoimmunity
parameters in Sjögren’s syndrome patients. Rheumatology 51, 482–490 (2012).
17. Chaudhury, N. M. A., Proctor, G. B., Karlsson, N. G., Carpenter, G. H. & Flowers, S. A. Reduced mucin-7 (muc7) sialylation and altered saliva rheology in Sjögren’s syndrome associated oral dryness. Mol. Cell. Proteomics 15, 1048–
1059 (2016).
18. Jahn, S. & Klein, J. Hydration lubrication: the macromolecular domain. Macromolecules 48, 5059–5075 (2015).
19. Wolff, M. & Kleinberg, I. Oral mucosal wetness in hypo- and normosalivators. Arch. Oral Biol. 43, 455–462 (1998).
20. Lee, S.-K., Lee, S.-W., Chung, S.-C., Kim, Y.-K. & Kho, H.-S. Analysis of residual saliva and minor salivary gland secretions in patients with dry mouth. Arch. Oral Biol. 47, 637–641 (2002).
21. Dawes, C. How much saliva is enough for avoidance of xerostomia? Caries Res. 38, 236–240 (2004).
22. Hahnel, S., Behr, M., Handel, G. & Bürgers, R. Saliva substitutes for the treatment of radiation-induced xerostomia: a review. Support. Care Cancer 17, 1331–1343 (2009).
23. Furness, S., Worthington, H. V, Bryan, G., Birchenough, S. & McMillan, R. Interventions for the management of dry mouth: topical therapies. Cochrane database Syst. Rev. (2011). doi:10.1002/14651858.CD009603.pub2
24. Vissink, A., Waterman, H. A., S-Gravenmade, E. J., Panders, A. K. & Vermey, A. Rheological properties of saliva substitutes containing mucin, carboxymethylcellulose or polyethylenoxide. J. Oral Pathol. 13, 22–28 (1984).
25. Madsen, F., Eberth, K. & Smart, J. D. A rheological assessment of the nature of interactions between mucoadhesive polymers and a homogenised mucus gel. Biomaterials 19, 1083–1092 (1998).
26. Ruissen, A. L. A. et al. Effects of carbohydrate polymers applicable in saliva substitutes on the anti-Candida activity
of a histatin-derived peptide. Arch. Oral Biol. 47, 749–756 (2002).
27. Preetha, A. & Banerjee, R. Comparison of artificial saliva substitutes. Trends Biomater. Artif. Organs 18, 178–186 (2005).
6
109
General Discussion
145468 Vinke BNW.indd 109
110
28. Hatton, M. N., Levine, M. J., Margarone, J. E. & Aguirre, A. Lubrication and viscosity features of human saliva and commercially available saliva substitutes. J. Oral Maxillofac. Surg. 45, 496–499 (1987).
29. Chen, J. & Stokes, J. R. Rheology and tribology: Two distinctive regimes of food texture sensation. Trends Food Sci. Technol. 25, 4–12 (2012).
30. Lee, H. -S. et al. Competitive adsorption of polyelectrolytes onto and into pellicle-coated hydroxyapatite investigated
by QCM-D and force s pectroscopy. ACS Appl. Mater. Interfaces 9, 13079–13091 (2017).
31. Veeregowda, D. H. et al. Role of structure and glycosylation of adsorbed protein films in biolubrication. PLoS One 7,
(2012).
32. Vinke, J., Kaper, H. J., Vissink, A. & Sharma, P. K. Dry mouth: saliva substitutes which adsorb and modify existing salivary condition films improve oral lubrication. Clin. Oral Investig. (2020). doi:10.1007/s00784-020-03272-x
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