Insufficient or absence of lubrication at articulating body parts results in pain, irritation and discomfort, e.g. in patients with Sjögren’s syndrome [1]. Restoring biolubrication in patients with Sjögren’s syndrome is important as prolonged insufficient lubrication will lead to persisting dryness, damage of tissues and bones and necessitate intervention using artificial lubricants or biomaterials to restore function. Although Sjögren’s syndrome is known to affect 0.5‐1% of the total population, surprisingly there is no comprehensive study on biolubrication that has succeeded in the development of effective therapeutics for Sjögren’s syndrome covering dry mouth, dry eyes and arthritis. Oral dryness can also occur due to the excessive use of drugs and pharmaceutical products and is a common problem in the elderly, who are often on multiple medication. In order to be able to develop effective therapeutics for dryness at articulating body parts, we have chosen the oral cavity as a model for understanding the mechanisms of biolubrication. The oral model was used to investigate the importance of biolubrication in tactile perception in vivo, and look for clues enabling the development of biomimetic, artificial lubricants that can alleviate pain and discomfort due to dryness in vivo. We have used various surface analytical techniques to elucidate the molecular mechanism of biolubrication by saliva, including quartz crystal microbalance with dissipation monitoring (QCM‐D), colloidal probe atomic force microscopy (AFM), X‐ray photoelectron spectroscopy (XPS), contact angle measurements and particulate microelectrophoresis.
Oral cavity as a model for biolubrication
One of the major disadvantages in biolubrication research is the limited availability and difficult access to lubricating biofluids. Synovial fluid and tears are known for their lubricating properties, but practically it is impossible to collect synovial fluid or tears in quantities (approximately 12 ml) required for
General discussion
143 biolubrication research. However, saliva can be collected from human volunteers in sufficiently large quantities for research purposes, while moreover the oral cavity allows certain techniques, like contact angle measurements, to be performed in volunteers [2]. An added advantage of the oral cavity is the possibility to collect parotid, submandibular and sublingual saliva separately, yielding the opportunity to identify the role of individual proteins and other components in biolubrication.
In chapters 2, 3 and 4 we demonstrated in vitro that the use of oral hygiene products influences the structure and composition of salivary conditioning films, including their lubricating properties with an impact on in vivo perception.
Lubrication of hard surfaces by salivary conditioning films adsorbed from the saliva produced by submandibular and sublingual glands, containing predominantly glycosylated mucins, was better than of films adsorbed from saliva excreted by the parotid glands. However, human whole saliva provided the best lubrication, indicating that lubrication by the glycosylated mucins is optimised by adsorption of proteins from other (parotid) glands. Similarly, glycosylated proteins like lubricins and ocular mucins in synovial fluid and tears, respectively, may be pivotal for effective biolubrication.
Amongst all biofluids, saliva as a natural boundary lubricant faces the hardest challenge because of the high contact pressures between oral hard tissues.
Synovial fluids lubricate cartilage surfaces articulating at a contact pressure of 7.5 MPa [3]. Salivary conditioning films lubricate both soft and hard tissues. Molar surfaces can exert a contact pressure of 87 MPa [4], which is 10 times higher than at knee joints. Overall, salivary conditioning films can provide sustained lubrication and prevent abrasion at high contact pressures.
Tactile perception in the oral cavity and consumer product design
Naturally built‐in sensing capabilities within our fingers and tongue can differentiate micro‐textures that cannot be perceived by our eyes. For example, we can differentiate the quality of paper and clothes by sliding our fingers over the products, sense the smoothness by sliding the tongue after brushing over a tooth surface and differentiate between texture and rheology of liquid foods.
Tactile perception is attributed to interfacial friction which is known to be controlled by surface roughness [5, 6]. Several interfacial parameters other than roughness can also influence the friction and hence, trigger tactile perception.
Revealing these parameters will be helpful in tuning interfacial friction generating the required perception in many consumer products. A smooth tooth feeling in vivo as perceived after brushing is related to friction behaviour of chemically and mechanically perturbed salivary conditioning films, as measured by colloidal probe atomic force microscopy (chapters 3 and 4). Note that the relationship between the physical parameters i.e. roughness, adhesion and repulsive forces and friction is similar to the relationship demonstrated in classical nanoscale tribology, as also described in chapter 1.
A brief guide into bio‐mimetic lubricants for boundary lubrication applications Biolubricative polymers are used as relatively cheap therapeutics for immediate relief in patients suffering from oral dryness, but hamper substantive action.
Natural saliva renews existing salivary conditioning films and films perturbed by toothpaste detergents or brushing. Sodium hexametaphosphate (NaHMP) influenced the architecture of salivary conditioning films and provided low friction, due to a combination of a rigid base layer and extended glycosylated outer surface of the conditioning film. This type of naturally occurring architecture is also involved in providing a smooth feeling in vivo (chapter 3). Therefore, we
General discussion
145 suggest mimicking this type of lubricous architecture in biomimetic lubricants to assist in better biolubrication in the oral cavity.
Some biomimetic lubricants, like polymethylmethacrylate block copolymers and polyzwitterionic brushes, provide very low coefficient of friction, i.e. 0.0004 [7, 8].
None of these products has made it to the market, however. This also suggest that the coefficient of friction of the biomimetic lubricants alone is not the only criteria for application as artificial lubricants. The interaction between biomimetic lubricants and the naturally occurring films from biofluids is important for their in vivo application, which is generally ignored in the development of biomimetic lubricants. In our opinion, a better strategy is to develop artificial lubricants that work together with an existing biolubrication system to enhance its lubricating capability rather than replacing the existing natural system.
Structural softness, degree of glycosylation and coefficient of friction for salivary conditioning films formed ex vivo (chapter 3) are considered as a reference for healthy oral condition, as shown in Figure 1. In case of dryness, the use of recombinant supercharged unfolded proteins (SUPs) with 36 (K36) and 72 (K72) positive charges based on elastin‐like polypeptides and cationic stannous ions from SnF2 containing mouthrinses (chapter 6) can improve the degree of glycosylation and reduce the coefficient of friction in salivary conditioning films.
However, the key‐parameters proposed did not match with the healthy reference (Fig. 1), but only move to the healthy corner of the graph. Note that this can be due to the use of reconstituted saliva from a number of individuals as a substitute for fresh saliva from one individual where some of the mucins might be lost or disintegrated during the centrifugation (chapter 3). Nevertheless, one of the important leads here, is that there is a linear relationship between the degree of
glycosylation and co‐efficient of friction. Overall this research work has set a pathway to provide better therapeutics for dry mouth that may be applicable for dry eyes and arthritis too.
Future research
1. We have only studied lubrication of salivary conditioning films adsorbed from saliva of healthy volunteers. In order to develop an effective therapeutic for dry mouth syndrome, we should extend this study to salivary conditioning films adsorbed from saliva of dry mouth patients. Furthermore, lubrication effects of the artificial salivas available on the market need to be investigated.
2. In vitro friction force and in vivo smooth mouth feeling have only been evaluated for a small number oral products and it would be of interest to include food products and beverages as well. Astringent feelings after consuming tea and wine are well known, but it is not clear whether biolubrication by salivary conditioning films is involved in astringency.
3. The entire thesis might be extended to evaluate mechanisms of biolubrication at diseased knee joints, despite the difficulties of obtaining a sufficient amount of synovial fluid.
4. Inclusion of the Tribochemist, an instrument combining infrared spectroscopy and microtribometry to provide real‐time molecular and structural analysis of lubricating films during friction, will help to reveal molecular events during friction.
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147
Figure 1 Degree of glycosylation (%Oglyco; closed symbols), structural softness (open symbols) and co‐efficient of friction (CoF) for salivary conditioning film (SCF) formed from fresh whole saliva (whole saliva P2), salivary conditioning films formed from reconstituted saliva (R‐SCF), R‐SCF with cationic recombinants with 72 (K72) and 36 (K36) charges, and R‐SCF with stannous cations from a stannous fluoride (SnF2) containing mouthrinse. SCF formed ex vivo from a donor (whole saliva‐P2) is assumed to possess properties for optimum natural biolubrication condition (healthy state?). CoF is determined from the slope of friction force and the normal force (applied upto 35 nN). Note that all the SCFs were sheared over an area of 5 µm x 5 µm.
REFERENCES
1. Bowman SJ (2010) Sjögren's syndrome. Medicine 38:105‐108.
2. Vissink A, De Jong HP, Busscher HJ, Arends J, 's‐Gravenmade EJ (1986) Wetting properties of human saliva and saliva substitutes. J Dent Res 65:1121‐1124.
3. Morrell KC, Hodge WA, Krebs DE, Mann RW (2005) Corroboration of in vivo cartilage pressures with implications for synovial joint tribology and osteoarthritis causation. P Natl Acad Sci USA 102:14819‐14824.
4. Dejak B, Mlotkowski A, Romanowicz M (2003) Finite element analysis of stresses in molars during clenching and mastication. J Prosthet Dent 90:591‐
597.
5. Mate CM, Carpick RW (2011) Materials science: A sense for touch. Nature 480:189‐190.
6. Jones CS, Billington RW, Pearson GJ (2004) The in vivo perception of roughness of restorations. Br Dent J 196:42‐47.
7. Raviv U et al. (2003) Lubrication by charged polymers. Nature 425:163‐165.
8. Chen M, Briscoe WH, Armes SP, Klein J (2009) Lubrication at physiological pressures by polyzwitterionic brushes. Science 323:1698‐1701.
Summary
Insufficient biolubrication represents a major healthcare burden that is facing greater pressure and impact on the quality of life with increasing age and life expectancy. Insufficient biolubrication can yield severe discomfort, and rather frequently occurs in the elderly, in patients using drugs or subjected to head‐neck radiotherapy and in patients with Sjögren’s syndrome, a syndrome which includes dryness of the mouth impeding proper speech and mastication, dry and irritated eyes, vaginal dryness and, in its secondary form, excessive friction and wear of articulating cartilage surfaces in hips and knees. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication in the elderly and diseased.
First, we decided to select a model system for our biolubrication research. As described in chapter 1, we selected the oral cavity as a model system, mainly due to its ease of accessibility and availability of its lubricating fluid, i.e. saliva.
Adsorbed salivary conditioning films (SCFs) in the oral cavity are known to provide boundary lubrication, which can be permanently hampered due to disease but also temporarily perturbed by the use of oral hygiene products. Using the simple daily dynamics of perturbation of SCFs, we have tried to provide a comprehensive analysis of biolubrication in the oral cavity at a molecular level and to identify the role of SCFs in lubrication and oral tactile perception. Influences of chemical and mechanical perturbation of SCFs on biolubrication were analyzed and effects of recombinant proteins adsorbed into adsorbed SCFs on biolubrication were determined to provide a clue to improve current saliva substitutes.
SCFs are formed on all oral surfaces exposed to saliva and protect the oral surfaces against its often hostile environment. Oral hygiene products, including toothpastes, are mainly designed for biofilm control, but their detergents and other active ingredients also affect the general properties of adsorbed SCFs. In
Summary
151 chapter 2, the kinetics of SCF formation, its hydrated thickness and visco‐elasticity are determined using a Quartz Crystal Microbalance with Dissipation (QCM‐D).
Two hour old in vitro adsorbed SCFs were 43.5 nm thick and its characteristic frequency was 9.4 MHz, whereas the dehydrated thickness, measured using X‐ray photoelectron spectroscopy, was 2.4 nm. Treatment with toothpaste slurries decreased the film thickness depending on fluoride‐detergent combination involved. Secondary exposure to saliva replenished the perturbed SCFs and increased the film thickness to much of its original thickness, although no relation existed between hydrated and dehydrated film thicknesses indicating differences in film structure. Treatment with SnF2‐SLS containing toothpaste slurries yielded a strong, immediate two‐fold increase in characteristic film frequency with respect to untreated films, indicating cross‐linking in adsorbed salivary‐protein‐films by Sn2+ that was absent when SLS was replaced by NaHMP. Secondary exposure to saliva of SCFs treated with SnF2 caused a strong six‐fold increase in characteristic frequency compared with primary salivary‐protein‐films, regardless whether SLS or NaHMP was the detergent. This suggests that ionized stannous, is not directly available for cross‐linking in combination with highly negatively charged NaHMP, but becomes slowly available after initial treatment to cause cross‐linking during secondary exposure to saliva.
Detergents like SLS and NaHMP in toothpastes not only influence the structure and composition of the SCFs, but also affect the lubrication by SCFs and sensory perception in the volunteers, as determined in chapter 3. Using different surface analytical techniques like atomic force microscopy (AFM), QCM‐D, X‐ray photoelectron spectroscopy (XPS) and contact angle measurements we demonstrated that adsorbed SCFs in vitro are more lubricious when their hydrophilicity and degree of glycosylation increases, meanwhile decreasing their
structural softness. High‐molecular‐weight, glycosylated proteins adsorbing in loops and trains, are described as necessary scaffolds impeding removal of water during loading of articulating surfaces. Comparing in vitro and in vivo water contact angles measured intra‐orally, the sensory‐perception in human volunteers could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces.
Lubrication by SCFs and sensory perception in volunteers are not only affected by detergents, but also by the different modes of mechanical brushing, as shown in chapter 4. Boundary lubrication by SCFs was influenced by different modes of tooth brushing, which corresponds to changes in SCFs roughness, dehydrated layer thickness and degree of glycosylation. Coefficient of frictions (COFs) on 16 hours old SCFs after manual, rotary‐oscillatory and sonically‐driven brushing were measured using colloidal probe AFM. AFM was also used to assess the roughness of SCFs prior to and after brushing. Dehydrated layer thicknesses and glycosylation of the SCFs were determined using XPS. Mouthfeel after manual and rotary‐oscillatory and sonically‐driven brushing was evaluated employing a split‐
mouth design. Compared with unbrushed and manually or sonically‐driven brushed SCFs, powered rotary‐oscillatory brushing lead to deglycosylation of the SCF, loss of thickness and a rougher protein film. Concurrently, due to deglycosylation and its increased roughness, the COF of a powered rotary‐
oscillatory brushed SCF increased strongly by a factor of ten with respect to an unbrushed SCF. Volunteers reported a slightly preferred mouthfeel after sonic‐
brushing as compared to powered rotating‐oscillating brushing. Overall, powered rotary‐oscillatory brushing can deglycosylate a SCF, leading to a rougher protein film as compared with manual and sonic‐brushing, therefore decreasing the
Summary
153 lubricative function of the SCF. This is consistent with clinical mouthfeel evaluation after different modes of brushing.
We have shown that biolubrication is influenced by the structure and glycosylation of adsorbed protein films, providing an important clue to design effective therapeutics to restore biolubrication in patients with insufficient biolubrication. In chapter 5, we apply recombinant supercharged unfolded proteins (SUPs) with 36 (K36) and 72 (K72) positive charges based on elastin‐like polypeptides to improve lubrication of adsorbed SCF. Adsorbed K36 and K72 interact with glycosylated mucins in SCFs to form a rigid film, which increases with the number of positive charges. Renewed exposure to saliva after adsorption of cationic SUPs recruits additional negatively charged glycosylated mucins to create a soft, hydrated film, especially when K72 is involved. These hydrated and rigid films improve lubrication and maintain their structural integrity upon high contact pressures. Current generations of artificial salivas are inadequate to restore oral lubrication on a lasting basis. Therefore, cationic SUPs represent a potential novel therapeutic modality to restore lubrication when availability of naturally occurring proteins is reduced.
In healthy persons, adsorbed SCFs are known for protecting the tooth surfaces against abrasion and also erosion. The structure and glycosylation of the SCFs influencing the lubrication behaviour or abrasion resistance can be also expected to influence the erosion protection by the SCFs. As shown in chapter 6, we use a SnF2 containing mouthrinse to demonstrate the importance of structural and glycosylation changes in SCFs, as induced by Sn2+ ions in the protection of enamel surfaces against erosion and abrasion. QCM‐D showed that SCFs became rigid after exposure to a SnF2 containing mouthrinse, which we attributed to cross‐
linking of adsorbed proteins by Sn2+ ions. During renewed exposure to saliva, the SnF2 treated SCF recruited more salivary proteins, thereby increasing the adsorbed mass and degree of glycosylation in the SCF, as determined from QCM‐D and XPS, respectively. The renewed adsorbed film on a SnF2 treated SCF provided a lower friction than when formed on an untreated SCF. Moreover, such rigid, more heavily glycosylated and lubricious SCFs yielded a lower calcium loss during exposure to a citric acid solution than untreated SCFs. Therewith, this is the first study to demonstrate physical changes in SCFs due to Sn2+ adsorption that can be related to the control of erosion and abrasion of enamel surfaces in vitro.
In chapter 7, we emphasize the advantages of using the oral cavity as a model system for biolubrication studies. Also, we highlight the role of biolubrication in tactile perception which can be of benefit for consumer based design of oral health care products. In the end, we provide details regarding naturally occurring SCFs lubricous architectures which can be important for biomimetic lubrication research to develop artificial lubricants that can provide better wetting of oral surfaces, reducing the sensation of pain due to oral dryness and improving the oral function like chewing, swallowing and speech.
Samenvatting
Onvoldoende biolubricatie (natuurlijke smering) is een vervelend ziektebeeld, die qua impact op de kwaliteit van leven toeneemt met toenemende leeftijd en levensverwachting. Onvoldoende biolubricatie kan leiden tot ernstig ongemak en komt frequent voor bij ouderen, patiënten die medicijnen gebruiken of zijn blootgesteld aan radiotherapie in het hoofd‐hals gebied, of patiënten met Sjögren’s syndroom. Sjögren’s syndroom is een ziekte waarbij droogheid van de mond spraak‐ en kauwproblemen veroorzaakt en wordt mede gekenmerkt door droogheid en irritatie van de ogen, vagina en, in zijn secundaire vorm, overmatige wrijving in en slijtage van gewrichtsoppervlakken in de heupen en knieën. Op dit moment is onze kennis wat betreft biolubricatie onvoldoende om een effectieve therapie te kunnen ontwikkelen om biolubricatie in ouderen en zieken voldoende te kunnen herstellen.
Bij aanvang van dit onderzoek hebben we allereerst een geschikt model systeem gekozen om biolubricatie te bestuderen. De mondholte is als model gekozen zoals beschreven in hoofdstuk 1, voornamelijk vanwege de toegankelijkheid en beschikbaarheid van de vloeistof die hierin voor lubricatie zorgt, namelijk het speeksel. Het is bekend dat geadsorbeerde speeksellagen (SCFs) in de mondholte zorgen voor smering, welke permanent verstoord kan worden door ziekte, maar dagelijks ook tijdelijk verstoord wordt door het gebruik van verschillende mondverzorgingsproducten. Door gebruik te maken van de simpele dagelijkse dynamiek van verstoringen van de SCFs hebben we een analyse gemaakt van de
Bij aanvang van dit onderzoek hebben we allereerst een geschikt model systeem gekozen om biolubricatie te bestuderen. De mondholte is als model gekozen zoals beschreven in hoofdstuk 1, voornamelijk vanwege de toegankelijkheid en beschikbaarheid van de vloeistof die hierin voor lubricatie zorgt, namelijk het speeksel. Het is bekend dat geadsorbeerde speeksellagen (SCFs) in de mondholte zorgen voor smering, welke permanent verstoord kan worden door ziekte, maar dagelijks ook tijdelijk verstoord wordt door het gebruik van verschillende mondverzorgingsproducten. Door gebruik te maken van de simpele dagelijkse dynamiek van verstoringen van de SCFs hebben we een analyse gemaakt van de