Role of structure and glycosylation of adsorbed protein films in biolubrication

Abstract

Water forms the basis of lubrication in the human body, but is unable to provide sufficient lubrication without additives. The importance of biolubrication becomes evident upon aging and disease, particularly under conditions that affect secretion or composition of body fluids. Insufficient biolubrication, may impede proper speech, mastication and swallowing, underlie excessive friction and wear of articulating cartilage surfaces in hips and knees, cause vaginal dryness, and result in dry, irritated eyes. Currently, our understanding of biolubrication is insufficient to design effective therapeutics to restore biolubrication. Aim of this study was to establish the role of structure and glycosylation of adsorbed protein films in biolubrication, taking the oral cavity as a model and making use of its dynamics with daily perturbations due to different glandular secretions, speech, drinking and eating, and tooth brushing. Using different surface analytical techniques (a quartz crystal microbalance with dissipation monitoring, colloidal probe atomic force microscopy, contact angle measurements and X-ray photo-electron spectroscopy), we demonstrated that adsorbed salivary conditioning films in vitro are more lubricious when their hydrophilicity and degree of glycosylation increase, 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, these findings were extrapolated to the in vivo situation. Accordingly, lubricating properties of teeth, as perceived in 20 volunteers comprising of equal numbers of male and female subjects, could be related with structural softness and glycosylation of adsorbed protein films on tooth surfaces. Summarizing, biolubrication is due to a combination of structure and glycosylation of adsorbed protein films, providing an important clue to design effective therapeutics to restore biolubrication in patients with insufficient biolubrication.

Figure 1. Ex vivo adsorption of proteins from salivas collected from different salivary glands.

Example of the QCM-D response to salivary protein adsorption on hydroxyapatite crystals as a function of time, expressed in changes in third harmonic frequency (Δf3, thick line) and dissipation (ΔD3, thin line), together with AFM images of the topography of the adsorbed salivary conditioning films as observed at the end of an experiment. The QCM-D chamber is initially filled with buffer till a stable base-line is observed, after which saliva is introduced. After 30 min of salivary protein adsorption, the chamber is perfused again with buffer. (A) parotid saliva (PAR) from a single donor, (B) submandibular saliva (SM) from a single donor, (C) fresh whole saliva (WS) from a single donor, (D) reconstituted whole saliva (RWS) from a pool of donors.

Figure 2. Structural softness, lubricity and repulsive forces of ex vivo adsorbed salivary films from different volunteers.

(A) Day-to-day and person-to-person variations in the structural softness of 30 min old adsorbed films on hydroxyapatite-coated quartz crystals from parotid (PAR), submandibular (SM), fresh whole (WS) and reconstituted whole saliva from a pool of donors (RWS), taken as the ratio of dissipation (ΔD3) and frequency shift (Δf3) of the third harmonic resonance frequency of the QCM crystal. (B) The coefficient of friction (COF) as a function of the normal force applied between a colloidal probe and films formed from salivas collected from different salivary glands and volunteers (see also Figure S1). Data pertaining to salivas collected at different days are indicated by multiple lines, with colors corresponding with the labels in Figure 2A. (C) Repulsive force as a function of the approach distance between a colloidal probe and films formed from salivas collected from different glands and volunteers. Data pertaining to salivas collected at different days are indicated by multiple lines, with colors corresponding with the labels in Figure 2A. The distance D of the repulsive forces (inserts) is taken as the distance where the colloidal probe starts to experience a repulsive force >0.1 nN.

Figure 3. Influence of detergents on desorption and adsorption of salivary proteins.

Example of the QCM-D response to protein adsorption from reconstituted whole saliva (RWS) on hydroxyapatite crystal surfaces, perturbation and continued adsorption of salivary proteins as a function of time, expressed in changes in third harmonic frequency (Δf3, thick line) and dissipation (ΔD3, thin line), together with AFM images of the topography of the adsorbed films as observed at the end of an experiment. Perturbation was established by exposure to (A) buffer, (B) SLS, (C) NaHMP. The QCM-D chamber is initially filled with buffer till a stable base-line is observed, after which RWS is introduced for 2 h to allow salivary protein adsorption, after which a buffer rinse is applied, followed by 2 min perturbation by buffer or a detergent, intermediate buffer rinsing for 15 min and continued perfusion of the chamber with RWS and at the end there was a final buffer rinsing, as indicated in the figure.

Figure 4. Structure and lubricity of the salivary conditioning films formed after perturbation by detergents.

(A) Structural softness of salivary films adsorbed on conditioning films exposed to buffer (RWS), SLS (SLS+RWS) and NaHMP (NaHMP+RWS) and after final buffer rinsing, i.e. at the very end of an adsorption experiment (see also Figure 3). Error bars represent the standard deviations over five independent measurements of the structural softness. Statistically significant (p<0.05, two tailed Student t-test) differences in properties of the SLS+RWS and NaHMP+RWS films with respect to RWS are indicated by *signs. Differences in the properties of the NaHMP+RWS with respect to SLS+RWS is indicated by #sign. (B) Coefficient of friction as a function of the normal force applied for salivary films adsorbed on films exposed to buffer (RWS), SLS (SLS+RWS) and NaHMP (NaHMP+RWS). The COF during compression and de-compression of the films is represented by closed and open symbols, respectively. Error bars represent the standard deviations over 18 independent COF measurements. (C) Example of the repulsive force as a function of the approach distance for the different films.

Figure 5. Glycosylation, clinically registered intra-oral contact angles and sensory perception of tooth surfaces in vivo.

(A) The %O_glyco of salivary films adsorbed on conditioning films exposed to buffer followed by reconstituted whole saliva (RWS), SLS (SLS+RWS) and NaHMP (NaHMP+RWS). Error bars represent the standard deviations over three independent XPS measurements on differently prepared samples. Statistically significant (p<0.05, two tailed Student t-test) differences of the SLS+RWS and NaHMP+RWS films with respect to RWS films is indicated by *sign. (B) Water contact angles in vitro for the films described in Figure 5a and clinically registered, water contact angles measured on the front incisors of human volunteers prior to and after brushing with a SLS or NaHMP containing formulation (represented by hatched columns). Error bars represent the standard deviations over twelve independent contact angle measurements. For the in vitro contact angles, statistically significant (p<0.05, two tailed Student t-test) differences with respect to RWS films are indicated by *signs, while #sign indicates significant differences of NaHMP+RWS films compared with SLS+RWS ones. Similarly, for the in vivo contact angles *signs are used to show the significant difference with respect to unbrushed films and #sign indicates significant differences of brushed films with NaHMP toothpaste compared with brushed films with SLS toothpaste. (C) Mouthfeel scores prior to and after brushing with an SLS or NaHMP containing toothpaste formulation. The mouthfeel questionnaire involved the following questions: 1. How do you like the smoothness of your teeth? 2. How do you like the clean feeling of your teeth? 3. How do you like the moist feeling of your teeth? 4. Overall, how do you like the feeling of your mouth? Scoring was done on a five point scale according to: −2 = extremely bad smoothness, −1 = bad smoothness, 0 = neutral, 1 = good smoothness, and 2 = extremely good smoothness. Error bars represent the standard deviations over the scores obtained from 64 volunteers after use of an SLS containing toothpaste and 12 volunteers after use of a NaHMP containing toothpaste. Statistically significant (p<0.1) differences with respect to pre-brushing are indicated by *signs for each formulation, while #sign indicates significant differences between the SLS and NaHMP containing toothpaste.

Figure 6. Schematic architecture of salivary conditioning films prior to and after chemical perturbation.

(A) unperturbed salivary conditioning film, showing glycosylated mucins adsorbed in loops and trains over a layer of densely packed low-molecular weight proteins, including proline-rich proteins, histatins and lysozymes. (B) salivary conditioning film after exposure to detergents, showing removal of hydrophobic, smaller proteins and partial detachment of high molecular weight glycosylated mucins. Due to their larger size and multiple adsorption sites, larger proteins do not fully detach like the smaller ones. (C) salivary conditioning film after continued salivary flow over films exposed to detergents. Due to their smaller size, low molecular weight proteins adsorb faster than higher-molecular weight mucins, yielding a denser layer of adsorbed low-molecular weight proteins on the surface causing a greater structural softness and extended loops of glycosylated mucins, leaving the surface more hydrophilic. Smaller proteins have less chance to adsorb after exposure to SLS because more trains are left that occupy the substratum surface than after exposure to NaHMP.