Several previous studies have investigated the rheological properties of CF sputum samples and shown that they have viscoelastic behaviour, which can be studied by oscillatory rheometry (microbead rheology or shear rheology) [8]. Nonetheless, rheological measurements of sputum samples face a number of challenges. Samples from spontaneous expectoration are normally diluted by saliva, thus pre-separation is required to assess the properties of the sputum itself. Sputum samples may exhibit significant variability, even from the same individual. There are many possible explanations for this, for example colonization with different bacteria, differences in ASL composition, various degrees of contamination by saliva [14, 15], and different intensities or frequencies of repetitive voluntary cough. Any of these factors can alter the solid content and the hydration level of the sputum and thus its viscoelastic properties [16]. The small sample size and heterogeneity within the sample can also complicate the analysis.
In the present study, we investigated some of the methodological factors influencing the rheological measurements. Sputum samples were separated from saliva by using the centrifugation technique described previously in the literature [4, 17]. After separation, the sputum gel phase was gently homogenized by stirring. For rheological analysis, a resting measurement phase was introduced to investigate whether pre-homogenization, the mechanical effects of sample agitation and insertion into the rheometer, or storage temperature could affect the rheological parameters and their change over time. Our results clearly demonstrate that the introduction of the resting phase into the measurement protocol is recommended because sputum samples undergo significant changes once inserted into the rheometer, which could be due to the unavoidable but disruptive effects of sample operation. The length of this section can be optimized for a given instrument and preparatory operation. It should be long enough for stabilization of the parameters. However, efforts should be made to limit the preparation time as short as possible to avoid the dehydration and degradation of the samples. In our experiments we established this time period of 30 min.
The technique used to mix the sputum with additives, such as mucolytics, can significantly alter the initial rheological properties of the sample. For example, the gel structure was broken down after ultrasonication and did not recover during the test stage. In contrast, pre-warming the samples did not appear to change the gel structure.
The rheological properties of sputum can be influenced by a number of factors, such as solid content, the degree of hydration, pH, electrolyte concentrations, the quantity and types of mucins, extracellular materials (e.g. extracellular DNA and F-actin), and interactions between mucin chains or with other molecules [18]. Mucolytic agents mainly target these interactions, but they may also influence the pH and/or hydration state of the ASL.
The use of hypertonic saline (HS) solution is an inexpensive, safe, and effective additional therapy in CF patients with stable lung function [19]. The inhalation of HS solution can improve mucociliary clearance due to its hyperosmolarity, where water transport into the airways is driven by osmosis, resulting in a deeper periciliary fluid layer and enhanced mucus hydration [20, 21]. Elkins and co-workers reported significant benefits of hypertonic saline inhalation in a 48‐week parallel group study, in which 164 CF patients were randomised to receive either 7% HS (300 mmol/L NaCl) or placebo (150 mmol/L NaCl) [21]. It was demonstrated that FEV1 was approximately 3% higher in the HS than in the placebo group. The effects of other osmotic agents, mannitol [22] and xylitol [23, 24], have also been studied in CF patients.
The inhalation of bicarbonate-containing solutions could be another useful adjuvant therapy in CF. NaHCO3 is an effective, safe and well-tolerated therapeutic agent in CF and possibly in other chronically infected lung diseases [7, 25, 26]. Gomez et al. have recently demonstrated that the inhalation of hypertonic NaHCO3 increased the pH of the ASL and decreased the gel strength of the sputum, which could be explained by greater expansion of mucins and DNA. This weakening effect of bicarbonate on gel strength has also been reported by Stigliani et al. [4] who showed that elastic and viscous moduli, as well as complex viscosity, were reduced after in vitro treatment. This group has also investigated the dissolution and permeation properties of ketoprofen lysinate (Klys) in CF sputum. Interestingly, CF sputum treated with NaHCO3 exhibited more rapid Klys dissolution and permeation than sputum without added bicarbonate [4].
In our study, the effects of NaCl and NaHCO3 solutions (300 mmol/L) were investigated on CF sputum samples in vitro. As a reference treatment, water was added to some sputum samples, which can be regarded as an indicator of the hydrating effect of the additive solution without the salt or bicarbonate. The overall aim of the present work was to develop an in vitro method to predict the efficacy of topically applied mucolytics. This could help in selecting the most appropriate inhalation formulation for the disease status of each CF patient. However, it must be kept in mind that the method presented here is not suitable for assessing the beneficial in vivo osmotic effects of hypertonic solutions.
Storage and loss modulus as well as the loss factor can be used to compare the efficacy of different in vitro treatments. The gel structure and its viscoelastic characteristics can be well characterized by measuring these rheological parameters. Mucus clearance is known to be influenced by viscoelasticity [5]. Two main mucus clearance processes can be distinguished: (i) mucociliary clearance (MCC) and (ii) cough clearance (CC). The efficacy of these clearance processes depends upon the rheological properties of sputum. In previous studies, MCC was simulated by using low frequency deformations (1 rad/s), while CC was mimicked by applying 100 rad/s. In micro-rheology, these frequencies correspond to the beating frequency of epithelial cilia and coughing, respectively [5, 27]. In shear rheology, it has been suggested that higher frequencies should be avoided because the inertia of the instrument strongly affects the torque response of the soft CF sputum. Since it has been recommended to limit frequencies to no higher than 10 rad/s [14, 28], we compared the rheological parameters (G′, G″ and tanδ) of the untreated and treated sputum at 1 and 10 rad/s.
The sputum samples showed great variability based on the elastic modulus at 1 rad/s of the untreated samples. The range of these elastic moduli correspond to values described in a previous study [4] in which the same sputum preparation method was applied. Importantly, we detected 3 distinct categories: (i) highly elastic (G′ > 100,000 Pa), (ii) elastic (100,000 Pa > G′ > 1000 Pa), and (iii) viscoelastic (G′ < 1000) sputum samples. Visually the highly elastic samples were very compact and behaved as a solid material. Elastic samples showed remarkable elasticity, but they were deformable, while viscoelastic samples presented liquid characteristics. The changes in the rheological parameters, with time and additive pre-treatment, were therefore assessed in each category separately.
In this study, we did not assess the clinical status of the patients, thus the conclusions were drawn on the basis of the rheological properties of the sputum samples alone. They were treated either at 10:4 (n = 61) or 10:1 (n = 15) sputum/additive solution ratios. We used a ratio of 10:1 (sputum/additive) based on methods published by Stigliani et al. [4]. In addition, we have chosen a ratio of 10:4 which may mimic the luminal environment following application of the hypertonic saline and water secretion.
When using the larger volume of additive (10:4), the gel strength of the sputum decreased because the viscoelastic moduli decreased and the loss factor increased at each frequency. The pronounced variability of the samples is reflected in the large standard deviation values, which are even more noticeable in the treated samples. Importantly, we found that the added solutions significantly reduced the gel strength of the sputum considering all investigated samples, but the most pronounced changes (lowest p values) were observed for NaHCO3 (p < 0.001). Samples with high elasticity (G′ > 100,000 Pa) exhibited the greatest changes in the parameters, suggesting that dilution of the gel structure may result in the greatest structural breakdown in samples of this type. It is remarkable that even more significant effects were detected when the samples were treated by NaHCO3.
For less elastic samples, a weakening of the gel structure was also observed when they were treated with distilled water or NaHCO3, but not with NaCl solution, where there were no significant changes in the parameters. For viscoelastic samples with a low elastic content, the additives did not cause significant changes in the parameters.
For all samples at 10:4 ratio, the effect of additives significantly reduces the rheological parameters of the sputum, but when the changes within the categories are analysed, it is clear that the reducing effect is mostly limited to samples with significant elasticity (highly elastic and elastic samples), at low elasticity no significant effect can be observed.
When a lower additive volume (10:1 ratio) was used, the mean values of the rheological parameters usually decreased, especially in the case of the highly elastic sputum samples following treatment with either NaHCO3 or NaCl solutions. A decrease in modulus was also observed in this additive ratio. The effects of NaCl were the most remarkable (but not significant) in the most elastic samples, while the efficacy of NaHCO3 was highest in the middle category (lower mean values observed in the case of the treated samples, in Table 2). The beneficial effects of NaCl could also be observed in the sputum samples with the lowest elastic content (G′ < 1000 Pa). Considering all samples treated with 10:1 sputum to additive ratio, the bicarbonate treated samples showed the lowest mean rheological parameters (and thus the more effective treatment), but within the categories this tendency is not prevailed, the effect of additives is different in the case of samples with different rheological profiles. However, it should be noted that no significance was detected in this sputum/additive ratio.
When comparing the alternative sputum/additive ratios used here, it is evident that larger amounts of added water alone can significantly reduce the gel character of the sputum samples. Based on the rheological properties of the initial sputum samples, it may be appropriate to categorize the treatment efficacy by each additive. The difference between the categories suggests that it is advisable to evaluate each sample individually. These efficacy measurements in vitro may be suitable for such an assessment.
In accordance with previously published observations, the standard deviations of the mean values were also high in the present work, which rendered the statistical analysis of the data difficult. However, the introduction of the resting phase, or the equilibration of the samples immediately before measurements may reduce the inaccuracy of the data.