Sources of support: Supported by institutional sources and by Sunstar Inc.
Evaluation of a moisturising micro-gel spray for prevention of cell dryness in oral mucosal cells: an in vitro study and evaluation in a clinical setting
Article first published online: 23 APR 2012
© 2012 Blackwell Publishing Ltd
European Journal of Cancer Care
Volume 21, Issue 6, pages 728–734, November 2012
How to Cite
OTA, Y., MORITO, A., FUJISAWA, K., NISHIDA, M., HATA, H., UENO, T., YURIKUSA, T. and MURATA, T. (2012), Evaluation of a moisturising micro-gel spray for prevention of cell dryness in oral mucosal cells: an in vitro study and evaluation in a clinical setting. European Journal of Cancer Care, 21: 728–734. doi: 10.1111/j.1365-2354.2012.01349.x
Re-use of this article is permitted in accordance with the Terms and Conditions set out at http://wileyonlinelibrary.com/onlineopen#OnlineOpen_Terms
- Issue published online: 19 OCT 2012
- Article first published online: 23 APR 2012
- Accepted 22 February 2012
- dry mouth;
- micro-gel spray;
- moisturising product;
- oral mucosal cells
OTA Y., MORITO A., FUJISAWA K., NISHIDA M., HATA H., UENO T., YURIKUSA T. & MURATA T. (2012) European Journal of Cancer Care
Evaluation of a moisturising micro-gel spray for prevention of cell dryness in oral mucosal cells: an in vitro study and evaluation in a clinical setting
A moisturising micro-gel spray for prevention of dryness was compared with commercial products and artificial saliva in vitro and in a clinical setting in patients with cancer. Survival of cultured human gingival epithelial cells was evaluated after treatment with each product for 15 min. A dry test was performed for products giving a 50% survival rate, in which cell survival was measured after drying of cells treated with each product. The survival rates of cells treated with the micro-gel spray and artificial saliva were significantly higher than those of control cells. The micro-gel spray was then evaluated for 1 week in patients with symptoms of dry mouth caused by cancer treatment. There was significant improvement of these symptoms at night and on awakening and of subjective symptoms of decreased salivary volume (P < 0.05). Mean visual analogue scale scores also significantly decreased (P < 0.01). These data suggest that evaluation of moisturising products for dryness prevention can be performed in cultured cells, since products that performed well in vitro also showed good efficacy for symptoms of dry mouth. The micro-gel spray was particularly effective for relieving symptoms of dry mouth in patients with cancer.
Damage to the mucous membrane (mucositis) can occur either as a consequence of direct effects of the chemotherapy and the radiotherapy in head and neck cancer on the epithelial cells or as diminution of the protective effect of saliva (Epstein et al. 2002; Potting et al. 2006; Hong et al. 2009; Mencoboni et al. 2011). White discoloration of the mucous membranes mostly precedes the redness, oedema and lesions. Because of the pain, potential nutritional deficit and requirement for parenteral nutrition, significant reduction in the quality of life and the risk of mucosal infection and subsequent septicaemia, oral mucositis is an important clinical problem and needs a special attention especially in the case of malignancies (Harrold 2010).
Symptoms of dry mouth (xerostomia) occur in patients with severe diseases such as Sjögren syndrome and salivary gland disorders caused by radiation therapy for cancer, as well as with ageing, stress and drug administration. Under such conditions, the mucosa is continuously rubified, resulting in inflammation (Guggenheimer & Moore 2003; Wick 2007). Pilocarpine hydrochloride and cevimeline hydrochloride, which work as stimulators of muscarinic and cholinergic receptors on the exocrine surface, are used in many countries as prescription drugs for treatment of radiation-induced xerostomia (Chambers et al. 2007; Chitapanarux et al. 2008; Jha et al. 2009). However, these drugs have side effects such as gastrointestinal dysfunction and sweating that limit their use (Chambers et al. 2007; Berk 2008; Jha et al. 2009; Nakamura et al. 2009). Under these circumstances, alternative therapy is given for dry mouth, with use of moisturising gels, rinses and sprays, and in-hospital prescription drugs (Davies 2000; Dirix et al. 2007; Gil-Montoya et al. 2008; Hahnel et al. 2009).
A new method has recently been developed for evaluation of moisture retention based on the survival rate of dried oral mucosal cells as an objective marker for comparison of moisturising ingredients (Mori et al. 2010; Morito et al. 2011). We have used this approach to develop a new moisturising product, which we refer to as a micro-gel spray (Morito et al. 2011). Here, we compared the effects of the micro-gel spray on cell survival and dryness prevention in vitro with those of commercial moisturising products and artificial saliva. We then evaluated the performance of the micro-gel spray in a clinical setting in patients with symptoms of dry mouth caused by cancer treatment.
MATERIALS AND METHODS
In vitro evaluation
Human gingival squamous cancer-derived Ca9-22 cells were cultured with 10% foetal bovine serum in Eagle's Minimum Essential Medium (Sigma, USA) at 37°C under 5% CO2. The cells were cultured in a 96-well plate for 2 days until they became confluent. After removing the medium, the cells were washed with Phosphate Buffered Saline and treated at 37°C under 5% CO2 for 15 min with the moisturising products shown in Table 1. The test samples were then aspirated and the cells were washed with Phosphate Buffered Saline. The cell survival rates were measured in an Alamar Blue assay using a microplate spectrometer (Gemini XPS; Molecular Devices, San Jose, CA, USA) with absorption and excitation wavelengths set at 560 nm and 590 nm respectively. The survival rate was calculated relative to an assumed survival rate of 1 with Phosphate Buffered Saline, using the following formula: Cell survival rate = Fluorescence level of the treated group/Fluorescence level of the Phosphate Buffered Saline (control) group.
|Product||Active and moisturising ingredients|
|Mouth wash A||Xanthan gum, polyvinylpyrollidone, carboxymethylcellulose|
|Mouth wash B||Lactoferrin, lysozyme, lactoperoxidase|
|Mouth wash C||Lactoprotein, lactoferrin, aloe vera|
|Mouth wash D||Hyaluronate|
|Mouth wash E||Xanthan gum, glycerine, hyaluronate|
|Mouth wash F||Polyglutaminate|
|Spray G||Glycerine, hyaluronate|
|Spray H||Sodium chloride, potassium chloride, calcium chloride hydrate, carmellose sodium|
|Micro-gel spray||Gellan gum, glycerine, glycosyltrehalose|
Next, we evaluated the cell viability after drying for products with a relative cell survival rate of ≥50%. The cell culture and product treatment were performed as described above. After the 15-min culture, the medium and samples were completely aspirated, and the cells were kept at 20°C and humidity of 30% for 6 to 8 min (Sanyo, Osaka, Japan). Then, 200 µL of Phosphate Buffered Saline was added to each well and the cell survival rate was determined using the Alamar Blue assay described above.
Subjects in evaluation of the micro-gel spray
The subjects were recruited from inpatients and outpatients with subjective symptoms of dry mouth after receiving head and neck radiotherapy and/or chemotherapy at Shizuoka Cancer Center from July to October 2008. The subjects were aged ≥20 years old. Patients prescribed drugs to improve salivary flow before the study and those who were determined to be inappropriate for participation in the study by the investigator due to a risk of aspiration were excluded. All patients received oral and written explanations of the objectives of the study, and written informed consent was obtained before participation in the study. This clinical evaluation was performed after review and approval by the ethical committee of Shizuoka Cancer Center.
Baseline characteristics of the subjects
Age, gender, primary cancer site and symptoms of dry mouth (stimulated salivary volume and subjective symptoms of oral dryness) were recorded as baseline characteristics at the start of the study. Subjective symptoms of dry mouth and accompanying symptoms were determined using 13 items on a questionnaire that the subjects answered by selecting a score of 1 to 5 points (Table 2). Symptoms of dry mouth were also evaluated using a 100-mm visual analogue scale (VAS). Stimulated salivary volume was measured after chewing of gum for 5 min.
|Items on the dry mouth questionnaire||1||2||3||4||5|
|Do you usually feel dryness in the mouth?||Not at all||Seldom||Partial||Yes||Severely|
|Do you often drink water?||Seldom||Less||Partial||Often||Very often|
|Do you suffer from dry mouth at night or on awakening?||Not at all||Seldom||Partial||Yes||Severely|
|Do you have difficulty swallowing when you eat dry foods such as a cracker?||Not at all||Seldom||Partial||Yes||Very often|
|Do you feel oral dryness when you eat a meal?||Not at all||Seldom||Partial||Yes||Very often|
|Do you feel stickiness in the mouth?||Not at all||Seldom||Partial||Sticky||Very often|
|Do you feel that you have mouth odour?||Not at all||Seldom||Partial||Yes||Very often|
|Is speech difficult?||Not at all||Seldom||Partial||Yes||Very often|
|Do you have difficulty in swallowing food?||Not at all||Seldom||Partial||Yes||Very often|
|Do you often suffer from stomatitis?||Not at all||Seldom||Partial||Yes||Very often|
|Do you have difficulty in tasting?||Not at all||Seldom||Partial||Yes||Very often|
|Do you have a pain in your tongue or mouth?||Not at all||Seldom||Partial||Yes||Very often|
|Do you feel that you have less salivary volume?||Not at all||Seldom||Partial||Yes||Very often|
The micro-gel spray was used as required for 1 week. Use of other products and drugs for prevention of oral dryness was prohibited during this period. Changes in subjective symptoms of dry mouth were evaluated based on an interview and changes in VAS scores obtained at baseline and after use of the gel spray for 1 week. Upon completion of the study, the subjects were asked about their general impressions of the micro-gel spray to evaluate its utility and effect duration.
Differences in the in vitro cell survival rate were evaluated by anova and a Tukey multiple comparison test. Subjective symptoms of dry mouth before and after the trial were compared by Wilcoxon signed-rank test. For the 100-mm VAS scale, a t-test was performed to examine the difference between data at baseline and after use of the micro-gel spray for 1 week. All data were analyzed using spss ver.13 (IBM Inc., USA). A two-sided P-value <0.05 was considered to indicate a significant difference.
In vitro evaluation
The survival rates after cells were immersed in individual moisturising products (saliva substitutes) for 15 min are shown in Figure 1. The cell survival rates with six commercial products were <50% compared with Phosphate Buffered Saline treatment (assuming a survival rate with Phosphate Buffered Saline of 1), and these products were excluded from further analysis. The other three products (two commercial products and the micro-gel spray) showed survival rates of ≥50% and were used in a drying test. The relative survival rates (Fig. 2) obtained after drying treatment for 6 to 8 min were 0.63 and 0.64 for product H and the micro-gel spray respectively. These rates were significantly higher than those with Phosphate Buffered Saline and product D (P < 0.01).
Evaluation in a clinical setting
Twenty subjects began use of the micro-gel spray. However, one subject could not eat food and showed symptoms of taste disorder, and thus the product was discontinued. A second subject was excluded from the analysis due to incorrect answers in the evaluation. Therefore, the final analysis was performed for 18 subjects. The background factors at baseline in these subjects are shown in Table 3. The subjects were 11 male and seven female patients and their average age was 61.4 years old (range: 32 to 77 years old). The mean stimulated salivary volume over 5 min at the start of the study was 1.5 mL (range: 0 to 6.0 mL).
|Patient||Age||Gender||Tumour site||Treatment||Stimulated salivary volume (mL/5 min)|
|1||32||Female||Benign tongue angioma||Radiotherapy||1|
|3||57||Male||Unknown primary tumour||Chemoradiotherapy||6|
|7||48||Female||Cancer of the tongue||Surgery||1|
|12||60||Male||Oropharyngeal cancer||Surgery, radiotherapy||3.5|
|14||77||Male||Gastric cancer/cancer of the tongue||Surgery, chemoradiotherapy||1|
|15||66||Female||Laryngeal/hypopharyngeal cancer||Surgery, radiotherapy||1.5|
The mean scores for subjective symptoms of dry mouth at baseline and on completion of the study are shown in Table 4. Significant improvement in dry mouth at night or on awakening and salivary flow occurred after 1 week of gel spray administration (P < 0.05). The mean VAS scores at baseline and at the end of the study are shown in Figure 3. The VAS score for oral dryness after treatment was significantly decreased by use of the micro-gel spray (P < 0.01 by t-test).
|Subjective symptoms of dry mouth||Day 0 (SD)||Day 7 (SD)||P-value|
|Dryness in the oral cavity||4.11 (0.90)||3.67 (0.97)||0.054|
|Fluid intake||3.78 (1.26)||3.33 (1.46)||0.054|
|Dry mouth at night or on awakening||4.28 (0.90)||3.72 (1.07)||0.026*|
|Difficulty of swallowing dry food||4.11 (1.08)||3.72 (1.07)||0.319|
|Feeling of oral dryness during eating||3.94 (1.11)||3.39 (1.15)||0.133|
|Sticky feeling in the oral cavity||3.67 (1.14)||3.50 (1.25)||0.408|
|Subjective feeling of mouth odour||2.22 (0.94)||2.22 (1.22)||0.951|
|Difficulty with speech||3.50 (0.86)||3.56 (0.98)||0.725|
|Difficulty swallowing||3.61 (1.15)||3.50 (1.10)||0.914|
|Stomatitis||2.39 (1.20)||2.39 (1.15)||1.000|
|Difficulty with tasting||3.17 (1.15)||2.94 (1.16)||0.248|
|Pain in tongue and oral cavity||2.44 (0.78)||2.44 (1.10)||1.000|
|Awareness of having less salivary flow||4.39 (0.70)||3.89 (1.08)||0.041*|
The results of evaluation of the micro-gel spray on a 5-point scale are shown in Table 5. Single use of the micro-gel spray resulted in a mean effect duration of 30 to 60 min. The scores for ease and pleasance of use, mucosal sensitivity, refreshment, change of dry mouth feeling and willingness to continue use were 3.9, 3.7, 3.5, 4.1 and 4.1 respectively.
|Duration of effect||–||0.5–1 h||0.5–1 h||<0.5–2to3 h|
|Ease and pleasance of use||3.9||4||4||1–5|
|Change of dry mouth feeling||4.1||4||4||2–5|
|Willingness to continue use||4.1||4||5||2–5|
The micro-gel spray was developed as a moisturising product for dry mouth caused by cancer treatment. In this study, the effects and utility of this product were evaluated. The results showed that the gel spray increased cell survival under dry conditions in vitro. In contrast, treatment of cells with six of eight commercial products resulted in cell survival rates of <50% after 15-min immersion and some products caused alteration of cellular morphology in this time period. This suggests that these products might cause direct damage to the cells.
Many products aimed at moisture retention and moistening have been developed for treatment of patients with symptoms of dry mouth and several of these products physically coat oral tissues. These moisturising products often contain carboxymethyl cellulose as a thickener and mucin and xanthan gum as moisturising and viscoelastic materials respectively. The micro-gel spray contains glycerine, gellan gum and glycosyltrehalose, which were selected by screening for ingredients with protective effects on cells (Morito et al. 2011). Trehalose has been shown to protect corneal epithelial cells against death by drying (Matsuo 2007). These effects may be caused by the trehalose sugar stabilising lipids and proteins on the cell membrane under conditions of oral dryness (Crowe et al. 1987).
The micro-gel spray significantly improved symptoms of dry mouth at night and on awakening and reduced salivary flow in patients with dry mouth caused by cancer treatment. A significant improvement in VAS scores for a post-treatment feeling of oral dryness was also obtained. The VAS score has been widely used for evaluation of symptoms of dry mouth (Shahdad et al. 2005; Dirix et al. 2007). In the current study, improved VAS scores corresponded with the results of a questionnaire survey, in which approximately 90% of the subjects answered that their symptoms of dry mouth were improved. In contrast, there was no improvement in swallowing of dry food, feeling of oral dryness during eating, sticky feeling in the oral cavity, subjective feeling of mouth odour, difficultywith speech and swallowing, development of stomatitis, difficulty with tasting, and pain in the tongue and oral cavity after use of the micro-gel spray. In a study of Aloe Vera Gel, carboxymethyl cellulose spray, porcine stomach mucin spray and rapeseed oil, symptoms of dry mouth after radiotherapy were also improved, but there were no significant improvements in difficulty with swallowing during eating, the need for water drinking, and difficulty with tasting (Momm et al. 2005).
One of the 20 subjects discontinued treatment with the micro-gel spray on the third day of use due to a bad taste. However, this subject might have had a taste disorder due to coated tongue due to the absence of oral food intake in the terminal phase. Approximately 80% of the subjects who continuously used the micro-gel spray felt that the usability was good or better, and only one subject felt that it was bad. The mean stimulated salivary volume in 5 min at the start of the study (baseline) was very low (1.5 mL), and thus the subjects had severe dry mouth. Oral symptoms caused by cancer treatment include oral mucositis, dentin hypersensitivity and oral dysgeusia, in addition to dry mouth (Fisher et al. 2003). Patients with these conditions require mild salivary substitute products with reduced sweetness (artificial saliva). Approximately 80% of the subjects were satisfied with the usability of the micro-gel spray, which suggests that the product has high tolerability for patients with oral symptoms. The median effect duration after single use of the micro-gel spray was 30 to 60 min, and the longest duration was 2 to 3 h. The effect duration of commercial moisturising products has also been reported to be about 30 to 60 min, although this depends on individual patients and symptoms (Regelink et al. 1998).
A more detailed evaluation of the effects of the micro-gel spray on dry mouth will require a clinical trial with a control group and a greater number of subjects. However, the product had a protective action against cell death in dry oral mucosal cells, and improved symptoms of dry mouth and showed high tolerability in patients receiving cancer treatment. The consistency of the in vitro and clinical results also suggests that the cell evaluation method is useful for assessment of moisturising products.
- 2008) Systemic pilocarpine for treatment of xerostomia. Expert Opinion on Drug Metabolism and Toxicology 4, 1333–1340. (
- 2007) Cevimeline for the treatment of postirradiation xerostomia in patients with head and neck cancer. International Journal of Radiation Oncology, Biology, Physics 6884, 1102–1109. , , , , , , , & (
- 2008) Effect of oral pilocarpine on post-irradiation xerostomia in head and neck cancer patients: a single-center, single-blind clinical trial. Journal of the Medical Association of Thailand 91, 1410–1415. , , , , , , , , & (
- 1987) Stabilization of dry phospholipid bilayers and proteins by sugars. The Biochemical Journal 242, 1–10. , , & (
- 2000) A comparison of artificial saliva and chewing gum in the management of xerostomia in patients with advanced cancer. Palliative Medicine 14, 197–203. (
- 2007) Efficacy of the BioXtra dry mouth care system in the treatment of radiotherapy-induced xerostomia. Supportive Care in Cancer 15, 1429–1436. , , , & (
- 2002) The role of salivary function in modulating chemotherapy-induced oropharngeal mucositis: a review of the literature. Oral Surgery, Oral Medicine, Oral Pathology, Oral Radiology, and Endodontics 94, 39–44. , , & (
- 2003) Phase III quality-of-life results: impact on patients’ quality of life to reducing xerostomia after radiotherapy for head-and-neck cancer-RTOG 97-09. International Journal of Radiation Oncology, Biology, Physics 56, 832–836. , , , , , , , , , & (
- 2008) Evaluation of the clinical efficacy of a mouthwash and oral gel containing the antimicrobial proteins lactoperoxidase, lysozyme and lactoferrin in elderly patients with dry mouth – a pilot study. Gerontology 25, 3–9. , & (
- 2003) Xerostomia etiology, recognition and treatment. The Journal of the American Dental Association 134, 61–69. & (
- 2009) Saliva substitutes for the treatment of radiation-induced xerostomia – a review. Supportive Care in Cancer 17, 1331–1343. , , & (
- 2010), Effective management of adverse effects while on oral chemotherapy: implications for nursing practice. European Journal of Cancer Care 19, 12–20. (
- 2009) recombinant human epidermal growth factor treatment of radiation-induced severe oral mucositis in patients with head and neck malignancies. European Journal of Cancer Care 18, 636–641. , , , , , & (
- 2009) Phase III randomized study: oral pilocarpine versus submandibular salivary gland transfer protocol for the management of radiation-induced xerostomia. Head and Neck 31, 234–243. , , , , , , , , , , & (
- 2007) Trehalose protects corneal epithelial cells from death by drying. The British Journal of Ophthalmology 85, 610–612. (
- 2011) Induction chemotherapy in head and neck cancer patients followed by concomitant docetaxel-based radiochemotherapy. European Journal of Cancer Care 20, 503–507. , , , , , , , , , , & (
- 2005) Different saliva substitutes for treatment of xerostomia following radiotherapy. Strahlentherapie und Onkologie 181, 231–236. , , & (
- 2010) Trehalose inhibits oral dryness by protecting the cell membrane. International Journal of Oral and Maxillofacial Surgery 39, 916–921. , , , , & (
- 2011) Protective effects of polysaccharides and polyhydric alcohols in a dry mouth model in cultured cells. Supportive Care in Cancer 20, 725–731. , , & (
- 2009) Oral pilocarpine (5 mg t.i.d.) used for xerostomia causes adverse effects in Japanese. Auris, Nasus, Larynx 36, 310–313. , , , , , , , , , & (
- 2006) The effectiveness of commonly used mouthwashes for the prevention of chemotherapy-induced oral mucositis: a systemic review. European Journal of Cancer Care 15, 431–439. , , & (
- 1998) Efficacy of a synthetic polymer saliva substitute in reducing oral complaints of patients suffering from irradiation-induced xerostomia. Quintessence International 29, 383–388. , , & (
- 2005) A double-blind, crossover study of Biotene Oralbalance and BioXtra systems as salivary substitutes in patients with post-radiotherapy xerostomia. European Journal of Cancer Care 14, 319–326. , , , & (
- 2007) Xerostomia: causes and treatment. The Consultant Pharmacist 22, 985–992. (