TECNOLOGÍA IDIRECT

PROCEDURES

As the oesophagus is continually bathed in saliva this fluid may have an important role in the adhesion of a substance onto the oesophagus. This indicates that saliva is an essential material within the investigations performed. However, saliva is a physiological fluid that has a composition that varies in response to many external factors. As this fluid is an important component in this piece of research the variability and steps taken to minimise this diversity are described within this chapter. Mucin is thought to be an important component of saliva when investigating adhesion at interfaces, as rheological synergy between mucin and polymers has been linked to mucoadhesive strength (Mortazavi et al, 1992). Natural saliva, artificial saliva and mucin solutions are discussed below.

2.4.1 Natural saliva

Using natural human saliva within scientific experiments raises several issues associated with the natural variations of the composition of this physiological fluid. The problems associated with the use of saliva are fully discussed in papers by Dawes (1974) and Rudney (1995). Differences associated with age, gender, time of day and even seasonal diversity can affect the composition of saliva and these differences may lead to results that are hard to analyse.

The natural saliva used throughout the study was collected from healthy, non­ smoking volunteers at least one hour after eating. All saliva samples were collected as required and used within one hour of collection. Unstimulated saliva was collected from subjects. Saliva was retained within the oral cavity for approximately 60 seconds then ejected into clean vials for immediate use. In ejecting saliva, minimal force was used to prevent collection of excessive oral debris.

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2.4.2 Artificial saliva

Artificial saliva provides a physiological medium devoid of the variability of saliva from its natural source. Artificial saliva was used in the retention model and in rheological studies to gain an insight into the interaction between salivary components and alginate solutions. Many different artificial salivas are available commercially and also there are many different formulations quoted in the literature. Comparative studies and evaluations of such formulations have been performed (e.g. Vissink et al, 1983). Table 2.4 compares typical formulations of artificial salivas; a commercially available saliva used for the treatment of dry mouth (British National Formulary, 2000), an ionically formulated artificial saliva based on the values found in the Geigy tables of scientific data (Lentner, 1981) and the artificial saliva formulation listed in the Dental Practitioners’ Formulary (1998).

Table 2.4. Comparison of three different formulations for artificial saliva

Commercial saliva Ionic artificial saliva Artificial saliva DPF

(Saliva Orthana®)

(per 1 0 0ml) (per 1 0 0ml) (per 1 0 0ml)

0.35 g gastric mucin 0.27 g gastric mucin 3 g sorbitol

0 . 2 g xylitol 42 mg sodium carbonate 0.65 g carmellose sodium

0.42 mg sodium 43 mg sodium chloride 80.38 mg dibasic potassium

fluoride phosphate

Plus preservatives and 0.149 g potassium chloride 62.5 mg potassium chloride flavouring agents

2 2 mg calcium chloride 36.62 mg monobasic potassium phosphate 91 mg di-sodium hydrogen 16.62 mg calcium chloride

orthophosphate

5.88 mg magnesium chloride 0.43 mg sodium fluoride + preservatives and colours

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Table 2.4 illustrates the many differences found between formulations of artificial saliva. Commercially available artificial salivas are usually employed to retain moisture within the oral cavity and their ionic formulation has little bearing on natural saliva. Studies that investigate the interactions of dental materials with saliva often use an artificial medium that is ionically similar to natural saliva yet which may have no rheological similarity to the latter (Leung & Darvell, 1997). Levine et al (1987) reviewed many different artificial saliva formulations found in the literature. As yet there is no substitute that mimics both the ionic and rheological properties of saliva; this project would require an artificial saliva that acts in both of these ways.

Two different artificial saliva formulations were chosen from the array available in the literature for this study. The first artificial saliva chosen was based on a formulation used widely within the dental world. Table 2.5 shows the formulation of this artificial saliva (Embleton et al, 1998).

Table 2.5. Formulation of artificial saliva I

Chemical Amount (g/L) Supplier

”Lab-Lemco” Powder 1 . 0 Oxoid

Yeast extract 2 . 0 Oxoid

Proteose peptone 5.0 Oxoid

Hog gastric mucin 2.5 Sigma

NaCl 0.35 Sigma

CaCl2 0 . 2 Sigma

KCl 0 . 2 Sigma

This solution was heated to 80 °C for 30 minutes prior to addition of 12.5 ml of filter sterilised 40% urea (Sigma).

The second artificial saliva used in the study was formulated based on the ionic environment of natural saliva. This formulation was prepared as per Documenta Geigy handbook (Lentner, 1981). Table 2.6 shows the formulation of this saliva.

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Table 2,6. Formulation of artificial saliva II

Chemical Amount (g/L) Supplier

NaHCOs 0.42 BDH

NaCl 0.43 BDH

KCl 1.49 BDH

CaCl2.2H2 0 0 . 2 2 BDH

NaH2P0 4.H2 0 0.91 BDH

Porcine gastric mucin 2.70 Sigma

(type II)

Deionised water to 1 0 0 0ml

From here onwards the artificial salivas will be referred to as artificial saliva I or II. Artificial saliva II compares favourably to the ionic content of natural saliva although artificial saliva I shows more similar rheological properties. The rheological properties of both artificial salivas and natural saliva are compared in Appendix I.

2,4.2.1 Preparation o f artificial sal iva

The artificial salivas used were prepared in one-litre batches according to the formulations listed in Tables 2.5 and 2.6. The chemicals were added to deionised water and stirred at a vigorous speed using a magnetic stirrer. The artificial salivas were prepared and used within an eight hour period. When not in use the solutions were stored in a refrigerator.

2.4.3 Preparation of mucin solutions

Mucins or mucoproteins are responsible for the lubricating properties of saliva. They provide saliva with the rheological properties necessaiy to coat and retain moisture on mucosal surfaces (Tabak et al, 1982). They are usually defined as glycoproteins containing more than 40 % carbohydrate with a protein core and oligosaccharide side chains attached by 0-glycosidic linkages. The length of the carbohydrate chain

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varies according to the origin of the mucin. Salivary mucins have carbohydrate chain lengths of 6 - 8 sugars compared to gastric mucins that have 19 sugars per chain (Allen & Pearson, 1993). Mucins are negatively charged due to the ester sulfate and sialic acid residues of the carbohydrate side chains although the protein core also has substantial amounts of acidic amino acids. The shape of mucin molecules has been described as being “bottle-brush”. This is shown schematically in Figure 2.4.

Heavily glycosylated

Protein core

Figure 2,4. Schematic diagram representing the bottle-brush shape of mucin molecules

The negative charge on the mucin molecules acts to increase the stiffness of this "bottle-brush" structure and leads to a large hydration sphere of the mucin in solution. Cloning of mucin genes has provided much information on the nature of the different structural properties of mucin molecules (Strous et al, 1992).

This study investigates the presence of mucin foimd within the unstirred water layer resident on œsophageal tissue and also examines the effect that this mucin may have on the interaction between the tissue and the alginate. This area is discussed more fully in Chapter 3, which probes the œsophageal tissue surface.

Within this study, two commercially available mucins (Sigma, UK) were used, porcine gastric mucin (PGM) and bovine submaxillaiy mucin (BSM). When investigating the interaction between alginate and a mucin solution it is important to consider the chemical properties of the mucin involved. Table 2.7 compares the properties of PGM and BSM.

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Table 2.7. Comparison of the composition of porcine gastric mucin and bovine submaxillary mucin Component PGM% B S M % Protein 43.9 57.6 Neuraminic acid 1 . 2 6.4 Hexosamine 16.0 6 . 2 Hexose 19.9 2.7

Mucin chemistry is discussed in further detail in Chapters 3 and 6. Previous studies have shown a difference in the interaction between polymers and mucin according to the type of mucin used (Hagerstrom et al, 2000). This phenomenon will be discussed in greater detail in the relevant experimental chapters.

2.4.3.1 Procedure for the preparation o f mucin solutions

Studies were performed investigating the interactions between alginate and solutions of mucin. Crude porcine gastric mucin (type II) supplied from Sigma was used in the studies investigating the interaction between mucin and alginate. This mucin was hydrated in deionised water over a suitable period of time using a magnetic stirrer to aid dispersion. Bovine submaxillary mucin was also investigated in combination with alginate. Solutions of bovine submaxillary mucin were prepared in the same manner as the solutions of porcine gastric mucin.