Introduction
The mucociliary system of the airways plays a decisive role in the defence mechanism for cleaning the air of bacteria, allergens, dust and other foreign particulate matter. It, therefore, has an important protective function for the whole organism. Moreover, it regulates the temperature and humidity of the inhaled air, which are important parameters for the function of the lung.
Because of the importance of these functions, it is vital to possess an effective and reliable evaluation method for observing under constant conditions the reaction of the mucociliary system to the influence of different noxious substances as well as protecting or healing agents.
Until recently, our study group has evaluated these influences using in vitro cultured human nasal mucosa. Increasing legal restrictions on the removal of human tissue and changing surgical techniques have led to a lack of this material. In the past, experiments on the in vitro cultured mucociliary system have been accomplished in several mammals, however most of these are not suitable either because of similar legal problems, as in monkeys, or because they are herbivores and therefore, not genetically very close to human beings. Furthermore, to gain fresh mucosa in order to culture mucociliary tissue, it is inevitable that all these experimental animals must be sacrificed.
We, therefore, developed an alternative model by using porcine mucosa from the trachea, which has the advantages, that the pig as an omnivore has a similar biology in comparison to the human being and that the tracheal mucosa is available as a normal by-product after slaughtering.
In order to prove the suitability of the in vitro culturing of this porcine mucosa, we investigated the capability to develop cilia, the minimal time range of their constant beating frequency and the reaction of the cilia to different drugs compared to the reaction of human mucosa.
Discussion
There are growing limitations of the removal of human tissue. Following legislation, it is permitted only with written consent of the patients, which increases administrative costs. Further, changing surgical techniques may lead to a lack of human tissue. In order to judge the effect of medicines on ciliary beat frequencies, a sufficient number of primary cultures must always be available.
In the past 30 years (since 1978), experiments on in vitro cultured mucociliary cells have been conducted on the following mammals: monkey [
23], cow [
30‐
35], sheep [
8,
15,
24‐
27], rabbit [
4,
13,
29,
36,
37], ferret [
16,
19], hamster [
14] and rat [
5,
7,
12,
22,
28].
Cows, ovines, rabbits and hamsters are all herbivore and are therefore not very close to human beings genetically. Closest to the humans are monkeys, but the use of them for experimental studies is also very restricted and not practical for tissue culturing.
Pigs, however, are omnivore and their close biological relationship to humans has been postulated in several studies [
2,
3]. Therefore, they have also been applied especially in experimental xenotransplantion [
6]. On the other hand, pigs have been used up to now only three times for in vitro culturing of mucosa [
9‐
11].
To gain fresh mucosa in order to culture mucociliary tissue, it is usually unavoidable that the experimental animals must be sacrificed. The use of porcine–trachea however, has the incomparable advantage that the tissue is a normal by-product of slaughtering of the pigs for food production. Their tracheas are either usually used for dog food or simply discarded.
We, therefore, postulated that pigs might be ideal donors for studying on in vitro cultured mucosa function.
The functional morphology and, especially, the cytoskeleton of the cilia as well as the molecular biology of the ciliated mucosa cells have been intensively investigated by numerous researchers. An overview is given by the famous textbook of Alberts et al. [
1]. These authors also point out that the fine structure of the cilia “…is found in almost all forms of eucaryotic flagella and cilia from protozoans to humans.” This substantial similarity therefore applies to the whole upper and lower respiratory tract of all mammalians. In order to prove the reliability for testing the comparability of in vitro cultured porcine tracheal mucosa cells with human nasal mucosa cells, we therefore concentrated on the investigation of (a) the capability of developing beating cilia, (b) the constancy of the beat frequency over a sufficient time and (c) the comparability of the reaction of the cilia to different drugs.
The capability of developing beating cilia
In comparison with our previous investigations of in vitro cultured human nasal mucosa cells, we found the following characteristics in the cultured porcine tracheal mucosa:
-
The speed of cell proliferation until adhesion to the coated tissue culture dishes happened within approximately 48 h and was similar to human cells;
-
The formation of cell conjugations began as irregular clusters and became increasingly confluent after a few days. This process was also comparable to the human mucosa;
-
The proliferation of the cells was associated with a simultaneous
growth of cilia as in the human cultures and showed the same appearance (Fig.
1). We did not observe a delayed proliferation of cilia;
-
The
survival time of its cells with a constant beating frequency is at least as long as the one of human mucosa (Fig.
2).
The constancy of the beat frequency over a sufficient time
From our human experiments, we knew that a maximum investigation period of 180 min is sufficient for analyzing the effects of naphazoline and benzalkonium chloride. However, to make sure that the cilia of the porcine mucosa hold up the constancy of beat frequency over a time sufficient for testing the influence of various pharmacological substances in this species, we primarily measured the CBF of the porcine tracheal cells up to 360 min. It could be demonstrated, that the CBF was absolutely constant during the whole test period. Therewith we could show that the survival time of the porcine mucosa cells is by far sufficient for those experimental studies.
Moreover, in our previous studies, the human nasal mucosa showed a constant beating frequency of 7.3 ± 1.4 Hz over the chosen investigation time of 180 min, whereas the CBF of the porcine mucosa was 7.57 ± 1.39 Hz, thus demonstrating no statistical difference between both tissues. These findings also confirm that the porcine tracheal mucosa provides optimal prerequisites for investigating the influence of harmful or healing substances on the human mucosa.
The comparability of the reaction of the cilia to different drugs
The main goal of our study was to answer the question whether the reaction of porcine and human cilia to different drugs is comparable. Therefore, we initially tested benzalkonium chloride, which we found in previous studies on human mucosa to be highly toxic on the cilia and their beat frequency. The functional and morphological reactions of the porcine ciliated cells to this substance were exactly the same as the reaction observed in the in vitro cultured human mucosa.
From the findings in our experiments with the human mucosa, we demonstrated, on the other hand, that naphazoline as a pure substance has no toxic influence on the survival of the cilia of human mucosa and their beat frequency with different concentrations. The average CBF was 105% compared with the mean equilibration frequency.
When testing the effect of naphazoline on the porcine mucosa we measured a CBF of 103.7% using a concentration of 0.01%, which was statistically equal to the CBF of the human mucosa.
With a concentration of 0.1%, the porcine CBF decreased to 92.5%. This value was significantly different to the equilibration period, but it was not significantly different to the human mucosal cells.
After rinsing out the substance, there was a rapid recovery of ciliary function. Whereas the first measurement after 20 min of rinsing out still showed the same CBF as at the end of exposure to naphazoline 0.1%, the CBF had already recovered to 96.8% (±19.6%) in the second measurement after 40 min.
Although there is no statistical difference, these initial data suggest that the porcine mucosal cells might have a slightly higher sensitivity than the human mucosa cells.
In conclusion, it became evident that the porcine tracheal mucosa can serve as an ideal tissue for the investigation of pharmacological effects on the sensitive physiology of mucosal cilia.
Our future investigations will also include hurtful substances causing neoplastic alteration or allergic reactions, for which this model also seems to be suitable.
In order to achieve comparable results to the earlier investigations with the human mucosa cells, in this study the DMEM culture medium has still been used. Due to our present studies, the airway epithelial cell growth medium (PromoCell) leads to a higher differentiation of cilia and therefore, will be used in our future experiments.