No abstract

Human nasal respiratory cells lose cilia in submerged cultures. This study compares the effect of extracellular matrix (ECM) molecules of the basal lamina on ciliogenesis in submerged cell cultures to ECM-free suspension cultures. Respiratory mucosa of nasal turbinates was the routine source for the cultures of nasal epithelial cells. For the submersion cultures, enzymatically isolated cells were seeded either on a layer of lethally irradiated ((60)Co, 60 Gy) murine 3T3-feeder fibroblasts or on an ECM-coated culture flask. For suspension cultures, the flasks were rotated for 3 days after cell seeding. In ECM-coated flasks, epithelial cell attachment and confluence was promoted and always much better than in cultures on a feeder layer. Respiratory cells lost cilia during the first 5 weeks in submerged cultures. Genesis of new, actively beating cilia was seen after 5-6 weeks when plastic culture dishes were coated with ECM molecules. Cells grown on uncoated plastic dishes together with 3T3-fibroblasts showed no ciliogenesis. Spheroids of epithelial cells in suspension cultures lost cilia during the 1st week and developed new cilia after 1-2 weeks in vitro. Our results suggest that ECM molecules are not the only signal for ciliary differentiation of respiratory cells in vitro, because suspension cultures are ECM free. However, the presence of ECM molecules in submerged cell cultures promotes the attachment and early confluence of seeded epithelial cells with a high density of cuboidal epithelial cells. The specific cellular shape and intense intercellular contact of these cuboidal cells may be among the most important signals inducing terminal differentiation and ciliogenesis.

Several ways to evaluate the sound transmission properties of middle ear implants are now established. Besides computer-based simulations using acoustic and electrical analog circuits or finite element analysis, measurements can be performed with temporal bone preparations. Experiments with these preparations consider various anatomical properties, but a large number of parameters influence the outcome of measurements. To facilitate standardized measurements, a mechanical middle ear model was developed that allows comparison of the transfer function of middle ear implants on defined conditions. The model approximates the impedances of the tympanic membrane and inner ear with the aid of thin, flexible membranes. The implants are fit between the membranes, and displacement at an artificial stapes foot-plate is measured with an optical probe. Fundamental influences on the sound transmission properties of nine different middle ear implants (total ossicular replacement prostheses) were examined. Although the material and shape were different, some of the prostheses revealed very similar transfer functions. The mass of the implant showed the largest influence on sound conduction. With a higher mass, the frequency area above approximately 1 kHz was found to be significantly deteriorated. The lightest implant used was 4 mg and showed the best overall results. These findings show that middle ear prostheses should be as light as possible for optimum high-frequency transmission.

It has been previously shown that fresh mucosa from diVerent mammals could serve as raw material for in vitro culturing with the diVerentiation of cilia, which are the most important morphological structures for the function of the mucociliary system. Increasing legal restrictions on the removal of human tissue and changing surgical techniques have led to a lack of fresh human mucosa for culturing. Most of the animals that have been used as donors up to now are genetically not very close to human beings and must all be sacriWced for such studies. We, therefore, established a modiWed system of culturing mucosa cells from the trachea of pigs, which is available as a regular by-product after slaughtering. With respect to the possibility of developing "beating" cilia, it could be shown that the speed of cell proliferation until adhesion to the coated culture dishes, the formation of conjunctions of cell clusters and the proliferation of cilia were comparable for porcine and human mucosa. Moreover, it could be demonstrated that the porcine cilia beat frequency of 7.57 § 1.39 Hz was comparable to the human mucosa cells beat frequency of 7.3 § 1.4 Hz and that this beat frequency was absolutely constant over the investigation time of 360 min. In order to prove whether the reaction to diVerent drugs is comparable between the porcine and human cilia, we initially tested benzalkonium chloride, which is known to be toxic for human cells, followed by naphazoline, which we found in previous studies on human mucosa to be non-toxic. The results clearly showed that the functional and morphological reactions of the porcine ciliated cells to these substances were similar to the reaction we found in the in vitro cultured human mucosa.

With the aid of a mechanical middle ear model (MMM) the sound transmission properties of different middle ear implants were investigated. Input of the MMM involved a broad-band signal from 100 to 5000 Hz that was supplied by a miniaturized loudspeaker. Displacement of an artificial stapes footplate was measured by a fiberoptic probe. The transfer functions of four different total ossicular replacement prostheses (TORPs) of different materials and shapes were compared. Three of the devices revealed similar transfer functions which corresponded to the typical curve of the normal middle ear. One of the prostheses demonstrated a high-frequency deterioration of approximately 5 dB. This effect was explained by a 3- to 6-times higher mass of the implant when compared to the others. Altogether, the weight of the prosthesis seems to have the most marked impact on sound transmission to the inner ear, whereas stiffness of the implant itself is less crucial as long as it can be regarded as a rigid body.