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Introduction

Advanced methods for regeneration of tissue or bone are receiving significant attention in recent years in almost all biomedical disciplines, including the area of dentistry and dental materials . From the bioengineering perspective, a focus of ongoing research is on identifying general and specific effects of mechanical stimulation on cells and multi-cellular aggregates . In a number of studies on cells, e.g. human and animal gingival fibroblasts, anti-apoptotic effects and activation of proliferation were determined as result of mechanical stimulation . Most of the experiments investigating the effect of mechanical stimulation on human gingival fibroblasts (hGFs) were carried out using devices/concepts for mechanically inducing deformation to biological cells through artificial substrate materials . In principle, the devices deploy flexible materials acting as substrate for initial cell seeding, as support for multi-cellular aggregates, and for transducing mechanical stimuli to adherent cells . Application of flexible artificial substrates commonly results in comparably homogeneous deformation that is transduced to the adherent cells and allows for evaluation of the deformation . The advantages of such devices are not doubted, however, artificial substrate materials and the homogeneous deformation result in conditions that are significantly different from that prevailing in vivo and are prone to side effects obscuring the response to the mechanical stimulus . Thus, drawing conclusions about the in vivo response of the cells and especially tissue-like multi-cellular aggregates based on the results of such in vitro experiments may be venturous. In the human body, distributions of strain and compression are highly inhomogeneous and localized, occasionally exhibiting steep gradients. Additionally, transducing mechanical load via a flexible artificial substrate to cells is different compared to the transduction from cell to cell or cell to extracellular matrix. For example, the reorientation of cells on flexible artificial substrates with respect to the direction of the tensile strain is restricted in multi-cellular aggregates due to the lower degree of freedom of movement of the cells.

The presented work aims to further develop fundamental experimental approaches for investigating the effects of mechanical load on cells and multi-cellular aggregates closer to in vivo conditions. A new experimental approach for generating vital multi-cellular aggregates omitting artificial substrates is developed and presented. hGFs are seeded onto glass slides that, after the cells adhere to their edges, are slowly pulled apart using precision threads and a piezoelectric element. The multi-cellular films fabricated by this method are evaluated according to their size and shape and an approach to determine the probability for successful stimulation or disintegration/collapse upon further stimulation is presented. The potential for application for tissue regeneration in medical practice is discussed.