## 4 Discussion  
In the study at hand, we employed spider silk to stabilize collagen gels for tissue engineering of TLCs. To further improve cell distribution and induce mechanotransduction, cyclic uniaxial strain was applied. The frequency of the cyclic strain was with 1 Hz lower than the stride frequency found in equine center, which is approximately 2 Hz, but comparable to the strain used by other studies, in particular for the promotion of stem cell differentiation towards tenocytes [31; 32; 33]. While some studies applied strain for as little as 15 to 20 min per 24 h, as 15 min per 24 h were sufficient for a notable effect regarding mechanical induction, longer application periods were suggested [33; 34]. To mimic the mechanical requirements of daily life activities, we chose much longer periods (i.e., 8 h per day), however, an interesting concept for future research might be the investigation of changes due to different periods of strain application between 15 min and 8 h or several periods per day. Figure 7: Results of the immunohistochemical staining. The cell nuclei were stained with 4′,6-diamidino-2-phenylindole (DAPI) and can be recognized by a blue fluorescence. The target proteins each show a green fluorescence. The spider silk shows a red autofluorescence. The measurement bars represent 200 micrometers. (**A**) Results of experimental group A. (**B**) Results of experimental group B. (**C**) Results of experimental group C. (**D**) Results of experimental group D.  
Biomechanical analysis of the TLCs in terms of traditional tensile testing was intended to be performed with a tensile test machine but turned out to be unsuccessful as TLCs displayed a fragile, soft and wet morphology and, therefore, were irrevocably damaged on loading in the tensile testing machine as it was described by other authors [33; 35]. This is clearly a limitation of the feasibility study at hand which needs to be overcome by future studies further characterizing spider silk-based TLCs as a biomechanical similarity to the natural tissue is necessary. A possible solution might be involvement of dynamic shear analysis, dynamic scanning calorimetry or Fourier-transform infrared spectroscopy [26]. However, spectroscopic analysis might be susceptible to interference of autofluorescence of spider silk as described before [30]. Nevertheless, mechanical properties close to or surpassing the mechanical properties of natural tendon tissue can be assumed as spider silk (which functions as a stabilizing backbone for the constructs in this study) has been shown to possess extraordinary properties. For example, bundles of native spider silk were investigated as a tendon tissue suture and showed tensile strength comparable to polypropylene sutures and a superior resistance to mechanical fatigue [36]. Concerning the viability, most of the cells in the TLC were viable. Interestingly, further analysis revealed differences regarding cell morphology and distribution dependent on whether axial stimulation was present or not. In both TLCs with or without spider silk, cell distribution and morphology in constructs without mechanical stimulation were more likely to show a higher cell density with round cell bodies. In contrast, mechanical stimulation led to a lower cell density with tenocyte-like morphology. In mature tendons, two different zones can be distinguished: the gliding zone with chondrocyte-like cells at regions of pressure at the hypomochlin and the traction zone with elongated tenocytes parallel to the collagen fibers [37]. In the TLCs in this study, areas without mechanical stimulation displayed histological features of a gliding zone in tendons while those areas where uniaxial strain was applied displayed histological features of a traction zone. Therefore, the whole construct may be regarded as traction zone equivalent. A red fluorescence was noticed in the LIVE/DEAD-Assay as well as in the immunofluorescence staining (Figures 5 and 7).