## 4 Discussion
This fluorescence might be caused either by autofluorescence of collagen type I or autofluorescence of spider silk. While spider silk displayed autofluorescence in the wavelength spectrum of 488 to 546 nm probably due to its amount of phosphorus [29; 38], the autofluorescence spectrum of collagen caused by the amino acids tyrosine and tryptophan lies more in wavelength spectrum of 305-380 nm [39]. Cell contraction in collagen gels has been described before [40], but could be only divided into two phases contrary to the three phases of native cells. As in the study at hand, mesenchymal MSC in collagen gels were found to show higher contraction rates than described for fibroblasts [41]. Collagen gel contraction is commonly used as a measure of cell-mediated matrix reorganization [42], therefore our theorem of a differentiation process in the TLC is further supported. Among the specific markers described to be expressed by tendon tissue are scleraxis (Scx), tenomodulin (TNMD), thrombospondin-4 (TSP-4), tenascin-C (TNC), type I collagen (Col I) and type III collagen (Col III). However, they show different expression level and pattern dependent from kind of tissue or duration of cultivation ex vivo [43]. Specific markers for a differentiation towards tenocytes were found in TLCs differentiated from MSCs. Col I is of utmost importance for the stability of tendons as well as remodeling of the ECM during tendon healing as it makes up more than 90% of the structural elements in normal tendon tissue [44]. Expression of Col I and III is up-regulated as a response to mechanical loading, which is consistent with our study as we found qualitatively more Col I- and Col III-positive cells in TLC with mechanical stimulation [45; 46]. This is probably due to adaption by strengthening of the ECM. Regarding the course of markers, it has to be considered that ASCs as a stem cell population were deployed to differentiate into tendon tissue instead of mature tenocytes or tendon progenitor cells. It has been reported that there are certain differences whether tendon tissue samples or tenocytes in cell cultures were investigated. When tenocytes were isolated and cultivated, only the markers Col I, Col III and TNC were significantly higher than in MSC cultures while Scx, TNMD, TSP-4 dropped immediately after plating isolated tenocytes into Passage 1 [43]. As we investigated TLC derived from differentiated ASC and not in vivo tissue samples, it was not surprising that these markers were positive at all. While they have been used to engineer tendon tissue, either co-cultures or addition of growth factors (i.e., bone morphogenetic protein-12) were applied to achieve TLCs [47; 48]. ASCs have been described to support tendon healing in vivo by inducing neovascularization [49]. To our knowledge, this is the first study using ASC for TLC engineering without supplementary addition of growth factors. However, one limitation is the use of ASC that came from only one fat tissue donor. As described elsewhere, the age of the donor, the localization of adipose tissue harvesting for stem cell isolation, and other factors play a crucial role for the multipotency of ASC [25; 50]. The presented study was approved by the ethics committee of Hannover Medical School therefore no changes could be made to the experimental setup. For future experiments, cell pooling would be useful to eliminate donor-specific differences. Spider silk is known as the world's toughest biomaterial surpassing the tensile strength of many biomaterials and the breaking strength of any artificial material reaching up to \(520\) MJ/kg\({}^{3}\)[51]. In earlier studies utilizing spider silk as tendon suture material, no significant decrease in failure load was reported after 1000 fatigue cycles of strain [36]. Therefore, it seems predestined for the use of tendon tissue engineering.