*3.3. Cell Viability*

GelMA electrospun mats were tested in vitro to determine the biocompatible character of the process (Figure 3d). Cell viability percentages of all samples were above 90%. The high number of living cells indicates that the crosslinking process and the modified gelatin were compatible with the used cell-line MG-63. Thus, gelatin based electrospun mat with micro- and nanotopographical features have a great potential for tissue engineering.

#### **4. Discussion**

The use of gelatin as a biopolymer scaffolding material for tissue engineering applications is directly related to its high biocompatibility, hydrophilicity, and bioactivity associated with specific peptide sequences. Electrospinning is a very attractive, complex, and versatile technique to prepare advanced functional nanofibrous scaffolds for a variety of tissue engineering and cell culture applications. Current efforts are focused on preparing electrospun scaffolds with controlled multilevel hierarchical structures. GelMA electrospun scaffolds have been reported using synthetic polymers and/or hazard, expensive solvents [27–29]. However, a pure gelatin scaffold that mimics the important features of natural ECM obtained by electrospinning is still pending. In this work, GelMA nanofibers without defects were obtained using acetic acid, a low toxic potential solvent. Furthermore, in order to overcome the limitations of the low stability of gelatin in aqueous media as well as the possible toxicity of chemical crosslinkers, the as-spun scaffolds were crosslinked by UV irradiation at different exposition times. The same strategy, but while varying the photoinitiator concentration, was reported by Lai et al. for cultivation of limbal epithelial cells [30].

The GelMA matrix was soluble in PBS before UV irradiation (NG-0UV) while crosslinked GelMA nanofibers were stable in PBS (NG-6UV, NG-9UV and NG-12UV). The mechanical properties of UV irradiated mats (NG-9UV) increased 7 times related to mats unexposed to UV (NG-0UV), indicating the efficiency of crosslinking.

In the last few decades, surface engineering technologies have been used as important tools to clarify the effects of the microenvironment on cellular behavior [3,11–14]. The fabrication of the surface topographies with geometrical micro and nanopatterns, like channels, pillars and pits with controlled dimensions have been possible through the use of various methods as photolithography, electron beam lithography and microfluidics. These geometric and topographical factors can have an influence in cell adhesion, migration, differentiation, and the shape of cells. To modulate cell behavior through surface engineering methods is not only useful to stimuli stem cell differentiation, but also to generate a favorable response of the implant. In order to control substrate topography, the micropatterns molding with different geometries and dimensions, were developed to fabricate scaffolds. Molds were obtained by photolithography and they were used as collector in electrospinning process. Therefore, GelMA electrospun mats were obtained with nano structure and microroughness. The SEM images and roughness analysis showed that micropatterns were successfully copied over GelMA electrospun mats. To the best of our knowledge, this is the first report in literature in which GelMA nanofibrous scaffolds with micropattern topographical features were designed and prepared.

By combining molding and electrospinning processing, it was possible to obtain scaffolds which mimic ECM. Thus, this simple and easy technique can be useful for developing sophisticated and complex materials for tissue engineering and cell culture. In future works, we will study deeply how different patterns influence the behavior of specific cell lines.

#### **5. Conclusions**

In summary, an inexpensive and rapid method for the fabrication of well-defined micro- and nanotopographic features on uniform bead-free electrospun GelMA fibers were developed in order to design 3D scaffolds that mimic ECM. The synthesis of GelMA and its processing by electrospinning was optimized, so therefore, defect-free GelMA nanofibrous mats were obtained. The results showed that crosslinking took place successfully and it could modulate the final properties. Furthermore, the use of micropatterned molds obtained by photolithography as a collector in the electrospinning process allowed controlling the roughness of mats. Taking into account the cell viability results, the methodology used in this study is a valuable tool to develop patterned GelMA based nanofibrous scaffolds for cell culture and tissue engineering.

**Author Contributions:** Ana Agustina Aldana performed the experimental work and wrote the first drafts of the manuscript and final version, Laura Malatto designed and prepared micropatterned molds, Muhammad Atiq Ur Rehman measured the roughness, Aldo Roberto Boccaccini advised during the project progress and collaborated in writing the manuscript and Gustavo Abel Abraham contributed to the conception of the project and collaborated in writing the manuscript.

**Funding:** This research was funded by CONICET (grant UE73) and MINCYT-DAAD (DA/16/02 Project). The APC was funded by University of Erlangen-Nuremberg.

**Acknowledgments:** The authors would like to thank Alina Grünewald and Vanesa Fuchs for technical support in cell viability and X-ray diffraction studies, respectively.

**Conflicts of Interest:** The authors declare no conflict of interest.
