Date Published: April 29, 2019
Publisher: Public Library of Science
Author(s): Nathalie Raffel, Ralf Dittrich, Tobias Bäuerle, Lisa Seyler, Amir Fattahi, Inge Hoffmann, Aldo Leal-Egaña, Matthias W. Beckmann, Aldo R. Boccaccini, Liliana Liverani, Thomas Webster.
Reproductive tissue engineering (REPROTEN) has been recently defined as the application of the tissue engineering approach targeting reproductive organs and several research works are focusing on this novel strategy. Being still an innovative field, most of the scaffold characterization techniques suitable for other tissue targets give inappropriate results, and there is the need to evaluate and investigate novel approaches. In particular the focus of this paper is the evaluation of the infiltration of ovarian follicles inside patterned electrospun scaffolds. Beyond the standard techniques, for the first time the use of magnetic resonance imaging (MRI) for this purpose is proposed and specific protocols for scaffold preparation are reported. Positive results in terms of evaluation of scaffolds incorporating follicles confirm this technique as highly effective for further applications in this field.
Great interest has been recently reported in development of tissue engineering-based approaches for the regeneration of reproductive organs and tissues [1–4]. Considering that these approaches are related to the treatment of infertility in relation to oncological pathologies, they have a relevant impact on the improvement of the patients’ quality of life. In fact, they can be potentially suitable also for pediatric oncological patients, increasing the relevance of focusing research works on this topic .
Morphological assessment to prove the adhesion and the growth of follicles on the patterned electrospun scaffolds was performed initially with light microscopy. The top view and the cross-section of the samples stained with H&E were analyzed and reported in Fig 3. It is possible to notice that the H&E staining combined with the light microscopy is effective to confirm the presence and the development of the follicles, as clearly shown in Fig 3A and 3B. Respect to previous work based on 3D printed scaffolds , it is possible to notice that the follicles adhesion and growth is not just related to the presence of the main struts of the pattern, but the presence of fibers in the middle of the macropores promoted the adhesion of more follicles also in the interior of the pores. From the cross-section analysis reported in Fig 3C and 3D it is not possible to obtain any information regarding the infiltration of the follicles inside the scaffolds. It is only possible to observe the scaffold, which is characterized by the white and pink color (due to H&E staining). Therefore we can conclude that this technique (H&E staining and light microscope analysis) is not suitable for the evaluation of follicles infiltration into scaffolds.
In conclusion, H&E staining with subsequent light microscopy or SEM analysis is not suitable for the evaluation of follicles infiltration in electrospun scaffolds; this is also true for SEM analysis alone. Nevertheless, SEM is effective to investigate fiber morphology as well as to precisely measure scaffold thickness; using critical point drying is strongly advisable, as it keeps the three-dimensional structure intact. Promising results were obtained by the use of MRI to assess follicles infiltration in the scaffolds. Precedent agarose embedding should be performed to fixate the specimens; this fixation technique further preserves the original morphology of scaffolds and follicles. In conclusion, the obtained results confirm that MRI is a promising technique with high perspectives for further applications in this field.