Investigation of the micro- and nano-structure of liver cells cultured on biodegradable silk fibroin-based scaffolds using scanning probe optical nanotomography

Objective: to analyze the 3D micro- and nano-structure and quantitative morphological parameters of liver cells
cultured on biodegradable silk fibroin-based film scaffolds.

Materials and methods. Samples of biodegradable silk fibroin-based scaffolds with cultured Wistar rat liver cells were obtained for the study. The 3D structure of liver cells cultivated on the scaffolds was studied by scanning probe optical nanotomography using an experimental setup combining an ultramicrotome and a scanning probe  microscope in correlation with fluorescence  microscopy.

Results. Nanoscale images and 3D nanotomographic reconstructions of rat liver cells cultured on scaffold were obtained. The morphological parameters of liver cells (average roughness, specific effective area) were determined. The average surface roughness of the liver cells Ra was found to be 124.8 ± 8.2 nm, while the effective surface area σ was 1.13 ± 0.02. Analysis of the volume distribution of lipid droplets showed that they occupy 28% of the cell volume.

Conclusion. Scanning probe optical nanotomography can successfully analyze the nanostructure and quantify the nanomorphology of liver cells cultured on biodegradable scaffolds.

1. Caplan J, Niethammer M, Taylor RMII, Czymmek KJ. The Power of Correlative Microscopy: Multi-modal, Multi-scale, Multi-dimensional. Curr Opin Struct Biol. 2011; 21 (5): 686–693. doi: 10.1016/j.sbi.2011.06.010.

2. Balashov V, Efimov A, Agapova O, Pogorelov A, Agapov I, Agladze K. High resolution 3D microscopy study of cardiomyocytes on polymer scaffold nanofibers reveals formation of unusual sheathed structure. Acta Biomaterialia. 2018; 68 (1): 214–222. doi: 10.1016/j.actbio.2017.12.031.

3. Efimov AE, Agapov II, Agapova OI, Oleinikov VA, Mezin AV, Molinari M et al. A novel design of a scanning probe microscope integrated with an ultramicrotome for serial block-face nanotomography. Review of Scientific Instruments. 2017; 88 (2): 023701. doi: 10.1063/1.4975202.

4. Mochalov KE, Chistyakov AA, Solovyeva DO, Mezin AV, Oleinikov VA, Molinari M et al. An instrumental approach to combining confocal microspectroscopy and 3D scanning probe nanotomography. Ultramicroscopy. 2017; 182: 118–123. doi: 10.1016/j.ultramic.2017.06.022.

5. Efimov AE, Moisenovich MM, Bogush VG, Agapov II. 3D nanostructural analysis of silk fibroin and recombinant spidroin 1 scaffolds by scanning probe nanotomography. RSC Adv. 2014; 4: 60943–60947. doi: 10.1039/c4ra08341e.

6. Efimov AE, Agapova OI, Safonova LA, Bobrova MM, Agapov II. 3D analysis of the micro- and nanostructure of lung tissue by scanning probe nanotomography. Russian Journal of Transplantology and Artificial Organs. 2020; 22 (3): 143–148. [In Russ, English abstract]. doi: 10.15825/1995-1191-2020-3-143-148.

7. Efimov AE, Agapova OI, Safonova LA, Bobrova MM, Parfenov VA, Koudan EV et al. 3D scanning probe nanotomography of tissue spheroid fibroblasts interacting with electrospun polyurethane scaffold. Express Polymer Letters. 2019; 13 (7): 632–641. doi: 10.3144/expresspolymlett.2019.53.

8. Kim HH, Park JB, Kang MJ, Park YH. Surface-modified silk hydrogel containing hydroxyapatite nanoparticle with hyaluronic acid–dopamine conjugate. Int J Biol Macromol. 2014; 70: 516–522. doi: 10.1016/j.ijbiomac.2014.06.052.

9. Gil ES, Panilaitis B, Bellas E, Kaplan DL. Functionalized Silk Biomaterials for Wound Healing. Adv Healthc Mater. 2013; 2: 206– 217. doi: 10.1002/adhm.201200192.

10. Zhua M, Wanga K, Meia J, Lib C, Zhanga J, Zhenga W et al. Fabrication of highly interconnected porous silk fibroin scaffolds for potential use as vascular grafts. Acta Biomater. 2014; 10 (5): 2014–2023. doi: 10.1016/j.actbio.2014.01.022.

11. Gu Y, Zhu J, Xue C, Li Z, Ding F, Yang Y, Gu X. Chitosan/silk fibroin-based, Schwann cell-derived extracellular matrix modified scaffolds for bridging rat sciatic nerve gaps. Biomaterials. 2014; 35 (7): 2253–2263. doi: 10.1016/j.biomaterials.2013.11.087.

12. Kasoju N, Bora U. Silk fibroin based biomimetic artificial extracellular matrix for hepatic tissue engineering applications. Biomed Mater. 2012; 7 (4): 1–12. doi: 10.1088/1748-6041/7/4/045004.

13. She Z, Jin C, Huang Z, Zhang B, Feng Q, Xu Y. Silk fibroin/chitosan scaffold: preparation, characterization, and culture with HepG2 cell. J Mater Sci: Mater Med. 2008; 19: 3545–3553. doi: 10.1007/s10856-008-3526-y