Regeneration of TiO2 Nanotube Arrays after Long-Term Cell and Tissue Culture for Multiple Use \u2013 an Environmental Scanning Electron Microscopy (ESEM) Survey of Adult Pig Retina and beyond

Sabrina Friebe,Stefan Mayr

doi:10.1186/s12575-019-0090-4

Abstract

Long-term organotypic culture of adult tissues not only open up possibilities for studying complex structures of explants in vitro, but also can be employed e.g. to investigate pathological changes, their fingerprints on tissue mechanics, as well as the effectiveness of drugs. While conventional culture methods do not allow for survival times of more than a few days, we have demonstrated recently that TiO2 nanotube arrays allow to maintain integrity of numerous tissues, including retina, brain, spline and tonsils, for as long as 2 weeks in vitro. A mystery in culturing has been the interaction of tissue with these substrates, which is also reflected by tissue debris after liftoff. As the latter reveals fingerprints of tissue adhesion and impedes with nanotube array reuse, we address within the present environmental scanning electron study debris nature and the effectiveness of cleaning approaches of distinct physical and chemical methods, including UV-light irradiation, O2 plasma treatment and application of an enzyme-based buffer. This will lays the foundation for large-scale regeneration and reuse of nanotube arrays in science and clinical research.

Highlights

Nanotubes of different materials have attracted tremendous scientific interest during the past two decades due to their high versatility for a variety of applications [1, 2]
Cell Proliferation on Nanotube Arrays In a first step we employed mouse fibroblasts that were cultured for 7 days on tuned nanotube arrays tube diameter (32 ± 3) nm) as model system to address within an environmental scanning electron microscope (ESEM) study cell adherence, lift-off and nanotube regeneration
Compared to cells grown in standard culture wells which show a doubling time of 22 h, L929 cells grow significantly slower on nanostructured surfaces

Summary

Introduction

Nanotubes of different materials have attracted tremendous scientific interest during the past two decades due to their high versatility for a variety of applications [1, 2]. Depending on their chemical and mechanical properties they can be used, e.g. as carrier material in catalysts or for drug delivery [3]. It is well known how to sterilize implant surfaces with distinct methods like UV-light or O2-plasma [20–22] These methods are partially suitable to clean the TiO2 nanotubes surface from biological contamination as we will show. For qualitative analysis we used an environmental SEM to investigate the cleaning characteristics of treatments with UV-light, O2-plasma and proteinase K

Methods

Results

Discussion

Conclusion