Research Article: Biochemical profiling of rat embryonic stem cells grown on electrospun polyester fibers using synchrotron infrared microspectroscopy

Date Published: April 18, 2018

Publisher: Springer Berlin Heidelberg

Author(s): Ernesto Doncel-Pérez, Gary Ellis, Christophe Sandt, Peter S. Shuttleworth, Agatha Bastida, Julia Revuelta, Eduardo García-Junceda, Alfonso Fernández-Mayoralas, Leoncio Garrido.

http://doi.org/10.1007/s00216-018-1049-z

Abstract

Therapeutic options for spinal cord injuries are severely limited; current treatments only offer symptomatic relief and rehabilitation focused on educating the individual on how to adapt to their new situation to make best possible use of their remaining function. Thus, new approaches are needed, and interest in the development of effective strategies to promote the repair of neural tracts in the central nervous system inspired us to prepare functional and highly anisotropic polymer scaffolds. In this work, an initial assessment of the behavior of rat neural progenitor cells (NPCs) seeded on poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) fiber scaffolds using synchrotron-based infrared microspectroscopy (SIRMS) is described. Combined with a modified touch imprint cytology sample preparation method, this application of SIRMS enabled the biochemical profiles of NPCs on the coated polymer fibers to be determined. The results showed that changes in the lipid and amide I–II spectral regions are modulated by the type and coating of the substrate used and the culture time. SIRMS studies can provide valuable insight into the early-stage response of NPCs to the morphology and surface chemistry of a biomaterial, and could therefore be a useful tool in the preparation and optimization of cellular scaffolds.

Partial Text

The intrinsic characteristics of the central nervous system (CNS) are a major impediment to its spontaneous recovery in response to injury and, as a consequence, lesions cause permanent functional deficits that depend on their location and extent. In fact, complete functional repair of a spinal cord injury (SCI) cannot generally be achieved without precise and significant aid [1]. Thus, SCI is a global problem that not only affects the physical and psychological well-being of patients and their families, but also places an enormous burden on the economic resources of developed countries and increases the mortality rates in developing nations [2]. To give an example, the incidence of traumatic SCI in Western Europe was recently reported to be between 218 and 316 cases (around half of which are due to traffic accidents) per million habitants, whereas for North America (the US and Canada), the mean is over three times that [3]. In Canada alone, the annual cost of SCI in 2012 was reported to be over 2 billion €, at least 32% of which was ascribed to attendant care. In 2017, the National Spinal Cord Injury Statistical Center in the USA estimated that, depending on the severity of the SCI, the average lifetime cost of treatment and care for a person who suffers a SCI at the age of 25 was 1.4–4.3 M€ [4]. Hence, there is a great deal of interest in improving this situation.

The FTIR spectrum of NPCs on P(HB-co-HHx) fibers showed a marked decrease in lipid band intensity at the longest culture time studied, 48 h. This observation is consistent with previous observations which showed a reduction in lipid content that was associated with a loss of stem cell pluri- or multipotency [29]. However, there is still no clear consensus about this, as the opposite behavior—an increase in lipid production with cell differentiation—has also been reported [31]. Several outcomes may be anticipated, depending on the differentiation path of the NPCs. NPC differentiation to an oligodendrocyte-like cell could increase the intensity of the lipid bands due to an increase in myelin. On the other hand, if NPCs differentiate to neuron or astrocyte-like cells, a reduction in the lipid signal intensity might be observed. In our case, previous results have shown that on standard culture plates with the culture medium used in this study, neurospheres differentiate to an intermediate stage, maintaining their differentiation potential to some extent [36].

Neural progenitor cells were cultured on electrospun P(HB-co-HHx) fiber substrates coated with either laminin or poly-L-lysine/laminin. At different times after cell seeding, the samples were fixed and the cellular material on the fibers was partly transferred onto IR windows using a modified touch imprint cytology method. This strategy proved to be very effective and enabled the biochemical profiles of the NPCs that evolved on the coated polymer fibers to be determined with SIRMS. The intensities of bands in the lipid and amide I–II regions were observed to be influenced by the substrate type and coating and the culture time. These results demonstrate the important insights that SIRMS studies can provide into the responses of NPCs to biomaterials (in this case to the morphology and surface chemistry of polymeric scaffolds), suggesting that such studies could be a fundamental tool in the preparation and optimization of cellular scaffolds for CNS tissue engineering and regenerative medicine.

 

Source:

http://doi.org/10.1007/s00216-018-1049-z

 

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