Eur J Neurosci. 2016 Feb;43(3):376-87.
Characterisation of cell-substrate interactions between Schwann cells and three-dimensional fibrin hydrogels containing orientated nanofibre topographical cues
Hodde D1, 2*, Gerardo-Nava J1, 2, 3*, Wöhlk V1, Weinandy S4, Jockenhövel S4, Kriebel A5, Altinova H1, 2, 6, Steinbusch HWM7, Möller M8, Weis J1, 2, Mey J3,5,7, 9 , Brook GA1,2,3
1: Institute of Neuropathology, Uniklinik RWTH Aachen University, Germany
2: Jülich-Aachen Research Alliance – Translational Brain Medicine (JARA Brain), Jülich, Germany
3: EURON – European Graduate School of Neuroscience
4: Department of Tissue Engineering and Textile Implants, AME – Helmholtz Institute for Biomedical Engineering and Uniklinik RWTH Aachen University, Germany
5: Institute of Biology II, RWTH Aachen University, Germany
6: Department of Neurosurgery, Uniklinik RWTH Aachen University, Germany
7: Department of Psychiatry and Neuropsychology, Division Neuroscience, Faculty of Health, Medicine and Life Sciences, Maastricht University, The Netherlands
7: Laboratorio de Regeneración Nerviosa, Hospital Nacional de Parapléjicos, Toledo, Spain
8: DWI−Leibniz Institute for Interactive Materials and Institute of Technical and Macromolecular Chemistry, RWTH Aachen University, Germany
9: Laboratorio de Regeneración Nerviosa, Hospital Nacional de Parapléjicos, Toledo, Spain
* These authors contributed equally in the production of this work.
The generation of complex three-dimensional bioengineered scaffolds that are capable of mimicking the molecular and topographical cues of the extracellular matrix found in native tissues is a field of expanding research. The systematic development of such scaffolds requires the characterisation of cell behaviour in response to the individual components of the scaffold. In the present investigation, we studied cell-substrate interactions between purified populations of Schwann cells and three-dimensional fibrin hydrogel scaffolds, in the presence or absence of multiple layers of highly orientated electrospun polycaprolactone nanofibres. Embedded Schwann cells remained viable within the fibrin hydrogel for up to 7 days (the longest time studied); however, cell behaviour in the hydrogel was somewhat different to that observed on the two-dimensional fibrin substrate: Schwann cells failed to proliferate in the fibrin hydrogel, whereas cell numbers increased steadily on the two-dimensional fibrin substrate. Schwann cells within the fibrin hydrogel developed complex process branching patterns, but, when presented with orientated nanofibres, showed a strong tendency to redistribute themselves onto the nanofibres, where they extended long processes that followed the longitudinal orientation of the nanofibres. The process length along nanofibre-containing fibrin hydrogel reached near-maximal levels (for the present experimental conditions) as early as 1 day after culturing. The ability of this three-dimensional, extracellular matrix-mimicking scaffold to support Schwann cell survival and provide topographical cues for rapid process extension suggest that it may be an appropriate device design for the bridging of experimental lesions of the peripheral nervous system.
Three dimensional (3D) bioengineering of scaffolds for tissue engineering and regenerative medicine is becoming increasingly sophisticated. In particular, scaffolds with precisely engineered 3D architectures, capable of reproducing many of the important characteristics of tissue extracellular matrix (ECM) has become an area of substantial interest in the field of peripheral nervous system (PNS) repair. Such scaffolds are showing great promise in being able to bridge physical gaps in traumatically damaged PNS, where they can support nerve fibre regeneration and the recovery of function (1). The most advanced scaffold designs combine both molecular signaling and a 3D topography to support of nerve repair.
Much of our understanding of how cells behave in response to signals in their environment is based on cell-substrate interactions in relatively simple 2D tissue culture systems (2) but this might not accurately reflect how cells respond when embedded within a more complex 3D environment, such as in tissues (3). In the context of developing a scaffold for PNS repair, based on orientated arrays of fine diameter synthetic (polycaprolactone, PCL) polymer fibres embedded within a fibrin hydrogel ECM, we performed a series of studies to learn how key PNS support cells (Schwann cells) respond to the elements of such an engineered structure. The fibres were designed to have diameters that were in the sub-micron range (i.e. nanofibres) to present the advantage of a large surface area to volume. Fibrin was chosen as the supporting ECM because it is matrix that is immediately deposited in the body where trauma causes tissue damage and bleeding, and it is the fibrin matrix that supports the earliest stages of cell migration during PNS repair across gaps.
Our first task was to prove that the embedding of the nanofibre arrays into the viscous fibrin solution wouldn’t cause them to coalesce or to destroy their particular orientation and spacing. We were able to show that although the fibres are extremely fine, they were also rather robust and showed no perturbation of spacing or orientation in the fibrin matrix. The next task was to see how Schwann cells would behave when completely surrounded by the fibrin matrix. The Schwann cells were seen to be evenly distributed throughout the fibrin, where they displayed rounded cell bodies (white arrow Fig 1A) with multiple, branching processes that grew quickly and extended in all directions (arrowheads, Fig. 1A). This behaviour could be seen throughout the full 7 days of tissue culturing and was surprising in that the classic morphology of Schwann cells, as seen many times in 2D tissue culture, is that of ovoid cell bodies with 1 or 2 elongated, thin, spindle shaped processes. Equally surprising was the behaviour of the Schwann cells in the orientated nanofibre-containing fibrin matrix. Here, almost al the cells rapidly became associated with the fibres, where they extended very long, simple processes (arrows, Fig. 1B) that closely followed the orientation of the nanofibre arrays (a single fibre is highlighted by multiple arrowheads, Fig. 1B). When viewed using 2 photon laser scanning microscopy, it was clear that most Schwann cells had vacated the fibrin-filled spaces between the nanofibre arrays and appeared as sheets of cells long, uniformly orientated process (Fig. 1C). Quantification of this behaviour confirmed the complex nature of Schwann cell growth within the fibrin matrix but also showed that process extension along the nanofibres was exceptionally fast, reaching maximal length by just 24 hours in tissue culture. This process growth was strikingly longer and faster than anything previously seen in our lab using the same nanofibres but in the absence of the fibrin hydrogel.
The importance of this study is two-fold:
Firstly, the control of cell behaviour by bioengineered scaffolds represents a crucial step in designing substrates that can support efficient and highly directional cell growth (and hopefully axon regeneration) across injury-induced lesion sites.
Secondly, when the Schwann cells were presented with a choice of substrates that they could follow (i.e. the homogeneous 3D fibrin matrix or the highly orientated nanofibre arrays), they almost always became closely associated with the nanofibres and faithfully followed their trajectory. This is striking because the fibrin molecules contain peptide signalling sequences that the Schwann cells can respond to. In contrast, the PCL nanofibres, do not possess any signalling molecules that the Schwann cells can respond to, but the cells still followed the fibres. This might support the notion of a hierarchy of substrate preferences, where cells to prefer to associate with the stiffer (i.e. PCL) substrate than the softer (fibrin substrate) even in the presence of matrix signalling sequences.
Figure 1. Schwann cell behaviour when embedded in fibrin matrix (A) or when embedded in fibrin matrix containing orientated nanofibre arrays (B, C). Cells showed a complex pattern of branching and non-direction growth within the fibrin matrix, but became highly orientated when presented with the nanofibre arrays.
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Acknowledgements: This study was supported by the Deutsche Forschungsgemeinschaft (DFG BR2299/4-1).