Development of biodegradable conducting scaffolds for treating peripheral nerve lesions taking into account the influence of mechanical vibrations on neurons regeneration in tissue engineering

Project Information

NERVE-REPAIR

Grant agreement ID: 622362

Project closed

Start date 27 February 2015

End date 27 June 2018

Funded under

• Specific programme "People" implementing the Seventh Framework Programme of the European Community for research, technological development and demonstration activities (2007 to 2013)

Total cost € 282 542,40

EU contribution € 282 542,40

Coordinated by

ECOLE POLYTECHNIQUE FEDERALE DE LAUSANNE
Switzerland

Final Report Summary - NERVE-REPAIR (Development of biodegradable conducting scaffolds for treating peripheral nerve lesions taking into account the influence of mechanical vibrations on neurons regeneration in tissue engineering)

• Summary description of the project objectives

The proposed project aims to develop engineered biodegradable conducting 3D scaffolds capable of promoting neuronal survival, as well as axon extension and guidance, for treating peripheral nerve lesions. Current approaches focus on the sensitivity of neurons to the surrounding environment, which includes surface topography, biochemical cues, and electrical activity. Constructs implanted in vivo are also subject to mechanical constrains that have an impact on tissue formation. These mechanical stimulations include 1) micro-movements resulting from the movements of the body when the individual is performing various daily activities, and 2) movements caused by the organs moving with blood pulse (heart beat). This project aims to investigate the influence of these mechanical stimulations on scaffolds and neuronal cells proliferation. Moreover, the mechanical constrains applied on engineered tissues inside the body are evaluated with flexible biodegradable strain and pressure sensors developed for this purpose. The envisioned long-term goal is to develop scaffolds with various geometries, taking advantage of the reaction of neural cells when mechanical stimulations are applied. This project proposes an additional strategy that can be combined to other therapies to improve nerve regeneration.

• Description of the work performed in the first phase of the project (Stanford)

During the period of March 2015 to June 2017 (outgoing phase performed at Stanford in the group of Prof. Zhenan Bao), Two partnership have been established with two Stanford research groups, with the objective to evaluate the impact of mechanical stimulations on scaffolds and neurons activity. These collaborations were key partnerships for the success of the project.
- Prof. Sarah Heilshorn’s biomaterials lab (design of materials that mimic the nano- and micro-scale order found in nature for applications mainly in regenerative medicine and tissue engineering).
- Prof. Beth Pruit’s Microsystems lab (development and response of cells to mechanical loading, measuring nanoscale mechanical behavior, and development of micro electro mechanical systems MEMS).

Based on these collaborations:
- The dorsal root ganglion cells extracted from chicks (DRG) were chosen as a peripheral nerve model. The advantage is the relative easy extraction (cells extracted from chicks fetus in eggs as compared to cells extracted e.g. from rats fetus), and the large size allowing for easy handling and manipulation (diameter around 200-500 um).
- It was decided to use the Elastin-like Polypeptide (ELP) Hydrogels developed in prof. Sarah Heilshorn’s lab as a scaffold material. Indeed, the study of the influence of mechanical stimulation on neurons can only be performed in a very controlled environment. The ELP gel system presents several advantages, including a control of the stiffness of the hydrogel, the density of the attachment sites for the cells. Moreover, the study can be performed both in 2D and in a more realistic 3D environment.

• Additional work performed at Stanford

In parallel to the above-described research, the fellow worked on the 4 projects below, resulting in first-author papers (2 already published, and 2 submitted):

Project 1: A Sensitive and Biodegradable Pressure Sensor Array for Cardiovascular Monitoring
Summary of the project: A new class of sensors made from completely biodegradable materials is currently emerging as environmentally friendly and biocompatible with the human body. These devices improve the efficacy of implantable systems and reduce waste associated with wearable and point-of-care diagnostics. Highly sensitive pressure sensors can measure pulse, arterial waveform, and pulse wave velocity (PWV), which provide important information about cardiac health. In this work, we report the development of a single-use biodegradable pressure sensor patch for application in cardiovascular monitoring. The sensors have a very high sensitivity of 0.76 kPa −1 and are capable of distinguish in a weight of 5 mg. Each patch contains an array of sensors to facilitate fast and easy positioning for cardiac health measurements.

Project 2: A stretchable and biodegradable strain and pressure sensor for orthopaedic application
Summary of the project: The ability to monitor, in real time, the mechanical forces on tendons after surgical repair could allow personalized rehabilitation programs to be developed for recovering patients. However, the development of devices capable of such measurements has been hindered by the strict requirements of biocompatible materials and the need for sensors with satisfactory performance. Here we report an implantable pressure and strain sensor made entirely of biodegradable materials. The sensor is designed to degrade after its useful lifetime, eliminating the need for a second surgery to remove the device. It can measure strain and pressure independently using two vertically isolated sensors capable of discriminating strain as small as 0.4% and the pressure exerted by a grain of salt (12 Pa), without them interfering with one another. The device has minimal hysteresis, a response time in the millisecond range, and an excellent cycling stability for strain and pressure sensing, respectively. We have incorporated a biodegradable elastomer optimized to improve the strain cycling performances by 54%. An in vivo study shows that the sensor exhibits excellent biocompatibility and function in a rat model, illustrating the potential applicability of the device to the real-time monitoring of tendon healing.

Project 3: Wireless Monitoring of Blood Flow via Biodegradable, Flexible, Passive Arterial Pulse Sensor
Summary of the project: The ability to monitor blood flow is critical to patient recovery and patient outcomes after complex reconstructive surgeries. Currently, only wired implantable monitoring technology is clinically available; however, these devices require careful fixation for accurate detection and need to be removed after use. This presents a burden for both patients and surgeons. Herein, we demonstrate a novel pressure sensor entirely made of biodegradable materials, which would degrade after its useful lifetime, eliminating the need for a removal. The sensor is based on fringe field capacitor technology and senses arterial blood flow both in contact and non-contact modes, allowing for easier mounting and reducing the risk of vessel trauma. The device can be operated wirelessly via inductive coupling. In addition, the sensor has minimal hysteresis, high robustness, fast response time, and excellent cycling stability. Sensor operation is demonstrated in vitro with a custom-made artificial artery model. Moreover in vivo studies demonstrate biocompatibility and function in a rat model, both in wired and wireless configurations, suggesting the applicability of this sensor to real-time post-operative monitoring of blood flow after reconstructive surgery.

Project 4: A hierarchically patterned, bio-inspired e-skin able to detect the direction of applied pressure for robotics
Summary of the project: Tactile sensing is required for the dexterous manipulation of objects in robotic applications. In particular, the ability to measure and distinguish in real time normal and shear forces is crucial for slip detection and interaction with fragile objects. Here we report a biomimetic soft electronic skin composed of an array of capacitors, capable of measuring and discriminating in real time both normal and tangential forces. The discrimination is enabled by a 3-dimensional structure that mimics the interlocked dermis-epidermis interface in human skin. Moreover, pyramid microstructures arranged along nature-inspired phyllotaxis spirals result in an e-skin with increased sensitivity, minimal hysteresis, excellent cycling stability and response time in the millisecond range. The e-skin is used to control a robot arm in various tasks, illustrating its potential application in robotics with tactile feedback.

• Description of the work performed in the second phase of the project (EPFL)

During the period of June 2017 to June 2018 (return phase performed at EPFL in the group of Prof. Stephanie Lacour, LSBI, Laboratory for Soft Bioelectronic Interfaces), it has been decided to reorient the project as follows:
- Studies performed at EPFL will be done on dorsal root ganglion cells extracted from rats instead of chicks. The reason for this choice is the fact that rats, that are mammalian, are a better animal model for human peripheral nerve than chicks.
- Studies performed at EPFL will use not only ELP as a substrate material for DRGs growth, but also collagen. The reason for this choice is the fact that collagen is one of main materials used clinically and commercially as nerve scaffold. In addition, other researchers at LSBI are working on rats DRGs growing on/into collagen scaffold. The results of this study will benefit to more researchers in the group if both ELP and collagen are investigated.

• Description of the main achieved results

- During the outgoing phase at Stanford: After learning 1) how to culture and extract the DRG cells, 2) how to extract ELP hydrogel, 3) how to ensure proper adhesion between the ELP gel and the silicone substrate used for mechanical stimulation, 4) how to correctly operate the mechanical stimulation system developed in prof. Beth Pruit’s Microsystems lab for applying a controlled mechanical deformation on the scaffold with neurons, the fellow was able to perform mechanical stretching tests on these cells. Both the overall growth shape and quantitative neurites orientation are investigated. More precisely, 3 sets of experiments were performed, with various mechanical stimulation frequency (0.1Hz and 1Hz), waveform shapes (triangle shape – constraint strain rate - versus sine shape – less abrupt movement), and max applied strain (5% and 10%). Each condition was investigated at least for 3 different wells, containing each 1 to 3 DRGs, and repeated twice on different experiments and days. All experiments were performed within Stanford facilities including cells staining and imaging. The data processing has been started at Stanford, showing promising results on the preferential growing of neurites along the direction of applied strain, and later finished at EPFL.

- During the return phase at EPFL: The data processing for the experiments done at Stanford has been performed, showing promising but not conclusive results yet (interesting trends but not statistically significant yet). It has been therefore decided to continue experiments at EPFL with a similar experimental setup for DRGs stretching, increasing the stimulation frequency (1-2Hz) and increasing the maximum strain applied (above 10%). The objective of these new experiments was 1) to observe if similar trends and observations can be made with rats DRGs model than with chicks DRGs models, 2) to observe if statistically significant results can be obtained, based on experiments including a larger number of samples for a given condition, and based on experiments where stronger stimulation on cells is applied. Cells staining/imaging and data processing for the experiments performed at EPFL will be done by the end of June 2018. During the return phase at EPFL, in addition to ELP gel, it was decided to investigate the growth of DRGs on collagen substrate. Moreover, DRGs extracted from chicks were replaced by DRGs extracted from rats, the mammalian model being considered to be closer to final application in human. Nerve scaffolds with microchannels to guide the neurites growth were fabricated, made of biodegradable elastomers, PGS (poly(glycerol sebacate)) and POMAC (poly(octamethylene maleate(anhydride) citrate)). Foamy versions of these scaffolds with microchannels were also fabricated.

• Conclusion

This project proposes an additional and new design strategy for scaffolds used in peripheral nerve repair. Scaffolds are used to reconnect peripheral nerves after an injury. The project started with studies on the impact of mechanical stimulations on neural cells proliferation. Studies performed at Stanford show the favorable impact of mechanical stimulations on cells growth, but the results are not statistically significant yet. Further investigations will be needed in the future to assess the best conditions to favor peripheral nerves growth, the long-term objective for the society being to help patients recover neural functions after nerve injuries.

Legend for Figure 1 – New design strategy for scaffolds used in peripheral nerve repair. (a) Scaffolds are used to reconnect peripheral nerves after an injury. (b) Investigations on nerve “stretch growth” in developing embryos and (c) clinical studies on applied therapeutic ultrasounds for nerve healing. (d) The project starts with studies on the impact of mechanical stimulations on neural cells proliferation. Preliminary studies performed at Stanford show the favorable impact of mechanical stimulations on cells growth. (e)-(f) Proposed strategy for the development of new scaffolds.

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