Development of a nerve stretching device to facilitate peripheral nerve repair

Abstract

Advancements in biomedical engineering have improved medical technology by redefining surgical standards and developing medical devices to carry out complex repairs at a tissue level; however, effective surgical management for segmental defects (nerve gaps) in peripheral nerves clinically, remains a challenging endeavour. Traditional nerve gap management surgical techniques cannot bridge longer defects effectively, while contemporary techniques using a variety of nerve conduits to facilitate nerve regeneration have consistently failed to reproduce reliable results. One of the root causes of these inconsistent results lies in the nerve’s inability to regrow expediently after injury and hence the ‘slow rate of nerve regeneration’ is one of the main factors in unsuccessful nerve gap management. This situation demands a satisfactory approach to address the aforementioned problem. In the past, researchers have attempted to increase the growth rate of axons by stretching them mechanically outside the body and have proved that axons can respond to mechanical stimulus where their growth rate depends on the type and degree of mechanical strain. These promising findings, however, are difficult to translate to in-vivo applications, because stretching a nerve in-vivo is not analogous to pulling it ex-vivo, and need appropriate resources to provide such translation, which limits the effectiveness of this technique clinically. This thesis aimed to address the slow rate of peripheral nerve regeneration post-trauma and presents a solution to expedite nerve growth rate. During this research work, a novel method of stretching a whole nerve in a conduit using a controlled negative pressure (vacuum) was developed. The rationale behind proposing negative pressure as a stretching agent was that, it would promote angiogenesis by drawing more blood, from the microvessels coursing in the epineurium, to nourish growing sprouts of axons. Appropriate bioengineering tools were fabricated to carry out the in-vivo nerve stretch which included building a vacuum generating device (nerve stretcher) and fabricating a synthetic T-shaped conduit to hold and stretch the transected nerve stumps using the generated vacuum. Firstly, safe limits of applying vacuum to nerve stumps were ascertained during a pilot study on cadaveric rats (Chapter 3), where, a surgical procedure was also developed to implant T-shaped conduits, and ability of the nerve stretcher in generating a stable vacuum was tested at various vacuum levels. Secondly, the developed technique for in-vivo nerve stretching was then preliminarily tested on live rats (Chapter 4) and based on the experimental observations, the strategy for implanting the nerve conduit for a longer post-surgical period was revised. Finally, the modified approach was tested on 30 rats to study the in-vivo mechanotransduction effect of the injured peripheral nerves in response to vacuum. These in-vivo experiments involved transecting sciatic nerves of rats, placing the respective nerve ends into T-shaped conduits and then applying vacuum. Nerve stumps were tractioned at various negative pressure levels for seven days. After seven days, nerves were excised, and each nerve stump was sectioned and stained using histological and immuno-histochemical staining methods, slides were analysed qualitatively and quantitatively to measure the extent of nerve growth in response to negative pressure. The results of in-vivo nerve stretch-growth showed that the three treatment groups displayed better outcomes in terms of absolute growth of nerve stumps, higher rates of angiogenesis, and a greater quantity of nissl substance in cytoplasm over the control group. First, absolute growth of the nerve stumps in all three treatment groups provided direct quantitative evidence of enhanced growth in sciatic nerves in response to mechanical stress acting in the form of negative pressure. Second, a higher quantity of blood vessels in the treatment group confirmed the efficacy of negative pressure in promoting angiogenesis. Third, the presence of nissl substance in greater amounts in the treatment groups signalled that the effect of nerve injury has started to resolve (on injury to a nerve, neurons deploy macrophages to clear up the cell debris to make room for Schwann cells that perform myelination of axons). During result analysis, each treatment group was compared against each other and the control group, and it was found that a negative pressure of 20 mmHg displayed the most superior outcomes, favouring both nerve lengthening and angiogenesis. The results of this research work successfully justified the hypothesis of this thesis (Chapter 2), that an in-vivo application of negative pressure acting directly to transected rat sciatic nerves will enhance nerve regrowth by promoting angiogenesis and nerve lengthening.