Pie chart illustrating the types of animal model used in ‘in vivo’ studies 

Context in source publication

Context 1

... engineering has advanced as an grafting in humans . However, complications integrative field, which incorporates cells, such as sensory loss, neuroma and scar growth factors, biomaterials and formation (4) may arise following peripheral engineering to produce an artificial section or nerve harvesting. Due to the resulting donor site system capable of replacing damaged human morbidity and graft mismatch, an alternative is tissue or to improve its functional effectiveness currently needed. Thus, the development of (1) . The nervous system is one of the numerous artificial nerve conduits began to progress areas in which tissue engineering is focusing on; toward replicating a nerve that may match the nerve conduits being a crucial element of that former’s functional capabilities. In that context, advancement (2,3) . nerve conduits’ function is being tested A nerve conduit is a guide tube manufactured throughout research for the last 50 years by from either natural or synthetic materials. It varying techniques and methods, yet, a standard aims to restore sensitivity to nerve gaps caused testing method does not exist (7,8) . by trauma, degenerative disease or tissue loss Nerve tubulation (conduits) was first introduced due to tumor resection (4) . Autologous nerve in the 19 th century by Gluck; he has proposed conduits are the current gold-standard tool (5) for the use of nerve conduits in 1880 whereby he repair of injured or diseased nerves, the sural employed the use of a bone as a tube for nerve nerve being the most commonly used for nerve repair (9) . Gluck has adapted his idea from issue engineering has advanced as an integrative field, which incorporates cells, growth factors, biomaterials and engineering to produce an artificial section or system capable of replacing damaged human tissue or to improve its functional effectiveness (1) . The nervous system is one of the numerous areas in which tissue engineering is focusing on; nerve conduits being a crucial element of that advancement (2,3) . A nerve conduit is a guide tube manufactured from either natural or synthetic materials. It aims to restore sensitivity to nerve gaps caused by trauma, degenerative disease or tissue loss due to tumor resection (4) . Autologous nerve conduits are the current gold-standard tool (5) for repair of injured or diseased nerves, the sural nerve being the most commonly used for nerve Neuber who had used a bone tube in 1879 to serve as a resorbable wound drain (9) . In current practice, the US Food and Drug Administration (FDA) and the conformity European (CE) approved the clinical use of four artificial nerve conduits; two are type 1 collagen nerve conduits and the other two conduits are synthetic polyester-based (10,11) . This review aims to summarize the existing testing methodologies of artificial nerve conduits in the setting of both in-vitro and in-vivo models and to analyze the outcome of these methods in order to attain a standardized method of research for future nerve conduit studies in the peripheral nervous system. The use of animal models for nerve conduit testing flourished in the past decade producing abundant volumes of published studies. A systematic review conducted by Angius et al in 2012 analyzed the methodologies of more than 416 published in-vivo nerve conduit studies and concluded there was genuine lack of consensus and consistency in researchers’ choice of methods (12) . The variability of methods included the choice of animal model, tested nerve, gap length, and assessment tool. The most popular choice of animal was rats, which accounted for up to 70% of all in-vivo studies (12) . The advantages of using rats included low maintenance cost, resilience to surgical intervention and infections, availability, and production of consistent assessment outcomes (13-15) . However, the drawbacks included the relatively small gap length compared to common human nerve lesions, the difference in neurophysiology to humans i.e., nerve axotomy produces full recovery in rats but not in human nerves and nerve regeneration is slower in humans (12,16,17) . Furthermore, there are different species of rats which have unknown variations in their physiological response to foreign materials for nerve regeneration (12) . The remaining 30% of animal models were accounted for by mouse, rabbits, dogs, cats and monkeys, with a few scattered studies on sheep and guinea pigs (12) (Fig. 1). The mouse model was used in 7.5% of all studies and shared similar advantages and disadvantages to the rat model. One unique advantage in the mouse model was the ability to genetically modify mouse to allow imaging of nerve regeneration of fluorescent-induced axons (18,19) . The major disadvantage of mouse model was its limited gap length of less than 13mm (12) . The rabbit model had been one of the more frequently used models amongst the larger animals (up to 7.5% of studies). The rabbit model facilitated testing of larger nerve gap lengths and produced reliable results from neuromorphometric and electrophysiological testing methods (12) . However, its disadvantages included cost, difficulty of care, limited molecular probes for mechanistic analysis and most importantly, the difference in anatomy e.g., hind limb muscle in rabbits functions to hop, this may reduce its strength for human clinical trials (12) . Nerve studies on dogs and cats also allowed large testing nerve gap, and commonly produced reliable neuromorphometric analysis (12) . One major advantage in the use of dogs was the ability to train the animal for functional motor and sensory analysis, however, major drawbacks, together with cats, included maintenance cost, ethical concerns in their role as domestic animals, and the lack of molecular probes present for mechanistic analysis (12) . There use of larger animals such as monkeys, sheep and guinea pigs in nerve conduit testing were less common (approximately 10, 4 and 1 reported cases, respectively). These animals allowed larger nerve gap length up to 60mm to be tested. However, these studies were restricted due to high cost and limited range of assessment tools available, including difficulties in training these species for functional testing compared to dogs (12) . Although the study of nonhuman primates i.e. monkey, would provide presumably the most reliable outcomes for a step toward human trials, recent reports from the Institute of Medicine had pledged their disagreement to nonhuman primates testing (20,21) . Overall, the selection of the animal type for clinical trials was essential, and researchers must consider the cost, availability, ethical issues and importantly, the physiology of the species e.g. lifespan, inter-variation of the species, susceptibly of infection and ability to withstand surgical interventions (22,23) . Furthermore, compatibility of the neurophysiology of the animal species to the human being must also be considered i.e., neuromicrostructure, inflammatory response, degeneration process (Wallerian), and regeneration capacity (24,25) . It is important to make aware that the testing model used will depend on the experimental question, thus most authors would agree that no single testing model will fit all, nevertheless, the call for a more standardized methodology and guidelines will aid research forward (13,23,26) . In addition, strict adherence to national regulation in animal-testing policies is vital (27) . The most commonly tested nerve was the sciatic nerve, accounted for over 70% of all studies (12) . The popular use of the sciatic nerve was likely to be due to its relatively anatomical accessibility and size compared to other peripheral nerves. The peroneal, tibial and facial nerve accounted for approximately 5-7% of the studies. A total of 17 different types of peripheral nerves that had been used for nerve conduits studies (12) (Fig. 2). Small volumes of individual studies used the median, radial, ulnar, alveolar, cavernous, saphenous, hypogastric, sural, optic, phrenic, recurrent laryngeal lingual and femoral nerves (12) . Overall, the selection of nerves was likely to be governed by resources, animal variability, and most importantly, intended purpose of the clinical trial. As previously described above, the length of nerve gap examined were influenced greatly by the selection of the tested animal: rats 1-50 mm, mouse 2-13 mm, rabbits 2-50 mm, dogs 10-90 mm, cats 1-50 mm, monkeys 1-50 mm, pigs 8mm and no nerve gap was examined in the sheep study (Table 1). The ideal nerve gap length studied would mimic distances commonly encountered in human nerve injuries, which vary tremendously. In most studies, the selection for gap lengths were >2 mm, which were decided upon the concept of critical length i.e., gap distance which regeneration would not occur unless nerve grafting or bridging occurs. Studies that conducted testing gap lengths < 2mm were not clear in the reason behind their selection. The range of gap lengths tested was from 1mm to 90mm. The most frequently used gap lengths were small distances of 1-5 mm and 6-10 mm, which accounted for 80% of all studies, followed by intermediate lengths of 11-15 mm, 16-20 mm and 24-30mm (25% of cases). Larger gap lengths of 40-90mm were less commonly tested. There were a vast number of available testing tools used to assess nerve recovery and function (Table ...