Introduction
Limb amputation due to diabetes and vascular disease is projected to double by 2050, highlighting the urgent need for advanced prosthetic solutions (Ziegler-Graham et al. Reference Ziegler-Graham, MacKenzie, Ephraim, Travison and Brookmeyer2008). Many additional amputations result from trauma or military combat injuries. Current artificial prostheses often fail to replicate the sensory and motor functions of natural limbs, leading to high abandonment rates, with many individuals using them for cosmetic purposes only (Biddiss and Chau Reference Biddiss and Chau2007).
We aimed to investigate the use of Normothermic Machine Perfusion (NMP) in the development of advanced neuroprostheses, specifically focusing on electrophysiological studies that evaluate muscle and nerve function. NMP mimics the in vivo physiological environment by delivering oxygenated blood and nutrients to preserve tissue viability. This technology may bridge the gap in preclinical testing of engineered interfaces.
Current state-of-the-art prostheses enabling real-time user control are based upon sensed muscle-controlled signals using surface electromyography as the communicating ‘interface’ between human and machine (Bergmeister et al. Reference Bergmeister, Vujaklija, Muceli, Sturma, Hruby, Prahm, Riedl, Salminger, Manzano-Szalai, Aman, Russold, Hofer, Principe, Farina and Aszmann2017). However, in complex injuries, muscle targets are absent or weak, and the quality of extracted signals is variable (Aszmann et al. Reference Aszmann, Roche, Salminger, Paternostro-Sluga, Herceg, Sturma, Hofer and Farina2015). The influence of neighbouring ‘noise’ from adjacent muscle contractions limits signal quality (Solomonow Reference Solomonow1984). Furthermore, surface EMG electrodes are for prosthetic control only and are unable to provide meaningful information about sensory feedback. Restoring a sense of ‘touch’ into artificial prostheses is a priority area for researchers and users (Raspopovic et al. Reference Raspopovic, Valle and Petrini2021). These are some of the reasons why thirty per cent of users abandon myoelectric prostheses resulting in emotional and psychological disturbance (Winslow et al. Reference Winslow, Ruble and Huber2018). Collectively, current advanced prostheses offer variable degrees of fine motor control, limited sensory discrimination and lack realism, depriving embodiment (Biddiss and Chau Reference Biddiss and Chau2007).
One attractive prospect would be to directly interface implantable bi-directional electrodes with the peripheral nervous system (Rapeaux et al. Reference Rapeaux, Syed, Cuttaz, Chapman, Green and Constandinou2022). Peripheral nerves have efferent (motor) and afferent (sensation via cutaneous mechanoreceptors, proprioceptors, thermoreceptors and nociceptors) signals. Distinct subpopulations of axons convey tactile, proprioceptive, thermal or noxious stimuli (Johansson and Vallbo Reference Johansson and Vallbo1979). As far back as the 1970s attempts were made to reproduce sensation via cuff or needle electrodes and to apply direct electrical stimulation (Clippinger et al. Reference Clippinger, Avery and Titus1974). With early attempts, subjects reported paraesthesia, vibrations, or a pulsing spread across their phantom hand, all deemed uncomfortable and unrealistic (Pylatiuk et al. Reference Pylatiuk, Kargov and Schulz2006). As mentioned, sensory feedback is crucial for human interaction, and this for example, may give feedback on how tightly objects can be held (Davis et al. Reference Davis, Wark, Hutchinson, Warren, O’Neill, Scheinblum, Clark, Normann and Greger2016). Understanding and interpreting how biological neuromuscular signals are exchanged in a meaningful bidirectional manner has yet to be elucidated.
Engineering and scientific barriers
Engineering barriers exist to developing implantable interfaces including the powering of implants (Biddiss and Chau Reference Biddiss and Chau2007), longevity, performance and interoperability (seamless data exchange and analysis). Other factors to consider are the regulatory components required for implant safety approval and the Research and Development costs required for testing prior to human implantation (Chakrabarty et al. Reference Chakrabarty, Chew, Güemes, Loeb, Sharma, Shivdasani, Sridhar and Yin2023). The notable preclinical challenges for the safe implantation of a bionic interface are summarised below (Table 1).
Research targets for the development of desirable implantable nerve electrode
Modelling barriers
Human implantation and interface design requires significant preclinical testing that conform with safety and efficacy laws (Pasquina et al. Reference Pasquina, Carvalho and Sheehan2015). Many studies have attempted to use in vivo and in vitro testing of explanted nerves (Andreis et al. Reference Andreis, Metcalfe, Janjua, Fazan, Jensen, Meijs and Nielsen2023). However, these have well known limitations, particularly around the use of laboratory animals. There is also limited translational relevance when using small animals (Aman et al. Reference Aman, Bergmeister, Festin, Sporer, Russold, Gstoettner, Podesser, Gail, Farina, Cederna and Aszmann2019). As a result, developing alternative approaches has become a ‘grand challenge’ for bioprosthetic research.
NMP serves to restore tissue viability by mimicking the in vivo environment via the delivery of a constant circulation to tissues. The core concept of perfusion technology is not novel and has been derived from clinical cardiopulmonary bypass circuits (CPB) and extracorporeal membrane oxygenation (ECMO), both of which have been used for decades. For example, a limb can be isolated from a food industry pig, connected to an advanced life support system and perfused with autologous blood and nutrients to simulate the biological environment more accurately (Amin et al. Reference Amin, Stone, Kerr, Geraghty, Joseph, Montero-Fernandez, Wong and Fildes2021). The approach is seen as being consistent with replacement, reduction and refinement efforts (Grimm et al. Reference Grimm, Biller-Andorno, Buch, Dahlhoff, Davies, Cederroth, Maissen, Lukas, Passini, Tornqvist, Olsson and Sandstrom2023) and offers greater translational relevance. Studies in both small and large animal models have primarily used this technology for the purposes of tissue preservation, for the interests of limb reconstruction, transplantation and pharmaceutical testing (Burlage et al. Reference Burlage, Lellouch, Taveau, Tratnig-Frankl, Pendexter, Randolph, Porte, Lantieri, Tessier, Cetrulo and Uygun2022).
Aims of this review
It has recently been acknowledged in a systematic review that there has been a significant upturn in the use of live animal models for interface development (Aman et al. Reference Aman, Bergmeister, Festin, Sporer, Russold, Gstoettner, Podesser, Gail, Farina, Cederna and Aszmann2019). However, our hypothesis was that despite increasing research activity, adoption of novel and advanced models has yet to occur. This is likely due to a lack of awareness of the approach in the bionic prostheses community. This is despite the fact that NMP offers a new tool for those developing bioelectronic devices to accelerate pre-clinical testing and ultimately shorten the routes to impact for patients impacted by amputation.
The aim of this article was to investigate the use of animal NMP models in advanced neuroprostheses development. This will consider electrophysiological studies targeted at muscle and peripheral nerves in both small and large animals for the development of implantable neuroprosthetic interfaces and discuss the barriers in their application when using NMP as a platform.
Methods
To investigate the use of animal NMP models in neuroprosthesis development, an electronic search was conducted in Ovid MEDLINE (1946 to October 2023) in accordance with PRISMA guidelines. The search targeted studies focusing on muscle and peripheral nerve interfacing with bionic prostheses using machine perfusion methods. This was followed by systematic screening of abstracts and full-text articles to identify relevant studies.
Search terms
An electronic search was conducted using Ovid MEDLINE (1946 to October 2023), following PRISMA guidelines (Liberati et al. Reference Liberati, Altman, Tetzlaff, Mulrow, Gotzsche, Ioannidis, Clarke, Devereaux, Kleijnen and Moher2009b). The research aimed to identify studies on animal limb machine perfusion specifically investigating muscle and peripheral nerve interfacing for bionic prostheses development. The search strategy, developed with librarian support from Manchester University Foundation Trust (MFT), included the following terms:
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(i) Limb AND (implantable nerve OR Electrode OR Prostheses OR Peripheral nerve) AND (Ex-vivo OR machine perfusion OR Perfusion)
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(ii) Limb AND (EMG OR ENG) AND (Ex-vivo OR machine perfusion OR Perfusion)
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(iii) Limb AND electrode testing AND Ex-vivo OR machine perfusion (iv) Limb AND (Ex-vivo OR machine perfusion OR Perfusion)
An electronic search was performed on Ovid MEDLINE (1946 to October 2023) in accordance with PRISMA guidelines (Liberati et al. Reference Liberati, Altman, Tetzlaff, Mulrow, Gotzsche, Ioannidis, Clarke, Devereaux, Kleijnen and Moher2009b). The research question was designed to identify animal limb machine perfusion studies specifically investigating muscle and peripheral nerve interfacing for the development of bionic prostheses. Our search strategy was designed with librarian support from Manchester University Foundation Trust (MFT). The search terms used were:
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(i) Limb AND (implantable nerve OR Electrode OR Protheses OR Peripheral nerve) AND (Ex-vivo OR machine perfusion OR Perfusion)
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(ii) Limb AND (EMG OR ENG) AND (Ex-vivo OR machine perfusion OR Perfusion)
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(iii) Limb AND electrode testing AND Ex-vivo OR machine perfusion
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(iv) Limb AND (Ex-vivo OR machine perfusion OR Perfusion)
This combination of search terms was based upon the inclusion criteria (Table 2).
Review protocol outlining the predetermined inclusion criteria for article selection
The protocol was registered with the PROSPERO international prospective register of systematic reviews, number CRD42022363528 available at (Page et al. Reference Page, Shamseer and Tricco2018). All articles were stored and reviewed using Rayyan.ai software (Ouzzani et al. Reference Ouzzani, Hammady, Fedorowicz and Elmagarmid2016). Two authors independently screened abstracts retrieved by all searches. After the initial assessment, full-text copies of all studies considered to be relevant were retrieved. Further screening of potentially relevant articles was performed via review of retrieved article reference lists.
Conclusions and perspectives
The initial search yielded 559 potential entries following deduplication (66 duplicates excluded). Full-text articles were sought for 44 studies. No studies using machine perfusion were identified directed solely towards the development of interfacing with muscle or peripheral nerves. Due to the paucity and heterogeneity of the included studies, a meta-analysis was not performed following the primary search. One single conference abstract was identified but contributed by our group as an abstract conference submission (Amin et al. Reference Amin, Stone, Cowey, Bowers, Smith, Entwistle, Casson and Fildes2023). Returned articles primarily comprised of interface imaging technologies and tracking of in vivo nerve health.
The initial search yielded 559 potential entries after deduplication (66 duplicates excluded). Full-text articles were sought for 44 studies. No studies using machine perfusion were identified that focused solely on the development of interfacing with muscle or peripheral nerves. Due to the paucity and heterogeneity of the included studies, a meta-analysis was not performed. One conference abstract, contributed by our group, was identified (Amin et al. Reference Amin, Stone, Cowey, Bowers, Smith, Entwistle, Casson and Fildes2023). The returned articles primarily involved interface imaging technologies and tracking in vivo nerve health.
The PRISMA flowchart summarises the selection process (Figure 1) (Liberati et al. Reference Liberati, Altman, Tetzlaff, Mulrow, Gotzsche, Ioannidis, Clarke, Devereaux, Kleijnen and Moher2009a). Our findings reveal that while several studies have explored electrical and muscle activity in NMP limb systems, none have employed this technology to advance bionic prostheses development. This underutilisation provides an opportunity, given the potential of NMP to enhance neuromuscular interfacing in bioelectronic devices. These findings align with prior research indicating limited application of NMP in neuroprosthetics (Aman et al. Reference Aman, Bergmeister, Festin, Sporer, Russold, Gstoettner, Podesser, Gail, Farina, Cederna and Aszmann2019). Future studies should explore ways to overcome barriers such as cost and technological complexity, which may explain this gap.
Selection process for systematic literature analyses as per PRISMA guidelines.
A systematic review by Aman et al. evaluated in vivo animal interface testing in rodents, cat, dog, pig and rabbits, comprising of direct muscle interfacing (23% of studies) and peripheral nerve interfacing (77% of studies). Studies were predominantly in rats. However, their rate of axonal regeneration is faster compared with humans, making them less comparable for clinical translation (Aman et al. Reference Aman, Bergmeister, Festin, Sporer, Russold, Gstoettner, Podesser, Gail, Farina, Cederna and Aszmann2019). Compared with large animals, small animals are cost-effective and allow for higher experimental turnover, but the acquisition of commercially available perfusion circuits is a limitation, resorting to self-manufactured circuits and oxygenators that are not clinically translatable (Berner et al. Reference Berner, Clement, Stadelmann, Kistler, Boone, Carrel, Tevaearai and Longnus2012).
Though not within the main aim of this review, and not accounting for our own NMP conference abstract, 44 ex vivo limb perfusion studies were retrieved in this review from rodents (7%), pigs (86%) and horses (7%). The indications were primarily for tissue preservation in the field of transplantation. Of all the pig studies, nine used muscle stimulation/contraction as a surrogate marker of viability. Pig models are considered the most translational model and share anatomical (muscle mass, vessel diameter and nerve calibre) (Sauerbrey et al. Reference Sauerbrey, Hindel, Maass, Kruger, Wissmann, Kramer, Nafz and Ludemann2014), and physiological characteristics that resemble humans (Dehoux and Gianello Reference Dehoux and Gianello2007; Kiermeir et al. Reference Kiermeir, Meoli, Muller, Abderhalden, Vogelin and Constantinescu2013). Non-Human Primates (NHP) have preclinical relevance, but ethical concerns remain for in vivo testing, and they are prohibitively expensive. Therefore, an overview of how this technology can be applied to emerging bionic implant testing would be useful given the advantages non in vivo studies have on animal welfare (Prescott and Lidster Reference Prescott and Lidster2017).
Use of NMP as a model
Despite the clear advantages of NMP in preserving tissue viability and neuromuscular function, our review identified a critical gap in its application for neuroprosthesis development. This underutilisation may stem from the complexity of NMP technology and the high costs of clinical-grade systems. However, integrating NMP into bioelectronic research could revolutionise preclinical testing by reducing reliance on live animal models, aligning with ethical guidelines, and potentially accelerating the development of advanced prosthetic technologies, ultimately improving patient outcomes.
Our lab has experience in developing physiological NMP protocols for pig limb perfusion (Amin et al. Reference Amin, Stone, Cowey, Bowers, Smith, Entwistle, Casson and Fildes2023; Amin et al. Reference Amin, Stone, Kerr, Geraghty, Joseph, Montero-Fernandez, Wong and Fildes2021). Two vital areas to control for using NMP as a model for bionic prosthetic development are ischaemia and maintenance of stable physiology. Critical ischaemia for muscle is 4 hours and nerve 8 hours, demonstrating the importance of limiting ischaemia (Muller et al. Reference Muller, Constantinescu, Kiermeir, Gajanayake, Bongoni, Vollbach, Meoli, Plock, Jenni, Banic, Rieben and Vogelin2013). Studies by others have reported loss of nerve conduction and muscle contractility immediately after amputation, later restored following NMP, with no contractility observed in cold-stored controls (Constantinescu et al. Reference Constantinescu, Knall, Xu, Kiermeir, Jenni, Gygax, Rieben, Banic and Vogelin2011; Duraes et al. Reference Duraes, Madajka, Frautschi, Soliman, Cakmakoglu, Barnett, Tadisina, Liu, Grady, Quintini, Okamoto, Papay, Rampazzo and Bassiri Gharb2018; Fahradyan et al. Reference Fahradyan, Said, Ordenana, Dalla Pozza, Frautschi, Duraes, Madajka-Niemeyer, Papay, Rampazzo and Bassiri Gharb2020; Said et al. Reference Said, Ordenana, Rezaei, Figueroa, Dasarathy, Brunengraber, Rampazzo and Gharb2020). Recently, we have applied the application of this concept for the development of advanced prosthetic interface technologies (Amin et al. Reference Amin, Stone, Cowey, Bowers, Smith, Entwistle, Casson and Fildes2023). This study highlights the potential of NMP to serve as a valuable preclinical model for advancing bioelectronics. The ability of NMP to preserve neuromuscular function post-amputation, alongside real-time electrical stimulation, offers new avenues for testing implantable interfaces without the ethical and practical constraints of live animal models. Nevertheless, further formal comparisons with in vivo and in vitro data is required. Future studies should focus on overcoming barriers such as cost and technological complexity to fully realise the potential of NMP in advancing bionic prostheses.
Though out of the remit of our search criteria, an interesting study using NMP of human cadaver upper limbs (Rezaei et al. Reference Rezaei, Ordenana, Figueroa, Said, Fahradyan, Dalla Pozza, Orfahli, Annunziata, Rohde, Madajka, Papay, Rampazzo and Bassiri Gharb2022) to preserve tissues for transplantation identified relevant findings. Contraction was preserved for approximately thirty hours. Neuromuscular function was significantly influenced by rising potassium (near limits expected to induce cardiac arrest in humans), decreasing calcium and acidosis. The cause of hyperkalaemia was unclear, but the authors suggested evaporative water loss, the absence of solid organs such as a kidney, intracellular loss from tissue injury and myocyte destruction (Rezaei et al. Reference Rezaei, Ordenana, Figueroa, Said, Fahradyan, Dalla Pozza, Orfahli, Annunziata, Rohde, Madajka, Papay, Rampazzo and Bassiri Gharb2022). In the presence of acidosis, potassium can transit from the intracellular to extracellular space to buffer falling pH and could be one explanation, aside from cell death, for the higher concentrations observed at the start of perfusion (Oster et al. Reference Oster, Perez and Vaamonde1978). To address this, we have developed a multi-organ system incorporating autologous organs including spleen, liver and kidney (Stone et al. Reference Stone, Amin, Geraghty, Kerr, Shaw, Dabare, Wong, Brough, Entwistle, Montero-Fernandez and Fildes2022).
To optimise perfusate physiology, perfusate exchange has been performed to account for electrolyte, red blood cell (RBC), lactate, creatine kinase (CK) and myoglobin derangements with time (Ozer et al. Reference Ozer, Rojas-Pena, Mendias, Bryner, Toomasian and Bartlett2015). It is acknowledged that both CK and myoglobin increase with NMP, yet macroscopic contractility is observed for hours and therefore its significance is unknown (Duraes et al. Reference Duraes, Madajka, Frautschi, Soliman, Cakmakoglu, Barnett, Tadisina, Liu, Grady, Quintini, Okamoto, Papay, Rampazzo and Bassiri Gharb2018; Fahradyan et al. Reference Fahradyan, Said, Ordenana, Dalla Pozza, Frautschi, Duraes, Madajka-Niemeyer, Papay, Rampazzo and Bassiri Gharb2020). Unsurprisingly, increasing limb weight (a marker of oedema) correlates with muscle injury and reduced contraction in pigs (Meyers et al. Reference Meyers, Pandey, Kopparthy, Sadeghi, Clark, Figueroa, Dasarathy, Brunengraber, Papay, Rampazzo and Bassiri Gharb2022). Ozer et al. performed a perfusate exchange at two hourly intervals to overcome rising potassium concentrations, but their analysis used single fibre muscle biopsies (Werner et al. Reference Werner, Alghanem, Rakestraw, Sarver, Nicely, Pietroski, Lange, Rudich, Mendias, Rojas-Pena, Magee, Bartlett and Ozer2017). Kuecklehaus et al. found blood-potassium continued to rise, and this was unresponsive to boluses of glucose and insulin (Kueckelhaus et al. Reference Kueckelhaus, Fischer, Sisk, Kiwanuka, Bueno, Dermietzel, Alhefzi, Aycart, Diehm and Pomahac2016), further demonstrating that the physiological environment is critical to neuromuscular function. This may explain why acellular solutions (Perfadex), frequently used clinically in solid organ perfusions demonstrates poor muscle contraction after 3 hours (Kueckelhaus et al. Reference Kueckelhaus, Dermietzel, Alhefzi, Aycart, Fischer, Krezdorn, Wo, Maarouf, Riella, Abdi, Bueno and Pomahac2017).
In one pig study, the median nerve was stimulated directly by electrodes (Kruit et al. Reference Kruit, Brouwers, van Midden, Zegers, Koers, van Alfen, Hummelink and Ulrich2021). The lowest voltage of two repeated measurements needed to obtain a visible flexor muscle contraction was recorded. The mean nerve stimulation threshold to obtain visible muscle contraction was comparable between limbs that were cooled using the gold standard preservation approach of Static Cold Storage (SCS; 4 hours) and NMP (18 hours with University of Wisconsin). Limbs were then replanted and observed for 12 hours. In vivo contraction was better preserved after NMP than SCS. Histology did not correlate with muscle function at 12 hours post-replantation, further questioning the relevance of histology and neuromuscular function. Contralateral limbs had a stable muscle contraction threshold throughout the entire follow-up period with values between 4.3 and 4.9 mV (Kruit et al. Reference Kruit, Brouwers, van Midden, Zegers, Koers, van Alfen, Hummelink and Ulrich2021). The mean contraction threshold after replantation of SCS limbs ranged between 4.0 and 5.4 mV and showed a small increase at 2 h after replantation. NMP perfused limbs had contraction thresholds between 4.1 and 9.3 mV. When comparing the muscle contraction thresholds at the end of the experiment, there were no differences in strength of stimulation needed to reach visible muscle contraction between the three groups (Kruit et al. Reference Kruit, Brouwers, van Midden, Zegers, Koers, van Alfen, Hummelink and Ulrich2021).
Advanced prostheses will not only rely upon motor stimulation, but also acquisition of sensory function data. No animal model to our knowledge has been capable of peripheral nerve afferent sensory signals. Studies have used functional MRI in NHPs via stimulation of the somatosensory cortex, but this is not cost-effective or practical (Flesher et al. Reference Flesher, Collinger, Foldes, Weiss, Downey, Tyler-Kabara, Bensmaia, Schwartz, Boninger and Gaunt2016). The adoption of NMP could revolutionise the preclinical testing landscape by reducing the reliance on live animal models, thus aligning with ethical guidelines and potentially accelerating the development of bionic prostheses.
Limitations and future directions of NMP as a model
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(i) Several limitations exist in the current use of NMP. First, the reliance on clinically approved life-support systems and consumables can increase costs, limiting its widespread adoption in research. Future efforts should focus on developing more affordable, simplified NMP circuits that are accessible to research labs. Second, small animal models such as rodents have limited translational relevance due to differences in anatomy and nerve regeneration compared to humans. Larger animals, such as pigs, offer more accurate modelling but come with increased ethical and financial considerations. Developing NMP protocols for these larger models could overcome these challenges, while refining in vitro methods or utilising cadaveric human limbs might further reduce reliance on live animal testing. Finally, signal extraction from peripheral nerves remains variable, often due to ‘noise’ from neighbouring tissues. More sophisticated electrode designs and improved signal processing algorithms may offer a way to address this.
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(ii) NMP requires clinically approved, advanced life-support systems and critical care consumables. Turbulent mechanical flow can damage intimal endothelium and provoke an inflammatory response (Matsuno and Kobayashi Reference Matsuno and Kobayashi2013), and NMP circuits cannot be constructed with uncoated surfaces made of foreign materials (plastic) that can activate the immune system and inflammatory cascades (Muller et al. Reference Muller, Constantinescu, Kiermeir, Gajanayake, Bongoni, Vollbach, Meoli, Plock, Jenni, Banic, Rieben and Vogelin2013). NMP is highly complex, with expertise in surgery, advanced perfusion and critical care needed.
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(iii) The optimisation of recirculating perfusate composition and experimental conditions requires further investigation.
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(iv) There is time-dependent deterioration in the recirculating blood-perfusate.
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(v) The pattern of nerve regeneration of the distal nerve after transection starts with that of peripheral nerve ‘Wallerian degeneration’, which results in phagocytosis of the axon and myelin remnants leaving a nerve scaffold. Macrophages stimulate Schwann cell proliferation in the proximal nerve attached to the cell body (NMP cannot account for this process in the amputated limb), later forming bands of Bungner that are essential for the regenerating axons to migrate towards the scaffold (Fu and Gordon Reference Fu and Gordon1997).
To advance this field, future research should focus on optimising the perfusate composition to enhance nerve preservation. Additionally, exploring the integration of real-time monitoring systems coupled with artificial intelligence and machine learning algorithms could provide deeper insights into tissue responses during NMP. This should be cost-effective and a simplified system that can be readily adopted for prosthetic development for not only interfacing but other aspects of research such as aiding signal transduction research. Interdisciplinary collaboration is essential to fully utilise the capabilities of NMP as a promising technology. This systematic review reports that studies have investigated the electrical and muscle activity present in NMP limb systems, but not utilised this technology to evaluate bionic technology. It may offer a new preclinical tool for those developing bioelectronic devices and ultimately accelerate the routes to impact for bioelectronics in medicine and healthcare.
Data availability statement
Data sharing not applicable, no new data generated.
Author contribution
KA, AC, JF conceptualised the review.
KA and EA were responsible for Data curation, formal analysis after agreeing a methodology with AC and JF.
All authors were responsible for writing the original draft as well as agreeing to the final publication.
Financial support
This research received no specific grant from any funding agency, commercial or not-for-profit sectors.
There are no financial declarations to report for this manuscript.
Competing interests
There are no conflicts of interest.
Open access
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