*1.2. Search Strategy*

The search strategy aimed to only find articles published in English or translated into English. There was no restriction on the date of publication. Articles were searched for between June–July 2020. Keywords used to search all databases and references sources were animal grimace score, animal grimace scale, animal pain assessment, animal pain indicators, animal pain face, animal pain scales and the NC3Rs website. All papers were retrieved and downloaded into Endnote with X8.0.1 with any duplicates removed. What is pain and why does it matter?

The International Association for the Study of Pain defines pain as: 'An unpleasant sensory and emotional experience associated with, or resembling that associated with, actual or potential tissue damage' [2]. Pain can be further categorised as acute, visceral or chronic. Acute pain serves an evolutionary and adaptive function to signal and avoid potential or actual damage to tissues. This type of pain may result from an injury or surgical wound [3–5]. Visceral pain is due to the activation of stretch or pressure receptors in visceral organs. Unalleviated or poorly treated acute pain can progress to chronic pain. The latter is the result of neuroplastic changes occurring within the nervous system, rendering the body more sensitive to pain and can even create sensations of pain without any external stimuli [3–7]. It is important to be able to manage and assess all types of pain in research mammals and avoid the inadvertent development of chronic pain. While the types of pain may manifest differently, research staff must be able to assess and alleviate pain to maintain optimal animal wellbeing (mental and physical).

There are moral, legal and ethical obligations that require those working with animals to manage pain [5,7–13]. The recognition, assessment and treatment of pain is an essential aspect in public support and acceptability in the use of animals for research [7,12,13]. Using the precautionary principle, animal ethics committees and research staff must acknowledge the potential for pain [5,10,12,14]. They must also consider the experimental and animal welfare consequences of pain and take steps to ensure pain is adequately managed during procedures [3–5,15]. Regulatory frameworks often apply, as a precaution, the anthropomorphic principle, by which any procedure causing, or expected to cause, pain in humans, may produce pain in animals. The European Union, United Kingdom and Australian regulations operate on this principle and require the alleviation of pain for research animals. Exceptions to the alleviation of pain may be granted for studies that measure pain and/or distress. However, even in these exceptional cases, there is always a maximum threshold level of pain before intervention is required [5,10,12,14–17]. Although management of pain and associated humane interventions will vary due to the nature of the experimental outcomes, researchers are required to intervene with predetermined criteria to alleviate pain or, if necessary, humanely euthanise animals. For practical purposes, animal users can only fulfil this obligation for humane intervention if they are able to identify pain rapidly, consistently and accurately in their target species.

Unalleviated pain results in alterations in animal behaviour, physiology, and physical states [18–20]. These changes can be identified in various ways through behavioural observation, biochemistry, haematology, endocrinology and physical alterations in locomotion or posture [4,21]. In addition to the suffering of the animal, these changes may impact experimental outcomes and become a confounder by increasing experimental variability and producing negative affective states. Conversely, positive emotional states of animals are linked to less experimental variability and more robust experimental results [22,23]. The full spectrum of potential confounders to unmitigated pain is not entirely understood; however, the literature supports that there are experimental and animal welfare benefits in identifying and subsequently treating pain and alleviating negative effects on animals used for research [3,17,19,21–24].

### *1.3. Pain Faces*

Humans are known to display a series of facial expressions linked to the experience of pain [25,26]. Those so-called 'pain faces' are used in human medicine to detect, as well as assess, pain in non-verbal humans (i.e., infants) [7,25,26]. These pain faces can be used to develop grimace scales and capitalise on the human propensity to focus on the facial area [27,28]. The conservation of these pain faces is also present in many non-human mammalian species [26,29–34] and are a naturally useful method in the identification and assessment of pain. However, as with any technique, Grimace scales have benefits and limitations. These are important to acknowledge and take into considerations before their use.

### *1.4. Pain Assessment Requirements*

There are a series of important considerations when determining if a method is an appropriate test in identifying and/or assessing pain. The testing method must reliably produce the same result independent of the observer and the number of times an animal is observed. These are, respectively, known as intraobserver and interobserver agreement. It should also be consistent between testing timepoints and observers [7,35,36]. An ideal method should be easy to train and not require specialist knowledge or equipment [7,33,36–38].

A suitable test must demonstrate validity by accurately determining or reflecting the presence or absence of pain [7,35,36,39]. To determine the validity of a pain assessment technique, we should test the animals before the painful stimulus, after the introduction of the painful stimulus and once pain relief has been provided. The test should demonstrate an absence of pain before the painful stimulus, an increase in pain at the introduction of the painful stimulus, and a subsequent reduction in observed pain on the delivery of an appropriate analgesic [7,36]. Ideally, the test should be able to demonstrate a dose–responsive curve to pain based on the administration of appropriate analgesia [40–44].

The specificity and sensitivity of a test are also crucial to ensure animals are correctly identified when pain or welfare concerns arise. If the specificity is too low, there is a risk of pain being incorrectly identified, potentially leading to unnecessary interventions such as pain relief or humane euthanasia [3,7,15,36]. Alternatively, if the sensitivity is too low, experimental animals may reach their threshold for intervention while being inaccurately identified as not painful, therefore remaining in pain possibly even beyond their humane endpoint. An appropriate method would demonstrate both high sensitivity and specificity to ensure correct assessment and correct management of arising pain or welfare issues [3,7,15,36].

Cage or pen-side pain identification techniques should rely on spontaneous rather than retrospective indicators of pain. It ensures humane intervention can be applied promptly with animals not left in distress for any extended length of time [9,45,46]. The assessment of pain should preferably be a non-invasive method, to avoid the risk of eliciting a pseudoanalgesic stress response, inhibiting the ability of the observer to detect pain accurately [47–50]. Techniques such as assessing the quality of nest-building in mice [3,15,18,51–53] or degree of burrowing in rodents are non-invasive, observatory, proxy measures to wellbeing and potentially pain [3,15,18,54,55].

### *1.5. Confounders to Pain Identification*

Some caveats must be maintained when selecting a pain assessment technique. Many pain assessment indicators may be ambiguous. The choice of a pain identification tool or methodology must be specific to the species and validated for the procedures or experimental work being performed [56–60]. It well accepted that not all animals demonstrate the same signs of pain, even for a similar nociceptive stimulus [36,61].  The types of procedures or experiment performed should not obscure the ability of the technique to detect pain [39,56–58,67]. Pain identification should be consistent across the species regardless of sex, strain or breed; however, differences in pain thresholds between sexes or strains may exist [67,68]. Additionally, some natural behaviours (i.e., flehmen response, aggression) [3,4,32,69–72] or physiological indicators (i.e., cortisol, heart rate) [3–5,7,15,17,21,66] may be equivocal and require differentiation. Whenever possible, the choice of technique should accurately identify an animal in pain, independent of the procedure or behaviour performed, species, affective or physiological state, sex, strain or breed.

### *1.6. Non-Grimace Scale Pain Assessment*

The individual expression, magnitude and experience of pain can vary between animals [67,68]. There are known difficulties in measuring the magnitude of a particular animal's pain or distress which can make the absolute measurement or degree of pain challenging to assess [4,21,62,68,73–75].

Before the development and use of grimace scales, a variety of indicators have been used in an attempt to identify and assess pain. These can be grouped into behavioural, physiological and physical indicators (Table 1). Typically, behavioural indicators have the benefit of being non-invasive, observational, requiring limited equipment and offering an opportunity to capture signs of pain in species or individuals that may hide signs of pain (i.e., prey) [3,21,50–55,66]. Many behavioural assessment techniques take time (>5 min), require extensive training, are more retrospective than spontaneous, and may be non-specific proxy indicators to pain [4,18,76–78]. Physiological indicators (neuroendocrine or sympathetic nervous system) are often non-specific markers related to stress or distress [3,5,21,79,80]. They do have the benefit of being relatively quantifiable but often require specialised equipment, are retrospective, usually require animal handling or restraint with the potential for a confounding pseudoanalgesic effect [47–49] or are a non-specific stress response [3,4,8,81]. Physical indicators such as changes in posture, locomotion and production yields, have been correlated with the presence of pain in animals [3,8,15,17,21,64,66,82–85]. However, physical indicators are just as often non-specific indicators of non-painful animal wellbeing or environmental factors [3,5,8,15,17,21,64,66,82,84].

Ideally, a pain assessment technique should ensure accurate pain identification and minimal opportunity for the confounding of experimental outcomes due to experimental procedures, sex, breed/strain, or species. At present, there is not a single non-invasive, low-cost behavioural, physical or physiological pain assessment technique that is spontaneous, pain-specific, easy to train and quick to use (Table 1) [5,15,16,21,38,46,53,78,86–89]. With the exception of some behavioural ethograms [63,78,90,91], other methods are unable to give a reliable dose-dependent response to pain. While many pain identification methods have their use and benefits, their use in the cage or pen-side management of animal pain and/or in the timely and appropriate application of humane intervention is limited.

Thus, a myriad of techniques has been developed in an attempt to assess and capture the various expressions of pain in animals. These tools usually revolve around three dimensions: behavioural, physiological, and physical [5,73]. Table 1 categorises and reviews some commonly used assessments [3,4,7,8,15,21,80] in terms of their dimension, ability to be timely (spontaneous), non-invasiveness, spontaneous, easiness to train, and low-cost with minimal or no equipment requirements.

**Table 1.** Behavioural, physiological, and physical pain assessment techniques.

\* Ultrasonic vocalization monitoring requires special equipment; \*\* e.g., Egg or Milk; \*\*\* Eggs can easily be counted; \*\*\*\* Obvious signs of lameness are easy to train but more subtle lameness may be more difficult.

### *Animals* **2020**, *10*, 1726

### *1.7. Grimace Scales in Animals*

Grimace scales are proving to be a useful methodology for the identification of pain in research that meets most of the prerequisites for identifying and assessing pain in research animals. A range of research species-specific grimace scores has been developed (Table 2) and used in a wide range of experimental studies and research settings (Table 3). The initial methodology in the mapping of pain and the development of a facial action coding system (FACS) was developed in humans [97,98]. A FACS is an anatomical classification system used to map facial movements and facial muscles areas involved in facial contraction and relaxation. Photographs and videos scored by blinded observers serve as the base of facial mapping for FACS. FACSs o ffers the ability to code and identify expressions of pain via the individual components of facial expressions known as facial action units (FAUs) [99]. FAUs consistent with the expression of pain can then be used to develop a pain face or 'grimace' [99]. Regions of the face that have been found to change during the expression of pain include the eye, nose, cheek, mouth, ear and whiskers [8,81,100]. The position or carriage of the head is also found to change in some species as well [33,64,68,85,101,102]. The FAUs related to the expression of a grimace face in mammalian animals used in research are included in Table 4. From this known 'grimace face', the severity of the pain experienced can be objectively scored from images and/or film of animals in a known naturally (i.e., lameness, mastitis) [37,82,85] or experimentally induced (i.e., plantar incision [41,44,103] state of pain.

Table 2 summarises many of the available studies that demonstrate a successful use of grimace scales in research animals. The table outlines which species-specific grimace scales have been validated, shown to be pain-specific, demonstrated a dose-dependent relationship, used in real-time and were easy to use. The di fferent pain states to which they are applicable is also listed. In all but one species (guinea pigs) [63,78,90], observers were found to correctly, reliably and objectively identify pain in animals when using facial expressions or facial action units.

**Table 2.** Summary of species-specific available grimace scores.

**Table 3.** Grimaces Scales Facial Action Units by Species.

**Table 4.** Grimace Scales by Experimental Study or Pain Type.

Control animals (negative or positive) were also included throughout this process and a simple species-specific grimace scale was developed [25,33,41,107]. The scoring system most commonly used in grimace scales is a three-point scale to determine if a specific FAU is not (score = 0), moderately (score = 1), or obviously present (score = 2) [41,45,100]. The scale must then demonstrate a

dose-dependent change in pain scores on the delivery of analgesia [7]. Further research is typically performed to ensure the applicability of the grimace scale across multiple pain scenarios or environments, sex, strain/breed, age, as well as type and length of painful stimuli [7]. The scoring system can be used three ways. Firstly, it can determine either the absence or presence of pain. Secondly, it can offer some distinction between the intensity of pain via the summation of total scores. A change by two or more points is considered to be a legitimate alteration in pain intensity [133]. Thirdly, a threshold score can be set to offer guidance to research staff as to when to intervene to provide pain alleviation or humane euthanasia of research animals. The process of developing a grimace scale is time intensive but once developed and validated is relatively easy to train research staff to use [7,38,70,85,101].

### **2. Advantages and Uses**

Grimace scales have been applied across numerous research models, species and environmental contexts [41,128] (Table 4). They are a technique that can also be used to detect pain in existing pain research models as well as analgesic drug studies [40–42,45,60,77,109,110,128–130]. Grimace scales offer the ability to detect and assess the severity of pain, determine the potential benefit of any analgesic intervention and assist in identifying humane interventions. The technique is of practical value as it can be used at the cage or pen-side level as a spontaneous indicator of pain [39,41,55,75,92]. As a methodology, it has the added benefit of being easy to teach to a range of observers including research staff, clinical veterinarians, animal scientists and undergraduate and graduate students [38,41,55,75,129]. Overall, the grimace scale methodology appears to be acceptably conserved and validated across a number of mammalian species and range of experiments. It is likely this technique has the capacity to be applied across an even greater range of mammalian species and experimental settings (Tables 3 and 4). However, a careful systematic assessment will always be required to ensure applicability, accuracy and validity.

Grimace scale facial expressions are proving to be a useful [81] complement to existing tools in the assessment of animal wellbeing. The scores generated from the grimace scale should be used in conjunction with the context in which the animal is scored, its history, the procedure performed and the general parameters for wellbeing and signalment (sex, strain, species). When used appropriately, it is an excellent method to identify pain and as an adjunct to maintaining animal wellbeing in research studies [3,64,70,82,85,87]. Using this technique has the potential to improve pain detection in research animals and enable observers (i.e., research staff) a better opportunity to provide analgesia, humane euthanasia or identify animals requiring reassessment. The use of these grimace scales can be a vital tool to enable mitigation of the experience of pain in animals and refine animal welfare outcomes [41,60,66,75,76,82,100,114,128]. Unlike other types of pain assessment, grimace scales are spontaneous and usable in real-time [7,45,55,76,87,91,92,101]. They can also be matched and corroborated against other known indicators of pain or painful diseases including, but not limited to, lameness [37,64,82,85], cortisol [70], behavioural ethograms [81,85,91,92], acute laminitis [37], mastitis and foot rot [82]. A future area for development and benefit is the use of software automation in the development and scoring of facial expressions. The use of scoring software along with the installation of video cameras into enclosures may be able to enhance and hasten the development of grimaces, offer highly accurate grimace scores for animals in pain but also allow the remote monitoring and scoring of affected animals [41,59,134,135].

Another benefit of the system is simplicity, as it enables staff to distinguish a painful face from a non-painful one. Using a three-point scale is thought to be very useful in the reduction in subjectivity and offers observers greater clarity, confidence and support as to when to administer pain relief or humane intervention [7,75,81]. Reduction in grimace scores has been shown to occur on the application of pain relief [33,35,41,45,82,85,100,102] in a dose-dependent manner [40,41,128]. Therefore, grimace scales have the potential to assess both the presence and severity of pain. The use of grimace scales can alert research staff to animal discomfort, which may require additional monitoring, assessment or analgesia.

Grimace scales are a non-invasive method in the detection of pain [7,81,100]. Many of the animals utilised in research are known 'prey' species with a high degree of stoicism and evolutionary adaptation to minimise expressions of pain or poor welfare states [50,63–65,74]. Consequently, an ideal pain identification and assessment should be non-invasive and should reduce the possibility for these prey animals to minimise their expression of pain or for the potential of stress-induced analgesia [47–50].

Both experienced and inexperienced observers can identify pain with a significant intraobserver and interobserver agreement [41,57,60,66,76,82,100,114]. A potential benefit of using grimace scales to identify and assess pain in animals is that extensive animal experience is not required. Observers varied in their background experience to research and animal work and their training in pain assessment techniques. The observers ranged from students (undergraduate and postgraduate), veterinarians, animal care professionals, and early to late-career researchers [33,38,70,75,101,106,114,116]. Another favourable outcome when using grimace scales is that a natural empathy or innate understanding of animal behaviour is not necessary nor is a belief in the ability of an animal to experience pain. Through the use of a grimace scale, pain identification and assessment can be more objective (for or against the presence of pain). It also requires research sta ff to formally record a score and monitor animals for signs of pain, which can o ffer a more precise framework to determine when humane intervention or pain relief is needed [36].

The apparent usefulness of grimace scales could be related to several factors. One of which is that it capitalises on the innate human tendency to focus on the facial area when observing an animal [28]. Interestingly, many FAUs (orbital tightening, ear position and cheek area) appear to be conserved across mammalian species [33,41,45,82,100] (Table 3) and may be tapping into an evolutionary conservation repertoire of known FAUs. It may help explain how even the single identification of a few potentially evolutionarily conserved FAUs can still be useful in detecting pain [35]. It is supported via statistical modelling which has identified the FAUs most strongly correlated with a pain face, thereby o ffering the potential to isolate which FAUs are critical for use in a grimace scale (i.e., statistically significant) and which ones may detract from the scale (i.e., equines have four and mice have two critical FAUs) [35]. It may explain why grimace scales are one of the few techniques proven to be robust across several di fferent mammalian species when compared to other pain assessment techniques [34]. However, by using only the minimum number required of FAUs to score pain, the ability to determine appropriate intervention thresholds and assess pain intensity may be reduced.

The use of FACS and subsequent combinations of FAUs appears to be an excellent method to identify changes in facial features, which are consistent with the experience of pain in animals [33,40,41,76,82,85,92,101,104,106,114,130]. The grimace scale method seems to meet many of the requirements for an ideal pain identification technique. It is known to be a reliable and validated method of assessing pain in many of the commonly used research animals [33,35,40,41,45,70,82,85,100,106,114].


*1.3.2 Secretory epithelium*

There are two types of glands—endocrine and exocrine.

There are irregular tight connective tissue sheath around the macroscopic glands which are parotid, submandibular, and sublingual gland. The connective tissue enters into the glands in the form of septa. The septa divide the gland into compartments. In the compartments, there may be acinus or tubules or both according to the type of the gland. Intralobulated duct channels are also included within the lobule. In the septa of connective tissue, interlobular channels are located. From these visible glands, the serous glands consist of acinus (alveolus), and the mucous glands are mucous tubules (**Figure 11a**–**d**). Exocrine glands can be subdivided into serous, mucous, and mixed glands according to the nature of the secretion they produce. Both serous- and mucous-secreting endpieces are covered with cubic epithelial cells which form intercalated (initial) ducts. Intercalated ducts combine to form the striated (intralobular) ducts. The intralobular ducts merge together to form the interlobular duct. These ducts are joined together and form "Stenson" in the parotid gland, "Wharton" in the submandibular gland, and "major sublingual" channels in the sublingual gland and open into the oral cavity. The most important features of intralobular ducts are the membrane folds extending from the cell basal to the nuclei. A large number of mitochondria were placed on the long axis of these folds. Such a location is particularly important for the salivary gland, which is functionally intense and consumes relatively more energy during its work. The parotid and the exocrine glands part of the pancreas are pure serous. The submandibular and sublingual glands are mixed glands [4–9].

**21**

*Morphology of Salivary and Lacrimal Glands DOI: http://dx.doi.org/10.5772/intechopen.84380*

apical portion is stained with eosin [4–8, 10] (**Figure 11c**).

The abundant zymogen granules in the serous glands are found in the upper part of the cell cytoplasm. Serous glands produce a smooth flow and protein secretion. The duct channels are narrower than the mucous glands. The nuclei of the cells are located in the middle and are round in shape. In the sections stained with hematoxylin–eosin, the basal part of the cell is stained with hematoxylin, and the

Mucous glands secrete carbohydrate properties. Since these granules are lost when preparing hematoxylin–eosin stained sections, these parts of the cells are often seen as empty. Therefore, these glands secrete in dense consistency and are lightly colored, and the discharge ducts are wider than the serous glands (**Figure 11d**) [4–9]. The epithelial cells of the gland are divided into three types according to the type

1.Merocrine secretion: The secretory product is transported to the apical surface of the cell by vesicles. The vesicles are combined with the cell membrane and give their contents to the external environment by exocytosis. As in the pancreas, the most common form of secretion in the body is the merocrine type.

2.Apocrine secretion: While the secretory product is separated from the apical part of the cell membrane, it takes some cell membrane with it. This secretion occurs in the mammary glands, the apocrine of the skin, the ciliary (Moll's) of

3.Holocrine secretion: Secretion product accumulates in the cell that continues to mature; then the secretory product is excreted into the outer environment together with the cell in which it is contained. The ovaries, the fat in the skin, and the Meibomian glands in the eyelid are the examples [4–8].

a.The parotid gland is surrounded by irregular tight connective tissue called a capsule. The parts separated from the capsule move into the gland and are called septa. The septa separate the gland into lobules (**Figure 12a**). Each lobule is composed of spherical acini, which empty their serous secretion into intercalated ducts, from where it flows into striated (intralobular) ducts. We will see these ducts in the lobule. The intralobular ducts combine to form interlobular ducts. The interlobular ducts are located in the septa. Interlobular ducts leave the lobule and together with muscular arteries, veins, nerve, and lymphatic vessels. The interlobular duct channels are also known as excretory duct. Excretory ducts are emerged to form the main duct, "the Stenson." Round-shaped acinus is a part of the serous gland. The acinus (acinar cell) is formed by laying a single-layer cubic or prismatic epithelium on the basal lamina (**Figure 12b**). Fat cells which are one of the connective tissue cells are located between acini. Myoepithelial cells are located between epithelial cells and basal lamina. Contraction of myoepithelial cells accelerates the flow of saliva. Acinar secretion with serous content and secretion passes to intercalated ducts. Several intercalated ducts form an intralobular (striated) duct.

the eyelid, and the seromusin glands of the outer ear canal.

**2. Structure of major salivary glands**

*1.3.3 Serous glands*

*1.3.4 Mucous glands*

of secretion:


**Small Diameter Cell-Free Tissue-Engineered Vascular Grafts: Biomaterials and Manufacture Techniques to Reach Suitable Mechanical Properties**

**María A. Rodríguez-Soto 1,\* , Camilo A. Polanía-Sandoval 2,3, Andrés M. Aragón-Rivera 1 , Daniel Buitrago 1 , María Ayala-Velásquez 1 , Alejandro Velandia-Sánchez 2,3 , Gabriela Peralta Peluffo 1 , Juan C. Cruz 1 , Carolina Muñoz Camargo 1 , Jaime Camacho-Mackenzie 2 , Juan Guillermo Barrera-Carvajal 2 and Juan Carlos Briceño 1,4,\***

	- 3 School of Medicine and Health Sciences, Universidad del Rosario, Bogotá 111711, Colombia
	- 4 Department of Research, Fundación Cardio Infantil Instituto de Cardiología, Bogotá 111711, Colombia
	- **\*** Correspondence<EMAIL_ADDRESS>(M.A.R.-S<EMAIL_ADDRESS>(J.C.B.); Tel.: +57-(1)-3394949 (ext. 1789) (M.A.R.-S.)

**Citation:** Rodríguez-Soto, M.A.; Polanía-Sandoval, C.A.; Aragón-Rivera, A.M.; Buitrago, D.; Ayala-Velásquez, M.; Velandia-Sánchez, A.; Peralta Peluffo, G.; Cruz, J.C.; Muñoz Camargo, C.; Camacho-Mackenzie, J.; et al. Small Diameter Cell-Free Tissue-Engineered Vascular Grafts: Biomaterials and Manufacture Techniques to Reach Suitable Mechanical Properties. *Polymers* **2022**, *14*, 3440. https:// doi.org/10.3390/polym14173440

Academic Editors: Domenico Acierno and Antonella Patti

Received: 1 June 2022 Accepted: 6 July 2022 Published: 23 August 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**Abstract:** Vascular grafts (VGs) are medical devices intended to replace the function of a blood vessel. Available VGs in the market present low patency rates for small diameter applications setting the VG failure. This event arises from the inadequate response of the cells interacting with the biomaterial in the context of operative conditions generating chronic inflammation and a lack of regenerative signals where stenosis or aneurysms can occur. Tissue Engineered Vascular grafts (TEVGs) aim to induce the regeneration of the native vessel to overcome these limitations. Besides the biochemical stimuli, the biomaterial and the particular micro and macrostructure of the graft will determine the specific behavior under pulsatile pressure. The TEVG must support blood flow withstanding the exerted pressure, allowing the proper compliance required for the biomechanical stimulation needed for regeneration. Although the international standards outline the specific requirements to evaluate vascular grafts, the challenge remains in choosing the proper biomaterial and manufacturing TEVGs with good quality features to perform satisfactorily. In this review, we aim to recognize the best strategies to reach suitable mechanical properties in cell-free TEVGs according to the reported success of different approaches in clinical trials and pre-clinical trials.

**Keywords:** tissue engineered vascular grafts; biomaterials; biodegradable; biomechanical stimulation; mechanical properties


**Roxana-Elena Tudoroiu 1, Mohammed Zaheeruddin 2, Nicolae Tudoroiu 3,\* and Sorin-Mihai Radu 4**

Received: 4 August 2020; Accepted: 13 August 2020; Published: 14 August 2020

**Abstract:** The purpose of this paper is to analyze the accuracy of three state of charge (SOC) estimators of a rechargeable Li-ion SAFT battery based on two accurate Li-ion battery models, namely a linear RC equivalent electrical circuit (ECM) and a nonlinear Simscape generic model, developed in Part 1. The battery SOC of both Li-ion battery models is estimated using a linearized adaptive extended Kalman filter (AEKF), a nonlinear adaptive unscented Kalman filter (AUKF) and a nonlinear and non-Gaussian particle filter estimator (PFE). The result of MATLAB simulations shows the efficiency of all three SOC estimators, especially AEKF, followed in order of decreasing performance by AUKF and PFE. Besides, this result reveals a slight superiority of the SOC estimation accuracy when using the Simscape model for SOC estimator design. Overall, the performance of all three SOC estimators in terms of accuracy, convergence of response speed and robustness is excellent and is comparable to state of the art SOC estimation methods.

**Keywords:** SAFT lithium-ion battery; Simscape model; 3RC ECM Li-ion battery model; state of charge; adaptive EKF SOC estimator; adaptive UKF SOC estimator; particle filter SOC estimator


**3.1 Kinetic study of the Cd2+ adsorption**

To determine the time required to reach the Cd2+ adsorption equilibrium, we monitored the change in Cd2+ concentration as a function of time. The results are shown in **Figure 3**.

This study shows that equilibrium is established rapidly for all adsorbents (**Figure 3**). It reaches its maximum for 30 and 20 minutes, respectively for Ccra and Ccre, while the adsorption kinetics of Cd2+ on Clan is relatively slow.

From these curves, it is also found that 50% of Cd2+ is adsorbed on the Ccre before the equilibrium reached 20 minutes. The equilibrium times (teq) are

**Figure 3.** *Kinetic study of Cd (II) adsorption on raw chitin.*

classified in the following order: teq Ccre < teq Ccra <teq Clan. This kinetics is very fast in comparison with other works cited in the literature concerning the adsorption of Cd2+ on mineral surfaces, which require a very large equilibrium time. For example, for the adsorption of Cd2+ on the illite, the equilibrium is reached after 54 days [1] and in the case of a natural clay [55, 56], the equilibrium is reached after 50 days. Melchor [39] having studied the adsorption of Cd2+ on chitin, mentions a time of equilibrium (40 minutes) comparable to our result.


*2.3. LTS with Synchronous Motors*

Various levitated LTSs using conventional synchronous motors based propulsion were described in [19], e.g., they are permanent-magnet-excited machines. A high-temperature superconducting linear synchronous motor was described in [20]. The application of superconducting linear flux-switching permanent magnet motors was discussed in [21].

#### *2.4. LTS with Induction Motors*

Linear induction machines were discussed as the most promising solution for LTSs in [1,15,17,18]. In the late 1940s, the British electrical engineer Eric Laithwaite, Professor at Imperial College London, developed the first full-size working prototype of the linear induction motor. The first commercial Maglev transport system in the world, the Birmingham Maglev, which opened in 1984, was also propelled with a linear induction motor. The latest problems related to the application of LIMs are discussed in Section 3.


*2.7. Sensory Evaluation*

Eight trained panelists (according to ISO standards) belonging to the staff of the Laboratory of Food Chemistry and Technology were recruited for assessing the plain and cherry-flavored yoghurts on days 1, 15 and 28 [21,22].

Two sessions per day (one in the morning and one in the afternoon) evaluating five formulations per session were conducted in the accredited according to ISO 17.025 Sensory Laboratory of NTUA (Athens, Greece) that has a standardized room (according to ISO standards) equipped with separate booths [23]. Samples were presented in plastic coded (three-digit random codes) cups, containing 50 mL of freshly removed from the refrigerator yoghurt samples. The panelists were asked to evaluate the samples using a 9-point intensity scale (1, lowest intensity; 9, highest intensity) based on pre-selected appearance (wheying-off, white color, yellowish color, cherry fruit color), tactile (ropy, uniform coagulum), orotactile (viscous, curdy, grainy), gustatory (sweet, bitter, sour, metallic) and olfactory (dairy flavor, sourmilk flavor, cherry flavor, rancid) sensory modalities. In addition, the panelists

were asked to rate the yoghurt samples using a 9-point hedonic scale (1: I do not like at all, 9: I like extremely). During the sensory evaluation sessions, the panelists were instructed to cleanse their palate with low sodium spring water (Zagori, Greece) and consume a small piece of unsalted bread. The data were collected in specifically designed ballots and the panelists were encouraged to write down any criticisms on the tested products.


Spinal Anesthesia in Pediatrics

*Enrique Hernández-Cortez, Yolanda M. Martínez-Barragán and Karen L. Iñiguez-Lopéz*

## **Abstract**

The survival of preterm newborn patients (PNB) depends in a great extent on the anesthetic technique used. Spinal anesthesia (SA) is considered the best-tolerated regional anesthetic method for highly unstable newborn infants (NB) with high risk of complications during the perioperative period. SA has been recommended for children at high risk for postoperative apnea due to general anesthetics or prematurity. Bronchopulmonary dysplasia (BPD) in the newborn is a disease that accompanies the patient to the operating room with a high incidence of mortality. SA in emergency surgery is a well-tolerated anesthetic procedure with proven effectiveness, with less hemodynamic and respiratory repercussions. At the same time, it produces greater protection against surgical stress in the NB weakened by the premature condition. Hemodynamic stability remains constant even in the newborn with heart disease.

**Keywords:** spinal anesthesia, regional anesthesia, newborn

### **1. Introduction**

The 8% of PNB require surgery to correct an organic birth defect in the first hours or days after birth. BPD in PNB is a serious disease with high mortality rates, which produces significant respiratory fragility with repercussions on the general condition of the patient. General anesthesia (GA) produces cardiovascular and hemodynamic changes that prolong intubation for hours or days after surgery, which complicates the underlying respiratory problem. Inhaled anesthetics, hypnotics, intravenous agents, and muscle relaxants can prolong and worsen the awakening of the NB.

 The fundamental premise for the administration of anesthetics is their reversibility, which implies that the brain, spinal cord and peripheral nerves are anatomically and physiologically the same before and after the administration of any anesthetic agent. Therefore, the pediatric anesthesiologist faces the dilemma of which anesthetic agents cause less neurocognitive damage. An alternative to the problem of neurocognitive damage is spinal anesthesia, because local anesthetics (LA) produce a neuroprotective effect on the central nervous system, with minimal physiological changes [1].

The most frequent surgical procedures in the NB are inguinal hernia repair, duodenal atresia, pyloromyotomy, myelomeningocele repair, and imperforate anus repair. In most of these urgent surgical procedures, SA helps us to solve the surgical procedure with proven efficacy and safety. Hemodynamic stability remains constant even in PNB with heart disease, in non-cardiac surgery [2].


**1. Introduction**

In the last few decades, wind storms have caused the most damage and economic losses in European forests, compared to all abiotic and biotic damage agents [1–4]. So far, winter storms have caused the most destructive damage in Western and Central Europe [3,5,6], e.g., storms like Vivian in 1990 (over 100 million m3 of timber), Lothar and Martin in 1999 (over 175 million m3), Kyrill in 2007 (54 million m3) and Klaus in 2009 (50 million m3), respectively. Damages have increased in recent years also in northern Europe [4,6,7], where in 2015 Gudrun damaged 70 million m3 and in 2007 Per damaged 12 million m3 of timber, mainly in Sweden. In Finland, over 25 million m3 of timber has been damaged during storms since 2000, the most in autumn storms in 2001 (Pyry and Janika, 7.3 million m3) and in summer storm in 2010 (Asta, Veera, Lahja and Sylvi, 8 million m3), respectively. The increasing amount of damages in European forests may at least partially be explained by increasing volume of growing stock and changes in forest structure (e.g., age, tree species) related to changes in forest management practices [1,5,8,9]. Forest disturbances may also amplify or even cancel out the expected increase in productivity of forests under changing climate [4,10].

Some recent studies indicate increased storminess for some regions in Europe (see e.g., review by [11]). However, the majority of studies point towards decadal variation in storminess without any clear trend for a direction or another [12–15]. In Finland, slight weakening of annual mean (−0.09 ms−1 decade−1) and maximum (−0.32 ms−1 decade−1) wind speeds across 33 weather stations have been observed in the period of 1959–2015 [16], which is in accordance with widespread weakening of terrestrial near-surface wind speeds [17,18]. For future projections, the change in the extreme wind speed during the coming decades is still a somewhat unsolved issue and the outcome is largely dependent on the climate model used for the simulation [11,19,20].

However, the risk of wind damage to forests may still increase in Northern Europe under climate change even if the frequency and severity of wind storms do not increase. This is due to the shortening of the frozen soil period, which improves tree anchorage during the windiest season of the year from late autumn to early spring [21–24]. Moreover, storms may be accompanied by heavier rainfall, leading to more saturated soils and increased risk of wind damage [5]. When estimating the forest wind damage risk it is thus essential to know whether the extreme wind speeds occur during the frozen or unfrozen soil conditions. Typically, the windiest season in Finland is from October to March [25] and soil frost season starts in October–November and ends in April–May [26]. However, there is large year-to-year and regional variation in soil frost duration.

Even a 20 cm thick frozen soil increases the anchorage of trees and reduces substantially the risk of uprooting [27,28]. According to tree-pulling experiments in Finland, under frozen soil the type of failure was stem breakage, whereas under unfrozen soil conditions, about 80% of trees uprooted, respectively [28]. From the three economically and ecologically most important boreal tree species in Finland, Norway spruce (*Picea abies*) with the shallow rooting is the most vulnerable to uprooting, followed by Silver and Downy birches (*Betula pendula* and *Betula pubescens*), and Scots pine (*Pinus sylvestris*), respectively [27,28]. However, from late autumn to early spring, birches (without leaves) are not vulnerable to wind damage and therefore excluded from this study.

For snow free surfaces, the soil frost modelling can be done using cold season frost sum and soil characteristics alone [29]. The presence of snow and vegetation complicates the modelling, and requires a more sophisticated approach including the modelling of heat and water transfer [23]. An example of a relatively simple approach accounting for the main controlling factors was published by [30]. It was further developed and tested in the Finnish conditions by [31] for the calculation of soil temperatures in three common combinations of soil and forest types in Finland, i.e., dense Norway spruce stands on clay or silt soil, Scots pine stands on sandy soil, and Scots pine stands on drained peatlands. Soil frost conditions can vary a lot, even up to few months in mean duration, depending on soil type. Peat is effective insulator compared to mineral soils, therefore having shorter soil frost periods in similar climatic conditions [31].

The estimation of the return levels of maximum wind speed values (extreme winds) can be done using observational data representing conditions at the observing station location or using reanalyzed data like ERA-Interim [32], representing a larger area's averaged value, respectively. When studying the high-resolution spatial variation of extreme winds, the data has to be either downscaled from the reanalyzed coarse grid to a local value or upscaled from station point observations to areas located between the stations. Downscaling can be done by applying various spatial statistical tools, e.g., [33,34], or complex airflow models like e.g., WAsP [35], which are typically applied for wind power potential predictions. GIS-based methods for mapping the areas having highest wind damage risk have also been introduced, e.g., [36–38]. One computationally feasible approach for the estimation of the return levels of extreme wind speeds for large geographical areas with very high spatial resolution is the wind multiplier approach [39–41]. In this method, return levels obtained, e.g., from the reanalysed data, are downscaled to local wind speeds with help of land cover (roughness) and topography data. By applying GIS-tools such as ArcGIS, QGIS or R, it is rather straightforward to produce the required multipliers.

The reliable high-resolution information on the spatial variation of extreme wind speeds can enhance wind damage risk management in forest planning and forestry. In the above context, the objective of this study was to produce spatially detailed estimates (maps) of the 10-year return level maximum wind speed under current climate for unfrozen and frozen soil conditions in some of the most common combinations of forest and soil types in Finland. By utilizing soil frost calculations of [31] to determine the duration of soil frost seasons, the wind speed return level calculations were done for dense Norway spruce stands on clay or silt soil, Scots pine stands on sandy soil, and Scots pine stands on drained peatland. The coarse resolution estimates of the 10-year return level of maximum wind speed were based on 1979–2014 ERA-Interim dataset [32]. Downscaling to a 20 m grid was done by applying the wind multiplier approach [41].