**1. Introduction**

Many linear DNA viruses possess terminal repeats (TRs) known to be critical for viral genome stability and propagation [1]. A parallel can be drawn with human chromosome telomeres that are composed of GC-rich repeat sequences of 5–10 nucleotides. In cells, telomeres are critical to maintain the linear structure of the chromosomes. They can adopt specific secondary structures, such as G-quadruplexes (G4), providing structural characteristics for protein binding and genomic stability. In addition, Cellular telomeres play a role in transcription regulation, chromatin compaction, subcellular localization, and chromosome segregation.

Similarly, *Parvoviridae* TRs have been demonstrated to be essential to several steps of the virus cycle. They vary in shape and size from approximatively 100 to 550

nucleotides [2]. Due to the presence of palindromic repeats, TRs can fold into T-, I-, J-, Y-, U- shape or simple hairpin-like structures. Up to now, the global shape has been named without any consensus, for example, Y-shape also being called "rabbit ears."

Viral genomes in some genera (*Ambi*- and *Itera-densovirus*; *Ave*-, *Dependo*-and *Erythro-parvovirus*) are homotelomeric meaning that both termini are similar but inverted, whereas, in other genera (*Brevi*- and *Hepan*-*densovirus*; *Amdo*-, *Boca*- and *Proto*-*parvovirus*), the 50 and 30 ends of the linear genome differ and therefore are called heterotelomeric. The strand polarity packed in viral capsids may be related to the left and right TRs dissimilarity. Indeed, most of the heterotelomeric parvoviruses encapsidate only one strand polarity, mainly negative. This preference may be due to inefficient nicking during replication or incomplete packaging signal at one TR [2]; for example, the minute virus of mice (MVM), a virus of the subfamily *Parvovirinae* and genus *Protoparvovirus*, harbors a Y-shape left end and a longer U-shape structure on its right end. After replication and ori resolution, the single-stranded DNA of minus polarity is preferentially displaced from the left TR and encapsidated. For parvovirus with both polarities, the proportion can range from 1 to 50% and may be influenced by the host cell in which the virus is produced [3].

Parvoviral TRs are involved in many steps of the virus life cycle. They contain most of the *cis*-acting information required for genome replication and encapsidation, including tetranucleotide repeats that serve as binding sites for NS1 (Rep) oligomer, a resolution site necessary for the completion of the DNA strand copy, and a packaging signal. Recognized as DNA double-strand breaks (DSB) in the host cell, TR can trigger a DNA damage response (DDR), leading to the circularization and concatemerization of the viral genomes either by non-homologous end-joining (NHEJ) or homologous recombination (HR) [4]. Finally, transcription regulation elements are contained in the genome ends. For example, the MVM TRs contain both symmetric and asymmetric binding sites for transcription factors that modulate expression from the adjacent P4 promoter [5] and the Acheta Domestica Densovirus TRs contain a TATA box used for transcription initiation of NS gene on one side and VP on the other side [6].

The TRs secondary structures, motifs composition, and their role in the virus-cell cycle have been under-examined. In this study, DNA secondary structures of the *Parvoviridae* TRs, including non-canonical secondary structures, have been predicted. We have shown a high diversity of parvovirus telomeres characteristics even within a genus. This chapter may provide significant knowledge for *Parvoviridae* classification and interaction with host cells*.*


**8. Minor salivary glands**

Minor salivary glands are present within the wall of the oral cavity and oropharynx, and have short ducts [48]. They are named on the basis of their location:

The minor salivary glands are present in all three forms, that is, serous, mucous, and seromucous, which are the contributors of serous and mucous secretions through secretary ducts to saliva. Secretary units exist in a variety of forms (i.e., acinar, tubuloacinar, or tubular). Mucous tubules surrounded by the serous acini frequently develop serous demilunes; however striated ducts (the small ducts) are not its characteristics. The epithelium present in it is the simply squamous to low-cuboidal in shape. Later in the oral cavity, it is changed into stratified squamous form. Glands in the cheeks lie in the middle, dorsal, and ventral rows. The glands of the labial regions are assemblage as superior (nasolabial glands), commissural labial (under the skin along the mouth angle), and inferior labial glands which are connected to the commissural labial glands [15].

The composition of the submandibular gland varies in terms of saliva secretion among different species. The mandibular salivary gland is responsible for the production of a major portion of saliva. These are situated in the ventral and caudal part of the angle of the mandible and are irregularly triangular in shape. The gland has three angles, two surfaces, and three borders. The lateral surface is covered partly by the ventral part of the parotid gland. The medial surface is related to retrophyrangeal lymph node, the pharynx, larynx, and the lingual artery. The mandibular salivary gland is cream-colored or pale yellow, weighing from 5 g to 11 g. It is tubule-alveolar in composition. A thin connective tissue capsule covers the gland and the gland is derived into lobes and lobules by connective tissue septa emerging from the capsule. These connective tissue septa contain ducts, blood vessels, lymphatic, and nerves. The mixed alveoli (seromucous) are more abundantly present in the parenchyma than the other two forms. The duct system of the gland consists of intercalated, striated, and excretory duct forms [49].

Like other ruminants, goats are known to process saliva that acts mainly as a bicarbonate phosphate buffer, which aids in rumination and maintaining electrolyte and water balance, thus the saliva has a role in producing alkaline activity and evenness of the food contents within the sac of rumen and reticulum [50]. After suckling, the milk is overstepped by the reticulo-rumen through the esophageal groove into the true stomach (abomasums) in goat kids. The rumen remains very small in kids due to consuming liquid contents only until and unless the animal receives the fibrous diet. The juvenile animal relies on milk to neutralize the acidic environment in the stomach.


**23. Less common wrist related pathologies in a golfing population**

#### **23.1 Dupuytren's contracture**

Dupuytren contracture (**Figure 20b**) is a benign, myeloproliferative progressive disease of the palmar fascia which results in shortening, thickening, and fibrosis of the fascia and aponeurosis of the palm. It results in nodular formation on the palmar fascia which creates fibrosis resulting in one or more fingers become permanently bent in a flexed position. Dupuytrens contracture is caused by progressive

*Leading Wrist Injuries in a Golfing Population. Golf Swing Biomechanics a Significant Cause… DOI: http://dx.doi.org/10.5772/intechopen.96979*

#### **Figure 20.**

*Dupuytren's contracture caused by shortening, thickening, and fibrosis of the fascia and aponeurosis of the palm, results in nodular formation and a flexion deformity at the 4th and 5th digits, making a consistent grip of a golf club difficult.*

thickening and shorting of the palmar fascia. This occurs due to slowly progressing fibrosis in the fascia that results in a flexion deformity at slowly the metacarpophalangeal (MCP) and proximal interphalangeal (PIP) joints usually affecting the 4th and 5th digits. The disease begins in the palm as painless nodules that form along longitudinal lines of tension. The nodules form cords that produce contracture deformities within fascial bands and tissues of the hand.

The disorder has varying pattern of genetic predisposition across different regions and populations and is also known as the Viking disease, and Celtic hand, with 30% of the over 60-year-old male Norwegian population and 20% of a similar British population suffering from this condition [51].

It is expressed in an autosomal dominant fashion. This *condition* is *most* commonly seen in *populations* of *Northern European*/*Scandinavian descent* [52]**.** It is *relatively uncommon* in *Southern European* and *South American populations*. *Males* are *affected* by a *2:1 ratio compared* to *women*.

The condition is associated with diabetes, seizure disorders, smoking, alcoholism, HIV, and vascular disease Ectopic manifestations beyond the hand can be seen in Ledderhose disease of the plantar fascia Peyronie disease (Dartos fascia of the penis), and Garrod disease (dorsal knuckle pads) [51–53].

Numerous authors going back as far as the 17th century have noted the association between traumatic events and the appearance of Dupuytren's contracture. Initially by Plater in 1614, Goyrand in1835 and, Guillaume. Dupuytren, a French Surgeon in 1833 who the condition is named after [53–56].

Golf has never specifically been cited as a caused of the condition but is a common disability encountered in the older golfer population. The disability causes technical issues gripping and swinging a golf club due to its anatomical location at the base of the wrist and the role of the 4th and 5th digit in gripping a club. Fatigue and hand pain has been reported in elite golfers with this condition and an inability to grip the club consistently.

In a 2017 survey of 504 Dupuytren's sufferers, the Dupuytren's society reported a significant proportion described difficulty golfing due to the pathology. In this

observational study 8% of sufferers without a contracture reported a difficulty, 11% of single hand contracture and 23% of bilateral contracture suffers reported disfunction while golfing [57].

Up to one-fifth of patients seeking treatment for primary Dupuytren's contracture were reported to suffer from an injury-induced Dupuytren's contracture. It was noted that the injury to the wrist and hand seems to trigger the development of less progressive form Dupuytren's contracture in younger age group [58].

In diagnosing Dupuytren Contracture the clinician needs to distinguish the condition from other diseases of the hand including stenosing flexor tenosynovitis, ganglion cysts, ECU tendon subluxation, Guyon's Canal Syndrome and soft tissue masses. Diabetes, seizure disorders, smoking, alcoholism, HIV, and vascular disease should be considered during a careful history due their association with this condition.

Clinically the condition usually progresses at a slow rate over the course of several years and individuals may not be aware of the condition until it starts to cause functional disability. Pits and grooves in the palm of the hand are an early sign followed by the development of nodules in the medial palm. These nodules are often painless. Pain may be present distally at the knuckles pads of the proximal interphalangeal (PIP) as contracture evolve. The disorder is not always progressive and in at least 50–70% of patients, it may stabilise or even regress.

Investigations include radiology, which is usually normal and serology to out-rule metabolic or infective pathologies which are associated such as Diabetes Mellitus, Alcoholism and HIV infection, if there is a clinical suspicion.

Ultrasound [38] is the diagnostic tool with the highest yield as it confirms the presence of thickening of the palmar fascia and nodule formation.

Treatment includes physical therapy during the early stage of the disease. Some patients may also benefit from a brace to stretch the digits and maintaining range of motion of the fingers is necessary to prevent adhesions. This is particularly important in the golfing population. Corticosteroid injections may be beneficial and should be performed using Ultrasound guidance. Needle aponeurotomy is typically reserved for mild contractures. Collagenase injection which is a relatively new, minimally invasive treatment derived from Clostridium histolyticum has shown good initial results. The treatment is not available in all jurisdictions and should only be performed by a hand surgeon who can deal with any potential side effects of this treatment. Surgical fasciectomy is reserved for those cases who have failed conservative therapy and have a persisting disability.

A significant proportion of older golfers suffer from this disability that causes pain, discomfort and impairs the player's ability to consistently grip a golf club, and regularly interferes with the enjoyment of the game. Golf due to trauma may provoke the injury and once present exacerbates the condition. Early identification, finger stretching, as well as the use of topical anti-inflammatory medication assist in reducing symptoms in golfers with mild or non- progressive disease.

Golfers frequently continue to play with this condition. In these instances, the Dupuytren's sufferer should undergo a careful assessment of equipment. Golf shaft weight and grips should be reviewed by a PGA golf professional. In particular, correct or augmented golf grips can facilitate safe and enjoyable golf for the Celtic Hand golfer. Thickening grips can help mitigate overactive hands through the hitting zone thus reducing stress on the palmar aponeurosis. Golf grips come in 4 basic diameters and can be refined by a golf professional by the addition of wraps under the grip, further customising the all-important handle of the golf club. Larger grips also improve shock absorption and reduce transition of force to an already compromised palmar fascia. Small grips result in an increase in grip pressure and a propensity to grip the club in the palm. Holding the club too high across the palm increases the risk of hand injury or the exacerbation of an existing condition. The golfer

*Leading Wrist Injuries in a Golfing Population. Golf Swing Biomechanics a Significant Cause… DOI: http://dx.doi.org/10.5772/intechopen.96979*

should ensure his grip is biomechanically correct and the club is held in the fingers rather than the palm of the hand. This can be achieved by regripping the club in the last three fingers of the leading hand at address, prior to swinging. This helps to stabilises the club at impact and limits the stress on the palm of the hand. These small manageable changes will contribute to lessening the affect that this condition has on recreational and elite golfers.


*Article* **Reliability and Responsiveness of a Novel Device to Evaluate Tongue Force**

**Marta Carlota Diaz-Saez 1,2,3, Alfonso Gil-Martínez 1,2,4,\* , Inae Caroline Gadotti 5 , Gonzalo Navarro-Fernández 2 , Javier Gil-Castillo 6 and Hector Beltran-Alacreu 7**

**Abstract:** Background: Measurements of tongue force are important in clinical practice during both the diagnostic process and rehabilitation progress. It has been shown that patients with chronic temporomandibular disorders have less tongue strength than asymptomatic subjects. Currently, there are few devices to measure tongue force on the market, with different limitations. That is why a new device has been developed to overcome them. The objectives of the study were to determine the intraand inter-rater reliability and the responsiveness of a new low-cost device to evaluate tongue force in an asymptomatic population. Materials and Methods: Two examiners assessed the maximal tongue force in 26 asymptomatic subjects using a developed prototype of an Arduino device. Each examiner performed a total of eight measurements of tongue force in each subject. Each tongue direction was measured twice (elevation, depression, right lateralization, and left lateralization) in order to test the intrarater reliability. Results: The intrarater reliability using the new device was excellent for the measurements of the tongue force for up (ICC > 0.94), down (ICC > 0.93) and right (ICC > 0.92) movements, and good for the left movement (ICC > 0.82). The SEM and MDC values were below 0.98 and 2.30, respectively, for the intrarater reliability analysis. Regarding the inter-rater reliability, the ICC was excellent for measuring the tongue up movements (ICC = 0.94), and good for all the others (down ICC = 0.83; right ICC = 0.87; and left ICC = 0.81). The SEM and MDC values were below 1.29 and 3.01, respectively, for the inter-rater reliability. Conclusions: This study showed a good-to-excellent intra- and inter-reliability and good responsiveness in the new device to measure different directions of tongue force in an asymptomatic population. This could be a new, more accessible tool to consider and add to the assessment and treatment of different clinical conditions in which a deficit in tongue force could be found.

**Keywords:** feedback; muscle strength; neurofeedback; rehabilitation; reproducibility of results tongue

#### **1. Introduction**

The tongue is a muscle that is part of the stomatognathic system and plays an important role in phonation, breathing, and eating [1,2]. The tongue has been classified as a muscular hydrostat structure due to its ability of movement and deformation without

**Citation:** Diaz-Saez, M.C.; Gil-Martínez, A.; Gadotti, I.C.; Navarro-Fernández, G.; Gil-Castillo, J.; Beltran-Alacreu, H. Reliability and Responsiveness of a Novel Device to Evaluate Tongue Force. *Life* **2023**, *13*, 1192. https://doi.org/10.3390/ life13051192

Academic Editors: Zuzanna Nowak and Aleksandra Nitecka-Buchta

Received: 13 March 2023 Revised: 21 April 2023 Accepted: 11 May 2023 Published: 16 May 2023

**Copyright:** © 2023 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/).

a bone holder preserving its volume [2]. The tongue performs five main movements: elevation, depression, protrusion, right lateralization, and left lateralization, and their multiple combinations. These movements are important during eating function for the intraoral manipulation of food and swallowing [3]. During chewing, the tongue muscles must coordinate with the masticatory muscles and the temporomandibular joints (TMJ). During breathing, tongue extrinsic muscles move the hyoid bone in the craniocaudal direction, allowing the pharynx to open. In addition, tongue muscles are coordinated with suprahyoid and infrahyoid muscles during swallowing and phonation. Therefore, muscle balance, understood as a phenomenon in which agonist and antagonist muscles work in coordination during voluntary movements or unexpected body perturbations, is important for tongue function. This phenomenon allows maintaining neural control of movements on a specific functional level and avoid excessive muscle control work. Likewise, it seems that coactivation could increase stiffness and movement speed, increasing the stability in kinematic chain cases. Thereby, any disturbance in muscle balance or dysfunction related to the tongue can lead to dysphagia, dysarthria or breathing difficulties [4].

Different studies have shown that the function of the tongue can be compromised in different clinical conditions such as oropharyngeal cancers [5], post-stroke sequalae [6], sequalae associated with Parkinson Disease [7], scleroderma [4], and chronic temporomandibular disorders (TMDs) [8]. For example, tongue weakness was shown to be associated with TMD patients with orofacial restricted mobility [9]. In addition, the aging process may also contribute to tongue disturbances. In this way, it has been reported that older adults have less tongue force than younger adults [10] and it is well known that deficient tongue force can compromise the behavior and efficiency of mastication and swallowing [11].

Due to the above-described factors, measurements of tongue force are important in clinical practice during both the diagnostic process and rehabilitation process [7,12,13]. Currently, there are few devices to measure tongue force on the market. The four most used and studied devices are the Kay Swallowing Workstation (KSW) [14], the Madison Oral Strengthening Therapeutic (MOST) [15], the Iowa Oral Performance Instrument (IOPI) [16], and the OroPress device [17]. The KSW (KayPENTAX Corporation, Lincoln Park, NJ, USA) device is a computerized system with three sensors which allow performing multiple simultaneous measurements in different tongue positions on the palate including pressure measurements during swallowing [18]. However, it is not portable due to its large size and it is very expensive. White et al. reported an excellent intrarater reliability in the KSW device in a healthy population (ICC = 0.92) [19]. The MOST device is a portable device with four or five sensors inside one intraoral piece with a small amount of pliable Reprosil Dental Putty (DENTSPLY International, York, PA, USA), which allows measurements of tongue isometric pressure against the hard palate in five different positions (anterior, middle, posterior, right, and left). The intraoral piece provides stability to the sensors, and it is easy to use by patients. The MOST device has not demonstrated its reliability and validity yet [20]. However, due to its price, it is less accessible for professionals and patients. Likewise, the IOPI (IOPI Northwest Company, LLC, Carnation, WA, USA) is the most used device in research because it is easy to use, it is portable, and has a silicon air-filled bulb, which allows measuring isometric tongue pressure against the hard palate. However, it has poor sensor stability which may cause measurement errors [21], and to date, there are no studies on validity and inter-rater reliability with the use of this device. Although a good inter-rater reliability to measure the maximum isometric tongue force with the IOPI was found (ICC > 0.75) [22], it has been shown to be less reliable than the other devices due to artifacts on the measurements [21]. Finally, the OroPress device is composed of a biomedical interface pressure transducer (BIPT-MS58 series, Measurement Specialities Ltd., Bevaix, Switzerland), an earpiece, and a wireless transmission module which transmits data to a remote laptop or notebook computer for real-time viewing and recording. This system measures the isometric and swallowing pressure applied by the tongue directly at the sensor tongue interface compared to those which apply the pressure indirectly through

a column of air or fluid. It is characterized as being portable, having low-cost sensors, and being able to capture pressure while swallowing food or fluids. Oropress demonstrated good-to-excellent ICC values (ICC = 0.86) for its reliability [17]. Nevertheless, this device only has a pilot study trying to demonstrate its validity [17]. A bigger sample is needed to develop a good study of validity, and an intra- and inter-rater reliability study must also be carried out. This is very important to corroborate the safety, the psychometric properties, and the clinical utility of all these devices.

In order to overcome the limitations of the devices and improve the features, a validated, portable, handy, and lower-cost prototype device to measure tongue force was developed. The prototype has an intuitive interface, and it has been developed to assess and train tongue force in different movements, allowing its use not only for professionals, but also for patients for clinical and home rehabilitation. The software includes videogames with biofeedback for training at home which could increase the patient's adherence to the treatment [23]. The new device promotes patient independence in the rehabilitation process and reduces social and health care costs [24]. This new instrument, unlike the others developed up to now, proposes accurate assessments and future treatments based on gamification (Table 1). Moreover, compared to current tongue force instruments, this new device has already demonstrated good validity values and a high intrarater reliability, ensuring its safe use in the clinic [25]. Nevertheless, as a first step in the validation process, good inter-rater reliability for this device is also needed. Reliability is defined as the probability that a system, instrument, or device could perform a specific function in certain circumstances. It refers not only to the agreement but also the consistency between measurements. Moreover, random and systematic errors are needed to obtain reliability data and ensure accurate results. For this reason, devices must demonstrate a good stability and reliability before their use or commercialization. This makes the device safer during its use in a variety of clinical and research settings as well as by any type of person (professionals, patients, or patients' relatives) and ensures the security to be used with patients and different environmental conditions. According to this, it is established that this type of study should be developed in healthy subjects at first for trying to protect vulnerable individuals and could ensure that the device is safe for its condition. After that, reliability studies must be performed in patients for demonstrating the clinical usefulness [26].

**Table 1.** Feature comparison between the new device, the IOPI, the KSW, and the MOST instruments.

The main objectives of this study were to determine the intra- and inter-rater reliability and the minimum detectable change (responsiveness) in the maximum tongue force measurements using a newly developed device. The authors of the study wanted to demonstrate that this device could measure with the same reliability independently of the professional or patient who is using it. Since current commercial systems do not have enough evidence of their validity or inter-rater reliability and due to the high costs of technologies such as fluoroscopy, currently applied screening techniques are very subjective and depend on the training and experience of the therapist. This makes the devices less reliable. For this reason, demonstrating the reliability and sensitivity of our system would help in developing more objective assessments, regardless of the therapist performing the measurements.

#### **2. Materials and Methods** in previous studies [28–31]. An ICC of 0.90 was estimated based on the hypothesis. A sample of 26 subjects with 2 measurements per subject was needed to achieve 80% power (β =

sampling.

*2.1. Subjects*

ing the measurements.

**2. Materials and Methods**

An intra- and inter-rater reliability single-blind study with repetitive measurements was conducted based on the guidelines for reporting reliability and agreement studies (GRRAS) [27]. This study was approved by the Ethics Committee from the Centro Superior de Estudios Universitarios La Salle (CSEULS) of the Universidad Autónoma de Madrid (project code: CSEULS-PI-036/2019). Subjects were recruited from the CSEULS of the Universidad Autónoma de Madrid. Participants were recruited through nonprobability sampling. 0.2) to detect an ICC of 0.90, with a significance level of 0.05. Subjects were excluded if they presented TMD, cancer, or an active infection of the neck/head/mouth, had a history of orofacial or cervical surgery, had temporomandibular/orofacial/cervical acute pain before or during the test, were undergoing physical ther-

*Life* **2023**, *13*, x FOR PEER REVIEW 4 of 13

less reliable. For this reason, demonstrating the reliability and sensitivity of our system would help in developing more objective assessments,regardless of the therapist perform-

An intra- and inter-rater reliability single-blind study with repetitive measurements

A total of 26 asymptomatic subjects older than 18 participated in this study. The sam-

ple size was calculated based on the intraclass correlation coefficient (ICC) values obtained

was conducted based on the guidelines for reporting reliability and agreement studies (GRRAS) [27]. This study was approved by the Ethics Committee from the Centro Superior de Estudios Universitarios La Salle (CSEULS) of the Universidad Autónoma de Madrid (project code: CSEULS-PI-036/2019). Subjects were recruited from the CSEULS of the Universidad Autónoma de Madrid. Participants were recruited through nonprobability

#### *2.1. Subjects* apy for the neck or craniofacial region, had more than 6 points out of 10 on the subjective

A total of 26 asymptomatic subjects older than 18 participated in this study. The sample size was calculated based on the intraclass correlation coefficient (ICC) values obtained in previous studies [28–31]. An ICC of 0.90 was estimated based on the hypothesis. A sample of 26 subjects with 2 measurements per subject was needed to achieve 80% power (β = 0.2) to detect an ICC of 0.90, with a significance level of 0.05. perception of fatigue scale, or had neurological disorders and rheumatic systemic disorders. *2.2. Instrumentation*

Subjects were excluded if they presented TMD, cancer, or an active infection of the neck/head/mouth, had a history of orofacial or cervical surgery, had temporomandibular/orofacial/cervical acute pain before or during the test, were undergoing physical therapy for the neck or craniofacial region, had more than 6 points out of 10 on the subjective perception of fatigue scale, or had neurological disorders and rheumatic systemic disorders. The new low-cost prototype device, introduced in a previous article [23], was specifically designed and developed to measure tongue force objectively and accurately. The device consists of a physical part and associated software. The physical part consists of a

#### *2.2. Instrumentation* hardware system that measures the pressure exerted on a piezoelectric sensor (FSR 402,

The new low-cost prototype device, introduced in a previous article [23], was specifically designed and developed to measure tongue force objectively and accurately. The device consists of a physical part and associated software. The physical part consists of a hardware system that measures the pressure exerted on a piezoelectric sensor (FSR 402, Interlink Electronics Inc., Irvine, CA, USA) [32] and transmits the information with an Arduino UNO via a wired connection to a personal computer, where the software is located (Figure 1). This type of sensor is a very thin and flexible piezoelectric that does not cause any discomfort to the patient. The software is responsible for processing and displaying the information in real time. In addition, the software facilitates the recording of the demographic information of the subjects and the information recorded by the sensor is stored in a database for the subsequent extraction of reports (Figure 2). Interlink Electronics Inc., Irvine, South California, EEUU) [32] and transmits the information with an Arduino UNO via a wired connection to a personal computer, where the software is located (Figure 1). This type of sensor is a very thin and flexible piezoelectric that does not cause any discomfort to the patient. The software is responsible for processing and displaying the information in real time. In addition, the software facilitates the recording of the demographic information of the subjects and the information recorded by the sensor is stored in a database for the subsequent extraction of reports (Figure 2).

**Figure 1.** Prototype tongue force device (inferior and upper views). Small casing protecting the backplane, extralong cable, and piezoelectric sensor. **Figure 1.** Prototype tongue force device (inferior and upper views). Small casing protecting the backplane, extralong cable, and piezoelectric sensor.

**Figure 2.** Spanish prototype device interface view. (**a**): Configuration screen view for the main characteristics and personal data of the patient, and the duration of the experiment; (**b**): Measurement screen view for the specific movement that is being measured with the time, maximum force, force exerted during each second, and a feedback representation of the force exerted. **Figure 2.** Spanish prototype device interface view. (**a**): Configuration screen view for the main characteristics and personal data of the patient, and the duration of the experiment; (**b**): Measurement screen view for the specific movement that is being measured with the time, maximum force, force exerted during each second, and a feedback representation of the force exerted.

The interface has a user-centered design for ease of use in the clinical environment. The device can measure the pressure exerted on the sensor by placing it in different positions. Depending on the positioning of the sensor, it is possible to measure the force exerted in the following movements: lip to lip, tongue elevation (tongue against the anterior part of the hard palate), tongue depression (tongue against the jaw), right tongue lateralization (tongue against the right cheek), left tongue lateralization (tongue against the left cheek), and their combinations. The interface has a user-centered design for ease of use in the clinical environment. The device can measure the pressure exerted on the sensor by placing it in different positions. Depending on the positioning of the sensor, it is possible to measure the force exerted in the following movements: lip to lip, tongue elevation (tongue against the anterior part of the hard palate), tongue depression (tongue against the jaw), right tongue lateralization (tongue against the right cheek), left tongue lateralization (tongue against the left cheek), and their combinations.

#### *2.3. Procedure 2.3. Procedure*

Two experienced physical therapists with more than 3 years' experience working in the cervico-craniofacial area were trained on how to perform the maximum tongue force test and the whole intervention. The biomedical engineer that developed the device specifically helped and trained both physical therapists on how to use the new device. The tongue force test was performed on each participant in a sitting position for the tongue movements mentioned above in Section 2.2. Two measurements of each tongue movement were performed by each rater. The GraphPad Quickcals website was used to randomize which assessor had to go first on the measurements. The measurements were performed on the same day for both raters. Each rater was blind to the other rater's measurements. The subjects and raters were not able to see the results between the 2 measurements performed for each movement. A single-use hypoallergenic protective measure made of nitrile was used to cover the Two experienced physical therapists with more than 3 years' experience working in the cervico-craniofacial area were trained on how to perform the maximum tongue force test and the whole intervention. The biomedical engineer that developed the device specifically helped and trained both physical therapists on how to use the new device. The tongue force test was performed on each participant in a sitting position for the tongue movements mentioned above in Section 2.2. Two measurements of each tongue movement were performed by each rater. The GraphPad Quickcals website was used to randomize which assessor had to go first on the measurements. The measurements were performed on the same day for both raters. Each rater was blind to the other rater's measurements. The subjects and raters were not able to see the results between the 2 measurements performed for each movement.

sensor during the measurements for each subject (Figure 3). The single-use protection was not changed during the whole test, only between different participants. The subjects were asked to sit with their back against the chair, feet on the ground, and head in its natural position. The tongue sensors were placed by the subjects following the instructions given by the rater according to the movement tested. During the maximum tongue force test, the subjects were then asked to exert the maximum tongue force against the sensor for 10 s. A 5 min resting period was used between each measurement. Firstly, for the lip-to-lip movement, the sensor was placed between the lips, not including the teeth. Secondly, the sensor was placed behind the superior incisors in the anterior part of the hard palate for the tongue elevation movement. Thirdly, the subjects placed the sensor behind the inferior incisors in the jaw for the tongue depression movement. Finally, right and left tongue lateralization movements were developed by placing the sensor in the anterior part of the right and left cheeks, respectively. The whole procedure is described in Figure 4. A single-use hypoallergenic protective measure made of nitrile was used to cover the sensor during the measurements for each subject (Figure 3). The single-use protection was not changed during the whole test, only between different participants. The subjects were asked to sit with their back against the chair, feet on the ground, and head in its natural position. The tongue sensors were placed by the subjects following the instructions given by the rater according to the movement tested. During the maximum tongue force test, the subjects were then asked to exert the maximum tongue force against the sensor for 10 s. A 5 min resting period was used between each measurement. Firstly, for the lip-to-lip movement, the sensor was placed between the lips, not including the teeth. Secondly, the sensor was placed behind the superior incisors in the anterior part of the hard palate for the tongue elevation movement. Thirdly, the subjects placed the sensor behind the inferior incisors in the jaw for the tongue depression movement. Finally, right and left tongue lateralization movements were developed by placing the sensor in the anterior part of the right and left cheeks, respectively. The whole procedure is described in Figure 4.

**Figure 3.** Single-use hypoallergenic protection covering the whole sensor to protect it. **Figure 3.** Single-use hypoallergenic protection covering the whole sensor to protect it. **Figure 3.** Single-use hypoallergenic protection covering the whole sensor to protect it.

*Life* **2023**, *13*, x FOR PEER REVIEW 6 of 13

**Figure 4.** Descriptive graphic of the procedure. **Figure 4.** Descriptive graphic of the procedure.

#### **Figure 4.** Descriptive graphic of the procedure. *2.4. Analysis and Sample Size 2.4. Analysis and Sample Size*

*2.4. Analysis and Sample Size* The sample size was calculated based on the intraclass correlation coefficient (ICC) The sample size was calculated based on the intraclass correlation coefficient (ICC) values obtained in previous studies [28–31]. An ICC of 0.90 was estimated based on the hypothesis. A sample of 26 subjects with 2 measurements per subject was needed to achieve 80% power (β = 0.2) to detect an ICC of 0.90, with a significance level of 0.05. The sample size was calculated based on the intraclass correlation coefficient (ICC) values obtained in previous studies [28–31]. An ICC of 0.90 was estimated based on the hypothesis. A sample of 26 subjects with 2 measurements per subject was needed to achieve 80% power (β = 0.2) to detect an ICC of 0.90, with a significance level of 0.05.

values obtained in previous studies [28–31]. An ICC of 0.90 was estimated based on the hypothesis. A sample of 26 subjects with 2 measurements per subject was needed to achieve 80% power (β = 0.2) to detect an ICC of 0.90, with a significance level of 0.05. The interclass correlation coefficient and standard error of measurement (SEM) were used to calculate the reliability. The ICC3,1 was designated as the two-way analysis of variance mixed model for the absolute agreement of single measures. The ICC3,2 was designated the same way as the ICC3,1 but using the average of the two measures of each rater to determine the inter-rater reliability [33]. Intraclass correlation coefficient values greater The interclass correlation coefficient and standard error of measurement (SEM) were used to calculate the reliability. The ICC3,1 was designated as the two-way analysis of variance mixed model for the absolute agreement of single measures. The ICC3,2 was designated the same way as the ICC3,1 but using the average of the two measures of each rater to determine the inter-rater reliability [33]. Intraclass correlation coefficient values greater than 0.75 indicate good reliability, those between 0.50 and 0.75 indicate moderate agreement, and those below 0.50 indicate poor agreement [33]. A 95% confidence interval (CI) was also calculated, and *p* < 0.05 was used as the level of statistical significance. The interclass correlation coefficient and standard error of measurement (SEM) were used to calculate the reliability. The ICC3,1 was designated as the two-way analysis of variance mixed model for the absolute agreement of single measures. The ICC3,2 was designated the same way as the ICC3,1 but using the average of the two measures of each rater to determine the inter-rater reliability [33]. Intraclass correlation coefficient values greater than 0.75 indicate good reliability, those between 0.50 and 0.75 indicate moderate agreement, and those below 0.50 indicate poor agreement [33]. A 95% confidence interval (CI) was also calculated, and *p* < 0.05 was used as the level of statistical significance.

than 0.75 indicate good reliability, those between 0.50 and 0.75 indicate moderate agreement, and those below 0.50 indicate poor agreement [33]. A 95% confidence interval (CI) was also calculated, and *p* < 0.05 was used as the level of statistical significance. Bland–Altman plots were constructed using mean differences between measurements [34]. Limits of agreement (LOA) were calculated as mean differences ± (standard

deviation multiplied by 1.96) [35]. Calculation of the occurrence of systematic or random changes in the data means that it was performed through a calculation of 95% confidence intervals (CI) of the mean differences between the values of the measurements.

The responsiveness was determined with minimal detectable change at 90%, which was calculated as SEM × 1.65 × √ 2 [36,37]. The MDC90 expresses the minimal change required to be 90% confident that the change observed between two measurements reflects a real change (sensitive measure) and not a measurement error.

#### **3. Results**

A total of 26 subjects were included in the reliability analysis (57.7% men and 42.3% women). The average age of the sample was 25.69 years old with a standard deviation of 7.46 years old. In relation to body mass index, it was 26.1 (25.7–26.5; 95%CI) in men and 24.1 (23.7–24.7; 95%CI) in women. In addition, the percentage of participants with or in the process of completing tertiary education was 63%. According to the Shapiro–Wilk test, the data were normally distributed (*p* > 0.05).

#### *3.1. Intrarater Reliability Results*

The descriptive data for intrarater reliability, ICC3,1, SEM, MDC90, and Bland–Altman analysis with the 95%CI and LOA are summarized in Table 2. Good-to-excellent intrarater reliability for all tongue movements was found for both raters (ICC3,1 ≥ 0.80). The SEM was <0.70 for rater A and <0.98 for rater B. The MDC was between 1.10 and 1.64 for rater A and between 0.96 and 2.30 for rater B.

**Table 2.** Intrarater reliability (*n* = 26).

Abbreviations: ICC: intraclass correlation coefficient; CI: confidence interval; SEM: standard error of measurement; MDC: minimum detectable change; SD: standard deviation; LOA: limits of agreement.

#### *3.2. Inter-Rater Reliability Results*

The descriptive data for inter-rater reliability, ICC3,2, SEM, MDC, and Bland–Altman analysis with the 95%CI and LOA are summarized in Table 3. Good-to-excellent intrarater reliability for all tongue movements was found for both raters (ICC3,2 ≥ 0.80). The SEM was <1.29. The MDC was between 1.20 and 3.01. Graphical representations of the Bland–Altman plot are shown in Figure 5.

**Table 3.** Inter-rater reliability (*n* = 26).

Abbreviations: ICC: intraclass correlation coefficient; CI: confidence interval; SEM: standard error of measurement; MDC: minimum detectable change; SD: standard deviation; LOA: limits of agreement. <1.29. The MDC was between 1.20 and 3.01. Graphical representations of the Bland–Altman plot are shown in Figure 5.

**Figure 5.** Graphical representations of the Bland–Altman plot. The red line is the mean difference. The green lines represent the Limits of Agreement (LOA). **Figure 5.** Graphical representations of the Bland–Altman plot. The red line is the mean difference. The green lines represent the Limits of Agreement (LOA).

Abbreviations: ICC: intraclass correlation coefficient; CI: confidence interval; SEM: standard error of measurement; MDC: minimum detectable change; SD: standard deviation; LOA: limits of agree-

Elevation 6.89 ± 4.07 6.32 ± 4.38 0.94 (0.86–0.97) 1.03 2.40 Depression 6.05 ± 3.37 5.08 ± 2.85 0.83 (0.57–0.93) 1.29 3.01 Right 2.52 ± 1.77 2.24 ± 1.66 0.87 (0.73–0.94) 0.61 1.43 Left 2.18 ± 1.26 1.97 ± 1.10 0.81 (0.63–0.91) 0.51 1.20 Bland–Altman Mean difference ± SD 95%CI LOA (Inf-Sup)

**Table 3.** Inter-rater reliability (*n* = 26).

Elevation 0.57 ± 1.43 (0.02 to 1.12) (−2.23 to 3.37) Depression 0.97 ± 1.65 (0.33 to 1.60) (−2.26 to 4.20) Right 0.27 ± 0.86 (−0.06 to 0.60) (−1.42 to 1.96) Left 0.21 ± 0.71 (−0.06 to 0.48) (−1.18 to 1.60)

ment.

**Mean ±SD Mean ±SD**

**Outcome Measurements (in Newtons)**

#### **4. Discussion**

As far as the authors know, this is the first study evaluating the maximum tongue force in four different directions of tongue movement. According to the results, a good-toexcellent intra- and inter-rater reliability was found for all movements. The measurements were also responsive to detect real changes.

This was also the first study testing the reliability of a device with a force-sensitive resistor (FSR) sensor to measure the maximum tongue force. Although the MOST device is composed of the same type of sensor, its reliability has not been tested [20]. There is currently no gold standard for maximum tongue force outcome measurements. That is why the results from this study are compared with the devices that are often used in clinical practice and research.

The present study has demonstrated an excellent intrarater reliability for maximum tongue force measurements of the superior, inferior, and right tongue movements (ICC3,1 > 0.93) and a good intrarater reliability for measurements of the left tongue movement (ICC3,1 > 0.82). The measurements of the superior tongue movement obtained the highest ICC3,1 values (>0.95). These values were slightly greater than those found for the reliability measurements of tongue force in superior movements using the IOPI device, which ranged from 0.77 to 0.90 [38]. Likewise, better ICC values were obtained when compared to the study by White et al., who reported an excellent intrarater reliability for the KSW device in a healthy population (ICC = 0.92) [19]. In reference to the Oropress reliability results, similar ICC values were found (ICC = 0.86) when compared to the present study [17].

An excellent inter-rater reliability for measurements of the tongue force in elevation (ICC3,2 = 0.94) and a good inter-rater reliability for measurements of the tongue force in depression and right and left lateralization (ICCs3,1 = 0.83, 0.87 and 0.81, respectively) were found in the current study. Youmans and Stierwalt (2006) obtained a 94% inter-rater agreement (r = 0.94) during the maximum isometric force measurement using the IOPI device [39]. The IOPI device is commonly used; however, it is only used to measure tongue force in one direction (tongue elevation). Additionally, the IOPI analysis protocols are different from the ones utilized in the present study. While the common IOPI protocol for analysis uses the highest value obtained during the three tests or the mean of the two best tests, the current study used the mean of the two measurements. Nevertheless, researchers cannot define the analysis with any of the devices since there is no defined protocol. Similarly to the IOPI device, the KSW instrument only measures superior tongue force movement and commonly collects the higher measure of the three tests performed.

The inter-rater reliability of tongue force measurements using the IOPI device in subjects with different conditions was reported to be good to excellent (ICC > 0.75) [22], with the exception of a study evaluating dysarthria patients in which a moderate reliability was found (ICC = 0.535) [22,38]. However, there are no recent studies available on the evaluation of the inter-rater reliability for the IOPI in healthy subjects, and the authors of this paper believe that this should be the first step prior to measurement and use in patients. Likewise, there is no inter-rater reliability research for measuring tongue force with the KSW device. The KSW device uses the same type of sensor as the IOPI, a silicon air-filled bulb. The main difference is that the KSW bulb is fixed to the palate, providing more stability and reliability. Probably due to its multiple functions, the KSW device is used more for research evaluating tongue force during swallowing. According to Fei et al., the KSW device is more reliable than the IOPI when evaluating tongue force during the function of swallowing [40].

Regarding responsiveness, the SEM values were low for elevation, depression, and right and left tongue movements (1.03, 1.09, 0.60, and 0.51, respectively). The MDC values were also low for elevation, depression, and right and left tongue movements: 2.40, 3.01, 1.43, and 1.20, respectively. Therefore, the new device was able to capture real change in tongue measures in all directions. Although we can assure good reliability and responsiveness for the device presented in this study in an asymptomatic population, we cannot guarantee the same findings in symptomatic subjects yet. Only one previous study

determined the SEM and the MDC of the IOPI device in asymptomatic subjects [38]. This study estimated these values using standard deviation (SD), while the present study based the calculation on the root mean square (RMS) [40]. The SD was used to estimate the SEM, avoiding possible uncertainties due to the selected ICC type [35]. Therefore, the evaluation of the SEM varies between studies. Additionally, a Bland–Altman method was used to evaluate agreement, including the LOA. A good LOA was found, and the SEM, MDC, and LOA revealed a good level of concordance. These values are very important for the use of the device in clinical practice as they ensure that any improvement in tongue force is due to the treatment rather than measurement errors.

This study demonstrated that the newly developed tongue force device is reliable for measuring the maximum tongue force in different directions within and in between professionals. The new device overcomes some limitations from the tongue devices commonly used in the literature. This validated, safe, portable, and easy-to-use device can allow patients to perform tongue exercises at home, and the ability of the device to display the tongue activity in real time may increase their motivation to progress with their rehabilitation program. All these features add to the fact that it is a low-cost instrument. We recommend that future studies are needed to test the tongue force device including both healthy subjects and patients. Additionally, future studies must include in silico/computational simulation to ensure that the force data used from the device is accommodated correctly [41].

#### *4.1. Limitations*

This study presents some limitations. Nonprobability sampling is always a limitation of a study. Ideally, a sufficiently large population would have been accessible for probability sampling. The reliability of the developed tongue force device was tested on healthy young subjects mainly (at an average of 25.7 years of age) and, therefore, these results should be taken with caution when transferring them to other populations. Further studies should test the device in different age groups in order to generalize the results. Likewise, future studies should include subjects with different health conditions. The results showed a significant statistical difference in some values of the Bland–Altman plot. These differences are close to 0 and all mean difference values are below the MCD in all cases. This led us to assume those results are statistically significant but not clinically relevant. The minimal clinically important difference (MCID) should be evaluated in future studies. Likewise, the values of other populations must be established and validated in future studies as with any measurement device or questionnaire.

#### *4.2. Clinical Implications*

From a neurophysiological point of view, it is known that the cerebral cortex has areas where information (input and output) from the V (trigeminal nerve), VII (facial nerve), and XII (hypoglossal nerve) cranial nerves is integrated [42]. In this way, these cranial nerves control the muscles of mastication, facial gestures, and the tongue, respectively, in order to achieve the optimal functionality of the entire system during speech and mastication, among other functions. Additionally, we have already published an observational study which showed significant differences in the maximum tongue force between asymptomatic women and those with chronic temporomandibular disorder, corroborating the necessity for the assessment of the tongue force in this pathology [43]. In this article, a decrease in tongue strength of about 30% on average across all directions was found in the group of patients with chronic TMD. In line with this, clinical experience shows that many patients with TMD (especially the chronic type) have lingual alternations both in terms of flexibility (length) and strength in various directions.

This new device to measure tongue force allows obtaining objective measurements of tongue force in clinical practice in order to help clinicians with the diagnosis process and treatment progression. This will give clinicians and patients real data to observe the changes during the treatment. Moreover, the new tongue force device has a diagnostic interface and treatment interface with different games to train the force at home and in the clinic. This training with games will motivate the patients and increase the adherence to the treatment. This offers an accessible device for patients and clinicians due to the fact that the few that are available in the market have this limitation and are much more expensive. Moreover, its validity has been proved in a previous study that has been recently published [25]. This could be a new tool to consider and add for the assessment and treatment of these patients. Likewise, as a new tool in the treatment of TMD, it could decrease the sociosanitary costs that this pathology implies for the sanitary system due to its chronicity.

#### **5. Conclusions**

This study showed a good-to-excellent intra- and inter-reliability for the newly developed device to measure the maximum tongue force in four different directions in an asymptomatic population. The measurements with the new device were also able to detect real changes, suggesting a more sensitive measure (good responsiveness in the device). These results confirmed that the device is suitable for objective and precise tongue measurements independently of the subject that is using this tool. The new prototype device seems to be an improved tongue force measurement tool that is safe, validated, and more accessible than others on the market.

**Author Contributions:** Conceptualization, A.G.-M.; methodology, A.G.-M. and J.G.-C.; software, J.G.-C.; formal analysis, G.N.-F.; investigation, M.C.D.-S.; data curation, G.N.-F. and J.G.-C.; writing original draft preparation, M.C.D.-S.; writing—review and editing, H.B.-A., A.G.-M. and I.C.G.; visualization, H.B.-A. and A.G.-M.; supervision, H.B.-A. and A.G.-M.; project administration, H.B.-A. and A.G.-M. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** The study was conducted in accordance with the guidelines for reporting reliability and agreement studies (GRRAS) [27]. This study was approved by the Ethics Committee from the Centro Superior de Estudios Universitarios La Salle (CSEULS) of the Universidad Autónoma de Madrid (project code: CSEULS-PI-036/2019). Subjects were recruited from the Centro Superior de Estudios Universitarios La Salle (CSEULS) of the Universidad Autónoma de Madrid.

**Informed Consent Statement:** Informed consent was obtained from all subjects involved in the study. Written informed consent has been obtained from the patient(s) to publish this paper.

**Data Availability Statement:** Not applicable.

**Acknowledgments:** The authors would like to thank Estela Sánchez and Mª Dolores Pérez (members of Innovation Support Unit in Hospital Universitario La Paz) who assisted us in drafting and submitting the application for protection for this new device. Moreover, we want to thank Daniel Mañoso for his help with the English translation.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

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**Acknowledgements**

The authors are thankful to the University of Technology Nowshera, Pakistan for providing the financial support for the publication of this chapter.

**Author details**

\*, Anwar Ali1

provided the original work is properly cited.

1 University of Technology, Nowshera, Pakistan

, Haider Ali1

\*Address all correspondence to<EMAIL_ADDRESS>
2 Escola d'Enginyeria, Autonomous University of Barcelona, Barcelona, Spain

*Static Var Compensator with Fractional Order Dynamics for Enhanced Stability and Control*

*DOI: http://dx.doi.org/10.5772/intechopen.79615*

3 Departamento de Ingeniería, Universidad de Bogotá Jorge Tadeo Lozano, Bogotá,

© 2018 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

, Asier Ibeas2 and Jorge Herrera3

Nasim Ullah1

Colombia

**281**

*Static Var Compensator with Fractional Order Dynamics for Enhanced Stability and Control DOI: http://dx.doi.org/10.5772/intechopen.79615*


**1. Cellular Microenvironment**

The cellular microenvironment is characterized by a mixture of extracellular matrix proteins, soluble signaling factors, neighboring cells, and the physical properties of the niche that affect cell behavior through direct or indirect biomechanical and biochemical signals [1,2]. Properties of the microenvironment, such as stiffness and composition, have been shown to direct cell physiology and lineage [3,4]. Such findings have inspired research to define the cellular microenvironment and its links to cellular behaviors using in vitro tissue models that can mimic biomechanical conditions [5,6]. Here, we will discuss the cellular niche, namely, the biological and mechanical properties of the extracellular matrix (ECM), how cells sense these properties, and the dysregulation of cell-ECM interactions in various disease states. How these factors, which are involved in crosstalk with cells, contribute to cellular activities and overall health, will also be presented through a review of the various research publications on the topic. This review will provide an in-depth

**Citation:** Sahan, A.Z.; Baday, M.; Patel, C.B. Biomimetic Hydrogels in the Study of Cancer Mechanobiology: Overview, Biomedical Applications, and Future Perspectives. *Gels* **2022**, *8*, 496. https://doi.org/10.3390/ gels8080496

Academic Editors: Kiat Hwa Chan and Yang Liu

Received: 30 May 2022 Accepted: 2 July 2022 Published: 10 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/).

overview of what is known and what is unknown about the biomechanics involved in cellmicroenvironment interactions, how the use of biomimetic hydrogel models can fill these gaps in the knowledge, and the utility of biomimetic hydrogels in biomedical applications.

### *1.1. Extracellular Matrix*

Tissues may be described as having two components: cellular and non-cellular. The extracellular matrix is the non-cellular component and is composed of proteins, polysaccharides, growth factors, signaling molecules, proteases, and water [7] (Figure 1). These components are distributed heterogeneously rather than homogenously, resulting in unique niche microenvironments for each cell as well as tissue-specific mechanical, physical, and biochemical properties [7]. These play a large role in regulating and mediating cell behaviors. While the ECM is a mixture of many components, a large portion of it is composed of proteins. These include proteoglycans such as hyaluronan, and fibrous proteins such as collagens, elastins, fibronectins, and laminins [8]. These proteins function to anchor cells to the ECM via focal adhesions and aid propagating signals between cells [8]. ECM composition also affects physical properties such as elasticity, stiffness, porosity, static architecture, and dynamic deformations of the matrix [9]. For instance, based on their concentration, assembly, and crosslinking densities, the structural collagens and elastins of the ECM significantly alter its mechanical properties such as composite strength, elasticity, and mechanical resistance [10,11]. ECM properties are not static; they undergo dynamic changes as the ECM is continuously being remodeled through protein degradation, deposition, or modifications that can be self-contained or caused by cellular activity [10]. In cases such as tissue repair, the activity of growth factors and cytokines in the ECM cause matrix metalloproteinases to activate for ECM remodeling [12]. Wound healing and tissue remodeling processes activate growth factors through mechanical and biochemical stimuli to change ECM composition [13]. There are also specialized forms of ECMs that have proven to be important regulators of tissue and cell behavior. For instance, basement membrane is important structurally and functionally for blood vessels because of its involvement in angiogenesis [12]. The ECM has also been shown to mediate or regulate stem cell fate, cell proliferation, cell differentiation, cell migration, and tissue regeneration [11,14–16].

### 1.1.1. Cell-ECM Interactions

The cell and its ECM are involved in a dynamic reciprocity, through which cues from the ECM and cellular activities are in crosstalk to maintain a healthy state [17]. Signaling processes are one of the ways that the cell-ECM interactions are facilitated. Cell-matrix adhesion sites, or focal contacts, enable communication between cells and the ECM through physical connections of cellular integrins and cadherins to ligands in the ECM [15,16]. Focal contacts are important to cellular processes that require physical attachment to the ECM, such as migration and angiogenesis [18]. Engagement of integrins and cadherins to certain ligands can activate signaling pathways such as that of the Rho family of GTPases to stimulate structural changes in the cell or induce other processes, therefore serving as an important step in biochemical cell-ECM interactions [6,16].

Proteoglycans present in the ECM have functions such as inducing aggregation and participating in ECM structure by adhering to structural proteins [19]. Some proteoglycans reside on the surfaces of epithelial cells, where they act similar to cell-adhesion molecules and bind collagens and fibronectin to anchor cells to the ECM [20,21]. In addition to these vital functions, proteoglycans are active co-receptors that mediate cellular signaling by binding soluble ligands in the ECM and encouraging the formation of receptor complexes on cell surfaces [22,23]. Proteoglycan co-receptors are vital to various developmental processes, and the loss of co-receptor function or mutation has been implicated in diseases such as cancer and ischemic heart disease [23]. Therefore, the ECM also plays a crucial role in mediating cell–cell communication, which will be discussed more thoroughly in Section 1.2 of this review.

**Figure 1.** Extracellular matrix (ECM) and cellular mechanotransduction. ECM components such as collagens can alter mechanical properties to induce changes in cellular signaling and gene or protein expression via mechanoreceptors and mechanotransduction proteins. Legend on the bottom left shows which ECM component is represented by each symbol. (Created with BioRender.com, ac-**Figure 1.** Extracellular matrix (ECM) and cellular mechanotransduction. ECM components such as collagens can alter mechanical properties to induce changes in cellular signaling and gene or protein expression via mechanoreceptors and mechanotransduction proteins. Legend on the bottom left shows which ECM component is represented by each symbol. (Created with BioRender.com, accessed on 29 May 2022).

### cessed on 29 May 2022)*.* Mechanobiology of the Cellular Microenvironment

1.1.1. Cell-ECM Interactions The cell and its ECM are involved in a dynamic reciprocity, through which cues from the ECM and cellular activities are in crosstalk to maintain a healthy state [17]. Signaling processes are one of the ways that the cell-ECM interactions are facilitated. Cell-matrix adhesion sites, or focal contacts, enable communication between cells and the ECM through physical connections of cellular integrins and cadherins to ligands in the ECM [15,16]. Focal contacts are important to cellular processes that require physical attachment to the ECM, such as migration and angiogenesis [18]. Engagement of integrins and cadherins to certain ligands can activate signaling pathways such as that of the Rho family of GTPases to stimulate structural changes in the cell or induce other processes, therefore serving as an important step in biochemical cell-ECM interactions [6,16]. Proteoglycans present in the ECM have functions such as inducing aggregation and participating in ECM structure by adhering to structural proteins [19]. Some proteoglycans reside on the surfaces of epithelial cells, where they act similar to cell-adhesion molecules and bind collagens and fibronectin to anchor cells to the ECM [20,21]. In addition The ECM is involved in mechanical crosstalk with cells [24], which relies on mechanotransduction proteins that help to regulate intracellular tensile response to mechanical forces from the ECM [25]. Mechanical stimuli that cells may receive include shear stress, membrane tension, force, strain, stiffness, and drag force [25]. These stimuli are listed in Table 1 with the mechanotransduction proteins identified to be involved in recognizing the stimuli and eliciting the response in cells. While the extracellular matrix of many cancers, including colon, breast, and prostate cancer, is stiffer than that of healthy tissues [26,27], cell deformability or reduced stiffness has been correlated to increased metastatic potential and invasiveness in cancer. In a study of ovarian cancer cells of varying invasiveness, Xu et al. found that the more-invasive ovarian cancer cell line was more deformable compared to the less-invasive cell line [28]. In another study, Hayashi and Iwata confirmed that cancer cells are softer (i.e., lower Young's modulus) than normal cells using atomic force microscopy [29]. Another study reported that cisplatin treatment caused decreased stiffness and invasiveness of prostate cancer cells [30], suggesting that various cell lines may have varied mechanical properties in the cancer state. Further controversy exists within the field of glioblastoma biomechanics. Gliomas are highly variable; therefore, measurements of tumor stiffness may vary depending on location of measurement [31]. Although some GBM tissues were stiffer than healthy reference tissues, GBM tissues, on average, were less

diseases such as cancer and ischemic heart disease [23]. Therefore, the ECM also plays a

to these vital functions, proteoglycans are active co-receptors that mediate cellular signaling by binding soluble ligands in the ECM and encouraging the formation of receptor stiff than healthy tissues [31–33]. The controversy regarding GBM tumor stiffness could by fueled by differences in methods of measurement, as there is currently no standard practice in the field [34–36].

Mechanical stimuli coming from the extracellular environment can be processed by cells through mechanotransduction pathways. The proteins of these pathways translate mechanical cues to induce biochemical and genetic responses. For instance, integrins and focal adhesion proteins are mechanotransduction proteins that communicate mechanical forces to the cell cytoskeleton, and there have been some studies that show force-dependent integrin activation [37–39]. Yes-associated protein (YAP) and other transcription factors have been shown to translocate to the nucleus in stiffer substrates [40]. Similarly, E-cadherin is a mechanotranducer of shear stress [41–43]. Many mechanotransduction proteins have been studied in the context of specific cell types, including platelet endothelial cell adhesion molecule-1 (PECAM-1) in skeletal muscle cells, G protein-coupled receptors in endothelial cells, and vascular endothelial growth factor receptor 2 (VEGFR2) in chondrocytes [44–47]. Cell surface receptors also transduce mechanical signals to cells upon recognition of a ligand that sustained force from the ECM, which can then cause conformational changes in the mechanoreceptor to activate a protein signaling pathway to alter cellular processes [48]. Such membrane proteins are not the sole propagators of mechanical force; studies have shown that there are mechanotransduction systems in cells that enable the progression of force through a long distance [49]. Src and Rac1 have been shown to be activated at distances from 30-60 µm from the original area the force was applied to, via cytoskeletal mediation of the force [49,50]. Mechanical signals can also be transmitted throughout the cytoskeleton to the nucleus via proteins such as linker of nucleo- and cyto-skeleton (LINC) complex 9 to change chromatin structure and cause nuclear stiffening [49,51]. Mechanotransduction systems translate the numerous mechanical cues from the ECM into biochemical signals interpreted by the cell that lead to signaling cascades that control transcription, proliferation, migration, and many other cellular processes [52–55] (Figure 2). For instance, smooth muscle cells can migrate along gradients of substrate stiffness through durotaxis [56]. ECM biomechanics is not only vital for cellular processes, but also regulates tissue and organlevel processes such as tissue differentiation, morphogenesis, and development [57–60]. Therefore, elucidating the interplay of ECM biomechanical and biochemical signals with mechanotransduction proteins and pathways is critical to understanding diverse aspects of cellular and tissue health. *Gels* **2022**, *8*, x FOR PEER REVIEW 5 of 36 ECM biomechanical and biochemical signals with mechanotransduction proteins and pathways is critical to understanding diverse aspects of cellular and tissue health.

**Figure 2.** Application of extracellular stress leads to increased intracellular calcium concentrations. Matthews et al. applied high levels of stress to cells (**A**) and found that when imaged via Fura-2AM ratio imaging, it led to a transient increase in calcium concentrations, as shown in pseudocolor images ranging from blue to yellow (**B**) that is quantified (**C**) as a function of time for control and gadolinium chloride-treated cells. (Figure reprinted/adapted with permission from Ref. [61]. Copyright 2006, National Academy of Sciences*.* **Figure 2.** Application of extracellular stress leads to increased intracellular calcium concentrations. Matthews et al. applied high levels of stress to cells (**A**) and found that when imaged via Fura-2AM ratio imaging, it led to a transient increase in calcium concentrations, as shown in pseudocolor images ranging from blue to yellow (**B**) that is quantified (**C**) as a function of time for control and gadolinium chloride-treated cells. (Figure reprinted/adapted with permission from Ref. [61]. Copyright 2006, National Academy of Sciences.

**Table 1.** Mechanotransducers of various mechanical properties and human cellular responses.

**ducers Mechanical Signal Examples of Cellular Responses**

creased cell stiffness [62,63]

maturation [40]

Implicated in development of mechanical unloading-induced diaphragm weakness [64]

regulate assembly of filaments [65]

membrane fusion promotion [67]

signaling [15]

Force Transmit tension to other proteins,

Force Regulate junction dynamics [68]

Shear Stress Altered cell morphology [69]

Integrins Force RhoA activation leading to in-

Vinculin Force Transmit tensile force [66]

Piezo1 Force Vascular structure [70] Lamin A Rigidity Nuclear mechanics [71,72]

Focal Adhesions Force Integrin convergence [73] Fibronectin Force Altered integrin binding [74]

Integrins Force Tyrosine Phosphorylation, MAPK

Myosin II Force Increased cortical tension and cell

Focal Adhesions Force Actin polymerization [55]

Stress Fibers (actin filaments, myosin II, etc.)

Vasodilator stimulated phosphoprotein (VASP), zyxin, and Testin LIM domain protein (TES)

Neurogenic locus notch homolog protein 1 (NOTCH1)

Titin Force

**Mechano-Trans-**

**Functional Category**

Cell Mechanical and Physical Properties

Alters Signaling Pathways

**Table 1.** Mechanotransducers of various mechanical properties and human cellular responses.

**Table 1.** *Cont.*

### 1.1.2. Cell-ECM Interactions in Cancer

Problems in mechanotransduction can result from changes in ECM mechanical properties and defects in proteins involved in mechano-sensitivity [101]. Since mechanotransduction is essential for modulating cellular homeostasis, its failure is linked to metastasis and cancer progression [96]. For instance, there are many proteins implicated in mechanotransduction in glioblastoma (GBM). Talin1 inhibition has been observed to decrease cell spreading and limit cell stiffness changes of glioma cells in response to ECM stiffness, proving its role as a mechanosensory [76]. Non-muscle myosin II depletion reduced the effect of matrix confinement on GBM cell motility [102]. Constitutive activation of RhoA GTPase caused lower sensitivity to matrix stiffness of GBM cells in toxicity assays [55]. Increased matrix stiffness was correlated to Hras, RhoA, and rho-associated, coiled-coilcontaining *protein* kinase 1 (ROCK1) upregulation, which are mechanosensor proteins that are implicated in migration and proliferation in cancers in general [61,87]. Integrins are also particularly important in mechanotransduction by relaying signals from the ECM to the cell actin cytoskeleton and are essential to cell migration and cell-matrix adhesion in cancer [103].

Identifying proteins involved in mechanotransduction and their roles in cancer progression can be an essential part of developing therapeutic strategies to hinder cancer progression and malignancy. Certain studies have shown the potential effects of targeting mechanotransduction proteins on cancer cells. Knock-down of CD44 led to decreased structural microtubule, vimentin, and glial fibrillary acidic protein expression and decreased migration and cell stiffness [104]. Targeted inhibition of integrins in the tumor microenvironment has been shown to reduce angiogenesis and inhibit tumor growth [103]. While these studies show partial inhibition of mechanical sensitivity as decreasing invasive properties of tumor cells, other studies have shown that tumor initiating cells are mostly insensitive to mechanical cues from the ECM and that mechanically-insensitive cells have increased motility and invasiveness in vitro [28,105–109]. A complex approach is needed to target mechanical sensitivity in cells through mechanotransduction-based therapeutics for it to become a promising mode of cancer treatment. Therefore, extensive research in mechanotransduction and cell-ECM crosstalk is essential.

### *1.2. Neighboring Cells and Secreted Factors*

In addition to the ECM components, cells are surrounded by heterogeneous populations of neighboring cells that are unique to the tissue and sub-location within the tissue [110]. How cells communicate and influence one another is crucial to maintaining homeostasis and coordinating processes that require several cells, such as tissue formation and regeneration.

Cells can interact through secreted signals that are recognized by membrane-bound receptors through either paracrine signaling, between cells, or autocrine signaling, which is from one cell to itself. In fact, cancer cells are often able to "override" signals from neighboring cells through autocrine pro-survival and proliferation signals. It is crucial to better understand the specific interactions between cells that promote healthy conditions or lead to disease states. For instance, Zervantonakis et al. found that fibroblasts in the tumor microenvironment of HER2 positive breast-cancer cells reduced drug sensitivity through paracrine signaling that activates mechanistic target of rapamycin (mTOR, anti-autophagic) and anti-apoptotic signals [111]. In addition to elucidating mechanisms of tumor resistance, cell–cell interaction dynamics can provide insights to developing self-assembled multicellular structures in vitro. Mueller et al. demonstrated that by utilizing pulsed light activation to control engineered photo-switchable cell-cell interactions, they were able to control the spatial organization of multicellular structures without a scaffold [112]. This highlights the importance of maintaining cell interactions in efforts to mimic physiological conditions in vitro.

Cells are also involved in communications through physical contacts with one another. This is another way that mechanical stimuli may play a role in influencing cellular processes. Physical contacts are also involved in collective migration, which has been exhibited by metastatic and invasive cancer cells [113]. The cytoskeletal tension at cell-cell contacts can serve as a significant regulator of mechanotransduction pathways, and there is a wide field of study on the mechano-sensing implications of cell-cell contacts such as focal adhesions and adherens [114]. One method in particular, physically interacting cell sequencing (PICseq), is a novel sequencing approach that combines cell sorting, RNA-sequencing, and computational modeling to describe complex cellular interactions in different contexts [115]. However, the heterogeneity and complexity of the interactions between cells and with the ECM need to be better recapitulated in vitro to provide a more accurate understanding of cellular communication networks.

### *1.3. Hydrogels as In Vitro Models of the Cellular Microenvironment*

Three-dimensional hydrogel models of the cellular microenvironment have gained interest in recent years for their improved mimicry of in vivo conditions as opposed to two-dimensional cell cultures (Table 2). These hydrogel models have been extensively studied and compared to 2D culture and in vivo conditions. Cells have been shown to have differing spatio-physical properties in 3D and conventional 2D culture conditions [116–119]. Hsieh and colleagues showed that cells cultured in 2D exhibit greater drug sensitivity than in vivo, and 3D cultures exhibit chemosensitivities comparable to solid tumors provided that they have similar cell density [120]. Expanding the cellular environment from 2D to 3D has also been shown to affect proliferation and metabolism [120–122]. Using 3D cultures has led to significant advancements in the understanding of cell migration strategies as well, since some of these could not be observed in 2D cultures [123–125]. For instance, fibroblasts have several different migration strategies that are utilized in different microenvironmental conditions, demonstrating cellular plasticity [125]. Such findings prompt questions about the mechano- and bio-sensing abilities of cells in relation to their environments. In this section, we will elaborate on the different methods utilized thus far to improve cell-microenvironment mimicry in the form of hydrogel models, the cellular response to such models in contrast with conventional cell-culture methods, and factors to be considered in crafting a hydrogel model.

**Table 2.** 2D versus 3D cultures [117–119,123,126–128].