**4.1 Using animal models**

120 Otolaryngology

Canine models are used extensively in phonation studies. Comparing vocal fold structure across dogs, monkeys, pigs and human models using histology and laryngeal videostroboscopy, Garrett et al (Garrett, Coleman et al. 2000) found that unlike the human vocal fold, which has a higher elastin concentration in the deeper layers of the lamina propria, both pig and dog had a thin band of elastin concentrated just deep to the epithelial basement membrane zone. Just deep to this thin band, collagen and the elastin were less concentrated as in humans. The mucosal wave on stroboscopy was most similar between humans and canines and it was concluded that dog vocal folds were the most ideal for use in surgical studies due to its similarity in size, histology and mucosal wave. However, Fleming et al (Fleming, McGuff et al. 2001) noted that the slight differences in vocal fold structure like the thicker lamina propria and the lack of a well defined vocal ligament would have implications on its vibratory characteristics. Also, the higher cost and ethical considerations of using a companion animal for experimental studies are practical issues that need to be decided upon. Nevertheless, Fleming et al argued that as canine vocal fold healing was found to be similar to humans and similar human pathological conditions have been found to occur in canine models, they are still suitable for use in vocal fold microsurgery. Hahn et al (Hahn, Kobler et al. 2005; Hahn, Kobler et al. 2006; Hahn, Kobler et al. 2006) also compared collagen and elastin distribution in human, dog, pig and ferret larynges. They found that canine lamina propria collagen levels were most similar to those of humans, but on quantitative histology, elastin and collagen distribution in the human

Pigs are also common models for vocal fold studies. Based on our experience with pig models, the dimensions of the larynx in a 30 to 40 kg pig are similar to that of the adult human (Garrett, Coleman et al. 2000; Jiang, Raviv et al. 2001). The vocal folds have a similar configuration, and the intrinsic muscles and distribution of the recurrent laryngeal nerve is similar as demonstrated by detailed dissections of cadaveric porcine laryngeal neuromuscular anatomy (Knight, McDonald et al. 2005). Other phonatory characteristics such as rotational mobility of the cricothryoid joint, and relative size and innervation of the cricothyroid muscle have also been studied and found to be similar to that of humans (Jiang, Raviv et al. 2001); although

An important difference between the pig and human vocal folds is that the pig has an additional fold in the vertical plane separated by a ventricle. The presence of a superior and inferior fold could relate to the thyroarytenoid muscle having two separate bellies (Knight, McDonald et al. 2005). It has been suggested that the inferior fold is the true vocal fold and the superior fold is akin to the ventricular fold in humans. However this remains a subject for debate as there is a further ventricle above the superior fold. It is suggested that vibration occurs at both folds as well as in the supraglottic structures during phonation

The pig larynx also differs in the structure of the arytenoid complex. The arytenoid cartilages have been described as fused across the posterior commissure, making laryngoscopic exposure more difficult (Garrett, Coleman et al. 2000). In addition to this, the arytenoids are also positioned more superiorly resulting in a steeper angle to the vocal folds.

these features are not of direct relevance to endoscopic laryngeal microsurgery.

lamina propria was best matched by the porcine vocal fold.

(Kurita, Nagata et al. 1983; Alipour and Jaiswal 2008).

**3.2 Canine models** 

**3.3 Porcine models** 

Extensive ex-vivo experiments have been carried out for phonation studies (Regner and Jiang ; Jiang, Zhang et al. 2003; Skodacek, Arnold et al. 2011), for modeling the vibratory dynamics of the vocal folds. These experiments allow precise and independent control of various parameters affecting phonation, enabling systematic investigation and measurements of vocal fold vibrations.

A typical setup of such experimental systems consists of a mounting assembly, a pseudo lung, humidifiers, thermometers, flow and pressure meters. The mounting assembly where the excised larynx is housed consists of one lateral pronged micromanipulator sutured to the anterior tip of the thyroid cartilage and two other micromanipulators attached bilaterally to the arytenoid cartilages. This allows the elongation of the vocal folds to be controlled precisely. Airflow is generated by either an internal building source or a conventional compressor and is conditioned by heaters/humidifiers in order to prevent the larynges from drying out. The excised larynx is clamped directly to a tube from the pseudo lung and flowmeters and pressure meters are used to measure subglottal airflow and pressure before entry to the larynx. This experimental system can be easily adapted for use in ex-vivo surgical experimentation and can provide a platform to assess the effects of surgical procedures on vocal fold vibration.

## **4.2 Mechanical models**

Alternatively, a mechanical model was proposed by Choo et al (Choo, Lau et al. 2010) specifically for the simulation of experiments on the vocal fold. In their design, they proposed the use of agarose as a material substitute for human vocal folds, mapping the mechanical properties of agar concentrations to that of vocal fold cover and ligament. By repeated casting of different concentrations of agarose into a mould, the phantom vocal folds were designed to mimic the layered structure of the vocal fold. In addition, vocal fold vibration was actuated externally with the use of vibrators, allowing the control of the vocal fold vibration frequency. Glottal gap and airflow could also be customized.

Investigation of Experimental Wound Closure Techniques in Voice Microsurgery 123

introduction of ligating clips manufactured from novel polymers such as polydioxanone in laparoscopic cholecystectomy helped to address these limitations. These clips are completely absorbed in the process of ester bond hydrolysis over a period of 180 days and the by-products are excreted by urine. Moreover, these clips produce minimal tissue reactivity with good

Earlier investigations using clips constructed from such polymers proved unsuitable for our requirements, as they could not provide adequate structural strength due to the minute size of the clips. As such, we are investigating the potential of using magnesium as the main

There are many reviews on the potential and viability of magnesium as a biomaterial (Pietak, Staiger et al. 2006; Witte, Hort et al. 2008; Zeng, Dietzel et al. 2008). Most of these studies focused on the use of magnesium in orthopaedic implants and bio-absorbable vascular stents, concentrating on improving its mechanical properties by alloying with various elements. Zhang et al. (Zhang and Yang 2008) reported significant improvement of both biocompatibility and mechanical properties with use of Zn as an additional alloying element to Mg-Si. Gu et al. (Gu, Zheng et al. 2009) reported good biocompatibility of magnesium with various alloying elements, recommending Al and Y for stents and Al, Ca, Zn, Sn, Si and Mn for orthopaedic implants. Drynda et al. (Drynda, Hassel et al. 2010) developed and evaluated fluoride coated Mg-Ca alloys for cardiovascular stents, reporting good biocompatibility and better degradation behaviour. However, as pure magnesium has been found to corrode too quickly in the low pH environment of physiological systems, much effort has also been placed into developing alloys or coatings to limit its degradation behaviour (Zeng, Dietzel et al. 2008). Rosalbino et al. (Rosalbino, De Negri et al. 2010) reported improved corrosion behaviour of Mg-Zn-Mn alloys for orthopaedic implants. Kannan et al. (Kannan and Raman 2008) studied the corrosion of AZ series (Al and Zn) magnesium alloys with the further addition of Ca, reporting significantly improved corrosion resistance with a reduction in mechanical properties (15% ultimate tensile strength and 20% elongation before fracture). Zhang et al. (Zhang, Zhang et al. 2009) reported the use of dual layer coatings of hydroxyapatite to considerably slow down the degradation of 99.9% pure magnesium substrates without

Based on the good biocompatibility and healing results demonstrated by these previous studies, we hypothesized that a bio-absorbable magnesium clip will be able to hold the wound site more securely and facilitate better healing as compared to surgical glue adhesives. Furthermore, with a design specifically aimed to reduce technical complexity in achieving apposition of epithelial flaps, a specifically designed prototype applicator could improve the ease of handling and speed of insertion, possibly translating to improved surgical outcomes. Due to the difficulty of simulating the vocal fold environment for both mechanical and bioabsorbability studies, in-vivo experiments were carried out to evaluate

A 30-40 kg pig has upper airway dimensions that provide reasonable approximation to that of an adult human. Using this in-vivo model we were able to approach the larynx

the feasibility of the clips in accordance to an approved protocol.

**5.1 In-vivo evaluation of microclips** 

adhesion and are radiolucent (Klein, Jessup et al. 1994).

bioabsorbable material to construct such microclips.

heat treatment.

Using stroboscopy, Choo et al observed vibratory dynamics in their mechanically driven model similar to that of the mucosal wave in human vocal folds. After simulating a microflap and then subjecting the vocal fold phantom to vibration, cracks were found propagating radially outwards. Both these features suggested that the setup had potential for surgical experimentation.

Fig. 2. Mechanical larynx setup courtesy of Choo et al (Choo, Lau et al. 2010).
