**2.2.1 Microflap technique**

116 Otolaryngology

Common causes of voice problems include benign vocal fold lesions such as nodules, polyps and cysts. While these lesions are non-cancerous, they may result in impaired vocal fold closure and vibration, and reduction of voice quality. Removal of these lesions is often

Current voice microsurgery techniques are based on Hirano's discovery of the layered structure of the vocal folds (Hirano 1974; Bleach, Milford et al. 1997; James B. Snow and Ballenger 2003). Based on his microscopic work, the vocal fold was found to have three well defined layers - the epithelium, lamina propria and vocalis muscle. The lamina propria was further subdivided into 3 layers, the superficial layer of the lamina propria (SLLP),

In the SLLP, elastin and collagen fibres are loosely arranged within a matrix, whereas dense elastin fibres make up most of the intermediate layer. Collagen is densely packed in the deep layer, providing most of the support for the lamina propria (James B. Snow and Ballenger 2003). Hirano also proposed a cover-body concept, providing an explanation for the vibratory characteristics of the vocal fold. Based on his theory, the cover (consisting of stratified squamous epithelium and the underlying SLLP) is attached to the body (consisting of the vocalis and thyroarytenoid muscles) by an elastic interface or ligament (composed of the intermediate and deep layers of the lamina propria), with an increasing stiffness from superficial to deep. This allows the cover to oscillate independently due to its elastic characteristics, resulting in the mucosal wave seen on stroboscopy and most of the vibratory

Early treatments for benign vocal fold lesions consisted of stripping (de-epithelialization) of the entire vocal fold (Sataloff, Spiegel et al. 1995). The healing process after this method of treatment often resulted in significant vocal fold scar formation which causes a change in the stiffness and viscoelastic layered structure of the lamina propria. This inhibits normal vibration of the vocal fold, and can cause significant dysphonia and possible glottic incompetence. However with the discovery by Hirano of the layered structure of the vocal fold and its implications on healing, treatment is now focused on preserving as much of the normal vocal fold structures as possible (Hochman and Zeitels 2000; Fleming, McGuff et al. 2001; Thekdi and Rosen 2002; Burns, Hillman et al. 2009). Avoiding injury to the deeper structures is important during voice microsurgery to minimize vocal fold scarring and

Current methods in voice microsurgery are divided into two main categories based on the surgical instruments used – either laser surgery or cold surgery. In laser surgery, a CO2 laser is used to ablate tissue and for coagulation of the target region (Yan, Olszewski et al. 2010). Together with a micro-manipulator for precise cutting, the reduced blood loss during laser surgery enables a relatively clear view of the surgical field. Although studies have found no significant difference in surgical outcomes between laser and cold surgery (Zeitels 1996; Hormann, Baker-Schreyer et al. 1999; Benninger 2000), risk of thermal damage to surrounding tissues is still dependent on familiarity with the equipment and surgical

dynamics required for good voice production and phonation (Hirano 1974).

carried out surgically using microlaryngoscopic techniques.

**2. Background** 

**2.1 Structure of the vocal fold** 

intermediate layer and deep layer.

**2.2 Wound creation** 

persistent post-operative hoarseness.

The microflap technique has been accepted as the standard approach for cold surgical removal of benign vocal fold lesions (Ford 1999; Hochman and Zeitels 2000; Lee and Chiang 2009), achieving the main principles of vocal fold surgery by minimal tissue excision, minimal trauma to SLLP and epithelium. This technique typically involves the initial creation of an epithelial incision beside the lesion. Blunt dissection is used to elevate the microflap while taking care to minimise trauma to the deeper layers of the lamina propria. Only pathologic tissue is excised and the microflap is then reapproximated (Sataloff, Spiegel et al. 1995) as seen in Figure 1.

Fig. 1. Microflap technique in practise, (Left) after removal of benign lesion and (right) redraping of microflap.

#### **2.3 Wound closure**

Following excision of the lesion, the microflap is redraped to promote primary healing (Hochman and Zeitels 2000). If there is loss of epithelium or dislodgement of the microflap,

Investigation of Experimental Wound Closure Techniques in Voice Microsurgery 119

sufficient tensile strength to withstand rupture of its bond (Woo 1995). As the vocal folds vibrate at high frequencies during speech, constant shearing against the adhesive glue causes wear and the resultant debris may impede the vibratory properties of the vocal fold

Experimentation on live humans is not possible or ethical in most situations. Cadaveric human larynges are also difficult or expensive to obtain. Hence, when studying a new technique or device, an animal model can provide a systematic platform for the experimentation and validation. However due to differences in vocal fold size and structure,

Depending on the research question to be addressed and the methodological approach, these differences can limit applicability of experimental results. Selection of an appropriate animal model needs careful consideration. Practical issues like size, availability of animal, availability of the facilities to house or carry out the procedures, procurement cost and maintenance of the animal for the duration of the study can restrict researchers from

Characteristics of particular interest when considering operative techniques include the size, shape and position of the larynx and other upper airway structures to simulate surgical access. Similarity of vocal fold shape and location is essential for testing microsurgical techniques, while similar tissue composition is necessary when assessing in-vivo behaviour

Various animal models have been used extensively in vocal fold studies with their results compared across models (Garrett, Coleman et al. 2000; Jiang, Raviv et al. 2001; Titze and Alipour 2006; Alipour and Jaiswal 2007; Alipour and Jaiswal 2008; Bless and Welham 2010; Alipour, Jaiswal et al. 2011). Three of the more commonly used models in operative studies are rabbits (Thibeault, Gray et al. 2002; Thibeault, Bless et al. 2003; Branski, Rosen et al. 2005; Carneiro and Scapini 2009; Campagnolo, Tsuji et al. 2010), dogs (Garrett, Coleman et al. 2000; Fleming, McGuff et al. 2001; Rousseau, Hirano et al. 2003; Karajanagi, Lopez-Guerra et al. 2011) and pigs (Blakeslee, Banks et al. 1995; Garrett, Coleman et al. 2000; Jiang, Raviv et

Due to their docile nature, relatively abundant numbers, ease of housing and management, rabbits are popular animal models. Rabbits are often used in immunological studies and exhibit similar vocal fold histology to humans. However access to rabbit vocal folds by standard suspension laryngoscopy is limited due to the smaller size of the rabbit larynx. Carneiro et al (Carneiro and Scapini 2009) used rabbits to study vocal fold grafts by exposing their vocal folds via a neck incision and laryngofissure. Branski et al (Branski, Rosen et al. 2005) studied the healing process of rabbit vocal fold after injury, using a neonatal laryngoscope to access the vocal fold. Campagnolo et al (Campagnolo, Tsuji et al. 2010) studied the healing effects of injectable corticosteroids after vocal fold surgery using a

or result in secondary intention healing and a broader scar.

one animal may not suit all research requirements.

**3. Selection of animal models** 

acquiring their ideal animal model.

**3.1 Rabbit models** 

custom made laryngoscope for access.

of implanted materials and tissue responses.

al. 2001; Alipour and Jaiswal 2008; Fonseca, Malafaia et al. 2010).

then healing can occur by secondary intention. In this case granulation tissue formation and epithelial migration occur (Woo 1995), and there is correspondingly more scar tissue formation Voice rest is usually prescribed after surgery (Ishikawa and Thibeault 2010), but even with a totally compliant patient, apposition of epithelial flaps edges can be difficult to maintain. Thus various methods like micro-sutures and fibrin glue (Bleach, Milford et al. 1997; Flock 2005; Kitahara, Masuda et al. 2005; Finck, Harmegnies et al. 2010; Skodacek, Arnold et al. 2011) have been used to improve wound closure and minimize scar tissue formation.

#### **2.3.1 Microsutures**

The use of microsutures in vocal fold wound closure was proposed by Woo et al in 1995, hypothesizing that microsutures would allow precise positioning of wound edges and maintenance of the approximation (Woo 1995). This would reduce exposure of the wound site and permit primary healing to occur. They carried out the procedure in 18 patients, finding improved voice results after surgery. As there was no control group and basis for comparison in Woo et al's study, Fleming et al attempted to compare the amount of scar formation with and without microsutures in a canine model (Fleming, McGuff et al. 2001). A small sample group of 4 dogs were used, with bilateral microflap defects created in each dog. 6-0 fast absorbing gut sutures were used to close the microflap on only one side, leaving the contralateral side unclosed. The amount of scar was evaluated between 39 and 49 days post surgery. Un-sutured vocal folds were found to have at around 75% larger scar formation than sutured vocal folds, concurring with Woo et al's hypothesis that the use of microsutures improves postoperative wound healing.

Fleming et al also identified the length of time required for suture placement as the main disadvantage of this technique, suggesting that practice and familiarization with the technique using larger sutures before actual surgery could help mitigate the learning curve.

Tsuji et al recently proposed an improvement to the microsuture technique (Tsuji, Nita et al. 2009) by pre-tying a small length of 4-0 non-absorbable nylon suture to the free end of a 7-0 absorbable suture. The nylon acted as an anchor at the epithelial surface, preventing the thread from escaping and removing the need for an assistant surgeon to maintain tension on the free end of the suture. This improved the ease of performing the technique. Their new technique was tested on human cadaveric larynges for a total of 10 sutures and they reported a placement time of 5 to 7 minutes per suture.

#### **2.3.2 Tissue adhesives**

Despite good wound healing results demonstrated by micro-sutures, many surgeons prefer using adhesives to hold down epithelial flaps to achieve wound closure. Tissue adhesives such as cyanoacrylates and fibrin glue have been used (Flock 2005) and may be easier to apply than that of sutures. A potential limitation of tissue adhesives includes increased scar tissue formation if glue accumulates between the epithelial edges preventing proper approximation, or by adhering the epithelium to the underlying connective tissue without proper reformation of the intervening layered structure. Rapid curing can also restrict the surgeon from re-apposing malpositioned flaps. Lack of tensile strength of the adhesive is another concern. Fibrin glue can take several minutes for initiation of curing and several hours to develop its full strength. Especially during the curing phase it may not possess sufficient tensile strength to withstand rupture of its bond (Woo 1995). As the vocal folds vibrate at high frequencies during speech, constant shearing against the adhesive glue causes wear and the resultant debris may impede the vibratory properties of the vocal fold or result in secondary intention healing and a broader scar.
