Fig. 4.1
The chemical structure of hyaluronic acid
In recent years, HA has aroused the interest of phonosurgeons as its viscoelastic properties, widely studied, are very similar to those of the true human vocal cord.
In addition to the purposes of type augmentation and medialization to correct glottic insufficiency of various origins, HA is used for its adhesive properties and is suggestive of its application to complete interventions where it is useful to avoid excessive scarring (cordotomy and removal of intracordal lesions, releasing of the mucosa, removal of Reinke’s edema).
Numerous scientific papers have been published in recent years that investigate the function of HA.
It is hypothesized that HA influences the biomechanical features, and in particular the viscoelasticity, of the extracellular matrix (ECM). This hypothesis has been widely demonstrated in the past. In a study published by Chan et al., conducted on human vocal cords, a statistically significant reduction of elasticity and increased cordal stiffness are reported after selective degradation of HA with bovine hyaluronidase [27].
It is also believed that HA plays a key role in the healing process of vocal cord damage. To clarify this role, and possibly to use HA for the purpose of biomedical phonosurgery, different animal studies (rabbit, dog, pig) were performed to identify variations in the concentration and distribution of HA after cordal injury.
The density of HA in an animal model of the rabbit in the acute stage of cicatrization was detected as being increased at 3 and 10 days after the cordal damage (with a peak at 5 days) and reduced after 15 days [28]. Similarly, the concentration of HA was reduced after cordectomy in a pig compared with the healthy control group at days 3, 10, and 15 postoperatively.
Its half-life is too short in the natural state to be used in surgery; it is rapidly metabolized in vivo by the hyaluronidase and its half-life is 0.5–4 days.
This explains the considerable efforts made in recent years to try to stabilize the molecule to obtain a material that is more persistent at the injection site.
Historically, at the industrial level, HA was extracted from rooster feathers, structures with a very high concentration of HA (7.5 mg/ml). The collection of feathers and procedures of extraction and purification, however, resulted in long production times with disproportionate costs. Also, in animal tissues, the HA forms complexes with other proteoglycans and is frequently contaminated by degrading enzymes. This makes it difficult to isolate highly purified HA. Alternatively, HA is extracted from some groups of streptococci type A and C, able to create a thick coating of HA to avoid the host’s immune system. This capsule of HA, presumably, performs other functions, including as a barrier against oxidizing reagents released from leukocytes and the ability to migrate through the epithelial layers within the tissues.
Despite these advantages for the pathogenicity of the bacteria, only a few species have acquired the ability to biosynthesize HA: Streptococcus equistimilis, S. pyogenes, S. uberis, and P. multocida. This can probably be explained by the presence of some of the disadvantages of the production of HA: the significant energy consumption and deprivation precursors of sugars with inhibition of cell wall synthesis. Industrially, the HA has been produced through the fermentation of group C streptococci since the early 1980s. Different bacterial strains have been used, including natural bacteria and mutant bacteria, which are high yielders of HA; considerable efforts in recent years have been made to increase the amount of HA obtainable and to produce a HA of a high quality.
Currently, the turning point for the optimal production of HA appears to be represented by the genetic engineering using the genome complete for some streptococci, including S. pyogenes, S. mutans, and S. agalactiae. Further progress in this field is expected from metabolic engineering, which will probably further improve the production of HA.
The purified HA obtained by the methods described above is a highly viscous, hydratable gel with a short half-life such as that present in tissues. In recent years, research has been aimed at changing this natural material and making it usable for biomedical purposes.
The two main techniques used to improve the chemical molecule of HA to obtain a more stable structure are cross-linking and coupling.
Numerous chemical agents have been used for cross-linking, and all the materials thus obtained, in which the two specific groups of chain (carboxylic and N-acetyl) are not altered, are grouped under the name hylan. The agents most frequently used include multifunctional epoxides, photoreactive agents, bis carboiimide, and carbodiimide. Most of the materials thus obtained are gels that are not soluble in water, with better viscosity and chemical stability than purified HA. They are also highly hydratable in aqueous solutions.
The polymers obtained by autocrosslinking are inter- and intramolecular esters of HA in which a portion of the carboxyl group is esterified with hydroxyl groups of the same or different polysaccharide molecules, forming a set of lactones and intermolecular bridges of esters. The level of crosslinking can be modified by modulating the conditions of the reaction.
The second chemical reaction used is coupling, in which specific functional groups are modified by chemical reactions such as esterification, amidation, and sulfation. These changes may, however, have important effects on the chemical–physical–biological features of the original molecule.
We are used to employing a self-crosslinked gel (ACP-based gel; Fidia Advanced Biopolymers, Abano Terme, Padua, Italy) derived from purified HA obtained through bacterial fermentation processes and stabilized with a process of self-crosslinking without the introduction of external bridging molecules. The product is presented as a sterile gel, transparent, highly viscous, and has a fairly prolonged persistence in the tissues; the commercial name is Sinil® gel.
This molecule has specific strong anti-adhesion properties; it also presents a peculiar stickiness that maintains adhesion of the mucosal flaps after phonosurgical operations involving a cordotomy or a scar. The high viscosity makes the injection problematic because of the resistance offered during the passage of fine needles, which are necessary for the hydrodissection of cordal mucosal adhesions. We have therefore developed a particular injection system that allows us to use a needle of 27 G, i.e., the oro-tracheal laryngeal injector (Medtronic, USA) and a syringe of 1 cc in which the piston is replaced with a metal one, which allows adequate force to be applied without bending (Fig. 4.2).
Fig. 4.2
The injection system of hyaluronic acid
Through a three-way junction, the material is poured into a syringe of 1 cc from that containing the Sinil® gel.
For the hydrodissection of cordal mucosal adhesions, in addition to Sinil gel, the Aminogam® Sterile Gel (Errekappa) is used too. It is composed of vials of 2 ml containing sodium hyaluronate at fast absorption, with a lower anti-adhesional and “glue” effect compared with Sinil® gel, but which also contains amino acids (glycine, l-proline, l-leucine, l-lysine) that facilitate the healing processes of the surgical wound. Aminogam® gel can be injected easily even without a high pressure gun; it can also be used in fiberendoscopy, unlike Sinil® gel, which does not pass through the endoscopic flexible needles even with an high pressure gun, because of its high density.
Instead, when a longer lasting mass effect is required, more stable HA needs to be employed. The HA most frequently used for this purpose is Restylane® (Q-Med), which remains at the implant site for about 6 months. We tested different types of Restylane® to find a material that no only lasted a long time, but also had flow characteristics by allowing the use of thin needles and even flexible endoscopy needles.
These features were specific to the omologous collagen Cosmoplast® (Allergan), which is unfortunately no longer commercially available.
We chose the Restylane® Lipp, an HA that is stable at the concentration of 20 mg/ml in sterile vials of 1 ml. As for autologous fat, this material is poured into the gun at high pressure through a three-way junction.
These materials are used for “off label” intracordal heterologous injection, unlike the Vox® implants and Radiesse® Voice; thus, patients should be informed of this when providing informed consent to laryngoplasty injection.
4.2.2 Autologous Fat
Autologous fat is one of the most frequently used materials in laryngoplasty injection, both in Italy and worldwide. Adipose tissue should not be considered exclusively an energetic reserve for the organism, but rather as a vital organ. The vascular stroma of adipose tissue contains a population of pluripotent stem cells of type [29, 30]. There are numerous international studies that investigate the ability of these cells to differentiate [31, 32]. Early works on phonosurgical procedure considered a sampling of the subcutaneous adipose tissue from the lower periumbilical region by using a cannula connected to an aspirator [9] or by using a scalpel to remove “blocks” [33]. After washing the material with a saline solution and drying it on a sterile gauze, adipose tissue was inserted by means of bayonet forceps into the classic high-pressure Brüning’s syringe [34]. Subsequently, Coleman proposed a procedure that, in addition to a greater simplicity, guaranteed lower resorption of the implanted fat [35]. This procedure, with some small changes [36–38], considers:
1.
The preparation of a solution of a local anesthetic and vasoconstrictor as follows: from a bottle of saline 100 cc remove 20 cc of saline and insert two vials of 10 cc of lidocaine 2 %, half a vial with 1 cc of adrenaline 1/1000, 3 cc of aqueous solution of bicarbonate sodium (1 M).
2.
This solution fills two 20-cc syringes with a luer-lock joint in which a needle is inserted (caliber 22 G, length 8 cm). The needle is driven into the inner part of the lower edge of the umbilicus and 30–40 cc of the solution infiltrate the lower subcutaneous periumbilical region, in a triangular area whose summit is the navel and the suprapubic region is the base.
3.
About 20 min should pass to obtain good local anesthesia and vasoconstriction before proceeding to liposuction using a 10-cc syringe with a luer-lock into which a needle is inserted (caliber 14 G, length 8 cm). The needle is driven into the area previously infiltrated with the solution of anesthetic and vasoconstrictor. The original technique of Coleman [35], however, makes use of a metallic cannula with a blunted tip and side opening with sharp edge (Fig. 4.3), which is inserted subcutaneously through a breach created with a scalpel. This gap is then closed with a suture.
Fig. 4.3
Syringe (10 cc) with luer-lock connected to a 14-G needle (top) and the Coleman cannula (bottom)
Liposuction (Fig. 4.4) is carried out with fast to-and-fro movements, while the syringe is held in maximum suction by means of an automatic locking system or by manual continuous pressure (which allows the degree of “tightness” of the syringe to be controlled). Two syringes are filled with subcutaneous adipose tissue mixed with blood and a solution of anesthetic and vasoconstrictor.
Fig. 4.4
Liposuction from the lower subcutaneous periumbilical area
The needle from the two 10-cc syringes filled with the aspirated material is removed and the luer-lock is closed tightly.
4.
The two syringes are then inserted into a centrifuge (opposite one to another to balance the weights) and centrifugation for 3 min at 3000 rpm is performed. In this way separation of the adipose tissue from the serum and red blood cells is achieved.
Holding the syringe upright, the following separate centrifuged layers can be recognized, which are, from top to bottom: the oily component that originates from the lysis of adipocytes, the vital adipocytes, and finally the water content with the cellular component of blood and the local anesthetic injected (Fig. 4.5). The bottom and top layers are removed, leaving only the concentrated fat.
Fig. 4.5
The layers of needle aspiration after centrifugation
5.
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Using a three-way junction the syringe is connected to an injector pistol, leaving the original serum supernatant in the syringe (Fig. 4.6).
Fig. 4.6
Pouring of concentrated fat into the syringe contained in the gun at high pressure (Ricci Maccarini and De Rossi), by means of a three-way junction
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