Vocal Fold Wound Healing: A Review for Clinicians *Ryan C. Branski, †‡§k{Katherine Verdolini, ‡k{Vlad Sandulache, †‡§{Clark A. Rosen, and ‡§k{#Patricia A. Hebda *New York, New York, and †‡§k{#Pittsburgh, Pennsylvania Summary: The basic science of wound healing is largely omitted from the curriculum of many voice clinicians. This fact is relatively disheartening as most therapeutic manipulation in the realm of laryngology and voice disorders deals with injured tissue. Therefore, the selection of therapeutic tasks for persons with vocal injury should ideally be informed by basic science in wound healing. Recently, several investigators have initiated lines of research to determine the course of vocal fold wound healing and the potential role of therapeutic agents, including behavioral agents. The current review seeks to provide a foundation of basic wound healing science and present the most current data regarding the wound healing process in the vocal folds. Key Words: Injury—Review—Vocal fold—Wound healing. the population has some type of voice dysfunction at any given point in time.1 Although the precise etiologic factors causing these problems remain relatively unknown, a likely cause is the response to injurious stimuli in a preponderance of cases. Injuries to the vocal folds, including phonotrauma as well as mechanical and chemical trauma, often result in changes to the lamina propria, benign vocal fold lesions, or vocal fold scar. This speculation is consistent with results from a recent study suggesting that approximately 22% of patients seeking treatment for voice disorders present with organic vocal fold lesions, resulting in dysphonia.2 Although vocal fold trauma cannot be implicated in all cases, there is a high likelihood that the primary etiology of these lesions is the inherent response to some sort of injury. In these cases, the goal of therapy via surgery or behavioral voice treatment must focus on (1) ceasing the injurious activity and (2) modulating wound healing or managing tissue that has undergone reparative processes. The goal of treatment, therefore, is restoration of the biomechanical function of the INTRODUCTION Voice disorders seem to be the most common communication disorder across the lifespan. Estimates indicate that anywhere from 3% to 9% of Accepted for publication August 10, 2005. From the *Department of Head and Neck Surgery, Memorial Sloan-Kettering Cancer Center, New York, New York; †Department of Communication Science and Disorders, University of Pittsburgh, Pittsburgh, Pennsylvania; ‡Department of Otolaryngology, University of Pittsburgh, Pittsburgh, Pennsylvania; §University of Pittsburgh Voice Center, University of Pittsburgh, Pittsburgh, Pennsylvania; kOtolaryngology Wound Healing Laboratory, Department of Pediatric Otolaryngology, Children’s Hospital of Pittsburgh, Pittsburgh, Pennsylvania; {McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, Pennsylvania; and the #Department of Cell Biology and Physiology, University of Pittsburgh, Pittsburgh, Pennsylvania. Address correspondence and reprint requests to Ryan C. Branski, PhD, Head and Neck Surgery, Memorial SloanKettering Cancer Center, New York, New York 10021. E-mail: branskir@mskcc.org Journal of Voice, Vol. 20, No. 3, pp. 432–442 0892-1997/$32.00 Ó 2006 The Voice Foundation doi:10.1016/j.jvoice.2005.08.005 432 VOCAL FOLD WOUND HEALING tissue. Therefore, treatment should be founded in the science of wound healing. However, discussion of wound healing is typically omitted from curricula of voice care clinicians. The current review seeks to present up-to-date information regarding the general tenets of wound healing as well as emerging literature regarding wound healing in the vocal folds. WOUND HEALING: AN OVERVIEW Wound healing is a dynamic, interactive process involving cells and extracellular matrix. The wound healing cascade, when uninterrupted, is typically organized temporally into three major, overlapping phases: inflammation, extracellular matrix (ECM) deposition and epithelialization, and remodeling.3 Although a gross oversimplification of the wound healing process, these phases serve as a basic framework for discussion. Disruptions in the normal wound healing sequence can produce less-than-desirable outcomes. In cases of impaired wound healing, reestablishment of tissue structure and function is diminished. Excessive and/or prolonged inflammation subsequent to tissue injury has been shown to result in extended tissue damage in conditions such as osteoarthritis. Systemic conditions such as diabetes, Cushing’s syndrome, malnutrition, and sepsis can also disrupt the normal healing process and lead to nonhealing wounds or, at the other end of the spectrum, excessive fibrosis.3 Inflammation Injury causes blood vessel disruption leading to leakage of blood into the wound area. Immediately after injury, the inflammatory response is critical for (1) stopping blood flow at the site of the wound, (2) filling tissue deficits, (3) providing a provisional matrix for subsequent cell migration into the wound bed, (4) sterilization of the wound bed, and (5) signaling other cells to come to the wound region and rebuild the ECM. A fibrin-rich clot plugs damaged vessels, fills tissue deficits, and serves as a provisional matrix for subsequent cellular invasion.4 Inflammatory cells such as macrophages and neutrophils disinfect the wound site by enzymatic clearance of contaminants. Such cells also 433 stimulate reepithelization of the wound and encourage replacement of the extracellular matrix.5 ECM deposition As inflammation subsides, emphasis is switched to fibroblast and epithelial cell infiltration of the wound bed and reconstitution of the ECM (deposition of fibronectin and collagen). Fibroblasts migrate into the wound area between 48 and 72 hours after injury.6 Fibroblasts are responsible for secreting new matrix materials such as collagen and hyaluronic acid in response to injury. Fibroblast activity in the wound bed leads to granulation tissue formation in dermal wounds, approximately 4 days after injury. Granulation tissue has high myofibroblast density, a transitional cell type with properties of both fibroblasts and smooth muscle cells. Myofibroblasts are responsible for wound contraction. Contraction is required to maintain tissue continuity, to reduce the size of the wound, and to facilitate scar production.7 Upon epithelialization (to be described in the next section), granulation tissue resolves. The end product of fibroblast activity is an approximation of the preinjury tissue structure and function. However, although most ECM components are present, they are poorly organized. During wound healing, fibroblasts produce large amounts of collagen and elastin.8 Glycosaminoglycans and proteoglycans are also produced.9 Glycosaminoglycans are large space-filling molecules found in the ECM of many organs including the skin and vocal folds. The specific signals responsible for glycosaminoglycan and proteoglycan production are relatively unknown. However, insight into the stimulus for proteoglycan production, for example, might prove to be useful in antifibrotic therapy. Under certain conditions, wound healing continues unabated with excessive scar formation, resulting in dermal pathologies such as hypertrophic scars and keloids.10 Both are characterized by an increased inflammatory response and ECM production11 as well as an abnormal fibroblast function.12,13 Epithelialization One major goal of wound healing is reconstitution of the epithelium as a functional barrier. Clinically, a wound is reepithelialized when a Journal of Voice, Vol. 20, No. 3, 2006 434 RYAN C. BRANSKI ET AL water-impermeable seal is present at the site of the wound.5 In dermal wound healing, epithelial cell migration into the site of injury after stimulation by growth factors14,15 begins 24 to 48 hours after the injury occurs.16 Tissue remodeling Wound healing and tissue repair continue long after a functional epidermal barrier has been restored. These events are collectively referred to as the remodeling phase of wound healing and refer to the deposition and reorganization of matrix components over time. Scar remodeling is typically thought to be the final stage of the wound healing cascade. Early scar, between 1 to 3 months after injury, is thick and stiff. In contrast, a more mature scar is typically more thin and pliable. This observation has significant implications for tissue elasticity. The pronounced differences between immature and mature scar are thought to be due, at least in part, to the reorganization of collagen fibers along lines of stress as well as to changes in proteoglycan content.17 Collagen content has been found to become relatively stable approximately 21 days after injury. However, both collagen deposition and remodeling are dynamic processes17,18 and are thought to continue up to 12 months after injury demonstrating increased degradation and deposition organized along lines of stress.17 VOCAL FOLD WOUND HEALING The vocal folds, unlike most structures in the body, are subjected to nearly continuous mechanical stresses due to phonation for many waking hours daily. Given this mechanically stressful state, it is surprising that the vocal folds are not structurally and physiologically compromised more often. There are two possible explanations for this apparent resistance to mechanical stress. First, the vocal folds may have an enhanced reparative capacity, by which any microstructural damage to the lamina propria and overlying epithelium can be repaired without a full-scale wound healing response. Second, the microstructure of the vocal fold lamina propria may be organized to accommodate more mechanical stress than a tissue such as dermis. It is likely that both hypotheses are correct given Journal of Voice, Vol. 20, No. 3, 2006 the microstructural elements present in the vocal folds that might explain increased vocal fold accommodation of mechanical stresses. In addition, if vocal folds can accommodate some level of continuous mechanical stress, it stands to reason that a threshold would exist, past which a full-scale wound healing response is required for tissue repair. The current review categorizes vocal fold injury into four types based on the source and chronicity of the injury: acute phonotrauma, chronic phonotrauma, nonphonatory mechanical injury, and chemical injury. Acute phonotrauma It is speculated that acute phonotraumatic injury not only disrupts the vascular network, but it also causes damage to the basement membrane zone and ECM. Clinically, acute phonotrauma manifests as edema of the vocal folds or laryngitis. Many patients may present with focal regions of inflammation at the midpoint of the musculomembranous vocal folds. This site is the region of greatest impact stress and the most common site of mass lesions associated with continued phonotrauma.19 Frequently, edema associated with phonotrauma likely resolves without specific intervention, and voice quality improves. Description of the magnitude and temporal pattern of the acute inflammatory process in the vocal folds may help elucidate the subsequent pathological events resulting in long-term vocal fold damage. Recent studies have described the acute inflammatory response in the vocal folds by measuring levels of inflammatory mediators. Marked shifts in IL-1b, TNF-a, and matrix metalloproteinase-8 levels in secretions collected from the surface of the vocal folds have been reported after an episode of acute phonotrauma.20 These data may prove useful for future investigations into the acute wound healing response in the vocal folds. Chronic phonotrauma Multiple episodes of acute phonotrauma can result in long-standing tissue damage. Although often termed chronic phonotrauma, these episodes likely encompass recurrent acute phonotraumatic (RAP) events. The result is a relatively permanent state of tissue repair or scarring, which may manifest VOCAL FOLD WOUND HEALING as a benign vocal fold lesion(s) and/or vocal fold scar. It is also likely that there is a continuum of responses associated with RAP whereby focal edema gives way to mass lesions and, finally, vocal fold scar based on the chronicity of injury. Mass lesions of the vocal folds are poorly defined. No standardized nomenclature system exists to facilitate both scholarly and clinical communication regarding voice disorders.21 One particular lesion, a fibrous mass, has not yet been described in the literature, but it has gained clinical popularity. Although the histology of this lesion has not been characterized, clinically it is referred to as a unilateral lesion of the midpoint of the musculomembranous vocal folds.22 Entire volumes could be dedicated to the description and nomenclature of benign mass lesions of the vocal folds. A vocal fold scar should not be thought of in terms of more commonly encountered dermal or mucosal scars. Although the dermis and airway mucosa are relatively static structures, the vocal folds are subjected to continuous mechanical stress. As a result, the process of vocal fold scar formation is likely different from that encountered in other tissues. In addition, a continuum of vocal fold scar exists ranging from focal to diffuse and exophitic to endophitic (sulcus vocalis). Three general types of long-standing vocal fold structural abnormalities have been described: nodules, polyps, and cysts. The extent to which each of these pathologies is truly a ‘‘scar’’ is yet unclear.21 Furthermore, the lack of standardized nomenclature for organic vocal fold lesions presents a challenge to describing these entities consistently in the literature.21 Vocal fold nodules are the most commonly diagnosed lesions of the vocal folds and are thought to be a consequence of repetitive of vocal trauma. Nodules are a disruption of the basement membrane zone with separation of the epithelium from the underlying extracellular matrix.23 Kotby et al24 described vocal fold nodules as having intercellular junction gaps, disruption and duplication of the basement membrane zone, and focal collagen deposition. Increased levels of fibronectin have also been identified at sites of nodular lesions.25 It has been hypothesized that the disruption of the basement membrane zone associated with vocal fold 435 nodules may place the tissue at increased risk for repeated injury, resulting in stiffening or scarring of the vocal folds over the long term.23 Polyps are thought to be a more acute vascular injury characterized by less fibronectin deposition and basement membrane zone disruption as compared with nodules.25 Kotby et al24 proposed that nodules and polyps represent a relative continuum of vocal fold injury, with critical variables being the chronicity of the wound and the focal or diffuse nature of the injury. Furthermore, polyps may represent the result of an arrest in the wound healing process involving stasis in the inflammatory phase. In contrast, nodules are likely a result of complete wound healing with deposition of fibronectin as a precursor to scar formation. Vocal fold cysts are typically less commonly encountered than other benign vocal fold lesions. It is unclear whether cysts are a result of a reparative process associated with phonation. Courey et al25 described cysts as having a basement membrane zone thickness between the thickness found for polyps and nodules. Varying cell types may line the lesion. Cysts may be lined with either columnar or squamous epithelium.26 The implications for these findings remain unclear. However, it is speculated that cysts located at the midpoint of the membranous vocal folds are likely due, at least in part, to injury associated with high intracordal impact stress. As with acute vocal fold injury, the use of biochemical markers of the wound healing process may provide some insight into the nature of more long-standing vocal fold lesions. Patients with active epithelial disease such as recurrent respiratory papilloma and squamous cell carcinoma exhibit markedly increased levels of IL-1b, suggesting an active inflammatory response to invasive disease processes.27 In contrast, patients with chronic pathological conditions of the vocal folds (cysts and polyps) do not seem to have significant IL-1b upregulation.27 These data suggest that benign vocal fold lesions represent the end product of the wound healing cascade, or at least an arrest of the process beyond the acute, active inflammatory phase. In addition, patients with established vocal fold lesions seem to have increased prostaglandin-E2 (PGE-2) levels. PGE-2 is an inflammatory mediator and is Journal of Voice, Vol. 20, No. 3, 2006 436 RYAN C. BRANSKI ET AL relatively ubiquitous in wound healing. Markers of wound healing such as PGE-2 be valuable in the description and differentiation of vocal fold lesions.27 Nonphonatory mechanical injury The vocal folds typically withstand regular mechanical trauma associated with phonation. However, the folds are also subjected to nonphonatory mechanical trauma. Examples include endotracheal intubation, phonomicrosurgery, and external laryngeal trauma. Phonomicrosurgery is common for the removal of vocal fold lesions and can result in disruption of the epithelium, basement membrane, and underlying lamina propria depending on the depth of the incision and horizontal involvement of the procedure. In the most severe case that involves complete ablation of the mucosa, a fibrinous clot forms at the site of the wound within 1 day after injury, in a rabbit model. By 3 days after injury, sparse epithelial coverage is present. More importantly, massive cellular infiltration and neo-lamina propria deposition is noted by 3 days after injury. Complete epithelial coverage is achieved by 5 days after injury. Neolamina propria deposition increases significantly 7 days after injury. By 21 days after surgical injury, the trilayered structure of the lamina propria is not yet regained.28 The precise sequence of events that follows surgical injury of the vocal fold can only be elucidated using animal models. Decreased collagen density in the scarred vocal fold as compared with control folds was reported 60 days after forcep biopsy taken from the midpoint of the membranous vocal folds, in a rabbit model.29 The relevant study suggested that scar in the vocal folds was associated with a loss of normal collagen architecture, not increased collagen density. However, that investigation may describe ‘‘immature’’ scar, as new matrix materials may not be stable until after 60 days postinjury. The concentration of many ECM components has been described after vocal fold surgical injury in an animal model. Decreased elastin content has been reported 60 days after surgical injury. Elastin in scarred vocal folds is composed of short, compact fibers. Although hyaluronic acid (HA) was found predominately in the deep lamina propria Journal of Voice, Vol. 20, No. 3, 2006 of the uninjured vocal folds at 60 days, HA was distributed throughout the scarred lamina propria. No significant difference was found between HA density of scarred versus normal vocal folds.29 Thibeault et al30 found significantly decreased levels of decorin 60 days after surgical injury in a rabbit model. Decorin, a proteoglycan found primarily in the superficial layer of the lamina propria, binds collagen and alters the kinetics of fibril formation.31 Decreased levels of decorin have been reported in the hypertrophic scar in the skin.32,33 Decreased decorin levels may be responsible for the altered collagen structure associated with vocal fold scar. In skin, decorin levels increase to subnormal levels over time after burn injuries.32 It is unclear whether this process takes place in vocal folds injury. However, it is likely that decorin levels increase with time as well as tissue motion. Decorin content in other tissues has been shown to increase with exercise.34 Decorin is an interesting vocal fold protein that warrants investigation as a potential antifibrotic agent. In addition, decreased levels of fibromodulin have been found in vocal fold scar (60 days after injury).30 Fibromodulin has been shown to inhibit transforming growth factor-beta (TGF-b)-induced collagen synthesis. Decreased levels of fibromodulin have been implicated in scar formation in the skin.35 However, the findings in the vocal fold are puzzling as an increase in collagen synthesis should correspond with decreased fibromodulin expression. Sixty days after injury, this is not the case, suggesting a potential imbalance in the wound healing response associated with vocal fold scar formation.29 Like decorin, the role of fibromodulin in the scar formation deserves investigation and may be a target for antifibrotic therapies. Not surprisingly, increased levels of fibronectin were reported in immature scar as described by Thibeault et al.30 This increase corresponds with the previous investigation regarding the role of fibronectin in dermal scar formation.36 Fibronectin has been implicated in the formation of adhesion proteins required for epidermal cell/basement membrane attachment as well as epidermal cell migration and replication. Therefore, Thibeault et al30 suggested that increased fibronectin would be expected after injury, supporting epithelialization. VOCAL FOLD WOUND HEALING The authors, however, did not mention these findings in the context of vocal nodules. As mentioned, vocal nodules are characterized by a disruption of the basement membrane zone. Therefore, it is a logical assumption that increased fibronectin depositions at the site of nodules are a component of the reparative process associated with reattachment of the epidermal/basement membrane zone complex. Rousseau et al37 described the development of a vocal fold scar 6 months after surgical injury. As early as 2 months after surgical removal of the epithelium and lamina propria in a canine model, no significant difference in collagen density was noted. Instead, the collagen was arranged in thick bundles of disorganized fibers. At 6 months after injury, collagen density was significantly increased in surgically injured as compared with normal vocal folds.37 Although animal models can be used to investigate the sequence of wound healing events that accompany vocal fold injury, human studies will be required for subsequent translation to clinical management choices. For obvious reasons, human studies cannot involve a precise examination of vocal fold micro-architectural changes during wound healing. Noninvasive techniques for monitoring wound healing hold the best promise for human studies. Recent studies have attempted to monitor the inflammatory phase of vocal fold repair using a noninvasive technique. Branski et al38 reported differential temporal expression of IL-1b and PGE-2 after surgical injury in a rabbit model. Maximal expression of IL-1b occurred 1 day after injury. Resolution to baseline levels was observed by seven days after injury. In contrast, PGE-2 did not significantly increase immediately after injury. Maximal PGE-2 expression occurred 7 days after injury. Preinjury levels were not achieved by 21 days after injury, the endpoint of the study. This type of investigation may yield insight into the wound healing process and provide therapeutic targets to minimize scar formation. Chemical/thermal injury Given their role of gatekeepers to the airway, the vocal folds are exposed to numerous irritants. Clinically, the most common agents of this type are cigarette smoke, inhaler treatment for asthma, 437 and laryngopharyngeal reflux. However, several reports in the laryngology literature also describe thermal injuries associated with the aspiration of hot liquid.39,40 In addition, there are emerging reports of upper airway burns from cocaine pipe screen ingestion.41,42 Obviously, vocal function is not the primary target of therapy in severe cases that may yield impaired respiratory function. More commonly, patients’ voice complaints seem to be related to exposure to airborne irritants such as cigarette smoke.43 Reinke’s edema (RE), a common clinical entity, is associated with prolonged exposure to the irritants in cigarette smoke. Clinically, patients with this condition present with vast, diffuse edema and erythema of the true vocal folds. RE is associated with hemorrhage, increased fibrin deposition, edematous lakes, and thickening of the basement membrane zone. Reinke’s edema likely represents an arrest in the normal reparative process due to prolonged exposure to inflammatory stimuli.44 In addition to smoke inhalation, the vocal folds are subjected to many other airborne irritants. Most commonly, patients experiencing such exposure present with vocal fold irritation and inflammation associated with prolonged use of inhaled b-agonists and/or steroids for the treatment of restrictive pulmonary disease. Areas of vocal fold hyperemia with plaque-like changes of the vocal fold surface have been noted in patients using combination corticosteroid and bronchodilator therapy.45 It is unclear whether the laryngitis is due to medicinal effects or due to the carrier. In addition, the literature is fraught with case reports of dysphonia associated with chemical fume exposure. Dysphonia has been connected to prolonged exposure to Freon, formaldehyde, and mercury among others. Even the performing arts community is not immune to exposure. Richter et al46 identified potentially toxic substances that may affect professional opera singers. These include formaldehyde, cobalt, aromatic diisocyanates, and other agents. It seems unclear why certain patients may be more susceptible to injury from airborne irritants than others. One hypothesis may be that individual thresholds for the elicitation of a wound healing response in the vocal folds are variable among humans. This notion is completely Journal of Voice, Vol. 20, No. 3, 2006 438 RYAN C. BRANSKI ET AL hypothetical at this point, but it warrants further investigation. Likely the most researched source of chemical vocal fold injury is laryngopharyngeal reflux (LPR). Approximately 50% of patients with laryngeal and voice disorders have pH-documented LPR, gastroesophageal reflux disease, or both.47,48 Clinically, this population presents with diverse symptomology and endoscopic findings. Most commonly, patients present with local edema, which ranges from mild to severe Reinke’s edema, pseudocyst, erythema, subglottic edema, and posterior commissure hypertrophy.49 There is emerging data to suggest that laryngeal epithelial defenses to refluxate are different from those in the esophagus.50 WOUND HEALING THERAPY Phonation is crucial for oral communication, and proper vocal fold function is a requisite of phonation. Therefore, the clinician interested in addressing a phonation deficit associated with vocal fold trauma must primarily address the structural issues associated with vocal fold injury, or attempt to control the vocal fold wound healing process. To date, no studies or methods have demonstrated a consistent method for controlling vocal fold wound healing. Historically, rest has been the primary treatment prescribed for most voice problems. A recent study has partially validated this approach. The authors reported more rapid reestablishment of the basement membrane zone in a canine model of surgical injury after a voice rest condition as compared with a phonation condition.51 However, poor patient compliance along with the social ramifications associated with aphonia limit voice rest as a therapeutic option in many cases. In addition, there is emerging evidence to suggest that low levels of mechanical stress may attenuate the inflammatory response in other tissue. To address these issues, ongoing studies are attempting to determine whether moderate voice use involving large-amplitude, low-impact stress vocal fold oscillations typically described as Resonant Voice can result in improved wound healing outcomes. Specifically, vocalization under the direction of a speech pathologist may allow for the requisite communication while Journal of Voice, Vol. 20, No. 3, 2006 facilitating optimal tissue mechanics. In addition to the potential antiinflammatory actions, the long-term tissue organization after injury may be altered in response to mechanical stress.52 Investigation is currently underway to examine these issues in the vocal folds. In the case of an established scar, the orthopedic literature suggests utility in prolonged stretching or the application of tension to the fibrotic region to regain functional mobility at the wound site. Arem and Madden53 were the first to describe scar elongation after low-load prolonged stress (LLPS). Several studies have since attempted to determine the optimal duration of LLPS in the form of stretch or splinting to yield the best outcomes. Brand54 suggested that holding the tissue in a moderately lengthened position for a ‘‘significant time’’ should induce functionally positive scar remodeling. Although ‘‘significant time’’ is not specific, it has spawned investigation to determine the duration of tension that yields optimal outcomes. This time is referred to as total end range time (TERT). It seems that end range must be maintained for at least 6 to 12 hours per day in order to receive maximal improvement.55,56 Specifically, Prosser56 reported that a mean TERT of 10 hours daily yielded functionally superior results compared with less time spent at end range. These data suggest the potential for the application of such techniques to the vocal folds through the use of prolonged vocal fold elongation associated with high pitches. However, the time and specific tasks required for optimal tissue outcomes remain unknown. Although voice therapy may be able to influence the acute inflammatory component of vocal fold wound healing, such therapy still relies heavily on appropriate patient compliance. An alternative approach to behavioral exercises is the chemical inhibition of the inflammatory phase of acute vocal fold injury. A long-standing technique in this regard involves the use of steroids. Systemic corticosteroids, either intramuscular or oral, are commonly prescribed to reduce vocal fold inflammation associated with prolonged phonotrauma in high-caliber voice users whose careers may be limited by the presence of subtle vocal fold edema. A case report in 2001 found marked improvement in vocal function after a 6-day course of oral methyl VOCAL FOLD WOUND HEALING prednisolone in a male professional singer.57 Several investigators have shown a marked reduction in vocal fold inflammation after either steroid injection into the lamina propria or even topical treatment with inhalation aerosol.58,59 There may also be some utility for steroid injection in the presence of established vocal fold lesions. In one study, 27 patients underwent endoscopic injection of triamcinolone acetonide to the site of bilateral vocal fold lesions. Postprocedure examination (1 to 3 weeks) revealed complete lesion resolution in 17 patients and marked decrease in lesion size in the remaining 10.60 The described study was poorly controlled and defined, but it supports the concept of targeted therapy to reduce the inflammatory phase after vocal fold injury. However, steroid use, particularly in the pediatric population, remains a controversial issue. As a result, various soluble mediators of wound healing are currently under investigation for their potential for vocal fold therapy. Hepatocyte growth factor (HGF), a powerful antifibrotic agent that modulates collagen formation and TGF-b expression has been considered as a potential therapeutic agent for vocal fold scar.61,62 Investigators have characterized the therapeutic potential of HGF in vivo after acute surgical injury to the vocal folds in a rabbit model. Histological investigation revealed improved wound healing with decreased fibrosis. Rheological assessment revealed that HGF treatment decreased vocal fold stiffness, improved mucosal wave propagation, phonation threshold pressure, vocal efficiency, and glottal closure.63 These findings are thought to be due to the antifibrotic effects of HGF on vocal fold fibroblast activity. In addition, HGF increases hyaluronic acid synthesis and decreases collagen type I synthesis in vocal fold fibroblasts in vitro.64–66 Mitomycin-C, a chemotherapeutic agent, has been used to improve the results of vocal fold wound healing. In addition to chemotherapeutic indications, mitomycin-C has also been shown to limit fibroblast activity and limit fibrosis associated with tracheal injury. This agent is gaining widespread use to prevent restenosis of the upper airway after airway-expanding surgery.67 However, topical application to the vocal folds after surgical injury had negative consequences on vocal fold vibratory 439 behavior as assessed via laryngeal videostroboscopy. As expected, mitomycin-C reduced fibroblast proliferation within the wound area, limiting the connective tissue response after injury.68 Although theoretically sound, the use of mytomycin-C does not seem to yield nonfibrotic, biomechanically sound tissue. Rosen69 suggested that the vocal fold scar is one of the most challenging voice problems clinicians face. The treatment of the scar is difficult due to the significant communication deficits associated with the pathology as well as the inherent difficulties associated with rehabilitation.70 In cases of an established vocal fold scar, several surgical techniques rely primarily on implantation of biomaterials that alter vocal fold pliability, compliance, and volume. A concern is that treatment does not directly augment the wound healing process, and moreover, implantation not only yields a localized vocal fold injury, but also it may elicit an inherent immune response to the injected material. For example, the foreign body response to Teflon injection into the vocal fold has been well documented.71 However, the use of autologous fat seems to elicit a minimal inflammatory response, little epithelial reaction, and minimal unexpected fibrosis.72 Numerous augmentation materials have been described in the literature. Augmentation materials should not only attempt to restore the biomechanical integrity of scarred vocal folds but also elicit a limited immune response that may alter the long-term function of the vocal folds. CONCLUSION Wound healing is a complex process that is partially directed by the structural and functional requirements placed on the tissue type in question. In the vocal fold, wound healing typically occurs in the context of exposure to pathogenic agents and continuous mechanical stresses associated with phonation. In addition, the precise architectural arrangement of the layered ECM presents a unique wound healing scenario. Each of these factors is important in determining the outcome of vocal fold wound healing. Appropriate management of vocal fold injury must account for each of these factors. Specifically, inflammation must be controlled and Journal of Voice, Vol. 20, No. 3, 2006 440 RYAN C. BRANSKI ET AL mechanical injury reduced. Cellular activity resulting in appropriate ECM restoration must be closely monitored and directed. Appropriate therapeutic control of each of these processes should result in optimal vocal fold wound healing. Although the existing vocal fold literature does not offer precise clinical management strategies, great strides have been made in recent years toward developing a comprehensive approach to vocal fold wound healing. Future approaches in the clinic must be based on a thorough understanding of the underlying cellular and molecular processes associated with vocal fold repair. This review offers a small insight into available data regarding the basic science of vocal fold repair. REFERENCES 1. Berry DA, Verdolini K, Montequin DW, Hess MM, Chan RW, Titze IR. A quantitative output-cost ratio in voice production. J Speech Lang Hear Res. 2001;44:29–37. 2. Coyle SM, Weinrich BD, Stemple JC. Shifts in relative prevalence of laryngeal pathology in a treatment-seeking population. J Voice. 2001;15:424–440. 3. Clark RAF. Wound repair. Overview and general considerations. In: Clark RAF, ed. The Molecular and Cellular Biology of Wound Repair. New York: Plenum Press; 1998. 4. Trowbridge HO, Emling RC. Inflammation. 5th ed. Chicago, IL: Quintessence Publishing Co, Inc.; 1997. 5. Hackam DJ, Ford HR. Cellular, biochemical, and clinical aspects of wound healing. Surg Infect. 2002;3:S23–S35. 6. VanLis JM, Kalssbeek GL. Glycosaminoglycans in human skin. Br J Dermatol. 1973;88:355–361. 7. Gabbiani G. The myofibroblast in wound healing and fibrocontractive diseases. J Pathol. 2003;200:500–503. 8. Woodley DT, O’Keefe EJ, Prunieras M. Cutaneous wound healing: a model for cell-matrix interactions. J Am Acad Dermatol. 1985;12:420–433. 9. Kirsner RS, Eaglstein WH. The wound healing process. Dermatologic Clin. 1993;11:629–640. 10. Tredget EE, Nedelec B, Scott PG, Ghahary A. Hypertrophic scars, keloids, and contractures. The cellular and molecular basis for therapy. Surg Clin North Am. 1997; 77:701–730. 11. Bettinger DA, Yager DR, Diegelmann RF, Cohen IK. The effect of TGF-beta on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg. 1996;98:827–833. 12. Gadson PF, Russell JD, Russell SB. Glucocorticoid receptors in human fibroblasts derived from normal dermis and keloid tissue. J Biol Chem. 1984;259:11236–11241. Journal of Voice, Vol. 20, No. 3, 2006 13. Bettinger DA, Yager DR, Diegelmann R, Cohen IK. The effect of TGF-B on keloid fibroblast proliferation and collagen synthesis. Plast Reconstr Surg. 1996;98:827–833. 14. Martin P. Wound healing—aiming for perfect skin. Science. 1997;276:75–81. 15. Werner S. Keratinocyte growth factor: a unique player in epithelial repair processes. Cytokine Growth Factor Rev. 1998;9:153–165. 16. O’Toole EA. Extracellular matrix and keratinocyte migration. Experiment Dermatol. 2001;26:525–530. 17. Ehrlich HP. Collagen considerations in scarring and regenerative repair. In: Longaker MT, ed. Scarless Wound Healing. New York: Marcel Dekker; 2000:99–113. 18. Martins-Green M. The dynamics of cell-ECM interactions with implications for tissue engineering. In: Lanza R, Langer R, Chick W, eds. Principles of Tissue Engineering. New York: R.G. Landes Company; 1997. 19. Jiang JJ, Titze IR. Measurement of vocal fold intraglottal pressure and impact stress. J Voice. 1994;8:132–144. 20. Verdolini K, Rosen CA, Branski RC, Hebda PA. Cytokine and protease shifts in laryngeal secretions associated with phonotrauma. Presented at: Combined Otolaryngology Spring Meetings, American Bronchoesophageal Association; Nashville, TN; 2003. 21. Rosen CA, Murry T. Nomenclature of voice disorders and vocal pathology. In: Rosen CA, Murry T, eds. The Otolaryngologic Clinics of North America. Philadelphia, PA: W.B. Saunders Company; 2000. 22. Rosen CA, Simpson CB, Postma GA, Courey MA, Sataloff RT. Lumps and bumps: a paradigm for vocal fold pathology. Presented at: Annual Meeting of the Academy of Otolaryngology, Head and Neck Surgery; Orlando, FL; 2003. 23. Gray SD, Hammond EH, Hanson D. Benign pathologic response of the larynx. Ann Otol Rhinol Laryngol. 1995; 104:8–13. 24. Kotby MN, Nassar AM, Seif EI, Helal EH, Saleh MM. Ultrastructural features of vocal fold nodules and polyps. Acta Otolaryngol. 1988;105:477–482. 25. Courey MS, Shohet JA, Scott MA, Ossoff RH. Immunohistochemical characterization of benign laryngeal lesions. Otol Rhinol Laryngol. 1996;105:525–531. 26. Shvero J, Koren R, Hadar T, Yaniv E, Sandbank J, Feinmesser R, Gal R. Clinicopathologica study and classification of vocal cord cysts. Pathol Res Pract. 2000;196: 95–98. 27. Branski RC, Verdolini K, Rosen CA, Hebda PA. Markers of wound healing in vocal fold secretions from patients with laryngeal pathology. Ann Otol Rhinol Laryngol. 2004;113:23–29. 28. Branski RC, Rosen CA, Verdolini K, Hebda PA. Acute vocal fold wound healing in a rabbit model. Ann Otol Rhinol Laryngol. 2005;114:19–24. 29. Thibeault SL, Gray SD, Bless DM, Chan RW, Ford CN. Histologic and rheologic characterization of vocal fold scarring. J Voice. 2002;16:96–104. VOCAL FOLD WOUND HEALING 30. Thibeault SL, Bless DM, Gray SD. Interstitial protein alterations in the rabbit vocal fold with scar. J Voice. 2003;17:376–382. 31. Pawlak AS, Hammond T, Hammond E, Gray SD. Immunocytochemical study of proteoglycans in vocal folds. Ann Otol Rhinol Laryngol. 1996;105:6–11. 32. Sayani K, Dodd CM, Nedelec B, Shen YJ, Ghahary A, Tredget EE, Scott PG. Delayed appearance of decorin in healing burn scars. Histopathology. 2000;36:262–272. 33. Scott PG, Dodd CM, Tredget EE, Ghahary A, Rahemtulla F. Immunohistochemical localization of the proteoglycans decorin, biglycan and versican and transforming growth factor-beta in post-burn hypertrophic and mature scars. Histopathology. 1995;26:423–431. 34. Visser NA, de Koning MH, Lammi MJ, Hakkinen T, Tammi M, van Kampen GP. Increase of decorin content in articular cartilage following running. Connect Tissue Res. 1998;37:295–302. 35. Soo C, Hu FY, Zhang X, Wang Y, Beanes SR, Lorenz HP, et al. Differential expression of fibromodulin, a transforming growth factor-B modulator, in fetal skin development and scarless repair. Am J Pathol. 2000; 157:423–433. 36. Kirscher CW, Hendrix MJC. Fibronectin in hypertrophic scars and keloids. Cell Tissue Res. 1983;231:29–37. 37. Rousseau B, Hirano S, Scheidt TD, Welham NV, Thibeault SL, Chan RW, Bless DM. Characterization of vocal fold scarring in a canine model. Laryngoscope. 2003;113:620–627. 38. Branski RC, Rosen CA, Hebda PA, Verdolini K. Cytokine analysis of acute wound healing in the larynx: A rabbit model. Presented at: The Voice Foundation’s 32nd Annual Symposim: Care of the Professional Voice; Philadelphia, PA; 2003. 39. Rosen D, Avishai-Eliner S, Borenstein A, Leviav A, Tabachnik E. Life-threatening burns in toddlers following hot liquid aspiration. Acta Pediatr. 2000;89:1018–1020. 40. Einav S, Braverman I, Yatsiv I, Avital A, Rothschild M. Airway burns and atelectasis in an adolescent following aspiration of molten wax. Ann Otol Rhinol Laryngol. 2000;109:687–689. 41. Haim DY, Lippmann ML, Goldberg SK, Walkenstein MD. The pulmonary complications of crack cocaine. A comprehensive review. Chest. 1995;107:233–240. 42. Ludwig WG, Hoffner RJ. Upper airway burn from crack cocaine pipe screen ingestion. Am J Emerg Med. 1999; 17:108–109. 43. Marcotullio D, Magliulo G, Pezone T. Reinke’s edema and risk factors: clinical and histopathologic aspects. Am J Otolaryngol. 2002;23:81–84. 44. Dikkers FG, Nikkels PG. Benign lesions of the vocal folds: histopathology and phonotrauma. Ann Otol Rhinol Laryngol. 1995;104:698–703. 45. Mirza N, Kasper Schwartz S, Antin-Ozerkis D. Laryngeal findings is users of combination corticosteroid and bronchodilator therapy. Laryngoscope. 2004;114:1566–1569. 441 46. Richter B, Lohle E, Knapp B, Weikert M, SchlomicherThier J, Verdolini K. Harmful substances on the opera stage: possible negative effects on singers’ respiratory tracts. J Voice. 2002;16:72–80. 47. Koufman JA. The otolaryngologic manifestations of gastroesophageal reflux disease (GERD): a clinical investigation of 225 patients using ambulatory 24-hour pH monitoring and an experimental investigation of the role of acid and pepsin in the development of laryngeal injury. Laryngoscope. 1991;101:1–78. 48. Koufman JA, Amin MR, Panetti M. Prevalence of reflux in 113 consecutive patients with laryngeal and voice disorders. Otolaryngol Head Neck Surg. 2000;123:385–388. 49. Belafsky PC. Abnormal endoscopic pharyngeal and laryngeal findings attributable to reflux. Am J Med. 2003;115: 90S–96S. 50. Johnston N, Bulmer D, Gill GA, Panetti M, Ross PE, Pearson JP, et al. Cell biology of laryngeal epithelial defenses in health and disease: further studies. Ann Otol Rhinol Laryngol. 2003;112:481–491. 51. Cho SH, Kim HT, Lee IJ, Kim MS, Park HJ. Influence of phonation on basement membrane zone recovery after phonomicrosurgery: a canine model. Ann Otol Rhinol Laryngol. 2000;109:658–666. 52. Xu Z, Buckley MJ, Evans CH, Agarwal S. Cyclic tensile strain acts as an antagonist of IL-1B actions in chrondrocytes. J Immunol. 2000;165:453–460. 53. Arem A, Madden J. Effects of stress on healing wounds: intermittent noncyclical tension. J Surg Res. 1976;20: 93–102. 54. Brand P. Clinical Mechanics of the Hand. St. Louis, MO: Mosby; 1985. 55. Glasgow C, Wilton J, Tooth L. Optimal daily total end range time for contracture: Resolution in hand splinting. J Hand Ther. 2003;16:207–218. 56. Prosser R. Splinting in the management of proximal interphalangeal joint flexion contracture. J Hand Ther. 1996;9: 378–386. 57. Watts CR, Clark R, Early S. Acoustic measures of phonatory improvement secondary to treatment by oral corticosteriods in a professional singer: a case report. J Voice. 2001;15:115–121. 58. Moesgaard NV, Hojslet PE. Topical treatment of Reinke’s oedema with beclomethasone dipropionate (BDP) inhalation aerosol. J Laryngol Otol. 1987;101:921–924. 59. Tateya I, Omori K, Kojima H, Hirano S, Kaneko K, Ito J. Steroid injection for Reinke’s edema using fiberoptic laryngeal surgery. Acta Otolaryngol. 2003;122:417–420. 60. Tateya I, Omori K, Kojima H, Hirano S, Kaneko K, Ito J. Steroid injection to vocal nodules using fiberoptic laryngeal surgery under topical anesthesia. Eur Arch Oto-rhino-laryngol. 2004;261:489–492. 61. Matsumoto K, Nakamura T. Hepatocyte growth factor (HGF) as a tissue organizer for organogenesis and regeneration. Biochem Biophys Res Comm. 1997;239: 639–644. Journal of Voice, Vol. 20, No. 3, 2006 442 RYAN C. BRANSKI ET AL 62. Bataller R, Brenner DA. Hepatic stellate cells as a target for the treatment of liver fibrosis. Semin Liver Dis. 2001; 21:437–451. 63. Hirano S, Bless DM, Rousseau B, Welham N, Montequin D, Chan RW, Ford CN. Prevention of vocal fold scarring by topical injection of hepatocyte growth factor in a rabbit model. Laryngoscope. 2004;114:548–556. 64. Hirano S, Bless D, Heisey D, Ford C. Roles of hepatocyte growth factor and transforming growth factor B1 in production of extracellular matrix by canine vocal fold fibroblasts. Laryngoscope. 2003;113:144–148. 65. Hirano S, Bless DM, Heisey D, Ford CN. Effect of growth factors on hyaluronan production by canine vocal fold fibroblasts. Ann Otol Rhinol Laryngol. 2003;112:617–624. 66. Hirano S, Bless DM, Massey RJ, Hartig GK, Ford CN. Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor. Ann Otol Rhinol Laryngol. 2003;112:1026–1033. 67. Hartnick CJ, Hartley BE, Lacy PD, Liu J, Bean JA, Willging JP, et al. Topical mitomycin application after laryngotracheal reconstruction: a randomized, double-blind, Journal of Voice, Vol. 20, No. 3, 2006 68. 69. 70. 71. 72. placebo-controlled trial. Arch Otolaryngol Head Neck Surg. 2001;127:1260–1264. Garrett CG, Soto J, Riddick J, Billante CR, Reinisch L. Effect of mitomycin-C on vocal fold healing in a canine model. Ann Otol Rhinol Laryngol. 2001;110:25–30. Rosen CA. Vocal fold scar. In: Rosen CA, Murry T, eds. The Otolaryngologica Clinics of North America. Voice Disorders and Phonosurgery. Philadelphia, PA: W.B. Saunders; 2000. Benninger MS, Alessi D, Archer S, Bastian R, Ford C, Koufman J, et al. Vocal fold scarring: current concepts and management. Otolaryngol Head Neck Surg. 1996; 115:474–482. Lewy RB, Millet D. Immediate local tissue reactions to Teflon vocal cord implants. Laryngoscope. 1978;88: 1339–1342. Duprat Ade AC, Costa HO, Lancelotti C, Ribeiro de Almeida R, Caron R. Histologic behavior of the inflammatory process in autologous fat implantation in rabbit vocal folds. Ann Otol Rhinol Laryngol. 2004; 113:636–640.