Biosimulation of inflammation and healing in surgically injured vocal folds.

OBJECTIVES: The pathogenesis of vocal fold scarring is complex and remains to be deciphered. The current study is part of research endeavors aimed at applying systems biology approaches to address the complex biological processes involved in the pathogenesis of vocal fold scarring and other lesions affecting the larynx. METHODS: We developed a computational agent-based model (ABM) to quantitatively characterize multiple cellular and molecular interactions involved in inflammation and healing in vocal fold mucosa after surgical trauma. The ABM was calibrated with empirical data on inflammatory mediators (eg, tumor necrosis factor) and extracellular matrix components (eg, hyaluronan) from published studies on surgical vocal fold injury in the rat population. RESULTS: The simulation results reproduced and predicted trajectories seen in the empirical data from the animals. Moreover, the ABM studies suggested that hyaluronan fragments might be the clinical surrogate of tissue damage, a key variable that in these simulations both is enhanced by and further induces inflammation. CONCLUSIONS: A relatively simple ABM such as the one reported in this study can provide new understanding of laryngeal wound healing and generate working hypotheses for further wet-lab studies.

ELR-negative CXC chemokine CXCL11 (IP-9/I-TAC) facilitates dermal and epidermal maturation during wound repair.

In skin wounds, the chemokine CXCR3 receptor appears to play a key role in coordinating the switch from regeneration of the ontogenically distinct mesenchymal and epithelial compartments toward maturation. However, because CXCR3 equivalently binds four different ELR-devoid CXC chemokines (ie, PF4/CXCL4, IP-10/CXCL10, MIG/CXCL9, and IP-9/CXCL11), we sought to identify the ligand that coordinates epidermal coverage with the maturation of the underlying superficial dermis. Because CXCL11 (IP-9 or I-TAC) is produced by redifferentiating keratinocytes late in the regenerative phase when re-epithelialization is completed and matrix maturation ensues, we generated mice in which an antisense construct (IP-9AS) eliminated IP-9 expression during the wound-healing process. Both full and partial thickness excisional wounds were created and analyzed histologically throughout a 2-month period. Wound healing was impaired in the IP-9AS mice, with a hypercellular and immature dermis noted even after 60 days. Re-epithelialization was delayed with a deficient delineating basement membrane persisting in mice expressing the IP-9AS construct. Provisional matrix components persisted in the dermis, and the mature basement membrane components laminin V and collagen IV were severely diminished. Interestingly, the inflammatory response was not diminished despite IP-9/I-TAC being chemotactic for such cells. We conclude that IP-9 is a key ligand in the CXCR3 signaling system for wound repair, promoting re-epithelialization and modulating the maturation of the superficial dermis.

Delayed reepithelialization and basement membrane regeneration after wounding in mice lacking CXCR3.

Wound healing is a complex, orchestrated series of biological events that is controlled by extracellular components that communicate between cell types to re-establish lost tissue. We have found that signaling by ELR-negative CXC chemokines through their common CXCR3 receptor is critical for dermal maturation during the resolving phase. In addition there needs to be complete maturation of the epidermis and regeneration of a delineating basement membrane for proper functioning. The role of this ligand-receptor system appears confounding as one ligand, CXCL4/(PF4), is present during the initial dissolution and two others, CXCL10/(IP-10) and CXCL11/(IP-9/I-TAC), are expressed by keratinocytes in the later regenerative and resolving phases during which the basement membrane is re-established. We examined CXCR3 signaling role in healing using a mouse lacking this receptor, as all three ligands act solely via the common receptor. Reepithelialization was delayed in CXCR3-deficient mice in both full and partial-thickness excisional wounds. Even at 90 days postwounding, the epidermis of these mice appeared less mature with lower levels of E-cadherin and cytokeratin 18. The underlying basement membrane, a product of both dermal fibroblasts and epidermal keratinocytes, was not fully established with persistent diffuse expression of the matrix components laminin 5, collagen IV, and collagen VII throughout the wound bed. These results suggest that CXCR3 and its ligands play an important role in the re-establishment of the basement membrane and epidermis. These studies further establish the emerging signaling network that involves the CXCR3 chemokine receptor and its ligands as a key regulator of wound repair.

Prostaglandin E2 differentially regulates contraction and structural reorganization of anchored collagen gels by human adult and fetal dermal fibroblasts.

Contraction and remodeling of granulation tissue by fibroblasts is a crucial component of dermal wound healing. Postnatal wounds heal with imperfect repair and scar formation, whereas tissue repair in fetal wounds is regenerative. Prostaglandin E2 (PGE2) modulates the behavior of fibroblasts in the wound bed. This study was designed to investigate the mechanism by which PGE2 regulates an in vitro model of granulation tissue, anchored collagen gels, by human adult and fetal dermal fibroblasts. We hypothesized that PGE2 differentially regulates contraction and remodeling of anchored collagen gels by these fibroblast phenotypes. These results indicate that once tension was generated, fetal fibroblasts exerted lower contractile forces resulting in less collagen contraction. This coincided with less prominent stress fibers, yet fetal fibroblasts were able to substantially remodel the collagen architecture. This mechanism was differentially modulated by PGE2 and was mimicked with a PGE2 receptor agonist, indicating a cyclic adenosine monophosphate (cAMP)-dependent mechanism through the EP2 receptor. However, direct up-regulation of cAMP led to decreases in contraction and remodeling by both fibroblast phenotypes indicating an altered signaling pathway. Therefore, targeting cAMP via the EP2 receptor could potentially decrease adult fibroblast contractile forces to the levels of the fetal fibroblast phenotype in order to decrease dermal scarring.

Identification of a pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), and its co-expression with fibronectin in fetal and postnatal rabbit airway mucosal and skin wounds.

Fibronectin (FN) is a multi-functional, adhesion protein and involved in multi-steps of the wound healing process. Strong evidence suggests that FN protein diversity is controlled by alternative RNA splicing; a coordinated transcription and RNA processing that is development-, age-, and tissue/cell type-regulated. We previously demonstrated that fetal rabbit airway mucosal healing is regenerative and scarless. Expression, regulation, and biological function of the FN gene and various spliced forms in this model are unknown. Airway and skin incisional wounds were made in fetal (gestation days 21-23), weanling (4-6 weeks) and adult (>6 months) rabbits. Non-wounded and wounded tissues were collected at 12 h (all age groups), 24 h and 48 h (weanling only) post-wounding. Expression profiles were obtained using mRNA differential display and cDNAs of interest were cloned, sequenced and validated by real-time PCR. Here, we report two rabbit cDNAs that showed similar expression patterns after wounding. One encodes a rabbit fibronectin gene, Fn1, and another shares a high sequence homology to a human pre-mRNA splicing factor, arginine/serine-rich 3 (Sfrs3), coding for a RNA binding protein, SRp20. Both Fn1 and Sfrs3 mRNAs were suppressed in fetal wounds but induced in postnatal wounds 12 h post-wounding. The increased levels of both Fn1 and Sfrs3 transcripts were sustained up to 48 h in weanling airway mucosal wounds. The augmentations of the two genes in postnatal airway mucosal wounds were more prominent than that in skin wounds, indicating that the involvement of Sfrs3 and Fn1 genes in postnatal airway mucosal wounds is tissue-specific. Literature provides evidence that SRp20 is indeed involved in the alternative splicing of FN and that the embryonic FN variants reappear during adult wound healing. A connection between the enhanced molecular activity of Sfrs3 and the regulation of the FN gene expression through alternative splicing during the early events of postnatal airway mucosal wound repair was proposed.

Fetal dermal fibroblasts retain a hyperactive migratory and contractile phenotype under 2-and 3-dimensional constraints compared to normal adult fibroblasts.

Fetal dermal fibroblasts participate in a dramatically different wound healing process compared to their adult counterparts, and it is thought that their intrinsic phenotype contributes to the unique properties of fetal repair. In particular, fibroblast migratory and contractile properties have been shown to be important in the development or lack of fibrosis/scarring. Despite extensive study to date, and multiple experimental techniques utilized by various laboratories, the precise differences between fetal and adult dermal fibroblasts remain unclear. We characterized the migratory and contractile dynamics of fetal dermal fibroblasts at the individual cell and population levels under both 2-dimensional (2D) and 3-dimensional (3D) constraints. Data indicate that (1) individual fetal fibroblasts attach and locomote quicker than adult fibroblasts, resulting in faster migration at the population level; (2) use of a 2D bioactive matrix (collagen) dramatically speeds up the transition from attachment to locomotion; and (3) fetal fibroblasts compact 2D collagen matrices faster than adult fibroblasts. These characteristics are maintained inside of a novel 3D construct, which approximates some in vivo tissue repair dynamics. Specifically, fetal fibroblasts invade this construct faster than adult fibroblasts, likely through more dynamic interactions with surrounding collagen fibers. In conclusion, the hyperactive migratory and contractile dynamics of fetal fibroblasts are qualitatively and quantitatively conserved despite transitions from individual cells to whole populations and from 2D to 3D constraints. We conclude that fetal fibroblasts display a robust phenotype, which is only partially altered by changes in substrate and geometric constraints. This phenotype likely is important in dictating the dynamics of fetal tissue repair.

Evaluation of microbial RNA extractions from Streptococcus pneumoniae.

The mechanisms that control Streptococcus pneumoniae's ability to colonize the nasopharynx or to invade the middle ear and cause acute otitis media are not understood. Focused study of these mechanisms requires efficient methods for the extraction of microbial RNA from minute clinical samples. Several lysis/extraction methods were tested and compared to determine the optimal conditions for isolating intact total RNA from pneumococcal cells. The sensitivity and efficiency of the extractions were evaluated by reverse transcription polymerase chain reaction (RT-PCR). Compared to other methods, mechanical homogenization in TRIZOL was the most efficient for releasing microbial RNA, and addition of polyinosinic acid (Poly I) as an RNA carrier increased the assay sensitivity to 10(2) colony forming units when detected by RT-PCR amplification of 16S ribosomal RNA or messenger RNA for penicillin binding protein 2b. Quantitative results were confirmed using a ribonuclease protection assay. Penicillin binding protein 2b was also detected in rat middle ear mucosa recovered 5 weeks after middle ear challenge with S. pneumoniae. This study describes a useful core methodology for use in identifying pneumococcal virulence genes from small titer samples and has promising applications in clinical studies of pneumococcal nasopharyngeal colonization and otitis media pathogenesis.

Effects of ciprofloxacin-dexamethasone on myringotomy wound healing.

OBJECTIVE: To evaluate the effects of the ciprofloxacin-dexamethasone (CDX) combination ototopical treatment after myringotomy on tympanic membrane (TM) healing in ears with eustachian tube obstruction (ETO) and unobstructed ears. STUDY DESIGN: Prospective, randomized, masked, controlled. METHODS: ETO was created in the left ear of 30 rats to induce a model of otitis media with effusion (OME). After 3 weeks, bilateral myringotomy was performed (day 0). Animals were randomized into three groups to receive no treatment or bilateral once daily ototopical treatment with balanced salt solution (BSS, vehicle) or CDX for 13 days. Bilateral otomicroscopy was performed on days 7, 14, and 28. On day 14, five randomly selected animals per group were humanely euthanized and the TM harvested for histology. Three additional rats provided normal negative control ears for histologic comparisons. RESULTS: On day 14, TM perforation healing rates were 100% in all ears of untreated and BSS-treated animals, 89% (8/9) in CDX-treated obstructed ears, and 30% (3/10) in CDX-treated unobstructed ears (P < .05 vs. BSS). On day 28, 100% (5/5) of the CDX-treated unobstructed ears and 80% (4/5) of the CDX-treated obstructed ears were healed. Histology showed initial TM thickening postmyringotomy in all ears but no significant qualitative differences between groups on day 28. CONCLUSION: Myringotomy healing was transiently modulated by treatment with CDX but proceeded normally after CDX discontinuation. This early modulation might enhance middle ear drainage and middle ear concentrations of CDX when tympanostomy tube surgery is performed in patients with active OME and ETO, thus potentially reducing otorrhea and preventing or treating infection. It would not be expected to increase the risk of premature tube extrusion or adversely affect normal healing of the TM after usual spontaneous extrusion.

Age-dependent differential expression of fibronectin variants in skin and airway mucosal wounds.

OBJECTIVE: To delineate age-dependent and tissue-specific molecular activities of the variant-inclusion fibronectin transcripts in fetal and postnatal skin and airway mucosal wounds during early events of the wound healing process. Fibronectin is involved in multiple steps of the wound healing process. The functional complexity of fibronectin is carried through its protein diversity, which is controlled in part by alternative RNA splicing, a coordinated transcription and RNA processing. From a rabbit model of airway mucosal wound healing, we isolated and cloned an RNA splicing factor, SRp20, that was coexpressed with Fn1 complementary DNA and suppressed in fetal wounds but induced in postnatal wounds. Previous studies revealed a link between the inclusion and/or exclusion of the alternatively spliced Fn1 variants (extra domain A [EDA], extra domain B [EDB], and a variable region [V]) and outcomes of wound repair. DESIGN: Skin and airway mucosal incisional wounds were made in fetal (gestational day 21-23), weanling (4-6 weeks), and adult (>6 months) rabbits. Tissues (nonwounded and wounded) were collected at 12 hours (all age groups), 24 hours, and 48 hours (weanling only) after wounding. The expression levels of the 3 Fn1 spliced domain (EDA, EDB, and V)-containing messenger RNA (mRNA) species were assessed by real-time polymerase chain reaction. RESULTS: Fn1 spliced variants were either suppressed or showed no change in fetal skin and airway mucosal wounds 12 hours after injury, whereas the spliced mRNAs were induced in postnatal wounds. The augmented molecular activities of Fn1 spliced variants were more prominent in airway mucosal wounds than in skin wounds. Furthermore, the EDA variant was dominantly selected in adult airway mucosal wounds (6-fold increase), which was strikingly different from the adult skin wounds (1-fold). CONCLUSION: Our study suggests that the age-dependent activation of Fn1-EDA mRNA may play a fundamental role in differentiating fetal wound regeneration from postnatal wound scar formation during the early events of airway mucosal wound healing.

Graded carbon dioxide laser-induced subglottic injury in the rabbit model.

OBJECTIVE: To conduct an endoscopic and histologic analysis of the subglottic effects of various carbon dioxide laser-induced injuries in the rabbit model. DESIGN: Animals were assigned to either a control (cricothyroidotomy only) group or 4 (cricothyroidotomy and posterior subglottic laser) groups that were injured using varying systematically controlled carbon dioxide laser power exposures (5 W, 8 W, and 12 W), with durations of 2 or 4 seconds, and surface area exposures (25% or 40%). SUBJECTS: Twenty-seven New Zealand white rabbits. INTERVENTIONS: The subglottis was approached via cricothyroidotomy. Control airways were immediately closed, while injured airways were subjected to graded carbon dioxide laser exposures prior to closure. Airways were endoscopically monitored preoperatively, immediately postoperatively, and on postoperative days 1, 7, 14, and 21, after which the animals were humanely killed and subglottic tissue harvested for histological evaluation. RESULTS: Clinical observation revealed no significantly obstructive (acute) stenosis during the duration of the study. Endoscopic visualization revealed the formation of posterior subglottic scarring. Histological analysis of the mucosa revealed that use of carbon dioxide laser resulted in a statistically significant (unpaired, 2-tailed t test, P<.05) proportional thickening of the lamina propria layer, without significant changes in the epithelial and cartilaginous layers. In addition, mucosal blood vessel size increased proportional to the power of the laser delivered to the area (P<.05). CONCLUSIONS: Carbon dioxide laser-induced injury to the subglottis caused localized scarring, lamina propria thickening, and increased vascularity, which resolved with time and was not associated with significant airway obstruction. This model describes a systematic, controlled, and reproducible method of investigating subglottic injury.

Elucidating the role of interleukin 1beta and prostaglandin E2 in upper airway mucosal wound healing.

OBJECTIVES: To determine whether (1) inflammatory mediators IL-1beta (interleukin 1beta) and prostaglandin E2 (PGE2) in mucosal secretions correlate with subglottic mucosal injury; and (2) mucosal fibroblasts contribute to PGE2 production during mucosal healing. DESIGN: The subglottic mucosa in rabbits was wounded by means of varied carbon dioxide laser power and duration. Subglottic fibroblasts were exposed to IL-1beta and assayed for production of PGE2. SUBJECTS: Thirty-eight New Zealand white rabbits were used. Fibroblasts from normal and pathologic human subglottic tissues were grown in culture. INTERVENTIONS: Subglottic injury was established in 29 rabbits, and 9 rabbits were sham-wounded. Subglottic mucosal secretions were collected at baseline and days 1, 3, 7, 14, and 21 postoperatively and assayed for IL-1beta and PGE2 by enzyme-linked immunosorbent assay. Tissue was analyzed using quantitative polymerase chain reaction. Fibroblast cultures were exposed to IL-1beta and analyzed for PGE2 and its synthetic enzymes. RESULTS: Subglottic injury was associated with increased levels of IL-1beta and PGE2 in secretions. More extensive mucosal injury resulted in higher PGE2 levels at earlier times. Levels of IL-1beta were maximal after lesser damage. Expression of IL-beta and cyclo-oxygenase 2 was elevated after mucosal injury. Fibroblast treatment with IL-1beta resulted in translocation of nuclear factor kappaB, up-regulation of PGE2 synthetic enzymes, and increased production of endogenous PGE2. CONCLUSIONS: Mucosal injury is associated with up-regulation of inflammatory genes and parallel increases in secretion levels of IL-1beta and PGE2, key mediators of inflammation and healing. Subglottic mucosal fibroblasts are a potential source of inflammatory mediators after injury or other trauma.

Dynamic biomechanical strain inhibits IL-1beta-induced inflammation in vocal fold fibroblasts.

Despite the fact that vocal folds are subjected to extensive mechanical forces, the role of mechanical strain in vocal fold wound healing has been overlooked. Recent studies on other tissues have demonstrated that low physiological levels of mechanical forces are beneficial to injured tissues, reduce inflammation, and induce synthesis of matrix-associated proteins essential for enhanced wound healing. In this study, we speculated that mechanical strain of low magnitudes also attenuates the production of inflammatory mediators and alters the extracellular matrix synthesis to augment wound healing in cultured vocal fold fibroblasts. To test this hypothesis, fibroblasts from rabbit vocal folds were isolated and exposed to various magnitudes of cyclic tensile strain (CTS) in the presence or absence of interleukin-1beta (IL-1beta). Results suggest that IL-1beta activates proinflammatory gene transcription in vocal fold fibroblasts. Furthermore, CTS abrogates the IL-1beta-induced proinflammatory gene induction in a magnitude-dependent manner. In addition, CTS blocks IL-1beta-mediated inhibition of collagen type I synthesis, and thereby upregulates collagen synthesis in the presence of IL-1beta. These findings are the first to reveal the potential utility of low levels of mechanical signals in vocal fold wound healing, and support the emerging on vivo data suggesting beneficial effects of vocal exercise on acute phonotrauma.

The Contractile Phenotype of Dermal Fetal Fibroblasts in Scarless Wound Healing.

PURPOSE OF REVIEW: Injured skin in the mammalian fetus can heal regeneratively due to the ability of fetal fibroblasts to effectively reorganize the extracellular matrix (ECM). This process occurs without fetal fibroblasts differentiating into highly contractile myofibroblasts which cause scarring and fibrosis in adult wounds. Here, we provide a brief review of fetal wound healing and the evidence supporting a unique contractile phenotype in fetal fibroblasts. Furthermore, we discuss the biomechanical role of the ECM in driving myofibroblast differentiation in wound healing and the implications for new clinical modalities based on the biophysical properties of fetal fibroblasts. RECENT FINDINGS: We and others have found that fetal fibroblasts are refractory to the environmental stimuli necessary for myofibroblast differentiation in adult wound healing including mechanical stress. SUMMARY: Understanding the biomechanical mechanisms that regulate the contractile phenotype of fetal fibroblasts may unlock new avenues for anti-scarring therapies that target myofibroblast differentiation of adult fibroblasts.

Primed fibroblasts and exogenous decorin: potential treatments for subacute vocal fold scar.

OBJECTIVE: To investigate hepatocyte growth factor (HGF) primed fibroblasts and decorin application on skin and vocal fold fibroblasts in vitro and in vivo in rabbit vocal fold scar model. STUDY DESIGN AND SETTING: Vocal fold and skin fibroblasts underwent five in vitro treatment conditions: control, epidermal growth factor, HGF, both decorin and HGF, and decorin alone. Hyaluronic acid and collagen enzyme-linked immunosorbent assays were performed. In vivo, 12 rabbits underwent unilateral vocal fold stripping. Injured vocal folds were then injected with skin fibroblasts, HGF, HGF-primed fibroblasts and decorin, or decorin. Outcomes included histologic and lamina propria height analyses. RESULTS: In vitro, HGF increased hyaluronic acid synthesis in vocal fold fibroblasts (P<0.001). HGF and decorin treatment diminished collagen secretion (P<0.01). In vivo, histologic findings indicated minimal difference in collagen amount between treatment groups. CONCLUSION: HGF and decorin together may decrease collagen production by skin and vocal fold fibroblasts. Fibroblast transplantation into scarred vocal folds has equivocal benefit.

Vocal fold wound healing: a review for clinicians.

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.

Adult-fetal fibroblast interactions: effects on cell migration and implications for cell transplantation.

Wound healing is a complex process involving close cooperation between multiple cell types. During wound healing, fibroblasts are primarily responsible for synthesis of the replacement extracellular matrix. Fibroblast therapy is under investigation in this and other laboratories for its potential use to modulate the final outcome of the wound-healing process. This study addresses the potential interactions between transplanted and host fibroblasts, using a two-dimensional mixed culture model. Our results show that fibroblasts of two different phenotypes, fetal and adult, exhibit different speeds of in vitro migration. These migration speeds are conserved in mixed cocultures, suggesting that the migratory response is an intrinsic property of the fibroblast rather than a response to juxtacrine or paracrine signals. These results have relevance for cell-based therapies in that they demonstrate that donor fibroblasts of a different phenotype may at least partially retain that phenotype in the host environment and in the presence of endogenous fibroblasts.

Mucosal biofilm formation on middle-ear mucosa in a nonhuman primate model of chronic suppurative otitis media.

BACKGROUND: An increased awareness of bacterial biofilms and their formation has led to a better understanding of bacterial infections that occur in the middle ear. Perhaps the best studied pathogen for its propensity toward biofilm formation is Pseudomonas aeruginosa, also the primary pathogen in chronic suppurative otitis media (CSOM). OBJECTIVE: The aim of this study was to determine whether P. aeruginosa forms a biofilm in the middle ear in the setting of CSOM in a nonhuman primate model. METHODS: Cynomolgus monkeys underwent perforation of the tympanic membrane and inoculation of the middle ear with a known biofilm-forming strain of P. aeruginosa. The contralateral ear was used as an internal control and was neither perforated nor infected. At the end of the study period, both ears were irrigated to remove planktonic bacteria, and the middle ear mucosa was removed and examined ultrastructurally using scanning electron microscopy (SEM) for determination of the presence or absence of biofilm formation. MAIN OUTCOME MEASURE: The identification of middle ear biofilm containing rod-shaped bacteria. RESULTS: SEM revealed that P. aeruginosa formed bacterial biofilm in vivo on the middle ear mucosal surface, seen only in the infected ear. Interestingly, biofilm formation caused by cocci was also seen in both the experimental as well as the control ear. CONCLUSION: P. aeruginosa forms biofilms in the middle ear in CSOM in primates. To our knowledge, this is the first report of disease-associated bacterial biofilm in a nonhuman primate model of CSOM. Such a model lays a foundation for much needed study into the role of biofilms in the pathophysiology of CSOM. Should CSOM be caused by biofilms, which is uncertain at this time, development of novel strategies for treatment and prevention may be possible. The finding of both rods and cocci forming biofilms also warrants further investigation.

Fibroblast transplantation in the airway: implications for subglottic stenosis.

OBJECTIVE: Because subglottic stenosis (SGS) represents one of the most challenging pathologies confronting the pediatric otolaryngologist, our laboratory is investigating the role fibroblasts play in mucosal scar formation in the course of SGS development. Our objective is to establish cell transplantation into the subglottic mucosal wound bed as a viable tool for examining the cellular processes that underlie the development of SGS. DESIGN: A series of 2 animal experiments, with animals assigned to a control, vehicle-only, or cell-treated group. SETTING: John G. Rangos Sr Research Center, Children's Hospital of Pittsburgh, Pittsburgh, Pa. SUBJECTS: Twenty-six New Zealand white rabbits. This animal model has been well established in the study of SGS formation. INTERVENTIONS: Fluorescently labeled exogenous fibroblasts were transplanted into the injured subglottis of the rabbits. RESULTS: Exogenous fibroblasts derived from fetal and adult dermis and subglottic mucosa were successfully transplanted into the injured subglottic mucosa of adult rabbits. Transplanted fibroblasts survived into the latter stages of wound healing (at 14 and 21 days) and appeared to be associated with a mild inflammatory cell influx and active remodeling of the mucosal wound bed. CONCLUSION: Cell transplantation is a viable tool for the study of fibroblast activity in the mucosal wound bed.