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.

Role of steroids in acute phonotrauma: A basic science investigation.

OBJECTIVES/HYPOTHESIS: Steroids are used for the treatment of laryngitis in vocal performers and other individuals despite the absence of evidence demonstrating their impact on vocal fold inflammation. Our objective was to examine laryngeal secretion cytokine inflammatory profile changes associated with corticosteroid treatment in a human phonotrauma model. STUDY DESIGN: Prospective, individual, randomized, double-blinded, controlled trial. METHODS: Participants included 10 healthy females who were randomized to either treatment with oral hydrocortisone or placebo, each given in three doses over 20 hours after the experimental induction of acute phonotrauma. Cytokines associated with inflammation and healing (interleukin [IL]-1ß, IL-6, IL-10) were measured in laryngeal secretions before and after vocal loading and at 4 and 20 hours after treatment. RESULTS: Proinflammatory mediators IL-1ß and IL-6 were doubled in the controls versus the steroid treatment group at 21 hours following induction of acute vocal fold inflammation. Anti-inflammatory cytokine IL-10 showed a 6.3-fold increase in the steroid treatment group versus the controls, indicating anti-inflammatory modulation by steroid treatment. CONCLUSIONS: This study provides biologic evidence supporting the use of steroids for acute vocal fold inflammation associated with phonotrauma. LEVEL OF EVIDENCE: 1b.

Vocal exercise may attenuate acute vocal fold inflammation.

OBJECTIVES/HYPOTHESES: The objective was to assess the utility of selected "resonant voice" (RV) exercises for the reduction of acute vocal fold inflammation. The hypothesis was that relatively large-amplitude, low-impact vocal fold exercises associated with RV would reduce inflammation more than spontaneous speech (SS) and possibly more than voice rest. STUDY DESIGN: The study design was prospective, randomized, and double blind. METHODS: Nine vocally healthy adults underwent a 1-hour vocal loading procedure, followed by randomization to a SS condition, vocal rest condition, or RV exercise condition. Treatments were monitored in clinic for 4 hours and continued extraclinically until the next morning. At baseline (BL), immediately after loading, after the 4-hour in-clinic treatment, and 24 hours post-BL, secretions were suctioned from the vocal folds bilaterally and submitted to enzyme-linked immunosorbent assay to estimate concentrations of key markers of tissue injury and inflammation: interleukin (IL)-1ß, IL-6, IL-8, tumor necrosis factor α, matrix metalloproteinase (MMP)-8, and IL-10. RESULTS: Complete data sets were obtained for three markers--IL-1ß, IL-6, and MMP-8--for one subject in each treatment condition. For these markers, results were poorest at 24-hour follow-up in the SS condition, sharply improved in the voice rest condition, and was the best in the RV condition. Average results for all markers and responsive subjects with normal BL mediator concentrations revealed an almost identical pattern. CONCLUSIONS: Some forms of tissue mobilization may be useful to attenuate acute vocal fold inflammation.

Cyclooxygenase-2 inhibition for the prevention of subglottic stenosis.

OBJECTIVE: To evaluate the role of targeted cyclooxygenase-2 inhibition in reducing scarring associated with a subglottic airway mucosal injury. DESIGN: Thirty-four New Zealand white rabbits underwent anterior cricothyroidotomy. Subglottic stenosis (SGS) was created by carbon dioxide laser injury. INTERVENTION: Treatment consisted of intraperitoneal injection of celecoxib or vehicle for 4 days. Endoscopies were performed to assess injury and healing. Subglottic mucosal secretions were collected with Gelfoam swabs (Pfizer Inc) before and after injury and at subsequent time points. Animals were humanely killed at 3 or 8 weeks after injury and airways were excised, followed by gross examination and histologic analysis to assess the severity of SGS. Secretions were analyzed for interleukin-1ß, prostaglandin E2 (PGE2), and matrix metalloproteinase-8 by enzyme-linked immunosorbent assays. RESULTS: Endoscopy showed mild to moderate stenosis in the celecoxib group, but mild to severe stenosis in the vehicle group. Histologic assessment confirmed and quantified reduction in stenosis and scarring as well as advanced reepithelialization. In the healing tissue, mucosal thickening (stenosis) was reduced significantly (P = .02) in celecoxib-treated animals compared with those treated with vehicle, at 3 and 8 weeks (decrease in thickness by 32% and 49%, respectively). Collagen density (fibrosis) was also reduced 25% at both 3 and 8 weeks but the difference was not statistically significant (P = .20). Reduced level of PGE2 in the subglottic mucosal secretions was correlated with mucosal thickness at 8 weeks (P = .02). CONCLUSION: Short-duration, anti-inflammatory therapy resulted in reduced stenosis and fibrosis with correlation of PGE2 levels in subglottic mucosal secretions.

Novel rat model of tympanostomy tube otorrhea.

OBJECTIVE: Tympanostomy tube otorrhea (TTO), caused by the presence of pathogenic bacteria in the middle ear, is the most common complication of TT insertion. No studies have described a reproducible animal model of TTO. We aimed to develop a rat model of TTO which, in turn, could be used to assay the levels of TNF-α and IL-1ß through the course of the infection. METHODS: The left Eustachian tubes of 55 male Sprague-Dawley albino rats were occluded with gutta-percha (ETO=Eustachian Tube Occlusion). Middle ear (ME) effusion was ascertained by weekly otomicroscopy. At 3 weeks tympanostomy tubes were placed bilaterally and the MEs were inoculated bilaterally with Streptococcus pneumoniae through the tubes. The rats were randomly assigned to one of two daily ototopical treatments: ciprofloxacin/dexamethasone (CDX) or placebo. The animals in each of the two treatment groups were further divided to receive 1, 2, 5 or 7 days of treatment. The rats were sacrificed after treatment was finished. The rates of otorrhea, positive middle ear (ME) cultures, and levels of TNF-α and IL-1ß in the ME fluid were measured. RESULTS: Left ETO followed by ME inoculation with S. pneumoniae and treatment with placebo resulted in persistent infection (100% culture-positive ME fluid at 10 days) and otorrhea (85.7%). Persistent infection of the left ear was accompanied by significantly elevated the levels of IL-1ß and TNF-α. Ears treated with CDX had lower rates of otorrhea at all time points and lower levels of IL-1ß and TNF-α. CONCLUSIONS: This study is the first to describe a reproducible animal model of acute TTO. Surgical obstruction of the ET, followed by TT placement and ME inoculation with S. pneumoniae induced persistent otorrhea and infection. Both IL-1ß and TNF-α appear to be potential markers of persistent middle ear infection. This novel model may be used in future studies of the pathogenesis and therapy of TTO.

Biosimulation of acute phonotrauma: an extended model.

OBJECTIVES/HYPOTHESIS: Personalized, preemptive, and predictive medicine is a central goal of contemporary medical care. The central aim of the present study was to investigate the utility of mechanistic computational modeling of inflammation and healing to address personalized therapy for patients with acute phonotrauma. STUDY DESIGN: Computer simulation. METHODS: Previously reported agent-based models (ABMs) of acute phonotrauma were extended with additional inflammatory mediators as well as extracellular matrix components. The models were calibrated with empirical data for a panel of biomarkers--interleukin (IL)-1ß, IL-6, IL-8, IL-10, tumor necrosis factor-α and matrix metalloproteinase-8--from individual subjects following experimentally induced phonotrauma and a randomly assigned voice treatment namely voice rest, resonant voice exercise, and spontaneous speech. The models' prediction accuracy for biomarker levels was tested for a 24-hour follow-up time point. RESULTS: The extended ABMs reproduced and predicted trajectories of biomarkers seen in experimental data. The simulation results also agreed qualitatively with various known aspects of inflammation and healing. Model prediction accuracy was generally better following individual-based calibration as compared to population-based calibration. Simulation results also suggested that the special form of vocal fold oscillation in resonant voice may accelerate acute vocal fold healing. CONCLUSIONS: The calibration of inflammation/healing ABMs with subject-specific data appears to optimize the models' prediction accuracy for individual subjects. This translational application of biosimulation might be used to predict individual healing trajectories, the potential effects of different treatment options, and most importantly, provide new understanding of health and healing in the larynx and possibly in other organs and tissues as well.

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.

Subglottic stenosis examined as a fibrotic response to airway injury characterized by altered mucosal fibroblast activity.

OBJECTIVE: To investigate the association between mucosal fibroblast activity and subglottic stenosis (SGS) development. DESIGN: Prospective study of an animal model of SGS. SETTING: Academic research laboratory. SUBJECTS: New Zealand white rabbits were assigned to either the cricothyroidotomy and carbon dioxide laser injury group or the cricothyroidotomy and silver nitrate injury group. Airways were excised for histologic analysis and the establishment of primary fibroblast cultures. Lesions from surgical excision of established SGS and subglottic tissue were used to analyze SGS recurrence. INTERVENTIONS: The subglottis was approached via cricothyroidotomy and was subjected to either carbon dioxide laser or silver nitrate injury before closure. The SGS lesions were excised at 8 to 10 weeks and were used to establish explants for fibroblast culture. The animals underwent recovery for an additional 14 days to follow recurrence of SGS. After 14 days, all the animals were killed humanely, and subglottic tissue was harvested for histologic evaluation. Rates of migration and contraction of SGS and normal airway fibroblasts were assayed using established in vitro methods under basal conditions and with prostaglandin E(2) treatment. MAIN OUTCOME MEASURES: For in vivo studies, injury, healing, and scarring of the mucosa and cartilage were the primary measures. For cultured fibroblast experiments, cellular responses of fibroblasts from normal and stenosed mucosa were compared and contrasted. RESULTS: Mucosal injury resulted in acute fibroplasia and chronic SGS, surgical excision of mature SGS at 8 weeks resulted in rapid recurrence of stenosis, and SGS-derived fibroblasts were relatively refractory to the effects of prostaglandin E(2) on migration and contraction. CONCLUSIONS: Subglottic stenosis represents a fibrotic airway repair process that involves fibroblasts that produce recurrent, excessive scar formation. We suggest that SGS development and recurrence may be partially dictated by altered fibroblast responsiveness to antifibroplastic signals during mucosal repair.

Translational systems biology and voice pathophysiology.

OBJECTIVES/HYPOTHESIS: Personalized medicine has been called upon to tailor healthcare to an individual's needs. Evidence-based medicine (EBM) has advocated using randomized clinical trials with large populations to evaluate treatment effects. However, due to large variations across patients, the results are likely not to apply to an individual patient. We suggest that a complementary, systems biology approach using computational modeling may help tackle biological complexity in order to improve ultimate patient care. The purpose of the article is: 1) to review the pros and cons of EBM, and 2) to discuss the alternative systems biology method and present its utility in clinical voice research. STUDY DESIGN: Tutorial. METHODS: Literature review and discussion. RESULTS: We propose that translational systems biology can address many of the limitations of EBM pertinent to voice and other health care domains, and thus complement current health research models. In particular, recent work using mathematical modeling suggests that systems biology has the ability to quantify the highly complex biologic processes underlying voice pathophysiology. Recent data support the premise that this approach can be applied specifically in the case of phonotrauma and surgically induced vocal fold trauma, and may have particular power to address personalized medicine. CONCLUSIONS: We propose that evidence around vocal health and disease be expanded beyond a population-based method to consider more fully issues of complexity and systems interactions, especially in implementing personalized medicine in voice care and beyond.

Prostaglandin E2 is activated by airway injury and regulates fibroblast cytoskeletal dynamics.

OBJECTIVES/HYPOTHESIS: To characterize the activation of cyclooxygenase (COX)-2/prostaglandin (PG) E2 signaling during airway mucosal repair and its subsequent role during the wound healing process. STUDY DESIGN: Prospective animal study. METHODS: The subglottis was approached via cricothyroidotomy. Sham airways were closed, and wounded airways were subjected to laser injury and closed. Subglottic tissue was harvested at 12 hours, 24 hours, 48 hours, and 72 hours postinjury. Secretions were collected preoperatively and at time of sacrifice. Inflammatory gene expression was analyzed using quantitative reverse transcriptase polymerase chain reaction. Subglottic/tracheal explants were exposed to exogenous IL-1beta in the presence or absence of COX inhibitors. Explant-produced PGE2 levels were assayed using enzyme linked immunoassays. Human airway fibroblast migration and collagen contraction were assayed in the presence or absence of prostaglandin E2. RESULTS: Laser injury triggers a rapid, dose-dependent increase in mucosal IL-1beta and COX-2 gene expression, with an anatomical distribution proportional to the distance from the site of injury. Gene upregulation correlates with dose-dependent increases in PGE2 mucosal secretion levels. Ex vivo analysis indicates IL-1beta is responsible for the activation of the COX-2 / PGE2 pathway. Prostaglandin E2 differentially inhibits airway fibroblast migration and contraction in a specific, dose-dependent manner. CONCLUSIONS: PGE2 is activated during mucosal inflammation and acts to decrease fibroplastic activity in the mucosal wound bed. During subglottic stenosis (SGS) development, the levels of PGE2 generated in response to injury may be insufficient to blunt the intrinsically fibroplastic phenotype of SGS fibroblasts, resulting in excessive scarring.

Repair of the tympanic membrane with urinary bladder matrix.

OBJECTIVES/HYPOTHESIS: To test urinary bladder matrix (UBM) as a potential treatment for tympanic membrane (TM) healing and regeneration. STUDY DESIGN: This prospective pilot study was designed to provide both qualitative and semiquantitative assessment of temporal and spatial healing events in the chinchilla model of chronic TM perforations with and without UBM patching. METHODS: Bilateral myringotomies were performed and repeated as necessary to create subtotal perforations over an 8-week period. Myringoplasty was then performed, with left TMs serving as controls and right TMs receiving UBM patches. TMs were excised at 4 weeks, 8 weeks, and 12 weeks. Fixed tissue samples were characterized for gross morphology, then processed for microscopic evaluation. RESULTS: Chronic perforations were maintained with one or more repeated myringotomies. Although both control and patched TMs were thicker than native tissue, patched TMs were transparent and uniform in thickness without any inclusions. UBM patches were readily degraded and replaced by newly deposited and organized host tissue that recapitulated the native TM layers. CONCLUSIONS: UBM scaffolds were an effective biological scaffold for TM closure and tissue remodeling, leading to thicker than normal anatomy but otherwise normal morphology. Future studies are required to determine functional and temporal outcomes as well as alternative patch orientations. The results show particular promise as a superior alternative means of reconstructing not only chronic TM perforations but also dimeric TMs associated with retraction pockets and atelectasis. Laryngoscope, 2009.

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.

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.

A patient-specific in silico model of inflammation and healing tested in acute vocal fold injury.

The development of personalized medicine is a primary objective of the medical community and increasingly also of funding and registration agencies. Modeling is generally perceived as a key enabling tool to target this goal. Agent-Based Models (ABMs) have previously been used to simulate inflammation at various scales up to the whole-organism level. We extended this approach to the case of a novel, patient-specific ABM that we generated for vocal fold inflammation, with the ultimate goal of identifying individually optimized treatments. ABM simulations reproduced trajectories of inflammatory mediators in laryngeal secretions of individuals subjected to experimental phonotrauma up to 4 hrs post-injury, and predicted the levels of inflammatory mediators 24 hrs post-injury. Subject-specific simulations also predicted different outcomes from behavioral treatment regimens to which subjects had not been exposed. We propose that this translational application of computational modeling could be used to design patient-specific therapies for the larynx, and will serve as a paradigm for future extension to other clinical domains.

Acute and chronic changes in the subglottis induced by graded carbon dioxide laser injury in the rabbit airway.

OBJECTIVE: To investigate the repair process following carbon dioxide laser injury to the upper airway mucosa (UAM) during the development of chronic subglottic stenosis (SGS). DESIGN: Animals were assigned to either sham control (cricothyroidotomy only) or injured (cricothyroidotomy and posterior subglottic laser) groups using various carbon dioxide laser exposures (8, 12, and 16 W) for 4 seconds. SUBJECTS: Twenty-four New Zealand white rabbits. INTERVENTIONS: The subglottis was approached via cricothyroidotomy. Sham control airways were immediately closed, whereas injured airways were subjected to graded carbon dioxide laser exposures prior to closure. Airways were endoscopically monitored preoperatively, postoperatively, and on postoperative days 7, 14, 28, 42, 56, 70, and 84. Animals were killed at 14 and 84 days. Subglottic tissue was harvested for histologic evaluation (reepithelialization, extracellular matrix, vascularity, and inflammation). MAIN OUTCOME MEASURES: Endoscopic visualization and histologic analysis. RESULTS: (1) Increases in UAM thickness (up to 5 times the thickness of normal mucosa) were observed but were limited primarily to the lamina propria. The mucosal epithelium regenerated without chronic changes. Focal areas of cartilage repair were encountered acutely after injury and to a greater extent in the chronic phases of repair. (2) Acutely, the thickened lamina propria comprised poorly organized extracellular matrix components and demonstrated increases in blood vessel size and number. (3) Histologic changes present in the acute phase only partially resolved in progression to chronic SGS. Chronic SGS was characterized by thick collagen fiber bundles extending into the remodeled subglottic cartilage. CONCLUSIONS: The carbon dioxide laser induces acute changes to lamina propria architecture and vascularity that persist chronically. Elucidating responsible signaling pathways may facilitate the development of therapeutic agents to prevent or reduce the formation of SGS.