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Objectives: To investigate the role of periostin (POSTN) and the transforming growth factor β (TGF-β) pathway in the formation of laryngotracheal stenosis (LTS) scar fibrosis and to explore the specific signaling mechanism of POSTN-regulated TGF-β pathway in tracheal fibroblasts., Methods: Bioinformatics analysis was performed on scar data sets from the GEO database to preliminarily analyze the involvement of POSTN and TGF-β pathways in fibrosis diseases. Expression of POSTN and TGF-β pathway-related molecules was analyzed in LTS scar tissue at the mRNA and protein levels. The effect of POSTN on the biological behavior of tracheal fibroblasts was studied using plasmid DNA overexpression and siRNA silencing techniques to regulate POSTN expression and observe the activation of TGF-β1 and the regulation of cell proliferation and migration via the TGF-β/RHOA pathway., Results: The bioinformatics analysis revealed that POSTN and the TGF-β pathway are significantly involved in fibrosis diseases. High expression of POSTN and TGF-β/RHOA pathway-related molecules (TGFβ1, RHOA, CTGF, and COL1) was observed in LTS tissue at both mRNA and protein levels. In tracheal fibroblasts, overexpression or silencing of POSTN led to the activation of TGF-β1 and regulation of cell proliferation and migration through the TGF-β/RHOA pathway., Conclusion: POSTN is a key molecule in scar formation in LTS, and it regulates the TGF-β/RHOA pathway to mediate the formation of cicatricial LTS by acting on TGF-β1. This study provides insights into the molecular mechanisms underlying LTS and suggests potential therapeutic targets for the treatment of this condition., Level of Evidence: NA Laryngoscope, 134:4078-4087, 2024., (© 2024 The American Laryngological, Rhinological and Otological Society, Inc.)

Aim: We aimed to confirm the inhibitory effect of nicotinamide on fibrotic scar formation following spinal cord injury in mice using functional metabolomics., Methods: We proposed a novel functional metabolomics strategy to establish correlations between gene expression changes and metabolic phenotypes using integrated multi-omics analysis. Through the integration of quantitative metabolites analysis and assessments of differential gene expression, we identified nicotinamide as a functional metabolite capable of inhibiting fibrotic scar formation and confirmed the effect in vivo using a mouse model of spinal cord injury. Furthermore, to mimic fibrosis models in vitro, primary mouse embryonic fibroblasts and spinal cord fibroblasts were stimulated by TGFβ, and the influence of nicotinamide on TGFβ-induced fibrosis-associated genes and its underlying mechanism were examined., Results: Administration of nicotinamide led to a reduction in fibrotic lesion area and promoted functional rehabilitation following spinal cord injury. Nicotinamide effectively downregulated the expression of fibrosis genes, including Col1α1, Vimentin, Col4α1, Col1α2, Fn1, and Acta2, by repressing the TGFβ/SMADs pathway., Conclusion: Our functional metabolomics strategy identified nicotinamide as a metabolite with the potential to inhibit fibrotic scar formation following SCI by suppressing the TGFβ/SMADs signaling. This finding provides new therapeutic strategies and new ideas for clinical treatment., (© 2024 The Author(s). CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Metabolic disorders are risk factors for stroke exacerbating subsequent complications. Rapidly after brain injury, a glial scar forms, preventing excessive inflammation and limiting axonal regeneration. Despite the growing interest in wound healing following brain injury, the formation of a glial scar in the context of metabolic disorders is poorly documented. In this study, we used db/db mice to investigate the impact of metabolic perturbations on brain repair mechanisms, with a focus on glial scarring. First, we confirmed the development of obesity, poor glucose regulation, hyperglycaemia and liver steatosis in these mice. Then, we observed that 3 days after a 30-min middle cerebral artery occlusion (MCAO), db/db mice had larger infarct area compared with their control counterparts. We next investigated reactive gliosis and glial scar formation in db/+ and db/db mice. We demonstrated that astrogliosis and microgliosis were exacerbated 3 days after stroke in db/db mice. Furthermore, we also showed that the synthesis of extracellular matrix (ECM) proteins (i.e., chondroitin sulphate proteoglycan, collagen IV and tenascin C) was increased in db/db mice. Consequently, we demonstrated for the first time that metabolic disorders impair reactive gliosis post-stroke and increase ECM deposition. Given that the damage size is known to influence glial scar, this study now raises the question of the direct impact of hyperglycaemia/obesity on reactive gliosis and glia scar. It paves the way to promote the development of new therapies targeting glial scar formation to improve functional recovery after stroke in the context of metabolic disorders., (© 2024 The Authors. European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Objectives: To aim of the study was to characterize the molecular profile and functional phenotype of idiopathic subglottic stenosis (iSGS)-scar epithelium., Methods: Human tracheal biopsies from iSGS scar (n = 6) and matched non-scar (n = 6) regions were analyzed using single-cell RNA sequencing (scRNA-seq). Separate specimens were used for epithelial cell expansion in vitro to assess average growth rate and functional capabilities using transepithelial-electrical resistance (TEER), fluorescein isothiocyanate-dextran flux permeability assay, ciliary coverage, and cilia beating frequency (CBF). Finally, epithelial tight junction protein expression of cultured cells was quantified using immunoblot assay (n = 4) and immunofluorescence (n = 6)., Results: scRNA-seq analysis revealed a decrease in goblet, ciliated, and basal epithelial cells in the scar iSGS cohort. Furthermore, mRNA expression of proteins E-cadherin, claudin-3, claudin-10, occludin, TJP1, and TJP2 was also reduced (p < 0.001) in scar epithelium. Functional assays demonstrated a decrease in TEER (paired 95% confidence interval [CI], 195.68-890.83 Ω × cm 2 , p < 0.05), an increase in permeability (paired 95% CI, -6116.00 to -1401.99 RFU, p < 0.05), and reduced epithelial coverage (paired 95% CI, 0.1814-1.766, fold change p < 0.05) in iSGS-scar epithelium relative to normal controls. No difference in growth rate (p < 0.05) or CBF was found (paired 95% CI, -2.118 to 3.820 Hz, p > 0.05). Immunoblot assay (paired 95% CI, 0.0367-0.605, p < 0.05) and immunofluorescence (paired 95% CI, 13.748-59.191 mean grey value, p < 0.05) revealed E-cadherin reduction in iSGS-scar epithelium., Conclusion: iSGS-scar epithelium has a dysfunctional barrier and reduced structural protein expression. These results are consistent with dysfunctional epithelium seen in other airway pathology. Further studies are warranted to delineate the causality of epithelial dysfunction on the downstream fibroinflammatory cascade in iSGS., Level of Evidence: NA Laryngoscope, 134:374-381, 2024., (© 2023 The American Laryngological, Rhinological and Otological Society, Inc.)

Aim: To investigate the effect of apigenin on fibrous scar formation after mouse spinal cord injury (SCI)., Methods: The pneumatic impactor strike method was used to establish an SCI model. Mice were intraperitoneally injected with 5 mg/kg or 20 mg/kg apigenin daily for 28 days after SCI. The Basso Mouse Scale (BMS) score, hematoxylin-eosin staining, and immunohistochemical staining were used to assess the effect of apigenin on scar formation and motor function recovery. Western blotting and qRT-PCR were used to detect the expression of fibrosis-related parameters in spinal cord tissue homogenates. NIH-3 T3 cells and mouse primary spinal cord fibroblasts, α-Smooth muscle actin (α-SMA), collagen 1, and fibronectin were used to evaluate apigenin's effect in vitro. Western blotting and immunofluorescence techniques were used to study the effect of apigenin on TGFβ/SMADs signaling., Results: Apigenin inhibited fibrous scar formation in the mouse spinal cord and promoted the recovery of motor function. It reduced the expression of fibroblast-related parameters and increased the content of nerve growth factor in vivo, decreasing myofibroblast activation and collagen fiber formation by inhibiting TGFβ-induced SMAD2/3 phosphorylation and nuclear translocation in vitro., Conclusion: Apigenin inhibits fibrous scar formation after SCI by decreasing fibrosis-related factor expression through TGFβ/SMADs signaling., (© 2022 The Authors. CNS Neuroscience & Therapeutics published by John Wiley & Sons Ltd.)

Adipose-derived stem cells (ASC) are said to have a pivotal role in wound healing. Specifically, ASC-secreted extracellular vesicles (EV) carry diverse cargos such as microRNAs (miRNAs) to participate in the ASC-based therapies. Considering its effects, we aimed to investigate the role of ASC-EVs in the cutaneous wound healing accompanied with the study on the specific cargo-medicated effects on wound healing. Two full-thickness excisional skin wounds were created on mouse dorsum, and wound healing was recorded at the indicated time points followed by histological analysis and immunofluorescence staining for CD31 and α-SMA. Human skin fibroblasts (HSFs) and human microvascular endothelial cells (HMECs) were co-cultured with EVs isolated from ASC (ASC-EVs), respectively, followed by the evaluation of their viability and mobility using CCK-8, scratch test and transwell migration assays. Matrigel-based angiogenesis assays were performed to evaluate vessel-like tube formation by HMECs in vitro. ASC-EVs accelerated the healing of full-thickness skin wounds, increased re-epithelialization and reduced scar thickness whilst enhanced collagen synthesis and angiogenesis in murine models. However, miR-486-5p antagomir abrogated the ASC-EVs-induced effects. Intriguingly, miR-486-5p was found to be highly enriched in ASC-EVs, exhibiting an increase in viability and mobility of HSFs and HMECs and enhanced the angiogenic activities of HMECs. Notably, we also demonstrated that ASC-EVs-secreted miR-486-5p achieved the aforesaid effects through its target gene Sp5. Hence, our results suggest that miR-486-5p released by ASC-EVs could be a critical mediator to develop an ASC-based therapeutic strategy for wound healing., (© 2020 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Scar formation is the most common cause for failure of glaucoma filtration surgery because of increased fibroblast proliferation and activation. We have now examined the effect of Y-27632, a Rho-associated protein kinase (ROCK) inhibitor, on postsurgical scarring formation in human Tenon fibroblasts (HTFs). Collagen gel contraction assay was used to compare contractility activity of Y-27632 with several antiglaucoma drugs. Immunofluorescence and western blotting were used to examine expression of scar formation-related factors. We found that Y-27632 inhibited collagen gel contraction, as well as α-smooth muscle actin and vimentin expression; these were promoted by treatment with latanoprost, timolol, or transforming growth factor (TGF)-β. To investigate the effect of Y-27632 in postsurgical scarring, we mimicked TGF-β secretion by stimulating HTFs with TGF-β prior to Y-27632 treatment. HTFs cultured in the presence of TGF-β significantly increased gel contraction. In contrast, when HTFs were treated with 10μM Y-27632, contraction was significantly inhibited. Furthermore, Y-27632 reduced TGF-β-induced phosphorylation of mitogen-activated protein kinase signalling. These results suggest that ROCK inhibitors may inhibit fibrosis by inhibiting transdifferentiation of Tenon fibroblasts into myofibroblasts and by inhibiting TGF-β signalling after surgery through mitogen-activated protein kinase pathway suppression. These results implicate that ROCK inhibitors may improve outcomes after filtering surgery with a potential antiscarring effect, while latanoprost and timolol may induce fibrosis. SIGNIFICANCE OF THE STUDY: Scar formation is the primary cause of failure after glaucoma filtration surgery. A ROCK inhibitor, Y-27632, has been introduced as a novel potential antiglaucoma treatment to reduce intraocular pressure. The aim of our study was to elucidate the effect of Y-27632 on scarring formation after glaucoma filtration surgery, in direct comparison with other antiglaucoma drugs. Our findings thus suggested that Y-27632 may inhibit fibrosis and improve outcome after glaucoma filtration surgery through inhibition of transdifferentiation of Tenon fibroblasts into myofibroblasts, and the TGF-β and MAPK signalling after surgery, while latanoprost and timolol may induce fibrosis., (© 2019 John Wiley & Sons, Ltd.)

Objective: Management of laryngotracheal stenosis (LTS) remains primarily surgical, with a critical need to identify targets for adjuvant therapy. Laryngotracheal stenosis scar fibroblasts exhibit a profibrotic phenotype with distinct metabolic shifts, including an increased glycolysis/oxidative phosphorylation ratio. This study examines the effects of the glutamine antagonist 6-diazo-5-oxo-l-norleucine (DON) on collagen production, gene expression, proliferation, and metabolism of human LTS-derived fibroblasts in vitro., Method: Paired normal and scar-derived fibroblasts isolated from subglottic and proximal tracheal tissue in patients with iatrogenic laryngotracheal stenosis (iLTS) were cultured. Proliferation rate, gene expression, protein production, and cellular metabolism were assessed in two conditions: 1) fibroblast growth medium, and 2) fibroblast growth medium with 1 × 10 -4 M DON., Results: DON treatment reduced cellular proliferation rate (n = 7, P =  0.0150). Expression of genes collagen 1 and collagen 3 both were reduced (n = 7, P =  0.0102, 0.0143, respectively). Soluble collagen production decreased (n = 7, P =  0.0056). As measured by the rate of extracellular acidification, glycolysis and glycolytic capacity decreased (n = 7, P =  0.0082, 0.0003, respectively). adenosine triphosphate (ATP) production and basal respiration decreased (n = 7, P = 0.0045, 0.0258, respectively), determined by measuring the cellular rate of oxygen consumption., Conclusion: The glutamine antagonist DON reverses profibrotic changes by inhibiting both glycolysis and oxidative phosphorylation in iLTS scar fibroblasts. In contrast to untreated iLTS scar fibroblasts, collagen gene expression, protein production, metabolic rate, and proliferation were significantly reduced. These results suggest DON and/or its derivatives as strong candidates for adjuvant therapy in the management of iatrogenic laryngotracheal stenosis. Enzymes involved in glutamine metabolism inhibited by DON offer targets for future investigation., Level of Evidence: NA. Laryngoscope, 128:E59-E67, 2018., (© 2017 The American Laryngological, Rhinological and Otological Society, Inc.)

Aims: Astroglial-fibrotic scar formation following central nervous system injury can help repair blood-brain barrier and seal the lesion, whereas it also represents a strong barrier for axonal regeneration. Intensive preclinical efforts have been made to eliminate/reduce the inhibitory part and, in the meantime, preserve the beneficial role of astroglial-fibrotic scar., Methods: In this study, we established an in vitro system, in which coculture of astrocytes and meningeal fibroblasts was treated with exogenous transforming growth factor-β1 (TGF-β1) to form astroglial-fibrotic scar-like cell clusters, and thereby evaluated the efficacy of RNAi targeting ephrin-B2 in preventing scar formation from the very beginning. We further tested the effect of RNAi-based mitigation of astroglial-fibrotic scar on spinal axon outgrowth on a custom-made microfluidic platform., Results: We found that siRNA targeting ephrin-B2 significantly reduced both the number and the diameter of cell clusters induced by TGF-β1 and diminished the expression of aggrecan and versican in the coculture, and allowed for significantly longer extension of outgrowing spinal cord axons into astroglial-fibrotic scar as assessed on the microfluidic platform., Conclusions: These results suggest that astroglial-fibrotic scar formation and particularly the expression of aggrecan and versican could be mitigated by ephrin-B2 specific siRNA, thus improving the microenvironment for spinal axon regeneration., (© 2017 John Wiley & Sons Ltd.)

The intermolecular recognition and interaction between human transforming growth factor β-1 (TGF-β1) and its cognate receptor TβRII have been implicated in the pathological condition of hypertrophic scarring (HS). Here, we attempted to rationally derive peptide inhibitors from the complex interface of TGF-β1 with TβRII to disrupt such interaction for the suppression of fibroblast activation involved in HS. A synthetic strategy that integrated computational design and fluorescence-based assay was described to examine the structural basis and energetic property of TGF-β1-TβRII crystal structure, from which a small peptide segment in the complex binding site was stripped artificially. Molecular dynamics simulations revealed that the linear peptide possesses a large intrinsic disorder that would incur considerable entropy penalty upon binding to TβRII; the peptide segment was then extended and cyclized by introducing a disulfide bond across its terminal residues that were premutated to cysteine. Normal mode analysis indicated that, as expected, the peptide flexibility was largely reduced upon the cyclization, and thus, the entropy penalty was minimized substantially, consequently promoting the spontaneous binding of peptide to TβRII. Fluorescence polarization assay confirmed that all linear peptides are typical non-binders of TβRII (K d  = ND), while the designed cyclic peptides exhibit moderate or high affinity with K d at micromolar level., (© 2017 John Wiley & Sons A/S.)

Reactive astrocytes play an essential role in determining the tissue response to ischaemia. Formation of a glial scar can block the neuronal outgrowth that is required for restoration of damaged tissue. Therefore, regulation of astrocyte activation is important; however, the mediator of this process has not been fully elucidated. Apoptosis signal-regulating kinase 1 (ASK1) is an early responder to oxidative stress, and plays a pivotal role in the intracellular signalling pathway of apoptosis, inflammation, and differentiation. To confirm whether ASK1 mediates astrocyte activation and leads to glial scar formation after cerebral ischaemia, we conducted in vivo and in vitro experiments. C57BL/6 mice were subjected to occlusion of the middle cerebral artery, and astrocyte cultures were exposed to oxygen-glucose deprivation. After silencing of ASK1 , astrocyte-associated genes were downregulated, as seen with the use of microarrays. The glial fibrillary acidic protein (GFAP) level was decreased, and correlated with the reduction in the ASK1 level. In astrocytes, reduction in the ASK1 level decreased the activity of the p38 pathway, and the levels of transcription factors for GFAP and GFAP transcripts after hypoxia. In the chronic phase, ASK1 depletion reduced glial scar formation and conserved neuronal structure, which may lead to better functional recovery. These data suggest that ASK1 may be an important mediator of ischaemia-induced astrocyte activation and scar formation, and could provide a potential therapeutic target for treatment after ischaemic stroke., (© 2016 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Epidural fibrosis might occur after lumbar discectomy and contributes to failed back syndrome. Transforming growth factor (TGF)-β has been reported to influence multiple organ fibrosis, in which connective tissue growth factor/cysteine-rich 61/nephroblastoma overexpressed 2 (CCN2) and CCN5 are involved. However, the effect of CCN2 and CCN5 on TGF-β induced fibrosis has not yet been elucidated. This study reports that CCN2 and CCN5 play opposing roles in cell proliferation and transdifferentiation of human skin fibroblasts or rabbit epidural scar-derived fibroblasts exposed to TGF-β. We observed that TGF-β1 induced fibroblasts proliferation and differentiation in a dose-dependent manner (from 0 μg/L to 20 μg/L). Meanwhile, CCN2 expression is up-regulated while CCN5 expression is inhibited by TGF-β1 exposure. Furthermore, it is demonstrated that CCN2 overexpression leads to promoted proliferation and elevated collagen and α-smooth muscle actin (α-SMA) expression, which are inhibited by CCN5 overexpression. Moreover, it is shown that the cysteine knot (CT) domain, present in CCN2 but absent in CCN5, plays an essential part in fibroblast proliferation and differentiation. Additionally, enhanced TGF-β and CCN2 expression but decreased CCN5 expression is found in rabbit epidural scar-derived fibroblasts. Overall, the results show the opposing effects of CCN2 and CCN5 on fibroblast proliferation and transdifferentiation induced by TGF-β., (© 2015 Wiley Publishing Asia Pty Ltd.)

Failure of surgery for glaucoma is usually due to post-surgical scarring (fibrosis), a process in which fibroblasts play a prominent role. We investigated the molecular mechanisms of such scarring by examining the expression of matrix metalloproteinases and cytokines in Tenon fibroblasts isolated from rats after glaucoma surgery. Filtration surgery was performed in one eye and implant surgery in the other; and Tenon fibroblasts were isolated from the tissue surrounding the bleb after surgery. The cells were cultured and examined for the expression of matrix metalloproteinases (MMPs) by reverse transcription-polymerase chain reaction, immunoblot and gelatin zymographic analyses. Culture supernatants were also assayed for cytokines with a multiplex array. The amounts of MMP-1 and MMP-3 mRNAs and proteins were greater in cells isolated after implant surgery than in those isolated after filtration surgery, with the progression of scar formation being more complete after the former surgery. The secretion of interleukin-6 (IL-6) by cells isolated after filtration surgery was greater than that for cells isolated after implant surgery. Depletion of IL-6 by RNA interference in cells isolated after filtration surgery increased the expression of MMP-1 and MMP-3 in these cells. These results thus suggest that the expression of MMP-1 and MMP-3 in Tenon fibroblasts is regulated by IL-6 during, and may play an important role in, scar formation after glaucoma surgery., (Copyright © 2012 John Wiley & Sons, Ltd.)

Objectives/hypothesis: In vitro modeling of cell-matrix interactions that occur during human vocal fold scarring is uncommon, as primary human vocal fold scar fibroblast cell lines are difficult to acquire. The purpose of this study was to characterize morphologic features, growth kinetics, contractile properties, α-smooth muscle actin (α-SMA) protein expression and gene expression profile of human vocal fold fibroblasts derived from scar (sVFF) relative to normal vocal fold fibroblasts (nVFF)., Study Design: In vitro., Methods: We successfully cultured human vocal fold fibroblasts from tissue explants of scarred vocal folds from a 56-year-old female and compared these to normal fibroblasts from a 59-year-old female. Growth and proliferation were assessed by daily cell counts, and morphology was compared at 60% confluence for 5 days. Gel contraction assays were evaluated after seeding cells within a collagen matrix. α-SMA was measured using western blotting and immunocytochemistry (ICC). Quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) was used to assess differential extracellular matrix gene expression between the two cell types., Results: sVFF were morphologically indistinguishable from nVFF. sVFF maintained significantly lower proliferation rates relative to nVFF on days 3 to 6 (day 3: P = .0138; days 4, 5, and 6: P < .0001). There were no significant differences in contractile properties between the two cell types at any time point (0 hours: P = .70, 24 hours: P = .79, 48 hours: P = .58). ICC and western blot analyses revealed increased expression of α-SMA in sVFF as compared with nVFF at passages 4 and 5, but not at passage 6 (passage 4: P = .006, passage 5: P = .0015, passage 6: P = .8860). Analysis of 84 extracellular matrix genes using qRT-PCR revealed differential expression of 15 genes (P < .01)., Conclusions: nVFF and sVFF displayed differences in proliferation rates, α-SMA expression, and gene expression, whereas no differences were observed in contractile properties or morphology. Further investigation with a larger sample size is necessary to confirm these findings., (Copyright © 2013 The American Laryngological, Rhinological, and Otological Society, Inc.)

Failure of glaucoma filtration surgery (GFS) is commonly attributed to scarring at the surgical site. The human Tenon's fibroblasts (HTFs) are considered the major cell type contributing to the fibrotic response. We previously showed that SPARC (secreted protein, acidic, rich in cysteine) knockout mice had improved surgical success in a murine model of GFS. To understand the mechanisms of SPARC deficiency in delaying subconjunctival fibrosis, we used the gene silencing approach to reduce SPARC expression in HTFs and examined parameters important for wound repair and fibrosis. Mitomycin C-treated HTFs were used for comparison. We demonstrate that SPARC-silenced HTFs showed normal proliferation and negligible cellular necrosis but were impaired in motility and collagen gel contraction. The expression of pro-fibrotic genes including collagen I, MMP-2, MMP-9, MMP-14, IL-8, MCP-1 and TGF-β(2) were also reduced. Importantly, TGF-β(2) failed to induce significant collagen I and fibronectin expressions in the SPARC-silenced HTFs. Together, these data demonstrate that SPARC knockdown in HTFs modulates fibroblast functions important for wound fibrosis and is therefore a promising strategy in the development of anti-scarring therapeutics., (© 2011 The Authors Journal of Cellular and Molecular Medicine © 2011 Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.)

Background: Hyaluronan (HA) is a major component of the extracellular matrix (ECM) with increased synthesis during tissue repair. Tumour necrosis factor-stimulated gene-6 (TSG-6) is known to catalyze the covalent transfer of heavy chains (HC1 and HC2) from inter-α-inhibitor (IαI) onto HA, and resultant HC•HA complexes have been implicated in physiological and pathological processes related to remodelling and inflammation., Objective: The aims of this study were to determine the expression of HA, TSG-6 and the IαI polypeptides in unscarred skin, normal scars and keloid scars., Methods: Formalin-fixed paraffin-embedded sections of unscarred skin, normal scars and keloid scars were prepared from patient samples collected during scar revision surgery. Haematoxylin and eosin, as well as immunofluorescent staining for HA, TSG-6 and the three polypeptide chains of IαI (i.e. HC1, HC2 and bikunin) were performed., Results: All skin types stained positive for TSG-6, HC1, HC2 and bikunin, associated with keratinocytes, fibroblasts and skin appendages all in close proximity to HA. Keloid lesions showed altered HA organization patterns compared with unscarred skin and normal scars. TSG-6 staining was significantly more intense in the epidermis compared with the dermis of all sample types. There was a significant reduction in TSG-6 levels within keloid lesions compared with the dermis of unscarred skin (P=0.017)., Conclusion: TSG-6 is expressed in unscarred skin, where its close association with HA and IαI could give rise to TSG-6-mediated HC•HA formation within this tissue. A reduction in the beneficial effects of TSG-6, caused by diminished protein levels in keloid lesions, could contribute to this abnormal scarring process., (© 2010 The Authors. Journal of the European Academy of Dermatology and Venereology © 2010 European Academy of Dermatology and Venereology.)

Objectives/hypothesis: To explore adipose-derived stem cell/fibroblast interactions as a potential remodeling pathway for vocal fold scar., Study Design: Fibroblasts and adipose-derived stem/stromal cells (ASCs) were cultured alone and in combination in a cell-contact-independent paracrine system. Analyses of cell proliferation, and the production of hyaluronic acid (HA) and collagen were performed on samples collected on days 1, 3, and 7., Methods: Normal fibroblasts (NFs) were isolated bilaterally from the subepithelial lamina propria of two normal ferret vocal folds. Scar fibroblasts (SFs) were isolated from vocal folds that were electrocauterized 2 weeks before harvest. ASCs were isolated from lipoaspirated subcutaneous abdominal fat of two ferrets. A transwell cell-contact-independent cell communication culturing system was used for coculture experiments. Cells were seeded at 50,000/well in both monoculture and coculture experiments., Results: In monoculture, SFs proliferated faster and produced less HA and more collagen than NFs at day 7 (P < .05). In SF/ASC coculture, SF proliferation was diminished and collagen production at day 7 decreased (P < .05). HA production did not differ between monoculture and coculture conditions., Conclusions: Normal and scar-tissue-derived vocal fold fibroblasts maintain phenotypic differences in culture, thus validating this in vitro scar model. In co-culture, contact-independent crosstalk occurs between SFs and ASCa, leading to less collagen secretion. The data support the hypothesis that ASCs can induce favorable remodeling of scarred vocal folds in vivo by their interactions with endogenous fibroblasts.

Background Information: The activation of fibroblasts into myofibroblasts is a crucial event in healing that is linked to remodelling and scar formation, therefore we determined whether regulation of myofibroblast differentiation via integrins might affect wound healing responses in populations of patient-matched HOFs (human oral fibroblasts) compared with HDFs (human dermal fibroblasts)., Results: Both the HOF and HDF cell types underwent TGF-beta1 (transforming growth factor-beta1)-induced myofibroblastic differentiation [upregulation of the expression of alpha-sma (alpha-smooth muscle actin)], although analysis of unstimulated cells indicated that HOFs contained higher basal levels of alpha-sma than HDFs (P<0.05). Functional blocking antibodies against the integrin subunits alpha 5 (fibronectin) or alpha v (vitronectin) were used to determine whether the effects of TGF-beta1 were regulated via integrin signalling pathways. alpha-sma expression in both HOFs and HDFs was down-regulated by antibodies against both alpha 5 and alpha v. Functionally, TGF-beta1 inhibited cell migration in an in vitro wound model and increased the contraction of collagen gels. Greater contraction was evident for HOFs compared with HDFs, both with and without stimulation by TGF-beta1 (P<0.05). When TGF-beta1-stimulated cells were incubated with blocking antibodies against alpha 5 and alpha v, gel contraction was decreased to that of non-stimulated cells; however, blocking alpha v or alpha 5 could not restore cellular migration in both HOFs and HDFs., Conclusions: Despite intrinsic differences in their basal state, the cellular events associated with TGF-beta1-induced myofibroblastic differentiation are common to both HOFs and HDFs, and appear to require differential integrin usage; up-regulation of alpha-sma expression and increases in collagen gel contraction are vitronectin- and fibronectin-receptor-dependent processes, whereas wound re-population is not.

Objectives/hypothesis: The objective was to measure the level of hyaluronan during the first 15 days after vocal fold biopsy in a rabbit model., Study Design: Experimental, nonrandomized prospective study., Methods: Twenty-eight rabbits underwent unilateral vocal fold biopsy. The contralateral vocal fold was preserved as a control sample. On days 3, 5, 10, and 15 after biopsy, hyaluronan levels in the injured and normal vocal folds were measured immunohistologically and with an ELISA assay., Results: Hyaluronan levels in the injured vocal fold were lowest on day 3 and highest on day 5 after injury. Statistical analysis revealed that the injured vocal fold demonstrated significantly lower levels of hyaluronan on day 3 than on days 5, 10, and 15. On day 5, the injured vocal fold demonstrated significantly greater levels of hyaluronan than those observed on days 3, 10, and 15. Compared with the normal vocal fold, the injured vocal fold demonstrated significantly lower levels of hyaluronan on days 3, 10, and 15. No differences were observed between the injured and normal vocal fold on day 5, when the hyaluronan level was maximized in the injured vocal fold., Conclusion: Maximizing hyaluronan levels in the early stages of wound repair may have therapeutic potential for decreasing the incidence of vocal fold scar. Decreased hyaluronan levels may provide a less than optimal environment for normal tissue regeneration and may contribute to the formation of scar tissue in vocal fold lamina propria.

Objectives/hypothesis: The authors sought to determine the role of the plasminogen pathway in wound healing. They hypothesized that decreased fibrin degradation may lead to increased collagen deposition. Presuming that the degree of histopathological abnormality correlates with the aesthetic appearance of the scar, we conducted a study that attempted to determine the histopathological appearance of scar tissue in mice with and without impaired function of the plasminogen pathway., Study Design: Mice with and without deficiencies in the plasminogen pathway underwent surgery. The role of the plasminogen pathway in wound healing was studied by analysis of scar tissue formation using the methods described., Methods: A 2-cm incision was made on the dorsum of mice with and without specified genetic deficiencies in the plasminogen pathway. After the animals were killed, the tissue was harvested, fixed, and prepared using hematoxylin and eosin as well as trichrome stains. Histopathological analysis and scoring were performed by two separate investigators in a blinded manner. Student's t test was used to determine statistical significance between groups., Results: A statistically significant difference in collagen orientation was noted between mice with impaired plasminogen pathway function and the wild-type (control) group (P =.0163). A statistical trend toward improved wound healing for plasminogen-deficient mice was found for overall histomorphological score (P =.0706)., Conclusion: The role of the plasminogen pathway in wound healing is one that should be noted and may lead to the development of new therapies that reduce scar tissue formation. Hence, the role of other thrombolytic and anti-thrombolytic agents in wound healing should be further investigated to precisely identify agents that play the most significant role in scar tissue formation.

1. Aldosterone has been considered a key hormone in the regulation of water, sodium and potassium metabolism, thus influencing blood pressure regulation. More recently, several studies have demonstrated that aldosterone is also produced in extra-adrenal tissues (e.g. the heart), suggesting a paracrine effect for aldosterone, such as to increase collagen synthesis in the heart. 2. Because aldosterone production in the heart increases after myocardial infarction (MI), we investigated the effect of chronic administration of spironolactone, an aldosterone receptor antagonist, in rats after MI compared with the effects produced by losartan and hydralazine. 3. Myocardial infarction was produced in male Wistar rats by surgical ligature of the left coronary artery. Sham-operated animals were used as controls. 4. Spironolactone (20 mg/kg per day), losartan (15 mg/kg per day) or hydralazine (20 microg/kg per day) were administered after MI and used for 1 month. 5. At the end of the treatment period, animals underwent haemodynamic recording (arterial and intraventricular pressures). The collagen content of the heart was evaluated by measuring the hydroxyproline (OH-Pro) concentration in right (RV) and left ventricle (LV) muscle fragments. 6. Infarct size was unaffected by drug treatments. The increased LV end-diastolic pressure observed in MI rats was prevented by losartan and remained unchanged following administration of spironolactone or hydralazine. 7. Losartan prevented RV and LV hypertrophy, as well as collagen proliferation in both ventricles, after MI. The postinfarction hypertrophy observed in RV and LV after MI remained unchanged in infarcted groups treated with spironolactone or hydralazine. 8. The OH-Pro concentration was significantly reduced in LV muscle in the MI group treated with spironolactone (682 +/- 40 vs 557 +/- 21 microg/g for MI vs MI + spironolactone, respectively; P < 0.05), an effect not observed in the hydralazine-treated group. 9. These data suggest that spironolactone prevents collagen proliferation in the surviving myocardium by mechanisms independent of the loading conditions of the heart chambers. Control of postinfarction hypertrophy and collagen accumulation produced by losartan seems to depend on the reduction in loading conditions of the heart chambers.

Objective: The objective was to assess the histological and viscoelastic shear tissue properties of the scarred vocal fold lamina propria at 2 and 6 months postoperatively in a canine model., Study Design: Experimental, nonrandomized prospective study., Methods: Six canine larynges were injured using a vocal fold stripping procedure. At 2 and 6 months postoperatively, histological analyses of the scarred and control lamina propria samples were completed for collagen, procollagen, elastin, and hyaluronic acid., Results: In canines killed at 2 months, scarred tissue samples contained increased procollagen and decreased elastin. Elastin fibers in the scarred lamina propria were characteristically tangled and disorganized. In canines killed at 6 months, scarred tissue samples showed decreased elastin and increased collagen. Collagen fibers formed thick, disorganized bundles, and elastin fibers were disorganized throughout the entire scarred vocal fold lamina propria. Viscoelastic shear tissue measurements revealed increased stiffness and viscosity in one of three cases at 2 months and in all three cases at 6 months, indicating increased stiffness and resistance to shear flow during oscillatory shear deformation for scarred tissue samples. No differences were observed between the two postoperative times., Conclusions: Results indicated that viscoelastic tissue changes may take place before scar maturation in the scarred vocal fold lamina propria and that, although abundant collagen deposition may influence viscoelastic shear tissue properties, disorganization of collagen and elastin fibers, thick bundle collagen formation, or the interplay of several of these factors might also play a contributing role.

In this study we investigated whether CNS axons regenerate following attenuation of scar formation using a combination of antibodies against two isoforms of transforming growth factor beta (TGFbeta). Anaesthetized adult rats were given unilateral mechanical lesions of the nigrostriatal tract. Implantation of transcranial cannulae allowed wounds to be treated with a combination of antibodies against TGFbeta1 and TGFbeta2 once daily for 10 days postaxotomy. Eleven days post-transection brains from animals under terminal anaesthesia were recovered for histological evaluation. Gliosis, inflammation and the response of dopaminergic nigral axons were assessed by immunolabelling. Treatment with antibodies against TGFbeta1 and TGFbeta2 attenuated (but did not abolish) the response of glial fibrillary acidic protein (GFAP)-immunoreactive astrocytes and of NG2-immunoreactive glia but did not attenuate the response of CR3-immunoreactive microglia and macrophages. However, this reduction in scar formation was not accompanied by growth of cut dopaminergic nigral axons. We conclude that treatment of injured adult rat brain with a combination of antibodies against TGFbeta1 and TGFbeta2 results in a reduction of scar formation but that this is not sufficient to enhance spontaneous long distance CNS axon regeneration.

A histochemical study using a Biotin-Streptavidin procedure for demonstrating interleukin 1 (IL-1) in burn scar specimens is described. Among seventeen scar specimens, five positive reactions to IL-1 were observed in the epidermis. No positive reactions in the dermal tissue, however, were detected. The twenty specimens of normal skin used as controls showed no positive reaction in either the dermis or the dermis.

It is essential that surgeons treating soft tissue wounds about the head and neck have a basic understanding of skin anatomy and physiology. The quality as well as the rate of healing is usually dependent on the type and extent of the original injury. Having a practical working knowledge of the pathophysiology of the various types of head and neck soft tissue injuries allows an intelligent choice of the most appropriate approach to a particular wound. The best functional and cosmetic results routinely are achieved by the wound's undergoing primary healing. Skin tapes and various sutures each exhibit certain benefits and disadvantages in primary closure of wounds. Skin tapes cause less inflammatory reaction but fail to close the subepithelial wound spaces. The inflammatory reaction and subsequent scarring caused by the various sutures depends upon the size of the suture needle, the diameter of the suture material and whether it is a monofilament or a woven suture. The essential nutrition of the wound must be maintained with the body providing adequate amounts of carbohydrates, proteins, trace elements and vitamins. The lack of adequate wound tissue perfusion by a normal or reversed oxygen gradient will lead to tissue necrosis and infection. The size of the inoculum of micro-organisms, the virulence of the organisms and host antimicrobial defense mechanisms determine if an infection will develop. The pharmacological action of each antibiotic must be understood in order to choose the proper antibiotic, its route of administration and to avoid unwanted side effects. Crushing tissue injuries, high velocity projectile tissue injuries and major burns of tissue may require several days to exhibit the true scope of the original injury. Most of these injuries must be treated by the open delayed method of wound treatment until the proper tissue conditions exist for healing. The proper timing and correct choice of autografts and tissue flaps from the surgeon's personal experience, study and conferring with other qualified surgeons. Upon gaining insight into the cellular and humoral antimicrobial defense system, the surgeon is able to provide the best conditions to allow these systems to function properly. Studies in the ultrastructures of skin along with the recently developed microbioassay techniques will allow a closer monitoring during the process of wound healing that will provide the basis for future techniques in the beneficial manipulation of wound healing.