Cryotherapy has antifibrotic and regenerative effects on human vocal fold fibroblasts

Abstract

Objectives/Hypothesis Vocal fold scarring remains a major treatment challenge, and scar prevention without residual lesions remains a dilemma. Cryotherapy has shown cosmetic outcomes on skin lesions with minimal scarring. The aim of this study was to clarify the beneficial effects of cryotherapy for the prevention and the treatment of vocal fold scarring. Study Design In vitro. Methods Primary cultures of human vocal fold fibroblasts (VFFs) were used in this study. Myofibroblast differentiation was stimulated by transforming growth factor β1 (TGF‐β1). We mimicked the cryotherapy effect on vocal fold healing in vivo by freezing VFFs ± TGF‐β1 in vitro. The influence of freezing on cell viability, proliferation, migration, and contractile properties were analyzed. The expression of collagen I, collagen III, fibronectin, TGF‐β1, matrix metallopeptidase 1 (MMP1), hyaluronan synthase 1 (HAS1) were investigated by real‐time polymerase chain reaction (RT‐PCR), and the expression of alpha smooth muscle actin (α‐SMA) and decorin were investigated by RT‐PCR and Western blot. Results Freezing was found to modify extracellular matrix (ECM) synthesis and differentiation of VFFs. Expression of collagen I, collagen III, fibronectin, α‐SMA, and TGF‐β1 was downregulated, and MMP1 was upregulated in VFFs + TGF‐β1 (myofibroblast) by freezing. HAS1 and decorin were upregulated in both VFFs ± TGF‐β1 by freezing. Freezing VFFs + TGF‐β1 (myofibroblast) with fast thawing had a lower expression of α‐SMA when compared with slow thawing. Freezing reduced the migration and collagen contraction of VFFs + TGF‐β1 (myofibroblast). Conclusion Cryotherapy induces antifibrotic and regenerative ECM alterations in VFFs. These data provide insight into the prevention and the treatment of vocal fold scarring with cryotherapy in phonomicrosurgery. Level of Evidence NA Laryngoscope, 129:E143–E150, 2019

Phlorotannins ameliorate extracellular matrix production in human vocal fold fibroblasts and prevent vocal fold fibrosis via aerosol inhalation in a laser‐induced fibrosis model

“…Due to excessive scarring, VF lamina propria tissue is replaced by fibrotic tissue with a disorganized ECM (Erndt‐Marino et al, 2018). VFFs are known to be central to fibrogenesis via the regulation of the synthesis and expression of ECM components, which involves complicated interactions with various cell lines (Gong, Zhang, Kang, Lamb, & Jiang, 2019; Inoue‐Mochita et al, 2018). During fibrogenesis, VFFs are overactivated and undergo a transition into myofibroblasts, resulting in the overproduction of the ECM and strong migration to damaged sites (Gong et al, 2019; Moran‐Salvador et al, 2019).…”

“…VFFs are known to be central to fibrogenesis via the regulation of the synthesis and expression of ECM components, which involves complicated interactions with various cell lines (Gong, Zhang, Kang, Lamb, & Jiang, 2019; Inoue‐Mochita et al, 2018). During fibrogenesis, VFFs are overactivated and undergo a transition into myofibroblasts, resulting in the overproduction of the ECM and strong migration to damaged sites (Gong et al, 2019; Moran‐Salvador et al, 2019). Therefore, the targeted inhibition of ECM synthesis and myofibroblast differentiation offers a promising approach to the prevention of VF fibrosis.…”

Current approaches to management of vocal fold scar

“…Gong et al examined the difference in response to injury of the rabbit VF when injured by VF stripping and then allowed to heal naturally vs after treatment with cryotherapy [9 ▪ ]. In animals treated immediately by cryotherapy following stripping there was reduced transcription of IL-6, collagen I and collagen III, TGF-β and α-smooth muscle actin (SMA) genes (pro-fibrotic genes) but up-regulation of HAS1 and MMP1 genes, favouring antifibrotic ECM remodelling [9 ▪ ]. This may offer a potential treatment aimed at dampening the fibrotic cascade after elective surgical procedures on the glottis.…”

Porcine Vocal Fold Lamina Propria-Derived Biomaterials Modulate TGF-β1-Mediated Fibroblast Activation in Vitro

“…Approaches to VFLP replacement with synthetic materials do not appear to be clinically viable, leaving a wide range of patients without options for regenerative therapy. − VF injury triggers the release of a variety of cytokines and signaling cascades aimed at promoting tissue recovery in which transforming growth factor β1 (TGF-β1) represents a major player during fibrosis. Since the fibrotic response alters the local protein composition affecting the function of the VFs, methods such as cryotherapy, polyethylene glycol (PEG), and cell therapies have been used to target TGF-β1 signaling to enhance VF wound healing. − Nevertheless, current biomaterials and therapeutic approaches fail to capture the specific composition of the VF tissue or have limited ability to modulate inflammatory and fibrotic responses underlying scar formation and altered VF function …”