Human Vocal Fold Fibroblasts Research Articles

The Human Umbilical Vein with Wharton's Jelly as an Allogeneic, Acellular Construct for Vocal Fold Restoration

This study investigated the potential of the decellularized human umbilical vein (HUV) as an allogeneic, acellular extracellular matrix (ECM) scaffold for engineering the vocal fold lamina propria in vitro. HUV specimens with Wharton's jelly on the abluminal surface were uniformly dissected from native umbilical cords using an automated procedure and subjected to a novel saline-based decellularization treatment for removal of potentially antigenic epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the resulting acellular constructs and cultured for 21 days. The structures of decellularized and fibroblast-repopulated HUV constructs and the attachment, proliferation, and infiltration of fibroblasts were examined with light microscopy and scanning electron microscopy. Changes in the relative densities of collagen in the constructs associated with decellularization and recellularization were quantified using digital image analysis. In addition, fibroblasts infiltrating the scaffolds were released by cell recovery and quantified by counting. Viscoelastic properties of the scaffolds were measured using a linear, simple-shear rheometer at phonatory frequencies. Results showed that an acellular ECM construct with an intact three-dimensional structure of Wharton's jelly was fabricated. Vocal fold fibroblasts readily attached on the abluminal surface of the construct with high viability, with significant cellular infiltration up to approximately 600 microm deep into the construct. A significant increase in collagen expression was observed with recellularization. The elastic modulus and dynamic viscosity of the fibroblast-repopulated scaffolds were comparable to those of the human vocal fold lamina propria. These findings supported the potential of the construct as a possible surgical allograft for vocal fold restoration and reconstruction.

Cigarette smoke and reactive oxygen species metabolism: Implications for the pathophysiology of Reinke's edema

Oxidative damage mediates the lower airway response to cigarette smoke (CS). In the vocal folds, the tissue phenotype is both distinct and largely uncharacterized. We sought to investigate the effects of CS on the oxidative status and fibroblast phenotype in the vocal folds. Specifically, we hypothesized that CS would induce a hypermetabolic fibroblast phenotype and altered oxidative metabolism potentially providing insight into the relationship among CS, Reinke's edema (RE), and malignancy. In vivo/in vitro. Heme oxygenase (HO)-1 gene expression was examined in human tissue. In vitro, the effects of cigarette smoke condensate (CSC) on HO-1 gene expression and secretion was assayed. In addition, CS-mediated intracellular reactive oxygen species synthesis was quantified, and compared to the response in pulmonary fibroblasts (HFL). We then examined the effects of CSC on migration and proliferation in human vocal fold fibroblasts (HVOX). : HO-1 expression was approximately 4-fold higher in RE samples versus vocal fold polyps. CSC induced HO-1 gene expression and secretion in a time- and dose-dependent fashion in vitro. CSC also increased intracellular ROS in both HVOX and HFL. CSC decreased HVOX proliferation and migration in a dose-dependent manner. These data suggest that the fibroblast phenotype is influenced by smoke. Our data suggest that the antioxidant response in the vocal fold tissue may be one mechanism of chemoprotection, a putative explanation for the observation that RE rarely transforms to malignancy. In addition, CSC does not appear to induce a hypermetabolic fibroblast phenotype as expected.

Effects of Transforming Growth Factor-β1 on Human Vocal Fold Fibroblasts

We studied the effect of transforming growth factor (TGF)-beta on immortalized human vocal fold fibroblasts. Normal human vocal fold fibroblasts were subjected to sequential lentiviral transduction with genes for human telomerase (hTERT) and SV40 large T antigen in order to produce an "immortalized" cell line of normal phenotype. After confirmation of vocal fold fibroblast transfection, these cells, referred to as HVOX, were treated with various concentrations of exogenous TGF-beta1 and assayed for collagen secretion, migration, and proliferation. In addition, components of the TGF-beta signaling pathway were examined in this cell line. TGF-beta stimulated collagen secretion and migration without altering proliferation of HVOX. HVOX constitutively expressed type I and II TGF-beta receptors, as well as messenger RNA for the Smad signaling proteins and for all TGF-beta isoforms. Exogenous TGF-beta1 induced temporally dependent alterations in Smad2 and Smad3 gene expression. TGF-beta increased Smad7 expression at both 4 and 24 hours. Prolonged exposure to TGF-beta decreased TGF-beta1 gene expression. Insight into the underlying pathophysiology of vocal fold fibrosis is likely to yield improved therapeutic strategies to mitigate vocal fold scarring. Our data suggest that TGF-beta signaling may be both paracrine and autocrine in this vocal fold fibroblast cell line, and we therefore propose that TGF-beta may be a reasonable target for therapies to prevent and/or treat vocal fold fibrosis, given its putative role in both acute and chronic vocal fold injury, as well as its effects on vocal fold fibroblasts.

Characteristics of Age‐Related Changes in Cultured Human Vocal Fold Fibroblasts

Normal human vocal fold fibroblast (hVFF) primary cell lines are unavailable commercially and are very difficult to acquire, subsequently little is known about their characteristics. The purpose of this study was to compare the morphological and proliferation characteristics and gene expression of hVFFs from different aged donors. In vitro. We developed three normal hVFF primary cell lines from donors aged 21 (21T), 59 (59T) and 79 (79T) years. We characterized their morphological features, proliferative abilities, telomere lengths, and their functional gene expression by quantitative real-time PCR. The 21T line maintained a typical spindle shape until passage 14 whereas 59T and 79T changed morphology to wider, shorter cells at passage 7. Proliferation rates were constant for the 21T through passage 14; 59T's proliferative half-life was passage 9, whereas 79T maintained lower proliferation rates from passage 4. Gene expression levels for fibronectin, collagen I, collagen VI, procollagen I and elastin demonstrated similar patterns for all lines, however, relative amounts decreased with the age of donor. Telomere lengths did not show differences related with donor age. hVFF primary cultures have limited proliferative capacity. The morphology, proliferation, differentiation, and gene expression levels of VFF can be affected by age, but senescence patterns were similar across the ages.

A Biodegradable, Acellular Xenogeneic Scaffold for Regeneration of the Vocal Fold Lamina Propria

A novel method for preparing an acellular xenogeneic extracellular matrix scaffold for tissue engineering was developed. Bovine vocal fold lamina propria specimens were treated with high-concentration sodium chloride, nucleic acid digestion, and ethanol dehydration for decellularization and removal of immunogenic foreign epitopes. Human vocal fold fibroblasts from primary culture were seeded onto the acellular scaffolds and cultured for 21 days. The decellularized and the recellularized scaffolds were examined by light microscopy, fluorescent microscopy, and scanning electron microscopy. Collagen synthesis and release by fibroblasts were quantified by the Sircol assay, whereas the synthesis and release of hyaluronic acid, decorin, and fibronectin were assessed by enzyme-linked immunosorbent assays. Viscoelastic shear properties of the scaffolds were quantified by a simple-shear rheometer at frequencies of up to 250 Hz. Preliminary results showed that a biodegradable, acellular extracellular matrix scaffold with an intact basement membrane and 3-dimensional structure of the matrix proteins was engineered. Vocal fold fibroblasts readily attached to and infiltrated the scaffold with high viability and active protein synthesis, demonstrating the biocompatibility. The elastic shear modulus and dynamic viscosity of the acellular scaffold and the fibroblast-repopulated scaffold were comparable to those of the human vocal fold cover. These findings support the potential of the scaffold as a xenograft for vocal fold reconstruction and regeneration.

Effects of Growth Factors on Extracellular Matrix Production by Vocal Fold Fibroblasts in 3-Dimensional Culture

Culturing cells in 3-dimensional (3D) systems is important in tissue engineering and in fundamental studies of cellular mechanisms that are sensitive or specific to the 3D environment. To guide the engineering of artificial vocal fold lamina propria tissue, we developed 3D cultures containing human vocal fold fibroblasts (hVFFs) dispersed in a synthetic peptide hydrogel matrix. Growth factors were added to the culture to examine their influence on extracellular matrix (ECM) synthesis, cell proliferation, and matrix contraction. The hVFF-hydrogel constructs were treated with transforming growth factor-beta 1 (TGF-beta1), basic fibroblast growth factor (bFGF), or hepatocyte growth factor (HGF), and the culture was maintained for 21 days. TGF-beta1 induced matrix contraction and enhanced collagen and sulfated glycosaminoglycan production, bFGF effectively increased cell proliferation, and HGF stimulated synthesis of hyaluronic acid and elastin with less collagen accumulation than other conditions. Of the growth factors tested, HGF appears to be most useful for stimulating essential tissue components for restoring vocal fold pliability. The results also suggest that multiple growth factors might be employed sequentially or in combination to program the makeup of cell-hydrogel constructs for vocal fold tissue repair.

Design and validation of a bioreactor for engineering vocal fold tissues under combined tensile and vibrational stresses

Criteria are outlined for the design of a bioreactor that can simulate the vibrational stresses in vocal fold movement during speech. Requirements are 0–1 mm amplitudes in the 20–200 Hz frequency range, a variable on–off stress regime, and maintenance of tissue viability over several days. The bioreactor uses dual drivers, one for low frequency (or static) strains, and another for high-frequencies vibrational strains. Response is linear at the driving end for an input of 0–5 V. The amplitude decreases linearly with frequency at constant input voltage, and the phase changes by nearly 180° over the 20–200 Hz range. Human vocal fold fibroblasts were cultured in a polymer substrate and subjected to static and vibrational forces. The results indicate that vibratory strain alters the expression levels of many extracellular matrix-related genes, as well as the spatial distribution of cells and matrix.

Morphological and functional changes of human vocal fold fibroblasts with hepatocyte growth factor.

Fibroblasts produce extracellular matrix and play an important role in wound healing and scarring. Hepatocyte growth factor (HGF) has strong antifibrotic activity, and has been suggested to have therapeutic potential for treatment of fibrotic diseases. In the present in vitro study, morphological and functional changes of human vocal fold fibroblasts with HGF were examined by transmission electron microscopy and enzyme-linked immunosorbent assay to help clarify the potential use of HGF in the prevention or treatment of vocal fold scarring. The HGF stimulated the production of hyaluronic acid (HA) and decreased the production of collagen type I from the fibroblasts in Reinke's space (FbRS), whereas fibronectin production was not affected. The HGF also changed the shape of the FbRS from an oval shape toward a spindle and stellate shape, and developed Golgi apparatus (GA) and rough endoplasmic reticulum (rER) in the FbRS. The fibroblasts in the macula flava (FbMF) presented with much more production of HA and collagen type I than did FbRS, and were more frequently formed in a stellate shape with well-developed GA and rER. The HGF decreased the production of collagen type I from the FbMF, but barely affected the FbMF in terms of the shape of the cells, the development of GA and rER, or the production of HA. These results were interpreted to suggest that the FbMF are not as susceptible to HGF as are FbRS. On the contrary, HGF appeared to activate the FbRS and modify the function. The increased HA and decreased collagen type I production from the FbRS suggest that HGF may be useful in the prevention or treatment of fibrotic vocal fold scarring.