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Gel-based injectable biomaterials have significant potential for treating vocal fold defects such as scarring. An ideal injectable for vocal fold lamina propria restoration should mimic the microenvironment of the lamina propria to induce scarless wound healing and functional tissue regeneration. Most current synthetic or natural injectable biomaterials do not possess the same level of complex, tissue-specific constituents as the natural vocal fold lamina propria. In this study we present a newly-developed injectable gel fabricated from decellularized bovine vocal fold lamina propria. Blyscan assay and mass spectrometry indicated that the vocal fold-specific gel contained a large amount of sulfated glycosaminoglycans and over 250 proteins. Gene Ontology overrepresentation analysis revealed that the proteins in the gel dominantly promote antifibrotic biological process. In vivo study using a rabbit vocal fold injury model showed that the injectable gel significantly reduced collagen density and decreased tissue contraction of the lamina propria in vocal folds with chronic scarring. Furthermore, this acellular gel only elicited minimal humoral immune response after injection. Our findings suggested that the tissue-specific, injectable extracellular matrix gel could be a promising biomaterial for treating vocal fold scarring, even after the formation of mature scar. STATEMENT OF SIGNIFICANCE: Vocal fold lamina propria scarring remains among the foremost therapeutic challenges in the management of patients with voice disorders. Surgical excision of scar may cause secondary scarring and yield inconsistent results. The present study reports an extracellular matrix-derived biomaterial that demonstrated antifibrotic effect on chronic scarring in vocal fold lamina propria. Its injectability minimizes the invasiveness of the delivery procedure and the degree of mucosal violation. In this work we also describe a new methodology which can more accurately identify proteins from the complex mixture of an acellular extracellular matrix gel by excluding interfering peptides produced during the enzymatic digestion in gel fabrication.

Objectives/Hypothesis To develop a vocal fold scarring model using an ablative laser in the rabbit as a platform for testing bioengineered therapies for missing or damaged lamina propria. Study Design Prospective controlled animal study. Methods An optimal laser energy level was first determined by assessing the depths of vocal fold injury created by a Holmium:YAG laser at various energy levels on fresh cadaveric rabbit larynges. The selected energy level was then used to create controlled unilateral injuries in vocal folds of New Zealand white rabbits, with the contralateral folds serving as uninjured controls. After 4 weeks, the larynges were harvested and subjected to excised-larynx phonation with high-speed imaging and immunohistochemical staining for collagen types I and III, elastin, and hyaluronic acid (HA) with quantitative histological analysis. Results A total of 1.8 joules produced full-thickness injury of the lamina propria without extensive muscle injury. After 4 weeks, the injured vocal folds vibrated with reduced amplitude (P = 0.036) in excised-larynx phonation compared to normal vocal folds. The injured vocal folds contained a higher relative density of collagen type I (P = 0.004), higher elastin (P = 0.022), and lower HA (P = 0.030) compared to normal controls. Collagen type III was unchanged. Conclusions With its potential for higher precision of injury, this laser vocal fold scarring model may serve as an alternative to scarring produced by cold instruments for studying the effects of vocal fold lamina propria bioengineered therapies. Level of Evidence N/A. Laryngoscope 124:2321–2326, 2014

This study examined the replicative senescence of primary-culture human vocal fold fibroblasts, in terms of changes in gross chromosomal structure with sub- culturing, and population doubling time (PDT). The mRNA expressions of 14 target genes were also examined. The objectives were to identify the onset of senescence for establishing the acceptable limit of sub-culturing, and to better understand the effect of cellular aging on matrix protein regulation in the vocal fold. Gross chromosomal changes in vocal fold fibroblasts from a 58-year-old woman were karyotyped with Giemsa stain. Proliferation of the fibroblasts was determined with cell recovery and PDT calculation. Transcript levels of the target genes were found by RT-PCR. Onset of significant chromosomal anomalies was seen with passage 5. For mRNA expressions, significant increases with passaging were observed in collagenase, macrophage elastase, lysyl oxidase and fibromodulin, whereas significant downregulation was detected in decorin, procollagen I, hyaluronic acid-synthase 2 and collagen III. This modulation pattern suggested that fibroblasts underwent in vitro aging, consistent with the significant increase in PDT. The inception of senescence did not occur until passage 5. These findings may facilitate the development of representative in vitro models for testing tissue engineering approaches involving primary-culture fibroblasts.

With a rat model of vocal fold injury, this study examined the in vivo host response to an acellular xenogeneic scaffold derived from the bovine vocal fold lamina propria, and the potential of the scaffold for constructive tissue remodeling. Bilateral wounds were created in the posterior vocal folds of 20 rats, and bovine acellular scaffolds were implanted into the wounds unilaterally, with the contralateral vocal folds as control. The rats were humanely sacrificed after 3 days, 7 days, 1 month, and 3 months, and the coronal sections of their larynges were examined histologically. Expressions of key matrix proteins including collagen I, collagen III, elastin, fibronectin, hyaluronic acid, and glycosaminoglycans were quantified with digital image analysis. Significant infiltration of host inflammatory cells and host fibroblasts in the scaffold implant was observed in the acute stage of wound repair (3 days and 7 days post-surgery). The mean relative densities of collagen I, collagen III, and glycosaminoglycans in the implanted vocal folds were significantly higher than those in the control after 3 days, followed by gradual decreases over 3 months. Histological results showed that the scaffolds were apparently degraded by 3 months, with no fibrotic tissue formation or calcification. These preliminary findings suggested that the bovine acellular scaffold could be a potential xenograft for vocal fold regeneration.

A bovine acellular scaffold was found to facilitate tissue remodeling in a rat model of vocal fold injury, whereas hepatocyte growth factor (HGF) has been shown to have an anti-scarring effect in the larynx. This study examined the loading and release kinetics of HGF in vitro, and the potential of the acellular scaffold as a timed-release system for the delivery of HGF in vivo. Bilateral wounds were created in the posterior vocal folds of 20 rats, with HGF-loaded acellular scaffolds implanted into the wounds unilaterally, and scaffolds without HGF implanted into the contralateral vocal folds as control. The rats were humanely sacrificed after 3, 7, 30, and 90 days and their larynges were examined histologically and immunohistochemically. Expressions of key matrix proteins in the vocal fold coronal sections were quantified by digital image analysis. Results demonstrated a gradual, sustained release of HGF for at least 7 days in vitro, consistent with the detection of glycosaminoglycans inherent of the scaffold. In rat vocal folds implanted with HGF-loaded scaffolds, apparently fewer inflammatory cells were observed 3 days after surgery when compared to the control. The mean relative densities of collagen III and hyaluronic acid were significantly lower than those of the control 7 days after surgery. Scaffold implants were apparently degraded by 3 months in all animals, with no evidence of fibrosis or calcification. These data suggested that the bovine acellular scaffold could be promising for the exogenous delivery of select growth factors in vivo.

INTRODUCTION The hyper-proliferative activity of stromal smooth muscle (SM) cells is believed to be responsible for the pathogenesis of benign prostatic hyperplasia (BPH). We have observed that those stromal cells can differentiate into unrelated specialized cells. We thus hypothesize that stromal cells derived from adults prostate specimens may contain adult stem cells. To test this hypothesis, human prostate stromal primary cultures were established and used for characterization of their stem cell properties. METHODS Immunoblotting, immunohistochemistry, RT-PCR, and tissue culture techniques were used to characterize the primary cultured human prostate-derived stromal cells for their stem cell and differentiation properties. The plasticity of these stromal cells was analyzed using cell culture and histology techniques. RESULTS Primary cultured prostate stromal cells from BPH patient possess polygonal and elongated fibroblast/myofibroblast cellular morphology. They are positive in CD30, CD34, CD44, NSE, CD133, Flt-1, stem cell factor (SCF), and neuron-specific enolase (NSE), but negative in C-Kit, stem cell antigen (SCA), SH2, CD11b. Expression of SM myogenic markers in these cells may be induced by sodium butyrate (NaBu) treatment. Induction to osteogenic and adipogenic differentiation in these cells is also evident. CONCLUSIONS Our study on primary stromal cells from BPH patients have yielded many interesting findings that these prostate stroma cells possess: (1) mesenchymal stem cell (MSC) markers; (2) strong proliferative potential; and (3) ability to differentiate or transdifferentiate to myogenic, adipogenic, and osteogenic lineages. These cell preparations may serve as a potential tool for studies in prostate adult stem cell research and the regulation of benign prostatic hyperplasia. Prostate 67: 1265–1276, 2007. © 2007 Wiley-Liss, Inc.