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An in vitro assessment of the response of THP-1 macrophages to varying stiffness of a glycol-chitosan hydrogel for vocal fold tissue engineering applications.

ABSTRACT: The physical properties of a biomaterial play an essential role in regulating immune and reparative activities within the host tissue. This study aimed to evaluate the immunological impact of material stiffness of a glycol-chitosan hydrogel designed for vocal fold tissue engineering. Hydrogel stiffness was varied via the concentration of glyoxal cross-linker applied. Hydrogel mechanical properties were characterized through atomic force microscopy and shear plate rheometry. Using a transwell setup, macrophages were co-cultured with human vocal fold fibroblasts that were embedded within the hydrogel. Macrophage viability and cytokine secretion were evaluated at 3, 24, and 72 hr of culture. Flow cytometry was applied to evaluate macrophage cell surface markers after 72 hr of cell culture. Results indicated that increasing hydrogel stiffness was associated with increased anti-inflammatory activity compared to relevant controls. In addition, increased anti-inflammatory activity was observed in hydrogel co-cultures. This study highlighted the importance of hydrogel stiffness from an immunological viewpoint when designing novel vocal fold hydrogels.

SUBMITTER: Coburn PT

PROVIDER: S-EPMC8079542 | biostudies-literature | 2021 Aug

REPOSITORIES: biostudies-literature

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An in vitro assessment of the response of THP-1 macrophages to varying stiffness of a glycol-chitosan hydrogel for vocal fold tissue engineering applications.

Coburn Patrick Thomas PT Herbay Alexandre Camille AC Berrini Mattia M Li-Jessen Nicole Y K NYK

Journal of biomedical materials research. Part A 20201106 8

The physical properties of a biomaterial play an essential role in regulating immune and reparative activities within the host tissue. This study aimed to evaluate the immunological impact of material stiffness of a glycol-chitosan hydrogel designed for vocal fold tissue engineering. Hydrogel stiffness was varied via the concentration of glyoxal cross-linker applied. Hydrogel mechanical properties were characterized through atomic force microscopy and shear plate rheometry. Using a transwell set ...[more]

PMID: 33112473

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Project description:There is a critical need for the establishment of an engineered model of the vocal fold epithelium that can be used to gain understanding of its role in vocal fold health, disease, and facilitate the development of new treatment options. Toward this goal, we isolated primary vocal fold epithelial cells (VFECs) from healthy porcine larynxes and used them within passage 3. Culture-expanded VFECs expressed the suprabasal epithelial marker cytokeratin 13 and intercellular junctional proteins occludin, E-cadherin, and zonula occludens-1. To establish the engineered model, we cultured VFECs on a hyaluronic acid-derived synthetic basement membrane displaying fibronectin-derived integrin-binding peptide (RGDSP) and/or laminin 111-derived syndecan-binding peptide AG73 (RKRLQVQLSIRT). Our results show that matrix stiffness and composition cooperatively regulate the adhesion, proliferation, and stratification of VFECs. Cells cultured on hydrogels with physiological stiffness (elastic shear modulus, G' = 1828 Pa) adopted a cobblestone morphology with close cell-cell contacts, whereas those on softer matrices (G' = 41 Pa) were spindle shaped with extensive intracellular stress fibers. The development of stratified epithelium with proliferating basal cells and additional (1-2) suprabasal layers requires the presence of both RGDSP and AG73 peptide signals. Supplementation of cytokines produced by vimentin positive primary porcine vocal fold fibroblasts in the VFEC culture led to the establishment of 4-5 distinct cell layers. The engineered vocal fold epithelium resembled native tissue morphologically; expressed cytokeratin 13, mucin 1, and tight/adherens junction markers; and secreted basement membrane proteins collagen IV and laminin 5. Collectively, our results demonstrate that stiffness matching, cell-matrix engagement, and paracrine signaling cooperatively contribute to the stratification of VFECs. The engineered epithelium can be used as a versatile tool for investigations of genetic and molecular mechanisms in vocal fold health and disease.

Project description:The biomechanical function of the vocal folds (VFs) depends on their viscoelastic properties. Many conditions can lead to VF scarring that compromises voice function and quality. To identify candidate replacement materials, the structure, composition, and mechanical properties of native tissues need to be understood at phonation frequencies. Previously, the authors developed the torsional wave experiment (TWE), a stress-wave-based experiment to determine the linear viscoelastic shear properties of small, soft samples. Here, the viscoelastic properties of porcine and human VFs were measured over a frequency range of 10-200 Hz. The TWE utilizes resonance phenomena to determine viscoelastic properties; therefore, the specimen test frequency is determined by the sample size and material properties. Viscoelastic moduli are reported at resonance frequencies. Structure and composition of the tissues were determined by histology and immunochemistry. Porcine data from the TWE are separated into two groups: a young group, consisting of fetal and newborn pigs, and an adult group, consisting of 6-9-month olds and 2+-year olds. Adult tissues had an average storage modulus of 2309±1394 Pa and a loss tangent of 0.38±0.10 at frequencies of 36-200 Hz. The VFs of young pigs were significantly more compliant, with a storage modulus of 394±142 Pa and a loss tangent of 0.40±0.14 between 14 and 30 Hz. No gender dependence was observed. Histological staining showed that adult porcine tissues had a more organized, layered structure than the fetal tissues, with a thicker epithelium and a more structured lamina propria. Elastin fibers in fetal VF tissues were immature compared to those in adult tissues. Together, these structural changes in the tissues most likely contributed to the change in viscoelastic properties. Adult human VF tissues, recovered postmortem from adult patients with a history of smoking or disease, had an average storage modulus of 756±439 Pa and a loss tangent of 0.42±0.10. Contrary to the results of some other investigators, no significant frequency dependence was observed. This lack of observable frequency dependence may be due to the modest frequency range of the experiments and the wide range of stiffnesses observed within nominally similar sample types.

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Project description:Glycol chitosan (GC) is a hydrophilic chitosan derivative, known for its aqueous solubility. Previously, we have demonstrated the feasibility of preparing injectable, enzymatically crosslinked hydrogels from HPP [3-(4-Hydroxyphenyl)-propionic acid (98%)]-modified GC. However, HPP-GC gels showed very slow degradation, which presents challenges as an in vivo protein delivery vehicle. This study reports the potential of acetylated HPP-GC hydrogels as a biodegradable hydrogel platform for sustained protein delivery. Enzymatic crosslinking was used to prepare injectable, biodegradable hydrogels from HPP-GC with various degrees of acetylation (DA). The acetylated polymers were characterized using Fourier transform infrared and nuclear magnetic resonance spectroscopy. Rheological methods were used to characterize the mechanical behavior of the hydrogels. In vitro degradation and protein release were performed in the presence and absence of lysozyme. In vivo degradation was studied using a mouse subcutaneous implantation model. Finally, two hydrogel formulations with distinct in vitro/in vivo degradation and in vitro protein release were evaluated in 477-SKH1-Elite mice using live animal imaging to understand in vivo protein release profiles. The lysozyme-mediated degradation of the gels was demonstrated in vitro and the degradation rate was found to be dependent on the DA of the polymers. In vivo degradation study further confirmed that gels formed from polymers with higher DA degraded faster. In vitro protein release demonstrated the feasibility to achieve lysozyme-mediated protein release from the gels and that the rate of protein release can be modulated by varying the DA. In vivo protein release study further confirmed the feasibility to achieve differential protein release by varying the DA. The feasibility to develop degradable enzymatically crosslinked GC hydrogels is demonstrated. Gels with a wide spectrum of degradation time ranging from less than a week and more than 6 weeks can be developed using this approach. The study also showed the feasibility to fine tune in vivo protein release by modulating HPP-GC acetylation. The hydrogel platform therefore holds significant promise as a protein delivery vehicle for various biomedical and regenerative engineering applications. Impact statement The study describes the feasibility to develop a novel enzyme sensitive biodegradable and injectable hydrogel, where in the in vivo degradation rate and protein release profile can be modulated over a wide range. The described hydrogel platform has the potential to develop into a clinically relevant injectable and tunable protein delivery vehicle for a wide range of biomedical applications.

Dataset Information

An in vitro assessment of the response of THP-1 macrophages to varying stiffness of a glycol-chitosan hydrogel for vocal fold tissue engineering applications.

Publications

An in vitro assessment of the response of THP-1 macrophages to varying stiffness of a glycol-chitosan hydrogel for vocal fold tissue engineering applications.

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