Development of a hydrogel-based cellular model of the vocal fold

Introduction: The human vocal fold (VF) consists of a stratified squamous epithelium, a lamina propria (LP), and the vocalis muscle. In adult humans, the LP is further divided into superficial, intermediate, and deep layers, with a gradual increase in stiffness from the superficial to the deep layer.1 Damage to the vocal fold can lead to scarring and dysphonia that are difficult to treat.2 Clinically, there is increasing interest in mesenchymal stem cell (MSC)-based therapies targeting vocal fold scarring. From the development perspective, how the lamina propria evolves from a relatively homogeneous structure in fetal and newborn vocal folds to the multilayered tissue in adults remains unknow. To investigate how the tissue microenvironment influences the phenotype and function of MSCs, we developed a hydrogel-based cellular model of the vocal fold lamina propria.3 MSCs were encapsulated in a hyaluronic acid (HA)-based hydrogel that was cell adhesive, protease degradable and mechanically compliant. Zero or seven days later, the matrix was stiffened via interfacial bioorthogonal tetrazine (Tz) ligation with trans-cyclooctene (TCO) using a peptide crosslinker that is either susceptible or resistant to cleavage by matrix metalloproteases (MMPs). We found that delayed stiffening is conducive to fibroblast commitment.

Materials and

Methods: MSCs were cultured and passaged in MSCGM media according to the manufacturer's instructions (Lonza). Tetrazine modified HA (HA-Tz), norbornene (Nb) and TCO-tagged MMP degradable (SMR) and non-degradable (SMV) crosslinkers (SMR-bisNb, SMV-bisNb, SMR-bisTCO and SMV-bisTCO) and Nb conjugated cell adhesive peptide RGDSP (RGD-Nb) were synthesis following reported protocols.3 The initial construct (nLP) was prepared by dispersing MSCs in HA-Tz (1 or 2wt%), RGD-Nb and SMR-bisNb at a Tz/Nb molar ratio of 2.5/1 at a RGD concentration of 0.5 mM. The nLP construct was maintained in MSCGM for 0 or 7 days, then SMR-bisTCO was supplemented in the media to introduce additional crosslinking via diffusion controlled interfacial tetrazine ligation with TCO. Twenty-four hours later, the stiffened constructs (sLP0 and sLP7) were established. A bilayered construct (mLP) comprised a stiff top layer and a soft bottom layer was established immediately after cell encapsulation by allowing the diffusion-controlled reaction to occur halfway through the gel thickness (Fig 1). Hydrogels crosslinked and stiffened with the scrambled peptide (SMV) were included as the controls. Cultures were maintained for 18 days in MSCGM, and cell morphology and phenotype were analyzed.

Results, Conclusions, and Discussions: Previous work shows that matrix degradability mediates the development of cellular traction forces, which in turn, control MSC differentiation towards osteoblasts,.4 Here, we utilized a TCO-based MMP-degradable or non-degradable peptide crosslinker to stiffen the hydrogel during live cell culture. Rheological analysis revealed that the initial hydrogel prepared with 1 wt% HA-Tz had an average shear elastic modulus (G') of 200 Pa. After the interfacial crosslinking with SMR-bisTCO, the hydrogel had an average G' of 500 Pa. Hydrogels prepared with 2wt% HA had an initial G' of 700 Pa, which increased to 1100 Pa after interfacial crosslinking. MSCs maintained high viability in the HA gels under all 3D conditions. By day 18, cells in 1wt% HA gels adopted a spindle shaped morphology, whereas those in 2wt% gels were less elongated (Fig 1). Although matrix stiffening did not significantly alter cell morphology, gene array analyses revealed significant changes in the expression of genes encoding important ECM proteins and adhesion molecules. RT-qPCR analysis on day 18 confirmed the increased expression of fibroblastic marker proteins TNC, FAP and FSP10 in sLP7 constructs as compared to the nLP controls. Furthermore, stiffening of soft gel construct on day 7 leads to an upregulation of the mechanosensitive protein YAP.
Our results indicate that the shape and phenotype of MSCs are matrix stiffness and degradability of the primary and secondary crosslinking. This in vitro model, designed to mimic physiological conditions, offers insights into vocal fold development and diseases.

Acknowledgements (Optional): This work was supported by National Institutes of Health (R01 DC014461)