ROCK INHIBITION PROMOTES THE DEVELOPMENT OF POLARIZED ACINI STRUCTURES WITH SECRETORY FUNCTIONS IN HYALURONIC ACID-BASED HYDROGEL MATRICES

Introduction:: Radiation therapy, a common treatment for head and neck cancers, can cause irreversible damage to the saliva-producing acinar cells. As a result, patients experience xerostomia, a condition characterized by salivary gland hypofunction. This condition has a profound impact on a patient’s quality of life. Salivary gland tissue engineering holds great promise as a regenerative solution. Previously, we cultured primary human salivary stem/progenitor cells (hS/PCs) in hyaluronic acid (HA)-based matrices with varying proteolytic degradability. Encapsulated single hS/PCs grew into multicellular spheroids by day 15. We demonstrated that cell-mediated matrix degradation is necessary to maintain the pro-acinar stem/progenitor phenotype. However, multicellular structures developed under these conditions did not contain correctly polarized lumens. In the current study, we investigated the effects of Rho kinase (ROCK) inhibition on the phenotypic changes and polarization of hS/PCs. We further analysed cell stiffness via mitochondria tracking microrheology. Our results indicated that cells treated with the ROCK inhibitor were softer as compared to the untreated controls on day 1 and day 7. In addition, ROCK inhibition led to the establishment of multicellular structures that were correctly polarized, as evidenced by the apical localization of GM130, and basal localization of basement membrane proteins. We also saw an increased expression of acinar markers (AQP3, SLC12A2, AMY1A) at the gene and the protein level in ROCK-inhibited constructs. In summary, we show that ROCK inhibition promotes the formation of polarized acini structures in HA-based synthetic matrices. This finding is an important step forward in salivary gland tissue engineering.


Materials and Methods:: hS/PCs were isolated from human parotid gland tissues obtained from consented patients prior to RT following the reported procedure. [1] Hydrogels were prepared by mixing thiolated HA (HA-SH), maleimide functionalized fibronectin derived peptide (RGDSP-MI), and maleimide functionalized protease degradable PQG↓IWGQ (GIW-bisMI), and non-degradable GIQQWGG (GIQ-bisMI) crosslinker solutions at a SH/MI ratio of 1/1. Hydrogel degradability was tuned by varying the molar ratio of GIW-bisMI and GIQ-bisMI crosslinkers while maintaining the total bisMI concentration constant. The resultant gels are referred to as 100DEG, 50DEG, and 0DEG, corresponding to gels containing 100%, 50%, and 0% GIW-bisMI crosslinker, respectively. hS/PCs were encapsulated in the hydrogels at a concentration of 3×10⁶ cells/mL and the constructs were cultured in HepatoStim media for 15 days. For ROCK inhibition studies, the media was supplemented with 20 µM ROCK inhibitor. Immunocytochemistry and qPCR analyses were performed on the constructs. For performing microrheology, the constructs were stained with Mitotracker Green (500 nM) for 16 h, and time-lapse confocal images were captured on day 1 and day 7 to monitor the movement of the mitochondria. The mean square displacement curves were plotted after tracking the mitochondria fluctuations on Trackmate ImageJ software.


Results, Conclusions, and Discussions:: Results and Discussions:

We formulated hyaluronic acid-based hydrogels of comparable storage moduli but varying proteolytic degradability (0DEG, 50DEG, and 100DEG hydrogels). hS/PCs were encapsulated as single cells and they formed multicellular spheroids in these matrices by day 15 (Fig. 1). Previously, we demonstrated that hS/PC-mediated matrix degradation is necessary for the maintenance of pro-acinar stem/progenitor phenotype (Fig. 1). However, the spheroids developed in these hydrogels did not show correct polarization. Correct apical-basal polarization is necessary to establish correct structural and functional domains and is critical in directing saliva secretion in the lumen. In the current study, we studied the effects of Rho kinase (ROCK) inhibition on the phenotype of hS/PCs encapsulated in the hydrogels. We performed mitochondria tracking microrheology to look at the cellular mechanics when the constructs were treated with ROCK inhibitor. The mitochondria were stained with Mitotracker green and the movement of the mitochondria was monitored using time-lapse imaging. We tracked the mitochondrial trajectory using the Trackmate plug-in in ImageJ software. Our results indicate that cells treated with ROCK inhibitor are softer as compared to untreated controls. Furthermore, we observed that ROCK-inhibited constructs show an increased number of correctly polarized spheroids (Fig. 2) as demonstrated by the apical localization of GM130 (an apical marker) and basal localization of basement membrane proteins (laminin α-1 and collagen IV) as characterized by confocal imaging. In addition, hS/PCs in ROCK-inhibited conditions demonstrated a higher expression of acinar markers (AQP3, SLC12A2, AMY1A) as compared to the untreated constructs at the gene (characterized by qPCR) and the protein level (characterized by immunocytochemistry and confocal imaging). Taken together, our results depict that ROCK inhibition promotes the formation of polarized acini structures in the HA-based synthetic matrices.

Conclusions:

In this work, we investigate the effects of ROCK inhibition on the phenotype of the cells encapsulated in HA-based hydrogels with varying degrees of proteolytic degradation. We demonstrate that hS/PCs in ROCK inhibited constructs form multicellular spheroids with correct polarity and pro-acinar phenotype. This is a major step forward towards engineering an implantable gland with the potential to restore salivary gland function for patients suffering from xerostomia.


Acknowledgements (Optional): : This work was supported by National Institutes of Health through R01 DE029655.


References (Optional): :
1. Pradhan S., et al, Tissue Engineering Part A, 2009, 15, 3309.
2. Ravikrishnan A., et al, ACS Biomaterials Science & Engineering, 2021, 7, 4305.