Abstract
Purpose :
The pathological mechanisms leading to aqueous deficient dry eye disease (ADDE) are still not fully understood, so current treatment is mostly palliative and lacks regenerative or reconstructive strategies. Tissue specific hydrogels (HG) derived from decellularized extracellular matrix have emerged as a promising substrate in tissue engineering due to their cell function promoting properties, therefore representing the ideal basis for an in vitro model to investigate ADDE pathology and treatment. Our aim was to produce a decellularized (d) lacrimal gland (LG) HG that ensures physiological cultivation of LG cells and meets the basic requirements for bioprinting a LG in vitro model.
Methods :
Porcine LG were decellularized and hydrolyzed with pepsin for 4, 12, 24, 48, or 96 h to obtain dLG-HG. dLG-HG samples (n=6) were analyzed rheologically (gelation kinetics and stiffness) and biochemically (collagen, glycosaminoglycan, and hyaluronan content). Proliferation of porcine LG epithelial cells (EC) grown on dLG-HG, Matrigel and collagen-I-HG was analyzed by MTT assay. EC mediated degradation of the different HG substrates was quantified by remaining HG area. Finally, the secretion capacity of EC was investigated by β-hexosaminidase assay.
Results :
Gelable dLG-HG with similar gelation kinetics and stiffness (all p≥0.798) were generated at all hydrolysis times. Hydrolysis time had no effect on collagen and glycosaminoglycan content (all p>0.856 and 0.970, respectively), while hyaluronan content increased in a time dependent manner (all p<0.001). EC proliferation on dLG-HG hydrolyzed for ≥24 h was higher than on Matrigel by 63.14±26.27% (p=0.04), 67.37±27.75% (p=0.039) and 77.97±38.35% (p=0.002), respectively. After 14 days of cultivation with EC, dLG-HG degraded to 6.99±2.55% (4 h) to 3.39±1.25% (96 h) of the original area (all p<0.0001). The secretion capacity of EC cultured on dLG-HG hydrolyzed for ≥24 h was increased by 50-76% (p≤0.006) compared to collagen-I and by 37-61% (p≤0.033) compared to Matrigel.
Conclusions :
dLG-HG hydrolyzed for ≥24 h positively affected proliferation and function of EC compared to collagen-I and Matrigel, thus representing a suitable substrate for a LG in vitro model. Due to its rheological and biochemical properties, dLG-HG meets the basic requirements of a bioink. However, the cell mediated degradation should be slowed down to enable longer cultivation.
This abstract was presented at the 2023 ARVO Annual Meeting, held in New Orleans, LA, April 23-27, 2023.