Modulation of Decellularized Lacrimal Gland Hydrogel Biodegradation by Genipin Crosslinking.

Purpose: Hydrogels derived from decellularized tissues are promising biomaterials in tissue engineering, but their rapid biodegradation can hinder in vitro cultivation. This study aimed to retard biodegradation of a hydrogel derived from porcine decellularized lacrimal glands (dLG-HG) by crosslinking with genipin to increase the mechanical stability without affecting the function and viability of lacrimal gland (LG)-associated cells. Methods: The effect of different genipin concentrations on dLG-HG stiffness was measured rheologically. Cell-dependent biodegradation was quantified over 10 days, and the impact on matrix metalloproteinase (MMP) activity was quantified by gelatin and collagen zymography. The viability of LG epithelial cells (EpCs), mesenchymal stem cells (MSCs), and endothelial cells (ECs) cultured on genipin-crosslinked dLG-HG was assessed after 10 days, and EpC secretory activity was analyzed by ß-hexosaminidase assay. Results: The 0.5-mM genipin increased the stiffness of dLG-HG by about 46%, and concentrations > 0.25 mM caused delayed cell-dependent biodegradation and reduced MMP activity. The viability of EpCs, MSCs, and ECs was not affected by genipin concentrations of up to 0.5 mM after 10 days. Moreover, up to 0.5-mM genipin did not negatively affect EpC secretory activity compared to control groups. Conclusions: A concentration of 0.5-mM genipin increased dLG-HG stiffness, and 0.25-mM genipin was sufficient to prevent MMP-dependent degradation. Importantly, concentrations of up to 0.5-mM genipin did not compromise the viability of LG-associated cells or the secretory activity of EpCs. Thus, crosslinking with genipin improves the properties of dLG-HG for use as a substrate in LG tissue engineering.

Cryopreservation in a Standard Freezer: -28 °C as Alternative Storage Temperature for Amniotic Membrane Transplantation.

Human amniotic membrane (hAM) is usually stored at -80 °C. However, in many regions, cryopreservation at -80 °C is not feasible, making hAM unavailable. Therefore, the possibility of cryopreservation at -28 °C (household freezer) was investigated. hAMs (n = 8) were stored at -80 °C or -28 °C for a mean time of 8.2 months. hAM thickness, epithelial integrity and basement membrane were assessed histologically. The collagen content, concentration of hepatocyte growth factor (HGF) and basic fibroblast growth factor (bFGF) were determined. Elastic modulus and tensile strength were measured. The mean thickness of hAM stored at -28 °C was 33.1 ± 21.6 µm (range 9.7-74.9); thickness at -80 °C was 30.8 ± 14.7 µm (range 13.1-50.7; p = 0.72). Mean collagen content, epithelial cell number and integrity score showed no significant difference between samples stored at -28 °C or -80 °C. Basement membrane proteins were well preserved in both groups. Mean tensile strength and elastic modulus were not significantly different. Concentration of bFGF at -28 °C was 1063.2 ± 680.3 pg/g (range 369.2-2534.2), and 1312.1 ± 778.2 pg/g (range 496.2-2442.7) at -80 °C (p = 0.11). HGF was 5322.0 ± 2729.3 pg/g (range 603.3-9149.8) at -28 °C, and 11338.5 ± 6121.8 pg/g (range 4143.5 to 19806.7) at -80 °C (p = 0.02). No microbiological contamination was detected in any sample. The cryopreservation of hAM at -28 °C has no overt disadvantages compared to -80 °C; the essential characteristics of hAM are preserved. This temperature could be used in an alternative storage method whenever storage at -80 °C is unavailable.