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ICE Krakow

ICE Krakow

ul. Marii Konopnickiej 17 30-302 Kraków


Assunção, Marisa (Regenerative Medicine Center Utrecht, Dept. of Orthopaedics, University Medical Center Utrecht)


"Introduction: Promoting and stabilizing microvascularization in engineered tissues and organs is still challenging for the available bioengineering technologies. Extracellular matrix (ECM)-based hydrogels have been extensively employed in microvascularization strategies thanks to their soft mechanics and native-like structure. Nonetheless, their bioactivity and physico-mechanical properties are difficult to control, given their biological complexity and batch-to-batch variability, which limit their application in tissue engineering. Norbornene-functionalized gelatin (GelNOR) gelates via thiol–ene click chemistry step-growth photopolymerization using multifunctional thiols as crosslinkers. Consequently, GelNOR physico-mechanical properties can be finely tuned. Additionally, as an endopeptidase cleavable ECM-derived material, GelNOR can be easily remodeled, facilitating cell-cell interactions and substitution by extracellular matrix derived from encapsulated cells. Here, we hypothesize that by using GelNOR we can obtain a hydrogel with optimal physico-mechanical properties, which will promote long-lasting microvascularization.
Methods: Hydrogels of variable compressive stiffness and crosslinking density were prepared by varying the GelNOR content (30 mg/mL, GelNOR[30]; 40 mg/mL, GelNOR[40]; and 50 mg/mL, GelNOR[50]) and crosslinker concentration (1-4 mM dithiothreitol, DTT) via UV light-mediated photopolymerization. The hydrogels, encapsulating human GFP-endothelial cells (ECs) and mesenchymal stromal cells (MSCs), were monitored over 14 days. Endothelial network formation was assessed by fluorescence microscopy and digitally quantified in terms of number of junctions and vascularized area.
Results: Increases in GelNOR content and DTT concentration resulted in a linear increase in compressive modulus and crosslinking density. Adjusting GelNOR:DTT ratio formed hydrogels between 0.67 ± 0.04 kPa (25% -NOR crosslinking in GelNOR[30]) and 3.29 ± 0.36 kPa (60% -NOR crosslinking in GelNOR[50]). All EC and MSC-laden hydrogels promoted endothelial network formation, exhibiting a peak in pre-vessel network density at day 5 (% of total area: GelNOR[30] ≈ 30 ± 3%, GelNOR[40] ≈ 25 ± 4%, GelNOR[50] ≈ 21 ± 2%). The pre-vascular networks prevailed until day 14 in all GelNOR hydrogels. GelNOR[50] led to stable vascular-like structures up to day 9, but denser endothelial networks (~2-fold increase in number of junctions) were observed in GelNOR[30] and GelNOR[40] gels, crosslinked by 1-2 mM DTT. When compared to the widely used methacrylated gelatin (GelMA), GelNOR hydrogels exhibited a strikingly superior performance (GelMA = 8 ± 1% microvascularized area); being GelNOR[30] hydrogels comparable to commercially available basement membrane-derived ECM (BME) in both vascularized area (BME = 35 ± 2%) and branching onset (day 3).
Conclusions: GelNOR was found to be a supportive material for neomicrovascularization that was optimized to meet the physico-mechanical requirements of ECs and MSCs for their self-assembly. GelNOR allows for the formation of hydrogels even at low crosslinking density and gelatin content that were found ideal for endothelial network formation. Our future work focuses on assessing the function of the observed vessel-like structures, as well as extending their stability. In our hands, GelNOR offers a versatile platform for the creation of fully customizable ECM-based hydrogels, where biological, biochemical, and mechanical properties can be easily tuned to match tissue-specific requirements."

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