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Introduction Hierarchically arranged self-organizing vasculature is a long-sought goal of tissue engineering. In native tissues, various factors such as fluid flow, biomechanical cues and biochemical cues (growth factors & cytokines) work in synergy to achieve high precision over vasculature. To this end, by employing spatiotemporally controlled growth factor’s availability within engineered tissues could help in guiding the developing vasculature. However, the conventional approaches for growth factor delivery often focuses on their immobilization or coupling within the engineered matrices (hydrogel), via various linker proteins or peptides. Even though it provides stable release rates, but imparts limitations upon upscaling with high specificity of multiple growth factors delivery. To overcome this limitation, the present study employed oligonucleotides based aptamers, that are affinity ligands designed to recognize proteins with high affinity and specificity.1 The developed aptamer-functionalized biomaterials were systematically studied for achieving patterned self-organizing microvascular networks in 3D microenvironments. Materials & Methods The aptamer-functionalized hydrogels were prepared via photo-polymerization of gelatin methacryloyl (GelMA) and acrydite functionalized aptamers having DNA sequence specific for binding to vascular endothelial growth factor (VEGF165). Visible light photoinitiator, tris(2,2′-bipyridyl)dichloro-ruthenium(II) hexahydrate with sodium persulfate was used. For patterning, aptamer-functionalized hydrogels 3D printing technique was employed. The human umbilical vein endothelial cells (HUVECs) and mesenchymal stem cells (MSCs) were used. The construct was 3D bioprinted as lines of aptamer-functionalized bio-ink next to plain GelMA lines (blue beads), making an interface. After 3D printing, the constructs were crosslinked and loaded with VEGF for 1 hr. It was expected that the 3D bioprinted aptamer lines would be able to sequester VEGF from the culture medium, compared to GelMA lines. To study the programmable/triggered growth factor (VEGF) release efficiency, the complementary sequences (CSs) were also added at specific time-points and their effect of microvascular network formation was studied. Results & Discussion The results obtained from physicochemical analysis of the aptamer-functionalized hydrogels confirmed the higher aptamer retention capacity of acrydite functionalized aptamers within the hydrogels, in comparison with the control aptamers for as long as 10 days at 37 °C. The VEGF ELISA experiments confirmed triggered release of VEGF from the aptamer functionalized hydrogels in response to CS addition. Without CS addition, these hydrogels could sustain a controlled release for until 10 days. Furthermore, in co-culture experiments, the developed patterned aptamer-functionalized hydrogels showed high cellular viability and ability to guide microvascular network formation (by HUVECs and MSCs) only within the aptamer-functionalized regions of the pattern, and not in GelMA regions, after 10 days of culture (Figure 1). However, differences in the microvascular organization was observed in the samples with triggered VEGF release on on day 5, compared to the samples without the VEGF release. These observations altogether confirmed the ability of patterned aptamer functionalized hydrogels in controlling self-organizing microvascular networks. Conclusions The present study confirms the potential of patterned aptamer-functionalized hydrogels in guiding self-organizing microvascular networks within 3D microenvironment, by spatiotemporally controlling VEGF bioavailability. Acknowledgements: This work is supported by an ERC Consolidator Grant under grant agreement no 724469. References 1. D. Rana, A. Kandar, N. Salehi-Nik, I. Inci, B. Koopman, J. Rouwkema. BioRxiv (2020) doi: https://doi.org/10.1101/2020.09.22.308619. |
