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Targeted therapies using biopharmaceuticals are of growing clinical importance in disease treatment. Currently, there are several limitations of protein-based therapeutics (biologicals), including suboptimal biodistribution, lack of stability, and systemic side effects. A promising approach to overcoming these limitations could be a therapeutic cell-loaded 3D construct consisting of a suitable matrix component that harbors producer cells continuously secreting the biological of interest. Here, the recombinant spider silk proteins eADF4(C16), eADF4(C16)-RGD, and eADF4(C16)-RGE have been processed together with HEK293 producer cells stably secreting the highly traceable reporter biological TNFR2-Fc-GpL, a fusion protein consisting of the extracellular domain of TNFR2, the Fc domain of human IgG1, and the luciferase of

OBJECTIVE Transplantation of prefabricated tissue-engineered flaps can be a potential alternative for healing large tissue defects. Providing adequate vascular supply for an engineered tissue construct is one of the key points in establishing successful tissue engineering based treatment approaches. In tissue engineering-based vascularization techniques like the AV loop, vascular grafts with high angiogenic potential can help to enhance neovascularization and tissue formation. Therefore, our study aimed to compare the angiogenic potential of vascular grafts from different locations in the rat. METHODS The angiogenic activity was investigated by an ex vivo vessel outgrowth ring assay using 1 mm height vascular segments embedded in fibrin for two weeks. RESULTS Maximum vessel outgrowth was observed on days 10-12. Upper extremity vessels exhibited stronger outgrowth than lower extremity vessels. Moreover, arterial vessels demonstrated higher angiogenic potential compared to venous vessels. CONCLUSION Collectively, our ex vivo findings suggest that upper extremity arterial vessels have a higher angiogenic capacity, which could be used to improve neovascularization and tissue formation in tissue engineering.

Due to its low immunogenic potential and the possibility to fine-tune their properties, materials made of recombinant engineered spider silks are promising candidates for tissue engineering applications. However, vascularization of silk-based scaffolds is one critical step for the generation of bioartificial tissues and consequently for clinical application. To circumvent insufficient vascularization, the surgically induced angiogenesis by means of arteriovenous loops (AVL) represents a highly effective methodology. Here, previously established hydrogels consisting of nano-fibrillary recombinant eADF4(C16) were transferred into Teflon isolation chambers and vascularized in the rat AVL model over 4 weeks. To improve vascularization, also RGD-tagged eADF4(C16) hydrogels were implanted in the AVL model over 2 and 4 weeks. Thereafter, the specimen were explanted and analyzed using histology and microcomputed tomography. We were able to confirm biocompatibility and tissue formation over time. Functionalizing eADF4(C16) with RGD-motifs improved hydrogel stability and enhanced vascularization even outperforming other hydrogels, such as fibrin. This study demonstrates that the scaffold ultrastructure as well as biofunctionalization with RGD-motifs are powerful tools to optimize silk-based biomaterials for tissue engineering applications.

Due to its low immunogenic potential and the possibility to fine-tune their properties, materials made of recombinant engineered spider silks are promising candidates for tissue engineering applications. However, vascularization of silk-based scaffolds is one critical step for the generation of bioartificial tissues and consequently for clinical application. To circumvent insufficient vascularization, the surgically induced angiogenesis by means of arteriovenous loops (AVL) represents a highly effective methodology. Here, previously established hydrogels consisting of nano-fibrillary recombinant eADF4(C16) were transferred into Teflon isolation chambers and vascularized in the rat AVL model over 4 weeks. To improve vascularization, also RGD-tagged eADF4(C16) hydrogels were implanted in the AVL model over 2 and 4 weeks. Thereafter, the specimen were explanted and analyzed using histology and microcomputed tomography. We were able to confirm biocompatibility and tissue formation over time. Functionalizing eADF4(C16) with RGD-motifs improved hydrogel stability and enhanced vascularization even outperforming other hydrogels, such as fibrin. This study demonstrates that the scaffold ultrastructure as well as biofunctionalization with RGD-motifs are powerful tools to optimize silk-based biomaterials for tissue engineering applications.

In situ tissue engineering is an emerging field aiming at the generation of ready-to-use three-dimensional tissues. One solution to supply a proper vascularization of larger tissues to provide oxygen and nutrients is the arteriovenous loop (AVL) model. However, for this model, suitable scaffold materials are needed that are biocompatible/non-immunogenic, slowly degradable, and allow vascularization. Here, we investigate the suitability of the known gelatin methacryloyl (GelMA)-based hydrogel for in-situ tissue engineering utilizing the AVL model. Rat AVLs are embedded by two layers of GelMA hydrogel in an inert PTFE chamber and implanted in the groin. Constructs were explanted after 2 or 4 weeks and analyzed. For this purpose, gross morphological, histological, and multiphoton microscopic analysis were performed. Immune response was analyzed based on anti-CD68 and anti-CD163 staining of immune cells. The occurrence of matrix degradation was assayed by anti-MMP3 staining. Vascularization was analyzed by anti-α-smooth muscle actin staining, multiphoton microscopy, as well as expression analysis of 53 angiogenesis-related proteins utilizing a proteome profiler angiogenesis array kit. Here we show that GelMA hydrogels are stable for at least 4 weeks in the rat AVL model. Furthermore, our data indicate that GelMA hydrogels are biocompatible. Finally, we provide evidence that GelMA hydrogels in the AVL model allow connective tissue formation, as well as vascularization, introducing multiphoton microscopy as a new methodology to visualize neovessel formation originating from the AVL. GelMA is a suitable material for in situ and in vivo TE in the AVL model.