Periodic Reporting for period 4 - VascArbor (Vascular Tree Formation in Multi-Structural Tissue Engineering)
Reporting period: 2021-10-01 to 2022-03-31
Engineered tissues offer great promise as an alternative for donor tissues, for which the supply is not meeting the demands. However, clinical application of engineered tissues is hampered. The integration of engineered tissues after implantation is limited due to the lack of a vascular network. Currently, strategies to include vascular networks rely on the spontaneous organization of vascular cells, or on the patterning of these cells. However, this results in either vascular networks that are not organized, or networks that lose their initial organization fast. This project will use a unique and novel approach to control vascular development and will therefore result in a vascular network with a controllable long-term organization. By allowing for anastomosis, and increasing nutrient delivery, this project will tackle an essential problem and will greatly enhance the clinical applicability of engineered tissues.
The main objective of VascArbor is to understand what factors control vascular organization, and to developed technology to control vascular organization within engineered tissues. Within VascArbor, fluid flows through engineered tissues were designed and controlled to guide vascular organization. Apart from that, growth factors were patterned both in space and time to further control vascular organization. In parallel, computational models were developed that can predict vascular organization and development based on processing parameters. This enables a direct link between a desired vascular organization, and the tissue construct geometry and processing conditions that are needed to acquire this organization.
VascArbor further developed technology for the fabrication of macro-scale tissue constructs, including a tissue building block approach and novel embedded 3D bio-printing technology. Within VascArbor, this technology was used to engineer vascularized cardiac muscle tissue based on the tissue organization guiding principles developed in this project.
The main objective of VascArbor is to understand what factors control vascular organization, and to developed technology to control vascular organization within engineered tissues. Within VascArbor, fluid flows through engineered tissues were designed and controlled to guide vascular organization. Apart from that, growth factors were patterned both in space and time to further control vascular organization. In parallel, computational models were developed that can predict vascular organization and development based on processing parameters. This enables a direct link between a desired vascular organization, and the tissue construct geometry and processing conditions that are needed to acquire this organization.
VascArbor further developed technology for the fabrication of macro-scale tissue constructs, including a tissue building block approach and novel embedded 3D bio-printing technology. Within VascArbor, this technology was used to engineer vascularized cardiac muscle tissue based on the tissue organization guiding principles developed in this project.
The project has focused on understanding how different factors, including patterned fluid flows and patterns of growth factors, influence vascular organization. Microfluidic platforms have been developed to investigate the combinatory effect of varying fluid flow profiles and growth factor gradients on vascular organization. To further understand what drives vascular organization, computational models were developed to predict organization based on multiple signals such as fluid flows and growth factors. These models were further refined and validated using data arising from cell culture experiments and from the observation of vascular organization in developing chicken egg models. These computational models enable a direct link between a desired vascular organization, and the tissue construct geometry and processing conditions that are needed to acquire this organization.
VascArbor has developed a novel technology based on aptamers to spatiotemporally pattern growth factors within a hydrogel tissue environment. Aptamers are small, single-stranded pieces of RNA that form unique 3D conformations based on their sequence. This enables selective binding to target biomolecules such as growth factors (GF) with high affinity and specificity. Complementary sequences (CS) having higher affinity towards the aptamers than the target GF can be added, resulting in demanded GF release upon aptamer-CS binding. This results in a temporal control over growth factor availability. As the hydrogels are compatible with photopatterning and 3D printing technology, spatial control over growth factor availability is also achieved. Based on this technology, an ERC Proof of Concept grant has been granted.
Additionally, two novel embedded bioprinting approaches have been developed. One is based on the printing of highly packed granular media. This technology is highly interesting to create patterned embedding baths. Multiple formulations of granular embedding baths have been investigated in terms of vascular network formation. Based on this technology, a patent has been filed and an ERC Proof of Concept grant has been granted. The other approach uses a viscous liquid as embedding bath. This approach allows for the concentration of cells within a 3D print, meaning that dilute cell suspensions can be printed which are then concentrated into a dense tissue. This has enabled the formation of macroscale beating cardiac tissue constructs.
Additionally, several perfusion bioreactor systems have been developed to accommodate tissue constructs of different sizes and shapes. These perfusion systems allow for control over fluid flow profiles within engineered tissue constructs in order to guide vascular organization and development. Combined, the project has resulted in new tools to tune the cellular environment within engineered tissue constructs, which is valuable for tissue engineering research in general. Additionally, by better understanding the signals that guide vascular organization within engineered tissues, combined with new technology to pattern these signals, the project has enabled a better control over vascular organization. Although full control to engineer a network organized as a vascular tree has not been achieved, important tools towards this goal have been developed.
VascArbor has developed a novel technology based on aptamers to spatiotemporally pattern growth factors within a hydrogel tissue environment. Aptamers are small, single-stranded pieces of RNA that form unique 3D conformations based on their sequence. This enables selective binding to target biomolecules such as growth factors (GF) with high affinity and specificity. Complementary sequences (CS) having higher affinity towards the aptamers than the target GF can be added, resulting in demanded GF release upon aptamer-CS binding. This results in a temporal control over growth factor availability. As the hydrogels are compatible with photopatterning and 3D printing technology, spatial control over growth factor availability is also achieved. Based on this technology, an ERC Proof of Concept grant has been granted.
Additionally, two novel embedded bioprinting approaches have been developed. One is based on the printing of highly packed granular media. This technology is highly interesting to create patterned embedding baths. Multiple formulations of granular embedding baths have been investigated in terms of vascular network formation. Based on this technology, a patent has been filed and an ERC Proof of Concept grant has been granted. The other approach uses a viscous liquid as embedding bath. This approach allows for the concentration of cells within a 3D print, meaning that dilute cell suspensions can be printed which are then concentrated into a dense tissue. This has enabled the formation of macroscale beating cardiac tissue constructs.
Additionally, several perfusion bioreactor systems have been developed to accommodate tissue constructs of different sizes and shapes. These perfusion systems allow for control over fluid flow profiles within engineered tissue constructs in order to guide vascular organization and development. Combined, the project has resulted in new tools to tune the cellular environment within engineered tissue constructs, which is valuable for tissue engineering research in general. Additionally, by better understanding the signals that guide vascular organization within engineered tissues, combined with new technology to pattern these signals, the project has enabled a better control over vascular organization. Although full control to engineer a network organized as a vascular tree has not been achieved, important tools towards this goal have been developed.
VascArbor has resulted in multiple developments well beyond the state of the art:
- The 3D printed granular bioactive embedding bath technology developed in this project has greatly enhanced the potential of embedded bioprinting technology. Where embedding baths are traditionally only used as a mechanical support to allow for the extrusion printing of bio-inks in any shape, the development of bioactive embedding baths adds the potential to locally control tissue development by including bioactive cues in the embedding bath. By making the bath itself printable, a high spatial control over the instructive signals is achieved.
- The aptamer based spatiotemporal growth factor patterning technology developed in this project allows for the spatiotemporal patterning of specific growth factors in a hydrogel tissue environment. This allows for an active control over spatial tissue development and organization. The platform allows for demanded release of the growth factors, and for multiple cycles of growth factor loading and release. As growth factor incorporation is based on affinity binding with the specific aptamer, no chemical modification of the growth factor is needed, resulting in optimal bioactivity of these molecules. Additionally, the platform allows for the sequestering of growth factors from the environment and does not rely on active loading of growth factors during the preparation of the hydrogels.
- The previous technologies developed within VascArbor, provide a high level of spatial control of tissue development. Using multiple controllable signals, including fluid flow shear stresses and the local availability of multiple angiogenic growth factors, has enabled a better control of vascular organization within engineered tissues. By combining this with computational models that can predict vascular organization based on the signals that are patterned within the tissue, a direct link between a desired vascular organization and the tissue construct geometry and processing conditions that are needed to acquire this organization has been achieved.
- The 3D printed granular bioactive embedding bath technology developed in this project has greatly enhanced the potential of embedded bioprinting technology. Where embedding baths are traditionally only used as a mechanical support to allow for the extrusion printing of bio-inks in any shape, the development of bioactive embedding baths adds the potential to locally control tissue development by including bioactive cues in the embedding bath. By making the bath itself printable, a high spatial control over the instructive signals is achieved.
- The aptamer based spatiotemporal growth factor patterning technology developed in this project allows for the spatiotemporal patterning of specific growth factors in a hydrogel tissue environment. This allows for an active control over spatial tissue development and organization. The platform allows for demanded release of the growth factors, and for multiple cycles of growth factor loading and release. As growth factor incorporation is based on affinity binding with the specific aptamer, no chemical modification of the growth factor is needed, resulting in optimal bioactivity of these molecules. Additionally, the platform allows for the sequestering of growth factors from the environment and does not rely on active loading of growth factors during the preparation of the hydrogels.
- The previous technologies developed within VascArbor, provide a high level of spatial control of tissue development. Using multiple controllable signals, including fluid flow shear stresses and the local availability of multiple angiogenic growth factors, has enabled a better control of vascular organization within engineered tissues. By combining this with computational models that can predict vascular organization based on the signals that are patterned within the tissue, a direct link between a desired vascular organization and the tissue construct geometry and processing conditions that are needed to acquire this organization has been achieved.