Periodic Reporting for period 1 - HYDROCUP (The hydrocup: a hollow electrospun scaffold for in vivo cell-laden hydrogel delivery)
Reporting period: 2022-09-01 to 2024-02-29
Secreted cytokines by transplanted cells can have positive effects such as immunomodulation, angiogenesis and tissue regeneration. However, transplanted cells are difficult to retain in a specific place to maximize the release of cytokines on site. Here, we developed a hollow electrospun scaffold with one open and one closed end for the in vivo delivery of cells as ?secretory factories? in a hydrogel, termed the hydrocup. Because of the closed end, a (cell-laden) hydrogel can easily be dispensed into the open end of the hydrocup, after which the open end is closed with sutures. As part of the ERC starting grant where this application stems from, we showed that adult stem cells encapsulated inside the hydrocups remained viable for 28 days in vitro and released functional cytokines similar to human mesenchymal stromal cells (hMSCs) in hydrogels alone. In addition, the hydrocups remained intact and fixed in place for 6 weeks after subcutaneous implantation in rats. In this ERC proof of concept proposal, we aim at further studying the hydrocup as a device for a wide variety of cell-based cytokine secretion or drug-laden hydrogel applications. We will further refine the device for applications in the cardiac regeneration market where stem cell based therapies suffer so far from low viability of cells after injection. We will make a market analysis study of competitors in the cell therapy field and propose an initial business plan for seeking further investments to create a spin-off company at the end of the grant.
Cytokines released by hMSCs have a wide variety of effects on other cells or local tissues, including immunomodulation, angiogenesis, chemoattraction, anti-scarring and supporting local stem cells in tissue regeneration. Hydrogels are widely used as extracellular matrix (ECM) to mimic cellular microenvironment because of their mechanical and structural similarity to native tissues. This motivated us to develop a “cell factory”, where hMSCs encapsulated in hydrogels would be injected in an electrospun scaffold to improve cell survival and retention. Although hydrogels formed by natural biopolymers well recapitulate the architecture and mechanical properties of the ECM, they are difficult to tailor and manipulate. Oppositely, synthetic hydrogels would give rise to uniquely tailorable materials. However, the mechanical properties of these materials are manipulated by typically varying the polymer concentration, which, however changes all other hydrogel properties simultaneously. Strategies to fabricate hydrogels with tailorable mechanical properties but without significantly changing the components are highly challenging.
Here, we started by employing the fully synthetic semi-flexible ethylene glycol-substituted PIC polymers. The thermal-responsive hydrogels are reversibly formed when an aqueous PIC solution is heated above the gelation temperature. The gel is unique as it forms fibrous networks in a non-covalent bundled architecture that closely mimics the architecture of biopolymer networks, such as those based on collagen, fibrin, and actin. PIC gels display the same characteristic strain-stiffening response as the biopolymer hydrogels, meaning the stiffness sharply increases upon deformation. The PIC polymer chains were equipped with azide groups for crosslinking. To introduce dynamic properties in this static fibrous hydrogels, we developed hydrogen bonding crosslinking and ionic crosslinking. The interactions between the polymer chains were achieved by a small spacer with, at one terminus, a dibenzocyclooctyne (DBCO) group that can covalently bind to the azide groups on the PIC chains and on the other terminus a dopamine moiety that forms a dynamic hydrogen bond or metal-coordination bond to the ion. The covalent crosslinking through a di-functional DBCO crosslinkers was introduced as a comparison. To promote cell-matrix interactions, the cell adhesive peptide GRGDS equipped with a DBCO-terminated PEG spacer was conjugated to azide-functionalized PIC polymers. Hence, we developed PIC hydrogels with four crosslinking approaches, including physical crosslinking, non-covalent (hydrogen bonding and ionic) crosslinking, and covalent crosslinking to study the effects on cell behaviors, where crosslinking density was sequentially increasing. As a control, alginate and collagen hydrogels were used to assess the effect of hydrogel types on cell activities. The mechanical properties of PIC hydrogels mixed with magnetic nanoparticles were extremely increased under an external magnetic field, owing to the strain-, or stress-stiffening properties of PIC hydrogels.
hMSCs were encapsulated in PIC hydrogels. We found that a high cell density promoted cell spreading, while a high polymer concentration decreased cell spreading. Live-dead assay indicated that cells had high affinity in RGD-conjugated PIC hydrogels. We also tested the effect of crosslinking density on cell behavior. Cell spreading in physical crosslinked hydrogels was highest, while in covalent crosslinked hydrogels was lowest. Therefore, we could control cell fate by simply varying the crosslinking approaches in hydrogels. hMSCs cytokine secretion was also modulated depending on the hydrogel-electospun cup combination used among the newly generated Hydrocup formulations.
Here, we started by employing the fully synthetic semi-flexible ethylene glycol-substituted PIC polymers. The thermal-responsive hydrogels are reversibly formed when an aqueous PIC solution is heated above the gelation temperature. The gel is unique as it forms fibrous networks in a non-covalent bundled architecture that closely mimics the architecture of biopolymer networks, such as those based on collagen, fibrin, and actin. PIC gels display the same characteristic strain-stiffening response as the biopolymer hydrogels, meaning the stiffness sharply increases upon deformation. The PIC polymer chains were equipped with azide groups for crosslinking. To introduce dynamic properties in this static fibrous hydrogels, we developed hydrogen bonding crosslinking and ionic crosslinking. The interactions between the polymer chains were achieved by a small spacer with, at one terminus, a dibenzocyclooctyne (DBCO) group that can covalently bind to the azide groups on the PIC chains and on the other terminus a dopamine moiety that forms a dynamic hydrogen bond or metal-coordination bond to the ion. The covalent crosslinking through a di-functional DBCO crosslinkers was introduced as a comparison. To promote cell-matrix interactions, the cell adhesive peptide GRGDS equipped with a DBCO-terminated PEG spacer was conjugated to azide-functionalized PIC polymers. Hence, we developed PIC hydrogels with four crosslinking approaches, including physical crosslinking, non-covalent (hydrogen bonding and ionic) crosslinking, and covalent crosslinking to study the effects on cell behaviors, where crosslinking density was sequentially increasing. As a control, alginate and collagen hydrogels were used to assess the effect of hydrogel types on cell activities. The mechanical properties of PIC hydrogels mixed with magnetic nanoparticles were extremely increased under an external magnetic field, owing to the strain-, or stress-stiffening properties of PIC hydrogels.
hMSCs were encapsulated in PIC hydrogels. We found that a high cell density promoted cell spreading, while a high polymer concentration decreased cell spreading. Live-dead assay indicated that cells had high affinity in RGD-conjugated PIC hydrogels. We also tested the effect of crosslinking density on cell behavior. Cell spreading in physical crosslinked hydrogels was highest, while in covalent crosslinked hydrogels was lowest. Therefore, we could control cell fate by simply varying the crosslinking approaches in hydrogels. hMSCs cytokine secretion was also modulated depending on the hydrogel-electospun cup combination used among the newly generated Hydrocup formulations.
Cytokine secretion was at high levels in hydrocups and the secreted cytokines were functional. However, the treatment for cardiac diseases is still changeling due to the lack of cardiac vascularization and conduction. To ensure success, we may need to introduce new functionalities to the hydrocups, e.g. developing electroconductive electrospun scaffolds or/and hydrogels for electrical remodeling.
For commercialization, proof of concept regeneration in an animal model is a crucial first step to generate key pre-clinical data before further developing Hydrocup business plan. This data set allows laying a solid foundation for the creation of a spin-off company that can focus on the further development steps, mobilise sufficient resources to execute first in human studies and create a clinical proof of concept data. A detailed value proposition to apply Hydrocup for cardiac regeneration will be developed in collaboration with a panel of envisioned end-users, evaluating the end-user jobs, pains and gains.
For commercialization, proof of concept regeneration in an animal model is a crucial first step to generate key pre-clinical data before further developing Hydrocup business plan. This data set allows laying a solid foundation for the creation of a spin-off company that can focus on the further development steps, mobilise sufficient resources to execute first in human studies and create a clinical proof of concept data. A detailed value proposition to apply Hydrocup for cardiac regeneration will be developed in collaboration with a panel of envisioned end-users, evaluating the end-user jobs, pains and gains.