Periodic Reporting for period 1 - BioTisSeal (Bioactive Tissue Sealant for Enhanced Wound Repair)
Reporting period: 2022-06-01 to 2023-11-30
Wound management is a routine part of medical practices worldwide, and delays in healing cause a significant clinical and economic burden. tissue adhesives/sealants that can adhere to wet tissue surfaces, can be used as barriers for blood leakage and to hold tissues together or fix implants onto the organ or tissue surfaces. However, most commercial sealants provide no bioactive properties, or at best rely on bulk growth factor (GF) loading for inducing cell infiltration or tissue regeneration. Angiogenic GFs are critically important for regulating the wound repair process. However, in case of chronic wounds, the excessive presence of proteases produced by inflammatory cells results in a shortage of endogenous GFs, which impairs the overall healing process. The overall objective of the project BioTisSeal has been to develop the technology for fabricating off-the-shelf bioactive tissue sealants that can stimulate wound healing by concentrating GFs involved in wound repair at the wound site. To achieve this objective, the following aims were defined:
- To optimize the conditions necessary for engineering bioactive tissue sealants, based on VEGF165 specific aptamers, its complementary sequence (CS) and gelatin methacryloyl hydrogels, for high extrudability, tissue adhesiveness and biocompatibility, in vitro.
- To investigate the potential of aptamer functionalized bioactive sealants for endogenous VEGF sequestration from the wound microenvironment as well as controlling its triggered release via CS binding in-vitro. Furthermore, to evaluate the angiogenic potential of the sequestered endogenous VEGF for enhancing wound healing angiogenesis, in vitro.
- To develop “off-the-shelf” formulations of the developed bioactive sealants by utilizing lyophilization and freeze-drying techniques that would increase its shelf life, transportability and commercialization prospects.
- To optimize the conditions necessary for engineering bioactive tissue sealants, based on VEGF165 specific aptamers, its complementary sequence (CS) and gelatin methacryloyl hydrogels, for high extrudability, tissue adhesiveness and biocompatibility, in vitro.
- To investigate the potential of aptamer functionalized bioactive sealants for endogenous VEGF sequestration from the wound microenvironment as well as controlling its triggered release via CS binding in-vitro. Furthermore, to evaluate the angiogenic potential of the sequestered endogenous VEGF for enhancing wound healing angiogenesis, in vitro.
- To develop “off-the-shelf” formulations of the developed bioactive sealants by utilizing lyophilization and freeze-drying techniques that would increase its shelf life, transportability and commercialization prospects.
The following work has been performed to reach the goals of the project:
GelMA tissue sealant development and evaluation:
GelMA was prepared using different concentrations of prepolymer, after which the gelling properties and storage moduli were determined and lap shear tests were performed. This showed that both 20% GelMA and 25% GelMA displayed sufficient mechanical strength and adhesive properties to be used as a tissue sealant. 20% GelMA showed good extrudability, while extrusion was more difficult with the 25% formulation. As such, 20% GelMA was chosen as the optimal formulation. Subsequently, burst pressure experiments were performed using porcine lung tissue. This revealed burst pressures in the order of 6 kPa. Experiments were repeated after the inclusion of aptamers in the GelMA hydrogels. Aptamers resulted in a slight increase in mechanical strength, but a slight decrease in lap-shear strength. However, the recorded values were still deemed sufficient for use as a tissue sealant.
VEGF and double-growth factor sequestration and controlled release:
The VEGF aptamer containing hydrogels were tested for their capacity to sequester VEGF from blood plasma, as a model to replicate the wound environment. This showed that the hydrogels were capable of concentrating VEGF, both with pure blood plasma and with blood plasma supplemented with additional VEGF. This served as a proof of principle that the developed hydrogel indeed has the capacity to sequester and concentrate GFs from plasma, thus potentially allowing for a bioactive effect. Subsequent release profiles upon the addition of complementary sequence aptamers were similar compared to the profiles of our previous study (1), which are included in Annex 1. Additional experiments also revealed that smaller portions of the sequestered GF could be released multiple times by adding smaller amounts of complementary sequence aptamer. Subsequent in vitro artificial wound model experiments showed a significant increase in wound vascularization and wound filling when using aptamer functionalized hydrogels compared to non-functionalized hydrogels, showing the biological activity of this approach.
Additional to what was planned in Annex 1, double-aptamer hydrogels were developed that can independently sequester and release two GFs (VEGF and PDGF) to increase the biological efficacy of the system. This serves as a proof-of-principle that multiple signals can be concentrated and released within a single hydrogel sealant, without interfering with the sequestration and release of the GFs.
Development of protocol for the lyophilization and reconstitution of GelMA tissue sealants:
Regarding the development of preservation protocols, a successful protocol has been developed to dry aptamer containing GelMA tissue sealants by infiltrating the hydrogel with a polysaccharide before lyophilization. Although the lyophilization resulted in shrinkage of the GelMA object and crack formation in the object, lyophilized GelMA sealants could be successfully reconstituted. Reconstituted sealants only showed a slight decrease in mechanical properties and adhesive properties, and were still able to sequester and release growth factors. This is an important step for the long term storage of these normally fast degrading hydrogels. Current work is focusing on determining the long-term stability of the dehydrated formulations and on determining the stability and long-term activity of aptamers included in the dehydrated gels.
1. Rana D, Kandar A, Salehi-Nik N, Inci I, Koopman B, Rouwkema J. Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues. Bioact Mater. 2022;12:71-84.
GelMA tissue sealant development and evaluation:
GelMA was prepared using different concentrations of prepolymer, after which the gelling properties and storage moduli were determined and lap shear tests were performed. This showed that both 20% GelMA and 25% GelMA displayed sufficient mechanical strength and adhesive properties to be used as a tissue sealant. 20% GelMA showed good extrudability, while extrusion was more difficult with the 25% formulation. As such, 20% GelMA was chosen as the optimal formulation. Subsequently, burst pressure experiments were performed using porcine lung tissue. This revealed burst pressures in the order of 6 kPa. Experiments were repeated after the inclusion of aptamers in the GelMA hydrogels. Aptamers resulted in a slight increase in mechanical strength, but a slight decrease in lap-shear strength. However, the recorded values were still deemed sufficient for use as a tissue sealant.
VEGF and double-growth factor sequestration and controlled release:
The VEGF aptamer containing hydrogels were tested for their capacity to sequester VEGF from blood plasma, as a model to replicate the wound environment. This showed that the hydrogels were capable of concentrating VEGF, both with pure blood plasma and with blood plasma supplemented with additional VEGF. This served as a proof of principle that the developed hydrogel indeed has the capacity to sequester and concentrate GFs from plasma, thus potentially allowing for a bioactive effect. Subsequent release profiles upon the addition of complementary sequence aptamers were similar compared to the profiles of our previous study (1), which are included in Annex 1. Additional experiments also revealed that smaller portions of the sequestered GF could be released multiple times by adding smaller amounts of complementary sequence aptamer. Subsequent in vitro artificial wound model experiments showed a significant increase in wound vascularization and wound filling when using aptamer functionalized hydrogels compared to non-functionalized hydrogels, showing the biological activity of this approach.
Additional to what was planned in Annex 1, double-aptamer hydrogels were developed that can independently sequester and release two GFs (VEGF and PDGF) to increase the biological efficacy of the system. This serves as a proof-of-principle that multiple signals can be concentrated and released within a single hydrogel sealant, without interfering with the sequestration and release of the GFs.
Development of protocol for the lyophilization and reconstitution of GelMA tissue sealants:
Regarding the development of preservation protocols, a successful protocol has been developed to dry aptamer containing GelMA tissue sealants by infiltrating the hydrogel with a polysaccharide before lyophilization. Although the lyophilization resulted in shrinkage of the GelMA object and crack formation in the object, lyophilized GelMA sealants could be successfully reconstituted. Reconstituted sealants only showed a slight decrease in mechanical properties and adhesive properties, and were still able to sequester and release growth factors. This is an important step for the long term storage of these normally fast degrading hydrogels. Current work is focusing on determining the long-term stability of the dehydrated formulations and on determining the stability and long-term activity of aptamers included in the dehydrated gels.
1. Rana D, Kandar A, Salehi-Nik N, Inci I, Koopman B, Rouwkema J. Spatiotemporally controlled, aptamers-mediated growth factor release locally manipulates microvasculature formation within engineered tissues. Bioact Mater. 2022;12:71-84.
The project has resulted in a clear proof-of-principle that aptamer-functionalized GelMA based tissue sealants can concentrate growth factors from the wound environment, offering the potential for a bioactive tissue sealant that can actively improve healing processes. It has further been shown that it is likely that the tissue sealant can be stored in a dehydrated state, which is a positive indicator for the shelf-life and the price of the product. During the project, a market analysis has been performed. Based on this analysis, it was concluded that BioTisSeal offers an interesting technology in a growing area of wound coverings and tissue sealants. However, given its higher price point compared to conventional wound dressing, entry to market was seen as difficult. Sealants for internal use could be a more interesting market, but given the higher risk, more research would be needed to establish the feasibility of the approach.
Regarding the IP protection of preservable hydrogel formulations, a Dutch patent application has been filed in June 2022.
Regarding the IP protection of preservable hydrogel formulations, a Dutch patent application has been filed in June 2022.