The effect of glycosaminoglycan content on polyethylenimine-based gene delivery within three-dimensional collagen-GAG scaffolds

Abstract

The design of biomaterials for increasingly complex tissue engineering applications often requires exogenous presentation of biomolecular signals. Integration of gene delivery vectors with a biomaterial scaffold offers the potential to bypass the use of expensive and relatively inefficient growth factor supplementation strategies to augment cell behavior. However, integration of cationic polymer based gene delivery vectors within three-dimensional biomaterials, particularly matrices which can carry significant surface charge, remains poorly explored. We examined the potential of polyethylenimine (PEI) as a gene delivery vector for three-dimensional collagen-glycosaminoglycan (CG) scaffolds under development for tendon repair. While acetylated versions of PEI have demonstrated improved transfection efficiency in 2D culture assays, we investigated translation of this effect to a 3D biomaterial that contains significant electrostatic charge. A reporter gene was used to examine the impact of polymer modification, polymer:DNA ratio, and the degree of sulfation of the biomaterial microenvironment on gene delivery in vitro. We observed highest transgene expression in acetylated and unmodified PEI at distinct polymer:DNA ratios; notably, the enhancement often seen in two-dimensional culture for acetylated PEI did not fully translate to three-dimensional scaffolds. We also found highly sulfated heparin-based CG scaffolds showed enhanced initial luciferase expression but not prolonged activity. While PEI constructs significantly reduced tenocyte metabolic health during the period of transfection, heparin-based CG scaffolds showed the greatest recovery in tenocyte metabolic health over the full 2 week culture. These results suggest that the electrostatic environment of three-dimensional biomaterials may be an important design criterion for cationic polymer-based gene delivery.

Increasing the strength and bioactivity of collagen scaffolds using customizable arrays of 3D-printed polymer fibers

“…In order to combat diffusive loss of these factors, additional efforts have described modifications to promote ubiquitous or spatially-patterned covalent immobilization of growth factors within the scaffold network [28–31]. More recently, we have also described the use of scaffold proteoglycan chemistry to promote transient sequestration of growth factors within the CG scaffold via non-covalent interactions [32,33]. …”

Cell number and chondrogenesis in human mesenchymal stem cell aggregates is affected by the sulfation level of heparin used as a cell coating

“…17–21 Such a concept would mimic the actions of heparin sulfate proteoglycans (HSPGs) typically found on the plasma membrane of a cell and within the ECM. 22,23 HSPGs play a role in regulation of signaling factor activity and can act as co-receptors for various growth factor receptors to lower receptor activation or alter duration of the signaling reactions.…”

Challenges and Opportunities to Harnessing the (Hematopoietic) Stem Cell Niche

“…Recent efforts for MSC expansion and musculoskeletal repair leveraging proteoglycan-inspired transient sequestration and molecular imprinting technologies demonstrate the importance of sequestration strategies, offering exciting opportunities to further control and refine stem cell response [99]. Here, charge-based or conformation-based biomolecule interactions within constructs could sequester exogenously-added or endogenously-produced biomolecular signals to enhance responses such as proliferation and fate specification [100–102]. As such, integrating charged proteoglycans such as heparin or chondroitin sulfate into 3D constructs have been shown to strengthen signaling pathways [99, 100] and amplify gene delivery [102] to boost MSC response.…”

“…Here, charge-based or conformation-based biomolecule interactions within constructs could sequester exogenously-added or endogenously-produced biomolecular signals to enhance responses such as proliferation and fate specification [100–102]. As such, integrating charged proteoglycans such as heparin or chondroitin sulfate into 3D constructs have been shown to strengthen signaling pathways [99, 100] and amplify gene delivery [102] to boost MSC response.…”