G. Lindberg (Christchurch, NZ)
University of Otago Christchurch Orthopeadic researchPresenter Of 1 Presentation
10.4.2 - Design of chondro-instructive hydrogels for high-throughput 3D-biofabrication of cartilage tissue modules
Abstract
Purpose
3D-bioassembly approaches combining high-throughput fabrication of tissue modules with automated extrusion-based 3D printing of reinforcing thermoplastic polymer scaffolds represents a promising strategy to gain translational relevance in the field of cartilage tissue engineering. However, a major bottleneck lies in designing cell-laden hydrogels that are compatible with 3D-biofabrication, tailorable and cell-instructive, all-in-one. This study thus aimed to develop versatile chondro-instructive hydrogels for high-throughput 3D-biofabrication of tissue modules. We focused on 1) formulating customizable and bioactive thiol-ene click hydrogels by photo-polymerising thiolated heparin (HepSH), a native cartilage glycosaminoglycan (GAG), with allylated gelatin (GelAGE), and 2) investigating if vitreous humor (VH), a highly hydrated tissue closely resembling the composition of cartilage, can be applied as unmodified, cell-instructive hydrogels; systematically studying biofabrication compatibility, micro-tissue self-assembly and capacity to facilitate chondrogenesis.
Methods and Materials
GelAGE-HepSH micro-spheres were biofabricated and photo-polymerised (450nm, 20wt.-%GelAGE, 0.5wt%HepSH) and equine VH was extracted. Chondrocyte-laden hydrogels (5-15x106 cells/ml) were cultured (3-5w, TGF-β1) and physico-chemical properties, HepSH retention, cellular health (Live/dead®, alamarBlue®) and tissue formation (GAG, DNA, IHC: Safranin-O, Agg/Cx43, Col I/II, PCR:Col I/II,Agg, Sox9) was characterized.
Results
GelAGE-HepSH was successfully biofabricated into Ø1mm micro-spheres with tailorable physico-chemical properties (7-98kPa) and good viability (>80%). GelAGE further allowed efficient conjugation of HepSH (>96%), yielding significantly greater differentiation (GAG/DNA=134g/g, PCR: collagen-type-II=1.4x, aggrecan:1.9x) compared to GelAGE alone (GAG/DNA=24g/g). Likewise was unmodified and intact VH successfully seeded with cells, subsequently self-assembling into Ø1mm micro-spheres displaying uniform distribution of GAGs and collagen type II with significantly greater expression of chondrogenic markers (collagen-type-II=2.2x, 3.3x, aggrecan=1.6x, 13.3x) as well as GAG/DNA (14.1±2.5g/g) compared to controls (micro-pellets=7.0±1.3g/g, collagen-type-1 hydrogels=1.6±0.6g/g).
Conclusion
This study demonstrated that developed GelAGE-HepSH and VH hydrogels can be favorably applied as multifunctional, customizable and cell-instructive biomaterials compatible with high-throughput 3D-bioassembly approaches for clinical relevance and practicality - concurrently demonstrating high shape fidelity, tailorable physico-chemical properties and improved bioactivity for functional cartilage repair.