Laminin-Mimetic Peptide-Functionalized Hydrogel Systems for the Phenotypic Modulation of Cells of the Nucleus Pulposus
Date of Award
Doctor of Philosophy (PhD)
The intervertebral disc (IVD) has been widely observed to undergo significant structural and biochemical changes with age and maturation. As degeneration progresses, changes in extracellular matrix composition and deposition, tissue cellularity, and metabolic activity have been characterized. Although the epidemiology of disc degeneration remains unclear, it is believed that the nucleus pulposus (NP) region of the IVD may be implicated in early degeneration. Specifically, cells of the nucleus pulposus have been observed to undergo a shift from their notochordal-like juvenile phenotype to a more fibroblast-like state in a manner concomitant to degenerative events. Because the disc has inherently little capacity for self-repair due to low vascularization and nutrient supply as well as a low native cell density, different strategies have been investigated towards the goal of intervertebral disc repair. It has been previously demonstrated that culture of degenerative NP cells upon soft (<1 kPa) full length laminin-111-containing hydrogels promotes increased levels of a panel of markers associated with the juvenile NP phenotype. Alternatively, culture of these cells upon a stiff (~10 kPa) laminin-functionalized material promoted expression of fibroblast-like markers. In this dissertation, a stiff, peptide-functionalized PEG-based hydrogel system was studied for inducing phenotypic shifts similar to those observed in cell culture upon soft full-length laminin functionalized systems. Specifically, integrin-binding IKVAV and syndecan-binding AG73 motifs isolated from the LG domains of laminin-111 were conjugated to an 8-arm maleimide terminated star-PEG chain through terminal cysteines via maleimide-thiol Michael-type addition reactions. Peptide-functionalized constructs were then used as systems for cell culture and cell encapsulation in vitro and in vivo. Following NP cell culture in 2D, findings reveal that a stiff hydrogel functionalized with the adequate adhesive ligand density promoted a shift towards increased expression of pro-NP phenotypic markers, with a parallel decrease in expression of markers associated with the fibroblast-like state. Translation from a 2D cell culture substrate into 3D cell encapsulation suggests that the developed hydrogel system supports cell viability, biosynthetic activity, and protein deposition in vitro. Delivery of a stiff, peptide-functionalized cell-laden hydrogel into the intradiscal space in an in vivo model of disc degeneration via disc puncture in caudal spines of rats demonstrated the polymeric material to support cell viability and retention in the intradiscal space up to 8 weeks following delivery. These data demonstrated the biomaterial’s ability to promote bioactivity in the delivered cells as observed by increased Saf-O staining and immunolabeling of BASP1, integrin α6, and N-cadherin within the disc space at the 8-week time point. Overall, the current work elaborates upon previous research outlining the effects of substrate stiffness and biofunctionalization on NP cell phenotype, presenting a meaningful approach for phenotypic modulation with increased spatial control of adhesive domains and targeted cellular microenvironmental interactions. This work expands upon previous knowledge by demonstrating an ability to guide NP cell differentiation via control of adhesive motif presentation rather than substrate stiffness alone. Furthermore, this system provides the ability to culture cells in 3D, allowing for the development of a deliverable for retaining the juvenile NP phenotype in vivo. The potential of the cell-laden bioactive injectable to support expression and maintenance of markers of the juvenile phenotype of NP cells in vivo may present a clinically relevant approach towards the treatment of degenerative discs. Furthermore, the ability to use stiff substrates with proper ligand presentation in order to promote levels of phenotypic control normally seen in soft substrates may present benefits in terms of mechanical support for such an implantable device.
Lori A. Setton
Jacob M. Buchowski, Nathaniel Huebsch, Amit Pathak, Simon Y. Tang,