Engineering synthetic hydrogels for directing adult stem cell function for ligament repair
Date
2016
Authors
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Journal ISSN
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Publisher
University of Delaware
Abstract
Anterior cruciate ligament (ACL) rupture is one of the most common knee injuries, and most ACL tears require surgery due to the poor natural healing capacity of the ACL. Current options for ACL repair are associated with high risks of donor site morbidity (i.e. an inability to kneel or knee pain) and osteoarthritis after surgery; consequently, the search for alternative treatments remains an active area of research. The goal of this dissertation is to develop strategies towards the use of adult human mesenchymal stem cells (MSCs) in ligament repair. We exploit the many advantages of synthetic hydrogels, or crosslinked hydrophilic polymer networks, to understand MSC-extracellular matrix interactions and control growth factor presentation with applications in ligament repair. ☐ Specifically, we first investigated the interactions between human MSCs and extracellular matrix (ECM) mimics using a statistical design of experiments (DOE) approach. Combinations of matrix modulus and chemical composition that mimic aspects of developing tendon/ligament tissue were examined, and their effects on human MSC tenogenic/ligamentogenic differentiation were evaluated through analysis of gene expression and protein production. In the presence of the growth factor BMP-13, increasing the elastic modulus (from 10 kPa to 50 kPa or 90 kPa) and collagen mimetic peptide (CMP) content (from 0 mM CMP to 2 mM CMP) of the synthetic ECM increased ligament-associated gene expression and protein production or retention (i.e. upregulation of scleraxis, collagen I, tenascin-C). These studies demonstrated the power of a DOE approach in probing cell-matrix interactions for MSC differentiation down difficult to achieve lineages, and these findings will inform the design of scaffolds for tendon/ligament regeneration. ☐ Next, we examine the use of synthetic poly(ethylene glycol) thiol–norbornene hydrogels for the release of various proteins, an initial step towards the release of growth factors that could direct stem cell function at the site of ligament repair. In preliminary studies, these hydrogels slowed the diffusion of a model protein, bovine serum albumin (BSA), causing delayed release when compared with diffusion through water. We then compared two theories commonly used to evaluate the mesh size, or pore size, of these networks (rubber elasticity theory and equilibrium swelling theory), to determine which theory was more suitable for predicting protein release from these networks. Although both theories gave comparable predictions of the average hydrogel mesh size, we found that the best predictor of therapeutic protein release was the release of a model protein of comparable size, likely due to defects that cause distributions of pore sizes in these PEG-based hydrogels. Specifically, BSA and the growth factor PDGF-BB have similar hydrodynamic diameters and are released from the hydrogel at comparable rates, with more than 80% of both proteins released within 48 hours after gel synthesis. A novel method for rapidly evaluating protein release rates from hydrogels, employing a cocktail of inexpensive model proteins and accessible proteomics methods (SDS-PAGE) is demonstrated; we anticipate that this method may be useful for predicting the release rates of more expensive, therapeutically-relevant proteins while reducing cost and increasing throughput. ☐ Finally, we evaluate the use of several non-viral gene delivery systems [polyethyleneimine (PEI), Lipofectamine, and H3/PEI] for gene transfer to human MSCs. The studies represent an initial step towards encapsulating these vectors in hydrogels to direct MSCs to locally produce growth factors, with eventual applications to produce growth factors in a spatially-specific manner towards tissue interface regeneration. Here, DOE approaches are used to optimize transfection of human MSCs with PEI polyplexes and H3/PEI polyplexes. H3/PEI polyplexes are able to transfect human MSCs leading to significant protein production and minimal cytotoxicity, suggesting the potential of these H3/PEI polyplexes in synthetic hydrogel-based scaffolds for both culture and translational applications with human stem cells. ☐ Collectively, the research described here highlights the significant potential that hydrogels have for modulating stem cell fate and controlling protein release towards improving ligament repair. These foundational studies provide insight into key MSC-extracellular matrix interactions for directing ligamentogenesis/tenogenesis and the design of materials for temporal presentation of growth factors to MSCs, with applications in each study towards improved ligament repair. We conclude by outlining several suggested directions that we anticipate will lead to translation of these findings into pre-clinical animal models for ligament injury and, in the long term, into clinical models for improved ligament repair.
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Keywords
Synthetic hydrogel, Adult stem cell, Ligament repair