Cell-Derived Extracellular Matrix Scaffolds Developed using Macromolecular Crowding
Cell-derived (CDM) matrix scaffolds provide a 3-dimensional (3D) matrix material that recapitulates a native, human extracellular matrix (ECM) microenvironment. CDMs are a heterogeneous source of ECM proteins with a composition dependent on the cell source and its phenotype. CDMs have several applications, such as for development of cell culture substrates to study stromal cell propagation and differentiation, as well as cell or drug delivery vehicles, or for regenerative biomaterial applications. Although CDMs are versatile and exhibit advantageous structure and activity, their use has been hindered due to the prolonged culture time required for ECM deposition and maturation in vitro. Macromolecular crowding (MMC) has been shown to increase ECM deposition and organization by limiting the diffusion of ECM precursor proteins and allowing the accumulation of matrix at the cell layer. A commonly used crowder that has been shown to increase ECM deposition in vitro is Ficoll, and was used in this study as a positive control to assess matrix deposition. Hyaluronic acid (HA), a natural crowding macromolecule expressed at high levels during fetal development, has been shown to play a role in ECM production, organization, and assembly in vivo. HA has not been investigated as a crowding molecule for matrix deposition or development of CDMs in vitro. This dissertation focused on 2 aims supporting the development of a functional, human dermal fibroblast-derived ECM material for the delivery deliver an antimicrobial peptide, cCBD-LL37, and for potentially promoting a pro-angiogenic environment. The goal of this thesis was to evaluate the effects of high molecular weight (HMW) HA as a macromolecular crowding agent on in vitro deposition of ECM proteins important for tissue regeneration and angiogenesis. A pilot proteomics study supported the use of HA as a crowder, as it preliminarily showed increases in ECM proteins and increased retention of ECM precursor proteins at the cell layer; thus supporting the use of HA as a crowder molecule. In the presence of HA, human dermal fibroblasts demonstrated an increase in ECM deposition comparable to the effects of Ficoll 70/400 at day 3 using Raman microspectroscopy. It was hypothesized that HA promotes matrix deposition through changes on ECM gene expression. However, qRT-PCR results indicate that HA and Ficoll 70/400 did not have a direct effect on collagen gene expression, but differences in matrix crosslinking and proteinase genes were observed. Decellularized CDMs were then used to assess CDM stiffness and endothelial sprouting, which indicated differences in structural organization of collagen, and preliminarily suggests that there are differences in endothelial cell migration depending on the crowder agent used in culture. Finally, the collagen retained in the decellularized CDM matrix prepared under MMC supported the binding of cCBD-LL37 with retention of antimicrobial activity when tested against E.coli. Overall, the differences in matrix deposition profiles in HA versus Ficoll crowded cultures may be attributed to crowder molecule-mediated differences in matrix crosslinking, turnover, and organization as indicated by differences in collagen deposition, matrix metalloproteinase expression, and matrix stiffness. MMC is a valuable tool for increasing matrix deposition, and can be combined with other techniques, such as low oxygen and bioreactor cultures, to promote development of a biomanufactured CDM-ECM biomaterial. Successful development of scalable CDM materials that stimulate angiogenesis and support antimicrobial peptide delivery would fill an important unmet need in the treatment of non-healing, chronic, infected wounds.