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Physical and biological characterization of a platform for cross-linking collagen allowing independent control of net charge and degradation time
Thesis

Physical and biological characterization of a platform for cross-linking collagen allowing independent control of net charge and degradation time

Liam Sweeney
Master of Science (M.S.), Drexel University
Jun 2024
DOI:
https://doi.org/10.17918/00010662
pdf
Sweeney_Liam_20242.56 MB
PDF Embargoed Access, Embargo ends: 31 Aug 2026

Abstract

Biomaterial Cross-linking Collagen Drug Delivery Systems Growth Factors Wound Healing
The ability to vary the charge and degradation time of biomaterials is advantageous for many aspects of tissue engineering and regenerative medicine across various treatment modalities. However, one of the most exciting opportunities is to take advantage of both features to specifically control the sequestration and delivery of important cytokines and factors for regenerative medicine. Presented here is a such a system for altering the net charge and degradation time of collagen, a critical extracellular matrix component for tissue regeneration. By oxidizing dextran polymers of various charges with peroxide, dextran-aldehyde cross-linkers were synthesized which can be used to cross-link collagen dispersions and ultimately alter the net charge of collagen foams. This platform is based on three different water-soluble solid cross-linkers of collagen: Dextran Aldehyde (neutrally charged), DEAE-Dextran Aldehyde (positively charged), and CM-Dextran Aldehyde (negatively charged). By incorporating these crosslinkers in a dispersion of bovine tendon derived collagen prior to freeze-drying and dehydrothermal treatment (DHT), the aldehyde groups of each dextran-aldehyde form cross-links with free amines available on the collagen peptide with a Schiff-base reaction. Importantly the system allows for independent control of net charge, through the type and concentration of cross-linker added to collagen dispersion, and degradation time, through the degree of oxidation and/or the concentration of cross-linker added to collagen dispersion. This work sought to characterize the physical properties of this material and understand its efficacy at binding specific factors relevant to regenerative medicine. We characterized the physical material properties through the following assessments: aldehyde functionalization, assessed through hydroxylamine hydrochloride titration, dross-linking density with through differential scanning calorimetry (DSC), and degradation time with enzymatic degradation Together, these metrics confirmed successful oxidation and control of cross-linking density. Moreover, we can spatially alter the net charge and degradation time within one single collagen material, opening an exciting list of future opportunities for drug delivery and biomimetic collagen biomaterials. Sequestration of relevant factors with opposite charges by different charged collagen foams was assessed through sequestration of epidermal growth factor (EGF) and platelet-derived growth factor (PDGF-bb) from a stock solution with enzyme-linked immunosorbent assays (ELISAs). Significantly more EGF, which is negatively charged (pI 4.6), was sequestered by the positively charged collagen, DEAE-Dextran Aldehyde cross-linked collagen (DEAE-DA Collagen (+)) than the neutral collagen, Neutral Dextran Aldehyde cross-linked collagen (Neutral-DA collagen), negatively charged collagen, CM-Dextran Aldehyde crosslinked collagen (CM-DA Collagen (-)), and control formaldehyde-cross-linked collagen. Similarly, significantly more PDGF-bb, which is positively charged (pI 9.8), was sequestered by the negatively charged collagen, CM-DA Collagen (-), than the neutral cross-linked collagen, Neutral-DA collagen, and the positively charged collagen DEAE-DA Collagen (+). Finally, we employed benchtop and animal models to understand the charged material's biocompatibility and efficacy for potential therapeutic and translational applications. In vitro biocompatibility, assessed using with human mesenchymal stromal cells (hMSCs), demonstrated reasonable viability across the majority of the charge formulations. Finally, a porcine wound healing model showed wound healing efficacy and in vivo biocompatibility. Interestingly, the charge of the material also appears to alter the cellular response during the early stages of wound healing, as demonstrated with histopathology analysis with Masson's Trichrome stained explants.

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