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Toward Better Recapitulation of Native Tissues and Tissue Environments

thesis
posted on 2024-06-24, 13:54 authored by Carly M BattistoniCarly M Battistoni

Tissue engineering utilizes polymers, cells, and other bioactive factors to promote regeneration within damaged tissue. The main works in this thesis employ naturally derived polymers for use in tissue engineering and explores ways to recapitulate native environments in vitro.

Collagen (col) is the most prevalent protein in the body. Col type I, II, and III are all fibril-forming collagens that provide structure to tissues. All three types polymerize in vitro to form hydrogels, and these hydrogels have often been studied for use in tissue engineering. Other applications include in vitro tissue models for studies on drug diffusion and drug delivery. Blending collagen types is of particular interest as col I is easier to source and is therefore cheaper than other collagen types. However, to confer biological signals to tissues where col II or III are more abundant (e.g., cartilage or cardiac tissue, respectively), col II or III can be added to col I to form col I/II or col I/III gels, respectively. Additionally, adding multiple types of col to hydrogel models better recapitulates the native environment and can better capture effects on drug diffusion. In this work, compared to col I alone, col I/II hydrogels polymerize more slowly, form more fibril bundles, result in softer hydrogels, and impede transport of larger macromolecules. On the other hand, col I/III gels polymerize at a similar rate to col I, create heterogenous fibril structures, are oftentimes stiffer than col I, and also impede transport of larger macromolecules. Additionally, this work explored the effect of polymerization temperature on blended gel polymerization and properties.

The second work evaluates col I/II hydrogels for a specific application: cartilage tissue engineering for osteoarthritic applications. Col II is the primary protein found in cartilage. Other components include: glycosaminoglycans, such as hyaluronic acid (HA) and chondroitin sulfate, chondrocytes (cartilage cells), and other small signaling molecules. Building on prior work in the group, high molecular weight hyaluronic acid (HA) was added to col I/II hydrogels, and cartilage differentiation of mesenchymal stem cells (MSCs) was assessed under ideal laboratory conditions and under pro-inflammatory, osteoarthritic conditions (i.e., cytokine-supplemented media of oncostatin M (OSM) at 10 ng/mL and tumor necrosis factor-α (TNF-α) at 20 ng/mL). The addition of HA did not dramatically impact cartilage differentiation of MSCs, however, HA did mitigate the effect of inflammation via downregulation of a degradative enzyme. HA had little impact on inflammatory cytokine production of interleukin (IL)-6 or IL-8, both of which are upregulated during osteoarthritis. However, a linear model suggests that HA and IL-8 are strongly correlated. Thus, this system should be explored further with different HA concentrations or presentations (e.g., chemically modified).

The last primary chapter of this thesis provides depth to the pro-inflammatory, osteoarthritic model used in the previous chapter. Different pro-inflammatory environments are studied using cytokines found in OA. MSC pellets (used in literature as controls to confirm chondrogenic potential of MSCs) were used to evaluate these inflammatory environments since MSCs are commonly used in tissue engineering. Six treatments were studied: negative control (without the chondrogenic growth factor TGF-β3), positive control (with the chondrogenic growth factor TGF-β3), and four cytokine treatments all with TGF-β3. First, IL-1β at 10 ng/mL was utilized as a comparison to literature. The other three cytokine groups used TNF-α at 20 ng/mL and OSM at 10 ng/mL individually or combined to form the main experimental group, OSM+TNF-α. All cytokine treatment groups limited cartilage production, but OSM decreased production to a statistically lesser extent than other cytokine groups. This trend was similar to observations made via immunostaining of cartilage matrix and gene expression analysis of aggrecan. Furthermore, OSM+TNF-α statistically lowered aggrecan gene expression. In terms of degradation, when compared to all other groups, OSM dramatically increased the protein expression of the degradative enzyme matrix metalloproteinase-13 (MMP-13). Evaluation of inflammatory markers (IL-6 and IL-8) revealed no signal for OSM-treated pellets. TNF-α yielded some signal after 1 week in culture but no signal after two weeks. IL-1β and OSM+TNF-α both resulted in sustained IL-6 and IL-8 expression, however, IL-1β exhibited large variance. Thus, each cytokine contributes to various pathways that are present in OA. Since the combination of OSM and TNF-α appeared to lower cartilage gene expression and resulted in sustained and reproducible IL-6 and IL-8 production, it may serve as a better model of OA than a single cytokine such as IL-1β.

History

Degree Type

  • Doctor of Philosophy

Department

  • Chemical Engineering

Campus location

  • West Lafayette

Advisor/Supervisor/Committee Chair

Julie Liu

Additional Committee Member 2

Alyssa Panitch

Additional Committee Member 3

John Morgan

Additional Committee Member 4

Xiaoping Bao

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