Date of Award

Spring 1-1-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemical & Biochemical Engineering

First Advisor

Stephanie J. Bryant

Second Advisor

Virginia L. Ferguson

Third Advisor

Kristi S. Anseth

Fourth Advisor

Jeffrey Stansbury

Fifth Advisor

John Kisiday

Abstract

Osteoarthritis (OA) is a debilitating joint disease that affects millions of Americans, young and old. This disease primarily involves the protective cartilage found on the ends of articulating bone surfaces in joints. Inherently, cartilage does not heal well, and the current clinical therapies available to treat cartilage injuries and OA patients often lead to healing with mechanically inferior fibrocartilage. Tissue engineering (TE) strategies could offer a viable alternative to the current therapies available. It important for tissue engineered cartilage to successfully integrate with the underlying subchondral bone, and attention must be given to the unique and complex interface that connects the bone and cartilage: the osteochondral interface. This research works towards developing an osteochondral tissue engineering strategy that utilizes a scaffold engineered to guide the concomitant differentiation of a single undifferentiated cell source down both chondrogenic and osteogenic lineages with the ultimate goal of synthesizing spatially organized bone, cartilage, and osteochondral interface extracellular matrix (ECM) molecules. Progress has been made towards this goal by investigating the response of human mesenchymal stromal cells (hMSCs) to external biochemical and biomechanical cues. The scaffolds selected for this research were poly(ethylene glycol) (PEG) based hydrogels modified with either a generic cell binding peptide (RGD), a cartilage ECM moiety (chondroitin sulfate), or a type I collagen analog peptide (P-15) as a bone ECM moiety. hMSCs encapsulated in these hydrogels were subjected to dynamic loading to impart biomechanical cues on the cells in combination with the biochemical cues from the modified scaffolds. Results indicated that RGD modified and ChS modified PEG scaffolds supported chondrogenic differentiation and the production of cartilage ECM matrix molecules including aggrecan, collagen II and collagen X. However, the application of a 15% intermittent dynamic compressive strain, whether applied immediately (RGD modified) or after an initial differentiation induction period (ChS) inhibited the production of the articular cartilage specific collagen II protein, suggesting that the 15% strain may be too large for guiding the hMSCs down an articular cartilage lineage. Further results indicated that hMSCs encapsulated in RGD modified scaffolds, in the absence of dynamic strain but in the presence of soluble osteogenic differentiation cues, produced significant collagen I, the primary collagen found in bone. Additional results suggested that P-15 modified hydrogels supported hMSC attachment, but did not offer enhanced production of bone biomarker molecules by encapsulated hMSCs. While optimal biochemical and biomechanical cues that guide hMSC differentiation remain to be elucidated, strategies to design multi-layer PEG based hydrogels were investigated and characterized. To mimic the variations in ECM and mechanical properties between bone and cartilage spanning the osteochondral interface, scaffolds were fabricated, characterized, and subjected to dynamic loading. When hMSCs were encapsulated in these scaffolds and cultured under free swelling or subjected to dynamic loading in osteochondral differentiation medium, the spatial presentation of biochemical and mechanical cues gave rise to characteristically different cartilage and bone protein expressions by hMSCs in each layer. These results indicate that it is possible to use the combination of biochemical and biomechanical cues to affect the spatial production of bone and cartilage specific ECM molecules in a scaffold with a single encapsulated cell source. As a better understanding of the cues that drive differentiation of adult human MSC differentiation become elucidated, findings from this research will aid in the development of complex 3D scaffolds for osteochondral tissue engineering strategies that are capable of delivering local cues to concomitantly guide a single cell source down bone, articular cartilage, and hypertrophic lineages within a single scaffold.

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