Date of Award

Fall 12-22-2009

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Graduate Group

Bioengineering

First Advisor

Steven B. Nicoll

Abstract

Low back pain may be caused by a direct, acute injury or degeneration of the intervertebral disc (IVD). Intradiscal replacement of the nucleus pulposus (NP) with a tissue engineered hydrogel scaffold may provide a biologic therapy capable of restoring the structure and mechanical function of the IVD. Therefore, the global objective of this dissertation was to develop and optimize a novel, cell-laden, covlently crosslinkable carboxymethylcellulose (CMC) hydrogel construct as a functional tissue engineered NP replacement. The versatility of the photocrosslinkable CMC system was explored by examining the resultant differences in material and mechanical properties due to varying the macromer concentration and molecular weight of the starting material. These biomaterials were shown to support NP cell viability and exhibited tunable material properties that may be easily tailored for specific applications. Culture conditions (medium formulation and TGF-beta3 supplementation) were also investigated in order to enhance matrix deposition and improve construct material and mechanical properties. Scaffolds cultured in serum-free medium supplemented with TGF-beta3 showed approximately a ten-fold increase in glycosaminoglycan (GAG) accumulation and a five-fold increase in mechanical properties (Ey). Given the load-bearing function of the NP, biomechanical stimulation, via hydrostatic pressurization, was utilized in conjunction with biochemical mediators to further augment tissue formation by engineered CMC constructs. However, TGF-beta3 supplementation alone was shown to have a more profound effect on the functional development of NP-seeded CMC constructs. Finally, the long-term effects of in vitro pre-conditioning with TGF-beta3 were examined in vitro, as well as in vivo, using a subcutaneous murine pouch model. Constructs maintained without TGF-beta3 exhibited no quantifiable changes in matrix content or mechanical properties over time. In contrast, TGF-beta3-treated scaffolds experienced a significant increase in matrix accumulation and Ey during the in vitro pre-conditioning period. TGF-beta3-treated scaffolds cultured in vitro following the pre-culture period were able to sustain these properties, while TGF-beta3-treated scaffolds maintained in vivo exhibited a significant loss in matrix accumulation and Ey, possibly due to scaffold stiffness and diffusion limitations. Although TGF-beta3 pre-conditioning produced long-term effects in vitro, the degradative properties of the CMC scaffold must be tailored for in vivo conditions. Taken together, cell-laden, covalently crosslinkable CMC hydrogel constructs may serve as potential NP tissue engineered replacements but will require further optimization prior to use in regenerative therapies.

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