Development of a Carboxymethylcellulose Hydrogel System for Stem Cell-Based Nucleus Pulposus Tissue Engineering

摘要

The intervertebral disc (IVD) is a heterogeneous tissue interposed between the vertebral bodies of the spine, comprised of the collagenous, lamellar annulus fibrosus (AF), and the hydrated, gelatinous nucleus pulposus (NP). Surgical procedures for IVD herniation, including discectomy or microdiscectomy, often give rise to alterations in disc structure, resulting in more complex injury or degeneration. Replacement of the lost NP material may prevent such post-surgical complications. Although synthetic implants have not been able to restore long-term disc function, tissue engineering strategies may provide functional IVD replacements. Several studies have reported the ability of hydrogels combined with mesenchymal stem cells (MSCs) and chondrogenic growth factors from the transforming growth factor (TGF-beta) family, specifically TGF-beta3, to mimic native NP tissue properties. Still, an optimal scaffold system has yet to be described. One candidate material is carboxymethylcellulose (CMC), a biocompatible, low-cost, negatively charged cellulose derivative, which is similar to the polyanionic glycosaminoglycans found in native NP tissue. In addition, growth factor dosage requirements and delivery methods have not been fully examined in order to enhance chondrogenic differentiation and growth factor bioavailability. These factors may impact the functional properties and eventual clinical translation of such engineered constructs. Therefore, the objective of this thesis was to create a novel, photocrosslinked CMC hydrogel supplemented with TGF-beta3 that will promote the production of functional NP-like matrix by encapsulated human MSCs (hMSCs). CMC hydrogels were shown to support the differentiation of encapsulated hMSCs in the presence of TGF-beta3. Altering bulk hydrogel material properties via CMC macromer concentration influenced the differentiation of hMSCs and subsequent deposition of mechanically functional extracellular matrix. Exposure time of tissue constructs to TGF-beta3 was also shown to impact hMSC differentiation and functional matrix deposition, where an early, transient exposure to the growth factor was able to induce differentiation. Finally, the ability to incorporate TGF-beta3 into the hydrogel prior to polymerization was analyzed by creating a photocrosslinked heparin-CMC hydrogel to sequester and improve the bioavailability of the soluble growth factor to encapsulated hMSCs. This work has provided insight into the effects of scaffold polymer composition and biochemical stimulation on functional matrix elaboration by hMSCs in these hydrogels to create NP tissue replacements.