Duration of TGF-β3 Exposure Impacts the Chondrogenic Maturation of Human MSCs in Photocrosslinked Carboxymethylcellulose Hydrogels

Intervertebral disc (IVD) herniation can be caused by both degeneration and traumatic injury, ultimately resulting in back pain or sciatica due to disc protrusion. Replacement of the nucleus pulposus (NP) tissue during surgical intervention post herniation could improve the long-term stability of the functional spinal unit. Tissue engineering strategies may potentially restore both biological and mechanical function of the NP. Recently, photocrosslinked carboxymethylcellulose (CMC) hydrogels were shown to support chondrogenic, NP-like extracellular matrix (ECM) elaboration by human mesenchymal stromal cells (hMSCs) when supplemented with TGF-beta 3. However, long-term preconditioning with soluble growth factors in vitro or the use of sustained growth factor delivery vehicles in vivo can be expensive and difficult to control. Transient supplementation with growth factors has been shown to maintain or improve maturation of tissue-engineered constructs. The objective of this study was to evaluate the influence of TGF-beta 3 exposure time on hydrogel bulk properties and NP-like matrix elaboration in hMSC-laden CMC hydrogels. Constructs were exposed to TGF-beta 3 for 2 weeks (Transient), 8 weeks (Continuous) or 0 weeks (controls). After 8 weeks of culture, both the Transient and Continuous groups exhibited increased ECM accumulation compared to 2 weeks and controls. The Transient group displayed significantly greater accumulation of collagens I and II, while GAG content was significantly higher in the Continuous group by 8 weeks. Distribution of ECM was more homogeneous in the Continuous group, while the Transient group exhibited more concentrated accumulation in the periphery of the hydrogel by 8 weeks. Mechanical properties improved over time in both groups, however, Continuous constructs demonstrated significantly more robust mechanical properties (equilibrium modulus and peak stress) compared to Transient gels at 8 weeks. Although the functional properties of Transient constructs did not surpass those achieved by Continuous scaffolds, they increased and were maintained upon growth factor removal at 2 weeks, and were greater than controls. Additionally, Transient construct mechanical properties (equilibrium modulus, % relaxation) were similar to those of native NP tissue. The differences seen in ECM distribution and subsequent construct functional maturation are likely due to the time available for diffusion of growth factors through the construct. Overall, these findings support the use of short-term TGF-beta 3 treatment to promote sufficient long-term tissue maturation in vitro in this hMSC-laden CMC hydrogel system.