Functional nucleus pulposus-like matrix assembly by human mesenchymal stromal cells is directed by macromer concentration in photocrosslinked carboxymethylcellulose hydrogels

Intervertebral disc (IVD) degeneration is associated with several pathophysiologic changes of the IVD, including dehydration of the nucleus pulposus (NP). Tissue engineering strategies may be used to restore both biological and mechanical function of the IVD following removal of NP tissue during surgical intervention. 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-β3; however, mechanical properties of these constructs did not reach native values. Fabrication parameters (i.e., composition, crosslinking density) can influence the bulk mechanical properties of hydrogel scaffolds, as well as cellular behavior and differentiation patterns. The objective of this study was to evaluate the influence of CMC macromer concentration (1.5, 2.5 and 3.5 % weight/volume) on bulk hydrogel properties and NP-like matrix elaboration by hMSCs. The lowest macromer concentration of 1.5 % exhibited the highest gene expression levels of aggrecan and collagen II at day 7, corresponding with the largest accumulation of glycosaminoglycans and collagen II by day 42. The ECM elaboration in the 1.5 % constructs was more homogeneously distributed compared to primarily pericellular localization in 3.5 % gels. The 1.5 % gels also displayed significant improvements in mechanical functionality by day 42 compared to earlier time points, which was not seen in the other groups. The effects of macromer concentration on matrix accumulation and organization are likely attributed to quantifiable differences in polymer crosslinking density and diffusive properties between the various hydrogel formulations. Taken together, these results demonstrate that macromer concentration of CMC hydrogels can direct hMSC matrix elaboration, such that a lower polymer concentration allows for greater NP-like ECM assembly and improvement of mechanical properties over time.