Introduction: The development of an injectable nucleus pulposus (NP) replacement with structural and mechanical resemblance to the native tissue would serve as a promising minimally invasive treatment for early intervertebral disc degeneration[1]-[3]. Carboxymethylcellulose (CMC) is a low-cost, biocompatible anionic polysaccharide that has been shown to support mesenchymal stem cell chondrogenesis[4],[5], and is a potential candidate material for NP repair. However, CMC lacks the negatively charged sulfate groups found on native glycosaminoglycans (GAG), which are important for tissue function. Moreover, GAG sulfation is essential for normal cartilage development and may enhance regenerative therapies[6],[7]. Therefore, the objective of this study was to engineer sulfated CMC hydrogels and investigate the effects of macromer concentration and sulfate composition on hydrogel properties.
Materials and Methods: Methacrylated CMC (2%, 3%, and 4% (w/v)) was prepared as previously described[4],[5], and the redox initiators, ammonium persulfate (APS) and tetramethylethylenediamine (TEMED), both at 10 mM, were added for radical polymerization[8]. Sulfation was achieved by the addition of 2-sulfoethylmethacrylate (2SEM) (0 mM, 1 mM, 5 mM, and 10 mM) to the reaction mixture. Sulfation was evaluated using the DMMB assay[4],[5], and the equilibrium Young’s modulus (EY) and the swelling ratio (QW) were measured as previously[4],[5]. To assess cytotoxicity, human dermal fibroblasts were encapsulated at 4 x 106 cells/mL and the PicoGreen assay was used to determine DNA content[4],[5]. A 2-way ANOVA with Tukey’s post-hoc test was used to determine the effects of macromer concentration and 2SEM concentration on hydrogel properties (n=5; p<0.05; mean±SD).
Results: For all macromer concentrations, DMMB staining intensity increased and the amount of GAG equivalent significantly increased with higher 2SEM concentration (Fig. 1). No significant differences were detected across macromer concentrations. QW significantly decreased while EY significantly increased with higher macromer concentration (Fig. 2). The concentration of 2SEM did not have significant effects on hydrogel mechanical properties. No significant differences were observed for cytotoxicity of hydrogels from all groups (Fig. 3).
Discussion: This is the first study to investigate an injectable, sulfated CMC hydrogel system as a potential material for NP replacement. Although previous studies have demonstrated the use of CMC hydrogels for NP tissue engineering, sulfated CMC hydrogels were fabricated in the present work to better mimic both the structural and mechanical properties of the native tissue[4],[5]. Sulfation of CMC hydrogels was confirmed and likely increased the fixed charge density based on the binding of cationic DMMB dye, but the degree of sulfation had no effect on functional properties (i.e., QW and EY). While the mechanical properties of all groups were within the range for native NP, 3% (w/v) gels were most similar and thus should be further explored[9].
Conclusion: Injectable sulfated CMC hydrogels were successfully fabricated and may serve as injectable, functional NP replacements.
NSF for funding; Devika Varma at The City College of New York for technical assistance
References:
[1] Francisco AT, et al (2013) Biomaterials 34:7381-8.
[2] Smith LJ, et al (2014) Tissue Eng Part A 20:1841-9.
[3] Sivan SS, et al (2014) Acta Biomater 10:1124-33.
[4] Gupta MS, et al (2011) Tissue Eng Part A 17:2903-10.
[5] Lin HA, et al (2015) J Biomed Mater Res A doi: 10.1002/jbm.a.25552.
[6] Dawson PA (2011) Semin Cell Dev Biol 22:653-9.
[7] Frith JE, et al (2013) Biomaterials 34:9430-40.
[8] Varma DM, et al (2014) ORS Annual Meeting
[9] Cloyd JM, et al (2007) Eur Spine J 16:1892-98.