Degeneration of the nucleus pulposus (NP) has been implicated as a major cause of low back pain. Tissue engineering strategies may provide a viable NP replacement therapy; however, culture conditions must be optimized to promote functional tissue development. In this study, a standard serum-containing medium formulation was compared to a chemically defined, serum-free medium to determine the effect on matrix elaboration and functional properties of NP cell-laden carboxymethylcellulose (CMC) hydrogels. Additionally, both media were further supplemented with transforming growth factor-beta 3 (TGF-beta(3)). Glycosaminoglycan (GAG) content increased in both TGF-beta(3)-treated groups and was highest for treated, serum-free constructs (9.46 +/- 1.51 microg GAG/mg wet weight), while there were no quantifiable GAGs in untreated serum-containing samples. Histology revealed uniform, interterritorial staining for chondroitin sulfate proteoglycan throughout the treated, serum-free constructs. Type II collagen content was greater in both serum-free groups and highest in treated, serum-free constructs. The equilibrium Young's modulus was highest in serum-free samples supplemented with TGF-beta(3) (18.54 +/- 1.92 kPa), and the equilibrium weight swelling ratio of these constructs approached that of the native NP tissue (22.19 +/- 0.46 vs. 19.94 +/- 3.09, respectively). Taken together, these results demonstrate enhanced functional matrix development by NP cells when cultured in CMC hydrogels maintained in serum-free, TGF-beta(3) supplemented medium, indicating the importance of medium formulation in NP construct development.
Tissue engineering strategies have the potential to improve upon current techniques for intervertebral disc repair. However, determining a suitable biomaterial scaffold for disc regeneration is difficult due to the complex fibrocartilaginous structure of the tissue. In this study, cells isolated from three distinct regions of the intervertebral disc, the outer and inner annulus fibrosus and nucleus pulposus, were expanded and seeded on resorbable polyester fiber meshes and encapsulated in calcium crosslinked alginate hydrogels, both chosen to approximate the native tissue architecture. Three-dimensional (3D) constructs were cultured for 14 days in vitro and evaluated histologically and quantitatively for gene expression and production of types I and II collagen and proteoglycans. During monolayer expansion, the cell populations maintained their distinct phenotypic morphology and gene expression profiles. However, after 14 days in 3D culture, there were no significant differences in morphology, gene expression, or protein production between all three cell populations grown in either alginate or polyester fiber meshes. The results of this study indicate that the culture environment may have a greater impact on cellular behavior than the intrinsic origin of the cells, and suggest that only a single-cell type may be required for intervertebral disc regenerative therapies.
Mechanical stimulation may be used to enhance the development of engineered constructs for the replacement of load bearing tissues, such as the intervertebral disc. This study examined the effects of dynamic hydrostatic pressure (HP) on outer and inner annulus (OA, IA) fibrosus cells seeded on fibrous poly(glycolic acid)-poly(L-lactic acid) scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material. scaffolds. Constructs were pressurized (5 MPa, 0.5 Hz) for four hours/day from day 3 to day 14 of culture and analyzed using ELISAs and immunohistochemistry (IHC) to assess extracellular matrix (ECM) production. Both cell types were viable, with OA cells exhibiting more infiltration into the scaffold, which was enhanced by HP. ELISA analyses revealed that HP had no effect on type I collagen production while a significant increase in type II collagen (COL II) was measured in pressurized OA constructs compared to day 14 unloaded controls. Both OA and IA dynamically loaded scaffolds exhibited more uniform COL II elaboration as shown by IHC analyses, which was most pronounced in OA-seeded scaffolds. Overall, HP resulted in enhanced ECM elaboration and organization by OA-seeded constructs, while IA-seeded scaffolds were less responsive. As such, hydrostatic pressurization may be beneficial in annulus fibrosus tissue engineering when applied in concert with an appropriate cell source and scaffold material.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.