This study examined the effects of continuous ribose treatment over various time windows during self-assembly of articular cartilage constructs. Experimental results supported the hypotheses motivating the study: 1) treatment of self-assembled constructs with ribose produced significant increases in biochemical and biomechanical properties; 2) week 2 was identified as the optimal treatment time window to produce the greatest improvements in constructs; and 3) continuous ribose treatment for the entire duration of culture had the greatest effect on construct properties, notably producing a 62% increase in compressive stiffness, a 66% increase in tensile stiffness, and a 126% increase in tensile strength compared to control. To the best of our knowledge, this is the first study not only to systematically compare ribose treatment over various time windows during in vitro tissue development, but also to examine the direct effects of ribose treatment on both cells and their surrounding ECM during tissue engineering. This study demonstrates the effectiveness of ribose as an agent to improve tissue engineered materials.
It was found that the optimal time window for ribose treatment is during week 2 (t
=8–14 days). Compared to controls, week 2 constructs exhibited significant improvements in GAG/WW (34% increase), collagen/WW (53% increase), compressive stiffness (40% increase), tensile stiffness (44% increase), and tensile strength (50% increase). To understand why intervening during week 2 can lead to such dramatic improvements in construct properties, it is important to consider the developmental milestones of self-assembling constructs. A previous study from our laboratory characterized matrix development during self-assembly [4
]. A principal finding was that collagen production reaches a plateau between days 10–14 of culture, after which GAG production predominates. It is thought that rapid production of GAG with no new collagen contributes to pre-stress within the fledgling ECM, thereby compromising the engineered tissue's tensile properties [6
]. Altering this imbalance between GAG and collagen has been shown to improve the tensile properties of self-assembled constructs [6
]. During week 2, before collagen production halts and GAG production ramps up, the developing ECM may be more susceptible to interventions like ribose that either reinforce existing matrix or induce new matrix biosynthesis. The beneficial effects of week 2 ribose treatment are corroborated by previous work showing that other stimuli also produce their maximal effects when applied to constructs during week 2 [5
One interesting finding is that the results for collagen content reflected trends seen for tensile strength but do not track as closely with tensile stiffness. It is understood that the tensile properties of cartilage are conferred by the tissue's collagen network. Collagen networks have complex structure-function relationships governed by peptide abundance, fibril organization, and crosslink presence [24
]. A possible explanation for the fidelity between tensile strength and collagen content is that collagen abundance may preferentially determine the tissue's failure point (and thus strength), whereas individual crosslinks within the network may determine the fiber bundle response to strain (and thus stiffness). Future studies on native cartilage should be undertaken to tease out structure-function relationships between collagen abundance, crosslinks, tensile stiffness, and tensile strength.
Increased GAG and compressive stiffness in ribose-treated constructs may be explained by the effects of crosslinking, as well. Glycation-mediated crosslinking may be trapping GAGs within the crosslinked network, thereby preventing GAG loss during culture. Higher compressive stiffness may be explained by tighter packing of GAG within the crosslinked network. One way to test this hypothesis in the future may be to examine the ratio of GAG to pentosidine, a molecule derived from ribose that is responsible for crosslinks between lysine and arginine residues in collagen [24
]. By studying correlations between GAG and pentosidine, inferences can be made about the effect of collagen crosslinking on GAG retention or loss.
Although the guiding principle underlying this study is that ribose induces crosslinking of the ECM, the observed changes in biochemistry and biomechanics may be further explained by cellular metabolism or osmotic stress. Ribose is known to affect cellular metabolism [15
] and therefore may influence chondrocyte biosynthesis during self-assembly. Additionally, ribose supplementation increases medium osmolarity. Cells undergo shrinkage in a hyperosmotic environment; such cellular strain is thought to alter chromatin condensation and nucleocytoplasmic transport [25
]. The downstream effect of these nuclear changes may be increased GAG or collagen. To test this concept in the future, one could examine the use of a non-reducing sugar such as sucrose to modulate osmolarity while preventing glycation.
Of note is that constructs treated with ribose during week 1 exhibited decreases in biochemical and biomechanical properties but increases in size and cell number compared to all other groups. During week 1, in a process resembling chondrogenesis in vivo
], our laboratory has shown that the nascent construct's efforts are focused primarily on cell condensation through N-cadherin upregulation rather than on ECM synthesis [4
]. As such, there is very little ECM available as a substrate for glycation. Thus, the effect of early ribose treatment may be metabolic or osmotic, rather than crosslink forming. Metabolically, ribose may have shunted cellular biosynthesis towards proliferation [15
], which may explain the 63% increase in cell number after week 1 treatment. Thus it is not surprising that, given the greater cell number, week 1 treated constructs are larger. Despite the greater cell number and size, these constructs were unable to keep up with ECM synthesis observed in other groups. Similar results were obtained in a previous study that investigated initial cell seeding density in self-assembly and revealed an upper limit for effective tissue engineering [27
Constructs treated with ribose throughout the entire 4 weeks exhibited the greatest improvements. It is possible that the beneficial effects seen with week 2 treatment were able to mitigate the negative effects seen with week 1 treatment. Most importantly, however, the success of these constructs demonstrates that treatment with 30 mM ribose can be used safely in tissue engineering strategies with no risk of in vitro
cytotoxicity. Future work is warranted to assess biocompatibility in vivo
, since previous work has suggested that AGEs may mark ECM proteins for targeted proteolysis [15
This work provides evidence that continuous treatment with ribose can significantly enhance the biochemical and biomechanical properties of self-assembled cartilage constructs. We have identified an optimal time window for ribose application. Additionally, we provide evidence that 30 mM ribose can be used safely in vitro without risk of cell death or other deleterious effects. Finally, a major innovation of this study is that it evaluated ribose application during self-assembly, a purely cell-mediated phenomenon, from which direct effects on both cells and ECM were ascertained for the first time.