Time and dose-dependent effects of chondroitinase ABC on growth of engineered cartilage

Abstract

Tissue engineering techniques have been effective in developing cartilage-like tissues in vitro. However, many scaffold-based approaches to cultivating engineered cartilage have been limited by low collagen production, an impediment for attaining native functional load-bearing tensile mechanical properties. Enzymatic digestion of glycosaminoglycans (GAG) with chondroitinase ABC (chABC) temporarily suppresses the construct's GAG content and compressive modulus and increases collagen content. Based on the promising results of these early studies, the aim of this study was to further promote collagen deposition through more frequent chABC treatments. Weekly dosing of chABC at a concentration of 0.15 U/mL resulted in a significant cell death, which impacted the ability of the engineered cartilage to fully recover GAG and compressive mechanical properties. In light of these findings, the influence of lower chABC dosage on engineered tissue (0.004 and 0.015 U/mL) over a longer duration (one week) was investigated. Treatment with 0.004 U/mL reduced cell death, decreased the recovery time needed to achieve native compressive mechanical properties and GAG content, and resulted in a collagen content that was 65 % greater than the control. In conclusion, the results of this study demonstrate that longer chABC treatment (one week) at low concentrations can be used to improve collagen content in developing engineered cartilage more expediently than standard chABC treatments of higher chABC doses administered over brief durations.

High seeding density of human chondrocytes in agarose produces tissue-engineered cartilage approaching native mechanical and biochemical properties

“…Unlike bovine constructs whose growth seemingly continues indefinitely (Nims et al, 2016; O’Connell et al, 2014), or canine constructs which have sustained growth for at least 42 days (Bian et al, 2010b), human constructs generally experienced no significant growth after four weeks in culture. This behavior is reflected by the regression analyses’ findings that only G* was positively correlated with culture duration after day 14 (Table 2).…”

“…This limitation is evident in the values of G*, which peak at ~1 MPa (Figure 5), whereas native levels in human cartilage are estimated to be five to ten times greater (Krishnan et al, 2003). We previously investigated various strategies to alleviate this limitation, by subjecting constructs to dynamic compressive loading to enhance functional properties (Bian et al, 2010b; Lima et al, 2007; Mauck et al, 2000), temporarily digesting GAGs with chondroitinase ABC to delay construct swelling and mitigate steric exclusion of synthesized collagen (Bian et al, 2009b; O’Connell et al, 2014), degrading the agarose scaffold with agarase to similarly mitigate steric effects (Ng et al, 2009b), or supplementing culture media with higher levels of amino acids to enhance collagen synthesis (Ng et al, 2008a). Most of these strategies have shown statistically significant improvements, though none have raised collagen content or dynamic modulus to native levels.…”

High fidelity visualization of cell-to-cell variation and temporal dynamics in nascent extracellular matrix formation

“…That is, the more permissive the microenvironment, the more readily newly-formed matrix could disperse throughout the construct. This finding may explain, in part, why the intentional removal of proteoglycans from developing cartilage constructs hastens subsequent construct growth27. Perhaps by decreasing the density of the pericellular microenvironment, mild digestion creates space for increased collagen deposition.…”

Matrix Production in Large Engineered Cartilage Constructs Is Enhanced by Nutrient Channels and Excess Media Supply

“…This hypothesis had previously motivated alternative strategies for enhancing collagen deposition by using chondroitinase ABC to enzymatically remove GAG and quell construct swelling. 46 The amount of glucose available in the culture media was adjusted by changing the MV per construct instead of modifying the glucose concentration. This choice was motivated by the desire to maintain the glucose concentration at the standard 25 mM of high-glucose DMEM and the concern that large changes in concentration might produce confounding effects due to alterations in osmolality and pH.…”