Chondrogenic capacity and alterations in hyaluronan synthesis of cultured human osteoarthritic chondrocytes

Abstract

During osteoarthritis there is a disruption and loss of the extracellular matrix of joint cartilage, composed primarily of type II collagen, aggrecan and hyaluronan. In young patients, autologous chondrocyte implantation can be used to repair cartilage defects. However, for more elderly patients with osteoarthritis, such a repair approach is contraindicated because the procedure requires a large expansion of autologous chondrocytes in vitro leading a rapid, perhaps irreversible, loss of the chondrocyte phenotype. This study investigates whether osteoarthritic chondrocytes obtained from older patients can be expanded in vitro and moreover, induced to re-activate their chondrocyte phenotype. A decrease in chondrocyte phenotype markers, collagen II, aggrecan and SOX9 mRNA was observed with successive expansion of cells in monolayer culture. However, chondrogenic induction in three-dimensional pellet culture successfully rescued the expression of all three marker genes to native levels, even with 4th passage cells—cells representing an approximate 625-fold expansion in cell number. This data supports the use of osteoarthritic cells for autologous implantation repair. In addition, another set of gene products were explored as useful markers of the chondrocyte phenotype. Differentiated primary chondrocytes exhibited a common pattern of hyaluronan synthase isoforms that changed upon cell expansion in vitro and, reverted back to the original pattern following pellet culture. Moreover, the change in isoform pattern correlated with changes in the molecular size of synthesized HA.

Culture Temperature Affects Human Chondrocyte Messenger RNA Expression in Monolayer and Pellet Culture Systems

“…However, in the pellet culture system, ACAN was notably suppressed compared to the monolayer culture system ( Fig 4C ). These results are inconsistent with those of previous reports [ 8 , 9 , 25 , 58 , 59 ]. However, some studies showed that, when chondrocytes are cultured in the pellet culture system in hypoxic conditions, ACAN expression was enhanced compared to that in the monolayer culture system.…”

“…When chondrocytes are isolated from the ECM and cultured in monolayer conditions, the chondrogenic phenotype disappears during de-differentiation and a fibroblastic phenotype appears [ 6 , 7 ]. De-differentiation occurs immediately in monolayer conditions [ 8 , 9 ]. Synthesis of type II collagen and aggrecan, which are specific markers of articular chondrocytes, decreases and type I collagen synthesis increases [ 6 , 7 ].…”

CRISPR/Cas9 knockout of HAS2 in rat chondrosarcoma chondrocytes demonstrates the requirement of hyaluronan for aggrecan retention

“…The effect of altered HA/aggrecan matrix assembly and retention was determined using two in vitro 3-D culture models often used for generating bioengineered cartilage [30-33]. First, RCS-Cas9 WT and RCS-Cas9 Has2 KO clones #3 and #7 were cultured in alginate beads for 7 days (Fig.…”

Minimal Sustainability of Dedifferentiation by ROCK Inhibitor on Rat Nucleus Pulposus Cells In Vitro

“…2C) for both treated and non-treated groups was observed with an increase in passage. Although unexpected, it is not uncommon to observe increased ACAN expression with advancing NP cell passage, as demonstrated by the work of Ono et al 29) Additionally, the notochordal cell type derived from rat NP tissue might have differentiated to a more chondrogenic phenotype in vitro monolayer culture conditions, resulting in enhanced ACAN expression 30,31) . As such, future studies might benefit from applying an animal model that lacks notochordal cells in their IVD 32) .…”