S100B + A1 CELISA: A Novel Potency Assay and Screening Tool for Redifferentiation Stimuli of Human Articular Chondrocytes

Abstract

During monolayer expansion, a necessary step in autologous chondrocyte implantation, human articular chondrocytes (HAC) dedifferentiate and lose their capacity to produce stable hyaline cartilage. Determining HAC potency and learning how to trigger their redifferentiation would improve cell-based cartilage regeneration therapies. We previously identified S100B and S100A1 proteins as markers of HAC redifferentiation potential. Here, we aimed to: (i) demonstrate a correlation between S100B + A1-positive HAC in monolayer culture and their neochondrogenesis capacity in pellet culture; (ii) develop an S100B + A1 cell-based ELISA, and (iii) prove that S100B + A1 induction in HAC increases their chondrogenic capacity. Expression patterns of S100A1 and S100B were investigated in HAC during dedifferentiation (monolayer) or redifferentiation (pellet or high-osmolarity/BMP4 treatment in monolayer) using qRT-PCR, immunocytochemistry, or immunohistochemistry. A cell-based ELISA (CELISA) was developed as a 96-well microplate multiplex assay to measure S100B + A1 (chondrogenesis), alkaline phosphatase (hypertrophy), and DNA amount (normalization), and applied to HAC, bone marrow-derived mesenchymal stem cells and the chondrocytic cell line ATDC5. The direct correlation between the percentage of S100B + A1-positive HAC in monolayer and their neochondrogenesis in pellets validates S100B + A1 as a marker of chondrogenic potency. The S100B + A1-CELISA accurately determines HAC differentiation status, allows identification of chondrogenic stimuli, and permits the simultaneous monitoring of the undesirable hypertrophic phenotype. This novel assay offers a high-throughput, comprehensive and versatile approach for measuring cell chondrogenic potency and for identifying redifferentiation factors/conditions. HAC improved neochondrogenesis in pellets-induced with high-osmolarity and BMP4 treatment in monolayer-suggests that cell instruction prior to implantation may improve cartilage repair. J. Cell. Physiol. 232: 1559-1570, 2017. © 2016 Wiley Periodicals, Inc.

S100B as an antagonist to block the interaction between S100A1 and the RAGE V domain

“…S100A1 and S100B are homogeneously expressed in all zones of normal articular cartilage [ 67 ] but are lost in osteoarthritic (OA) cartilage, together with proteoglycans and type II collagen [ 68 ]. The expression of S100A1 and S100B is high in human articular chondrocytes compared to osteophytic chondrocytes [ 69 ].…”

Shockwave Treatment Enhanced Extracellular Matrix Production in Articular Chondrocytes Through Activation of the ROS/MAPK/Nrf2 Signaling Pathway

“…These results were consistent with those of previous studies. 21,22 After 5 days of culture, shockwaves (500 impulses at EFDs of 0.07, 0.15, or 0.3 mJ/mm 2 ) were applied to the chondrocyte pellets every day for 5 days. Cell viability was measured using the alamarBlue test after the first, second, and third shockwave applications and 7 days after the final application.…”

S100A1 and S100B: Calcium Sensors at the Cross‐Roads of Multiple Chondrogenic Pathways

“…Our group revealed concomitant gene expression modulation of S100A1 and S100B with the classical chondrogenic markers collagen type II and aggrecan: down‐regulation during HAC dedifferentiation and up‐regulation upon treatment with the chondrogenic factor bone morphogenic protein‐4 (BMP‐4) (Diaz‐Romero et al, ). Importantly, we correlated the induction of S100A1 and S100B in monolayer cultured HAC with their increased chondrogenic capacity in pellets (Diaz‐Romero et al, ). Despite scarce studies about specific intracellular roles of S100A1 and S100B in chondrocytes, it seems likely that their protein targets might be conserved across different cell types.…”

“…S100A1 and S100B are present in late proliferative and prehypertrophic chondrocytes of mice growth plate (Saito et al, 2007). In humans, S100A1 and S100B are homogeneously expressed in all zones of normal articular cartilage (Diaz-Romero et al, 2014) but are lost in OA cartilage together with proteoglycans and collagen type II (Diaz-Romero et al, 2016). Additionally, expression of S100A1 and S100B is higher in human articular compared to osteophytic chondrocytes (Gelse et al, 2012).…”