Population doublings and percentage of S100‐positive cells as predictors of in vitro chondrogenicity of expanded human articular chondrocytes

Abstract

The aim of this study was to investigate the interconnection between the processes of proliferation, dedifferentiation, and intrinsic redifferentiation (chondrogenic) capacities of human articular chondrocyte (HAC), and to identify markers linking HAC dedifferentiation status with their chondrogenic potential. Cumulative population doublings (PD) of HAC expanded in monolayer culture were determined, and a threshold range of 3.57-4.19 PD was identified as indicative of HAC loss of intrinsic chondrogenic capacity in pellets incubated without added chondrogenic factors. While several specific gene and surface markers defined early HAC dedifferentiation process, no clear correlation with the loss of intrinsic chondrogenic potential could be established. CD90 expression during HAC monolayer culture revealed two subpopulations, with sorted CD90-negative cells showing lower proliferative capacity and higher chondrogenic potential compared to CD90-positive cells. Although these data further validated PD as critical for in vitro chondrogenesis, due to the early shift in expression, CD90 could not be considered for predicting chondrogenic potential of HAC expanded for several weeks. In contrast, an excellent mathematically modeled correlation was established between PD and the decline of HAC expressing the intracellular marker S100, providing a direct link between the number of cell divisions and dedifferentiation/loss of intrinsic chondrogenic capacity. Based on the dynamics of S100-positive HAC during expansion, we propose asymmetric cell division as a potential mechanism of HAC dedifferentiation, and S100 as a marker to assess chondrogenicity of HAC during expansion, of potential value for cell-based cartilage repair treatments.

Micromass co-culture of human articular chondrocytes and human bone marrow mesenchymal stem cells to investigate stable neocartilage tissue formation in vitro

“…Isolation and culture of HAC was carried out as previously described (Diaz-Romero et al, 2005;Giovannini et al, 2010). Briefly, cells were released from the extracellular matrix by sequential enzymatic digestion using 2.5mg/ml pronase (Roche, Rotkreuz, Switzerland) for 1h followed by 250μg/ ml collagenase-P (Roche) overnight.…”

“…In our previous study, we have established a threshold of HAC cumulative population doublings identifying cells with retained intrinsic chondrogenic potential i.e. the ability to re-differentiate into cartilage-like tissue in a defined serum-free 3D system devoid of chondrogenic factors, and possibly instruct other cells (Giovannini et al, 2010). In this study we investigated whether HAC with retained intrinsic chondrogenic potential could instruct human MSC towards chondrogenesis and could stabilize MSC phenotype by preventing hypertrophic differentiation.…”

“…Three characteristics were considered for the evaluation: (1) the uniformity and intensity of Safranin-O staining; (2) the distance between cells and the amount of matrix produced; (3) the cell morphology (condensed/necrotic/pycnotic bodies, spindle/fibrous, rounded chondrogenic morphology). Immunohistochemistry (IHC) for collagen type II, collagen type X and S100 was performed as previously described (Giovannini et al, 2010). After pretreatment with 0.1% trypsin for 20min at 37°C for collagen type II and S100, or 2mg/ml hyaluronidase for 60min followed by 0.02mg/ml Protease XXIV for 60min for collagen type X, the sections were incubated overnight with a monoclonal primary antibody against collagen type II (II-II6B3, Hybridoma Bank, University of Iowa) at a dilution of 1:500 or with a monoclonal antibody against collagen type X (clones X-34 and X-53, a kind gift from Dr. K. von der Mark) at a dilution of 1:100, in a humid chamber at 4°C.…”

“…Biochemical analysis of pellets was performed as previously described (Giovannini et al, 2010). Pellets were digested with proteinase K (0.25ml of 1mg/ml proteinase K in 50mM Tris) (Sigma) overnight at 56°C.…”

“…Thus, to obtain the required cell number necessary for re-implantation, human articular chondrocytes (HAC) need to be expanded in vitro, a process during which the cells de-differentiate and lose their chondrogenic capacities to re-build stable hyalinelike tissue upon re-implantation (Mandelbaum et al, 2007). Chondrocyte de-differentiation has been characterized by changes in cell morphology, gene expression, extracellular matrix synthesis, surface marker expression patterns (Von der Mark et al, 1977;Schnabel et al, 2002;Marlovits et al, 2004;Diaz-Romero et al, 2005), as well as a progressive loss of intrinsic potential to form stable cartilage-like tissue in vitro (Giovannini et al, 2010), and after intramuscular injection in nude mice (Dell'Accio et al, 2001). These observations could explain why fibrocartilaginous tissue, rather than hyaline cartilage, is often found in patients following ACI treatment.…”