Secretion of matrix metalloproteinase 3 by expanded articular chondrocytes as a predictor of ectopic cartilage formation capacity in vivo

Abstract

Objective. Monolayer expansion of human articular chondrocytes (HACs) is known to result in progressive dedifferentiation of the chondrocytes and loss of their stable cartilage formation capacity in vivo. For an optimal outcome of chondrocyte-based repair strategies, HACs capable of ectopic cartilage formation may be required. This study was undertaken to identify secreted candidate molecules, in supernatants of cultured HACs, that could serve as predictors of the ectopic cartilage formation capacity of cells.Methods. Standardized medium supernatants (n ‫؍‬ 5 knee cartilage samples) of freshly isolated HACs (PD0) and of HACs expanded for 2 or 6 population doublings (PD2 and PD6, respectively) were screened by a multiplexed immunoassay for 15 distinct interleukins, 8 matrix metalloproteinases (MMPs), and 11 miscellaneous soluble factors. Cartilage differentiation markers such as cartilage oligomeric matrix protein and YKL-40 were determined by enzyme-linked immunosorbent assay. HACs from each culture were subcutaneously transplanted into SCID mice, and the capacity of the chondrocytes to form stable cartilage was examined histologically 4 weeks later. Results. Whereas freshly isolated (PD0)HACs generated stable ectopic cartilage that was positive for type II collagen, none of the cell transplants at PD6 formed cartilaginous matrix. Loss of the ectopic cartilage formation capacity between PD0 and PD6 correlated with a drop in the secretion of MMP-3 to <10% of initial levels, whereas changes in the other investigated molecules were not predictive. Chondrocytes with MMP-3 levels of >20% of initial levels synthesized cartilaginous matrix, whereas those with low MMP-3 levels (<10% of initial levels) at PD2 failed to regenerate ectopic cartilage.Conclusion. Loss of the capacity for stable ectopic cartilage formation in the course of HAC dedifferentiation can be predicted by determining the relative levels of MMP-3, demonstrating that standardized culture supernatants can be used for quality control of chondrocytes dedicated for cell therapeutic approaches.

The effects of cyclic tensile strain on the organisation and expression of cytoskeletal elements in bovine intervertebral disc cells: an in vitro study

“…MMP-3, a highly expressed MMP in IVD, was signifi cantly elevated in response to tensile strain. MMP-3 has both anabolic and catabolic properties; it is important in cartilage formation (Pelttari et al, 2008) but can also activate other MMPs such as MMP-1 (Murphy et al, 1987), ultimately playing an important role in disc degeneration (Haro et al, 2000). In our study, tensile strain inhibited MMPs-1 and -9 mRNA expression in OAF cells, which coincided with a transient decline of the endogenous inhibitors -TIMP-1 and TIMP-2.…”

“…The observed increase in β-tubulin mRNA and protein synthesis in OAF cells may refl ect a response to meet cellular demands i.e., ECM turnover, as collagen types I and II transcription were induced by strain. Equally, increased β-tubulin synthesis may be necessary to promote and maintain an assembled microtubule network to protect the cell from mechanical damage, as tensile strain has previously been shown to increase microtubule assembly (Putnam et al, 2001). Although the precise mechanism of microtubule involvement in mechanosignalling is unclear, they are essential for transmitting stresses to activate other cytoplasmic proteins within the signalling cascade(s) (Putnam et al, 2003;Na and Wang, 2008).…”

Chondrogenic differentiation of human bone marrow-derived mesenchymal stem cells in a simulated osteochondral environment is hydrogel dependent

“…In vitro chondrogenesis of hBMSCs is known to be accompanied by hypertrophy (Pelttari et al, 2006;Pelttari et al, 2008;Hellingman et al, 2011). In strategies aiming for cartilage regeneration, terminal differentiation leading to mineralisation and ultimately to bone formation is undesirable (Farrell et al, 2009;Scotti et al, 2010;Farrell et al, 2011).…”

“…Human bone marrow-derived mesenchymal stem cells (hBMSCs) are one of the potential cell sources based on their multipotency, their expandability in vitro and the possibility to use autologous cells to treat patients (Pittenger et al, 1999).…”

“…This specific gel has been developed for future minimally invasive treatments allowing injection of the material followed by in situ gelation. So far, the HA-pNIPAM gel has been studied for possible use in nucleus pulposus tissue regeneration (Peroglio et al, 2012;Malonzo et al, 2013).…”