Simon R. Tew

Tissue engineering: chondrocytes and cartilage

Chapter summary Tissue engineering offers new strategies for developing treatments for the repair and regeneration of damaged and diseased tissues. These treatments, using living cells, will exploit new developments in understanding the principles in cell biology that control and direct cell function. Arthritic diseases that affect so many people and have a major impact on the quality of life provide an important target for tissue engineering. Initial approaches are in cartilage repair; in our own programme we are elucidating the signals required by chondrocytes to promote new matrix assembly. These principles will extend to other tissues of the musculoskeletal system, including the repair of bone, ligament and tendon.

Transduction of passaged human articular chondrocytes with adenoviral, retroviral, and lentiviral vectors and the effects of enhanced expression of SOX9.

Chondrocytes form and maintain the extracellular matrix of cartilage. The cells can be isolated from cartilage for applications such as tissue engineering, but their expansion in monolayer culture causes a progressive loss of chondrogenic phenotype. In this work, we have investigated the isolation of human articular chondrocytes from osteoarthritic (OA) cartilage at joint replacement, their expansion in monolayer culture, and their transduction with adenoviral, retroviral, and lentiviral vectors, using the gene encoding green fluorescent protein as a marker gene. The addition of growth factors (transforming growth factor beta(1), fibroblast growth factor 2, and platelet-derived growth factor BB) during cell culture was found to greatly increase cell proliferation and thereby to selectively enhance the efficiency of transduction with retrovirus. With adenoviral and lentiviral vectors the transduction efficiency achieved was 95 and 85%, respectively. Using growth factor-supplemented medium with a retroviral vector, efficiency in excess of 80% was achieved. The expression was stable for several months with both retrovirus and lentivirus when analyzed by fluorescence-activated cell-sorting flow analysis and immunoblotting. Transduction with SOX9 was investigated as a method to reinitiate cartilage matrix gene expression in passaged human OA chondrocytes. Endogenous collagen II expression (both mRNA and protein) was increased in monolayer culture using both adenoviral and retroviral vectors. Furthermore, collagen II gene expression in chondrocytes retrovirally transduced with SOX9 was stimulated by alginate bead culture, whereas in control chondrocytes it was not. These results demonstrated methods for rapid expansion and highly efficient transduction of human OA chondrocytes and the potential for the recovery of key features of chondrocyte phenotype by transduction with SOX9.

Cartilage, SOX9 and Notch signals in chondrogenesis

Cartilage repair is an ongoing medical challenge. Tissue engineered solutions to this problem rely on the availability of appropriately differentiated cells in sufficient numbers. This review discusses the potential of primary human articular chondrocytes and mesenchymal stem cells to fulfil this role. Chondrocytes have been transduced with a retrovirus containing the transcription factor SOX9, which permits a greatly improved response of the cells to three‐dimensional culture systems, growth factor stimulation and hypoxic culture conditions. Human mesenchymal stem cells have been differentiated into chondrocytes using well‐established methods, and the Notch signalling pathway has been studied in detail to establish its role during this process. Both approaches offer insights into these in vitro systems that are invaluable to understanding and designing future cartilage regeneration strategies.

Regulation of SOX9 mRNA in Human Articular Chondrocytes Involving p38 MAPK Activation and mRNA Stabilization

Human articular chondrocytes rapidly lose their phenotype in monolayer culture. Recently we have shown that overexpression of the transcription factor SOX9 greatly enhanced re-expression of the phenotype in three-dimensional aggregate cultures. Here we show that endogenous SOX9 mRNA can be rapidly up-regulated in subcultured human articular chondrocytes if grown in alginate, in monolayer with cytochalasin D, or with specific inhibition of the RhoA effector kinases ROCK1 and -2, which all prevent actin stress fiber formation. Disruption of actin stress fibers using any of these redifferentiation stimuli also supported the superinduction of SOX9 by cycloheximide. The superinduction was blocked by inhibitors of the p38 MAPK signaling pathway and involved the stabilization of SOX9 mRNA. Furthermore stimulation of chondrocyte p38 MAPK activity with interleukin-1β resulted in increased levels of SOX9 mRNA, and this was again dependent on the absence of actin stress fibers in the cells. In this study of chondrocyte redifferentiation we have provided further evidence of the early involvement of SOX9 and have discovered a novel post-transcriptional regulatory mechanism activated by p38 MAPK, which stabilized SOX9 mRNA.

Cellular methods in cartilage research: Primary human chondrocytes in culture and chondrogenesis in human bone marrow stem cells

Work in our laboratory has focused on the in vitro culture of both human articular chondrocytes and human mesenchymal stem cells to understand what controls their ability to synthesise an appropriate cartilage-like extracellular matrix containing a predominantly collagen type II fibrillar network embedded in an aggrecan-rich ECM. This review focuses on the methodologies that we have found to be successful with cartilage and bone marrow sources of human cells and comments on the many factors which may enable improved phenotypic performance once the cells are in a fully chondrogenic environment.

Human infrapatellar fat pad-derived stem cells express the pericyte marker 3G5 and show enhanced chondrogenesis after expansion in fibroblast growth factor-2.

IntroductionInfrapatellar fat pad (IPFP) is a possible source of stem cells for the repair of articular cartilage defects. In this study, adherent proliferative cells were isolated from digests of IPFP tissue. The effects of the expansion of these cells in fibroblast growth factor-2 (FGF-2) were tested on their proliferation, characterisation, and chondrogenic potential.MethodsIPFP tissue was obtained from six patients undergoing total knee replacement, and sections were stained with 3G5, alpha smooth muscle actin, and von Willebrand factor to identify different cell types in the vasculature. Cells were isolated from IPFP, and both mixed populations and clonal lines derived from them were characterised for cell surface epitopes, including 3G5. Cells were expanded with and without FGF-2 and were tested for chondrogenic differentiation in cell aggregate cultures.Results3G5-positive cells were present in perivascular regions in tissue sections of the IPFP, and proliferative adherent cells isolated from the IPFP were also 3G5-positive. However, 3G5 expression was on only a small proportion of cells in all populations and at all passages, including the clonally expanded cells. The cells showed cell surface epitope expression similar to adult stem cells. They stained strongly for CD13, CD29, CD44, CD90, and CD105 and were negative for CD34 and CD56 but were also negative for LNGFR (low-affinity nerve growth factor receptor) and STRO1. The IPFP-derived cells showed chondrogenic differentiation in cell aggregate cultures, and prior expansion with FGF-2 enhanced chondrogenesis. Expansion in FGF-2 resulted in greater downregulation of many cartilage-associated genes, but on subsequent chondrogenic differentiation, they showed stronger upregulation of these genes and this resulted in greater matrix production per cell.ConclusionThese results show that these cells express mesenchymal stem cell markers, but further work is needed to determine the true origin of these cells. These results suggest that the expansion of these cells with FGF-2 has important consequences for facilitating their chondrogenic differentiation.

Notch Signaling Through Jagged‐1 Is Necessary to Initiate Chondrogenesis in Human Bone Marrow Stromal Cells but Must Be Switched off to Complete Chondrogenesis

We investigated Notch signaling during chondrogenesis in human bone marrow stromal cells (hMSC) in three‐dimensional cell aggregate culture. Expression analysis of Notch pathway genes in 14‐day chondrogenic cultures showed that the Notch ligand Jagged‐1 (Jag‐1) sharply increased in expression, peaking at day 2, and then declined. A Notch target gene, HEY‐1, was also expressed, with a temporal profile that closely followed the expression of Jag‐1, and this preceded the rise in type II collagen expression that characterized chondrogenesis. We demonstrated that the shut‐down in Notch signaling was critical for full chondrogenesis, as adenoviral human Jag‐1 transduction of hMSC, which caused continuous elevated expression of Jag‐1 and sustained Notch signaling over 14 days, completely blocked chondrogenesis. In these cultures, there was inhibited production of extracellular matrix, and the gene expression of aggrecan and type II collagen were strongly suppressed; this may reflect the retention of a prechondrogenic state. The JAG‐1‐mediated Notch signaling was also shown to be necessary for chondrogenesis, as N‐[N‐(3,5‐difluorophenacetyl‐l‐alanyl)]‐(S)‐phenylglycine t‐butyl ester (DAPT) added to cultures on days 0–14 or just days 0–5 inhibited chondrogenesis, but DAPT added from day 5 did not. The results thus showed that Jag‐1‐mediated Notch signaling in hMSC was necessary to initiate chondrogenesis, but it must be switched off for chondrogenesis to proceed.

The epitope characterisation and the osteogenic differentiation potential of human fat pad-derived stem cells is maintained with ageing in later life

Some clinical settings are deficient in osteogenic progenitors, e.g. atrophic nonunited fractures, large bone defects, and regions of scarring and osteonecrosis. These benefit from the additional use of bone marrow-derived mesenchymal stem cells, but these cells exhibit an age-related decline in lifespan, proliferation and osteogenic potential. Therapeutic approaches for the repair of bone could be optimised by the identification of a stem cell source that does not show age-related changes. Fat pad-derived stem cells are capable of osteogenesis, but a detailed study of the effect of ageing on their epitope profile and osteogenic potential has so far not been performed. Fat pad-derived cells were isolated from 2 groups of 5 patients with a mean age of 57 years (S.D. 3 years) and 86 years (S.D. 3 years). The proliferation, epitope profile and osteogenic differentiation potential of cells from the 2 groups were compared. Cells isolated from the fat pad of both groups showed similar proliferation rates and exhibited a cell surface epitope profile similar but not identical to that of bone marrow-derived stem cells. The cells from both groups cultured in osteogenic medium exhibited osteogenesis as shown by a significant upregulation of alkaline phosphatase and osteocalcin genes, and significantly greater alkaline phosphatase enzyme activity compared to cells cultured in the control medium. The cells cultured in the osteogenic medium also showed greater calcium phosphate deposition on alizarin red staining. There was no significant difference between the osteogenic potential of the two age groups for any of the parameters studied. The fat pad is a consistent and homogenous source of stem cells that exhibits osteogenic differentiation potential with no evidence of any decline with ageing in later life. This has many potential therapeutic tissue engineering applications for the repair of bone defects in an increasingly ageing population.

Bone Marrow-Derived Mesenchymal Stem Cells Express the Pericyte Marker 3G5 in Culture and Show Enhanced Chondrogenesis in Hypoxic Conditions

Bone marrow‐derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in some cells. In this study, bone marrow‐derived stem cells were characterized and the effects of hypoxia on chondrogenesis investigated. Adherent bone marrow colony‐forming cells were characterized for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. The cells stained strongly for markers of adult mesenchymal stem cells, and a high number of cells were also positive for the pericyte marker 3G5. The cells showed a chondrogenic response in cell aggregate cultures and, in lowered oxygen, there was increased matrix accumulation of proteoglycan, but less cell proliferation. In hypoxia, there was increased expression of key transcription factor SOX6, and of collagens II and XI, and aggrecan. Pericytes are a candidate stem cell in many tissue, and our results show that bone marrow‐derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension. This has important implications for tissue engineering applications of bone marrow‐derived stem cells.

Differences in repair responses between immature and mature cartilage.

Cartilage has a poor reparative capacity although it is unclear as to what extent this may be dependent on age or maturation. In the current study, the cellular responses of chondrocytes to experimental wounding in vitro using embryonic, immature, and mature cartilage have been compared. In all cases, the response was consistent (a combination of cell death that included apoptosis and proliferation). The speed of response varied in terms of cell death with embryonic cartilage showing the most rapid response and mature cartilage showing the slowest response. Intrinsic repair as assessed by the ability to heal the lesion was not detected in any of the culture systems used. It was concluded that the poor repair potential of cartilage is not maturation dependent in the systems studied.