Timothy E. Hardingham

Proteoglycans: many forms and many functions.

Proteoglycans are produced by most eukaryotic cells and are versatile components of pericellular and extracellular matrices. They belong to many different protein families. Their functions vary from the physical effects of the proteoglycan aggrecan, which binds with link protein to hyaluronan to form multi‐molecular aggregates in cartilage; to the intercalated membrane protein CD44 that has a proteoglycan form and is a receptor and a cell‐binding site for hyaluronan; to heparan sulfate proteoglycans of the syndecan and other families that provide matrix binding sites and cell‐surface receptors for growth factors such as fibroblast growth factor (FGF). One feature that recurs in proteoglycan biology is that their structure is open to extensive modulation during cellular expression. Examples of protein changes are known, but a major source of structural variation is in the glycosaminoglycan chains. The number of chains and their length can vary, as well as their pattern of sulfation. This may result in the switching of different chain types with different properties, e.g., chondroitin sulfate and heparan sulfate, and it may also result in the selective expression of sulfated chain sequences that have specific functions. The control of glycosaminoglycan structure is not well understood, but it does appear to be used to change the properties of proteoglycans to suit different biological needs. Proteoglycan forms of proteins are thus important modifiers of the organization of the pericellular and extracellular matrices and modulators of the processes that occur there.—Hardingham, T. E., Fosang, A. J. Protoglycans: many forms and many functions. FASEB J. 6: 861‐870; 1992.

Chondrogenic Differentiation of Human Bone Marrow Stem Cells in Transwell Cultures: Generation of Scaffold-Free Cartilage

Human bone marrow stem cells (hMSCs) have been shown to differentiate in vitro into a number of cell lineages and are a potential autologous cell source for the repair and replacement of damaged and diseased musculoskeletal tissues. hMSC differentiation into chondrocytes has been described in high‐density cell pellets cultured with specific growth and differentiation factors. We now describe how culture of hMSCs as a shallow multicellular layer on a permeable membrane over 2–4 weeks resulted in a much more efficient formation of cartilaginous tissue than in established chondrogenic assays. In this format, the hMSCs differentiated in 14 days to produce translucent, flexible discs, 6 mm in diameter by 0.8–1 mm in thickness from 0.5 × 106 cells. The discs contained an extensive cartilage‐like extracellular matrix (ECM), with more than 50% greater proteoglycan content per cell than control hMSCs differentiated in standard cell pellet cultures. The disc constructs were also enriched in the cartilage‐specific collagen II, and this was more homogeneously distributed than in cell pellet cultures. The expression of cartilage matrix genes for collagen type II and aggrecan was enhanced in disc cultures, but improved matrix production was not accompanied by increased expression of the transcription factors SOX9, L‐SOX5, and SOX6. The fast continuous growth of cartilage ECM in these cultures up to 4 weeks appeared to result from the geometry of the construct and the efficient delivery of nutrients to the cells. Scaffold‐free growth of cartilage in this format will provide a valuable experimental system for both experimental and potential clinical studies.

Directed differentiation of human embryonic stem cells toward chondrocytes

We report a chemically defined, efficient, scalable and reproducible protocol for differentiation of human embryonic stem cells (hESCs) toward chondrocytes. HESCs are directed through intermediate developmental stages using substrates of known matrix proteins and chemically defined media supplemented with exogenous growth factors. Gene expression analysis suggests that the hESCs progress through primitive streak or mesendoderm to mesoderm, before differentiating into a chondrocytic culture comprising cell aggregates. At this final stage, 74% (HUES1 cells) and up to 95–97% (HUES7 and HUES8 cells) express the chondrogenic transcription factor SOX9. The cell aggregates also express cell surface CD44 and aggrecan and deposit a sulfated glycosaminoglycan and cartilage-specific collagen II matrix, but show very low or no expression of genes and proteins associated with nontarget cell types. Our protocol should facilitate studies of chondrocyte differentiation and of cell replacement therapies for cartilage repair.

Proteoglycans of articular cartilage: Changes in aging and in joint disease

In human osteoarthritis and animal models of degenerative joint disease, damage to the structure of cartilage proteoglycan is a central event. Loss of proteoglycan from the matrix alters the physicochemical properties of the tissue, but the pathological process and biochemical mechanisms that lead to this loss are poorly understood. This review examines the present state of knowledge regarding proteoglycan structure and the changes that occur in aging and osteoarthritis. It also discusses how these studies will influence the development of new methods for measuring cartilage breakdown.

Hypoxic conditions increase hypoxia-inducible transcription factor 2α and enhance chondrogenesis in stem cells from the infrapatellar fat pad of osteoarthritis patients

Stem cells derived from the infrapatellar fat pad (IPFP) are a potential source of stem cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we investigated the effects of hypoxia on gene expression changes and chondrogenesis in stem cells from the IPFP removed from elderly patients with osteoarthritis at total knee replacement. Adherent colony-forming cells were isolated and cultured from the IPFP from total knee replacement. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured for 14 days as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions. Gene expression analysis, DNA and glycosoaminoglycan assays and immunohistochemical staining were determined to assess chondrogenesis. IPFP-derived adherent colony-forming cells stained strongly for markers of adult mesenchymal stem cells, including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Cell aggregates of IPFP cells showed a chondrogenic response. In hypoxic conditions there was increased matrix accumulation of proteoglycan but less cell proliferation, which resulted in 3.5-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of hypoxia-inducible transcription factor (HIF)2α and not HIF1α, and the expression of key transcription factors SOX5, SOX6 and SOX9, and that of aggrecan, versican and collagens II, IX, X and XI, was also increased. These results show that cells with stem cell characteristics were isolated from the IPFP of elderly patients with osteoarthritis and that their response to chondrogenic culture was enhanced by lowered oxygen tension, which upregulated HIF2α and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of cells derived from the IPFP.

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.

An ELISA plate-based assay for hyaluronan using biotinylated proteoglycan G1 domain (HA-binding region).

An enzyme-linked absorbent assay for the determination of hyaluronan (HA) has been developed. The procedure is sensitive, simple and is based on a microtitre plate format. The assay involves competition between HA absorbed to the plate and HA free in solution for binding to biotinylated cartilage proteoglycan binding region (G1 domain). The range of the assay is 10-2500 ng/ml with 50% inhibition at about 200 ng/ml. The assay can be used in guanidine HCl up to 0.6 M and in 0.5% deoxycholate or 0.5% nonidet. This new technique involves fewer experimental steps and is simpler to perform than other methods.

Macromolecular Diffusion of Biological Polymers Measured by Confocal Fluorescence Recovery after Photobleaching

Fluorescence recovery after photobleaching with an unmodified confocal laser scanning microscope (confocal FRAP) was used to determine the diffusion properties of network forming biological macromolecules such as aggrecan. The technique was validated using fluorescein isothiocyanate (FITC)-labeled dextrans and proteins (molecular mass 4-2000 kDa) at 25 degrees C and with fluorescent microspheres (207 nm diameter) over a temperature range of 5-50 degrees C. Lateral diffusion coefficients (D) were independent of the focus position, and the degree and extent of bleach. The free diffusion coefficient (Do) of FITC-aggrecan determined by confocal FRAP was 4.25 +/- 0.6 x 10(-8) cm2 s-1, which is compatible with dynamic laser light scattering measurements. It appeared to be independent of concentration below 2.0 mg/ml, but at higher concentrations (2-20 mg/ml) the self-diffusion coefficient followed the function D = Do(e)(-Bc). The concentration at which the self-diffusion coefficient began to fall corresponded to the concentration predicted for domain overlap. Multimolecular aggregates of aggrecan ( approximately 30 monomers) had a much lower free diffusion coefficient (Do = 6.6 +/- 1.0 x 10(-9) cm2 s-1) but showed a decrease in mobility with concentration of a form similar to that of the monomer. The method provides a technique for investigating the macromolecular organization in glycan-rich networks at concentrations close to those found physiologically.

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.