Eric Steck

Induction of Intervertebral Disc–Like Cells From Adult Mesenchymal Stem Cells

The potential of adult mesenchymal stem cells (MSCs) to differentiate towards cartilage, bone, adipose tissue, or muscle is well established. However, the capacity of MSCs to differentiate towards intervertebral disc (IVD)‐like cells is unknown. The aim of this study was to compare the molecular phenotype of human IVD cells and articular chondrocytes and to analyze whether mesenchymal stem cells can differentiate towards both cell types after transforming growth factor β (TGFβ)‐mediated induction in vitro.

The use of mesenchymal stem cells for chondrogenesis

The application of autologous chondrocytes in cartilage repair procedures is associated with several disadvantages, including injury of healthy cartilage in a preceding surgery frequently resulting in formation of inferior fibrocartilage at defect sites. In order to improve the quality of regeneration, adult mesenchymal stem cells (MSC) are regarded as a promising alternative. The great challenge, when considering MSC for articular cartilage repair, is to generate cells with features of stable chondrocytes which are resistant to hypertrophy and terminal differentiation, as found in hyaline articular cartilage. Common in vitro protocols for chondrogenic differentiation of MSC successfully induce expression of multiple cartilage-specific molecules, including collagen type II and aggrecan, and result in a chondrocyte-like phenotype. However, in vitro chondrogenesis of MSC additionally promotes induction of fibrocartilage-like features such as expression of collagen type I, and hypertrophy, as demonstrated by up-regulation of collagen type X, MMP13 and ALP-activity. As a consequence, differentiated MSC pellets undergo mineralisation and vascularisation after ectopic transplantation in a process similar to endochondral ossification. This review discusses the complexity and entailed challenges when considering MSC from various sources for clinical application and the necessity to optimise chondrogenesis by repressing hypertrophy to obtain functional and suitable cells for cartilage repair.

Impact of growth factors and PTHrP on early and late chondrogenic differentiation of human mesenchymal stem cells.

Common in vitro protocols for chondrogenesis of mesenchymal stem cells (MSCs) induce an inadequate, hypertrophic differentiation cascade reminiscent of endochondral bone formation. We aimed to modify chondrogenic protocols in order to identify potent inducers, promotors, and inhibitors to achieve better chondrogenesis. Nine factors suspected to stimulate or inhibit chondrogenesis were used for chondrogenic in vitro induction of MSC. Differentiation was assessed by immunohistochemistry, alcian‐blue staining, qRT‐PCR, and quantification of alkaline phosphatase (ALP) activity. Pre‐differentiated pellets were transplanted subcutaneously into SCID mice to investigate stable cartilage formation. Transforming growth factor (TGF)‐β was always required for chondrogenic differentiation and deposition of a collagen‐type‐II‐positive extracellular matrix, while bone morphogenetic protein (BMP)‐2, ‐4, ‐6, ‐7, aFGF, and IGF‐I (10 ng/ml) were alone not sufficiently inductive. Each of these factors allowed differentiation in combination with TGF‐β, however, without preventing collagen type X expression. bFGF or parathyroid hormone‐like peptide (PTHrP) inhibited the TGF‐β‐responsive COL2A1 and COL10A1 expression and ALP induction when added from day 0 or 21. In line with a reversible ALP inhibition, in vivo calcification of pellets was not prevented. Late up‐regulation of PTH1R mRNA suggests that early PTHrP effects may be mediated by a receptor‐independent pathway. While TGF‐β was a full inducer, bFGF and PTHrP were potent inhibitors for early and late chondrogenesis, seemed to induce a shift from matrix anabolism to catabolism, but did not selectively suppress COL10A1 expression. Within a developmental window of collagen type II+/collagen type X− cells, bFGF and PTHrP may allow inhibition of further differentiation toward hypertrophy to obtain stable chondrocytes for transplantation purposes. J. Cell. Physiol. 223: 84–93, 2010.

Adenovirus-Mediated Gene Transfer of Growth and Differentiation Factor-5 into Tenocytes and the Healing Rat Achilles Tendon

Growth and differentiation factor-5 (GDF-5) is known to induce tendon tissue and stimulate tendon healing. The hypothesis was that adenoviral GDF-5 transfer leads to transitory transgene expression and improves Achilles tendon healing. In vitro experiments were first performed with rat tenocytes. Transgene expression was evaluated by RT-PCR, Western blotting and GDF-5-ELISA. In vivo virus dosage and transgene expression were examined by a marker gene transfer (LacZ and luciferase). In the main experiment in 131 rats, adenovirus particles (3× 1010) were injected into transected Achilles tendons. The time course of GDF-5 mRNA expression was assessed by real-time RT-PCR. Histology and biomechanical testing were used to evaluate tendon healing and tensile strength. In vitro GDF-5 was secreted with a maximum after 2 weeks (330 ng GDF-5/106 cells per 24 hr). In vivo GDF-5 transgene expression showed a maximum at 4 weeks. At 8 weeks, GDF-5 specimens were thicker (p < 0.05) with a trend to higher strength (p = 0,064). Histology showed greater cartilage formation in type II collagen stains than in controls. Injection of adenovirus particles successfully can deliver the GDF-5 gene in healing tendons and leads to thicker tendon regenerates after 8 weeks. This technique might become a new approach for nonsurgical treatment of tendon injuries.

Early and stable upregulation of collagen type II, collagen type I and YKL40 expression levels in cartilage during early experimental osteoarthritis occurs independent of joint location and histological grading

While morphologic and biochemical aspects of degenerative joint disease (osteoarthritis [OA]) have been elucidated by numerous studies, the molecular mechanisms underlying the progressive loss of articular cartilage during OA development remain largely unknown. The main focus of the present study was to gain more insight into molecular changes during the very early stages of mechanically induced cartilage degeneration and to relate molecular alterations to histological changes at distinct localizations of the joint. Studies on human articular cartilage are hampered by the difficulty of obtaining normal tissue and early-stage OA tissue, and they allow no progressive follow-up. An experimental OA model in dogs with a slow natural history of OA (Pond–Nuki model) was therefore chosen. Anterior cruciate ligament transection (ACLT) was performed on 24 skeletally mature dogs to induce joint instability resulting in OA. Samples were taken from different joint areas after 6, 12, 24 and 48 weeks, and gene expression levels of common cartilage molecules were quantified in relation to the histological grading (modified Mankin score) of adjacent tissue. Histological changes reflected early progressive degenerative OA. Soon after ACLT, chondrocytes responded to the altered mechanical conditions by significant and stable elevation of collagen type II, collagen type I and YKL40 expression, which persisted throughout the study. In contrast to the mild to moderate histological alterations, these molecular changes were not progressive and were independent of the joint localization (tibia, femur, lateral, medial) and the extent of matrix degeneration. MMP13 remained unaltered until 24 weeks, and aggrecan and tenascinC remained unaltered until 48 weeks after ACLT. These findings indicate that elevated collagen type II, collagen type I and YKL40 mRNA expression levels are early and sensitive measures of ACLT-induced joint instability independent of a certain grade of morphological cartilage degeneration. A second phase of molecular changes in OA may begin around 48 weeks after ACLT with altered expression of further genes, such as MMP13, aggrecan and tenascin. Molecular changes observed in the present study suggest that dog cartilage responds to degenerative conditions by regulating the same genes in a similar direction as that observed for chondrocytes in late human OA.

Chondrogenesis of mesenchymal stem cells in gel-like biomaterials in vitro and in vivo.

Gel-like carrier materials were introduced into cell therapy of cartilage lesions to improve chondrocyte retention and distribution in the defect. Mesenchymal stem cells (MSC) are now discussed as an alternative cell source for repair. We here asked whether distinct gel-like carriers can support chondrogenesis of MSC in vitro and lead to stable cartilage-like transplants in vivo. Chondrogenesis of MSC embedded in collagen type I gel, fibrin glue, Matrigel and PuraMatrix peptide hydrogel was assessed and gene expression analysis, proteoglycan content, and collagen synthesis were quantified. Differentiated constructs were transplanted subcutaneously into SCID mice. All carriers supported chondrogenesis in vitro, but displayed material-dependent differences on COL2A1 gene expression, total collagen synthesis and proteoglycan deposition. The undesired calcification and microossicle formation in ectopic transplants in vivo was consistently suppressed by Matrigel. In sum, gel-like biomaterials were suitable carriers for MSC and promoted chondrogenesis. Suppression of calcification by particular gel-like materials makes their use even more attractive for MSC-based tissue engineering approaches in cartilage repair.

Regulation of H19 and its encoded microRNA-675 in osteoarthritis and under anabolic and catabolic in vitro conditions.

Cartilage degeneration in the course of osteoarthritis (OA) is associated with an alteration in chondrocyte metabolism. In order to identify molecules representing putative key regulators for diagnosis and therapeutic intervention, we analyzed gene expression and microRNA (miR) levels in OA and normal knee cartilage using a customized cartilage cDNA array and quantitative RT-PCR. Among newly identified candidate molecules, H19, IGF2, and ITM2A were significantly elevated in OA compared to normal cartilage. H19 is an imprinted maternally expressed gene influencing IGF2 expression, whose transcript is a long noncoding (lnc) RNA of unknown biological function harboring the miR-675. H19 and IGF2 mRNA levels did not correlate significantly within cartilage samples suggesting that deregulation by imprinting effects are unlikely. A significant correlation was, however, observed for H19, COL2A1, and miR-675 expression levels in OA tissue, and functional regulation of these candidate molecules was assessed under anabolic and catabolic conditions. Culture of chondrocytes under hypoxic signaling showed co-upregulation of H19, COL2A1, and miRNA-675 levels in close correlation. Proinflammatory cytokines IL-1β and TNF-α downregulated COL2A1, H19, and miR-675 significantly without close statistical correlation. In conclusion, this is the first report demonstrating deregulation of an lncRNA and its encoded miR in the context of OA-affected cartilage. Stress-induced regulation of H19 expression by hypoxic signaling and inflammation suggests that lncRNA H19 acts as a metabolic correlate in cartilage and cultured chondrocytes, while the miR-675 may indirectly influence COL2A1 levels. H19 may not only be an attractive marker for cell anabolism but also a potential target to stimulate cartilage recovery.

Enhanced expression of the human chitinase 3-like 2 gene (YKL-39) but not chitinase 3-like 1 gene (YKL-40) in osteoarthritic cartilage

The knowledge of molecular alterations in osteoarthritic cartilage is important to identify novel therapeutic targets or to develop new diagnostic tools. We aimed to characterize the molecular response to cartilage degeneration by identification of differentially expressed genes in human osteoarthritic versus normal cartilage. Gene fragments selectively amplified in osteoarthritic cartilage by cDNA representational difference analysis included YKL-39 and the oesophageal-cancer-related-gene-4 (ECRG4). YKL-39 expression was significantly upregulated in cartilage from patients with osteoarthritis (n=14) versus normal subjects (n=8) according to real-time PCR (19-fold, p=0.009) and cDNA array analysis (mean 15-fold, p<0.001) and correlated with collagen 2 up-regulation. In contrast, the homologous cousin molecule YKL-40 (chitinase 3-like 1), which is elevated in serum and synovial fluid of patients with arthritis, showed no significant regulation in OA cartilage. Enhanced levels of YKL-40 may, therefore, be derived from synovial cells while modulation of YKL-39 and collagen 2 expression reflected the cartilage metabolism in response to degradation.

Stimulation of gene expression and loss of anular architecture caused by experimental disc degeneration--an in vivo animal study.

Study Design. An external compression model was used to evaluate gene and protein expression in intervertebral discs with moderate disc degeneration. Objective. To determine messenger ribonucleic acid and protein expression levels of relevant disc components. Summary of Background Data. An animal model of mechanically induced disc degeneration was developed and characterized histologically. However, little is known at the molecular level in moderate disc degeneration. Methods. There were 8 New Zealand white rabbits subjected to monosegmental posterior compression to induce moderate disc degeneration. Twelve animals served as controls or sham controls. Discs were analyzed using immunohistochemistry for collagen type 1 (COL1), COL2, aggrecan, and bone morphogenetic protein-2/4 (BMP-2/4). For gene analysis, conventional and quantitative polymerase chain reactions were used for COL1A2, COL2A1, aggrecan, BMP-2, biglycan, decorin, osteonectin, fibromodulin, fibronectin, matrix metalloproteinase-13 (MMP-13), and tissue inhibitor of MMP-1. Gene expression for nontreated, sham-treated, and compressed discs was quantified in relation to the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase. Results. Immunohistochemistry of compressed discs showed a loss of anular architecture, and a significant reduction of BMP-2/4 and COL2 positive cells. Gene expression analysis showed a significant up-regulation of COL1A2, osteonectin, decorin, fibronectin, tissue inhibitor of MMP-1, BMP-2, and MMP-13 in compressed discs. Conclusions. Experimental moderate disc degeneration is characterized by a loss of BMP-2/4 and COL2 positive cells, although gene expression of disc constituents, catabolic enzymes, and growth factors is stimulated to reestablish disc integrity.

Rapid regulation of collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of metalloproteinase 1, 2, 3 expression in response to mechanical loading of cartilage explants in vitro.

This study analyzes the molecular response of articular chondrocytes to short-term mechanical loading with a special focus on gene expression of molecules relevant for matrix turnover. Porcine cartilage explants were exposed to static and dynamic unconfined compression and viability of chondrocytes was assessed to define physiologic loading conditions. Cell death in the superficial layer correlated with mechanical loading and occurred at peak stresses >or=6 MPa and a cartilage compression above 45%. Chondrocytes in native cartilage matrix responded to dynamic loading by rapid and highly specific suppression of collagen expression. mRNA levels dropped 11-fold (collagen 2; 6 MPa, P=0.009) or 14-fold (collagen 1; 3 and 6 MPa, P=0.009) while levels of aggrecan, tenascin-c, matrix metalloproteinases (MMP1, 3, 13, 14), and their inhibitors (TIMP1-3) did not change significantly. Thus, dynamic mechanical loading rapidly shifted the balance between collagen and aggrecan/tenascin/MMP/TIMP expression. A better knowledge of the chondrocyte response to mechanical stress may improve our understanding of mechanically induced osteoarthrits.