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Inferior tendon healing can lead to scarring and tendinopathy. The role of complement in tendon healing is still unclear. The aim of this study was to understand tenocytes response to mechanical injury and whether complement is regulated by injury. Tenocytes were injured using an optimized automated scratch assay model. Using a self-assembled plotter system, 50 parallel lines of injury were created in a 6 cm diameter tenocyte cell layer. Tenocytes mitotic activity and survival post injury was assessed using FDA/ethidiumbromide assay. Furthermore, this injury model was combined with stimulation of the tenocytes with the complement split fragment C3a. Gene expression of C3aR, C5aR (CD88), CD46, CD55, tumor necrosis factor (TNF)α, interleukin (IL)-1β, matrix metalloproteinase (MMP)-1 was analyzed. Immunolabeling for C5aR and CD55 was performed. An enhanced mitotic activity and some dead cells were detected in the vicinity of the scratches. Gene expression of the C3aR was suppressed after 4 h but induced after 24 h post injury. C5aR was down-regulated at 24 h, CD46 and CD55 were induced at 24 h in response to injury and CD55 was also elevated at 4 h. MMP-1 was upregulated by injury but both proinflammatory cytokines remained mainly unaffected. Combination of injury with C3a stimulation led to an enhanced C3aR, CD55 and TNFα gene expression. According to the gene expression data, the protein expression of C5aR was reduced and that of CD55 induced. In summary, a specific response of complement regulation was found in mechanically injured tenocytes which may be involved in healing responses.

SummaryMyelodysplastic syndromes (MDSs) are a heterogeneous group of myeloid neoplasms with defects in hematopoietic stem and progenitor cells (HSPCs) and possibly the HSPC niche. Here, we show that patient-derived mesenchymal stromal cells (MDS MSCs) display a disturbed differentiation program and are essential for the propagation of MDS-initiating Lin−CD34+CD38− stem cells in orthotopic xenografts. Overproduction of niche factors such as CDH2 (N-Cadherin), IGFBP2, VEGFA, and LIF is associated with the ability of MDS MSCs to enhance MDS expansion. These factors represent putative therapeutic targets in order to disrupt critical hematopoietic-stromal interactions in MDS. Finally, healthy MSCs adopt MDS MSC-like molecular features when exposed to hematopoietic MDS cells, indicative of an instructive remodeling of the microenvironment. Therefore, this patient-derived xenograft model provides functional and molecular evidence that MDS is a complex disease that involves both the hematopoietic and stromal compartments. The resulting deregulated expression of niche factors may well also be a feature of other hematopoietic malignancies.

Tendon healing is a time consuming process leading to the formation of a functionally altered reparative tissue. Tissue engineering-based tendon reconstruction is attracting more and more interest. The aim of this study was to establish tenocyte expansion on microcarriers in continuous bioreactor cultures and to study tenocyte behavior during this new approach. Human hamstring tendon-derived tenocytes were expanded in monolayer culture before being seeded at two different seeding densities (2.00 and 4.00 3 106 cells/1000 cm2 surface) on CytodexTM type 3 microcarriers. Tenocytes’ vitality, growth kinetics and glucose/ lactic acid metabolism were determined dependent on the seeding densities and stirring velocities (20 or 40 rpm) in a spinner flask bioreactor over a period of 2 weeks. Gene expression profiles of tendon extracellular matrix (ECM) markers (type I/III collagen, decorin, cartilage oligomeric protein [COMP], aggrecan) and the tendon marker scleraxis were analyzed using real time detection polymerase chain reaction (RTD-PCR). Type I collagen and decorin deposition was demonstrated applying immunolabeling. Tenocytes adhered on the carriers, remained vital, proliferated and revealed an increasing glucose consumption and lactic acid formation under all culture conditions. “Bead-to-bead” transfer of cells from one microcarrier to another, a prerequisite for continuous tenocyte expansion, was demonstrated by scanning electron microscopy. Type I and type III collagen gene expression was mainly unaffected, whereas aggrecan and partly also decorin and COMP expression was significantly downregulated compared to monolayer cultures. Scleraxis gene expression revealed no significant regulation on the carriers. In conclusion, tenocytes could be successfully expanded on microcarriers. Therefore, bioreactors are promising tools for continuous tenocyte expansion.

Interplay between complement factors, regulatory proteins, anaphylatoxins and cytokines could be involved in tendon healing and scar formation. The expression and regulation of complement factors by cytokines or anaphylatoxins are completely unclear in tendon. Hence, the gene expression of the anaphylatoxin receptors C3aR, C5aR and cytoprotective complement regulatory proteins (CRPs) was analysed in human tendon, cultured primary tenocytes and to directly compare the general expression level, additionally in human leukocytes. Time-dependent regulation of complement by cytokines and the anaphylatoxin C3a was assessed in cultured tenocytes. Gene expression of the anaphylatoxin receptors C3aR, C5aR and the CRPs CD46, CD55 and CD59 was detected in tendon, cultured tenocytes and leukocytes, whereas CD35 could only be found in tendon and leukocytes. Compared with cultured tenocytes, complement expression was higher in tendon and compared with leukocytes C3aR, C5aR, CD35 and CD55, but not CD46 and CD59 gene expression levels were lower in tendon. C3aR mRNA was up-regulated by both TNFα and C3a in cultured tenocytes in a time-dependent manner whereby C5aR gene expression was only induced by C3a. IL-6 or C3a impaired the CRP gene expression. C3a stimulation lead to an up-regulation of TNFα and IL-1β mRNA in tenocytes. Degenerated tendons revealed an increased C5aR and a reduced CD55 expression. The expression profile of the investigated complement components in tendon and cultured tenocytes clearly differed from that of leukocytes. Tenocytes respond to the complement split fragment C3a with CRP suppression and enhanced pro-inflammatory cytokine gene expression suggesting their sensitivity to complement activation.

Synovial injury and healing are complex processes including catabolic effects by proinflammatory cytokines and anabolic processes by anti-inflammatory mediators. Here we examined the expression of pro- versus anti-inflammatory mediators in synovium of patients with diagnostic arthroscopy (control), joint trauma (JT), osteoarthritis (OA), and rheumatoid arthritis (RA). Synovial samples from these patients were subjected to RT-PCR and double immunofluorescence confocal microscopy of pro- and anti-inflammatory mediators as well as immune cell markers. Interestingly, pro- and anti-inflammatory mediators were expressed predominantly in granulocytes in patients with JT and in macrophages, lymphocytes, and plasma cells in patients with OA and RA. Interestingly, parallel to the severity of inflammation, proinflammatory mediators IL-1β, TNF-α, and 5-LOX specific mRNA as well as immunoreactive (IR) cells were significantly more abundant in patients with RA and JT than in those with OA. However, anti-inflammatory mediators 15-LOX, FPR2, and IL-10 specific mRNA as well as IR cells were significantly more abundant in patients with OA than in those with JT and RA. These findings show that upregulation of proinflammatory mediators contributes to the predominantly catabolic inflammatory process in JT and RA synovium, whereas upregulation of anabolic anti-inflammatory mediators counteracts inflammation resulting in the inferior inflammatory process in OA synovium.

Tissue trauma induces an inflammatory response associated with a cytokine release that may engage complement pathways. Cytokine-mediated complement expression may contribute to cartilage degradation. Hence, we analysed the complement expression profile in primary articular and non-articular chondrocytes and its interrelation with cytokines. The expression of the anaphylatoxin receptors (C3aR and C5aR) and the complement regulatory proteins (CPRs) CD35, CD46, CD55 and CD59 was studied in cultured articular, auricular and nasoseptal chondrocytes using RTD-PCR and immunofluorescence labelling. The complement profile of peripheral blood mononuclear cells (PBMCs) was opposed to the expression in articular chondrocytes. The time-dependent regulation (6 and 24 h) of these complement factors was assessed in articular chondrocytes in response to the cytokines TNFα, IL-10 or TNFα combined with IL-10 (each 10 ng/mL). C3aR, C5aR, CD46, CD55 and CD59 but almost no CD35 mRNA was expressed in any of chondrocyte types studied. The anaphylatoxin receptor expression was lower and that of the CRPs was higher in chondrocytes when compared with PBMCs. The majority of the studied complement factors were expressed at a significantly lower level in non-articular chondrocytes compared with the articular chondrocytes. TNFα significantly increased the C3aR expression in chondrocytes after 6 and 24 h. TNFα + IL-10 significantly downregulated C5aR and IL-10 significantly inhibited the CD46 and CD55 gene expression after 24 h. C5aR and CD55 could be localised in cartilage in situ. Anaphylatoxin receptors and CRPs are regulated differentially by TNFα and IL-10. Whether cytokine-induced complement activation occurs in response to cartilage trauma has to be further identified.

The availability of autologous articular chondrocytes remains a limiting issue in matrix assisted autologous chondrocyte transplantation. Non-articular heterotopic chondrocytes could be an alternative autologous cell source. The aims of this study were to establish heterotopic chondrocyte cocultures to analyze cell-cell compatibilities and to characterize the chondrogenic potential of nasoseptal chondrocytes compared to articular chondrocytes. Primary porcine and human nasoseptal and articular chondrocytes were investigated for extracellular cartilage matrix (ECM) expression in a monolayer culture. 3D polyglycolic acid- (PGA) associated porcine heterotopic mono- and cocultures were assessed for cell vitality, types II, I, and total collagen-, and proteoglycan content. The type II collagen, lubricin, and Sox9 gene expressions were significantly higher in articular compared with nasoseptal monolayer chondrocytes, while type IX collagen expression was lower in articular chondrocytes. Only β1-integrin gene expression was significantly inferior in humans but not in porcine nasoseptal compared with articular chondrocytes, indicating species-dependent differences. Heterotopic chondrocytes in PGA cultures revealed high vitality with proteoglycan-rich hyaline-like ECM production. Similar amounts of type II collagen deposition and type II/I collagen ratios were found in heterotopic chondrocytes cultured on PGA compared to articular chondrocytes. Quantitative analyses revealed a time-dependent increase in total collagen and proteoglycan content, whereby the differences between heterotopic and articular chondrocyte cultures were not significant. Nasoseptal and auricular chondrocytes monocultured in PGA or cocultured with articular chondrocytes revealed a comparable high chondrogenic potential in a tissue engineering setting, which created the opportunity to test them in vivo for articular cartilage repair.

Implantation of tissue-engineered heterotopic cartilage into joint cartilage defects might be an alternative approach to improve articular cartilage repair. Hence, the aim of this study was to characterize and compare the quality of tissue-engineered cartilage produced with heterotopic (auricular, nasoseptal and articular) chondrocytes seeded on polyglycolic acid (PGA) scaffolds in vitro and in vivo using the nude mice xenograft model. PGA scaffolds were seeded with porcine articular, auricular and nasoseptal chondrocytes using a dynamic culturing procedure. Constructs were pre-cultured 3 weeks in vitro before being implanted subcutaneously in nude mice for 1, 6 or 12 weeks, non-seeded scaffolds were implanted as controls. Heterotopic neo-cartilage quality was assessed using vitality assays, macroscopical and histological scoring systems. Neo-cartilage formation could be observed in vitro in all PGA associated heterotopic chondrocytes cultures and extracellular cartilage matrix (ECM) deposition increased in vivo. The 6 weeks in vivo incubation time point leads to more consistent results for all cartilage species, since at 12 weeks in vivo construct size reductions were higher compared with 6 weeks except for auricular chondrocytes PGA cultures. Some regressive histological changes could be observed in all constructs seeded with all chondrocytes subspecies such as cell-free ECM areas. Particularly, but not exclusively in nasoseptal chondrocytes PGA cultures, ossificated ECM areas appeared. Elastic fibers could not be detected within any neo-cartilage. The neo-cartilage quality did not significantly differ between articular and non-articular chondrocytes constructs. Whether tissue-engineered heterotopic neo-cartilage undergoes sufficient transformation, when implanted into joint cartilage defects requires further investigation.

Objective Recently, the "epigenetic molecular clock hypothesis" linked increasing DNA methylation in a distinct CpG island in the cardiac-specific homeobox gene ( CSX ) gene to relative mitotic cell age. To determine mitotic cell age in hematopoietic cells of myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) and acute lymphoblastic leukemia (ALL) patients, we assessed differential C SX methylation patterns in these diseases vs age-adjusted healthy controls. Materials and Methods We performed bisulfite pyrosequencing to analyze CSX methylation in CD34 + and bone marrow (BM) cells from 53 MDS, 62 AML, 77 ALL patients, and 37 controls. Results Analysis of MDS CD34 + and BM cells revealed significantly increasing methylation of CSX in controls CSX methylation between the CD34 + vs the unselected BM compartment were detected in matched MDS low-risk but not high-risk and AML samples. ALL samples displayed highly elevated CSX methylation levels as compared to controls. Conclusions Assessment of mitotic cell age by CSX methylation analysis could reveal novel insights into the distinct progression of hematologic diseases.

Objective Analysis of the histopathologic features of hip arthritis in patients with ankylosing spondylitis (AS) has revealed accumulation of infiltrating mononuclear cells in the bone end plate and presence of hyaline articular cartilage that is not found in areas of total cartilage destruction. This study was undertaken to assess whether chondrocytes attract lymphocytes and whether cartilage chondrocytes from patients with AS have the potential to directly stimulate T cells in an HLA-restricted manner. Methods Human HLA–B27+ T cell lines, specific for the Epstein-Barr virus–derived peptide EBNA258–266, and autologous chondrocytes, serving as nonprofessional antigen-presenting cells (APCs), were available for use in a model system to study chondrocyte functions in femoral head joint cartilage of patients with AS. Peptide functionality of cytotoxic T cells was assessed by flow cytometry, and cellular interactions were detected by fluorescence confocal microscopy. Results When maintained in an alginate matrix, chondrocytes isolated from the femoral heads of patients with AS constitutively expressed type II collagen and CD80. When pulsed with the EBNA258–266 peptide, autologous chondrocytes functioned as APCs and, specifically, induced interferon-γ production in CD8+ T cells. In mixed chondrocyte–T cell cultures, cell–cell contacts were dependent on the presence of the EBNA258–266 peptide. T cells adjacent to chondrocytes produced perforin and granzyme B; both molecules were found in focal aggregates, a prerequisite for antigen-specific lysis of target cells. Conclusion Antigen presentation through human chondrocytes allows the stimulation of peptide-specific CD8+ T cells. These results indicate that human chondrocytes can act as nonprofessional APCs, and suggest that there is an interferon-γ–triggered autocrine loop of immune cell–mediated chondrocyte activation in the already inflamed environment. Thus, local HLA-dependent activation of peptide-specific cytotoxic CD8+ T cells by chondrocytes might contribute to inflammatory processes in the spondylarthritides.

Gene transfer into cultured chondrocytes by using adenoviral vectors has potential applications in treating cartilage disorders. The present study was undertaken to compare and optimize two chondrocyte culture conditions for adenoviral transduction efficacy by using primary human articular chondrocytes cultivated either directly in a monolayer condition or as outgrowths from alginate-stored chondrocyte cultures. Isolated primary chondrocytes from human articular cartilage were either immediately transduced with an EGFP (enhanced green fluorescent protein)-gene-bearing adenoviral vector (1,000 and 3,000 virus particles/cell) or cultured in alginate before transduction. Immunohistochemistry and flow cytometric analysis were employed to determine the expression of extracellular matrix proteins and of the alphavbeta5 integrin receptor involved in adenoviral cell entry. Monolayer chondrocytes exhibited moderate transduction rates (mean 22.2% and 46.9% EGFP-positive cells at 1,000 and 3,000 virus particles/cell by 72 h post-transduction), whereas alginate-derived chondrocytes revealed significantly higher transduction efficacies (95.7% and 99%). Both monolayer and alginate-derived chondrocytes expressed alphavbeta5 integrin, type II collagen and cartilage proteoglycans. The mean fluorescence intensity of type II collagen was significantly higher in the alginate-derived chondrocytes, whereas that of alphavbeta5 integrin was higher in the monolayer chondrocytes. Our results indicate that transduction efficacy is independent of alphavbeta5 integrin expression levels in chondrocytes. Moreover, adenoviral transduction of alginate-derived chondrocytes is more efficient than that for monolayer chondrocytes and may be a suitable tool to achieve sufficient numbers of transduced and differentiated chondrocytes for experimental applications and cartilage repair.

Interleukin 1beta (IL-1beta) is a pleiotropic pro-inflammatory cytokine that plays a key role in mediating cartilage degradation in osteoarticular disorders such as osteoarthritis (OA) and rheumatoid arthritis (RA). At the cellular level, IL-1beta activates matrix degrading enzymes, down-regulates expression of matrix components and induces chondrocyte apoptosis. Curcumin (diferuloylmethane) is an anti-inflammatory phytochemical agent that has recently been shown to antagonize the pro-inflammatory effects of cytokines in chondrocytes and other cells. To test the hypothesis that curcumin also protects chondrocytes from morphological alterations induced by IL-1beta, we investigated its in vitro effects on apoptotic signalling proteins and key cartilage-specific matrix components in IL-1beta-stimulated chondrocytes. Human articular chondrocytes were pre-treated with 10 ng/mI IL-1beta alone for 30 min before being co-treated with IL-1beta and 50 microM curcumin for 5, 15 or 30 min, respectively. The ultrastructural morphology of chondrocytes was investigated by transmission electron microscopy. The production of collagen type II, the adhesion and signal transduction receptor beta1-integrin, the apoptosis marker activated caspase-3 was analysed by immunohistochemistry, immunoelectron microscopy and Western blotting. Transmission electron microscopy of chondrocytes stimulated with IL-1beta revealed early degenerative changes which were relieved by curcumin co-treatment. The suppression of collagen type II and beta1-integrin synthesis by IL-1beta was inhibited by curcumin. Additionally, curcumin antagonized IL-1beta-induced caspase-3 activation in a time-dependent manner. This study clearly demonstrates that curcumin exerts anti-apoptotic and anti-catabolic effects on IL-1beta-stimulated articular chondrocytes. Therefore curcumin may have novel therapeutic potential as an adjunct nutraceutical chondroprotective agent for treating OA and related osteoarticular disorders.

Curcumin (diferuloylmethane) is a nontoxic dietary pigment in tumeric and curry and a potent inhibitor of the common transcription factor Nuclear Factor kappaB (NF-kappaB) in several cell types. It is well established that some of the catabolic effects of the proinflammatory cytokines interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha in osteoarthritis are regulated by the activation of NF-kappaB. Therefore, the aim of this study was to determine whether curcumin modifies the catabolic response of chondrocytes to IL-1beta. Human articular chondrocytes were prestimulated with 10 ng/mL IL-1beta for 0, 4, 8, 12, or 24 h and then cotreated with 50 microM curcumin for 0, 12, 24, 36, or 48 h. Synthesis of the cartilage-specific collagen type II and matrix-degrading enzyme matrix metalloproteinase-3 (MMP-3) was investigated in chondrocytes by Western blot analysis. Activation and nuclear translocation of NF-kappaB were observed by immunofluorescence microscopy. IL-1beta induced a decrease in collagen type II and upregulation of MMP-3 in a time-dependent manner. Upregulation of MMP-3 was inhibited by curcumin in a time-dependent manner. In addition, IL-1beta-induced a decrease in type II collagen, which was relieved by curcumin treatment. In response to IL-1beta, NF-kappaB translocated to the nucleus, but translocation was inhibited by curcumin, as revealed by immunofluorescence microscopy. Taken together, these results confirmed an IL-1beta-mediated upregulation of proinflammatory MMP-3 in chondrocytes via an NF-kappaB activation mechanism. Curcumin protected chondrocytes from the catabolic effects of IL-1beta, such as MMP-3 upregulation, and interestingly also relieved cytokine-induced suppression of matrix protein synthesis. Therefore, curcumin antagonizes crucial catabolic effects of IL-1beta signaling that are known to contribute to the pathogenesis of osteoarthritis.

We previously have reported that the mitogen-activated protein kinase (MAPK) pathway is stimulated by adhesion of human chondrocytes to anti-beta(1)-integrin antibodies or collagen type II in vitro. These mechanisms most likely prevent chondrocyte dedifferentiation to fibroblast-like cells and chondrocyte death. To investigate whether this pathway plays an essential role for the differentiation, phenotype, and survival of chondrocytes, we blocked mitogen-activated protein kinase/extracellular signal-regulated kinase (Erk) (MEK), a kinase upstream of the kinase Erk by using U0126. Exposure of chondrocytes to U0126 caused activation of caspase-3 in a dose-dependent manner. Western blot analysis with an antibody specific for dually phosphorylated Erk shows that collagen type II induced phosphorylation of Erk1/2 was specifically blocked by U0126 in a dose-dependent manner. Immunohistochemical analysis showed that treated chondrocytes were caspase-3 positive. In treated chondrocytes, the cleavage of 116-kDa poly(ADP-ribose)polymerase resulted in the 85-kDa apoptosis-related cleavage fragment and was associated with caspase-3 activity. Analysis by electron microscopy showed typical morphological signs of apoptosis, such as crescent-shaped clumps of heterochromatin, and a degraded pericellular matrix. Thus, these results indicate that the MEK/Erk signal transduction pathway is involved in the maintenance of chondrocytes differentiation and survival. These data stimulate further investigations on the role of mitogen-activated protein kinase pathways in human chondrocytes.

We have examined the mechanism by which collagen-binding integrins co-operate with insulin-like growth factor-I (IGF-I) receptors (IGF-IR) to regulate chondrocyte phenotype and differentiation. Adhesion of chondrocytes to anti-β1 integrin antibodies or collagen type II leads to phosphorylation of cytoskeletal and signalling proteins localized at focal adhesions, including α-actinin, vinculin, paxillin and focal adhesion kinase (FAK). These stimulate docking proteins such as Shc (Src-homology collagen). Moreover, exposure of collagen type II-cultured chondrocytes to IGF-I leads to co-immunoprecipitation of Shc protein with the IGF-IR and with β1, α1 and α5 integrins, but not with α3 integrin. Shc then associates with growth factor receptor-bound protein 2 (Grb2), an adaptor protein and extracellular signal-regulated kinase. The expression of the docking protein Shc occurs only when chondrocytes are bound to collagen type II or integrin antibodies and increases when IGF-I is added, suggesting a collaboration between integrins and growth factors in a common/shared biochemical signalling pathway. Furthermore, these results indicate that focal adhesion assembly may facilitate signalling via Shc, a potential common target for signal integration between integrin and growth-factor signalling regulatory pathways. Thus, the collagen-binding integrins and IGF-IR co-operate to regulate focal adhesion components and these signalling pathways have common targets (Shc-Grb2 complex) in subcellular compartments, thereby linking to the Ras-mitogen-activated protein kinase signalling pathway. These events may play a role during chondrocyte differentiation.

Synovial fibroblasts (SF) contribute to the pathogenesis of osteoarthritis (OA), but the effects of intra-articular cytokines on SF are not completely understood. The aim of this study was to characterize the interplay between tumor necrosis factor (TNF)α and the anti-inflammatory interleukin (IL)-10. Non-immortalized human SF and SF of the human cell line K4IM were stimulated with recombinant TNFα, IL-10, or TNFα + IL-10 (10 ng/ml each) for 24 h or transduced with an adenoviral vector overexpressing human IL-10 (hIL-10) and subsequently treated with 10 ng/ml TNFα for 24 h. Effects on the gene expression and protein synthesis of IL-6, IL-10, matrix metalloproteinases (MMP)-1, −3, type I collagen, β1-integrin, and CD44 were investigated via real-time detection polymerase chain reaction, immunofluorescence labeling, flow cytometry, and Western blotting. IL-10 release by transduced SF was confirmed with enzyme-linked immunosorbent assay. Both cell populations were activated by TNFα and by TNFα + IL-10, increasing their gene expression and protein synthesis of IL-6, IL-10, MMP-1, and MMP−3 and altering the synthesis of type I collagen, β1-integrin, and CD44. hIL-10 overexpression greatly elevated the gene expression and protein synthesis of IL-10. However, transduction did not significantly affect the gene expression of IL-6, MMP-1, and MMP−3 in SF. The increased expression of pro-inflammatory and catabolic mediators in TNFα-activated SF indicates their role in OA pathogenesis, suggesting they are a potential therapeutic target. Although the vigorousness of the responses of non-immortalized SF and K4IM clearly differ, the K4IM cell line seems to be a suitable model for non-immortalized human SF.

Cartilage injury remains a challenge in orthopedic surgery as articular cartilage only has a limited capacity for intrinsic healing. Autologous chondrocyte transplantation (ACT) is a suitable technique for cartilage repair, but requires articular cartilage biopsies for autologous chondrocyte expansion. The use of heterotopic chondrocytes derived from non-articular cartilage sources such as auricular chondrocytes may be a novel approach for ACT. The aim of the study is to evaluate whether co-cultured articular/auricular chondrocytes exhibit characteristics comparable to articular chondrocytes. Analysis of the proliferation rate, extracellular cartilage matrix (ECM) gene and protein expression (type II and I collagen, elastin, lubricin), beta1-integrins and the chondrogenic transcription factor sox9 in articular/auricular chondrocytes was performed using RTD-PCR, flow cytometry, immunofluorescence microscopy and Western blot analysis. Additionally, three-dimensional (3D) chondrocyte mono- and co-cultures were established. The proliferative activity and elastin gene expression were lower and that of type II collagen and lubricin was higher in articular compared with auricular chondrocytes. The species generally did not influence the chondrocyte characteristics, with the exception of type I collagen and sox9 expression, which was higher in porcine but not in human articular chondrocytes compared with both types of auricular chondrocytes. beta1-integrin gene expression did not differ significantly between the chondrocyte types. The type II collagen gene and protein expression was higher in articular chondrocyte monocultures and was slightly higher in co-cultures compared with monocultured auricular chondrocytes. Both chondrocyte types survived in co-culture. Despite their differing expression profiles, co-cultures revealed some adjustment in the ECM expression of both chondrocyte types.

For lack of sufficient human cartilage donors, chondrocytes isolated from various animal species are used for cartilage tissue engineering. The present study was undertaken to compare key features of cultured large animal and human articular chondrocytes of the knee joint. Primary chondrocytes were isolated from human, porcine, ovine and equine full thickness knee joint cartilage and investigated flow cytometrically for their proliferation rate. Synthesis of extracellular matrix proteins collagen type II, cartilage proteoglycans, collagen type I, fibronectin and cytoskeletal organization were studied in freshly isolated or passaged chondrocytes using immunohistochemistry and western blotting. Chondrocytes morphology, proliferation, extracellular matrix synthesis and cytoskeleton assembly differed substantially between these species. Proliferation was higher in animal derived compared with human chondrocytes. All chondrocytes expressed a cartilage-specific extracellular matrix. However, after monolayer expansion, cartilage proteoglycan expression was barely detectable in equine chondrocytes whereby fibronectin and collagen type I deposition increased compared with porcine and human chondrocytes. Animal-derived chondrocytes developed more F-actin fibers during culturing than human chondrocytes. With respect to proliferation and extracellular matrix synthesis, human chondrocytes shared more similarity with porcine than with ovine or equine chondrocytes. These interspecies differences in chondrocytes in vitro biology should be considered when using animal models.

Articular cartilage is a highly specialized tissue that is vulnerable to injuries as well as to degenerative and inflammatory diseases. Irreversible cartilage damage occurs in the presence of a disturbed homeostasis of anabolic and catabolic processes. Synovial inflammation induced by cytokines and growth factors causes degradation of articular cartilage and finally programmed cell death (apoptosis) of chondrocytes which represent major components in the pathogenesis of arthritis. Interleukin-1 (IL-1) is the prototypic inducer of catabolic responses in chondrocytes. Interleukin-18 (IL-18) has structural similarities with the IL-1 family of proteins. Enhanced levels of IL-18 were detected in the serum of patients with rheumatoid arthritis, thus indicating a potential role of IL-18 during synovial inflammation. In view of the structural homology with IL-1, the present study investigates the effects of IL-18 on human cultured chondrocyte signaling pathways involved in apoptotic processes, where-as the inhibition of the mitogen-activated protein kinase (MAPK) pathway plays a key role in chondrocyte proliferation and lifespan. Human articular chondrocyte were cultured and incubated with IL-18. The cultures are investigated by Western blot analysis of MAPK. Treated chondrocytes showed degradation of collagen typ II and a significant increase of caspase-3 in a dose-dependent manner. These results indicate that the inhibition of the latter signal transduction pathway through IL-18 also blocks differentiation and reduces lifespan of chondrocytes through apoptotic processes.

Limited supplies of tendon tissue for use in reconstructive surgery require development of phenotypically stable tenocytes cultivated in vitro. Tenocytes in monolayer culture display an unstable phenotype and tend to dedifferentiate, but those in three-dimensional culture may remain phenotypically and functionally differentiated. In this study we established a three-dimensional high-density culture system for cultivation of human tenocytes for tissue engineering. Human tenocytes were expanded in monolayer culture before transfer to high-density culture. The synthesis of major extracellular matrix proteins and the ultrastructural morphology of the three-dimensional cultures were investigated for up to 2 weeks by electron microscopy, immunohistochemistry, immunoblotting and quantitative, real-time PCR. Differentiated tenocytes were able to survive over a period of 14 days in high-density culture. During the culture period tenocytes exhibited a typical tenocyte morphology embedded in an extensive extracellular matrix containing cross-striated collagen type I fibrils and proteoglycans. Moreover, expression of the tendon-specific marker scleraxis underlined the tenocytic identity of these cells. Taken together, we conclude that the three-dimensional high-density cultures may be useful as a new approach for obtaining differentiated tenocytes for autologous tenocyte transplantation to support tendon and ligament healing and to investigate the effect of tendon-affecting agents on tendon in vitro.

Objective: If dedifferentiated chondrocytes could be induced to redifferentiate in vitro , then we might thereby be furnished with a population of phenotypically stable cells for autologous implantation in reconstructive surgery. We therefore investigated the redifferentiation capabilities of chondrocytes which, having migrated from alginate beads to form a monolayer, were subsequently passaged. We also characterized the molecular traits of irreversibly dedifferentiated cells. Methods: Human chondrocytes that had migrated from alginate beads to form a monolayer (passage 1) were passaged seven times (passages 2–8). Cells from each passage were then recultivated in alginate beads. We assessed the synthesis of type-II collagen, cartilage-specific proteoglycans, adhesion molecules (integrins), signaling proteins (Src-homology collagen [Shc] and extracellular-signal-regulated kinase [Erk]) and the apoptosis marker ‘activated' caspase-3 in monolayer or secondary alginate cultures. Results: The synthesis of cartilage-specific type-II collagen, α3-integrin, Shc and activated Erk1/2 decreased rapidly after four passages in monolayer culture. Up to passage 4, cells redifferentiated in alginate culture. However, between passages 5 and 8, cells began to produce activated caspase-3; these cells not only failed to redifferentiate when recultivated in alginate, but underwent apoptosis. Conclusion: We conclude that the loss of chondrogenic potential by chondrocytes maintained in monolayer culture is associated with a decrease in the synthesis of cartilage markers and with a suppressed activation of key signaling proteins in the Ras-mitogen-activated protein kinase pathway (Shc and Erk1/2). These events lead to apoptosis. A decrease in Shc/Erk expression/interaction could serve as a recognition marker for irreversibly dedifferentiated chondrocytes in tissue engineering.

Background and objective Extracellular matrix proteins, such as collagen and fibronectin affect proliferation, differentiation, and morphogenesis of chondrocytes. The interactions between chondrocytes and matrix proteins are mediated largely by the β1 subfamily of integrins. There is evidence from previous studies that low level laser irradiation may influence synthesis rate of those specific cartilage matrix proteins.

High-density cultures are widely used as an in vitro model for studies of embryonic cartilage formation. In the present study we investigated the suitability of high-density cultures for the redifferentiation of dedifferentiated chondrocytes. When primary human chondrocytes were cultured in alginate beads, some cells emigrated into Petri dishes. These cells were cultured for one to eight passages (each passage lasting about 3 days) in monolayer culture. At each passage, monolayer cells were removed and allowed to grow in high-density cultures at the medium-air interface and subsequently investigated with morphological, immunolocalization and biochemical methods for the production of cartilage-specific markers, i.e. collagen type II and cartilage-specific proteoglycans. When such dedifferentiated chondrocytes from monolayer passages P1-P4 were introduced in high-density culture, they regained a chondrocyte phenotype and formed cartilage nodules surrounded by fibroblast-like cells. Cells were interconnected by typical gap junctions and after a few days in culturing produced cartilage-specific extracellular matrix, notably collagen type II and cartilage-specific proteoglycans. In contrast, cells taken from monolayer passages P5-P8 did not produce these chondrocyte-specific extracellular materials when grown in high-density culture. We conclude that the growth of dedifferentiated chondrocytes in high-density culture promotes their redifferentiation and reveals their chondrogenic potential. Such high-density cultures might serve as a model system to initiate and promote the redifferentiation of chondrocytes and to provide sufficient quantities of differentiated chondrocytes for autologous chondrocyte transplantation.