Adult Human Cartilage Research Articles

Heparanase is expressed in adult human osteoarthritic cartilage and drives catabolic responses in primary chondrocytes

The chondrocytes' pericellular matrix acts as a mechanosensor by sequestering growth factors that are bound to heparan sulfate (HS) proteoglycans. Heparanase is the sole mammalian enzyme with HS degrading endoglycosidase activity. Here, we aimed to ascertain whether heparanase plays a role in modulating the anabolic or catabolic responses of human articular chondrocytes. Primary chondrocytes were incubated with pro-heparanase and catabolic and anabolic gene expression was analyzed by quantitative polymerase chain reaction (PCR). MMP13 enzymatic activity in the culture medium was measured with a specific fluorescent assay. Extracellular regulated kinase (ERK) phosphorylation was evaluated by Western blot. Human osteoarthritis (OA) cartilage was assessed for heparanase expression by reverse-transcriptase PCR, by Western blot and by a heparanase enzymatic activity assay. Cultured chondrocytes rapidly associated with and activated pro-heparanase. Heparanase induced the catabolic genes MMP13 and ADAMTS4 and the secretion of active MMP13, and down-regulated the anabolic genes ACAN and COL2A1. PG545, a HS-mimetic, inhibited the effects of heparanase. Heparanase expression and enzymatic activity were demonstrated in adult human osteoarthritic cartilage. Heparanase induced ERK phosphorylation in cultured chondrocytes and this could be inhibited by PG545, by fibroblast growth factor 2 (FGF2) neutralizing antibodies and by a FGF-receptor inhibitor. Heparanase is active in osteoarthritic cartilage and induces catabolic responses in primary human chondrocytes. This response is due, at least in part, to the release of soluble growth factors such as FGF2.

Micromass cultures are effective for differentiation of human amniotic fluid stem cells into chondrocytes

OBJECTIVES:Articular cartilage is vulnerable to injuries and undergoes an irreversible degenerative process. The use of amniotic fluid mesenchymal stromal stem cells for the reconstruction of articular cartilage is a promising therapeutic alternative. The aim of this study was to investigate the chondrogenic potential of amniotic fluid mesenchymal stromal stem cells from human amniotic fluid from second trimester pregnant women in a micromass system (high-density cell culture) with TGF-β3 for 21 days.METHODS:Micromass was performed using amniotic fluid mesenchymal stromal stem cells previously cultured in a monolayer. Chondrocytes from adult human normal cartilage were used as controls. After 21 days, chondrogenic potential was determined by measuring the expression of genes, such as SOX-9, type II collagen and aggrecan, in newly differentiated cells by real-time PCR (qRT-PCR). The production of type II collagen protein was observed by western blotting. Immunohistochemistry analysis was also performed to detect collagen type II and aggrecan. This study was approved by the local ethics committee.RESULTS:SOX-9, aggrecan and type II collagen were expressed in newly differentiated chondrocytes. The expression of SOX-9 was significantly higher in newly differentiated chondrocytes than in adult cartilage. Collagen type II protein was also detected.CONCLUSION:We demonstrate that stem cells from human amniotic fluid are a suitable source for chondrogenesis when cultured in a micromass system. amniotic fluid mesenchymal stromal stem cells are an extremely viable source for clinical applications, and our results suggest the possibility of using human amniotic fluid as a source of mesenchymal stem cells.

The Role of Micrornas in Osteoarthritis and Ageing-Related Functional Decline in Joint Tissue Homeostasis

Purpose: Osteoarthritis is a degenerative disease associated with changes in the articular cartilage and bone, severely affecting patients' mobility and quality of life. A similar, but less acute decline is also seen during ageing. microRNAs (miRs) are novel post-transcriptional regulators of gene expression. microRNAs have been implicated in different aspects of musculoskeletal ageing through their differential expression, however the functional consequences of this are still not fully understood. Methods: Wild type C57BL6/J adult (3–6 months old) mice and human cartilage explants from knee replacement surgeries were used. To study microRNA function in vivo, modified microRNA antagomiR was delivered via intraarticular injection into knee joints of mice or via transfection into the human explants. Knee joints were dissected for histological assessment or RNA isolation. Human cartilage explants were cultured with IL-1β, miR mimic and/or antagomiR. Fixed joints and explants were subsequently decalcified and processed for wax embedding. Serial coronal sections were stained with Safranin O at regular intervals across the joint, and scored for articular cartilage degradation severity, using the OARSI grading system (from grades 0–6). RNA was used for microRNA and mRNA profiling using high-throughput sequencing and subsequently qPCR. Results: Our data shows dysregulation of miR-199 in human and mouse models of OA. Using qPCR and luciferase assays, we validated a selection of the microRNAs target genes differentially expressed in mouse femorotibial joints and human cartilage explants models of OA. miR-199 was further analysed using in vitro and in vivo gain- and loss-of-function approaches. Conclusions: In summary, we have shown the regulation of OA-associated target genes by miR-199 in the in vitro and in vivo models of OA and have shown that dysregulation of miR-199 expression may contribute to the pathogenic phenotype in the OA models. The authors would like to acknowledge financial support from the MRC-Arthritis Research UK Centre for Integrated research into Musculoskeletal Ageing (CIMA) and BBSRC.

Implications of trauma and subsequent articulation on the release of Proteoglycan-4 and tissue response in adult human ankle cartilage.

The purpose of this study was to investigate the effects of trauma and subsequent articulation on adult human ankle cartilage subjected to an injurious impact. Trauma was initiated through impaction on talar cartilage explants. Articulation and loading were applied in a joint bioreactor over 5 consecutive days. The early (24 h) effects of impaction included a reduced chondrocytes viability (51% vs. 81% for non-impacted; p = 0.03), increased levels of apoptosis (43% vs. 27%; p = 0.03), and an increase in the histopathology score (4.4 vs. 1.7; p = 0.02) as compared to non-impacted cartilage explants. One of the key findings was that damage also stimulated the PRG4 release (2.2 vs. 1.5 μg/ml). Subsequent articulation for 5 days did not lead to further changes in tissue histopathology and cell viability, neither for injured nor non-injured samples. However, articulation led to an increased apoptosis in the injured samples (p = 0.03 for the interaction term). Articulation also caused a significant increase of PG/GAG release into the culture medium (p = 0.04) for both injured and non-injured samples; however, the synthesis of PG was not affected by articulation (p = 0.45) though the PG synthesis was higher in injured samples (p < 0.01). With regard to the PRG4 release, impacted samples continued to show higher amounts (p = 0.01), adding articulation led to a reduction (p = 0.02). The current study demonstrated that adult human talar cartilage increases both the PRG4 release and biosynthetic activity as an immediate cellular response to injury. Articulation played a less contributing role to biosynthesis and remodeling, behaving mostly neutral, in that no further damage emerged. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:667-676, 2017.

Randomized double-blind study of a single intra-articular injection of allogeneic mesenchymal precursor cells + hyaluronan compared to hyaluronan alone to explore clinical and structural outcomes in patients post anterior cruciate ligament reconstruction

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Specific synovial joint progenitor cell populations uniquely contribute to articular cartilage growth and repair

s / Osteoarthritis and Cartilage 24 (2016) S8eS62 S16 differentiation. lncRNAs can be derived from both intergenic and overlapping protein-coding gene regions, are expressed in a highly tissue-specific manner and are emerging as key regulators of gene expression. In this study we establish a role for lncRNAs in chondrogenesis and cartilage. Methods: Paired end sequencing was performed by Illumina Genome Analyzer IIx on total RNA isolated from human articular cartilage and human MSCs undergoing chondrogenic differentiation. lncRNAs and novel transcripts were identified by aligning sequence reads to the transcriptome and genome respectively. Novel transcripts were confirmed by rapid amplification of cDNA ends (RACE) and expression validated by real-time RT-PCR in a panel of human tissues. Expression was assessed in the tri-lineage differentiation of MSCs into chondrocytes, osteoblasts and adipocytes. lncRNA expression was depleted with specific siRNAs to establish their role during differentiation. Overexpression of lncRNAs was performed by cloning and lentiviral transduction as well as CRISPR/Cas9-VP16 mediated endogenous promoter activation. Chondrocyte and cartilage development was assessed by gene and protein expression measured with standard molecular biology, biochemical and histological techniques. lncRNA-regulated genes were identified by microarray and lncRNA-regulated signalling pathways in chondrogenesis assessed by Western blotting and reporter activation. Results: RNA sequencing indicates that human adult cartilage contains upwards of 3000 lncRNAs (FPKM >1) and that 188 of these are induced during MSC chondrogenic differentiation (>2fold). Of particular interest are a group of lncRNAs upstream of the SOX9 locus, found in a gene desert on chromosome 17, which includes SOX9-AS1, LINC01152 and a novel 4 exon version of the predicted 3 exon RNA, ENS0430908. This novel lncRNA is chondrocyte specific and upregulated during chondrogenic differentiation of MSCs. RNAi targeting novel ENS0430908 prevented MSC chondrogenesis with a complete loss of cartilage matrix formation and significant reductions in chondrocyte gene expression. Therefore we named this novel lncRNA ROCR (Regulator Of Chondrogenesis RNA). Depletion of ROCR totally blocked the induction of SOX9 during MSC chondrogenesis, consistent with the reduced cartilage-specific gene expression. Microarray analysis indicates that loss of ROCR phenocopies the loss of SOX9 on gene expression changes during the early stages of chondrogenesis. Loss of ROCR may also disrupt chondrogenic cell signalling, in particular activation of the TGF-b pathway. In contrast to the effect during chondrogenesis loss of ROCR enhances osteoblastogenic differentiation and gene expression. Conclusions: The cartilage transcriptome contains numerous noncoding transcripts many of which may have essential functions in cartilage biology. ROCR is essential for cartilage development and appears to promote a chondrocyte cell fate for MSCs over an osteoblast cell fate. Because SOX9-expressing cells are progenitors for numerous tissues identifying chondrocyte-specific regulatory elements might aid understanding of differentiation of chondrocytes from MSCs, potentially useful in chondrocyte tissue engineering applications. 15 SPECIFIC SYNOVIAL JOINT PROGENITOR CELL POPULATIONS UNIQUELY CONTRIBUTE TO ARTICULAR CARTILAGE GROWTH AND REPAIR R.S. Decker y, H.-B. Um y, N.A. Dyment z, N. Cottingham y, E. Koyama y, M. Kronenberg z, P. Maye z, D.W. Rowe z, M. Pacifici y. yChildren's Hosp. of Philadelphia, Philadelphia, PA, USA; zUniv. of Connecticut, Farmington,

Cellular viability and outgrowth of cryopreserved particulated adult human allograft cartilage

Event Abstract Back to Event Cellular viability and outgrowth of cryopreserved particulated adult human allograft cartilage Alex Mcnally1, Kurt Sly1, Chris Chapman1, Lee Krengel1 and Steve Lin1 1 Exactech, Inc., Biologics, United States Introduction: Particulated cartilage autograft and fresh osteochondral allograft has been used clinically to treat cartilage defects[1]. However, both these current cartilage repair techniques present drawbacks. Autograft cartilage can cause donor site morbidity and there is a limit to the amount than can be harvested in order to fill a large defect. Fresh osteochondral allograft with fresh living cells has a short shelf-life making it difficult to manage inventory and resulting in tissue being discarded due to limited shelf-life. We propose cryopreservation of allograft cartilage to extend the shelf-life but retain viable and active chondrocytes within particulated cartilage fragments. In this study, we evaluated the viability and bioactivity of cryopreserved human cartilage fragments compared to fresh tissue. Materials and Methods: Human cartilage fragments were harvested from seven adult human condyles (LifeLink Tissue Bank) and sieved between 1-0.1mm. The fragments were then cryopreserved for 12, and 24 weeks. Fresh unfrozen fragments served as a control. A subset of the cartilage fragments at each time point was digested overnight with collagenase to dissociate the tissue and isolate chondrocytes. The viability of the chondrocytes was determined with nucleic acid binding dyes acridine orange (AO) and propidium iodide (PI). Live nucleated cells fluoresce green and the dead nucleated cells fluoresce red. The cellular viability was determined using the Cellometer K2 (Nexcelom). The remaining cartilage fragments were placed in 24-well transwell plates. All fragments were cultured at 37°C, 5% CO2 in complete media consisting of DMEM+GlutaMax (Gibco) supplemented with 10% FBS, 1X antibiotics, and 1X antimycotic for 8 weeks. After 8 weeks the constructs were encased in HistoGel (Thermo Fisher) and collected from the transwells. The constructs were fixed in 10% formalin and embedded in paraffin wax and 5μm sections were prepared. Sections were stained with hematoxylin and eosin (H&E). Results and Discussion: The viability of fresh cartilage was about 92%. The viability of the cryopreserved chondrocytes at 12 and 24 weeks was 84% and 82% respectively and was not significantly different compared to fresh tissue (Figure 1). Fresh cartilage fragments displayed hypercellular regions indicative of chondrocytes outgrowth and fragment fusion after 8 weeks of culture (Figure 2A). The cryopreserved fragments following 12 and 24 weeks of cryopreservation displayed cellular outgrowth and fragment fusion following 8 weeks of culture as well (Figure 2B-C). Conclusion: Several studies have shown that cartilage is a source of chondrocytes which can be used to treat cartilage defects. We investigated a processing technique for adult allograft cartilage which entails particulation and cryopreservation. In this study the cryopreserved fragments maintained high viability and both fresh and cryopreserved human cartilage fragments displayed robust outgrowth. Cryopreservation of adult cartilage with a prolonged shelf-life may provide an attractive alternative for treating cartilage defects. Future studies should evaluate the bioactivity of these fragments in-vivo.

Engineering physiologically stiff and stratified human cartilage by fusing condensed mesenchymal stem cells

For a long time, clinically sized and mechanically functional cartilage could be engineered from young animal chondrocytes, but not from adult human mesenchymal stem cells that are of primary clinical interest. The approaches developed for primary chondrocytes were not successful when used with human mesenchymal cells. The method discussed here was designed to employ a mechanism similar to pre-cartilaginous condensation and fusion of mesenchymal stem cells at a precisely defined time. The formation of cartilage was initiated by press-molding the mesenchymal bodies onto the surface of a bone substrate. By image-guided fabrication of the bone substrate and the molds, the osteochondral constructs were engineered in anatomically precise shapes and sizes. After 5weeks of cultivation, the cartilage layer assumed physiologically stratified histomorphology, and contained lubricin at the surface, proteoglycans and type II collagen in the bulk phase, collagen type X at the interface with the bone substrate, and collagen type I within the bone phase. For the first time, the Young’s modulus and the friction coefficient of human cartilage engineered from mesenchymal stem cells reached physiological levels for adult human cartilage. We propose that this method can be effective for generating human osteochondral tissue constructs.

Aggrecan nanoscale solid-fluid interactions are a primary determinant of cartilage dynamic mechanical properties.

Poroelastic interactions between interstitial fluid and the extracellular matrix of connective tissues are critical to biological and pathophysiological functions involving solute transport, energy dissipation, self-stiffening and lubrication. However, the molecular origins of poroelasticity at the nanoscale are largely unknown. Here, the broad-spectrum dynamic nanomechanical behavior of cartilage aggrecan monolayer is revealed for the first time, including the equilibrium and instantaneous moduli and the peak in the phase angle of the complex modulus. By performing a length scale study and comparing the experimental results to theoretical predictions, we confirm that the mechanism underlying the observed dynamic nanomechanics is due to solid-fluid interactions (poroelasticity) at the molecular scale. Utilizing finite element modeling, the molecular-scale hydraulic permeability of the aggrecan assembly was quantified (kaggrecan = (4.8 ± 2.8) × 10(-15) m(4)/N·s) and found to be similar to the nanoscale hydraulic permeability of intact normal cartilage tissue but much lower than that of early diseased tissue. The mechanisms underlying aggrecan poroelasticity were further investigated by altering electrostatic interactions between the molecule's constituent glycosaminoglycan chains: electrostatic interactions dominated steric interactions in governing molecular behavior. While the hydraulic permeability of aggrecan layers does not change across species and age, aggrecan from adult human cartilage is stiffer than the aggrecan from newborn human tissue.

Effects of insulin-like growth factor-1 and dexamethasone on cytokine-challenged cartilage: relevance to post-traumatic osteoarthritis

Interleukin-1 is one of the inflammatory cytokines elevated after traumatic joint injury that plays a critical role in mediating cartilage tissue degradation, suppressing matrix biosynthesis, and inducing chondrocyte apoptosis, events associated with progression to post-traumatic osteoarthritis (PTOA). We studied the combined use of insulin-like growth factor-1 (IGF-1) and dexamethasone (Dex) to block these multiple degradative effects of cytokine challenge to articular cartilage. Young bovine and adult human articular cartilage explants were treated with IL-1α in the presence or absence of IGF-1, Dex, or their combination. Loss of sulfated glycosaminoglycans (sGAG) and collagen were evaluated by the DMMB and hydroxyproline assays, respectively. Matrix biosynthesis was measured via radiolabel incorporation, chondrocyte gene expression by qRT-PCR, and cell viability by fluorescence staining. In young bovine cartilage, the combination of IGF-1 and Dex significantly inhibited the loss of sGAG and collagen, rescued the suppression of matrix biosynthesis, and inhibited the loss of chondrocyte viability caused by IL-1α treatment. In adult human cartilage, only IGF-1 rescued matrix biosynthesis and only Dex inhibited sGAG loss and improved cell viability. Thus, the combination of IGF-1 + Dex together showed combined beneficial effects in human cartilage. Our findings suggest that the combination of IGF-1 and Dex has greater beneficial effects than either molecule alone in preventing cytokine-mediated cartilage degradation in adult human and young bovine cartilage. Our results support the use of such a combined approach as a potential treatment relevant to early cartilage degradative changes associated with joint injury.

Characterization of colony-forming cells in adult human articular cartilage

Recent studies have shown that adult human articular cartilage contains stem-like cells within the native structure. In this study, we aimed to determine the localization of putative stem cell markers such as CD90, STRO-1, OCT-3/4, CD105 and CD166 in adult human articular cartilage tissue sections and demonstrate the expression of these markers within the expanded surface zone colony-forming (CF) cells and evaluate their differentiation potential. Biopsy samples were either fixed immediately for immunohistochemical analyses or processed for in vitro cell culture. Immunohistochemical and flow cytometry analyses were performed by using CD90, STRO-1, OCT-3/4, CD105 and CD166 antibodies. Isolated colony-forming (CF) cells were further stimulated, by using the appropriate growth factors in their pellet culture, to obtain cartilage, bone and adipose lineages. We observed that the expression of the stem cell markers were in various zones of the human adult cartilage. Flow cytometry results showed that in CF cells the expression of CD90 and CD166 was high, while OCT-3/4 was low. We also determined that CF cells could be stimulated towards cartilage, bone and adipose lineages. The results of this research support the idea that the resident stem-like cells in adult human articular cartilage express these putative stem cell markers, but further experimental investigations are needed to determine the precise localization of these cells.

A compositional analysis of cadaveric human nasal septal cartilage

To localize quantitatively the major biochemical constituents of native adult human septal cartilage across whole septa. Prospective, basic science. The nasal septa from seven cadavers were partitioned into 24 separate regions: six from caudal to cephalic and four from dorsal to ventral. Biochemical assays were used to determine the quantities, relative to wet weight, of the major constituents of cartilage: chondrocytes, collagen, and sulfated glycosaminoglycan. On average, each milligram of wet cartilage contained 24,900 cells, 73.9 μg collagen, and 17.1 μg sulfated glycosaminoglycan. Cell number showed no significant variation across the septa. In contrast, the caudal regions of the septa were associated with higher levels of collagen, the ventral regions correlated with higher levels of sulfated glycosaminoglycan, and the dorsal regions were associated with an elevated ratio of collagen to sulfated glycosaminoglycan. This study represents the first characterization of the biochemical composition of native human septal cartilage across whole septa. Quantities of collagen and sulfated glycosaminoglycan showed region-specific variation across the septum. The localized pattern of collagen and sulfated glycosaminoglycan deposition are consistent with the significance of preserving the L-strut during rhinoplasty and other nasal reconstructive procedures. In addition, it may assist in defining design goals for tissue-engineered septal neocartilage constructs to meet specific reconstructive needs in the future.

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

Human Articular Chondrocytes Express Multiple Gap Junction Proteins: Differential Expression of Connexins in Normal and Osteoarthritic Cartilage

The role of norepinephrine in adult human articular chondrocytes

Norepinephrine (NE) belongs to the catecholamine family of tyrosine-derived neurotransmitters of the sympathetic nervous system. Tyrosine-hydroxylase positive sympathetic nerve fibers have been identified in bone marrow, in the periosteum, and in bone-adherent ligaments indicating that growth and metabolic activity of bone and joint tissues is regulated by sympathetic neurotransmitters. It is known that NE can regulate cell proliferation or apoptosis in several cell types, such as osteoblasts. It is further described that NE modulates inflammation during rheumatoid arthritis and gut inflammation. Here, we aim to understand the role of NE in human osteoarthritic chondrocytes with regard to inflammation and its impact on metabolic activity. Human chondrocytes were isolated from post-surgery discarded human osteoarthritic articular cartilage. Expression of adrenergic receptor on articular cartilage chondrocytes was tested with standard end point PCR and immunohistochemical analysis. Employing 3D cell cultures in fibrin gel, effects of NE on IL-1β induced gene expression of pro-inflammatory cytokines and matrix metalloproteinases (MMP) were analyzed with quantitative real-time PCR. The impact of NE on cell proliferation was determined in monolayer culture with BrdU ELISA and FACS analyses, the effect on apoptosis with Caspase 3/7 ELISA. To test the adrenergic receptor involved in apoptosis or proliferation, the α1-adrenergic receptor antagonist doxazosin, the α2-adrenergic receptor antagonist yohimbine, and the β1/2/3-adrenergic receptor antagonist nadolol were included in BrdU/caspase-3/7-ELISAs. Chondrocytes cultured in monolayer and in 3D under non- and inflammatory conditions expressed α1D-, α2A/B/C- and β2-adrenergic receptors. β2-Adrenergic receptors were detected on protein level in human osteoarthritic cartilage as well. Stimulation with NE has significantly reduced IL-1β induced gene expression of IL-8 and MMP-13 in human osteoarthritic chondrocytes cultured in 3D fibrin gel. Notably, we were unable to detect an impact on IL-1β induced gene expression of interleukin-6, MMP-2 and MMP-3. Furthermore, NE inhibits BrdU incorporation compared to the controls. This effect was reversed by nadolol. In cell cycle analyses, we showed that NE increased the G1/G0-phase population and decreased S-phase population. Additionally, we observed an increase of caspase-3/7 activity after NE treatment that was reversed after addition of α1-adrenergic receptor antagonist doxazosin. Neurotransmitters of the sympathetic nervous system like NE presumably mediate an anti-inflammatory/chondroprotective effect in human osteoarthritic chondrocytes via reducing IL-1β induced IL-8 and MMP-13. Furthermore, NE is able to modulate the metabolic activity of chondrocytes by a cell cycle slow-down via β2-adrenergic receptor signaling and by induction of apoptosis via α1D-adrenergic receptor signaling. We therefore assume a yet unknown function of catecholaminergic neurotransmitters in adult human cartilage that might have an impact on osteoarthritis pathology.

Chondrogenesis, chondrocyte differentiation, and articular cartilage metabolism in health and osteoarthritis.

Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. Following chondrogenesis, the chondrocytes remain as resting cells to form the articular cartilage or undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue-engineering strategies is the inability of the resident chondrocytes to lay down a new matrix with the same properties as it had when it was formed during development. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. The pivotal proteinase that marks OA progression is matrix metalloproteinase 13 (MMP-13), the major type II collagen-degrading collagenase, which is regulated by both stress and inflammatory signals. We and other investigators have found that there are common mediators of these processes in human OA cartilage. We also observe temporal and spatial expression of these mediators in early through late stages of OA in mouse models and are analyzing the consequences of knockout or transgenic overexpression of critical genes. Since the chondrocytes in adult human cartilage are normally quiescent and maintain the matrix in a low turnover state, understanding how they undergo phenotypic modulation and promote matrix destruction and abnormal repair in OA may to lead to identification of critical targets for therapy to block cartilage damage and promote effective cartilage repair.

Chondrogenesis, joint formation, and cartilage metabolism

Chondrogenesis occurs as a result of mesenchymal cell condensation and chondroprogenitor cell differentiation. The chondrocytes then form the cartilage at the end of the opposing bones with the intervening interzones formed during cavitation or they undergo proliferation, terminal differentiation to chondrocyte hypertrophy, and apoptosis in a process termed endochondral ossification, whereby the hypertrophic cartilage is replaced by bone. A similar sequence of events occurs in the postnatal growth plate and leads to rapid growth of the skeleton. Human adult articular cartilage is a complex tissue of matrix proteins that varies from superficial to deep layers and from loaded to unloaded zones. A major challenge to efforts to repair cartilage by stem cell-based and other tissue engineering strategies is the inability of the resident chondrocytes to lay down new matrix with the same properties as it had when it was formed during development. This is particularly true of the collagen network, which is susceptible to cleavage once the proteoglycans are depleted. Thus, understanding and comparing the mechanisms of cartilage remodeling during development, osteoarthritis (OA), and aging may lead to more effective strategies for preventing cartilage damage and promoting repair. To identify and characterize mediators of cartilage remodeling common to these processes, we are using culture models of primary human and mouse chondrocytes and cell lines and mouse models to manipulate and compare gene expression with complementary approaches. MMP-13, the major type II collagen-degrading collagenase, is regulated by both stress and inflammatory signals that not only contribute to irreversible joint damage (progression) in OA, but importantly, also to the initiation/onset phase, wherein chondrocytes in articular cartilage leave their natural growth - and differentiation-arrested state. We and other investigators have found that there are common mediators of these processes in human OA cartilage and from early through late stages of OA in mouse models, including the surgical models (good matrix with abnormal loading) and the genetic models during aging (bad matrix with normal loading). We are validating our in vitro analyses of the signaling and transcriptional mechanisms that determine the expression and activities of these mediators by in vivo analyses of the consequences of knockout or transgenic overexpression of these genes in mouse models. In current studies, we are examining the epigenetic mechanisms and using proteomics and genomics approaches to map the signaling networks and microRNA targets that impact on gene expression programs during the onset and progression of OA in both human and murine cartilage. Since the chondrocytes in adult human cartilage are normally quiescent and maintain the matrix in a low turnover stated, understanding how they undergo phenotypic modulation and promote matrix destruction and abnormal repair in OA may to lead to identification of critical targets for therapy to block cartilage damage and promote effective cartilage repair.

The antiapoptotic gene survivin is highly expressed in human chondrosarcoma and promotes drug resistance in chondrosarcoma cells in vitro

BackgroundChondrosarcoma is virtually resistant to chemotherapy and radiation therapy. Survivin, the smallest member of the inhibitor of apoptosis protein family, is a critical factor for tumor progression and resistance to conventional therapeutic approaches in a wide range of malignancies. However, the role of survivin in chondrosarcoma has not been well studied. We examined the importance of survivin gene expression in chondrosarcoma and analysed its influences on proliferation, apoptosis and resistance to chemotherapy in vitro.MethodsResected chondrosarcoma specimens from which paraffin-embedded tissues could be extracted were available from 12 patients. In vitro experiments were performed in human chondrosarcoma cell lines SW1353 and Hs819.T. Immunohistochemistry, immunoblot, quantitative PCR, RNA interference, gene-overexpression and analyses of cell proliferation and apoptosis were performed.ResultsExpression of survivin protein was detected in all chondrosarcoma specimens analyzed, while undetectable in adult human cartilage. RNA interference targeting survivin resulted in a G2/M-arrest of the cell cycle and led to increased rates of apoptosis in chondrosarcoma cells in vitro. Overexpression of survivin resulted in pronounced resistance to doxorubicin treatment.ConclusionsThese findings indicate that survivin plays a role in the pathogenesis and pronounced chemoresistance of high grade chondrosarcoma. Survivin antagonizing therapeutic strategies may lead to new treatment options in unresectable and metastasized chondrosarcoma.

Computed tomography measurement of the auricle in Han population of north China

A large number of anthropometric studies of the human auricle have been conducted in various populations. However, data from Han Chinese population are currently not available. The morphology of the auricle is highly complex. Consequently, traditional direct measurement techniques are unable to provide sufficient detail and accuracy. The present study sought to provide anthropometrical data on the auricles of Han Chinese individuals using a three-dimensional (3D) computed tomography (CT) reconstruction technique. A total of 485 data sets were utilised for this study. This sample consisted of data from 241 females aged 18-75 years and 244 males aged 18-74 years. All participants were categorised by gender and further into three age groups. Twelve anthropometrical dimensions of normal auricles were measured with 3D CT. Auricular, tragal, lobular and conchal indices were calculated to further examine the auricles. Earlobes were classified into three basic groups according to the angle of the junction of the earlobe with the cheek. Few of the anthropometrical data showed significant bilateral differences in auricular dimensions. The height of the tragus, lobular length, lobular width, conchal width, protrusion at tragal level and inclination angle of auricles showed no significant differences between males and females. All the linear dimensions of auricles, except the height of the tragus, conchal length and width, increased with age for both males and females. Protrusion at the superaurale and tragal levels showed a decrease of between 4% and 9% for males and 4% and 10% for females across all age groups. This study is the first to report CT measurement of normal auricles in a northern Han Chinese population. We found that this method enabled the complexity of the ear to be measured easily and accurately. Increasing our knowledge of normal auricular dimensions will be beneficial for plastic surgeons.

SOX Gene Expression in Human Osteoarthritic Cartilage

Objective: While the developmental role of the SOX transcription factors in fetal chondrocyte differentiation is well documented, much less is known about the expression of SOX family members in normal and osteoarthritic adult cartilage. Therefore, the aim of the present study was to present a thorough analysis of SOX gene expression in normal and osteoarthritic human adult cartilage. Methods: RNA from normal and osteoarthritic knee cartilage from human adults was analyzed by gene expression profiling using GeneChip technology (Affymetrix) and quantitative real time PCR. Results: Most members of the SOX transcription factor family showed no or very low expression levels in normal and osteoarthritic cartilage from adults. In contrast, SOX9 expression was fairly high in normal cartilage, amounting to approximately 20% of GAPDH levels. SOX9 transcript levels were substantially reduced in osteoarthritis. SOX6 levels were reduced, albeit starting from a low basis expression in normal tissue. Conclusion: The presented data indicate that the role of the SOX transcription factor family in adult human cartilage is most probably restricted to a few members, with SOX9 being the most prominent. Furthermore, the reduction of SOX9 and SOX6 transcript levels in osteoarthritic chondrocytes might be responsible for the loss of phenotypic stability of osteoarthritic chondrocytes.

Marrow stimulation techniques

Due to the very low intrinsic activity of human adult cartilage, healing of chondral and osteochondral defects in patients cannot be expected. In treating symptomatic cartilage damage, marrow stimulation methods belong to the most frequently used methods, along with autologous chondrocyte transplantation (ACT) and mosaicplasty. These arthroscopic procedures are generally easy and the marrow stimulation treatment costs relatively little. In recent years, Pridie drilling has been increasingly replaced by the microfracture technique. This modification relies on the same biological principles of promoting resurfacing with the formation of fibro-cartilaginous repair tissue. For the treatment of smaller cartilage defects (<2.5 cm(2)), microfracture still remains the first choice for treatment. The clinical results after microfracture in the knee are age dependent. Younger and active patients (<40 years) with smaller isolated traumatic lesions on the femoral condyles have the best long-term results. The deterioration of the clinical results begins after 18 months and is significantly more pronounced in older patients with defects on the patella-femoral joint and tibia. The inferior quality of the repair tissue, partially incomplete defect filling and new bone formation in the defect area seem to be limitations of these methods. The AMIC (autologous matrix induced chondrogenesis) technique was developed to enable treatment of larger defects by the application of a collagen Type III/I membrane (Geistlich Pharma, Wolhusen, Switzerland), in particular when cell-engaged procedures such as ACT cannot be used for financial reasons or because it is not indicated. AMIC seems to be particularly suitable for treating damaged retropatellar cartilage, which is an advantage because these defects can be hard to treat with standard microfracturing alone. The results of the ongoing studies are awaited to establish whether better results with this technology are achievable in the long term.