Alcian Blue Staining Intensity Research Articles

Polydeoxyribonucleotide ameliorates IL-1β-induced impairment of chondrogenic differentiation in human bone marrow-derived mesenchymal stem cells

Osteoarthritis (OA) is a degenerative disease of the joints, prevalent worldwide. Polydeoxyribonucleotide (PDRN) is used for treating knee OA. However, the role of PDRN in IL-1β-induced inflammatory responses in human bone marrow-derived mesenchymal stem cells (hBMSCs) remains unknown. Here, we investigated the role of PDRN in IL-1β-induced impairment of chondrogenic differentiation in hBMSCs. hBMSCs treated with PDRN showed a large micromass, enhanced safranin O and alcian blue staining intensity, and increased expression of chondrogenic genes in IL-1β-induced inflammatory responses, in addition to regulation of catabolic and anabolic genes. In addition, PDRN treatment suppressed the expression of inflammatory cytokines and mitigated IL-1β-induced apoptosis in hBMSCs. Mechanistically, PDRN treatment increased the formation of cyclic adenosine monophosphate (cAMP) and upregulated the phosphorylation of cAMP-dependent protein kinase A (PKA)/cAMP response element binding protein (CREB) through the adenosine A2A receptor in hBMSCs and thus blocked the nuclear factor kappa-light-chain-enhancer of activated B cell (NF-κB) signaling pathway. Thus, IL-1β-induced expression of inflammatory cytokines in hBMSCs was directly reduced by adenosine A2A receptor activation. Based on our results, we suggest that PDRN may be a promising MSC-based therapeutic agent for OA.

Short preconditioning with TGFβ of equine adipose tissue-derived mesenchymal stem cells predisposes towards an anti-fibrotic secretory phenotype: A possible tool for treatment of endometrosis in mares

Endometrosis in mares is a disease resulting from chronic inflammation characterized by peri glandular fibrosis. There is no effective treatment so far, which opens the door for exploring the use of stem cells as a candidate. Transforming growth factor beta (TGFβ) is crucial for the establishment and progression of fibrosis in mare's endometrosis. We aimed to develop regenerative approaches to treat endometrosis by using mesenchymal stem cells (MSC), for which understanding the effect of TGFβ on exogenous MSC is crucial. We isolated and characterized equine adipose MSC from six donors. Cells were pooled and exposed to 10 ng/ml of TGFβ for 0, 4, and 24 h, after which cells were analyzed for proliferation, migration, mesodermal differentiation, expression of fibrosis-related mRNAs, and prostaglandin E2 secretion. At 24 h of exposition to TGFβ, there was a progressive increase in the contraction of the monolayer, leading to nodular structures, while cell viability did not change. Exposure to TGFβ impaired adipogenic and osteogenic differentiation after 4 h of treatment, which was more marked at 24 h, represented by a decrease in Oil red and Alizarin red staining, as well as a significant drop (p < 0.05) in the expression of key gene regulators of differentiation processes (PPARG for adipose and RUNX2 for osteogenic differentiation). TGFβ increased chondrogenic differentiation as shown by the upsurge in size of the resulting 3D cell pellet and intensity of Alcian Blue staining, as well as the significant up-regulation of SOX9 expression (p < 0.05) at 4 h, which reached a maximum peak at 24 h (p < 0.01), indicative of up-regulation of glycosaminoglycan synthesis. Preconditioning MSC with TGFβ led to a significant increase (p < 0.05) in the expression of myofibroblast gene markers aSMA, COL1A1, and TGFβ at 24 h exposition time. In contrast, the expression of COL3A1 did not change with respect to the control but registered a significant downregulation compared to 4 h (p < 0.05). TGFβ also affected the expression of genes involved in PGE2 synthesis and function; COX2, PTGES, and the PGE2 receptor EP4 were all significantly upregulated early at 4 h (p < 0.05). Cells exposed to TGFβ showed a significant upregulation of PGE2 secretion at 4 h compared to untreated cells (p < 0.05); conversely, at 24 h, the PGE2 values decreased significantly compared to control cells (p < 0.05). Preconditioning MSC for 4 h led to an anti-fibrotic secretory phenotype, while a longer period (24 h) led to a pro-fibrotic one. It is tempting to propose a 4-h preconditioning of exogenous MSC with TGFβ to drive them towards an anti-fibrotic phenotype for cellular and cell-free therapies in fibrotic diseases such as endometrosis of mares.

A bovine nucleus pulposus explant culture model.

Low back pain is a global health problem that is frequently caused by intervertebral disc degeneration (IVDD). Sulfated glycosaminoglycans (sGAGs) give the healthy nucleus pulposus (NP) a high fixed charge density (FCD), which creates an osmotic pressure that enables the disc to withstand high compressive forces. However, during IVDD sGAG reduction in the NP compromises biomechanical function. The aim of this study was to develop an ex vivo NP explant model with reduced sGAG content and subsequently investigate biomechanical restoration via injection of proteoglycan‐containing notochordal cell‐derived matrix (NCM). Bovine coccygeal NP explants were cultured in a bioreactor chamber and sGAG loss was induced by chondroitinase ABC (chABC) and cultured for up to 14 days. Afterwards, diurnal loading was studied, and explant restoration was investigated via injection of NCM. Explants were analyzed via histology, biochemistry, and biomechanical testing via stress relaxation tests and height measurements. ChABC injection induced dose‐dependent sGAG reduction on Day 3, however, no dosing effects were detected after 7 and 14 days. Diurnal loading reduced sGAG loss after injection of chABC. NCM did not show an instant biomechanical (equilibrium pressure) or biochemical (FCD) restoration, as the injected fixed charges leached into the medium, however, NCM stimulated proliferation and increased Alcian blue staining intensity and matrix organization. NCM has biological repair potential and biomaterial/NCM combinations, which could better entrap NCM within the NP tissue, should be investigated in future studies. Concluding, chABC induced progressive, time‐, dose‐ and loading‐dependent sGAG reduction that led to a loss of biomechanical function. Keywords biomechanics | intervertebral disc | matrix degradation | low back pain | proteoglycans

Generation of a Biomimetic Substitute of the Corneal Limbus Using Decellularized Scaffolds.

Patients with severe limbal damage and limbal stem cell deficiency are a therapeutic challenge. We evaluated four decellularization protocols applied to the full-thickness and half-thickness porcine limbus, and we used two cell types to recellularize the decellularized limbi. The results demonstrated that all protocols achieved efficient decellularization. However, the method that best preserved the transparency and composition of the limbus extracellular matrix was the use of 0.1% SDS applied to the half-thickness limbus. Recellularization with the limbal epithelial cell line SIRC and human adipose-derived mesenchymal stem cells (hADSCs) was able to generate a stratified epithelium able to express the limbal markers p63, pancytokeratin, and crystallin Z from day 7 in the case of SIRC and after 14–21 days of induction when hADSCs were used. Laminin and collagen IV expression was detected at the basal lamina of both cell types at days 14 and 21 of follow-up. Compared with control native limbi, tissues recellularized with SIRC showed adequate picrosirius red and alcian blue staining intensity, whereas limbi containing hADSCs showed normal collagen staining intensity. These preliminary results suggested that the limbal substitutes generated in this work share important similarities with the native limbus and could be potentially useful in the future.

Osteogenic protein-1 attenuates apoptosis and enhances matrix synthesis of nucleus pulposus cells under high-magnitude compression though inhibiting the p38 MAPK pathway.

Disc degeneration is correlated with mechanical load. Osteogenic protein-1 (OP-1) is potential to regenerate degenerative disc. To investigate whether OP-1 can protect against high magitude compression-induced nucleus pulposus (NP) cell apoptosis and NP matrix catabolism, and its potential mechanism; porcine discs were cultured in a bioreactor and compressed at a relatively high-magnitude mechanical compression (1.3 MPa at a frequency of 1.0 Hz for 2 h once per day) for 7 days. OP-1 was added along with the culture medium to investigate the protective effects of OP-1. NP cell apoptosis and matrix biosynthesis were evaluated. Additionally, activity of the p38 MAPK pathway is also analyzed. Compared with the control group, high magnitude compression significantly promoted NP cell apoptosis and decreased NP matrix biosynthesis, reflected by the increase in the number of terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL)-positive cells and caspase-3 activity, the up-regulated expression of Bax and caspase-3 mRNA and down-regulated expression of Bcl-2 mRNA, and the decreased Alcian Blue staining intensity and expression of matrix proteins (aggrecan and collagen II). However, OP-1 addition partly attenuated the effects of high magnitude compression on NP cell apoptosis and NP matrix biosynthesis. Further analysis showed that inhibition of the p38 MAPK pathway partly participated in this process. OP-1 can attenuate high magnitude compression-induced NP cell apoptosis and promoted NP matrix biosynthesis, and inhibition of the p38 MAPK pathway may participate in this regulatory process. The present study provides that OP-1 may be efficient in retarding mechanical overloading-exacerbated disc degeneration.

Evaluation of the Effects of Biodegradable Microspheres Loaded with Quercetin on Adipogenic and Chondrogenic Differentiation of Cellular Spheroids

This study was performed to evaluate the effects of quercetin application and the biodegradable poly(lactic-co-glycolic acid)-based microspheres loaded with quercetin on the adipogenic and chondrogenic differentiation of three-dimensional cells composed of gingiva-derived stem cells and osteoblast-like cells. Three-dimensional cell spheroids were fabricated using silicon elastomer-based concave microwells and cultured in adipogenic and chondrogenic media. Quercetin at 1 μg/mL (Q1) and microspheres loaded with quercetin at 1 μg/mL (M1) were used. Quantitative cellular viability was evaluated using Cell Counting Kit-8 assay. Adipogenesis was determined after oil red O staining and chondrogensis was evaluated by measuring relative intensity of Alcian blue staining. Spheroids were well maintained irrespective of quercetin application in silicon elastomer-based concave microwells. The relative Cell Counting Kit-8 assay values for unloaded, Q1, and M1 groups in adipogenic media on Day 1 were 100.0±7.6%, 153.6±11.3%, and 104.7±6.3%, respectively. Relative values of adipogenesis were 114.6%, 116.2%, and 113.4% for unloaded, Q1, and M1 groups on Day 7, respectively, and relative values of adipogenesis were 131.7%, 137.5%, and 133.6% for unloaded, Q1, and M1 groups on Day 14, respectively. The relative Cell Counting Kit-8 assay values for unloaded, Q1, and M1 groups in chondrogenic media on Day 1 were 100.0±3.2%, 162.1±13.0%, and 96.6±3.7%, respectively. Relative values of chondrogensis were 103.6%, 111.0%, and 103.7% for unloaded, Q1, and M1 groups on Day 7, respectively, and the relative values of chondrogensis were 157.4%, 165.3%, and 160.9% for unloaded, Q1, and M1 groups on Day 14, respectively. Application of quercetin produced quercetin at 1 μg/mL and promoted the viability of cell spheroids cultured in adipogenic and chondrogenic media. However, application of quercetin did not reach statistically significant enhancement of adipogenisis or chondrogenesis at the experimental condition.

Static compression down-regulates N-cadherin expression and facilitates loss of cell phenotype of nucleus pulposus cells in a disc perfusion culture.

Mechanical compression often induces degenerative changes of disc nucleus pulposus (NP) tissue. It has been indicated that N-cadherin (N-CDH)-mediated signaling helps to preserve the NP cell phenotype. However, N-CDH expression and the resulting NP-specific phenotype alteration under the static compression and dynamic compression remain unclear. To study the effects of static compression and dynamic compression on N-CDH expression and NP-specific phenotype in an in vitro disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days and subjected to static or dynamic compression (0.4 MPa for 2 h once per day). The noncompressed discs were used as controls. Compared with the dynamic compression, static compression significantly down-regulated the expression of N-CDH and NP-specific markers (laminin, brachyury, and keratin 19); decreased the Alcian Blue staining intensity, glycosaminoglycan and hydroxyproline contents; and declined the matrix macromolecule (aggrecan and collagen II) expression. Compared with the dynamic compression, static compression causes N-CDH down-regulation, loss of NP-specific phenotype, and the resulting decrease in NP matrix synthesis.

Lysophosphatidylcholine acyltransferase 4 is involved in chondrogenic differentiation of ATDC5 cells

Glycerophospholipids have important structural and functional roles in cells and are the main components of cellular membranes. Glycerophospholipids are formed via the de novo pathway (Kennedy pathway) and are subsequently matured in the remodeling pathway (Lands’ cycle). Lands’ cycle consists of two steps: deacylation of phospholipids by phospholipases A2 and reacylation of lysophospholipids by lysophospholipid acyltransferases (LPLATs). LPLATs play key roles in the maturation and maintenance of the fatty acid composition of biomembranes, and cell differentiation. We examined whether LPLATs are involved in chondrogenic differentiation of ATDC5 cells, which can differentiate into chondrocytes. Lysophosphatidylcholine acyltransferase 4 (LPCAT4) mRNA expression and LPCAT enzymatic activity towards 18:1-, 18:2-, 20:4-, and 22:6-CoA increased in the late stage of chondrogenic differentiation, when mineralization occurred. LPCAT4 knockdown decreased mRNA and protein levels of chondrogenic markers as well as Alcian blue staining intensity and alkaline phosphatase activity in ATDC5 cells. These results suggest that LPCAT4 plays important roles during the transition of chondrocytes into hypertrophic chondrocytes and/or a mineralized phenotype.

Long-term load duration induces N-cadherin down-regulation and loss of cell phenotype of nucleus pulposus cells in a disc bioreactor culture.

Long-term exposure to a mechanical load causes degenerative changes in the disc nucleus pulposus (NP) tissue. A previous study demonstrated that N-cadherin (N-CDH)-mediated signalling can preserve the NP cell phenotype. However, N-CDH expression and the resulting phenotype alteration in NP cells under mechanical compression remain unclear. The present study investigated the effects of the compressive duration on N-CDH expression and on the phenotype of NP cells in an ex vivo disc organ culture. Porcine discs were organ cultured in a self-developed mechanically active bioreactor for 7 days. The discs were subjected to different dynamic compression durations (1 and 8 h at a magnitude of 0.4 MPa and frequency of 1.0 Hz) once per day. Discs that were not compressed were used as controls. The results showed that long-term compression duration (8 h) significantly down-regulated the expression of N-CDH and NP-specific molecule markers (Brachyury, Laminin, Glypican-3 and Keratin 19), attenuated Alcian Blue staining intensity, decreased glycosaminoglycan (GAG) and hydroxyproline (HYP) contents and decreased matrix macromolecule (aggrecan and collagen II) expression compared with the short-term compression duration (1 h). Taken together, these findings demonstrate that long-term load duration can induce N-CDH down-regulation, loss of normal cell phenotype and result in attenuation of NP-related matrix synthesis in NP cells.

Excess maternal and postnatal thyroxine alters chondrocyte numbers and the composition of the extracellular matrix of growth cartilage in rats

ABSTRACTPurpose/Aim: The aim of this study was to evaluate the effects of excess maternal and postnatal thyroxine on chondrocytes and the extracellular matrix (ECM) of growth cartilage. Materials and Methods: We used 16 adult female Wistar rats divided into two groups: thyroxine treatment and control. From weaning to 40 days of age, offspring of the treated group (n = 8) received L-thyroxine. Plasma free T4 was measured. Histomorphometric analysis was performed on thyroids and femurs of all offspring. Alcian blue histochemical staining and real-time reverse transcription polymerase chain reaction measurements of gene expression levels of Sox9, Runx2, Aggrecan, Col I, Col II, Alkaline phosphatase, Mmp2, Mmp9, and Bmp2 were performed. Data were analyzed for statistical significance by student’s t-test. Results: Excess maternal and postnatal thyroxine reduced the intensity of Alcian blue staining, altered the number of chondrocytes in proliferative and hypertrophic zones in growth cartilage, and reduced the gene expression of Sox9, Mmp2, Mmp9, Col II, and Bmp2 in the growth cartilage of all offspring. Additionally, excess thyroxine altered the gene expression of Runx2, Aggrecan and Col I, and this effect was dependent on age. Conclusions: Excess thyroxine in neonates suppresses chondrocyte proliferation, stimulates chondrocyte hypertrophy and changes the ECM composition by reducing the amount of proteoglycans and glycosaminoglycans (GAGs). Prolonged exposure to excess thyroxine suppresses chondrocyte activity in general, with a severe reduction in the proteoglycan content of cartilage and the expression of gene transcripts essential for endochondral growth and characteristics of the chondrocyte phenotype.

In Vitro Engineering of High Modulus Cartilage-Like Constructs.

To date, the outcomes of cartilage repair have been inconsistent and have frequently yielded mechanically inferior fibrocartilage, thereby increasing the chances of damage recurrence. Implantation of constructs with biochemical composition and mechanical properties comparable to natural cartilage could be advantageous for long-term repair. This study attempted to create such constructs, in vitro, using tissue engineering principles. Bovine synoviocytes were seeded on nonwoven polyethylene terephthalate fiber scaffolds and cultured in chondrogenic medium for 4 weeks, after which uniaxial compressive loading was applied using an in-house bioreactor for 1 h per day, at a frequency of 1 Hz, for a further 84 days. The initial loading conditions, determined from the mechanical properties of the immature constructs after 4 weeks in chondrogenic culture, were strains ranging between 13% and 23%. After 56 days (sustained at 84 days) of loading, the constructs were stained homogenously with Alcian blue and for type-II collagen. Dynamic compressive moduli were comparable to the high end values for native cartilage and proportional to Alcian blue staining intensity. We suggest that these high moduli values were attributable to the bioreactor setup, which caused the loading regime to change as the constructs developed, that is, the applied stress and strain increased with construct thickness and stiffness, providing continued sufficient cell stimulation as further matrix was deposited. Constructs containing cartilage-like matrix with response to load similar to that of native cartilage could produce long-term effective cartilage repair when implanted.

MicroRNA-30a promotes chondrogenic differentiation of mesenchymal stem cells through inhibiting Delta-like 4 expression

AimsMicroRNAs (miRNAs) play important roles in chondrogenic differentiation of mesenchymal stem cells (MSCs). However, the regulation of miR-30a during such process has not yet been well understood. The aim of the study was to investigate the effects of miR-30a on chondrogenic differentiation of MSCs and explore the underlying mechanisms. Materials and methodsMSCs were isolated from rat bone marrow, and their immunophenotypes and multilineage differentiation potentials were identified. MiR-30a mimics or inhibitor were transfected into rat MSCs and SW1353 cells, respectively, and then the effects of miR-30a on chondrogenic differentiation were detected. The predicted target gene Delta-like 4 (DLL4, a ligand of the Notch signaling family) was verified by luciferase reporter assay, quantitative real time PCR and western blot. Key findingsMiR-30a was significantly up-regulated during chondrogenic differentiation of rat MSCs. Additionally, transfection of miR-30a mimics remarkably promoted the differentiation of rat MSCs into chondrocytes as evidence by the notably increased mRNA and protein expression levels of chondrogenic markers Collagen II and aggrecan as well as the enhanced alcian blue staining intensity, whereas inhibition of miR-30a obviously suppressed such process. Furthermore, during chondrogenesis, DLL4 expression was found to significantly decrease at both mRNA and protein levels, which was negatively regulated by miR-30a through directly targeting the 3'UTR of DLL4. SignificanceOur results indicate that miR-30a acts as a key promoter for chondrogenic differentiation of MSCs by down-regulating DLL4 expression, and provide a novel insight on miRNA-mediated MSC therapy for cartilage-related disorders including osteoarthritis.

Dexamethasone inhibits chondrocyte differentiation by suppression of Wnt/β-catenin signaling in the chondrogenic cell line ATDC5.

Glucocorticoids (GCs) regulate proliferation and differentiation in cultured mesenchymal cells through the modulation of various molecules. However, the relationship between growth factor signaling and GCs in differentiating chondrocytes has not been elucidated. In this study, we examined the effects of Wnt/β-catenin signaling on chondrocyte differentiation and the effects of a GC analogue, dexamethasone (Dex), on Wnt/β-catenin signaling activity by using a chondrocyte progenitor cell line ATDC5. Western blot analysis and TCF/LEF-optimized promoter EGFP (TOPEGFP) assay showed that both β-catenin protein levels and TCF/LEF transcription were up-regulated during insulin-transferrin-sodium selenite (ITS)-induced chondrogenic differentiation. Morphological analysis showed that TCF/LEF transcription activity was most prominent in cartilage nodule-like structures. Furthermore, a β-catenin mutant with constitutive transcriptional activity (ΔN90) showed increased Alcian blue staining intensity and mRNA expression of Sox9, Col2a, aggrecan, Col10, and alkaline phosphatase, even in the absence of ITS stimulation. In contrast, Dex suppressed formation of ITS-induced cartilage nodule-like structures, TCF/LEF-mediated transcription, and β-catenin protein levels. Real-time PCR analysis showed that Dex increased the mRNA expression levels of secreted frizzled-related protein 1 (sFRP1) and Axin2. Furthermore, treatment with a sFRP1 inhibitor or the ΔN90 β-catenin mutant transfection attenuated Dex-induced suppression of cartilage matrix production by increasing Sox9 mRNA levels. These results suggest that Dex inhibits chondrocyte differentiation via down-regulation of Wnt/β-catenin signaling, which promotes chondrocyte differentiation in ATDC5 cells.

Supplementation of strontium to a chondrogenic medium promotes chondrogenic differentiation of human dedifferentiated fat cells.

Dedifferentiated fat cells (DFAT cells) isolated from adipose tissue have been demonstrated to differentiate into chondrogenic cells in vitro. Nevertheless, an efficient method to facilitate its chondrogenic differentiation is still unexplored, hampering the extensive application of these cells in cartilage regeneration therapies. Here we provide the evidence that supplementation of strontium ions (Sr) in a chondrogenic medium (CM) significantly promotes early chondrogenic differentiation of DFAT cells. Human DFAT cells and the mesenchymal stem cell line (RCB2153) were subjected to the CM supplemented with/without Sr. After 14 days, alcian blue staining intensity significantly increased in DFAT cells, but not in RCB2153, subjected to CM with Sr. mRNA expression analysis revealed that the CM with 1.5 mM Sr increased the expression of chondrogenic marker, collagen type 2 alpha 1, whereas there was no significant change in osteogenic markers, collagen type 1 alpha 1, runt-related transcription factor 2, and osteocalcin, and hypertrophic chondrogenic marker, collagen type 10 alpha 1. Inhibitors for extracellular signal-regulated kinase 1/2 (ERK1/2), Akt, and calcium-sensing receptor (CaSR) pathways significantly diminished the alcian blue staining intensity, providing the first evidence that these signal pathways are associated with chondrogenic differentiation of DFAT cells. CaSR and ERK1/2 pathways independently induced Sr-mediated early chondrogenic differentiation. These results suggest that Sr supplementation into the CM may provide a powerful platform for preparing chondrogenically differentiated DFAT cells for cartilage regeneration.

Characterization of Human Endplate Biopsies: Preliminary Results on Cellular Composition, Metabolic Activity and Interactions with Disk Cells

Introduction Tissue alterations that result in an impaired endplate (EP) permeability are assumed to play a crucial role in the pathogenesis of intervertebral disk (IVD) degeneration. Therefore, disturbance of endplate permeability is one of the main problems that need to be solved for successful disk tissue engineering. However, the biological mechanisms underlying these alterations are poorly understood. To shed light on this aspect, a standardized analysis of EP structure and cellular composition is essential to understand the degeneration processes. Purpose of this study was the development of standardized protocols for a descriptive characterization of EP samples by histology, transmission electron microscopy (TEM), analysis of differentiation and metabolic activity of EP cells. In first preliminary experiments we also aimed to detect possible interactions of the EP with disk cells of the same donor patients in a bioassay with EP-conditioned media. The suitability of these methods for EP characterization and possible correlations with degenerative alterations is discussed. Materials and Methods Cartilaginous endplates obtained during spine surgery of total 27 human donors underwent the following procedures: (1) isolation of endplate cells, in vitro expansion and characterization of chondrogenic differentiation in 3D pellet cultures ( n = 6), (2) preparation of standardized EP-sections for histological and ultrastructural analyses ( n = 8), and (3) preparation of standardized endplate punches that were incubated for 24 hours in serum-free culture medium followed by determination of metabolic activity of endplate cells by MTT-test ( n = 13) and further analysis of conditioned media (CM) in a bioassay with NP cells of the same donor ( n = 2). Pellet cultures of EP cells were characterized for matrix protein expression by Alcian blue staining. Influences of CM on gene expression of matrix proteins, matrix degrading enzymes, and inflammation factors by NP cells were determined. Results Differentiation of endplate cells in pellet cultures varied strongly not only between the six individual donors, but also in different disk levels of the same patient. Part of the samples exhibited a very low proteoglycan production whereas others were strongly positive in the Alcian blue staining intensity suggesting high variations in the differentiation capacity of EP cells. Histological examination of the endplate samples allowed a characterization of both the endplates and an observation of the adjacent structures as the very thin endplate layer could not be clearly separated from adjacent tissue structures. High variations between tissue samples from different donors could be found. TEM images showed signs of degeneration both in the matrix and the EP cells whereas higher numbers of apoptotic cells and matrix degradation products could be found in stronger degenerated tissue samples. Standardized EP punches that were cultured for 24 hours in DMEM and incubated for 4 hours with MTT showed different staining intensities suggesting donor-specific variations in metabolic activity of EP cells. In first two experiments with CM of EP samples, a dose-dependent upregulation of inflammation-factors and matrix metalloproteinases (Interleukins IL-1β, IL-6, IL-8, MMP-3, and MMP13) by NP-cells of the same donors could be detected. The extent of this effect differed strongly between the two patients with different degeneration degree. Conclusion The present results suggest that a combination of different methods allows the characterization of EP tissue and possible interactions with the adjacent disk tissue. As the cartilaginous EP itself is very thin and with low cellularity, isolation of an almost homogeneous EP population is very difficult and the cell yield is very poor. Therefore, methods that directly analyze the cells in situ for example by laser capture micro-dissection might be more suitable to determine the gene expression profile of EP cells. Analysis of metabolic activity of standardized EP punches by MTT test is a promising approach provided that the samples are standardized with regard to diameter and height with no adjacent other tissue regions. TEM investigation is appropriate to investigate cellular details, but also with this method, the poor cellularity of EP tissue is a challenge. A very promising approach is the analysis of CM in a bioassay with NP cells of the same donor that allows the detection of possible interactions between both tissue areas. In ongoing investigations, we analyze higher numbers of EP samples with different degrees of degeneration to enable a correlation between the biological and clinical data of the patients. For this purpose, analyses of nondegenerated control samples are essential. In summary, all these methods together allow a holistic view on the mechanisms that contribute to the morphological and pathophysiological changes of the EP and potential interaction with the adjacent disk tissue. Acknowledgement Part of this research work was supported by the EU framework 7 project Geneodisc (FP7, 2007–2013) under grant agreement no. HEALTH-F2-2008-201626. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared

Polyhexanide and hydrogen peroxide inhibit proteoglycan synthesis of human chondrocytes

The use of local antiseptics is a common method in septic joint surgery. We tested polyhexanide and hydrogen peroxide, two of the most frequently used antiseptics with high efficacy and low toxicity. The purpose of this study was to evaluate the effects of both antiseptics on the extracellular cartilaginous matrix synthesis of human chondrocytes. Chondrocytes were isolated from donated human knee joints, embedded in alginate beads, and incubated for 10 and 30 minutes with polyhexanide (0·04%), hydrogen peroxide (3%), or phosphate-buffered saline (PBS) for control. Cartilaginous matrix production was quantified through light microscopic analysis of Alcian blue staining. Cell number and morphology were detected by histological analysis. Chondrocytes showed a decreased intensity of blue colouring after antiseptic treatment versus PBS. In contrast to that, neither the cell number per view field nor the cell morphology differed between the groups. Polyhexanide has more toxic potential than hydrogen peroxide. Based on the fact that the cell number and morphology was not altered by the substances at the examined concentrations, the lower intensity of Alcian blue staining of treated chondrocytes indicates a decreased cartilage-specific matrix synthesis by polyhexanide more than by hydrogen peroxide and control.

MicroRNA-221 Regulates Chondrogenic Differentiation through Promoting Proteosomal Degradation of Slug by Targeting Mdm2

MicroRNAs (miRNAs) are small RNAs that fulfill diverse functions by negatively regulating gene expression. Here, we investigated the involvement of miRNAs in the chondrogenic differentiation of chick limb mesenchymal cells and found that the expression of miR-221 increased upon chondrogenic inhibition. Blockade of miR-221 via peanut agglutinin-based antisense oligonucleotides reversed the chondro-inhibitory actions of a JNK inhibitor on the proliferation and migration of chondrogenic progenitors as well as the formation of precartilage condensations. We determined that mdm2 is a relevant target of miR-221 during chondrogenesis. miR-221 was necessary and sufficient to down-regulate Mdm2 expression, and this down-modulation of Mdm2 by miR-221 prevented the degradation of (and consequently up-regulated) the Slug protein, which negatively regulates the proliferation of chondroprogenitors. These results indicate that miR-221 contributes to the regulation of cell proliferation by negatively regulating Mdm2 and thereby inhibiting Slug degradation during the chondrogenesis of chick limb mesenchymal cells.

Nuclear factor E2 p45-related factor 2 negatively regulates chondrogenesis

The transcription factor nuclear factor E2 p45-related factor 2 (Nrf2) forms heterodimers with small musculoaponeurotic fibrosarcoma (Maf) proteins for the selective recognition of the antioxidant responsive element on target genes, followed by the regulation of gene expression of phase II detoxifying enzymes as well as oxidative-stress-inducible proteins in different tissues. In the present study, we investigated the role of Nrf2 in the regulation of chondrocyte differentiation as well as the expression pattern of Nrf2 in cartilage. In tibia from embryonic mice at E15.5, Nrf2 mRNA expression was restricted to both proliferating and pre-hypertrophic chondrocytes, with few signals in early and late hypertrophic chondrocytes expressing both type X collagen and osteopontin. On in situ hybridization analysis of tibia from neonatal mice at 1 day after birth, by contrast, Nrf2 was expressed in all chondrocytic layers in addition to osteoblasts attached to cancellous bone. In pre-chondrogenic cell line ATDC5 cells, furthermore, expression of Nrf2 mRNA was also confirmed together with mRNA expression of the Kelch-like ECH associating protein 1 and small Maf proteins. In ATDC5 cells stably transfected with Nrf2, significant inhibition was seen in the differentiation-dependent induction of alkaline phosphatase and increase in the Alcian blue staining intensity. Furthermore, stable overexpression of Nrf2 significantly decreased mRNA expression of several chondrocyte differentiation markers such as type II collagen, type X collagen and osteopontin. These data suggest that Nrf2 may be a negative regulator of the cellular differentiation toward maturation in chondrocytes.

Cyclic hydrostatic compression stimulates chondroinduction of C3H/10T1/2 cells

While the potential for intermittent hydrostatic pressure to promote cartilaginous matrix synthesis is well established, its potential to influence chondroinduction remains poorly understood. This study examined the effects of relatively short- and long-duration cyclic hydrostatic compression on the chondroinduction of C3H/10T1/2 murine embryonic fibroblasts by recombinant human bone morphogenetic protein-2 (rhBMP-2). Cells were seeded at high density into round bottom wells of a 96-well plate and supplemented with 25 ng/ml rhBMP-2. Experimental cultures were subjected to either 1,800 cycles/day or 7,200 cycles/day of 1 Hz sinusoidal hydrostatic compression to 5 MPa (applied 10 min on/10 min off) for 3 days. Non-pressurized control and experimental cultures were maintained in static culture for an additional 5 days. Cultures were then analyzed for alcian blue staining intensity, DNA and sulfated glycosaminoglycan (sGAG) content, and for the rate of collagen synthesis. Whereas cultures subjected to 1,800 pressure cycles exhibited no significant differences (statistical or qualitative) compared to controls, those subjected to 7,200 cycles stained more intensely with alcian blue, contained nearly twice as much sGAG, and displayed twice the rate of collagen synthesis as non-pressurized controls. This study demonstrates the potential for cyclic hydrostatic compression to stimulate chondrogenic differentiation of the C3H/10T1/2 cell line in a duration-dependent manner.

The effects of polyester (terylene) fibre implants on normal equine superficial digital flexor tendon

AIM: To determine the effect of polyester (terylene) fibre implants on normal equine superficial digital flexor (SDF) tendon structure. METHODS: Normal forelimb SDF tendons (n=24) of 12 horses were divided into unoperated, sham-operated, and implanted (terylene fibre implant) groups. Horses were assessed for lameness and ultrasonographic changes to SDF tendons at intervals up to 48 weeks post-operatively. After euthanasia, SDF tendons were collected for histological and ultrastructural examination. Histological sections were examined for alcian blue staining intensity, cellularity, fibril bundle alignment, fascicle separation and crimp morphology. Mass-average diameters (MADs) of collagen fibrils were calculated from electron micrographs and compared between treatment groups. RESULTS: Insertion of terylene fibre implants resulted in short-term (<8 weeks) lameness in implanted limbs. Ultrasonographically, the implants could be detected in 50% of implanted tendons, but were associated with tendon swelling and the presence of hypoechoic core lesions in 7/8 implanted limbs. There were significant alterations in alcian blue staining, cellularity and crimp morphology in the central fascicles of sham-operated and implanted tendons, and alteration in fibril alignment in the central fascicles of implanted tendons. Unoperated tendons remained histologically normal. MADs of collagen fibrils did not differ between sham-operated, implanted and unoperated limbs. CONCLUSIONS: Both the sham procedure and the insertion of terylene fibre implants led to alterations in tendon structure that persisted for up to 48 weeks. Persistence of disorganised connective tissue at the proximal and distal ends of the terylene fibre implants may predispose implanted tendons to continued risk of injury. CLINICAL RELEVANCE: It is unlikely that terylene fibre implants offer any advantage over standard non-surgical treatments for mild to moderate cases of SDF tendonitis in the horse.