Aggrecan Research Articles

A Wogonin-rich Fraction of scutellaria baicalensis Root Extract Exerts Chondroprotective Effects by Suppressing il-1β-induced Activation of AP-1 in Human OA Chondrocytes

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1β-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1β showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1β-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1β-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.

Hyaluronan oligosaccharides stimulate matrix metalloproteinase and anabolic gene expression in vitro by intervertebral disc cells and annular repair in vivo.

The role of hyaluronan (HA) oligosaccharides in disc cell-mediated matrix metalloproteinase (MMP) and anabolic gene expression in vitro and annular repair in vivo were examined. Monolayer and alginate bead cultures of ovine intervertebral disc cells were stimulated with 10-12 mer hyaluronan oligosaccharides (HA-oligos). Annulus fibrosus (AF) monolayers were poorly responsive to the HA-oligos, proMMP-2 levels were marginally elevated and levels were MMP-9 unaffected. ProMMP-2 displayed a strong dose-dependent increase in the nucleus pulposus (NP) monolayers. In AF alginate bead cultures, proMMP-2 and active MMP-9 increased up to day 10, in NP cultures proMMP-2 was progressively converted to active MMP-2 over days 7-10 and active MMP-9 levels were elevated on day 10. A steady decline in MMP-2 and MMP-9 activity was evident over days 2-10 in the non-stimulated NP cultures. Disc cell viabilities were ≥92±5% in all cultures indicating that the HA-oligo was not cytotoxic. Reverse-transcription polymerase chain reaction demonstrated an upregulation in MMP1, MMP113 and ADAMTS1 and the anabolic matrix repair genes ACAN, COL1A1 and COL2A1 in the NP by HA-oligos, whereas AF MMP13, ADAMTS1, ADAMTS4 and ADAMTS5, ACAN and COL2A1 were down-regulated; this differential regulation is expected to promote clearance of granulation/scar tissue from AF defects and matrix replenishment. The AF defect sites contained enlarged annular lamellae in vivo in response to the HA oligos, which is consistent with an active repair response. Masson trichrome and PicroSirius red histology and immunolocalization of type I collagen supported active remodelling in the outer lesion zone by the HA-oligo treatment but not the inner lesion. Copyright © 2016 John Wiley & Sons, Ltd.

A wogonin-rich-fraction of Scutellaria baicalensis root extract exerts chondroprotective effects by suppressing IL-1\u03b2-induced activation of AP-1 in human OA chondrocytes

Osteoarthritis (OA) is a common joint disorder with varying degrees of inflammation and sustained oxidative stress. The root extract of Scutellaria baicalensis (SBE) has been used for the treatment of inflammatory and other diseases. Here, we performed activity-guided HPLC-fractionation of SBE, identified the active ingredient(s) and investigated its chondroprotective potential. We found that the Wogonin containing fraction-4 (F4) was the most potent fraction based on its ability to inhibit ROS production and the suppression of catabolic markers including IL-6, COX-2, iNOS, MMP-3, MMP-9, MMP-13 and ADAMTS-4 in IL-1β-treated OA chondrocytes. OA chondrocytes treated with F4 in the presence of IL-1β showed significantly enhanced expression of anabolic genes ACAN and COL2A1. In an in vitro model of cartilage degradation treatment with F4 inhibited s-GAG release from IL-1β-treated human cartilage explants. The inhibitory effect of F4 was not mediated through the inhibition of MAPKs and NF-κB activation but was mediated through the suppression of c-Fos/AP-1 activity at transcriptional and post transcriptional levels in OA chondrocytes. Purified Wogonin mimicked the effects of F4 in IL-1β-stimulated OA chondrocytes. Our data demonstrates that a Wogonin-rich fraction of SBE exert chondroprotective effects through the suppression of c-Fos/AP-1 expression and activity in OA chondrocytes under pathological conditions.

Orally administered simvastatin partially preserves lumbar vertebral bone mass but not integrity of intervertebral discs in ovariectomized rats.

The present study aimed to investigate the effect of orally administered simvastatin on lumbar vertebral bone mass and intervertebral disc (IVD) degeneration in ovariectomized (OVX) rats. A total of 30 female Sprague-Dawley (SD) rats were subjected to either bilateral ovariectomy (n=20) or sham surgery (n=10). After 12 weeks, the OVX rats were orally administered either saline vehicle (OVX + V group; n=10), or 5 mg/kg/day simvastatin (OVX + SIM group; n=10). Following 12 weeks of treatment, necropsy was conducted and bone mineral density (BMD) was determined in the L5-6 vertebrae. Furthermore, the microstructure and biomechanical properties of the L3 vertebrae were detected by micro-computed tomography and compression testing, respectively. The L5-6 vertebrae were analyzed by measurement of IVD height, observation of histological changes by van Gieson staining, and evaluation of collagen-II (col-II), aggrecan (AGG) and collagen I (col-I) expression by immunohistochemical analysis. Rats in the OVX+V group had lower BMD, bone volume/trabecular volume ratio, maximum load and elastic modulus than the sham group. Rats in the OVX + SIM group had higher BMD and biomechanical strength values than the rats in the OVX+V group. Histological analysis showed that the OVX + V and OVX + SIM groups exhibited significantly higher disc degeneration scores and significantly lower IVD height than the sham group. Immunohistochemical analysis revealed lower expression levels of col-II and AGG, but higher levels of col-I in the annulus fibrosis and endplate in OVX+V rats compared with the sham group. The OVX + SIM group exhibited levels of col-II, AGG and col-I expression comparable with those of OVX+V rats, with the exception of an upregulation of col-II expression in the annulus fibrosis. These data demonstrate that simvastatin treatment partially prevented bone loss and the deterioration of biomechanical properties of lumbar vertebrae, but not the progression of IVD degeneration in OVX rats.

Cobalt chloride supplementation differently affects human mesenchymal stem cells isolated from dental pulp, umbilical cord and adipose tissues in their chondrogenic potential

Articular cartilage is an avascular tissue without innervations, characterized by low cell density and abundant extracellular matrix (ECM). These characteristics leave articular cartilage with very limited capacity of repair and regeneration. Multipotent stem/stromal cells (MSC) are considered promising for cartilage tissue engineering. Stem cells are resided in a special microenvironment known as the stem-cell niche, characterized by the presence of low oxygen concentration. Previous studies have reported that hypoxic conditions could enhance the chondrogenic differentiation of mesenchymal stem cells in the presence of an inductive medium. Cobalt chloride (CoCl2) imitates hypoxia in vitro by preventing hypoxia-inducible factor-alpha (HIF-a) from being destroyed by oxygen. However, the long-term hypoxic culture of stem cells is difficult and requires special attention to avoid cell death due to cobalt treatment. In this study we investigated if CoCl2 affected MSCs isolated from dental pulp, umbilical cord and adipose tissue in their potential to differentiate toward the chondrogenic phenotype. Cells were treated with concentrations of CoCl2 ranging from 50 to 400 uM. Cell proliferation, mRNA expression of stem-cell marker and chondrogenic associated genes were analyzed by RT-PCR and Real-time PCR. The results showed that the CoCl2 supplementation had no effect on the proliferation of all the three type of cells analyzed, while the up-regulation of chondrogenic markers such as aggrecan, sox9, and type II collagen, was dependent on the cellular source. This study shows that hypoxia induced by CoCl2 treatment can differently influence the behavior of MSCs of different sources in their chondrogenic potential. These findings should be taken into consideration in the treatment of cartilage repair and regeneration based on stem cell therapies.

Are genes encoding proteoglycans really associated with the risk of anterior cruciate ligament rupture?

Proteoglycans are considered integral structural components of tendon and ligament and have been implicated in the resistance of compressive forces, collagen fibrillogenesis, matrix remodelling and cell signalling. Several sequence variants within genes encoding proteoglycans were recently implicated in modulating anterior cruciate ligament ruptures (ACLR). This study aimed to test the previously implicated variants in proteoglycan and vascular epithelial growth factor encoding genes with risk of ACLR in a population from Poland. A case control genetic association study was conducted using DNA samples from 143 healthy participants without a history of ACL injuries (99 male and 44 females) (CON group) and 229 surgically diagnosed ACLR participants (158 males and 71 females). All samples were genotyped for the ACAN: rs1516797, BGN: rs1042103, rs1126499, DCN: rs516115 and VEGFA: rs699947 variants. Main findings included the (i) ACAN rs1516797 G/T genotype which was underrepresented in the CON group (CON: 36%, n=52, ACLR: 49%, n=112, p=0.017, OR=1.68, 95% CI 1.09 to 2.57) when all participants were investigated and (ii) the BGN rs1042103 A allele was significantly under-represented in the male CON group compared to the male ACLR group (CON: 39%, n=78, ACLR: 49%, n=156, p=0.029, OR=1.5, 95% CI 1.05 to 2.15). Furthermore, BGN inferred haplotypes were highlighted with altered ACLR susceptibility. Although the study implicated the ACAN and BGN genes (combination of genotype, allele and haplotype) in modulating ACLR susceptibility, several differences were noted with previous published findings.

MTORC1 Signaling Promotes Limb Bud Cell Growth and Chondrogenesis.

mTORC1 signaling has been shown to promote limb skeletal growth through stimulation of protein synthesis in chondrocytes. However, potential roles of mTORC1 in prechondrogenic mesenchyme have not been explored. In this study, we first deleted Raptor, a unique and essential component of mTORC1, in prechondrogenic limb mesenchymal cells. Deletion of Raptor reduced the size of limb bud cells, resulting in overall diminution of the limb bud without affecting skeletal patterning. We then examined the potential role of mTORC1 in chondrogenic differentiation in vitro. Both pharmacological and genetic disruption of mTORC1 significantly suppressed the number and size of cartilage nodules in micromass cultures of limb bud mesenchymal cells. Similarly, inhibition of mTORC1 signaling in chondrogenic ATDC5 cells greatly impaired cartilage nodule formation, and decreased the expression of the master transcriptional factor Sox9, along with the cartilage matrix genes Acan and Col2a1. Thus, we have identified an important role for mTORC1 signaling in promoting limb mesenchymal cell growth and chondrogenesis during embryonic development. J. Cell. Biochem. 118: 748-753, 2017. © 2016 Wiley Periodicals, Inc.

RNA Sequencing Reveals a Role of TonEBP Transcription Factor in Regulation of Pro-inflammatory Genes in Response to Hyperosmolarity in Healthy Nucleus Pulposus Cells: A HOMEOSTATIC RESPONSE?

Transcription factor tonicity-responsive enhancer-binding protein (TonEBP/NFAT5) is critical for osmo-adaptation and extracellular matrix homeostasis of nucleus pulposus (NP) cells in their hypertonic tissue niche. Recent studies implicate TonEBP signaling in inflammatory disease and rheumatoid arthritis pathogenesis. However, broader functions of TonEBP in the disc remain unknown. RNA sequencing was performed on NP cells with TonEBP knockdown under hypertonic conditions. 1140 TonEBP-dependent genes were identified and categorized using Ingenuity Pathway Analysis. Bioinformatic analysis showed enrichment of matrix homeostasis and cytokine/chemokine signaling pathways. C-C motif chemokine ligand 2 (CCL2), interleukin 6 (IL6), tumor necrosis factor (TNF), and nitric oxide synthase 2 (NOS2) were studied further. Knockdown experiments showed that TonEBP was necessary to maintain expression levels of these genes. Gain- and loss-of-function experiments and site-directed mutagenesis demonstrated that TonEBP binding to a specific site in the CCL2 promoter is required for hypertonic inducibility. Despite inhibition by dominant-negative TonEBP, IL6 and NOS2 promoters were not hypertonicity-inducible. Whole-disc response to hypertonicity was studied in an ex vivo organ culture model, using wild-type and haploinsufficient TonEBP mice. Pro-inflammatory targets were induced by hypertonicity in discs from wild-type but not TonEBP-haploinsufficient mice. Mechanistically, NF-κB activity increased with hypertonicity and was necessary for hypertonic induction of target genes IL6, TNF, and NOS2 but not CCL2 Although TonEBP maintains transcription of genes traditionally considered pro-inflammatory, it is important to note that some of these genes also serve anabolic and pro-survival roles. Therefore, in NP cells, this phenomenon may reflect a physiological adaptation to diurnal osmotic loading of the intervertebral disc.

Effects of cyclic tensile stress on metabolism and apoptosis of chondrocytes inrat

Objective To investigate the effect of cyclic tensile stress (CTS) on the metabolism inand apoptosis of rat chondrocytes. Methods Primary rat chondrocytes were cultured on a Bioflex plate for one day and then stretched cyclically for 24 hours at a frequency of 0.5 Hz using a Flexcell-5000T apparatus. The cells were divided into 5 groups according to their stretching ratio: 0% (the control group), 2%, 6%, 10% and 14%. After the stretching, Col II, Aggrecan, MMP-13 and ADAMTS-5 mRNA were measured using qPCRs, and the NO and PGE2 levels were measured using ELISA kits. Moreover, TUNEL staining and Annexin V-FITC/PI were used to analyze the apoptosis of chondrocytes. Results Compared with the control group, the average levels of Col II and Aggrecan mRNA decreasedin 10% and 14% groups [(0.738±0.11) and (0.58±0.13), (0.75±0.11) and (0.55±0.09)]. In those groups, the MMP-13 [(2.47±0.47) and (2.88±0.36)] and ADAMTS-5 mRNA level [(2.39±0.33) and (2.75±0.49)], the NO [(6.96±0.96) and (8.28±0.82)] and PGE2 level [(6.83±0.66) and (7.15±0.71)] had increased significantly. In the 6% group the average levels of Col II(1.76±0.30) and Aggrecan mRNA (1.93±0.14) of 6% group were significantly higher than the control group, but the NO level of the former (3.07±0.20) was significantly lower than the control group (3.89±0.33). The apoptosis rate of chondrocytesin 2% and 6% groups were (0.065±0.013) and (0.063±0.147), without significant differences to that of the control group (0.045±0.008). However, compared with the control group, apoptosis in the 10% and 14% groups [(0.135±0.026) and (0.184±0.020)] increased significantly. Conclusion The effect of cyclic tensile stress on chondrocyte metabolism and apoptosis was magnitude-dependent. Ten percent and 14% CTS can increase the catabolism and apoptosis of chondrocytes.Ten percent and 14% strain can increase the catabolism and apoptosis of chondrocytes.Cyclic 6% strain can increase the anabolism of chondrocytes, but 2% strain has no impact on metabolism or apoptosis. Key words: Cyclic tensile stress; Chondrocyte; Apoptosis; Metabolism; Rat

Study on the effects of gradient mechanical pressures on the proliferation, apoptosis, chondrogenesis and hypertrophy of mandibular condylar chondrocytes in vitro

ObjectiveTo investigate the effects of gradient mechanical pressure on chondrocyte proliferation, apoptosis, and the expression of markers of chondrogenesis and chondrocyte hypertrophy. MethodsMandibular condylar chondrocytes from 5 rabbits were cultured in vitro, and pressed with static pressures of 50kPa, 100kPa, 150kPa and 200kPa for 3h, respectively. The chondrocytes cultured without pressure (0kPa) were used as control. Cell proliferation, apoptosis, and the expression of aggrecan (AGG), collagen II (COL2), collagen X (COL10), alkaline phosphatase (ALP) were investigated. Ultrastructures of the pressurized chondrocytes under transmission electron microscopy (TEM) were observed. ResultsChondrocyte proliferation increased at 100kPa and decreased at 200kPa. Chondrocyte apoptosis increased with peak pressure at 200kPa in a dose-dependent manner. Chondrocyte necrosis increased at 200kPa. The expression of AGG increased at 200kPa. The expression of COL2 decreased at 50kPa and increased at 150kPa. The expression of COL10 and ALP increased at 150kPa. Ultrastructure of the pressurized chondrocytes under TEM showed: at 100kPa, cells were enlarged with less cellular microvillus and a bigger nucleus; at 200kPa, cells shrank with the sign of apoptosis, and apoptosis cells were found. ConclusionsThe mechanical loading of 150kPa is the moderate pressure for chondrocyte: cell proliferation and apoptosis is balanced, necrosis is reduced, and chondrogenesis and chondrocyte hypertrophy are promoted. When the pressure is lower, chondrogenesis and chondrocyte hypertrophy are inhibited. At 200kPa, degeneration of cartilage is implied.

Research progress of genetic susceptibility genes associated with intervertebral disc degeneration

The process of intervertebral disc degeneration, which could result in intervertebral disc structural and functional change, is a chronic one with multiple factors. The pathophysiologic process is still not completely find out. More and more research reports manifest that certain gene polymorphism also lead to increased risk of intervertebral disc degeneration except environmental factors. Discussions about related genetic factors and their pathophysiological role in the process of degeneration could have a further understanding to disease development. Elucidating genetic components which are associated with degeneration could not only provide insights into the mechanism of the process, but also have clinical significance for early diagnosis and prevention. In order to have a thorough understanding of functional role played by different genes, this paper summarize polymorphism and disease correlation by selecting 15 genes after reviewed the related literature published in recent years. Genetic polymorphisms in 15 genes have been analyzed in association with intervertebral disc degeneration, including aggrecan, collagen Types Ⅰ, Ⅸ and Ⅺ, fibronectin, HAPLN1, CILP, MMP-1, 2 and 3, PARK2, IL-1, 6 and VDR. Each genetic polymorphism codes for a protein which has a functional role in the pathogenesis of disease. Among the 15 genes analyzed, polymorphisms in aggrecan, Type Ⅸ collagen, MMP3, IL1, IL6 and VDR show the most promise as functional variants. Genetic studies are necessary for understanding the mechanism of the degeneration. Relevant genetic information could be used as a predictive model for determining individuals’ risk for intervertebral disc degeneration eventually.

Direct electrospinning of 3D auricle-shaped scaffolds for tissue engineering applications

Thirty-two poly(ε)caprolactone (PCL) scaffolds have been produced by electrospinning directly into an auricle-shaped mould and seeded with articular chondrocytes harvested from bovine ankle joints. After seeding, the auricle shaped constructs were cultured in vitro and analysed at days 1, 7, 14 and 21 for regional differences in total DNA, glycosaminoglycan (GAG) and collagen (COL) content as well as the expression of aggrecan (AGG), collagen type I and type II (COL1/2) and matrix metalloproteinase 3 and 13 (MMP3/13). Stress-relaxation indentation testing was performed to investigate regional mechanical properties of the electrospun constructs. Electrospinning into a conductive mould yielded stable 3D constructs both initially and for the whole in vitro culture period, with an equilibrium modulus in the MPa range. Rapid cell proliferation and COL accumulation was observed until week 3. Quantitative real time PCR analysis showed an initial increase in AGG, no change in COL2, a persistent increase in COL1, and only a slight decrease initially for MMP3. Electrospinning of fibrous scaffolds directly into an auricle-shape represents a promising option for auricular tissue engineering, as it can reduce the steps needed to achieve an implantable structure.

Biotechnological Chondroitin a Novel Glycosamminoglycan With Remarkable Biological Function on Human Primary Chondrocytes.

ABSTRACTCartilage tissue engineering, with in vitro expansion of autologus chondrocytes, is a promising technique for tissue regeneration and is a new potential strategy to prevent and/or treat cartilage damage (e.g., osteoarthritis). The aim of this study was (i) to investigate and compare the effects of new biotechnological chondroitin (BC) and a commercial extractive chondroitin sulfate (CS) on human chondrocytes in vitro culture; (ii) to evaluate the anti‐inflammatory effects of the innovative BC compared to extractive CS. A chondrogenic cell population was isolated from human nasoseptal cartilage and in vitro cultures were studied through time‐lapse video microscopy (TLVM), immunohistochemical staining and cytometry. In order to investigate the effect of BC and CS on phenotype maintainance, chondrogenic gene expression of aggrecan (AGN), of the transcriptor factor SOX9, of the types I and II collagen (COL1A1 and COL1A2), were quantified through transcriptional and protein evaluation at increasing cultivation time and passages. In addition to resemble the osteoarthritis‐like in vitro model, chondrocytes were treated with IL‐1β and the anti‐inflammatory activity of BC and CS was assessed using cytokines quantification by multiplex array. BC significantly enhances cell proliferation also preserving chondrocyte phenotype increasing type II collagen expression up to 10 days of treatment and reduces inflammatory response in IL‐1β treated chondrocytes respect to CS treated cells. Our results, taken together, suggest that this new BC is of foremost importance in translational medicine because it can be applied in novel scaffolds and pharmaceutical preparations aiming at cartilage pathology treatments such as the osteoarthritis. J. Cell. Biochem. 117: 2158–2169, 2016. © 2016 The Authors. Journal of Cellular Biochemistry Published by Wiley Periodicals, Inc.

Shear stresses modulate chondrogenic and osteogenic genes expression in human bone marrow derived stem cells

Background: Mechanical stimuli in the microenvironment may provide cues for differentiation, maintenance of phenotypes, and extracellular matrix homeostasis. This study investigated bone marrow-derived stem cells (BMSCs) response to mechanical stresses in terms of differentiation and the extracellular matrix/matrix metalloproteinase genes expression. Materials & Methods: Human BMSCs were exposed to shear stresses (20 rpm or 50 rpm) for 2, 5, or 10 days with or without osteogenic induction medium. The expression of chondrogenic (type II collagen and aggrecan) and osteogenic (type I collagen, alkaline phosphatase, osteopontin, osteocalcin) phenotypic genes and matrix metalloproteinase genes (aggrecanse, MMP-2, and MMP-9) were analyzed by reverse transcription polymerase chain reaction. In vitro differentiation of BMSCs after shear stress treatment were analyzed by von Kossa staining on monolayer cultures and safarin-O staining on pelleted cells. Results: Shear stress at 20 rpm significantly upregulatedexpression of type II collagen on the 5th day, type I collagen on the 2nd, 5th, and 10th day, osteopontin on the 2nd and 5th day, and osteocalcin on the 5th day as compared to shear stress at 50 rpm. Combination of osteogenic induction medium and shear stress at 20 rpm decreased the mRNA expression levels of chondrogenic genes (type II collagen on all time points and aggrecan on the 5th and 10th day) as compared with shear stress alone. It also decreased the expression of osteogenic genes (type I collagen, alkaline phosphatase, and osteopontin) on the 10th day as compared to osteogenic induction medium. Concomitantly, shear stress upregulated aggrecanase on the 10th day, MMP-2 on the 10th day, and MMP-9 on the 5th and 10th day as compared with osteogenic induction medium. Shear stress increased mineralization of the extracellular matrix as shown by von Kossa staining on the 2nd, 5th, and 10th day. Chondrogenic differentiation by cell pellet analysis found similar amount of proteoglycans but less organized extracellular matrix in shear stress-treated BMSCs as compared to control cells. Discussion: Although shear stress could modulate chondrogenic gene expression as well as osteogenic genes expression in BMSCs, the combination of shear stress inhibited the expression of osteogenic phenotypic genes induced by osteogenic induction medium. Parallel studies revealed that shear stress concomitantly modulate the expression of extracellular matrix genes and the matrix metalloproteinase genes. In vitro differentiation analysis found that shear stress may enhance osteogenesis but interfere with chondrogenesis. Our results may provide data for the design or development of in vitro bioreactor systems to control lineage differentiation of stem cells for tissue engineering. More studies on the mechanotransduction pathways are needed in the future.

Influence of acidity microenvironment of degenerative intervertebral disc on nucleus pulpous - derived mesenchymal stem cells and adipose- derived mesenchymal stem cells

Objective To compare the viability, proliferation and matrix metabolism of nucleus pulpous-derived mesenchymal stem cells (NPMSCs) compared with adipose-derived mesenchymal stem cells (ADMSCs) under intervertebral disc (IVD)like acidic conditions. Methods In our vivo experiment, ADMSCs and NPMSCs from Sprague Dawley rats were cultured under four different pH levels representing the standard condition (pH 7.4) and the normal, mildly degenerated and severely degenerated IVD (pH 7.1, 6.8 and 6.5, separately). By using annexin-V-FITC/propidium iodide staining and a CCK-8 assay, we examined cell viability and cell proliferation respectively. The expression of aggrecan, collagen-Ⅰ , collagen-Ⅱ, MMP-2, ADAMTS4 and TIMP-3 at mRNA and protein levels were measured by RT-PCR and Western blotting. Results Flow cytometry and CCK-8 assays revealed that the viability and proliferation of ADMSCs and NPMSCs decreased with increasing acidity from pH 7.4 to 6.5. Compared with ADMSCs, NPMSCs had significantly fewer apoptotic and necrotic cells under acidic conditions (P<0.05). Cell proliferation of NPMSCs was significantly greater than that of ADMSCs under IVD-like acidic conditions (P<0.05). RT-PCR and western blotting indicated that, in both cell types with increasing acidity, the expression of aggrecan, collagen-Ⅰ , collagen-Ⅱ and TIMP-3 at mRNA and protein levels decreased, while that of MMP-2 and ADAMTS4 increased. Compared with ADMSCs, NPMSCs expressed significantly higher levels of aggrecan and collagen-Ⅱ, and lower levels of collagen-Ⅰ , MMP-2 and ADAMTS4. Conclusion An acidic environment can significantly restrain the IVD regeneration by ADMSCs or NPMSCs. NPMSCs appeared less sensitive to inhibition by acidic pH, and might be promising candidates for cell-based IVD regeneration. Key words: Intervertebral disc degeneration; Mesenchymal stem cells; Cell proliferation

Change and Significance of RhoA/ROCK signaling pathway in the model with natural degeneration of the rat endplate chondrocytes

To explore the change and Significance of RhoA/ROCK signaling pathway in the model with natural degeneration of the rat endplate chondrocytes. Endplate chondrocytes were selected by enzyme digestion and cultured in vitro to divided into control (P2 cells), naturally passaged (P5 cells) groups and treatment group (P5+ROCK Inhibitor Y27632). The phenotype of endplate chondrocytes were identified by toluidine blue stains and F-actin stains. Type II collagen, aggrecan and SOX9 genes were examed by Real-time RT-PCR to verify the degeneration model. The RhoA/ROCK signaling pathway related gene ROCK-1, ROCK-2 were detected by RT-PCR and Western blot. The actived RhoA was examed by active-RhoA detection and Western blot. With the passaging,endplate chondrocytes completely lost the original cell morphology, the levels of type II collagen (P5/P2=0.248, P<0.001), aggrecan (P5/P2=0.172, P<0.001) and SOX9 (P5/P2 =0.499, P<0.001) significantly reduced. There is also a certain reduction of ROCK-1 (P5/P2=0.652, P<0.001), but ROCK-2 (P5/P2=2.527, P<0.001) expression increased significantly. And the active-RhoA were Significant increased too.ROCK-1 AND ROCK-2 were down-regulated in the treatment group. And type II collagen, aggrecan, SOX9 significantly increased. The degeneration of endplate chondrocytes with decreased ROCK-1 expression but increased active-RhoA and ROCK-2 expression suggest that RhoA/ROCK signaling pathway play an important role in the in vitro degeneration of endplate chondrocytes.Modulating the expression of RhoA/ROCK signaling pathway may be a new method of solving the problem of the degeneration of intervertebral disc.

Effects of Ghrelin on metabolic activity of equine cartilage affected with inflammatory degeneration: preliminary isolation, characterization of equine cultured chondrocytes and assessment of LPS-induced inflammatory stress.

Ghrelin, the natural ligand for GH secretagogue receptors, influences the activity of both human and rat joint chondrocytes, modulating the synthesis of eicosanoids in the cartilage 1 . This project aims to assess the possible protective effects of Ghrelin on equine cultured chondrocytes exposed to lipopolysaccharide (LPS), as a model of osteoarthritic joint. In the preliminary step of our study, we have characterized equine chondrocyte primary cell cultures and standardized the detrimental effects LPS on chondrocytes health. Equine articular cartilage was collected with aseptic procedure and equine chondrocytes were isolated by overnight incubation. Cells were cultured in monolayer culture to obtain third-passage (P3) chondrocytes and specific cartilaginous genes were identified by qualitative PCR. P3 chondrocytes were treated with increasing LPS concentrations (1-100 ng/ml) and the effects of LPS were assessed with the apoptosis-induction test and fluorescence microscopy after 1, 3, 6, 12, 24, 48 h of treatment. PCR evidenced type II collagenases and aggrecan expression in isolated cells confirming the isolation of chondrocytes. Healthy chondrocytes (HC) showed no fluorescence, necrotic chondrocytes (NC) red fluorescence, early apoptotic chondrocytes (EAC) green fluorescence and advanced apoptotic chondrocytes (AAC) mixed green-red fluorescence. In LPS-stimulated chondrocytes, there was a trend throughout an increased percentage of AAC that became equal to or overwhelmed the percentage of HC after 3 hours for all LPS treatments and lasted for 6 hours after the highest LPS concentration used. On the basis of these results, the concentration of LPS 100 ng/ml for 6 h will be used for subsequent experiments.

Directing Chondrogenesis of Primary Chondrocytes by Exposure to Glucose Concentrations

Since articular cartilage is avascular, both nutrient supply and metabolic waste excretion depend on diffusion. However, the major cause of the progression of articular cartilage defect is the poor inherent regenerative capacity of chondrocytes which limits the process of cartilage tissue repair. Creation of nutrient gradients in in vitro cell culture, however, can provide a clue on zonal distributions of cells and glycosaminoglycan synthesis throughout the tissue engineered cartilage. We hypothesized that glucose gradient, in combination with growth factors, could induce differences in matrix distributions for articular cartilage regeneration. Chondrocytes were harvested from bovine cartilage and expanded in monolayers. First, either p0 or p2 chondrocytes were differentiated in serum-free chondrogenic medium containing different glucose concentrations supplemented with TGFβ3/dex or IGF-1under hypoxic or normoxic conditions for 7 days in monolayer. The results indicate that cellular metabolism, cell numbers and glycosaminoglycan (GAG) content increased with increase in glucose concentration in all conditions. Aggrecan (AGC) expression consistently increased with decreasing glucose concentration in both normoxic and hypoxic conditions. COL II and COL I expressions increased with increasing glucose concentration up to 5mmol/L. The expression of COMP increased with increasing glucose concentration under hypoxic conditions and interestingly showed an opposite trend under normoxic conditions. However, comparing the chondrogenic capacity of p0 and p2 cells in the different glucose concentrations did not show differences, but the potential of p2 cells was in general lower compared to p0. Hypoxia had stimulatory effects on matrix production compared to normoxia in both passages. Therefore, supplemented glucose concentration in monolayer could induce differences in matrix production, but the chondrogenic potential remained equal. Therefore, this information could be use to a create gradients through a tissue-engineered cartilage.

Effect of autologous platelet-rich plasma on the chondrogenic differentiation of rabbit adipose-derived stem cells in vitro.

This study aimed to isolate rabbit adipose-derived stem cells (ADSCs) and explore the potential of platelet-rich plasma (PRP) in the chondrogenic differentiation of ADSCs, thereby potentially providing a new approach for the repair and regeneration of cartilage injury. Rabbit ADSCs were isolated and characterized by induction towards adipogenic, osteogenic and chondrogenic lineages in vitro. The isolated ADSCs were also cultured with or without 10% PRP. Immunofluorescence staining, toluidine blue staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR) were used to detect type II collagen (Col II) and aggrecan (AGC) expression. Col II immunofluorescence staining and toluidine blue staining indicated that following induction by autologous PRP, ADSCs manifested Col II and AGC expression. The expression of Col II and AGC mRNA was significantly upregulated in the PRP-treated cells when compared with that in control cells. Autologous PRP produced by laboratory centrifugation was able to promote the chondrogenic differentiation of rabbit ADSCs in vitro.

Influence of chondrocytes of different origin on early differentiation of bone marrow mesenchymal stem cells in co-culture system

Objective To investigate the influence of chondrocytes originating from different source on early chondrogenic differentiation of bone marrow mesenchymal stem cells (MSCs) in isolated co-culture system. Methods We applied hanging cell-culture system to culture chondrocytes of different origin (osteoarthritis chondrocyte cells, nomal chondrocyte cells, infant chondrocyte cells) and controls. These chondrocytes and MSCs of the same origin were cultured in the common medium in a separated condition, and observed by microscope at 3, 6, 9, 12 day after co-culture. Expression levels of aggrecan, collagen type Ⅱ (Col-2), cartilage-specific transcription factor (Sox-9) in MSCs of different origin were determined by Real-time PCR. Results MSCs showed obviously morphological differentiation induced by chondrocytes of different origin at 12 day after co-culture as compared with controls. Real-time PCR analysis showed that SOX9 mRNA level was stimulated by 1.7-fold, 1.6-fold and 1.2-fold (all P<0.05) and aggrecan mRNA level was increased by 2.8-fold, 2.2-fold and 1.3-fold (all P<0.05) in infant chondrocytes group, nomal chondrocytes group, osteoarthritis chondrocytes group respectively as compared with controls while COL2 mRNA level had no significant differences among the four groups. Corresponding protein signal level had obvious differences among the four groups, especially in infant chondrocytes as compared with osteoarthritis chondrocytes and nomal chondrocytes. Conclusions Isolated co-culture system may indirectly promote MSCs differentiation to chondrocytes by local micro-environment regulation. Chondrocytes of different origin have different effects on MSCs differentiation, but they could promote MSCs differentiation to chondrocytes. Key words: Chondrocytes; Cell differentiation; Coculture techniques; Mesenchymal stem cells