Retention Of Glycosaminoglycans Research Articles

STEM-01. DEVELOPMENT OF A HUMAN-SPECIFIC 3DIN VITRO PAEDIATRIC CEREBELLAR TUMOUR MODEL USING DECELLULARISED BRAIN AUTOPSY TISSUE

Abstract BACKGROUND Medulloblastoma (MB) and Atypical Teratoid/ Rhabdoid Tumours (ATRT) represent paediatric cerebellar tumours with a dismal prognosis. For ATRT in particular, 5-year survival remains at less than 32%. We have developed a 3D in vitro model termed ‘tumoursphere matrix’ which recapitulates in vivo crosstalk between tumour and reactive brain. METHODS AND RESULTS We co-cultured D283 (Group 3-MYC amplified MB) and BT12 (ATRT-MYC) cells with primary human cerebellar astrocytes within a PEGDA hydrogel containing decellularised cerebellar extracellular matrix (ECM) derived from non-disease human autopsy brain. Cerebellar ECM was extensively characterised, showing a significant reduction in cellular material whilst retaining ECM ligands. Reduction of DNA content to 174 ng/mg was corroborated by an absence of nuclei in immunohistochemical staining. Colorimetric assays indicated collagen and glycosaminoglycan retention in decellularised ECM, and Immunofluorescence confirmed retention of fibronectin, laminin and hyaluronic acid. Detection of all ECM ligands was cross-validated by Orbitrap-Secondary Ion Mass Spectrometry. A solubilised ECM/PEGDA hydrogel was used to coat an AggreWell™ plate. Primary human cerebellar astrocytes, D283 and BT12 cells were cultured as spheroids within the ECM/PEGDA hydrogel for 7 days with no significant decrease in cellular viability. Cytokine and Human Matrix Metalloproteinase (MMP) Antibody Arrays showed cytokine and MMP expression in all three cell lines was retained in the presence of ECM material. Furthermore, D283 and BT12 cells were co-cultured with primary astrocytes for 7 days prior to separation using fluorescence activated cell sorting to generate individual cell populations. CONCLUSIONS We have developed a 3D in vitro model which can co-culture cells for over 7 days, whilst recapitulating the in vivo tumour environment. RNA sequencing is currently in progress to identify differentially expressed genes in cerebellar tumour cells upon astrocytic and brain ECM crosstalk. We hypothesise that biochemical signalling underlying this communication will reveal putative therapeutic vulnerabilities.

Immunomodulatory potential of mesenchymal stromal cell-derived extracellular vesicles in chondrocyte inflammation.

Osteoarthritis (OA) affects a large percentage of the population worldwide. Current surgical and nonsurgical concepts for treating OA only result in symptom-modifying effects. However, there is no disease-modifying therapy available. Extracellular vesicles released by mesenchymal stem/stromal cells (MSC-EV) are promising agents to positively influence joint homeostasis in the osteoarthritic surroundings. This pilot study aimed to investigate the effect of characterized MSC-EVs on chondrogenesis in a 3D chondrocyte inflammation model with the pro-inflammatory cytokine TNFα. Bovine articular chondrocytes were expanded and transferred into pellet culture at passage 3. TNFα, human MSC-EV preparations (MSC-EV batches 41.5-EVi1 and 84-EVi), EVs from human platelet lysate (hPL4-EV), or the combination of TNFα and EVs were supplemented. To assess the effect of MSC-EVs in the chondrocyte inflammation model after 14 days, DNA, glycosaminoglycan (GAG), total collagen, IL-6, and NO release were quantified, and gene expression of anabolic (COL-II, aggrecan, COMP, and PRG-4), catabolic (MMP-3, MMP-13, ADAMTS-4 and ADAMTS-5), dedifferentiation (COL-I), hypertrophy (COL-X, VEGF), and inflammatory (IL-8) markers were analyzed; histological evaluation was performed using safranin O/Fast Green staining and immunohistochemistry of COL I and II. For statistical evaluation, nonparametric tests were chosen with a significance level of p < 0.05. TNFα supplementation resulted in catabolic stimulation with increased levels of NO and IL-6, upregulation of catabolic gene expression, and downregulation of anabolic markers. These findings were supported by a decrease in matrix differentiation (COL-II). Supplementation of EVs resulted in an upregulation of the chondrogenic marker PRG-4. All MSC-EV preparations significantly increased GAG retention per pellet. In contrast, catabolic markers and IL-8 expression were upregulated by 41.5-EVi1. Regarding protein levels, IL-6 and NO release were increased by 41.5-EVi1. Histologic and immunohistochemical evaluations indicated a higher differentiation potential of chondrocytes treated with 84-EVi. MSC-EVs can positively influence chondrocyte matrix production in pro-inflammatory surroundings, but can also stimulate inflammation. In this study MSC-EV 41.5-EVi1 supplementation increased chondrocyte inflammation, whereas MSC-84-EVi supplementation resulted a higher chondrogenic potential of chondrocytes in 3D pellet culture. In summary, the selected MSC-EVs exhibited promising chondrogenic effects indicating their significant potential for the treatment of OA; however, the functional heterogeneity in MSC-EV preparations has to be solved.

S-ALLYL-L-CYSTEINE-INDUCED ANTI-INFLAMMATORY AND ANTI-APOPTOTIC EFFECTS IN CHONDROCYTES IS ASSOCIATED WITH SUPPRESSION OF THE MITOCHONDRIAL INFLAMMATION PATHWAY

One major aspects to consider while dealing with osteoarthritis is oxidative stress. This deleterious oxidative stress is responsible for the increased production of Reactive Oxygen Species and triggers several inflammatory pathways, including Mitochondrial Inflammation Pathway (MIP), which leads the cell to apoptosis. Chondrocytes, under oxidative stress, are unable to synthesize cartilage efficiently. S-Allyl-L-Cysteine (SAC) is known to be a potent natural, water-soluble antioxidant derived from garlic whose antioxidant properties have been evaluated in several diseases at the molecular level; other than osteoarthritis. Herein, we investigated the potential of S-Allyl-L-Cysteine (SAC) preconditioning of chondrocytes against oxidative stress-mediated mitochondrial inflammation. SAC priming alleviated oxidative stress-induced injuries by significantly improved cell viability, morphology and activated cell migration. In addition, decreased lactate dehydrogenease, increased superoxide dismutase release and retention of glycosaminoglycans were observed.SAC preconditioning ameliorated the injurious effects of oxidative stress as revealed by significant downregulation in gene expression of hypoxia-induciblefactor 1α (Hif-1α), Xanthine Oxidase (XO), Caspase-9 (Casp-9), Caspase-3(Casp-3), Interleukin 1 beta (IL-1β) and inducible nitric oxide synthase (iNOS). These findings suggest that SAC preconditioning might enhance the antioxidant and anti-inflammatory efficacy of chondrocytes by regulating the MIP pathway and improving cellular responses.

Characterization of mesenchymal stem cells and fibrochondrocytes in three-dimensional co-culture: analysis of cell shape, matrix production, and mechanical performance.

BackgroundBone marrow mesenchymal stem cells (MSCs) have shown positive therapeutic effects for meniscus regeneration and repair. Preliminary in vitro work has indicated positive results for MSC applications for meniscus tissue engineering; however, more information is needed on how to direct MSC behavior. The objective of this study was to examine the effect of MSC co-culture with primary meniscal fibrochondrocytes (FCCs) in a three-dimensional collagen scaffold in fibrochondrogenic media. Co-culture of MSCs and FCCs was hypothesized to facilitate the transition of MSCs to a FCC cell phenotype as measured by matrix secretion and morphology.MethodsMSCs and FCCs were isolated from bovine bone marrow and meniscus, respectively. Cells were seeded in a 20 mg/mL high-density type I collagen gel at MSC:FCC ratios of 0:100, 25:75, 50:50, 75:25, and 100:0. Constructs were cultured for up to 2 weeks and then analyzed for cell morphology, glycosaminoglycan content, collagen content, and production of collagen type I, II, and X.ResultsCells were homogeneously mixed throughout the scaffold and cells had limited direct cell–cell contact. After 2 weeks in culture, MSCs transitioned from a spindle-like morphology toward a rounded phenotype, while FCCs remained rounded throughout culture. Although MSC shape changed with culture, the overall size was significantly larger than FCCs throughout culture. While 75:25 and 100:0 (MSC mono-culture) culture groups produced significantly more glycosaminoglycan (GAG)/DNA than FCCs in mono-culture, GAG retention was highest in 50:50 co-cultures. Similarly, the aggregate modulus was highest in 100:0 and 50:50 co-cultures. All samples contained both collagen types I and II after 2 weeks, and collagen type X expression was evident only in MSC mono-culture gels.ConclusionsMSCs shift to a FCC morphology in both mono- and co-culture. Co-culture reduced hypertrophy by MSCs, indicated by collagen type X. This study shows that MSC phenotype can be influenced by indirect homogeneous cell culture in a three-dimensional gel, demonstrating the applicability of MSCs in meniscus tissue engineering applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13287-016-0301-8) contains supplementary material, which is available to authorized users.

Glycosaminoglycan entrapment by fibrin in engineered heart valve tissues

Tissue engineered heart valves (TEHVs) may provide a permanent solution to congenital heart valve disease by permitting somatic valve growth in the pediatric patient. However, to date, TEHV studies have focused primarily on collagen, the dominant component of valve extracellular matrix (ECM). Temporal decreases in other ECM components, such as the glycosaminoglycans (GAGs), generally decrease as cells produce more collagen under mechanically loaded states; nevertheless, GAGs represent a key component of the valve ECM, providing structural stability and hydration to the leaflets. In an effort to retain GAGs within the engineered constructs, here we investigated the utility of the protein fibrin in combination with a valve-like, cyclic flexure and steady flow (flex–flow) mechanical conditioning culture process using adult human periodontal ligament cells (PLCs). We found both fibrin and flex–flow mechanical components to be independently significant (p<0.05), and hence important in influencing the DNA, GAG and collagen contents of the engineered tissues. In addition, the interaction of fibrin with flex–flow was found to be significant in the case of collagen; specifically, the combination of these environments promoted PLC collagen production resulting in a significant difference compared to dynamic and statically cultured specimens without fibrin. Histological examination revealed that the GAGs were retained by fibrin entrapment and adhesion, which were subsequently confirmed by additional experiments on native valve tissues. We conclude that fibrin in the flex–flow culture of engineered heart valve tissues: (i) augments PLC-derived collagen production; and (ii) enhances retention of GAGs within the developing ECM.

Chemical modification of collagen improves glycosaminoglycan retention of their co-precipitates

Being prevalent extracellular matrix components, collagen and glycosaminoglycan (GAG) are co-precipitated as scaffolds for tissue regeneration. However, the amount of GAG incorporated and its long-term retention present a persistent problem. In this study, chemical modifications, namely deamination, methylation and amination, were used to alter the net charge of collagen prior to fabrication of collagen–GAG co-precipitate. While most GAGs were lost in the untreated group and the deaminated group within 1day, methylation and amination of collagen retained over 20% and 40% GAG after 6days, respectively. Moreover, over 60% of GAG retention was achieved in the aminated group after cell seeding for 8days. Furthermore, amination of collagen increased the GAG/hydroxyproline ratio in the co-precipitate to >4.5, approaching that of native nucleus pulposus. Ultrastructural analysis showed that the aminated group contains abundant granular substances resembling the extracellular matrix of native nucleus pulposus. Despite lower initial cell adhesion than untreated, all modified scaffolds promoted proliferation of human mesenchymal stem cells (hMSCs) and showed >95% cell viability at all time points. Cell morphology was distinct among the different groups, being round in the untreated control and methylated groups but elongated in deaminated and aminated groups. hMSCs adhered to scaffolds via collagen receptor integrin α2β1 in all groups, while all but the aminated group showed extensive expression of the general matrix receptor integrin αv. This work reports an effective method, namely amination of collagen, to improve GAG incorporation and retention in collagen–GAG co-precipitates, facilitating the fabrication of GAG-rich collagenous scaffold for intervertebral disc tissue engineering.

Identification of potential biophysical and molecular signalling mechanisms underlying hyaluronic acid enhancement of cartilage formation

This study determined the effects of exogenous hyaluronic acid (HA) on the biomechanical and biochemical properties of self-assembled bovine chondrocytes, and investigated biophysical and genetic mechanisms underlying these effects. The effects of HA commencement time, concentration, application duration and molecular weight were examined using histology, biomechanics and biochemistry. Additionally, the effects of HA application on sulphated glycosaminoglycan (GAG) retention were assessed. To investigate the influence of HA on gene expression, microarray analysis was conducted. HA treatment of developing neocartilage increased compressive stiffness onefold and increased sulphated GAG content by 35 per cent. These effects were dependent on HA molecular weight, concentration and application commencement time. Additionally, applying HA increased sulphated GAG retention within self-assembled neotissue. HA administration also upregulated 503 genes, including multiple genes associated with TGF-β1 signalling. Increased sulphated GAG retention indicated that HA could enhance compressive stiffness by increasing the osmotic pressure that negatively charged GAGs create. The gene expression data demonstrate that HA treatment differentially regulates genes related to TGF-β1 signalling, revealing a potential mechanism for altering matrix composition. These results illustrate the potential use of HA to improve cartilage regeneration efforts and better understand cartilage development.

Effects of Perfusion and Dynamic Loading on Human Neocartilage Formation in Alginate Hydrogels

Dynamic loading and perfusion culture environments alone are known to enhance cartilage extracellular matrix (ECM) production in dedifferentiated articular chondrocytes. In this study, we explored whether a combination of these factors would enhance these processes over a free-swelling (FS) condition using adult human articular chondrocytes embedded in 2% alginate. The alginate constructs were placed into a bioreactor for perfusion (P) only (100 μL/per minute) or perfusion and dynamic compressive loading (PL) culture (20% for 1 h, at 0.5 Hz), each day. Control FS alginate gels were maintained in six-well static culture. Gene expression analysis was conducted on days 7 and 14, while cell viability, immunostaining, and mechanical property testing were performed on day 14 only. Total glycosaminoglycan (GAG) content and GAG synthesis were assessed after 14 days. Col2a1 mRNA expression levels were significantly higher (at least threefold; p<0.05) in both bioreactor conditions compared with FS by days 7 and 14. For all gene studies, no significant differences were seen between P and PL treatments. Aggrecan mRNA levels were not significantly altered in any condition although both GAG/DNA and (35)S GAG incorporation studies indicated higher GAG retention and synthesis in the FS treatment. Collagen type II protein deposition was low in all samples, link protein distribution was more diffuse in FS condition, and aggrecan deposition was located in the outer regions of the alginate constructs in both bioreactor conditions, yet more uniformly in the FS condition. Catabolic gene expression (matrix metalloproteinase 3 [MMP3] and inducible nitric oxide synthase [iNOS]) was higher in bioreactor conditions compared with FS, although iNOS expression levels decreased to approximately fourfold less than the FS condition by day 14. Our data indicate that conditions created in the bioreactor enhanced both anabolic and catabolic responses, similar to other loading studies. Perfusion was sufficient alone to promote this dual response. PL increased the deposition of aggrecan surrounding cells compared with the other conditions; however, overall low GAG retention in the bioreactor system was likely due to both perfusion and catabolic conditions created. Optimal conditions, which permit appropriate anabolic and catabolic processes for accumulation of ECM and tissue remodeling for neocartilage development, specifically for humans, are needed.

In Vitro Evaluation of a Thermoreversible Hyaluronan-Based Hydrogel Loaded with Cell-Seeded Polyurethane Particles for Nucleus Pulposus Repair

Introduction Thermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells. Materials and Methods Human bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test ( p &lt; 0.05). Results In the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups ( Fig. 1a ). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials ( Fig. 1B ). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels. [Figure: see text] Conclusion Human disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Mortisen D, et al. Biomacromolecules 2010;11:1261–1272 Peroglio M, et al. European Spine Journal Epub ahead of print Mauth C, et al. European Cells and Material 2009;18:27–38

In Vitro Evaluation of a Thermoreversible Hyaluronan-Based Hydrogel Loaded with Cell-Seeded Polyurethane Particles for Nucleus Pulposus Repair

IntroductionThermoreversible hyaluronan (HA) based hydrogels represent a promising carrier for IVD repair because of the mild gel formation allowing for encapsulation of viable cells and biological stimulation of cells by HA.1,2 Additionally, polyurethane (PU) foams can provide elasticity, which is of key importance for the restoration of IVD mechanical function, as the latter experiences repeated loading during the daily human activities. Finally, the addition of relevant cells may promote a faster restoration of the IVD function. Therefore, the aim of this study was to compare the single and combined effect of PU particles and HA hydrogel on the proliferation and differentiation of human IVD and mesenchymal stem cells. Materials and MethodsHuman bone marrow stromal cells (hMSCs) or human disk cells (hIVD cells) (P2-P4) were seeded on PU particles; after 12 hours, the seeded particles were suspended in either a 12% w/v thermoreversible hyaluronan-poly(N-isopropylacrylamide) hydrogel (HA-pNIPAM)2 or in a fibrinogen/thrombin solution.3 Each sample contained 50 µL hydrogel, 2.5 mg PU particles, and 2.5 × 105 cells. Controls included hMSC (or hIVD cells) seeded HA-pNIPAM, hMSC (or hIVD cells) seeded fibrin, and hMSC (or hIVD cells) seeded PU. After gelling for 1 hour at 37°C, samples were cultured in chondrogenic medium (DMEM high glucose, 1% ITS, 130 µM ascorbic acid, 100 nM dexamethasone and 10 ng/mL TGF-β1) for one week and analyzed for DNA (by Picogreen), sulfated glycosaminoglycan (GAG) (by 1,9 dimethylmethylene blue), mRNA expression, and histology (toluidine blue) at day 3 and 7. Three independent experiments were performed in triplicates for each group and cell type. After confirming the normality of data distribution and exclusion of the outliers, data were analyzed with a univariate general linear model and post hoc Bonferroni test (p < 0.05). ResultsIn the MSC cultures, a slight DNA increase was observed between day 3 and 7 for both PU/HA-pNIPAM and PU/fibrin samples. At day 7, the highest amounts of DNA were found for the PU/fibrin group, followed by fibrin and PU groups (Fig. 1a). The amount of DNA was higher in the PU group than PU/HA-pNIPAM and HA-pNIPAM, suggesting a lower cell proliferation in the presence of the thermoreversible hydrogel. A similar DNA profile was obtained when hIVD cells were used instead of hMSCs. A significantly increased amount of GAG was observed in hMSC-seeded PU/HA-pNIPAM samples (Fig.1B) and there was an increase of GAG from day 3 to day 7. All other groups had a much lower amount of glycosaminoglycans and there was only a slight increase of GAG from day 3 to day 7. The GAG retention (GAG in sample divided by the sum of GAG in the sample and GAG released in the medium) at day 7 was comprised between 50% and 80% for all groups. Disk cells produced significantly less GAG in the PU/HA-pNIPAM system compared to hMSC, indicating that MSCs were more responsive to the combination of these two biomaterials (Fig. 1B). Moreover, the GAG retention at day 7 was lower with human IVD cells (20–60%) compared to hMSCs in all groups. This suggests that an early and higher GAG accumulation is associated to a better GAG retention in the hydrogel. This finding may be related to the fact that while MSCs were obtained from patients undergoing various types of surgeries, IVD cells were obtained from (degenerate) disks of patients undergoing total IVD replacement. The gene expression profile confirmed MSC chondrogenic differentiation with a strong upregulation of collagen type II and, to a lower extent, of aggrecan and SOX9 (compared to cells collected on the day of seeding) without significant differences between the two gels. Histological sections revealed cell migration from PU particles to the hydrogels.Figure 1 Mean and standard deviation of DNA (a) and sulfated glycosaminoglycans (GAG) (b) in HA-pNIPAM and fibrin, with and without the addition of PU particles after 1 week of culture; * p < 0.05 relative to PU/Fibrin, o p < 0.05 relative to fibrin. ConclusionHuman disk cells from degenerating disks synthesized less GAG than hMSCs, indicating that MSCs may represent a suitable cell type for the biological repair of IVDs. A synergic effect for MSC differentiation was observed when a thermoreversible hyaluronan-based hydrogel was combined with polyurethane particles.I confirm having declared any potential conflict of interest for all authors listed on this abstractYesDisclosure of InterestNone declaredMortisen D, et al. Biomacromolecules 2010;11:1261–1272Peroglio M, et al. European Spine Journal Epub ahead of printMauth C, et al. European Cells and Material 2009;18:27–38

Biodegradable Electrospun Scaffolds for Annulus Fibrosus Tissue Engineering: Effect of Scaffold Structure and Composition on Annulus Fibrosus Cells in Vitro

Introduction Lumbar disk herniation is the pathological condition for which spinal surgery is most often performed. One significant shortcoming is the high recurrence rate after conventional discectomy. This is partly due to the fact that existing methods for annulus fibrosus repair are not able to restore normal tissue structure and biomechanical function. A biological annular repair may improve the surgical outcome by providing an environment which mimics the AF tissue mechanics and structure. A cell-seeded membrane patch is likely to be a promising approach for AF repair, though the adequate properties of a suitable membrane patch have not been described yet. In this study, the effect of structural and material properties of poly(ester-urethane) (PU) and poly(?-caprolactone) (PCL) based membranes, processed by electrospinning technology, on AF cells cultured onto their surface was investigated. The aim was to assess the potential of these membranes to support the regeneration of damaged AF. Materials and Methods PU (Mn = 500,000 g/mol), produced as previously reported,1 and PCL (Mn = 80,000 g/mol) were processed into nonoriented and oriented electrospun scaffolds to mimic collagen fiber alignment of AF tissue. The scaffolds were evaluated using scanning electron microscopy; image analysis software was used to quantitatively analyze fiber diameter and orientation. The mechanical behavior was characterized by uniaxial tensile testing and compared to reported values for human outer AF lamella.2 AF cells were isolated from bovine tail disks, seeded onto the scaffolds (7.8 × 104 cells/cm2) and cultured in DMEM containing 10% FCS and 50 µg/mL ascorbic acid for up to 21 days. Spin-coated flat membranes were used as controls. Analyses included DNA, glycosaminoglycan (GAG), collagen content, histology, and collagen type I immunohistochemistry. Gene expression levels of collagen type I, collagen type II, aggrecan, elastin, biglycan, versican, and decorin were determined. Results Mean fiber diameters were 3.10 ± 1.49 µm and 2.17 ± 1.53 µm for non-oriented and oriented PCL, whereas 0.92 ± 0.36µm and 0.84 ± 0.32µm were measured for nonoriented and oriented PU. The failure stress of an AF lamella is 5.6 to 10.3 MPa.2 The oriented PU scaffold was able to withstand similar forces (4.65 ± 0.42MPa) before reaching the yield point and was the only material reaching the range of the failure strain (26.81 ± 1.16%) of a native AF lamella (9.2 to 12.7%). Despite this, the Young's modulus of oriented PU (17.96 ± 0.66MPa) remained below that of a single lamella of the AF (82–136MPa). Only the oriented PCL (46.00 ± 1.09MPa) came close to the values of the native AF. An increase in DNA was observed after 14 days relative to day 1 for all groups. At day 7, the DNA content was higher in electrospun PU than PCL scaffolds. After 14 and 21 days, there was an overall higher DNA amount in electrospun compared to spin-coated surfaces. In all groups, the amounts of GAG and collagen increased between day 7 and 21. GAG retention in the scaffolds was higher in electrospun scaffolds compared to flat control surfaces. GAG contents were increased in nonoriented compared to oriented scaffolds (Fig. 1). Moreover, by day 21 the collagen content was enhanced in the electrospun scaffolds compared to the spin-coated surfaces. Relative gene expression of elastin was upregulated, while collagen type II and aggrecan expression was downregulated by day 21 compared to day 0. The remaining genes were unchanged over the culture time. There was no difference in gene expression between the different membranes. In all scaffolds, deposition of extracellular matrix (ECM) could be observed by Toluidine blue staining and immunohistochemistry. Cell and matrix ingrowth was increased in PCL compared to PU scaffolds, which may be due to the higher porosity of PCL materials. Fig. 1. GAG content after 7, 14, and 21 days of AF cell culture on spin-coated PCL (PCL sc), spin-coated PU (PU sc), nonoriented PCL (PCL no es), nonoriented PU (PU no es), oriented PCL (PCL o es), and oriented PU (PU o es). GAG retained in scaffolds and released into medium is shown as * p &lt; 0.05 for total GAG. Mean ± SD. N = 5 experiments (triplicates each) Conclusion Both PU and PCL membranes supported AF cell growth and ECM production and accumulation. Electrospinning improved the retention of collagen and GAG in the scaffolds. Based on their mechanical characteristics oriented electrospun PU scaffolds may be appropriate materials for AF tissue engineering. Further studies with an improved 3D environment to increase cell ingrowth and to prevent cell de-differentiation will be required. I confirm having declared any potential conflict of interest for all authors listed on this abstract Yes Disclosure of Interest None declared Boissard CI, et al. Acta Biomater 2009;5:3316–3327 Skaggs DL, et al. Spine (Phila Pa 1976.) 1994;19:1310–1319

Strategies for Enhancing the Accumulation and Retention of Extracellular Matrix in Tissue-Engineered Cartilage Cultured in Bioreactors

Production of tissue-engineered cartilage involves the synthesis and accumulation of key constituents such as glycosaminoglycan (GAG) and collagen type II to form insoluble extracellular matrix (ECM). During cartilage culture, macromolecular components are released from nascent tissues into the medium, representing a significant waste of biosynthetic resources. This work was aimed at developing strategies for improving ECM retention in cartilage constructs and thus the quality of engineered tissues produced in bioreactors. Human chondrocytes seeded into polyglycolic acid (PGA) scaffolds were cultured in perfusion bioreactors for up to 5 weeks. Analysis of the size and integrity of proteoglycans in the constructs and medium showed that full-sized aggrecan was being stripped from the tissues without proteolytic degradation. Application of low (0.075 mL min−1) and gradually increasing (0.075–0.2 mL min−1) medium flow rates in the bioreactor resulted in the generation of larger constructs, a 4.0–4.4-fold increase in the percentage of GAG retained in the ECM, and a 4.8–5.2-fold increase in GAG concentration in the tissues compared with operation at 0.2 mL min−1. GAG retention was also improved by pre-culturing seeded scaffolds in flasks for 5 days prior to bioreactor culture. In contrast, GAG retention in PGA scaffolds infused with alginate hydrogel did not vary significantly with medium flow rate or pre-culture treatment. This work demonstrates that substantial improvements in cartilage quality can be achieved using scaffold and bioreactor culture strategies that specifically target and improve ECM retention.

Skeletal advance and arrest in giant non‐metamorphosing African clawed frog tadpoles (Xenopus laevis: Daudin)

This study examines the skeletons of giant non-metamorphosing (GNM) Xenopus laevis tadpoles, which arrest their development indefinitely before metamorphosis, and grow to excessively large sizes in the absence of detectable thyroid glands. Cartilage growth is isometric; however, chondrocyte size is smaller in GNM tadpoles than in controls. Most cartilages stain weakly with alcian blue, and several cartilages are calcified (unlike controls). However, cartilages subjacent to periosteum-derived bone retain strong affinities for alcian blue, indicating a role for periosteum-derived bone in the retention of glycosaminoglycans during protracted larval growth. Bone formation in the head, limb, and axial skeletons is advanced in comparison with stage-matched controls, but arrests at various mid-metamorphic states. Both dermal and periosteum-derived bones grow to disproportionately large sizes in comparison to controls. Additionally, mature monocuspid teeth form in several GNM tadpoles. Advances in skeletal development are attributable to the old ages and large sizes of these tadpoles, and reveal unexpected developmental potentials of the pre-metamorphic skeleton.

Isolation of a native osteoblast matrix with a specific affinity for BMP2

During their commitment and differentiation toward the osteoblast lineage, mesenchymal stem cells secrete a unique extracellular matrix (ECM) that contains large quantities of glycosaminoglycans (GAGs). Proteoglycans (PGs) are major structural and functional components of the ECM and are composed of a core protein to which one or more glycosaminoglycan sugar chains (GAGs) attach. The association of BMP2, a member of the TGF-beta super-family of growth factors, and a known heparin-binding protein, with GAGs has been implicated as playing a significant role in modulating the growth factor's in vitro bioactivity. Here we have characterised an osteoblast-derived matrix (MX) obtained from decellularised MC3T3-E1 cell monolayers for its structural attributes, using SEM and histology, and for its functional ability to maintain cell growth and viability. Using a combination of histology and anion exchange chromatography, we first confirmed the retention of GAGs within MX following the decellularisation process. Then the binding specificity of the retained GAG species within the MX for BMP2 was examined using a BMP2-HBP/EGFP (BMP2 Heparin-Binding Peptide/Enhanced Green Fluorescent Protein) fusion protein. The results of this study provide further evidence for a central role of the ECM in the regulation of BMP2 bioactivity, hence on mesenchymal stem cell commitment to the osteoblast lineage.

Effects of collagen type on the behavior of adult canine annulus fibrosus cells in collagen–glycosaminoglycan scaffolds

The healing of intervertebral disc defects may be improved by the implantation of cell-seeded collagen-based scaffolds. The present study evaluated in vitro the effects of the collagen type (type I vs type II) from which the scaffolds were fabricated on the behavior of adult canine annulus fibrosus cell-seeded collagen-glycosaminoglycan (GAG) scaffolds. Protein and GAG biosynthesis continued through the 8-week period of the experiment, demonstrating the viability of the cells in type I and type II collagen-GAG scaffolds. Statistical analysis revealed no significant effect of the type of collagen that makes up the scaffold on the biosynthetic activity. For both scaffold types, the amount of retained newly synthesized proteins increased from 1 to 2 weeks and from 6 to the 8 weeks. In contrast, the rate of GAG retention increased to a maximum at 4 weeks for both types of scaffolds, then decreased to about 50% of that level after 8 weeks. The number of cells generally increased the first week but then decreased in type I scaffolds while remaining constant in type II scaffolds. It could be assumed that most newly synthesized protein was lost to the medium, as the quantity of protein and collagen remained constant. Cell-mediated contraction of the scaffolds contributed to folds that formed in the constructs and to create an oriented architecture tissue. These findings commend the use of a type II collagen-GAG scaffold for further study on the basis of its maintenance of cell number and the slightly higher accumulated GAG content.

Inhibition of transmembrane calcium influx induces decrease in proteoglycan synthesis in immature rat sertoli cells

Beyond increased cAMP synthesis, calcium influx has been involved in signal transduction triggered by the gonadotropin follicle-stimulating hormone (FSH), the main regulator of Sertoli cells functions. In order to delineate a possible involvement of calcium in the regulation of proteoglycan synthesis, we have examined the effect of low-voltage-activated calcium channel blocker verapamil on both [(35)S]-sulfate and [(3)H]-glucosamine incorporation into proteoglycan molecules neosynthesized by cultured Sertoli cells from 20-day-old rats. Verapamil induced a dose- and time-dependent decrease in labeling of both secreted and cell-associated proteoglycans, as determined by quantitative solid-phase assay. This effect was mimicked by the addition of the calcium chelator EGTA, suggesting that verapamil effect resulted from the inhibition of transmembrane calcium influx. The decrease in apparent proteoglycan synthesis appeared to be attributable primarily to a lowering of the glycanation process, as shown by experiments using an exogenous acceptor for glycosaminoglycan synthesis. Moreover, verapamil induced a decrease in relative proportion of heparan sulfate proteoglycans in the cell layer. Pulse-chase kinetics demonstrated that verapamil also altered proteoglycan catabolism, leading to glycosaminoglycan retention in the cell layer and inhibiting the proteoglycan desulfation step. We conclude that intracellular calcium is essential to maintain Sertoli cell proteoglycan expression and could thus be involved in the repression of Sertoli cell cAMP-dependent syntheses such as estradiol production.

Effect of tenidap on cartilage integrity in vitro.

The maintenance of articular cartilage integrity during long term treatment with non-steroidal anti-inflammatory drugs (NSAIDs) is of clinical importance. These experiments were set up to test the action of tenidap, naproxen, and diclofenac on bovine and porcine cartilage, matrix synthesis, and catabolism. Short term organ culture techniques were used to determine the effect of interleukin 1 (IL-1) on synthesis and degradation, and the action of tenidap and the other drugs on these parameters. The retention of glycosaminoglycans (GAGs) and the synthesis of GAGs by incorporation of sulphur-35 labelled sulphate was used to determine the chondrocyte metabolic activity. The action of human recombinant interleukin 1 alpha (hrIL-1 alpha) in increasing catabolic activity and inhibiting synthetic activity of the animal cartilages was confirmed. Tenidap was shown to give substantial and significant protection against the catabolic effects of hrIL-1 alpha and, to a lesser degree, against the inhibition of matrix synthesis by the cytokine. Neither diclofenac nor naproxen in doses expected to occur in the synovial fluid showed this action. Tenidap also inhibited the GAG loss from cocultures and, to a moderate degree, reversed the inhibition of synthesis by synovial tissue. Tenidap also stimulated cartilage repair activity during recovery from IL-1 treatment. The optimum concentration of the action against IL-1 was between 5 and 10 micrograms/ml. Above this concentration tenidap itself showed some inhibitory action on GAG synthesis. Bearing in mind the problems in extrapolating from in vitro work on animal cartilages to humans, it seems possible that tenidap may be useful in decreasing the deleterious action of cytokines such as IL-1 on cartilage integrity during arthritic disease and in stimulating chondrocyte repair processes.

The retention and ultrastructural appearances of various extracellular matrix molecules incorporated into three-dimensional hydrated collagen lattices

Artificial extracellular matrices composed of collagen, glycosaminoglycans (GAG), proteoglycans (PG), plasma fibronectin (FN), and a hyaluronate-binding protein (HABP) have been prepared that morphologically resemble embryonic extracellular matrices in vivo at the light and electron microscope level. The effect of each of the above matrix molecules on the structure and “self-assembly” of these artificial matrices was delineated. (1) Matrix components assembled in vitro morphologically resemble their counterparts in vivo, for the most part. Scanning and transmission electron microscopy indicate that under our assembly and fixation conditions, collagen forms striated fibrils that are 125 nm in diameter, FN forms 30- to 60-nm granules, chondroitin sulfate proteoglycan (CSPG) forms 27- to 37-nm granules, chondroitin sulfate (CS) assembles into 100- to 250-nm spheres, and hyaluronate (HA) appears either as granular mats when fixed with cetylpyridinium chloride (CPC) or as 1.5- to 3-nm microfibrils when preserved with ruthenium red plus tannic acid. These molecules are known to assume the same configurations in embryonic matrices when the same preservation techniques are used with the exception of FN, which generally forms fibrillar arrays. (2) Addition of various matrix molecules can radically change the appearance of the collagen gels. HA greatly expands the volume of the gel and increases the space between collagen fibrils. CSPG at low concentrations (<1 mg/ml) and CS at high concentrations (>20 mg/ml) bundle the collagen fibrils into twisted ropes. (3) A variety of assays were used to examine binding between various matrix components and retention of these components in the hydrated collagen lattices. These assays included solid-phase binding assays, negative staining of spread mixtures of matrix components, cryostat sections of unfixed mixtures of matrix components, and retention of radiolabeled matrix molecules in fixed and washed gels. A number of these binding interactions may play a role in the assembly and stabilization of the matrix. (a) HA, CSPG, and FN bind to collagen. CS appears to only weakly bind to collagen, if at all. (b) FN promotes the increased retention of HA, CSPG, and to a very small degree, CS, in collagen gels. Conversely, the GAG increase the retention of 3H-FN in the gels. Furthermore, FN binds to HA, CS, and CSPG as demonstrated by solid surface binding assays and morphological criteria. The increased retention of GAG and CSPG by the addition of FN may be due to both stabilization of binding to the collagen and trapping of matrix complexes within the gel. (c) HA binds to both CS and CSPG. Its association with CSPG monomers produces the typical “bead-on-a-string” appearance that has been well characterized in vivo. HA association with CS suggests yet another way that matrix can be stabilized. (d) A 70-kDa protein that has been shown to bind to both HA and collagen increases the retention of HA in the collagen gels and promotes HA fibril formation. Fibronectin, GAG, and CSPG spontaneously organize in the collagen matrices in a manner that is consistent both with binding characteristics that have been previously published and those that are reported here.