Cx43 regulates mechanotransduction mechanisms in human preterm amniotic membrane defects.

OBJECTIVE: The effects of mechanical stimulation in preterm amniotic membrane (AM) defects were explored. METHODS: Preterm AM was collected from women undergoing planned preterm caesarean section (CS) due to fetal growth restriction or emergency CS after spontaneous preterm prelabour rupture of the membranes (sPPROM). AM explants near the cervix or placenta were subjected to trauma and/or mechanical stimulation with the Cx43 antisense. Markers for nuclear morphology (DAPI), myofibroblasts (αSMA), migration (Cx43), inflammation (PGE2 ) and repair (collagen, elastin and transforming growth factor ß [TGFß1 ]) were examined by confocal microscopy, second harmonic generation, qPCR and biochemical assays. RESULTS: In preterm AM defects, myofibroblast nuclei were highly deformed and contractile and expressed αSMA and Cx43. Mechanical stimulation increased collagen fibre polarisation and the effects on matrix markers were dependent on tissue region, disease state, gestational age and the number of fetuses. PGE2 levels were broadly similar but reduced after co-treatment with Cx43 antisense in late sPPROM AM defects. TGFß1 and Cx43 gene expression were significantly increased after trauma and mechanical stimulation but this response dependent on gestational age. CONCLUSION: Mechanical stimulation affects Cx43 signalling and cell/collagen mechanics in preterm AM defects. Establishing how Cx43 regulates mechanosignalling could be an approach to repair tissue integrity after trauma.

Machine learning and disease prediction in obstetrics.

Machine learning technologies and translation of artificial intelligence tools to enhance the patient experience are changing obstetric and maternity care. An increasing number of predictive tools have been developed with data sourced from electronic health records, diagnostic imaging and digital devices. In this review, we explore the latest tools of machine learning, the algorithms to establish prediction models and the challenges to assess fetal well-being, predict and diagnose obstetric diseases such as gestational diabetes, pre-eclampsia, preterm birth and fetal growth restriction. We discuss the rapid growth of machine learning approaches and intelligent tools for automated diagnostic imaging of fetal anomalies and to asses fetoplacental and cervix function using ultrasound and magnetic resonance imaging. In prenatal diagnosis, we discuss intelligent tools for magnetic resonance imaging sequencing of the fetus, placenta and cervix to reduce the risk of preterm birth. Finally, the use of machine learning to improve safety standards in intrapartum care and early detection of complications will be discussed. The demand for technologies to enhance diagnosis and treatment in obstetrics and maternity should improve frameworks for patient safety and enhance clinical practice.

Cx43 mediates changes in myofibroblast contraction and collagen release in human amniotic membrane defects after trauma.

The wound healing capacity of the fetal membranes after spontaneous or iatrogenic membrane rupture is unclear. We examined the healing mechanisms in amniotic membrane (AM) defects after trauma. Traumatised human AM defects were cultured for 4 days. Markers for nuclear (DAPI), cell type (vimentin, αSMA) and healing (Cx43, TGFß1, collagen) were examined by immunofluorescence (IMF) confocal microscopy, Second Harmonic Generation (SHG) imaging and RT-qPCR. After trauma, AMCs and myofibroblasts migrated to the AM wound edge. Within four days, αSMA expressing myofibroblasts showed abundant Cx43 localized in the cytoplasmic processes. The highly contractile spindle-shaped myofibroblasts were present in the defect site and released collagen. In contrast, AMCs expressed vimentin and formed Cx43 plaques between cells found in the outer edges of the wound. Whilst AMCs were absent in the defect site, αSMA expressing myofibroblasts continued to elongate and polarize the collagen fibres. Both TGFß1 and Cx43 gene expression were significantly increased after trauma. Cx43 has differential effects on AM cell populations that increase cellularity, contraction and potentially migration to the wound edge resulting in collagen polarisation in the AM defect site. Establishing how Cx43 regulates AM cell function could be an approach to repair defects in the membranes after trauma.

The Mechanical Interplay Between Differentiating Mesenchymal Stem Cells and Gelatin-Based Substrates Measured by Atomic Force Microscopy.

Traditional methods to assess hMSCs differentiation typically require long-term culture until cells show marked expression of histological markers such as lipid accumulation inside the cytoplasm or mineral deposition onto the surrounding matrix. In parallel, stem cell differentiation has been shown to involve the reorganization of the cell's cytoskeleton shortly after differentiation induced by soluble factors. Given the cytoskeleton's role in determining the mechanical properties of adherent cells, the mechanical characterization of stem cells could thus be a potential tool to assess cellular commitment at much earlier time points. In this study, we measured the mechanical properties of hMSCs cultured on soft gelatin-based hydrogels at multiple time points after differentiation induction toward adipogenic or osteogenic lineages. Our results show that the mechanical properties of cells (stiffness and viscosity) and the organization of the actin cytoskeleton are highly correlated with lineage commitment. Most importantly, we also found that the mechanical properties and the topography of the gelatin substrate in the vicinity of the cells are also altered as differentiation progresses toward the osteogenic lineage, but not on the adipogenic case. Together, these results confirm the biophysical changes associated with stem cell differentiation and suggest a mechanical interplay between the differentiating stem cells and their surrounding extracellular matrix.

Potential sealing and repair of human FM defects after trauma with peptide amphiphiles and Cx43 antisense.

OBJECTIVE: We examined whether peptide amphiphiles functionalised with adhesive, migratory or regenerative sequences could be combined with amniotic fluid (AF) to form plugs that repair fetal membrane (FM) defects after trauma and co-culture with connexin 43 (Cx43) antisense. METHODS: We assessed interactions between peptide amphiphiles and AF and examined the plugs in FM defects after trauma and co-culture with the Cx43antisense. RESULTS: Confocal microscopy confirmed directed self-assembly of peptide amphiphiles with AF to form a plug within minutes, with good mechanical properties. SEM of the plug revealed a multi-layered, nanofibrous network that sealed the FM defect after trauma. Co-culture of the FM defect with Cx43 antisense and plug increased collagen levels but reduced GAG. Culture of the FM defect with peptide amphiphiles incorporating regenerative sequences for 5 days, increased F-actin and nuclear cell contraction, migration and polarization of collagen fibers across the FM defect when compared to control specimens with minimal repair. CONCLUSIONS: Whilst the nanoarchitecture revealed promising conditions to seal iatrogenic FM defects, the peptide amphiphiles need to be designed to maximize repair mechanisms and promote structural compliance with high mechanical tolerance that maintains tissue remodeling with Cx43 antisense for future treatment.

Multicomponent hydrogels for the formation of vascularized bone-like constructs in vitro.

The native extracellular matrix (ECM) is a complex gel-like system with a broad range of structural features and biomolecular signals. Hydrogel platforms that can recapitulate the complexity and signaling properties of this ECM would have enormous impact in fields ranging from tissue engineering to drug discovery. Here, we report on the design, synthesis, and proof-of-concept validation of a microporous and nanofibrous hydrogel exhibiting multiple bioactive epitopes designed to recreate key features of the bone ECM. The material platform integrates self-assembly with orthogonal enzymatic cross-linking to create a supramolecular environment comprising hyaluronic acid modified with tyramine (HA-Tyr) and peptides amphiphiles (PAs) designed to promote cell adhesion (RGDS-PA), osteogenesis (Osteo-PA), and angiogenesis (Angio-PA). Through individual and co-cultures of human adipose derived mesenchymal stem cells (hAMSCs) and human umbilical vascular endothelial cells (HUVECs), we confirmed the capacity of the HA-Tyr/RGDS-PA/Osteo-PA/Angio-PA hydrogel to promote cell adhesion as well as osteogenic and angiogenic differentiation in both 2D and 3D setups. Furthermore, using immunofluorescent staining and reverse transcription-quantitative polymerase chain reaction (RT-qPCR), we demonstrated co-differentiation and organization of hAMSCs and HUVECs into 3D aggregates resembling vascularized bone-like constructs. STATEMENT OF SIGNIFICANCE: This body of work presents a new approach to develop more complex, yet functional, in vitro environments for cell culture while enabling a high level of control, tuneability, and reproducibility. The multicomponent self-assembling bioactive 2D and 3D hydrogels with nanofibrous architecture designed to recreate key molecular and macromolecular features of the native bone ECM and promote both osteogenesis and angiogenesis. The materials induce endothelial cells towards large vascular lumens and MSCs into bone cells on/within the same platform and form vascularized-bone like construct in vitro. This strategy looks encouraging for lifelike bone tissue engineering in vitro and bone tissue regeneration in vivo.

Magnetically responsive layer-by-layer microcapsules can be retained in cells and under flow conditions to promote local drug release without triggering ROS production.

Nanoengineered vehicles have the potential to deliver cargo drugs directly to disease sites, but can potentially be cleared by immune system cells or lymphatic drainage. In this study we explore the use of magnetism to hold responsive particles at a delivery site, by incorporation of superparamagnetic iron oxide nanoparticles (SPIONs) into layer-by-layer (LbL) microcapsules. Microcapsules with SPIONs were rapidly phagocytosed by cells but did not trigger cellular ROS synthesis within 24 hours of delivery nor affect cell viability. In a non-directional cell migration assay, SPION containing microcapsules significantly inhibited movement of phagocytosing cells when placed in a magnetic field. Similarly, under flow conditions, a magnetic field retained SPION containing microcapsules at a physiologic wall shear stress of 0.751 dyne cm-2. Even when the SPION content was reduced to 20%, the majority of microcapsules were still retained. Dexamethasone microcrystals were synthesised by solvent evaporation and underwent LbL encapsulation with inclusion of a SPION layer. Despite a lower iron to volume content of these structures compared to microcapsules, they were also retained under shear stress conditions and displayed prolonged release of active drug, beyond 30 hours, measured using a glucocorticoid sensitive reporter cell line generated in this study. Our observations suggest use of SPIONs for magnetic retention of LbL structures is both feasible and biocompatible and has potential application for improved local drug delivery.

Targeting mechanotransduction mechanisms and tissue weakening signals in the human amniotic membrane.

Mechanical and inflammatory signals in the fetal membrane play an important role in extracellular matrix (ECM) remodelling in order to dictate the timing of birth. We developed a mechanical model that mimics repetitive stretching of the amniotic membrane (AM) isolated from regions over the placenta (PAM) or cervix (CAM) and examined the effect of cyclic tensile strain (CTS) on mediators involved in mechanotransduction (Cx43, AKT), tissue remodelling (GAGs, elastin, collagen) and inflammation (PGE2, MMPs). In CAM and PAM specimens, the application of CTS increased GAG synthesis, PGE2 release and MMP activity, with concomitant reduction in collagen and elastin content. Co-stimulation with CTS and pharmacological agents that inhibit either Cx43 or AKT, differentially influenced collagen, GAG and elastin in a tissue-dependent manner. SHG confocal imaging of collagen fibres revealed a reduction in SHG intensity after CTS, with regions of disorganisation dependent on tissue location. CTS increased Cx43 and AKT protein and gene expression and the response could be reversed with either CTS, the Cx43 antisense or AKT inhibitor. We demonstrate that targeting Cx43 and AKT prevents strain-induced ECM damage and promotes tissue remodelling mechanisms in the AM. We speculate that a combination of inflammatory and mechanical factors could perturb typical mechanotransduction processes mediated by Cx43 signalling. Cx43 could therefore be a potential therapeutic target to prevent inflammation and preterm premature rupture of the fetal membranes.

Tissuepatch is biocompatible and seals iatrogenic membrane defects in a rabbit model.

OBJECTIVE: To evaluate novel sealing techniques for their biocompatibility and sealing capacity of iatrogenic fetal membrane defects in a pregnant rabbit model. METHOD: At day 23 of gestation (term = d31), a standardized fetoscopy was performed through a 14G cannula. The resulting fetal membrane defect was closed with condensed collagen, collagen with fibrinogen, Tissuepatch, Duraseal, or a conventional collagen plug (Lyostypt) as reference. At d30, the fetuses were harvested and full thickness fetal membrane samples were analyzed. The study consisted of 2 consecutive parts: (1) biocompatibility testing by fetal survival, apoptosis, and infiltration of polymorphonuclear cells in the membranes and (2) the efficacy to seal fetal membrane defects. RESULTS: Three sealants (collagen with fibrinogen, Duraseal, or Lyostypt) were associated with a higher fetal mortality compared to control unmanipulated littermates and hence were excluded from further analysis. Tissuepatch was biocompatible, and amniotic fluid levels were comparable to those of control untouched littermates. Compared to the condensed collagen, Tissuepatch was also easier in surgical handling and induced limited cell proliferation. CONCLUSION: Tissuepatch had the best biocompatibility and efficacy in sealing an iatrogenic fetal membrane defect in the pregnant rabbit compared to other readily available sealants.

Trauma induces overexpression of Cx43 in human fetal membrane defects.

OBJECTIVE: We developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects. METHOD: Term human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining. RESULTS: After trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture. CONCLUSION: Cx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Connexin 43 is overexpressed in human fetal membrane defects after fetoscopic surgery.

OBJECTIVE: We examined whether surgically induced membrane defects elevate connexin 43 (Cx43) expression in the wound edge of the amniotic membrane (AM) and drives structural changes in collagen that affects healing after fetoscopic surgery. METHOD: Cell morphology and collagen microstructure was investigated by scanning electron microscopy and second harmonic generation in fetal membranes taken from women who underwent fetal surgery. Immunofluoresence and real-time quantitative polymerase chain reaction was used to examine Cx43 expression in control and wound edge AM. RESULTS: Scanning electron microscopy showed dense, helical patterns of collagen fibrils in the wound edge of the fetal membrane. This arrangement changed in the fibroblast layer with evidence of collagen fibrils that were highly polarised along the wound edge but not in control membranes. Cx43 was increased by 112.9% in wound edge AM compared with controls (p < 0.001), with preferential distribution in the fibroblast layer compared with the epithelial layer (p < 0.01). In wound edge AM, mesenchymal cells had a flattened morphology, and there was evidence of poor epithelial migration across the defect. Cx43 and COX-2 expression was significantly increased in wound edge AM compared with controls (p < 0.001). CONCLUSION: Overexpression of Cx43 in the AM after fetal surgery induces morphological and structural changes in the collagenous matrix that may interfere with normal healing mechanisms. © 2016 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.

Controlled release of C-type natriuretic peptide by microencapsulation dampens proinflammatory effects induced by IL-1ß in cartilage explants.

C-type natriuretic peptide (CNP) exhibits potent anti-inflammatory effects in chondrocytes that have the potential to repair cartilage damage observed in osteoarthritis (OA). However, treatments for OA have been challenging due to poor targeting and delivery of therapeutics. The present study fabricated polyelectrolyte microcapsules loaded with CNP and examined whether the layer-by-layer (LbL) approach could have protective effects in cartilage explants treated with the pro-inflammatory cytokine, interleukin-1ß (IL-1ß). SEM showed uniform, 2 to 3 µm spherical microcapsules with morphological characteristic similar to templates loaded with or without CNP. The protein was localized around the external surface of the microcapsules with encapsulation efficiencies >82.9%. CNP release profiles were broadly similar following 9 days of culture. The presence of CNP microcapsules did not significantly affect cell viability (80%) with DNA values that remained stable throughout the culture conditions. Confocal imaging showed clustering of microcapsules in chondrocytes to natriuretic peptide receptor (Npr) 2 and 3. Treatment of cartilage explants with CNP microcapsules led to concentration-dependent inhibition of NO release in response to IL-1ß and restoration of matrix synthesis. In summary, we demonstrate controlled delivery of CNP to dampen pro-inflammatory effects induced by IL-1ß in cartilage explants. The LbL approach has the potential to promote cartilage repair in vivo.

Low oxygen tension increased fibronectin fragment induced catabolic activities--response prevented with biomechanical signals.

INTRODUCTION: The inherent low oxygen tension in normal cartilage has implications on inflammatory conditions associated with osteoarthritis (OA). Biomechanical signals will additionally contribute to changes in tissue remodelling and influence the inflammatory response. In this study, we investigated the combined effects of oxygen tension and fibronectin fragment (FN-f) on the inflammatory response of chondrocytes subjected to biomechanical signals. METHODS: Chondrocytes were cultured under free-swelling conditions at 1%, 5% and 21% oxygen tension or subjected to dynamic compression in an ex vivo 3D/bioreactor model with 29 kDa FN-f, interleukin-1beta (IL-1ß) and/or the nitric oxide synthase (NOS) inhibitor for 6 and 48 hours. Markers for catabolic activity (NO, PGE2), tissue remodelling (GAG, MMPs) and cytokines (IL-1ß, IL-6 and TNFα) were quantified by biochemical assay. Aggrecan, collagen type II, iNOS and COX-2 gene expression were examined by real-time quantitative PCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse data. RESULTS: Both FN-fs and IL-1ß increased NO, PGE2 and MMP production (all P< 0.001). FN-f was more active than IL-1ß with greater levels of NO observed at 5% than 1% or 21% oxygen tension (P < 0.001). Whilst FN-f reduced GAG synthesis at all oxygen tension, the effect of IL-1ß was significant at 1% oxygen tension. In unstrained constructs, treatment with FN-f or IL-1ß increased iNOS and COX-2 expression and reduced aggrecan and collagen type II (all P < 0.001). In unstrained constructs, FN-f was more effective than IL-1ß at 5% oxygen tension and increased production of NO, PGE2, MMP, IL-1ß, IL-6 and TNFα. At 5% and 21% oxygen tension, co-stimulation with compression and the NOS inhibitor abolished fragment or cytokine-induced catabolic activities and restored anabolic response. CONCLUSIONS: The present findings revealed that FN-fs are more potent than IL-1ß in exerting catabolic effects dependent on oxygen tension via iNOS and COX-2 upregulation. Stimulation with biomechanical signals abolished catabolic activities in an oxygen-independent manner and NOS inhibitors supported loading-induced recovery resulting in reparative activities. Future investigations will utilize the ex vivo model as a tool to identify key targets and therapeutics for OA treatments.

Natriuretic peptide receptors regulate cytoprotective effects in a human ex vivo 3D/bioreactor model.

INTRODUCTION: The present study examined the effect of C-type natriuretic peptide (CNP) and biomechanical signals on anabolic and catabolic activities in chondrocyte/agarose constructs. METHODS: Natriuretic peptide (Npr) 2 and 3 expression were compared in non-diseased (grade 0/1) and diseased (grade IV) human cartilage by immunofluoresence microscopy and western blotting. In separate experiments, constructs were cultured under free-swelling conditions or subjected to dynamic compression with CNP, interleukin-1ß (IL-1ß), the Npr2 antagonist P19 or the Npr3 agonist cANF4⁻²³. Nitric oxide (NO) production, prostaglandin E2 (PGE2) release, glycosaminoglycan (GAG) synthesis and CNP concentration were quantified using biochemical assays. Gene expression of Npr2, Npr3, CNP, aggrecan and collagen type II were assessed by real-time qPCR. Two-way ANOVA and a post hoc Bonferroni-corrected t-test were used to analyse the data. RESULTS: The present study demonstrates increased expression of natriuretic peptide receptors in diseased or older cartilage (age 70) when compared to non-diseased tissue (age 60) which showed minimal expression. There was strong parallelism in the actions of CNP on cGMP induction resulting in enhanced GAG synthesis and reduction of NO and PGE2 release induced by IL-1ß. Inhibition of Npr2 with P19 maintained catabolic activities whilst specific agonism of Npr3 with cANF4⁻²³ had the opposite effect and reduced NO and PGE2 release. Co-stimulation with CNP and dynamic compression enhanced anabolic activities and inhibited catabolic effects induced by IL-1ß. The presence of CNP and the Npr2 antagonist abolished the anabolic response to mechanical loading and prevented loading-induced inhibition of NO and PGE2 release. In contrast, the presence of the Npr3 agonist had the opposite effect and increased GAG synthesis and cGMP levels in response to mechanical loading and reduced NO and PGE2 release comparable to control samples. In addition, CNP concentration and natriuretic peptide receptor expression were increased with dynamic compression. CONCLUSIONS: Mechanical loading mediates endogenous CNP release leading to increased natriuretic peptide signalling. The loading-induced CNP/Npr2/cGMP signalling route mediates anabolic events and prevents catabolic activities induced by IL-1ß. The CNP pathway therefore represents a potentially chondroprotective intervention for patients with OA, particularly when combined with physiotherapeutic approaches to stimulate biomechanical signals.

Biological vascular grafts for hemodialysis access.

Alternatives to autogenous arteriovenous hemodialysis (HD) access, such as synthetic arteriovenous bypass grafts and central venous catheters, are associated with a higher rate of complications. The evaluated article assessed the repeated cannulation challenges of HD in tissue-engineered blood vessels implanted in a bovine in vivo model (n = 15). Two groups were studied. A short-term group in which the graft was explanted and histologically examined (n = 7) and a second group in which the graft was left in for 6 months or until outflow venous stenosis occurred (n = 8). Two grafts from each group occluded 1-month postoperatively. Of the 11 remaining, cannulation was well tolerated with adequate hemostasis. Histological analysis demonstrated host cell repopulation of the outer surface in the short-term group (n = 5) and stable wall geometry in the long-term group. The authors concluded that their study proves the concept of using a scaffold-based approach to tissue-engineered blood vessels for HD access.

Biomechanical Conditioning Enhanced Matrix Synthesis in Nucleus Pulposus Cells Cultured in Agarose Constructs with TGFß.

Biomechanical signals play an important role in normal disc metabolism and pathology. For instance, nucleus pulposus (NP) cells will regulate metabolic activities and maintain a balance between the anabolic and catabolic cascades. The former involves factors such as transforming growth factor-ß (TGFß) and mechanical stimuli, both of which are known to regulate matrix production through autocrine and paracrine mechanisms. The present study examined the combined effect of TGFß and mechanical loading on anabolic activities in NP cells cultured in agarose constructs. Stimulation with TGFß and dynamic compression reduced nitrite release and increased matrix synthesis and gene expression of aggrecan and collagen type II. The findings from this work has the potential for developing regenerative treatment strategies which could either slow down or stop the degenerative process and/or promote healing mechanisms in the intervertebral disc.

Biomechanical signals and the C-type natriuretic peptide counteract catabolic activities induced by IL-1ß in chondrocyte/agarose constructs.

INTRODUCTION: The present study examined the effect of C-type natriuretic peptide (CNP) on the anabolic and catabolic activities in chondrocyte/agarose constructs subjected to dynamic compression. METHODS: Constructs were cultured under free-swelling conditions or subjected to dynamic compression with low (0.1 to 100 pM) or high concentrations (1 to 1,000 nM) of CNP, interleukin-1ß (IL-1ß), and/or KT-5823 (inhibits cyclic GMP-dependent protein kinase II (PKGII)). Anabolic and catabolic activities were assessed as follows: nitric oxide (NO) and prostaglandin E2 (PGE2) release, and [3H]-thymidine and 35SO4 incorporation were quantified by using biochemical assays. Gene expression of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), aggrecan, and collagen type II were assessed with real-time quantitative PCR (qPCR). Two-way ANOVA and the post hoc Bonferroni-corrected t tests were used to examine data. RESULTS: CNP reduced NO and PGE2 release and partially restored [3H]-thymidine and 35SO4 incorporation in constructs cultured with IL-1ß. The response was dependent on the concentration of CNP, such that 100 pM increased [3H]-thymidine incorporation (P < 0.001). This is in contrast to 35SO4 incorporation, which was enhanced with 100 or 1000 nM CNP in the presence and absence of IL-1ß (P < 0.001). Stimulation by both dynamic compression and CNP and/or the PKGII inhibitor further reduced NO and PGE2 release and restored [3H]-thymidine and 35SO4 incorporation. In the presence and absence of IL-1ß, the magnitude of stimulation for [3H]-thymidine and 35SO4 incorporation by dynamic compression was dependent on the concentration of CNP and the response was inhibited with the PKGII inhibitor. In addition, stimulation by CNP and/or dynamic compression reduced IL-1ß-induced iNOS and COX-2 expression and restored aggrecan and collagen type II expression. The catabolic response was not further influenced with the PKGII inhibitor in IL-1ß-treated constructs. CONCLUSIONS: Treatment with CNP and dynamic compression increased anabolic activities and blocked catabolic effects induced by IL-1ß. The anabolic response was PKGII mediated and raises important questions about the molecular mechanisms of CNP with mechanical signals in cartilage. Therapeutic agents like CNP could be administered in conjunction with controlled exercise therapy to slow the OA disease progression and to repair damaged cartilage. The findings from this research provide the potential for developing novel agents to slow the pathophysiologic mechanisms and to treat OA in the young and old.

Quantification of mRNA using real-time PCR and Western blot analysis of MAPK events in chondrocyte/agarose constructs.

In vitro models of chondrocyte mechanobiology have been used to compare the intracellular signalling pathways altered in normal and osteoarthritis-affected cartilage. However, differences in the model system and type of loading configuration have led to complicated pathways. This chapter is a follow-on of previous studies from our group utilising 3D agarose as a physiological model to study mechanotransduction pathways. Experimental methods are described to assess targets at the protein and gene expression level by Western blot analysis and real-time PCR, respectively. This chapter provides a quantitative gene expression approach to explore the intracellular pathways activated by both mechanical loading and inflammatory mediators and examine upstream phosphorylation events. Ultimately, development of methods used to analyse mechano-sensitive pathways will provide important information for the identification of appropriate pharmacological and physiotherapeutic agents for the treatment of osteoarthritis.

Biomechanical modulation of collagen fragment-induced anabolic and catabolic activities in chondrocyte/agarose constructs.

INTRODUCTION: The present study examined the effect of collagen fragments on anabolic and catabolic activities by chondrocyte/agarose constructs subjected to dynamic compression. METHODS: Constructs were cultured under free-swelling conditions or subjected to continuous and intermittent compression regimes, in the presence of the N-terminal (NT) and C-terminal (CT) telopeptides derived from collagen type II and/or 1400 W (inhibits inducible nitric oxide synthase (iNOS)). The anabolic and catabolic activities were compared to the amino-terminal fibronectin fragment (NH2-FN-f) and assessed as follows: nitric oxide (NO) release and sulphated glycosaminoglycan (sGAG) content were quantified using biochemical assays. Tumour necrosis factor-alpha (TNFalpha) and interleukin-1beta (IL-1beta) release were measured by ELISA. Gene expression of matrix metalloproteinase-3 (MMP-3), matrix metalloproteinase-13 (MMP-13), collagen type II and fibronectin were assessed by real-time quantitative polymerase chain reaction (qPCR). Two-way ANOVA and the post hoc Bonferroni-corrected t-test was used to examine data. RESULTS: The presence of the NT or CT peptides caused a moderate to strong dose-dependent stimulation of NO, TNFalpha and IL-1beta production and inhibition of sGAG content. In some instances, high concentrations of telopeptides were just as potent in stimulating catabolic activities when compared to NH2-FN-f. Depending on the concentration and type of fragment, the increased levels of NO and cytokines were inhibited with 1400 W, resulting in the restoration of sGAG content. Depending on the duration and type of compression regime employed, stimulation with compression or incubation with 1400 W or a combination of both, inhibited telopeptide or NH2-FN-f induced NO release and cytokine production and enhanced sGAG content. All fragments induced MMP-3 and MMP-13 expression in a time-dependent manner. This effect was reversed with compression and/or 1400 W resulting in the restoration of sGAG content and induction of collagen type II and fibronectin expression. CONCLUSIONS: Collagen fragments containing the N- and C-terminal telopeptides have dose-dependent catabolic activities similar to fibronectin fragments and increase the production of NO, cytokines and MMPs. Catabolic activities were downregulated by dynamic compression or by the presence of the iNOS inhibitor, linking reparative activities by both types of stimuli. Future investigations which examine the signalling cascades of chondrocytes in response to matrix fragments with mechanical influences may provide useful information for early osteoarthritis treatments.