Eleanor Jones

Increased expression of IL-6 family members in tendon pathology

Objectives. Histological examination of pathological tendon generally does not reveal signs of inflammation. However, the inflammatory cytokine IL-6 has been shown to be expressed in ruptured rotator cuff tendon. The aim of this study was to investigate the expression of IL-6 family members in painful posterior tibialis tendon (PTT) and in painful and ruptured Achilles tendon (AT) compared with normal tendon. Methods. AT samples were obtained from cadavers (normal) or from patients undergoing surgical procedures to treat chronic painful tendinopathy or ruptured tendon. PTT samples were obtained from patients undergoing surgery for other reasons (normal) and from patients with PTT dysfunction (painful). Total RNA was extracted and mRNA expression was analysed by quantitative real-time PCR. Results. Collagen type I α-chain I (COL1A1) expression was increased in both painful PTT and AT compared with normal. Ciliary neurotrophic factor levels were increased in painful PTT only. In the painful AT, cyclooxygenase-2 (COX2) and IL-6 expression increased compared with normal. In the ruptured AT, levels of VEGF A, COX2, oncostatin-M, leukaemia inhibitory factor and IL-6 expression were higher compared with both normal and painful AT. IL-6R expression decreased in both painful and ruptured AT compared with normal. Conclusion. Painful AT and PTT show different expression patterns, indicating a substantial difference between those two tendinopathies. Inflammatory markers are up-regulated in painful and particularly in ruptured AT, pointing towards a role of inflammation not only in rupture healing, but also in Achilles tendinopathy.

Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFβ

Tendinopathies are a range of diseases characterised by degeneration and chronic tendon pain and represent a significant cause of morbidity. Relatively little is known about the underlying mechanisms; however onset is often associated with physical activity. A number of molecular changes have been documented in tendinopathy such as a decrease in overall collagen content, increased extracellular matrix turnover and protease activity. Metalloproteinases are involved in the homeostasis of the extracellular matrix and expression is regulated by mechanical strain. The aims of this study were to determine the effects of strain upon matrix turnover by measuring metalloproteinase and matrix gene expression and to elucidate the mechanism of action. Primary Human Achilles tenocytes were seeded in type I rat tail collagen gels in a Flexcell™ tissue train system and subjected to 5% cyclic uniaxial strain at 1 Hz for 48 h. TGFβ1 and TGFβRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFβ protein levels were determined using a cell based luciferase assay. We observed that mechanical strain regulated the mRNA levels of multiple protease and matrix genes anabolically, and this regulation mirrored that seen with TGFβ stimulation alone. We have also demonstrated that the inhibition of the TGFβ signalling pathway abrogated the strain induced changes in mRNA and that TGFβ activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFβ activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis.

UNLABELLED Controlling the cell-substrate interactions at the bio-interface is becoming an inherent element in the design of implantable devices. Modulation of cellular adhesion in vitro, through topographical cues, is a well-documented process that offers control over subsequent cellular functions. However, it is still unclear whether surface topography can be translated into a clinically functional response in vivo at the tissue/device interface. Herein, we demonstrated that anisotropic substrates with a groove depth of ∼317nm and ∼1988nm promoted human tenocyte alignment parallel to the underlying topography in vitro. However, the rigid poly(lactic-co-glycolic acid) substrates used in this study upregulated the expression of chondrogenic and osteogenic genes, indicating possible tenocyte trans-differentiation. Of significant importance is that none of the topographies assessed (∼37nm, ∼317nm and ∼1988nm groove depth) induced extracellular matrix orientation parallel to the substrate orientation in a rat patellar tendon model. These data indicate that two-dimensional imprinting technologies are useful tools for in vitro cell phenotype maintenance, rather than for organised neotissue formation in vivo, should multifactorial approaches that consider both surface topography and substrate rigidity be established. STATEMENT OF SIGNIFICANCE Herein, we ventured to assess the influence of parallel groves, ranging from nano- to micro-level, on tenocytes response in vitro and on host response using a tendon and a subcutaneous model. In vitro analysis indicates that anisotropically ordered micro-scale grooves, as opposed to nano-scale grooves, maintain physiological cell morphology. The rather rigid PLGA substrates appeared to induce trans-differentiation towards chondrogenic and/or steogenic lineage, as evidence by TILDA gene analysis. In vivo data in both tendon and subcutaneous models indicate that none of the substrates induced bidirectional host cell and tissue growth. Collective, these observations indicate that two-dimensional imprinting technologies are useful tools for in vitro cell phenotype maintenance, rather than for directional neotissue formation, should multifactorial approaches that consider both surface topography and substrate rigidity be established.

The influence of anisotropic nano- to micro-topography on in vitro and in vivo osteogenesis

AIM Topographically modified substrates are increasingly used in tissue engineering to enhance biomimicry. The overarching hypothesis is that topographical cues will control cellular response at the cell-substrate interface. MATERIALS & METHODS The influence of anisotropically ordered poly(lactic-co-glycolic acid) substrates (constant groove width of ~1860 nm; constant line width of ~2220 nm; variable groove depth of ~35, 306 and 2046 nm) on in vitro and in vivo osteogenesis were assessed. RESULTS & DISCUSSION We demonstrate that substrates with groove depths of approximately 306 and 2046 nm promote osteoblast alignment parallel to underlined topography in vitro. However, none of the topographies assessed promoted directional osteogenesis in vivo. CONCLUSION 2D imprinting technologies are useful tools for in vitro cell phenotype maintenance.

MMP and TIMP temporal gene expression during osteocytogenesis

Osteocytes within bone differentiate from osteoblast precursors which reside in a mineralised extracellular matrix (ECM). Fully differentiated osteocytes are critical for bone development and function but the factors that regulate this differentiation process are unknown. The enzymes primarily responsible for ECM remodelling are matrix metalloproteinases (MMP); however, the expression and role of MMPs during osteocytogenesis is undefined. Here we used MLO-A5 cells to determine the temporal gene expressions of the MMP family and their endogenous inhibitors--tissue inhibitors of metalloproteinases (TIMPs) during osteocytogenesis. RT-qPCR revealed expression of 14 Mmps and 3 Timps in MLO-A5 cells. Mmp2, Mmp23 and Mmp28 were decreased concurrent with mineralisation onset (P < 0.05*). Mmp14 and Mmp19 mRNAs were also significantly increased at day 3 (P < 0.05*) before returning to baseline levels at day 6. Decreased expressions of Timp1, Timp2 and Timp3 mRNA were observed by day 6 compared to day 0 (P < 0.05*). To examine whether these changes are linked to osteocytogenesis, we determined Mmp/Timp mRNA expressions in mineralisation-limited conditions. RT-qPCR revealed that the previously observed decreases in Mmp2, Mmp23 and Mmp28 were not observed in these mineralisation-limited cultures, therefore closely linking these MMPs with osteocyte differentiation. Similarly, we found differential expression of Timp1, Timp2 and Timp3 mRNA in mineralisation-restricted cultures (P < 0.05*). In conclusion, we have identified several members of the MMP/TIMP families as regulators of ECM remodelling necessary for the acquisition of the osteocyte phenotype.

Corrigendum to “Cyclical strain modulates metalloprotease and matrix gene expression in human tenocytes via activation of TGFβ” [Biochim. Biophys. Acta (2013) 2596–2607]

DOI of original article: http://dx.doi.org/10.1016/j.bbamcr.2013.06.019. ⁎ Corresponding author at: School of Biological Sciences, University of East Anglia, Norwich, Norfolk NR4 7TJ, UK. Tel.: +44 1603 591785. E-mail address: Eleanor.R.Jones@uea.ac.uk (E.R. Jones). Table 2 Comparison of changes in gene expression with Strain and TGFβ. Fold changes with strain and TGFβ were calculated compared to controls (non-strained and non treated with TGFβ) Increases in gene expression are represented by +, decreases in gene expression are represented by –.

Data on in vitro and in vivo cell orientation on substrates with different topographies

This data article contains data related to the research article entitled “Substrate topography: A valuable in vitro tool, but a clinical red herring for in vivo tenogenesis” [1]. We report measurements on tenocyte viability, metabolic activity and proliferation on substrates with different topographies. We also report the effect of substrates with different topographies on host cells in a subcutaneous model.

78 Human Tenocyte Metabolism Under Pathological And Physiological Loading Conditions

Introduction Tendinopathies are common, debilitating tendon disorders, seen among both athletes and non-athletes. Due to the unclear aetiology of tendinopathy, treatment is often generalised, and efficacy limited. Changes in mechanical and cellular interactions after microdamage are thought to be key in developing tendinopathy (Arnoczky et al., 2007). Therefore understanding how the cell strain environment modulates matrix turnover and catabolism is essential. A unique fibre composite system has been developed for this purpose. It mimics the unique tenocyte environment by encapsulating cell-coated polyethylene glycol (PEG)-RGD rods within a PEG matrix, using UV light initiated polymerisation. By recapitulating the specific, tightly controlled strain conditions, seen by tenocytes in situ, multiple post-analysis techniques such gene expression can be explored. Materials and Methods PEG-RGD rods of two stiffness were made (20% and 60% PEG), and soaked for 10 or 60 min prior to encapsulation to generate four different shear:tension ratios for cells. Rods were seeded with either healthy or diseased human tenocytes (obtained following surgery with ethical permissions) prior to encapsulation; 12 composites for each strain condition. The local strain environment was characterised by straining composites whilst visualising rod extension and shear with brightfield microscopy and cell deformation with confocal microscopy. The cell response to strain was also characterised, applying 5% cyclic strain (1Hz) to samples for 24hrs, in custom-built chambers maintained in an incubator. Gene expression across the groups was compared via RT-qPCR. Results Tenocytes successfully attach to rods in all composite conditions and remain viable. Altering the materials chemistry of rods and surrounding PEG, adjusted the extent of rod strain in relation to the applied gross strain (Figure 1), creating a spectrum of local cell strain conditions, spanning physiological and pathological conditions. Preliminary results confirm that strain applied to the construct does result in cell deformation. Gene expression analysis has shown that human tenocytes respond to brief handling when encapsulated in composites, particularly with an up-regulation of MMP1, and it takes 24 h for gene expression to stabilise. Cells also respond to the range of shear:tension strain ratios, with MMP1 and COL1A1 differentially regulated by varying shear strains and rod stiffness. Abstract 78 Figure 1 Summary of Micromechanics showing rod extension (%) vs. gross strain (%) for cell seeded composites Discussion/ Conclusion This novel fibre composite material provides the first system able to investigate tenocyte mechanotransduction and physiological and pathological levels of cell shear and tension. Current experiments have begun identifying the cell strain conditions associated with catabolic and anabolic matrix turnover, providing an exciting avenue for further understanding tendinopathy. Data suggests that the shear-strain ratio experienced by cells could be an important factor regulating their behaviour. Acknowledgments Dharmesh Patel is supported by an Arthritis Research UK Studentship. Reference Arnoczky, et al. Int J Exp Pathol, 2007;88:217–26

The Expression of Collagen-Degrading Proteases Involved in Dupuytren’s Disease Fibroblast-Mediated Contraction

An imbalance of collagen deposition and degradation contributes to Dupuytren’s disease pathology. We have previously shown that the expression of a subset of matrix metalloproteinases (MMPs) and their inhibitors, the tissue inhibitors of metalloproteinases (TIMPs), was altered in Dupuytren’s disease tissue and correlated with post-operative recurrence. In this study, the expression of collagen-degrading enzymes was measured in a fibroblast-populated collagen lattice (FPCL) model. The expression of all MMPs measured increased in the collagen gel compared to monolayer culture with a subset displaying altered expression in response to tension and release. These results were observed despite high expression of inhibitors.