Activation of synovial fibroblasts from patients at revision of their metal-on-metal total hip arthroplasty.

BACKGROUND: The toxicity of released metallic particles generated in metal-on-metal (MoM) total hip arthroplasty (THA) using cobalt chromium (CoCr) has raised concerns regarding their safety amongst both surgeons and the public. Soft tissue changes such as pseudotumours and metallosis have been widely observed following the use of these implants, which release metallic by-products due to both wear and corrosion. Although activated fibroblasts, the dominant cell type in soft tissues, have been linked to many diseases, the role of synovial fibroblasts in the adverse reactions caused by CoCr implants remains unknown. To investigate the influence of implants manufactured from CoCr, the periprosthetic synovial tissues and synovial fibroblasts from patients with failed MoM THA, undergoing a revision operation, were analysed and compared with samples from patients undergoing a primary hip replacement, in order to elucidate histological and cellular changes. RESULTS: Periprosthetic tissue from patients with MoM implants was characterized by marked fibrotic changes, notably an increase in collagen content from less than 20% to 45-55%, an increase in α-smooth muscle actin positive cells from 4 to 9% as well as immune cells infiltration. Primary cell culture results demonstrated that MoM synovial fibroblasts have a decreased apoptosis rate from 14 to 6% compared to control synovial fibroblasts. In addition, synovial fibroblasts from MoM patients retained higher contractility and increased responsiveness to chemotaxis in matrix contraction. Their mechanical properties at a single cell level increased as observed by a 60% increase in contraction force and higher cell stiffness (3.3 kPa in MoM vs 2.18 kPa in control), as measured by traction force microscopy and atomic force microscopy. Further, fibroblasts from MoM patients promoted immune cell invasion by secreting monocyte chemoattractant protein 1 (MCP-1, CCL2) and induced monocyte differentiation, which could also be associated with excess accumulation of synovial macrophages. CONCLUSION: Synovial fibroblasts exposed in vivo to MoM THA implants that release CoCr wear debris displayed dramatic phenotypic alteration and functional changes. These findings unravelled an unexpected effect of the CoCr alloy and demonstrated an important role of synovial fibroblasts in the undesired tissue reactions caused by MoM THAs.

3D models of chondrocytes within biomimetic scaffolds: Effects of cell deformation from loading regimens.

BACKGROUND: Mechanical conditioning has been widely used to attempt to enhance chondrocyte metabolism for the evolution of functionally competent cartilage. However, although upregulation of proteoglycans have been reported through the application of uniaxial compression, minimal collagen has been produced. The study is designed to examine whether alternative loading regimens, equivalent to physiological conditions, involving shear in addition to compression can enhance collagen production. METHODS: Finite element models were developed to determine how the local chondrocyte environments within agarose constructs were influenced by a range of static and dynamic loading regimens. 3-D poro-viscoelastic models were validated against experimental data. In particular, these models were used to characterise chondrocyte deformation in compression with and without shear superimposed, with special reference to the formation of pericellular matrix around the cells. FINDINGS: The models of the hydrogel constructs under stress relaxation and dynamic cyclic compression conditions were highly correlated with the experimental data. The cell deformation (y/z) in the constructs was greatest in the centre of the constructs, increasing with magnitude of compression up to 25%. The superposition of shear however did not produce significant additional changes in deformation, with the presence of PCM reducing the chondrocyte deformation. INTERPRETATION: The use of FE models can prove important in the definition of appropriate, optimised mechanical conditioning regimens for the synthesis and organisation of mature extra cellular matrix by chondrocyte-seeded constructs. They will also provide insight into the mechanisms relating cell deformation to mechanotransduction pathways, thereby progressing the development of functionally competent tissue engineered cartilage.

Sub-toxic levels of Co2+ are anti-inflammatory and protect cartilage from degradation caused by IL-1ß.

BACKGROUND: Cobalt ions from some orthopaedic implants induce a dose-dependent cytotoxic and pro-inflammatory response. Recent studies show that sub-toxic levels of cobalt influence actin organisation regulating fibroblasts and macrophages behaviour. However little is known about the influence of sub-toxic levels of cobalt on articular cartilage biology and biomechanics. Previously, we have reported that IL-1ß signalling in chondrocytes, is regulated by primary cilia and associated intraflagellar transport. Since primary cilia expression is modulated by actin organisation, we set out to test the hypothesis that sub-toxic levels of cobalt regulate cilia expression and IL-1ß signalling thereby influencing articular cartilage degradation. METHODS: Isolated chondrocytes and bovine cartilage explants were subjected to Co2+ in the presence and absence of IL-1ß. Primary cilia were monitored by confocal immunofluorescence. Nitric oxide and PGE2 release were used to monitor IL-1ß signalling. Degradation of cartilage matrix was assessed by the release of sGAG and the biomechanical properties of the tissue in uniaxial unconfined compression. FINDINGS: Sub-toxic levels of Co2+ (50 µM) blocked IL-1ß-induced primary cilia elongation in isolated chondrocytes. This was associated with disruption of pro-inflammatory signalling in both isolated chondrocytes and cartilage explants, and inhibition of cartilage matrix degradation and loss of biomechanical properties. INTERPRETATION: This study reveals that low levels of cobalt ions are anti-inflammatory, preventing cartilage degradation in response to IL-1ß. This mechanism is associated with regulation of primary cilia elongation. These observations provide new insight into the potential beneficial role of cobalt and may lead to novel mechanisms for controlling cartilage inflammation.

Prediction of taper performance using quasi static FE models: The influence of loading, taper clearance and trunnion length.

The head-neck taper junction has been widely reported to corrode leading to adverse tissue reactions. Taper corrosion is a poorly understood phenomenon but has been associated with oxide layer damage and ingress of corrosive physiological fluids. Micromotion may damage the oxide layer; although little is understood about the prevailing stresses which cause this. The ingress of fluid around the joint space into the taper will depend on the taper contact position and the separation of the interfaces during loading. The current work reports on the effect of taper clearances and trunnion length on the taper surface stresses and the taper gap opening. These were determined for CoCr/Ti taper interfaces using FE under loading conditions including walking and stair climb as well as hip simulator load profiles. Shorter trunnions and stair climb loading were shown to generate the greatest taper gaps (82 µm) and also the largest surface stresses (1200 MPa) on the head taper. The largest taper gaps were associated with smaller taper contact areas. Clearances within ±0.1° had no effect on the taper gaps generated, as the tapers engaged over comparable lengths; the taper gap opening was dependent upon the taper engagement length rather than location (proximal or distal) of contact. The walking profile or variants applied by hip simulators, was insufficient to differentiate between taper designs and evaluate differences in the magnitudes of taper gaps. The use of more demanding activity such as stair climb during in vitro evaluations could provide better predictions of taper performance in vivo. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 107B: 138-148, 2019.

Mechanical loading induces primary cilia disassembly in tendon cells via TGFß and HDAC6.

This study used isolated human tenocytes to test the hypothesis that cyclic mechanical strain directly stimulates primary cilia disassembly, and to elucidate the mechanisms involved. Cells were seeded onto flexible membranes and strained at 0-3%; 1 Hz, for up to 24 hours. Cilia length and prevalence progressively reduced with increasing strain duration but showed full recovery within 2 hours of strain removal. The response to loading was not influenced by actin organisation as seen in other cell types. However, the loading response could be recreated by treatment with TGFß. Furthermore, treatment with the HDAC6 inhibitor Tubacin, or a TGFß receptor inhibitor both prevented strain induced cilia disassembly. These data are the first to describe primary cilia expression in isolated tenocytes, showing that mechanical strain regulates cilia expression independent of changes in tendon extracellular matrix. Furthermore, we show that cilia disassembly is mediated by the activation of TGFß receptors leading to activation of HDAC6. Previous studies have shown that cilia are required for TGFß signalling and that tendon mechanosignalling is mediated by TGFß. The present study therefore suggests a novel feedback mechanism whereby cilia disassembly inhibits prolonged TGFß activation in response to continuous cyclic loading.

Cobalt (II) ions and nanoparticles induce macrophage retention by ROS-mediated down-regulation of RhoA expression.

Histological assessments of synovial tissues from patients with failed CoCr alloy hip prostheses demonstrate extensive infiltration and accumulation of macrophages, often loaded with large quantities of particulate debris. The resulting adverse reaction to metal debris (ARMD) frequently leads to early joint revision. Inflammatory response starts with the recruitment of immune cells and requires the egress of macrophages from the inflamed site for resolution of the reaction. Metal ions (Co2+ and Cr3+) have been shown to stimulate the migration of T lymphocytes but their effects on macrophages motility are still poorly understood. To elucidate this, we studied in vitro and in vivo macrophage migration during exposure to cobalt and chromium ions and nanoparticles. We found that cobalt but not chromium significantly reduces macrophage motility. This involves increase in cell spreading, formation of intracellular podosome-type adhesion structures and enhanced cell adhesion to the extracellular matrix (ECM). The formation of podosomes was also associated with the production and activation of matrix metalloproteinase-9 (MMP9) and enhanced ECM degradation. We showed that these were driven by the down-regulation of RhoA signalling through the generation of reactive oxygen species (ROS). These novel findings reveal the key mechanisms driving the wear/corrosion metallic byproducts-induced inflammatory response at non-toxic concentrations. STATEMENT OF SIGNIFICANCE: Adverse tissue responses to metal wear and corrosion products from CoCr alloy implants remain a great challenge to surgeons and patients. Macrophages are the key regulators of these adverse responses to the ions and debris generated. We demonstrated that cobalt, rather than chromium, causes macrophage retention by restructuring the cytoskeleton and inhibiting cell migration via ROS production that affects Rho Family GTPase. This distinctive effect of cobalt on macrophage behaviour can help us understand the pathogenesis of ARMD and the cellular response to cobalt based alloys, which provide useful information for future implant design and biocompatibility testing.

Complex mechanical conditioning of cell-seeded agarose constructs can influence chondrocyte biosynthetic activity.

Articular cartilage with its inherently poor capacity for self-regeneration represents a primary target for tissue engineering strategies, with approaches focusing on the in vitro generation of neo-cartilage using chondrocyte-seeded 3D scaffolds subjected to mechanical conditioning. Although uniaxial compression regimens have significantly up-regulated proteoglycan synthesis, their effects on the synthesis of collagen have been modest. Articular cartilage is subjected to shear forces during joint motion. Accordingly, this study utilized an apparatus to apply biaxial loading to chondrocytes seeded within agarose constructs with endplates. The chondrocytes yielded a monotonic increase in proteoglycan synthesis both in free swelling culture up to day 8 and when the constructs were subjected to dynamic compression alone (15% amplitude at a frequency of 1 Hz for 48 h). However, when dynamic shear (10% amplitude at 1 Hz) was superimposed on dynamic compression, total collagen synthesis was also up-regulated, within 3 days of culture, without compromising proteoglycan synthesis. Histological analysis revealed marked collagen deposition around individual chondrocytes. A significant proportion (50%) of collagen was released into the culture medium, suggesting that it had only been partially synthesized in its mature state. The overall biosynthetic activity was enhanced more when the biaxial stimulation was applied in a continuous mode as opposed to intermittent loading. Results of the present study strongly suggest that proteoglycan and collagen synthesis may be triggered by uncoupled mechanosensitive cellular responses. The proposed in vitro model and the prescribed conditioning protocols demonstrated that a short pre-culture period is preferable to long free swelling culture condition as it enables a significantly higher up-regulation of collagen. Biotechnol. Bioeng. 2017;114: 1614-1625. © 2017 Wiley Periodicals, Inc.

Measurement outcomes from hip simulators.

Simulation of wear in total hip replacements has been recognised as an important factor in determining the likelihood of clinical success. However, accurate measurement of wear can be problematic with factors such as number and morphology of wear particles produced as well as ion release proving more important in the biological response to hip replacements than wear volume or wear rate alone. In this study, hard-on-hard (CoCr alloy, AgCrN coating) and hard-on-soft (CoCr alloy and CrN coating on vitamin E blended highly cross-linked polyethylene) bearing combinations were tested in an orbital hip simulator under standard and some adverse conditions. Gravimetric wear rates were determined for all bearings, with cobalt and where applicable, silver release determined throughout testing. Isolation of wear particles from the lubricating fluid was used to determine the influence of different bearing combinations and wear conditions on particle morphology. It was found that cobalt and silver could be measured in the lubricating fluid even when volumetric wear was not detectable. In hard-on-hard bearings, Pearson's correlation of 0.98 was established between metal release into the lubricating fluid and wear volume. In hard-on-soft bearings, coating the head did not influence the polyethylene wear rates measured under standard conditions but did influence the cobalt release; the diameter influenced both polyethylene wear and cobalt release, and the introduction of adverse testing generated smaller polyethylene particles. While hip simulators can be useful to assess the wear performance of a new material or design, measurement of other outcomes may yield greater insight into the clinical behaviour of the bearings in vivo.

The increase in cobalt release in metal-on-polyethylene hip bearings in tests with third body abrasives.

Hypersensitivity reactions in patients receiving metal-on-metal hip replacements have been attributed to corrosion products as observed by elevated cobalt and chromium ions in the blood. Although the majority of cases are reported in metal-on-metal, incidences of these reactions have been reported in the metal-on-polyethylene patient population. To date, no in vitro study has considered cobalt release for this bearing combination. This study considered four 28 mm and seven 52 mm diameter metal-on-polyethylene bearings tested following ISO standard hip simulator conditions as well as under established abrasive conditions. These tests showed measurable cobalt in all bearings under standard conditions. Cobalt release, as well as polyethylene wear, increased with diameter, increasing from 52 to 255 ppb. The introduction of bone cement particles into the articulation doubled polyethylene wear and cobalt release while alumina particles produced significant damage on the heads demonstrated by cobalt levels of 70,700 ppb and an increased polyethylene wear from a mean value of 9-160 mm(3)/mc. Cobalt release was indicative of head damage and correlated with polyethylene wear at the next gravimetric interval. The removal of third body particles resulted in continued elevated cobalt levels in the 52 mm diameter bearings tested with alumina compared to standard conditions but the bearings tested with bone cement particles returned to standard levels. The polyethylene wear in the bone cement tested bearings also recovered to standard levels, although the alumina tested bearings continued to wear at a higher rate of 475 mm(3)/mc. Cobalt release was shown to occur in metal-on-polyethylene bearings indicating damage to the metal head resulting in increased polyethylene wear. While large diameter metal-on-polyethylene bearings may provide an increased range of motion and a reduced dislocation risk, increased levels of cobalt are likely to be released and this needs to be fully considered before being widely adopted.

Deformation of uncemented metal acetabular cups following impaction: experimental and finite element study.

Predicting failure following the implantation of acetabular cups used in hip replacements is important in ensuring robust component designs. This study has developed 3D explicit dynamics finite element (FE) models to investigate the deformation of press-fit metal cups following insertion in the acetabular cavity. The cup deformation following insertion is clearly influenced by the forces encountered during insertion, the initial position of the cup in the cavity, the support provided by the underlying bone and the geometry of the cup itself. Experimentally validated explicit dynamics FE models were used to allow a physiologically relevant simulation of the impaction of cups, which is encountered in clinical practice, in comparison to previous studies that have used unrealistically high static forces to simulate a static press-fit insertion. Diametrical cup deformations were twice as large when the cup was tilted at 5° with respect to the cavity compared to when the poles of the cup and the cavity were aligned. The introduction of a non-uniform support to the cup increased deformations further by a factor of approximately 2.5. The greatest deformations established in the model were between 80 and 150 µm similar to typical cup-femoral head clearances. Increasing the thickness at the pole of the cup and reducing the cup diameter resulted in significantly smaller deformations being generated. These results suggest that small cup misalignments, which may not be noticeable in a clinical situation, may produce significant deformations after insertion especially when coupled with the non-uniform support found in the pelvis.

Design and validation of an in vitro loading system for the combined application of cyclic compression and shear to 3D chondrocytes-seeded agarose constructs.

Physiological loading is essential for the maintenance of articular cartilage by regulating tissue remodelling, in the form of both catabolic and anabolic processes. To promote the development of tissue engineered cartilage which closely matches the long term functionality of native tissue, bioreactors have been developed to provide a combination of loading modalities, which reflect the nature of normal physiological loads. This study describes the design and validation of an in vitro mechanical system for the controlled application of bi-axial loading regimes to chondrocyte-seeded agarose constructs. The computer-controlled system incorporates a robust gripping system, which ensures the delivery of precise values of cyclic compressive and shear strain to 3D cell-seeded constructs. Sample prototypes were designed, optimised using finite element analysis and validated performing compressive and shear fatigue mechanical tests. The horizontal and vertical displacements within the bioreactor are precisely controlled by a dedicated programme that can be easily implemented. The synchronisation of the orthogonal displacements was shown to be accurate and reproducible. Constructs were successfully loaded with a combined compressive and shear loading regimen at 1 Hz for up to 48 h with no appreciable loss of cell viability or mechanical integrity. These features along with the demonstrated high consistency make the system ideally suitable for a systematic investigation of the response of chondrocytes to a complex physiologically relevant deformation profile.

Simple isolation method for the bulk isolation of wear particles from metal on metal bearing surfaces generated in a hip simulator test.

Isolation and characterization of metal-on-metal (MoM) wear particles from simulator lubricants is essential to understand wear behaviour, ion release and associated corrosive activity related to the wear particles. Substantial challenges remain to establish a simple, precise and repeatable protocol for the isolation and analysis of wear particles due to their extremely small size, their tendency to agglomerate and degrade. In this paper, we describe a simple and efficient method for the bulk isolation and characterisation of wear particles from MoM bearings. Freeze drying was used to remove the large volume of water from the serum lubricant, enzymes used to digest the proteins and ultracentrifugation to finally isolate and purify the particles. The present study involved a total of eight steps for the isolation process and a wear particle extraction efficiency of 45% was achieved.

Functional analysis of tenocytes gene expression in tendon fascicles subjected to cyclic tensile strain.

Tenocytes are known to be mechanoresponsive and the present study tests the hypothesis that distinct mechanical stimulation regimes, associated with the short-term and extended application of cyclic tensile strain, alters the balance between anabolic and catabolic processes. Microarray technology has been used to provide a comprehensive analysis of alterations in gene expression within isolated tendon fascicles in response to cyclic tensile strain using a well-established model system. Isolated rat tail tendon fascicles were subjected to cyclic tensile strain (3% amplitude superimposed on a 2% static strain) for 1 or 24 hr. Messenger RNA expression level was assessed using Illumina microarray. The number of genes significantly altered in strained fascicles from the level of unstrained control fascicles was greater at 24 hr than 1 hr. The expression levels of many extracellular matrix components remained unchanged at both time points; however, a number of members of the matrix metalloproteinase (MMP) and a disintegrin and metalloproteinase with a thrombospondin (ADAMTS) families were significantly downregulated at 24 hr. Functional annotation revealed that upregulated genes were significantly associated with the regulation of transcription at 1 hr and translation at 24 hr. Downregulated genes were associated with inflammatory responses at 1 hr, and genes inhibited at 24 hr were significantly associated with cell apoptosis and a variety of metabolic functions. The present results suggest that the metabolic balance was shifted in favor of catabolism by the application of a small number of tensile strain cycles, whereas an extended number stimulates strong anti-catabolic effects.

Differential regulation of gene expression in isolated tendon fascicles exposed to cyclic tensile strain in vitro.

Mechanical stimulus is a regulator of tenocyte metabolism. The present study investigated temporal regulation of the expression of selected genes by tenocytes in isolated fascicles subjected to tensile strain in vitro. Cyclic tensile strain with a 3% amplitude superimposed on a 2% static strain was provided for 10 min, followed by either an unstrained period or continuous cyclic strain until the end of a 24-h incubation period. mRNA expression of selected anabolic and catabolic genes were evaluated with quantitative PCR at 10 min, 1 h, 6 h, and 24 h. The application of 6-h cyclic strain significantly upregulated type III collagen mRNA expression in strained fascicles compared with unstrained controls, but no alterations were observed in mRNA expression of type I collagen and biglycan. Significant downregulation in the expression of the decorin core protein was observed in fascicles subjected to 24-h cyclic strain. MMP3 and MMP13 expression levels were upregulated by the application of 10 min of cyclic strain, followed by a progressive downregulation until the end of the incubation period in both the absence and the presence of the continuing cyclic strain. Accordingly, alterations in the expression of anabolic genes were limited to the upregulation of type III collagen by prolonged exposure to cyclic strain, whereas catabolic genes were upregulated by a small number of strain cycles and downregulated by a prolonged cyclic strain. These findings demonstrate distinctive patterns of mechanoregulation for anabolic and catabolic genes and help our understanding of tenocyte response to mechanical stimulation.

Cell-generated forces influence the viability, metabolism and mechanical properties of fibroblast-seeded collagen gel constructs.

The aim of this study was to investigate the influence of the endogenous forces generated by fibroblast-mediated contraction, using four individual collagen gel models that differed with respect to the ability of the cells to contract the gel. Human neonatal dermal fibroblasts were seeded in type I collagen and the gels were cast in a racetrack-shaped mould containing a removable central island. Two of the models were mechanically stressed (20 mm and 10 mm), as complete contraction was prevented by the presence of a central island. The central island was removed in the third model (released) and the final model was cast in a Petri dish and detached, allowing full multi-axial contraction (SR). Cell viability was maintained in the 10 mm, released and SR models over a 6 day culture period but localized regions of cell death were evident in the 20 mm model. Cell and collagen alignment was developed in the 20 mm and 10 mm models and to a lesser extent in the released model, but was absent in the SR model. Cell proliferation and collagen synthesis was lower in the 20 mm model compared to the other systems and there was evidence of enhanced matrix metalloproteinase production. The mechanical properties of the 20 mm model system were inferior to the 10 mm and released systems. The 10 mm model system induced a high level of cell and matrix orientation and may, therefore, represent the best option for tissue-engineered ligament repair involving an orientated fibroblast-seeded collagen gel.

Development and validation of a novel bioreactor system for load- and perfusion-controlled tissue engineering of chondrocyte-constructs.

Osteoarthritis is a severe socio-economical disease, for which a suitable treatment modality does not exist. Tissue engineering of cartilage transplants is the most promising method to treat focal cartilage defects. However, current culturing procedures do not yet meet the requirements for clinical implementation. This article presents a novel bioreactor device for the functional tissue engineering of articular cartilage which enables cyclic mechanical loading combined with medium perfusion over long periods of time, under controlled cultivation and stimulation conditions whilst ensuring system sterility. The closed bioreactor consists of a small, perfused, autoclavable, twin chamber culture device with a contactless actuator for mechanical loading. Uni-axial loading is guided by externally applied magnetic fields with real-time feedback-control from a platform load cell and an inductive proximity sensor. This precise measurement allows the development of the mechanical properties of the cultured tissue to be monitored in real-time. This is an essential step towards clinical implementation, as it allows accounting for differences in the culture procedure induced by patient-variability. This article describes, based on standard agarose hydrogels of 3 mm height and 10 mm diameter, the technical concept, implementation, scalability, reproducibility, precision, and the calibration procedures of the whole bioreactor instrument. Particular attention is given to the contactless loading system by which chondrocyte scaffolds can be compressed at defined loading frequencies and magnitudes, whilst maintaining an aseptic cultivation procedure. In a "proof of principle" experiment, chondrocyte seeded agarose gels were cultured for 21 days in the bioreactor system. Intermittent medium perfusion at a steady flow rate (0.5 mL/min) was applied. Sterility and cell viability (ds-DNA quantification and fluorometric live/dead staining) were preserved in the system. Flow induced shear stress stimulated sGAG (sulfated glycosaminoglycan) content (DMMB assay) after 21 days, which was confirmed by histological staining of Alcian blue and by immunostaining of Aggrecan. Experimental data on mechanotransduction and long-term studies on the beneficial effects of combined perfusion and different mechanical loading patterns on chondrocyte seeded scaffolds will be published separately.

Configuration of anchorage holes affects cemented fixation of the acetabular component in total hip replacement: an in vitro study.

Although surgical fixation techniques are major contributing factors to the survivorship of total hip replacements, they vary considerably among orthopaedic surgeons. We investigated the effect of the following configuration of anchorage holes on the stability of acetabular component fixation: 3 x 12 mm, 3 x 6 mm, 6 x 6 mm, and 12 x 6 mm. The reconstructed acetabulae were tested to torque failure, whilst being subjected to a compressive load of 2.1 KN. Higher torque to failure values were obtained for specimens with three 12 mm anchorage holes, compared with six or more 6 mm anchorage holes and were in line with our computer simulation results. We propose that the longevity of cemented total hip replacements could be improved by drilling a few large anchorage holes.

Time dependence of cyclic tensile strain on collagen production in tendon fascicles.

Mechanical loading is a regulator of tissue metabolism in tendon, which may lead to alterations in structural and mechanical properties via mechanotransduction processes. The present study investigated specified responses of tenocyte metabolism in isolated tendon fascicles subjected to four loading regimes. Cyclic tensile strain of 3% amplitude superimposed on a 2% static strain was applied to the fascicles for 10min, 1, 6 or 24h of a 24-h incubation period. Collagen synthesis, assessed by [(3)H]-proline incorporation, was upregulated by the 24h straining regime, but was inhibited by the 10-min regime. Cyclic strain enhanced the retention of newly synthesised collagen within the matrix. More than 90% of the newly synthesised collagen was retained in all cases, but the long-term application of cyclic strain had less pronounced effects on the retention. These results indicate that collagen synthesis by tenocytes is controlled by a complex mechanosensitive process with a temporal component.

The influence of swelling and matrix degradation on the microstructural integrity of tendon.

Tendon is multi-level fibre composite material, responsible for the transmission of forces from muscles to the skeleton. It is composed of a hierarchical arrangement of collagenous units surrounded by a proteoglycan-rich matrix, arranged to support strain transfer, and thus contribute to the mechanical behaviour of tendon. This study examines the effect of swelling and enzymatic degradation on structural integrity at different levels of the tendon hierarchy. Biochemical and microstructural analysis are used to examine the effects of incubation on the composition and swelling of the matrix, prior to a mechanical characterisation of sample integrity. Results indicated significant swelling of tendon fibrils and interfibrillar matrix after incubation in phosphate buffered saline, leading to a reduction in ultimate tensile load, with failure initiated between fibrils and sub-fibrils. In contrast, incubation with the enzyme chondroitinase ABC resulted in a total removal of glycosaminoglycan from the samples, and a subsequent reduction in the extent of swelling. These fascicles also demonstrated an increase in failure loads, with failure predominating between fibres. The findings from this work confirm the importance of the non-collagenous matrix components in controlling strain transfer within tendon structures. It also highlights the necessity to maintain samples within a suitable and controlled environment prior to testing.