Stiffness post-total knee replacement: A proof of principle study investigating the effect of gene expression analysis of markers of fibrosis.

BACKGROUND: To establish proof of principle of a link between phenotypic expression and stiffness after TKR. METHODS: From 100 patients, genetic expression of markers of fibrosis was performed for 15 synovial samples from patients categorised as 'best post-operative range of movement (ROM)' and 15 samples from patients with 'worst ROM'. These markers included Matrix Metalloproteinases (MMPs), A Disintegrin and Metalloproteinases with Thrombospondin (ADAMTS) and Tissue Inhibitors of Matrix Metalloproteinases (TIMPs). Genetic marker data were compared to Oxford Knee Scores (OKS) and Pain Catastrophizing Scores (PCS). RESULTS: Quantitative markers for gene expression demonstrated more outliers in stiff compared to non-stiff knees, suggesting a greater imbalance in pro- and anti-fibrotic markers in stiff knees. Whilst there was a significant difference in the range of post-operative knee flexion (p = 0.001) and extension (p = 0.001), there was no statistically significant difference between stiff and non-stiff knees in pre-operative or post-operative OKS (p ≥ 0.06). There was no difference in the individual components of the individual PCS score items nor the PCS total scores when stiff and non-stiff knees were compared (p > 0.05). CONCLUSION: Biological factors, namely gene expression of MMPs, TIMPs and ADAMTS, may contribute towards post-TKR stiffness. This now warrants further investigation to better understand this relationship based on larger, multi-centre, cohorts. LEVEL OF EVIDENCE: Level 3.

Induction of Tendon-Specific Markers in Adipose-Derived Stem Cells in Serum-Free Culture Conditions.

IMPACT STATEMENT: Herein, we describe the tenogenic effect of bone morphogenetic protein-12 and transforming growth factor-ß1 in cultured adipose-derived stem cells (ADSCs) in serum-free conditions. This culture system provides an insight into serum-free culture conditions in stem cell differentiation protocols. A positive response of the ADSCs to the tenogenic induction was observed. In particular, the different growth factors used in this study displayed notable differences both on the gene and on the protein expression of the tendon-specific markers. The results underline the positive outcome of the serum removal in tenogenic differentiation protocols, contributing to the development of future cell-based therapies for tendon regeneration and repair.

An in vitro investigation into the effects of 10 Hz cyclic loading on tenocyte metabolism.

Tendinopathy is a prevalent, highly debilitating condition, with poorly defined etiology. A wide range of clinical treatments has been proposed, with systematic reviews largely supporting shock wave therapy or eccentric exercise. Characterizing these treatments have demonstrated both generate perturbations within tendon at a frequency of approximately 8-12 Hz. Consequently, it is hypothesized that loading in this frequency range initiates increased anabolic tenocyte behavior, promoting tendon repair. The primary aim of this study was to investigate the effects of 10 Hz perturbations on tenocyte metabolism, comparing gene expression in response to a 10 Hz and 1 Hz loading profile. Tenocytes from healthy and tendinopathic human tendons were seeded into 3D collagen gels and subjected to 15 minutes cyclic strain at 10 Hz or 1 Hz. Tenocytes from healthy tendon showed increased expression of all analyzed genes in response to loading, with significantly increased expression of inflammatory and degradative genes with 10 Hz, relative to 1 Hz loading. By contrast, whilst the response of tenocytes from tendinopathy tendon also increased with 10 Hz loading, the overall response profile was more varied and less intense, possibly indicative of an altered healing response. Through inhibition of the pathway, IL1 was shown to be involved in the degradative and catabolic response of cells to high-frequency loading, abrogating the loading response. This study has demonstrated for the first time that loading at a frequency of 10 Hz may enhance the metabolic response of tenocytes by initiating an immediate degradatory and inflammatory cell response through the IL1 pathway, perhaps as an initial stage of tendon healing.

Synthesis and Fabrication of Surface-Active Microparticles Using a Membrane Emulsion Technique and Conjugation of Model Protein via Strain-Promoted Azide-Alkyne Click Chemistry in Physiological Conditions.

The rapid surface immobilization of protein on monodispersed polyester microcarriers is reported. A model protein, functionalized with a dibenzocyclooctyne core, immobilizes on the surface of azide-terminal polycaprolactone microcarriers within 10 min compared to 12 h for other conjugation techniques, and it is conducted in physiological conditions and in the absence of coupling reagents.

Fascicles and the interfascicular matrix show decreased fatigue life with ageing in energy storing tendons.

Tendon is composed of rope-like fascicles bound together by interfascicular matrix (IFM). The IFM is critical for the function of energy storing tendons, facilitating sliding between fascicles to allow these tendons to cyclically stretch and recoil. This capacity is required to a lesser degree in positional tendons. We have previously demonstrated that both fascicles and IFM in energy storing tendons have superior fatigue resistance compared with positional tendons, but the effect of ageing on the fatigue properties of these different tendon subunits has not been determined. Energy storing tendons become more injury-prone with ageing, indicating reduced fatigue resistance, hence we tested the hypothesis that the decline in fatigue life with ageing in energy storing tendons would be more pronounced in the IFM than in fascicles. We further hypothesised that tendon subunit fatigue resistance would not alter with ageing in positional tendons. Fascicles and IFM from young and old energy storing and positional tendons were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results show that both IFM and fascicles from the SDFT exhibit a similar magnitude of reduced fatigue life with ageing. By contrast, the fatigue life of positional tendon subunits was unaffected by ageing. The age-related decline in fatigue life of tendon subunits in energy storing tendons is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms resulting in this reduced fatigue life will aid in the development of treatments and interventions to prevent age-related tendinopathy. STATEMENT OF SIGNIFICANCE: Understanding the effect of ageing on tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for age-related tendon injury. In this study, we demonstrate for the first time that the fatigue resistance of the interfascicular matrix decreases with ageing in energy storing tendons. This is likely to contribute to the increased risk of injury in aged tendons. Full understanding of the mechanisms that result in this reduced fatigue resistance will aid in the development of treatments and interventions to prevent age-related tendinopathy.

A Clinical, Biological, and Biomaterials Perspective into Tendon Injuries and Regeneration.

Tendon injury is common and debilitating, and it is associated with long-term pain and ineffective healing. It is estimated to afflict 25% of the adult population and is often a career-ending disease in athletes and racehorses. Tendon injury is associated with high morbidity, pain, and long-term suffering for the patient. Due to the low cellularity and vascularity of tendon tissue, once damage has occurred, the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Current treatment options focus on pain management, often being palliative and temporary and ending in reduced function. Most treatments available do not address the underlying cause of the disease and, as such, are often ineffective with variable results. The need for an advanced therapeutic that addresses the underlying pathology is evident. Tissue engineering and regenerative medicine is an emerging field that is aimed at stimulating the body's own repair system to produce de novo tissue through the use of factors such as cells, proteins, and genes that are delivered by a biomaterial scaffold. Successful tissue engineering strategies for tendon regeneration should be built on a foundation of understanding of the molecular and cellular composition of healthy compared with damaged tendon, and the inherent differences seen in the tissue after disease. This article presents a comprehensive clinical, biological, and biomaterials insight into tendon tissue engineering and regeneration toward more advanced therapeutics.

Fascicles and the interfascicular matrix show adaptation for fatigue resistance in energy storing tendons.

UNLABELLED: Tendon is composed of rope-like fascicles, bound together by interfascicular matrix (IFM). Our previous work shows that the IFM is critical for tendon function, facilitating sliding between fascicles to allow tendons to stretch. This function is particularly important in energy storing tendons, which experience extremely high strains during exercise, and therefore require the capacity for considerable inter-fascicular sliding and recoil. This capacity is not required in positional tendons. Whilst we have previously described the quasi-static properties of the IFM, the fatigue resistance of the IFM in functionally distinct tendons remains unknown. We therefore tested the hypothesis that fascicles and IFM in the energy storing equine superficial digital flexor tendon (SDFT) are more fatigue resistant than those in the positional common digital extensor tendon (CDET). Fascicles and IFM from both tendon types were subjected to cyclic fatigue testing until failure, and mechanical properties were calculated. The results demonstrated that both fascicles and IFM from the energy storing SDFT were able to resist a greater number of cycles before failure than those from the positional CDET. Further, SDFT fascicles and IFM exhibited less hysteresis over the course of testing than their counterparts in the CDET. This is the first study to assess the fatigue resistance of the IFM, demonstrating that IFM has a functional role within tendon and contributes significantly to tendon mechanical properties. These data provide important advances into fully characterising tendon structure-function relationships. STATEMENT OF SIGNIFICANCE: Understanding tendon-structure function relationships is crucial for the development of effective preventative measures and treatments for tendon injury. In this study, we demonstrate for the first time that the interfascicular matrix is able to withstand a high degree of cyclic loading, and is specialised for improved fatigue resistance in energy storing tendons. These findings highlight the importance of the interfascicular matrix in the function of energy storing tendons, and potentially provide new avenues for the development of treatments for tendon injury which specifically target the interfascicular matrix.

Distribution of proteins within different compartments of tendon varies according to tendon type.

Although the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the energetic cost of locomotion. To maximise energy storage and return, energy-storing tendons need to be more extensible and elastic than tendons with a purely positional function. These properties are conferred in part by a specialisation of a specific compartment of the tendon, the interfascicular matrix, which enables sliding and recoil between adjacent fascicles. However, the composition of the interfascicular matrix is poorly characterised and we therefore tested the hypothesis that the distribution of elastin and proteoglycans differs between energy-storing and positional tendons, and that protein distribution varies between the fascicular matrix and the interfascicular matrix, with localisation of elastin and lubricin to the interfascicular matrix. Protein distribution in the energy-storing equine superficial digital flexor tendon and positional common digital extensor tendon was assessed using histology and immunohistochemistry. The results support the hypothesis, demonstrating enrichment of lubricin in the interfascicular matrix in both tendon types, where it is likely to facilitate interfascicular sliding. Elastin was also localised to the interfascicular matrix, specifically in the energy-storing superficial digital flexor tendon, which may account for the greater elasticity of the interfascicular matrix in this tendon. A differential distribution of proteoglycans was identified between tendon types and regions, which may indicate a distinct role for each of these proteins in tendon. These data provide important advances into fully characterising structure-function relationships within tendon.

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

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 interfascicular matrix enables fascicle sliding and recovery in tendon, and behaves more elastically in energy storing tendons.

While the predominant function of all tendons is to transfer force from muscle to bone and position the limbs, some tendons additionally function as energy stores, reducing the cost of locomotion. Energy storing tendons experience extremely high strains and need to be able to recoil efficiently for maximum energy storage and return. In the equine forelimb, the energy storing superficial digital flexor tendon (SDFT) has much higher failure strains than the positional common digital extensor tendon (CDET). However, we have previously shown that this is not due to differences in the properties of the SDFT and CDET fascicles (the largest tendon subunits). Instead, there is a greater capacity for interfascicular sliding in the SDFT which facilitates the greater extensions in this particular tendon (Thorpe et al., 2012). In the current study, we exposed fascicles and interfascicular matrix (IFM) from the SDFT and CDET to cyclic loading followed by a test to failure. The results show that IFM mechanical behaviour is not a result of irreversible deformation, but the IFM is able to withstand cyclic loading, and is more elastic in the SDFT than in the CDET. We also assessed the effect of ageing on IFM properties, demonstrating that the IFM is less able to resist repetitive loading as it ages, becoming stiffer with increasing age in the SDFT. These results provide further indications that the IFM is important for efficient function in energy storing tendons, and age-related alterations to the IFM may compromise function and predispose older tendons to injury.

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.

Tendon extracellular matrix damage, degradation and inflammation in response to in vitro overload exercise.

The role of inflammation in tendon injury is uncertain and a topic of current interest. In vitro studies of tendon accelerated overload damage can serve as a valuable source of information on the early stages of tendinopathy. Viable fascicle bundles from bovine flexor tendons were subjected to cyclic uniaxial loading from 1-10% strain. Immuno-staining for inflammatory markers and matrix degradation markers was performed on the samples after mechanical testing. Loaded samples exhibited visible extracellular matrix damage, with disrupted collagen fibers and fiber kinks, and notable damage to the interfascicular matrix. Inflammatory markers COX-2 and IL-6 were only expressed in the cyclically loaded samples. Collagen degradation markers MMP-1 and C1,2C were colocalized in many areas, with staining occurring in the interfascicular matrix or the fascicular tenocytes. These markers were present in control samples, but staining became increasingly intense with loading. Little MMP-3 or MMP-13 was evident in control sections. In loaded samples, some sections showed intense staining of these markers, again localized to interfascicular regions. This study suggests that inflammatory markers may be expressed rapidly after tendon overload exercise. Interestingly, both inflammation and damage-induced matrix remodeling seem to be concentrated in, or in the vicinity of, the highly cellular interfascicular matrix.

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.

Early stage fatigue damage occurs in bovine tendon fascicles in the absence of changes in mechanics at either the gross or micro-structural level.

Many tendon injuries are believed to result from repetitive motion or overuse, leading to the accumulation of micro-damage over time. In vitro fatigue loading can be used to characterise damage during repeated use and investigate how this may relate to the aetiology of tendinopathy. This study considered the effect of fatigue loading on fascicles from two functionally distinct bovine tendons: the digital extensor and deep digital flexor. Micro-scale extension mechanisms were investigated in fascicles before or after a period of cyclic creep loading, comparing two different measurement techniques - the displacement of a photo-bleached grid and the use of nuclei as fiducial markers. Whilst visual damage was clearly identified after only 300 cycles of creep loading, these visual changes did not affect either gross fascicle mechanics or fascicle microstructural extension mechanisms over the 900 fatigue cycles investigated. However, significantly greater fibre sliding was measured when observing grid deformation rather than the analysis of nuclei movement. Measurement of microstructural extension with both techniques was localised and this may explain the absence of change in microstructural deformation in response to fatigue loading. Alternatively, the data may demonstrate that fascicles can withstand a degree of matrix disruption with no impact on mechanics. Whilst use of a photo-bleached grid to directly measure the collagen is the best indicator of matrix deformation, nuclei tracking may provide a better measure of the strain perceived directly by the cells.

Effect of fatigue loading on structure and functional behaviour of fascicles from energy-storing tendons.

Tendons can broadly be categorized according to their function: those that act purely to position the limb and those that have an additional function as energy stores. Energy-storing tendons undergo many cycles of large deformations during locomotion, and so must be able to extend and recoil efficiently, rapidly and repeatedly. Our previous work has shown rotation in response to applied strain in fascicles from energy-storing tendons, indicating the presence of helical substructures which may provide greater elasticity and recovery. In the current study, we assessed how preconditioning and fatigue loading affect the ability of fascicles from the energy-storing equine superficial digital flexor tendon to extend and recoil. We hypothesized that preconditioned samples would exhibit changes in microstructural strain response, but would retain their ability to recover. We further hypothesized that fatigue loading would result in sample damage, causing further alterations in extension mechanisms and a significant reduction in sample recovery. The results broadly support these hypotheses: preconditioned samples showed some alterations in microstructural strain response, but were able to recover following the removal of load. However, fatigue loaded samples showed visual evidence of damage and exhibited further alterations in extension mechanisms, characterized by decreased rotation in response to applied strain. This was accompanied by increased hysteresis and decreased recovery. These results suggest that fatigue loading results in a compromised helix substructure, reducing the ability of energy-storing tendons to recoil. A decreased ability to recoil may lead to an impaired response to further loading, potentially increasing the likelihood of injury.

Fascicles from energy-storing tendons show an age-specific response to cyclic fatigue loading.

Some tendons, such as the human Achilles and equine superficial digital flexor tendon (SDFT), act as energy stores, stretching and recoiling to increase efficiency during locomotion. Our previous observations of rotation in response to applied strain in SDFT fascicles suggest a helical structure, which may provide energy-storing tendons with a greater ability to extend and recoil efficiently. Despite this specialization, energy-storing tendons are prone to age-related tendinopathy. The aim of this study was to assess the effect of cyclic fatigue loading (FL) on the microstructural strain response of SDFT fascicles from young and old horses. The data demonstrate two independent age-related mechanisms of fatigue failure; in young horses, FL caused low levels of matrix damage and decreased rotation. This suggests that loading causes alterations to the helix substructure, which may reduce their ability to recoil and recover. By contrast, fascicles from old horses, in which the helix is already compromised, showed greater evidence of matrix damage and suffer increased fibre sliding after FL, which may partially explain the age-related increase in tendinopathy. Elucidation of helix structure and the precise alterations occurring owing to both ageing and FL will help to develop appropriate preventative and repair strategies for tendinopathy.

Functionally distinct tendon fascicles exhibit different creep and stress relaxation behaviour.

Most overuse tendinopathies are thought to be associated with repeated microstrain below the failure threshold, analogous to the fatigue failure that affects materials placed under repetitive loading. Investigating the progression of fatigue damage within tendons is therefore of critical importance. There are obvious challenges associated with the sourcing of human tendon samples for in vitro analysis so animal models are regularly adopted. However, data indicates that fatigue life varies significantly between tendons of different species and with different stresses in life. Positional tendons such as rat tail tendon or the bovine digital extensor are commonly applied in in vitro studies of tendon overuse, but there is no evidence to suggest their behaviour is indicative of the types of human tendon particularly prone to overuse injuries. In this study, the fatigue response of the largely positional digital extensor and the more energy storing deep digital flexor tendon of the bovine hoof were compared to the semitendinosus tendon of the human hamstring. Fascicles from each tendon type were subjected to either stress or strain controlled fatigue loading (cyclic creep or cyclic stress relaxation respectively). Gross fascicle mechanics were monitored after cyclic stress relaxation and the mean number of cycles to failure investigated with creep loading. Bovine extensor fascicles demonstrated the poorest fatigue response, while the energy storing human semitendinosus was the most fatigue resistant. Despite the superior fatigue response of the energy storing tendons, confocal imaging suggested a similar degree of damage in all three tendon types; it appears the more energy storing tendons are better able to withstand damage without detriment to mechanics.

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

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 1Hz for 48h. TGFß1 and TGFßRI inhibitor were added to selected cultures. RNA was measured using qRT-PCR and TGFß 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 TGFß stimulation alone. We have also demonstrated that the inhibition of the TGFß signalling pathway abrogated the strain induced changes in mRNA and that TGFß activation, rather than gene expression, was increased with mechanical strain. We concluded that TGFß activation plays an important role in mechanotransduction. Targeting this pathway may have its place in the treatment of tendinopathy.

Helical sub-structures in energy-storing tendons provide a possible mechanism for efficient energy storage and return.

The predominant function of tendons is to position the limb during locomotion. Specific tendons also act as energy stores. Energy-storing (ES) tendons are prone to injury, the incidence of which increases with age. This is likely related to their function; ES tendons are exposed to higher strains and require a greater ability to recoil than positional tendons. The specialized properties of ES tendons are thought to be achieved through structural and compositional differences. However, little is known about structure-function relationships in tendons. This study uses fascicles from the equine superficial digital flexor (SDFT) and common digital extensor (CDET) as examples of ES and positional tendons. We hypothesized that extension and recoil behaviour at the micro-level would differ between tendon types, and would alter with age in the injury-prone SDFT. Supporting this, the results show that extension in the CDET is dominated by fibre sliding. By contrast, greater rotation was observed in the SDFT, suggesting a helical component to fascicles in this tendon. This was accompanied by greater recovery and less hysteresis loss in SDFT samples. In samples from aged SDFTs, the amount of rotation and the ability to recover decreased, while hysteresis loss increased. These findings indicate that fascicles in the ES SDFT may have a helical structure, enabling the more efficient recoil observed. Further, the helix structure appears to alter with ageing; this coincides with a reduction in the ability of SDFT fascicles to recoil. This may affect tendon fatigue resistance and predispose aged tendons to injury.