Single cell multi-omics characterise discrete human tendon cells populations that persist in vitro and on fibrous scaffolds.

Chronic tendinopathy represents a growing healthcare burden in the ageing global population. Curative therapies remain elusive as the mechanisms that underlie chronic inflammation in tendon disease remain unclear. Identifying and isolating key pathogenic and reparative cells is essential in developing precision therapies and implantable materials for improved tendon healing. Multiple discrete human tendon cell populations have been previously described ex vivo. To determine if these populations persist in vitro, healthy human hamstring tenocytes were cultured for 8 d on either tissue culture plastic or aligned electrospun fibres of absorbable polydioxanone. Novel single-cell surface proteomics combined with unbiased single-cell transcriptomics (CITE-Seq) was used to identify discrete tenocyte populations. 6 cell populations were found, 4 of which shared key gene expression determinants with ex vivo human cell clusters: PTX3_PAPPA, POSTN_SCX, DCN_LUM and ITGA7_NES. Surface proteomics found that PTX3_PAPPA cells were CD10+CD26+CD54+. ITGA7_NES cells were CD146+ and POSTN_SCX cells were CD90+CD95+CD10+. Culture on the aligned electrospun fibres favoured 3 cell subtypes (DCN_LUM, POSTN_SCX and PTX3_ PAPPA), promoting high expression of tendon-matrix-associated genes and upregulating gene sets enriched for TNF-a and IL-6/STAT3 signalling. Discrete human tendon cell subpopulations persisted in in vitro culture and could be recognised by specific gene and surface-protein signatures. Aligned polydioxanone fibres promoted the survival of 3 clusters, including pro-inflammatory PTX3-expressing CD10+CD26+CD54+ cells found in chronic tendon disease. These results improved the understanding of preferred culture conditions for different tenocyte subpopulations and informed the development of in vitro models of tendon disease.

Cytokines in tendon disease: A Systematic Review.

OBJECTIVES: Emerging evidence indicates that tendon disease is an active process with inflammation that is critical to disease onset and progression. However, the key cytokines responsible for driving and sustaining inflammation have not been identified. METHODS: We performed a systematic review of the literature using MEDLINE (U.S. National Library of Medicine, Bethesda, Maryland) in March 2017. Studies reporting the expression of interleukins (ILs), tumour necrosis factor alpha (TNF-α) and interferon gamma in diseased human tendon tissues, and animal models of tendon injury or exercise in comparison with healthy control tissues were included. RESULTS: IL-1ß, IL-6, IL-10, and TNF-α are the cytokines that have been most frequently investigated. In clinical samples of tendinopathy and tendon tears, the expression of TNF-α tended not to change but IL-6 increased in tears. Healthy human tendons showed increased IL-6 expression after exercise; however, IL-10 remained unchanged. Animal tendon injury models showed that IL-1ß, IL-6, and TNF-α tend to increase from the early phase of tendon healing. In animal exercise studies, IL-1ß expression showed a tendency to increase at the early stage after exercise, but IL-10 expression remained unchanged with exercise. CONCLUSIONS: This review highlights the roles of IL-1ß, IL-6, IL-10, and TNF-α in the development of tendon disease, during tendon healing, and in response to exercise. However, there is evidence accumulating that suggests that other cytokines are also contributing to tendon inflammatory processes. Further work with hypothesis-free methods is warranted in order to identify the key cytokines, with subsequent mechanistic and interaction studies to elucidate their roles in tendon disease development.Cite this article: W. Morita, S. G. Dakin, S. J. B. Snelling, A. J. Carr. Cytokines in tendon disease: A Systematic Review. Bone Joint Res 2017;6:656-664. DOI: 10.1302/2046-3758.612.BJR-2017-0112.R1.

Resorbable electrospun polydioxanone fibres modify the behaviour of cells from both healthy and diseased human tendons.

Chronic tendinopathy in an active and ageing population represents an increasing burden to healthcare systems. Rotator cuff tendinopathy alone accounts for approximately 70 % of all shoulder pain. Tendinopathic tissue has a disorganised extracellular matrix, altered vasculature, and infiltration of fibroblasts and inflammatory cells. This altered biology may contribute to the limited success of surgical repair strategies. Electrospun resorbable scaffolds can potentially enhance endogenous repair mechanisms by influencing the tissue microenvironment. Polydioxanone (PDO) has an established safety profile in patients. We compared the response of healthy and diseased human tendon cells to electrospun PDO fibres using live cell imaging, proliferation, flow cytometry, and gene expression studies. Within 4 h of initial contact with electrospun PDO, healthy tendon cells underwent a marked transformation; elongating along the fibres in a fibre density dependent manner. Diseased tendon cells initially responded at a slower rate, but ultimately underwent a similar morphological change. Electrospun fibres increased the proliferation rate of diseased tendon cells and increased the ratio of type I:IIIcollagenmRNA expression. Flow cytometry revealed decreased expression of CD106, a marker of mesenchymal stem cells, and increased expression of CD10 on healthy versus diseased tendon cells. PDO electrospun scaffolds further promoted CD106negCD10pos expression of healthy tendon cells. Despite their behavioural differences, both healthy and diseased human tendon cells responded to electrospun PDO fibres. This encourages further work establishing their efficacy in augmenting surgical repair of diseased tendons.

The response of tenocytes to commercial scaffolds used for rotator cuff repair.

Surgical repairs of rotator cuff tears have high re-tear rates and many scaffolds have been developed to augment the repair. Understanding the interaction between patients' cells and scaffolds is important for improving scaffold performance and tendon healing. In this in vitro study, we investigated the response of patient-derived tenocytes to eight different scaffolds. Tested scaffolds included X-Repair, Poly-Tape, LARS Ligament, BioFiber (synthetic scaffolds), BioFiber-CM (biosynthetic scaffold), GraftJacket, Permacol, and Conexa (biological scaffolds). Cell attachment, proliferation, gene expression, and morphology were assessed. After one day, more cells attached to synthetic scaffolds with dense, fine and aligned fibres (X-Repair and Poly-Tape). Despite low initial cell attachment, the human dermal scaffold (GraftJacket) promoted the greatest proliferation of cells over 13 days. Expression of collagen types I and III were upregulated in cells grown on non-cross-linked porcine dermis (Conexa). Interestingly, the ratio of collagen I to collagen III mRNA was lower on all dermal scaffolds compared to synthetic and biosynthetic scaffolds. These findings demonstrate significant differences in the response of patient-derived tendon cells to scaffolds that are routinely used for rotator cuff surgery. Synthetic scaffolds promoted increased cell adhesion and a tendon-like cellular phenotype, while biological scaffolds promoted cell proliferation and expression of collagen genes. However, no single scaffold was superior. Our results may help understand the way that patients' cells interact with scaffolds and guide the development of new scaffolds in the future.

The relationship between in vivo limb and in vitro tendon mechanics after injury: a potential novel clinical tool for monitoring tendon repair.

REASONS FOR PERFORMING THE STUDY: Highly prevalent superficial digital flexor tendon (SDFT) injury results in compromised tendon function through fibrosis and high frequency of re-injury due to altered biomechanical function. This study investigated the consequences of SDF tendinopathy on limb mechanics in relation to the mechanical properties of injured tendon. OBJECTIVES: To develop and validate a noninvasive in vivo assessment of tendon mechanics to investigate the effect of recent SDFT injury on limb stiffness index, providing an objective method to assess quality of healing. HYPOTHESES: Limb stiffness index would reduce as a consequence of SDFT injury and progressively increase during tendon healing and correlate with in vitro mechanical properties of the respective SDFTs. METHODS: Kinematic analysis was performed at walk in 10 horses that had sustained career-ending SDFT injury. Stiffness index was derived from limb force recorded via a series of force plates and measurement of change in metacarpophalangeal joint angle using 3D motion analysis software. Horses were subjected to euthanasia 7 months after injury, the SDFTs removed and subjected to nondestructive in vitro mechanical testing. RESULTS: Limb stiffness index was reduced following SDFT injury in comparison with the contralateral limb and increased during the convalescent period, approximating that of the contralateral limb by 7 months post injury. There was a significant positive correlation between in vivo limb stiffness index and in vitro SDFT stiffness. CLINICAL RELEVANCE: The ability to assess and monitor SDFT mechanical competence through limb stiffness measurement techniques in horses recovering from SDFT injury and the possibility of corroborating this with functional tendon healing may permit a more objective and accurate assessment of optimal tendon repair in the horse. This technique may be a useful method for assessing the efficacy of treatment regimens for tendinopathy and could be utilised to predict time to safe return to performance or re-injury.

Osseous abnormalities associated with collateral desmopathy of the distal interphalangeal joint: part 1.

REASONS FOR PERFORMING STUDY: Osseous abnormalities associated with collateral ligament (CL) injury of the distal interphalangeal (DIP) joint have been documented using magnetic resonance imaging (MRI) but there is currently limited information about the frequency of osseous pathology associated with CL injury. OBJECTIVES: To determine the frequency of occurrence of osseous abnormality coexistent with CL injury of the DIP joint and describe the distribution and character of osseous lesions; and to establish if there was an association between osseous abnormality and increased radiopharmaceutical uptake (IRU). HYPOTHESES: There would be a higher incidence of osseous abnormality at the insertion of an injured CL than at the origin; and a relationship between the presence of osseous abnormality and duration of lameness. MATERIALS AND METHODS: Magnetic resonance images of 313 feet of 289 horses with foot pain and a definitive diagnosis of collateral desmopathy of the DIP joint were analysed retrospectively for presence and type of osseous abnormality in the middle and distal phalanges. Scintigraphic images were examined and the presence of IRU in the middle or distal phalanges recorded. RESULTS: Osseous abnormalities were detected in 143 (45.7%) feet, 27 (18.8%) of which had osseous and CL injury alone, while the remaining 116 had CL related osseous injury and multiple injuries within the hoof capsule. Entheseous new bone and endosteal irregularity of the middle and distal phalanges were the most frequent types of osseous abnormality. There was a higher incidence of osseous abnormalities medially than laterally and at the ligament insertion than at the origin. There was a significant association between presence of IRU and osseous injury. CONCLUSIONS: A variety of osseous lesions of differing severity are associated with CL injury. Normal radiopharmaceutical uptake does not preclude significant osseous pathology associated with CL injury. CLINICAL RELEVANCE: Further studies are necessary in order to determine if osseous abnormalities associated with CL injury influence prognosis for return to performance.

Osseous abnormalities associated with collateral desmopathy of the distal interphalangeal joint. Part 2: treatment and outcome.

REASONS FOR PERFORMING STUDY: There are currently few long-term follow-up data relating to recovery from injury of a collateral ligament (CL) of the distal interphalangeal (DIP) joint and limited information about the effect of associated osseous injury on prognosis. OBJECTIVES: To describe long-term follow-up results for horses with CL injury, with and without associated osseous injury; and to determine the effect of extracorporeal shock wave therapy (ECSWT) or radial pressure wave therapy (RPWT) on outcome. HYPOTHESES: Prognosis for return to performance for horses with CL-related osseous injury would be worse than for horses with CL injury alone. METHODS: Magnetic resonance images from 313 feet of 289 horses with foot pain and a definitive diagnosis of collateral desmopathy of the DIP joint were analysed retrospectively for presence of osseous abnormality associated with the ligament origin or insertion and the middle and distal phalanges. Horses were assigned to groups according to the combination of their injuries. Type of treatment was recorded and follow-up information obtained. Thirty-two horses with additional sources of lameness were excluded from analysis of outcome. RESULTS: Follow-up data were available for 182 horses, 55 of which had follow-up information for up to 2 years after presentation. Twenty-seven percent of horses with CL injury alone and 34% of horses with CL related osseous injury returned to their previous performance level. Prognosis for a combination of injuries to multiple soft tissue and osseous structures within the hoof capsule was substantially worse. There was no effect of ECSWT or RPWT on outcome. CONCLUSIONS: The presence of mild to moderate CL related osseous injury does not appear to influence prognosis compared with CL injury alone. CLINICAL RELEVANCE: Further studies of a larger number of horses are necessary in order to ascertain if specific types of osseous pathology influence return to performance levels.

How many positions can we perceptually encode, one or many?

Here we show that our sensitivity for discriminating relative position across the visual field is limited. In experiment 1 we show that we are much worse at detecting a texture defined by the relative position of elements within an array than would be expected if we had access to multiple estimates of relative position across the visual field. In experiment 2 we show that human performance is impaired for positional judgments when there is uncertainty as to which of a number of possible elements is misaligned. This impairment is greater than one would expect from an ideal observer model and greater than that found for a comparable task involving orientation. It is consistent with positional thresholds being determined by only one estimate of relative position. In experiment 3 we estimate the number of suprathreshold positional signals that can be pooled at the same time across the visual field using a standard summation variance paradigm. The results suggest that the human visual system is limited to one estimate of position, but additional estimates can be built up serially over time; however, this process is slow and probably cognitive in nature. These experiments taken as a whole suggest that only one estimate of relative position (i.e. relative to a predefined reference) at a time is accessible at the perceptual level.

Summation of concentric orientation structure: seeing the Glass or the window?

Rotational Glass patterns are discriminable from noise at substantially lower signal-to-noise levels than translational patterns, a finding that has been attributed to the operation of concentrically tuned units in cortical area V4 (Wilson, Wilkinson, & Asaad, Vis. Res. 37 (17) (1997) 2325; Wilson & Wilkinson, Vis. Res. 38 (19) (1998) 2933). Under experimental conditions similar to Wilson et al. we found this advantage to be largely contingent on the pattern being viewed through a circular aperture. Because rotation of a random dot set cannot lead to the presence of unmatched dots at the boundary of a circular aperture, the integrity of low spatial frequency information at the boundary reliably indicates the presence of rotational, but not translational, structure. When we removed this cue, either using a square aperture or surrounding a round aperture with noise dots, none of the nine subjects tested showed any statistically significant advantage for rotational Glass patterns (although at least two did take longer to master the task with translational compared to rotational patterns). We go on to show generally similar patterns of global integration for both rotational and translational patterns. We conclude that this paradigm presently offers no concrete psychophysical evidence for specialised concentric orientation detectors.

Snakes and ladders: the role of temporal modulation in visual contour integration.

We investigated temporal aspects of the cortical mechanisms supporting visual contour integration by measuring observers' efficiency at detecting fragmented contours, composed of Gabor micropatterns, embedded in a field of distractor elements. Gabors consisted of a static Gaussian enveloping a sinusoidal carrier which was temporally modulated by motion or counter-phase flicker. The elements forming the path could be oriented either parallel ('snakes') or perpendicular to the contour orientation ('ladders'). Sensitivity to contour structure (estimated by measuring the maximum tolerable element orientation jitter supporting contour detection) was increased when the elements were drifting or flickering. Snakes were more detectable than ladders under all conditions. The increase in sensitivity conferred by drifting carriers was present even when the elements in the same stimulus were drifting at a range of speeds spanning almost three octaves. These results lend further support to the notion that the contour integration system receives separate transient and sustained input.

Contour interaction in amblyopia: scale selection.

It has been known for some time that visual acuity in amblyopia is higher for single letters than for letters in a row (termed crowding). Early work showed that this could not be accounted for on the basis of the destructive interaction of adjacent contours (termed contour interaction), which was shown to be, in resolution units, normal in amblyopia. We have re-examined this issue using a letter stimulus that is modulated about a mean light level. This allows an examination of the effects of contrast polarity and spatial filtering within the contour interaction paradigm. We show that the majority of strabismic amblyopes that we investigated exhibit an anomalous contour interaction that, in some cases, was dependent on the contrast polarity of the flanking stimuli. Furthermore, we show that while amblyopes do select the optimum scale of analysis for unflanked stimuli, they do not select the optimum scale of analysis for flanked stimuli. For reasons that may have to do with their poorer shape discrimination, they select a non-optimal scale to process flanked stimuli.

Information limit on the spatial integration of local orientation signals.

Channel-based models of human spatial vision require that the output of spatial filters be pooled across space. This pooling yields global estimates of local feature attributes such as orientation that are useful in situations in which that attribute may be locally variable, as is the case for visual texture. The spatial characteristics of orientation summation are considered in the study. By assessing the effect of orientation variability on observers' ability to estimate the mean orientation of spatially unstructured textures, one can determine both the internal noise on each orientation sample and the number of samples being pooled. By a combination of fixing and covarying the size of textured regions and the number of elements constituting them, one can then assess the effects of the texture's size, density, and numerosity (the number of elements present) on the internal noise and the sampling density. Results indicate that internal noise shows a primary dependence on texture density but that, counterintuitively, subjects rely on a sample size approximately equal to a fixed power of the number of samples present, regardless of their spatial arrangement. Orientation pooling is entirely flexible with respect to the position of input features.

Local and global visual grouping: tuning for spatial frequency and contrast.

Glass patterns are visual textures composed of a field of dot pairs (dipoles) whose orientations are determined by a simple geometrical transformation, such as a rotation. Detection of structure in these patterns requires the observer to perform local grouping (to find dipoles) and global grouping to combine their orientations into a percept of overall shape. We estimated the spatial frequency tuning of these grouping processes by measuring signal-to-noise detection thresholds for Glass patterns composed of spatially narrow-band elements. Local tuning was probed by varying the spatial frequency difference between the two elements comprising each dipole. Global tuning was estimated using dipoles containing one spatial frequency and then estimating masking as a function of the spatial frequency of randomly positioned noise elements. We report that the tuning of local grouping is band-pass (ie, it is responsive to a narrow range of spatial frequencies), but that tuning of global grouping is broad and low-pass (ie, it integrates across a broader range of lower spatial frequencies). Control experiments examined how the contrast and visibility of elements might contribute to these findings. Local grouping proved to be more resistant to local contrast variation than global grouping. We conclude that local grouping is consistent with the use of simple-oriented filtering mechanisms. Global grouping seems to depend more on the visibility of elements that can be affected by both spatial frequency and contrast.

Impoverished second-order input to global linking in human vision.

Recent evidence points to the importance of global operations across spatial regions larger than individual cortical receptive fields. Studies of contour integration and motion trajectory detection suggest that network operations between local detectors underlie the encoding of extended contours in space and extended trajectories in motion. Here we ask whether such network operations also occur between second-order-detectors known to exist in visual cortex. We compared performance for stimuli composed of either first-order or second-order elements equated for visibility, and we show that unlike the first-order case, there is little or no linking interaction between local second-order detectors. Near chance performance was found for elements defined by second-order attributes when observers had to identify either an elongated spatial contour or an extended motion trajectory embedded in noise elements. This implies that the network operations thought to underlie these two global tasks receive, at best, an impoverished input from local detectors that encode second-order image attributes.

Contour interaction in fovea and periphery.

It has been known for some time that both foveal and peripheral visual acuity are higher for single letters than for letters in a row. Early work showed that this was due to the destructive interaction of adjacent contours (termed contour interaction). It has been assumed to have a neural basis, and a number of competing explanations have been advanced that implicate either high-level or low-level stages of visual processing. Our previous results for foveal vision suggested a much simpler explanation, one determined primarily by the physics of the stimulus rather than the physiology of the visual system. We show that, under conditions of contour interaction or crowding, the most relevant physical spatial-frequency band of the letter is displaced to higher spatial frequencies and that foveal vision tracks this change in spatial scale. In the periphery, however, beyond 5 degrees, the physical explanation is not sufficient. Here we show that there are genuine physiological lateral spatial interactions, which are due to changes in the spatial scale of analysis.

Sensitivity to contrast modulation depends on carrier spatial frequency and orientation.

We consider how the detection of second-order contrast structure depends on the orientation and spatial frequency of first-order luminance structure. For patterns composed of a bandpass noise carrier multiplied by a contrast envelope function, we show that sensitivity to the envelope varies in proportion to the spatial frequency of the carrier. For oriented carriers at low spatial-frequencies, detection of the contrast envelope is easier when the envelope and carrier are perpendicular, but this dependency diminishes as the spatial frequency of the carrier increases. These differences are not attributable to either the detection of side-bands, or the presence of spurious contrast structure in unmodulated carrier images. A final experiment measured envelope detection in the presence of noise masks. Results indicate that orientationally and spatially-band pass filtering precedes the detection of second-order structure.

The foveal 'crowding' effect: physics or physiology?

It has been known for some time that both foveal and peripheral visual acuity is higher for single letters than for letters in a row. Early work showed that this was due to the destructive interaction of adjacent contours (termed 'crowding' or contour interaction). It has been assumed to have a neural basis and a number of competing explanations have been advanced which implicate either high-level or low-level stages of visual processing. Our results suggest a much simpler explanation, one primarily determined by the physics of the stimulus rather than the physiology of the visual system. We show that, under conditions of contour interaction or 'crowding', the most relevant physical spatial frequency band of the letter is displaced to higher spatial frequencies and that foveal vision tracks this change in spatial scale.