Purpose. Collagen-rich structures of the knee are prone to damage through acute injury or chronic “wear and tear”. Collagen becomes more disorganised in degenerative tissue e.g. osteoarthritis. An alignment index (AI) used to analyse orientation distribution of collagen-rich structures is presented. Method. A healthy caprine knee was scanned in a Siemens Verio 3T Scanner. The caprine knee was rotated and scanned in nine directions to the main magnetic field B. 0. A 3D PD SPACE sequence with isotropic 1×1×1mm voxels (TR1300ms, TE13ms, FOV256mm,) was optimised to allow for a greater angle-sensitive contrast. For each collagen-rich voxel the orientation vector is computed using Szeverenyi and Bydder's method. Each orientation vector reflects the net effect of all the fibres comprised within a voxel. The assembly of all unit vectors represents the fibre orientation map. Alignment Index (AI) in any direction is defined as a ratio of the fraction of orientations within 20° (solid angle) centred in that direction to the same fraction in a random (flat) case. In addition, AI is normalised in such a way that AI=0 indicates isotropic collagen alignment. Increasing AI values indicate increasingly aligned structures: AI=1 indicates that all collagen fibres are orientated within the cone of 20° centred at the selected direction. AI = (nM - nRnd)/(nTotal - nRnd) if nM >= nRnd. AI = 0 if nM < nRnd. Where:. nM is a number of reconstructed orientations that are within a cone of 20° centred in selected direction. nRnd is a number of random orientations within a cone of 20° around selected direction. nTotal is a number of collagen reach voxels. By computing AI for a regular gridded orientation space we are able to visualise change of AI on a hemisphere facilitating understanding of the collagen fibre orientation distribution. Results. The patella tendon had an AI=0.6453. The Anterior Cruciate Ligament (ACL) had an AI=0.2732. The meniscus had an AI=0.1847. Discussion. The most aligned knee structure is the patella tendon where the collagen fibres align with the skeleton to transmit forces through bones and muscles. This structure had the AI closest to 1. The ACL had the second highest AI and is composed of two fibre bundles aligned diagonally across the knee. The meniscus acts as a shock absorber and is made up of vertical, radial and circumferential fibres which disperse forces more equally. The complexity of the meniscal structure resulted in the lowest AI. To date, this technique has only been performed with healthy tissue; the AI may become closer to zero if there is damage disrupting the collagen fibre alignment. The AI can further our understanding of collagen orientation distribution and could be used as a quantitative, non-invasive measure of structural health

Sprains and strains result from collagen fibre overextension. This study investigated changes in the molecular state of collagen due to overextension damage, thereby gaining insight into tissue degeneration and cellular detection of damage. Overextension results in intermolecular and intrafibrillar sliding, detected with x-ray diffraction. Tendon rupture results in increased susceptibility to proteolytic enzymes. These observations and contemporary theory concerning collagen fibre stability lead to the hypothesis that sub-rupture overextension should result in reduced thermal stability of fibrous collagen. Tendons were harvested from steer tails. Each provided a specimen for control and for overextension. Sub-rupture overextension at 1%/s strain rate was accomplished on a mechanical testing system, under the control of custom software, until the slope of the force-deformation curve was approximately zero (before complete failure). Two loading treatments were tested: one-cycle and five-cycles. Two specimen types were tested: native tendons ± NaBH4 crosslink stabilization. Tendons in each of the four groups (2x2) were paired by originating tail. Thermal stability was assessed in terms of denaturation temperature (Td) using hydrothermal isometric tension testing. Specimens were held at constant length and heated from ambient temperature to 90degC. Td was defined as the temperature where load suddenly increased due to molecular unraveling and attempted shrinkage. Overextension of native specimens reduced the thermal stability of the collagen (p< 0.0001) and five-cycles had a still greater effect (p=0.03). Td of controls was 64.5±1.0degC (mean±SD). After one-cycle, Td dropped to 63.2±1.0degC and, after five-cycles, Td dropped to 61.8±2.0degC. For stabilised tendons, the effect of multiple cycles was lost (p=0.08) but overstretching decreased Td by ~2degC (p< 0.0001). This study confirms that the molecular state of collagen is altered by overextension damage, reducing Td by up to 10% of the expected range (37–65degC) in our experiments. This is thought to occur due to intermolecular sliding that liberates specific domains on the molecules, lowering the activation energy for uncoiling. These domains may also be key targets in degeneration and cell-collagen signaling

Introduction: Elucidation of the exact cause of adolescent idiopathic scoliosis (AIS) remains an elusive goal. The intervertebral disc is one of the many areas that have been investigated in an effort to find a cause for this condition. We hypothesize that a qualitative change in the orientation of collagen fibers in the annular layers of the disc could cause the deformity seen in AIS. This paper presents a mathematical model of such a change and how it could produce appropriate deforming forces. Hypothesis: In the normal disc the collagen fibers are obliquely orientated. Fibers in adjacent lamellae are orientated in opposing directions. This means that as forces are transmitted from a compressed nucleus to the annular fibers there is no net force tending to rotate one vertebra with respect to its neighbour. If there is a preponderance of fibers running in one direction as the nucleus is compressed there will be a net resultant force perpendicular to the long axis of the spine tending to produce an intervertebral rotation. This intervertebral rotation, applied to successive spinal segments will cause a scoliotic deformity. Model: The highly oriented structure of the AF suggests the utility of an explicit representation of the collagen fibres and their mechanical contribution to disc function. In our study we have considered two groups of fibres, representing the clockwise and counter clockwise fibres in the disc. The AF is considered as a continuum containing two populations of fibres assumes to be of equal density and uniform distribution within an isotropic material as originally described by Spencer. Nuclear compression as a result of growth was modelled as a tendency to produce increased intervertebral separation of spinal segments and examined whether the resultant transformation that leads to a scoliotic pattern of deformity. Based on anatomical data from literature the positions of the 12 nodes that represent the thoracic vertebrae are applied to the model. The three-dimensional location of each vertebral body is defined. We store the coordinates of thoracic vertebrae in a three-dimensional matrix. In the present study in order to involve the translation operation in our transformation, we have used the homogeneous transformation matrix or Denavit & Hartenberg matrix. In the present model for the initial set of transformations the reference axis is chosen to be the lowest vertebral axis (T-12) and remains unchanged throughout the transformation. All elements of the spine above the reference axis are transformed (translated and rotated). After completion of this iteration and storing the values for the origin coordinate and vector values in the next level of the matrix, the next reference axis is chosen. For the second axis everything above the axis will be transformed in the same way with the current axis and the one preceding it remaining unchanged. Therefore for each transformation a new reference axis is taken and the transformations are applied to all vectors and origins above it leaving all elements preceding it unchanged by the transformation. Results: The first part of the model shows that rotational displacement increases linearly with changes in the fibre ratio. Rotational displacement on the other hand occurs independently of distraction of the vertebral bodies. When the rotational displacement is applied to a series of segments it produces alterations of curvature in the three planes. Specifically it produces a lateral curvature in the coronal plane and a hypokyphotic curvature in the saggital plane. The magnitude of these displacements varies with the imbalance in fibre ratio. Discussion: The proposed changes in annular fiber orientation have been modeled using accepted mathematical methods. These changes will produce an intervertebral rotation whose magnitude depends on the degree of fiber imbalance akin to that seen in AIS. When the displacements produced by this rotation being applied to a series of segments is modeled, it will produce a three dimensional deformity similar to that seen in AIS. Ongoing histological studies are being performed to see if the proposed imbalance can be identified in patients with AIS. Such a fiber orientation anomaly may be genetically determined by some fashion of directional sense gene and may be the aetiological basis for AIS

The anulus fibrosus of the human lumbar intervertebral disc has a complex, hierarchical structure comprised of collagens, proteoglycans and elastic fibres. Recent histological studies have suggested that the elastic fibre network may play an important functional role. In this study, it was hypothesised that elastic fibres enhance the mechanical integrity of the extracellular matrix transverse to the direction of the collagen fibres. Using a combination of biochemically verified enzymatic treatments and biomechanical tests, it was demonstrated that degradation of elastic fibres resulted in a significant reduction in both the initial modulus and the ultimate modulus, and a significant increase in the extensibility, of radially oriented anulus fibrosus specimens. Separate treatments and mechanical tests were used to account for any changes attributable to non-specific degradation of glycosaminoglycans. Additionally, histological assessments provided a unique perspective on structural changes in the elastic fibre network in radially oriented specimens subjected to tensile deformations. The results of this study demonstrate that elastic fibres play an important and unique role in the mechanical properties of the anulus fibrosus, and provide the basis for the development of improved material models to describe intervertebral disc mechanical behaviour

Aims. Extracellular matrix (ECM) is a critical determinant of tissue mechanobiology, yet remains poorly characterized in joint tissues beyond cartilage in osteoarthritis (OA). This review aimed to define the composition and architecture of non-cartilage soft joint tissue structural ECM in human OA, and to compare the changes observed in humans with those seen in animal models of the disease. Methods. A systematic search strategy, devised using relevant matrix, tissue, and disease nomenclature, was run through the MEDLINE, Embase, and Scopus databases. Demographic, clinical, and biological data were extracted from eligible studies. Bias analysis was performed. Results. A total of 161 studies were included, which covered capsule, ligaments, meniscus, skeletal muscle, synovium, and tendon in both humans and animals, and fat pad and intervertebral disc in humans only. These studies covered a wide variety of ECM features, including individual ECM components (i.e. collagens, proteoglycans, and glycoproteins), ECM architecture (i.e. collagen fibre organization and diameter), and viscoelastic properties (i.e. elastic and compressive modulus). Some ECM changes, notably calcification and the loss of collagen fibre organization, have been extensively studied across osteoarthritic tissues. However, most ECM features were only studied by one or a few papers in each tissue. When comparisons were possible, the results from animal experiments largely concurred with those from human studies, although some findings were contradictory. Conclusion. Changes in ECM composition and architecture occur throughout non-cartilage soft tissues in the osteoarthritic joint, but most of these remain poorly defined due to the low number of studies and lack of healthy comparator groups. Cite this article: Bone Joint Res 2024;13(12):703–715

Aims. Developmental dysplasia of the hip (DDH) is a complex musculoskeletal disease that occurs mostly in children. This study aimed to investigate the molecular changes in the hip joint capsule of patients with DDH. Methods. High-throughput sequencing was used to identify genes that were differentially expressed in hip joint capsules between healthy controls and DDH patients. Biological assays including cell cycle, viability, apoptosis, immunofluorescence, reverse transcription polymerase chain reaction (RT-PCR), and western blotting were performed to determine the roles of the differentially expressed genes in DDH pathology. Results. More than 1,000 genes were differentially expressed in hip joint capsules between healthy controls and DDH. Both gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses revealed that extracellular matrix (ECM) modifications, muscle system processes, and cell proliferation were markedly influenced by the differentially expressed genes. Expression of Collagen Type I Alpha 1 Chain (COL1A1), COL3A1, matrix metalloproteinase-1 (MMP1), MMP3, MMP9, and MMP13 was downregulated in DDH, with the loss of collagen fibres in the joint capsule. Expression of transforming growth factor beta 1 (TGF-β1) was downregulated, while that of TGF-β2, Mothers against decapentaplegic homolog 3 (SMAD3), and WNT11 were upregulated in DDH, and alpha smooth muscle actin (αSMA), a key myofibroblast marker, showed marginal increase. In vitro studies showed that fibroblast proliferation was suppressed in DDH, which was associated with cell cycle arrest in G0/G1 and G2/M phases. Cell cycle regulators including Cyclin B1 (CCNB1), Cyclin E2 (CCNE2), Cyclin A2 (CCNA2), Cyclin-dependent kinase 1 (CDK1), E2F1, cell division cycle 6 (CDC6), and CDC7 were downregulated in DDH. Conclusion. DDH is associated with the loss of collagen fibres and fibroblasts, which may cause loose joint capsule formation. However, the degree of differentiation of fibroblasts to myofibroblasts needs further study. Cite this article: Bone Joint Res 2021;10(9):558–570

To characterize the microstructural organization of collagen fibers in human medial menisci and the response to mechanical loading in relation to age. We combine high resolution imaging with mechanical compression to visualize the altered response of the tissue at the microscale. Menisci distribute the load in the knee and are predominantly composed of water and specifically hierarchically arranged collagen fibers. Structural and compositional changes are known to occur in the meniscus during aging and development of osteoarthritis. However, how microstructural changes due to degeneration affect mechanical performance is still largely unknown [1]. Fresh frozen 4 mm Ø plugs of human medial menisci (n=15, men, 20-85 years) with no macroscopic damage nor known diseases from the MENIX biobank at Skåne University Hospital were imaged by phase contrast synchrotron tomography at the TOMCAT beamline (Paul Scherrer Institute, CH). A rheometer was implemented into the beamline to perform in-situ stress relaxation (2 steps 15% and 30% strain) during imaging (21 keV, 2.75μm pixel size). 40s scans were acquired before and after loading, while 14 fast tomographs (5s acquisitions) were taken during relaxation. The fiber 3D orientations and structural changes during loading were determined using a structure tensor approach (adapting a script from [1]). The 3D collagen fiber orientation in menisci revealed alternating layers of fibers. Two main areas are shown: surfaces and bulk. The surface layers are a mesh of randomly oriented fibers. Within the bulk 2-3 layers of fibers are visible that alternate about 30° to each other. Structural degeneration with age is visible and is currently being quantified. During stress-relaxation all menisci show a similar behavior, with samples from older donors being characterized by larger standard deviation Furthermore, the behavior of the different layers of fibers is tracked during relaxation showing how fibers with different orientation respond to the applied loading. Acknowledgments: We thank PSI for the beamtime at the TOMCAT beamline X02DA, and funding from Swedish Research Council (2019-00953), under the frame of ERA PerMed, and the Novo Nordisk Foundation through MathKOA (NNF21OC0065373)

Damage to and repair of the acetabular labral-chondral complex are areas of clinical interest in the treatment of young adults with pain in the hip and in the prevention of degenerative arthritis of the hip. There are varying theories as to why most acetabular tears are located anterosuperiorly. We have studied the prenatal development of the human acetabular labral-chondral complex in 11 fetal hips, aged from eight weeks of gestation to term. There were consistent differences between the anterior and posterior acetabular labral-chondral complex throughout all ages of gestation. The anterior labrum had a somewhat marginal attachment to the acetabular cartilage with an intra-articular projection. The posterior labrum was attached and continuous with the acetabular cartilage. Anteriorly, the labral-chondral transition zone was sharp and abrupt, but posteriorly it was gradual and interdigitated. The collagen fibres of the anterior labrum were arranged parallel to the labral-chondral junction, but at the posterior labrum they were aligned perpendicular to the junction. We believe that in the anterior labrum the marginal attachment and the orientation of the collagen fibres parallel to the labral-chondral junction may render it more prone to damage than the posterior labrum in which the collagen fibres are anchored in the acetabular cartilage. The anterior intra-articular projection of the labrum should not be considered to be a pathological feature

To address the current challenge of anterior cruciate ligament (ACL) reconstruction, this study is the first to fabricate a braided collagen rope (BCR) which mimics native hamstring for ACL reconstruction. The study aims to evaluate the biological and biomechanical properties of BCR both in vivo and vitro. Rabbit ACL reconstruction model using collagen rope and autograft (hamstring tendon) was conducted. The histological and biomechanical evaluations were conducted at 6-, 12-, 18, 26-week post-operation. In vitro study included cell morphology analysis, cell function evaluation and RNA sequencing of the tenocytes cultured on BCR. A cadaver study was also conducted to verify the feasibility of BCR for ACL reconstruction. BCR displays satisfactory mechanical strength similar to hamstring graft for ACL reconstruction in rabbit. Histological assessment showed BCR restore ACL morphology at 26 weeks similar to native ACL. The superior dynamic ligamentization in BCR over autograft group was evidenced by assessment of cell and collagen morphology and orientation. The in vitro study showed that the natural collagen fibres within BCR enables to signal the morphology adaptation and orientation of human tenocytes in bioreactor. BCR enables to enhance cell proliferation and tenogenic expression of tenocytes as compared to hydrolysed collagen. We performed an RNA-Sequencing (RNA-seq) experiment where RNA was extracted from tenocyte seeded with BCR. Analysis of enriched pathways of the up-regulated genes revealed that the most enriched pathways were the Hypoxia-inducible factor 1-alpha (HIF1A) regulated networks, implicating the possible mechanism BCR induced ACL regeneration. The subsequent cadaver study was conducted to proof the feasibility of BCR for ACL reconstruction. This study demonstrated the proof-of-concept of bio-textile braided collagen rope for ACL reconstruction, and the mechanism by which BCR induces natural collagen fibres that positively regulate morphology and function of tenocytes

Orthopaedic soft tissues, such as tendons, ligaments, and articular cartilage, rely on their unique collagen fiber architectures for proper functionality. When these structures are disrupted in disease or fail to regenerate in engineered tissues, the tissues transform into dysfunctional fibrous tissues. Unfortunately, collagen synthesis in regenerating tissues is often slow, and in some cases, collagen fibers do not regenerate naturally after injury, limiting repair options. One of the research focuses of my team is to develop functional fiber replacements that can promote in vivo repair of musculoskeletal tissues throughout the body. In this presentation, I will discuss our recent advancements in electrowriting 3D printing of natural polymers for creating functional fiber replacements. This manufacturing process utilizes electrical signals to control the flow of polymeric materials through an extrusion nozzle, enabling precise deposition of polymeric fibers with sizes that cannot be achieved using conventional extrusion printing methods. Furthermore, it allows for the formation of fiber organizations that surpass the capabilities of conventional electrospinning processes. During the presentation, I will showcase examples of electrowritten microfiber scaffolds using various naturally-derived polymers, such as gelatin (a denatured form of collagen) and silk fibroin. I will discuss the functional properties of silk-based scaffolds and highlight how they exhibit restored β-sheet and α-helix structures [1]. This restoration results in an elastic response of up to 20% deformation and the ability to withstand cyclic loading without plastic deformation. Additionally, I will present our latest results on the compatibility of this technique with patterning cell-laden fiber structures [2]. This novel biofabrication process allows for the printing of biomimetic microscale architectures with high cell viability, and offers a promising approach to understanding how shear and elongation forces influence cell development of hierarchical (collagen) fibers. Acknowledgements: The author would like to thank the Reprint project (OCENW.XS5.161) and the program “Materials Driven Regeneration” (024.003.013) by the Netherlands Organization for Scientific Research for the financial support

Abstract. Objectives. The enthesis is a specialised structure at the interface between bone and tendon with gradual integration to maintain functionality and integrity. In the process of fabricating an in-vitro model of this complex structure, this study aims to investigate growth and maturation of bone, tendon and BMSC spheroids followed by 3D mini-tissue production. Methods. Cell spheroids Spheroids of differentiated rat osteoblasts (dRObs), rat tendon fibroblasts (RTFs) and bone marrow stem cells (BMSC) were generated by culturing in 96 well U bottom cell repellent plates. With dROb spheroids previously analysed [1], RTF spheroids were examined over a duration of up to 28 days at different seeding densities 1×10. 4. , 5×10. 4. , 1×10. 5. , 2×10. 5. in different media conditions with and without FBS (N=3). Spheroid diameter was analysed by imageJ/Fiji; Cell proliferation and viability was assessed by trypan blue staining after dissociating with accutase + type II collagenase mix; necrotic core by H&E staining; and extracellular matrix by picro-sirius red (RTFs) staining to visualise collagen fibres under bright-field and polarised light microscope. 3D mini-tissue constructs. 15 day old mineralised dROb spheroids (∼1.5mm diameter) were deposited in pillar array supports using a customised spheroid deposition system to allow 3D mini-tissue formation via fusion (N=3). Similarly BMSC and RTF spheroids were deposited after determining the seeding density that produced spheroid size equivalent to 15 day old dROb spheroids. Gentle removal of spheroids from supports was performed on day 2, 4 and 6 to assess spheroid fusion. Histological staining was performed to observe cellular arrangement and extracellular matrix. Results. RTF spheroids diameter reduced over the course of 28 days regardless of the seeding density. A substantial decline in cell numbers over time was observed and suggests lack of cell proliferation due to tenogenic differentiation. Absence of a necrotic core in RTF spheroids, in all seeding densities, reveals their inherent capacity to maintain cell viability in avascular conditions. Picro-sirius red staining demonstrated the presence of collagen type I fibres predominantly in peripheral regions of spheroids maintaining its shape. Small amounts of collagen type III were also noticed. The dROb spheroids fused rapidly within 2 days resulting in the formation of a mini-tissue. 2×10. 5. RTFs and 3×10. 5. BMSCs produced spheroids of ∼1.5mm on day 3 and day 1 respectively. When these spheroids were deposited in pillar array supports, they did not undergo fusion even up to 6 days. This suggests inadequate aggregation of spheroids and insufficient ECM production at this early stage. Conclusions. This study has demonstrated the ability of RTFs to produce necrotic core-free spheroids with collagen fibres maintaining their structural integrity. For mini-tissue formation, we predict a longer initial culture time of RTF and BMSC spheroids will allow increased cellular interaction and ECM production before deposition, and will facilitate spheroid fusion. These findings will be applied in producing heterogenous mini-tissues, serving as a 3D in-vitro enthesis model. Declaration of Interest. (a) fully declare any financial or other potential conflict of interest

The posterior capsule is variously incised and excised during total hip replacement (THR). There is no consensus on the direction of the capsulotomy and the need to repair the posterior capsule. The objective of this study was to determine the orientation of the collagen fibres and nerves in the posterior hip capsule in patients undergoing THR. Specimens from five patients with osteoarthrosis of the hip (with no fixed deformity) were obtained and fixed in 10% neutral buffered formalin. Sutures were placed to mark the head and trochanteric end before excising. A standard posterior approach was used. The samples were examined and reported by a pathologist. Samples were processed overnight in a VIP5 automatic tissue processor and embedded in paraffin wax, preserving the location of the suture sites on embedding. Sections were cut at 5 Ïm and routinely stained with haematoxylin and eosin. The van Gieson stain was used for collagen fibres. Nerve fibres were highlighted using immunohistochemistry for S100 protein and blood vessels using an antibody to CD34. The collagen bundles seen were predominantly parallel to the axis of the specimen. Dispersed within the collagen bundles were small vascular leashes that were parallel with the collagen fibres. The S100 staining revealed that these were neurovascular leashes, with small nerves running alongside the vessels and the collagen. Nerves that separate from the vessels were likely to serve proprioceptive and nociceptive functions. The direction of the capsulotomy during THR by posterior approach has been traditionally perpendicular to the direction of the capsular fibres. However, if possible, capsulotomy along the orientation of the collagen fibres may be advantageous. As this study demonstrates, it will result in less damage to the capsular collagen fibres, blood vessels and nerves resulting in better capsular repair and healing, and better conservation of pro-prioceptive and nociceptive functions

Intervertebral disc function and dysfunction is governed by its structural architecture of concentric layers of highly ordered collagen fibres. This architecture is important at the mm scale for overall mechanical performance of the disc; and at the micron scale for mechano-transduction signalling pathways of the disc cells that are responsible for matrix maintenance and therefore disc health. To understand such mechanical behaviour 3-dimensional collagen fibre architecture must be quantified in intact intervertebral discs. Conventional imaging modalities lack either the spatial resolution (e.g. x-ray diffraction) or penetration (e.g. optical, electron or confocal laser microscopy) to yield mechanically important information. Preliminary studies of scanning acoustic microscopy (SAM) at 50 MHz visualises alternating layers of fibre texture, however exactly what is being imaged requires both explanation and validation. Three-dimensional SAM data sets obtained from intact discs were compared to polarised-light and scanning electron micrographs of individual layers of fibres, peeled by micro-dissection from discs. The dimensions of the structural features were measured and recorded. Optical and electron microscopy revealed that each layer consisted of highly oriented collagen fibres of diameter 5 μm with regularly spaced splits between fibres with a spacing of approximately 20–30 μm. The SAM data sets showed layers with a uniform highly oriented fibre texture that reversed between adjacent layers. Resolution of the texture was limited by the acoustic system to approximately 30 μm. It is clear that SAM at 50 MHz cannot resolve and therefore image individual collagen fibres. However, the regular defects in the fibre layers can be visualised and convey complete information about local collagen fibre architecture. SAM therefore provides an effective way of quantifying the fibrous structure of intact, hydrated, unfixed intervertebral discs

1. The surface of mature adult human articular cartilage from the knee has been studied by electron microscopy in eleven patients ranging in age from thirty-seven to eighty-three years. The ultrastructural appearance varies from person to person and often from area to area in the same specimen. These variations range from an intact surface to one showing overt fibrillation visible with the light microscope. 2. In areas where the articular surface appears intact the underlying superficial matrix consists of closely packed collagen fibres with only a small amount of interfibrillary ground substance. The collagen fibres show a predominantly tangential orientation in this region of the cartilage. Osmophilic lipidic bodies are sometimes seen in the matrix very close to the joint surface. 3. The appearances under the electron microscope are altered in what is interpreted as an early ultrastructural change in the development of cartilage fibrillation. In the affected areas the collagen fibres show abnormally wide separation by an excessive amount of interfibrillary matrix. Collagen fibres become directly exposed to the joint cavity, and the surface can also show accumulations of finely granular material and sometimes tuft-like projections containing collagen fibres and fine fibrils. At a slightly later stage shallow clefts and steeply sloping curves are apparent at the surface. It is suggested that these various alterations precede the development of overt fibrillation visible under the light microscope. 4. Electron micrographs occasionally show small "blisters" at the articular surface. Electron microscopy has not given evidence of shedding of cells into the joint cavity from non-fibrillated areas of adult human articular cartilage. Cells can, however, sometimes become exposed at the surface in fibrillated areas

Residual strain development in biological tissue is believed to result from remodeling in response to repetitive loading. This study hypothesized that differences in in-vivo loading between levels of the bovine tail result in differences in intervertebral disc (IVD) annulus fibrosus (AF) microstructural remodeling. The hypothesis was tested by quantifying tail musculature using clinical computed tomography and tissue microstructure using collagen fiber crimp period, which has previously been correlated with residual strain. Three bovine tail segments (levels c1 through c6) were imaged using a clinical computed tomography (CT) scanner followed by removal of muscle and harvest of IVDs. The discs were frozen, and transverse cryosections were obtained. Additionally, tangential plane cryosections were obtained from the inner and outer zones of the AF. Transverse CT slices corresponding to each joint level thresholded for both disc and muscle tissue and analyzed in MATLAB. First, the centroid of the disc image was calculated to use as an origin. Then the disc area and moments of inertia about the flexion extension axis and lateral bending axis were calculated. Total muscle area was then calculated, along with muscle moments of inertia relative to the disc centroid. All muscle parameters were normalized by those of the corresponding disc. Cryosections were imaged using an inverted light microscope equipped with crossed polarizing filters and a digital camera. A MATLAB routine was used to perform Fourier transform analysis on user selected lines of interest in the transverse micrographs, yielding average fiber crimp period in the inner and outer AF. Micrographs from tangential sections were opened in ImageJ, and fiber orientation angles were measured manually. Muscle moments of inertia were analyzed using a two-way ANOVA with disc level and axis as dependent variables. Normalized muscle area was analyzed with a one-way ANOVA with disc level as a dependent variable. A two-way ANOVA, with disc level and zone (inner versus outer) was used to analyze collagen fiber crimp period and collagen fiber angle. Normalized muscle moment of inertia showed significant effects of both level and axis (p < 0 .001), decreasing at distal levels, and being lower about the flexion-extension axis than the lateral bending axis. Normalized muscle cross section showed a visible, but not significant (p=0.0721) decreasing trend with disc level. Fiber crimp period had significant effects of both level and zone (p < 0 .001), and was significantly longer in the outer zone than inner at all levels. Significant decrease in crimp period at distal levels were seen in the outer AF, but not the inner. While fiber angle was significantly (p < 0 .001) higher in the inner AF (36±6.6°) than outer AF (24±3.5°)), there was no significant effect of level. Fiber crimp period in the AF has previously been correlated with residual circumferential strain, with larger crimp period corresponding to increased residual tension. The present study suggests that at proximal levels of the tail, where peak compressive and bending stresses in the AF (as inferred from normalized muscle area and moments of inertia respectively) are greatest, there is more accumulation of residual strain

INTRODUCTION. In order to address high failure rates following rotator cuff repairs, a greater understanding is required of the underlying structural changes so that treatments can be appropriately targeted and biomarkers of failure can be identified. As collagen is the primary constituent of tendon and determines force transmission, collagen structural changes may affect responses to loading. For example changes in collagen 1 and 5 are associated with the hyperelastic Ehlers-Danlos syndrome, which is diagnosed by looking for pathopneumonic altered collagen fibres or ‘collagen flowers’ in skin using transmission electron microscopy (TEM). To date no study has been performed on the microstructure of torn human rotator cuff tendons using TEM. It was hypothesized that normal, small and massive human rotator cuff tendons tears will have altered microscopic structures. The unique study aimed to use TEM to compare the ultrastructure of small and massive rotator cuff tears, to normal rotator cuff tendons. METHODS. Samples from 7 human rotator cuff tendons repairs were obtained, including 4 massive (>5 cm) and 3 small (< 1 cm) tears, and 3 matched normal controls with no history of connective tissue disorders. Specimens were fixed in 4% glutaraldehyde in 0.1M phosphate buffer, processed and examined blind using routine TEM examination. To assess whether changes in the relative expression of collagen 1 and 5 (COL1A1, COL5A1 and COL5A2) occurred in all tears, qPCR was performed on another 6 phenotypically matched patients. RESULTS. The basic structure of the normal tendon consisted of tightly packed clumps of dense packed parallel running collagen fibers with few fibroblasts and small amounts of fine filamentous material between clumps. In contrast, torn samples were more variable with areas of less dense packing of collagen fibers and larger areas of filamentous material plus variable numbers of lipid droplets both within the fibroblast and between the collagen bundles. There was also evidence of twisting and random orientation of individual collagen fibers. All torn tendons showed evidence of a proportion of the fibers within the collagen bundles being enlarged with a serrated outline, similar in appearance to ‘collagen flowers’. Clear differences between the small and massive tears were not identified. qRT-PCR of torn rotator cuff tendon specimens demonstrated no altered collagen expression compared to normal tendons. DISCUSSION. This novel study has identified the previously unreported presence of atypical collagen fibers with focal swelling resulting in the appearance of ‘collagen flowers’ in torn rotator cuff tendons only. This appearance is considered pathognomonic of Ehlers-Danlos syndrome, classical type 1 and 2. Torn tendons also showed an increase in filamentous material, and infiltration with fat droplets. These novel findings may offer insight into the mechanisms of structural damage that contribute to rotator cuff failure. Further examination is required, to evaluate the significance of these observations

Summary. Objective assessment of tendon histomorphology, particularly in the context of tissue repair, requires comprehensive analyses of both cellular distribution and matrix architecture. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique provide objective, quantitative assessment of collagen fiber organization with high specificity. Concurrent nuclear staining allows simultaneous analyses of cell morphology and distribution. Introduction. Tendon injuries can be career-limiting in human and equine athletes, since the architectural organization of the tissues are lost in the course of fibrotic repair. Objective assessment of tendon repair is problematical, particularly in research addressing potential therapies. Fourier Transform analyses of histological images collected with second harmonic generation (SHG-FT) technique can provide objective, quantitative assessments of collagen fiber organization with high specificity. This study describes the use of SHG-FT with fluorescently-labelled tendon-derived cells (TDC) in an in-vivo model of equine tendinitis to assess the temporal and spatial effects of cell delivery on collagen fiber organization. Materials and methods. Collagenase-induced tendinitis was created in the mid-metatarsal region of one hindlimb superficial digital flexor tendons (SDFT) in two horses. SDFTs from two clinically normal adult horses and were also used as controls. Autogenous TDCs were isolated from the lateral digital extensor tendon of the contralateral hind limb. Four weeks post-collagenase injection, 10×10. 6. DiI-labeled TDCs were injected into the tendon lesions. Tendon samples were obtained for histologic evaluation following euthanasia, 2-weeks after cell injections. Tendon samples were cryo-sectioned to 25–30μ exposed to nuclear counter stains (DAPI and PI) and imaged immediately through a confocal microscope (Zeiss LSM 710) with a 2-photon laser source, to obtain backward SHG (bSHG) and forward SHG (fSHG) images. In addition, images with DiI and DAPI fluorescence were acquired using 500–550 nm (green) or 565–615 nm (orange) emission filters, respectively. Fourier analysis of the SHG images was carried out using imageJ software. Results. DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. This was possible because the excitation wavelength of the two-photon laser (780nm) and detection of emissions above 565nm do not interfere with the bSHG band (380–400nm). Images collected with bSHG included signals from DAPI-stained nuclei. In contrast, emissions from PI-labeled nuclei were acquired independently of SHG signals. The contrast generated by individual collagen fibers was higher in images collected with fSHG than bSHG. SHG-FT of fSHG images provided accurate assessment of collagen fiber orientation in repair tissue and normal tendon. Discussion/Conclusions. Objective assessment of collagen orientation, along with spatial distribution of cells within healing tendon serves as useful indices of healing. Injected DiI-labeled TDCs could be imaged successfully under two-photon fluorescence concurrently with SHG imaging. However, DiI fluorescence is susceptible to photo-bleaching during SHG acquisition. Use of an alternative nuclear counter stains, such as PI, that do not emit along with SHG signal should be considered to optimise data acquisition and support simultaneous analyses of collagen structure, cellular morphology and cell distribution. SHG-FT histologic analysis along with biochemical and biomechanical indices collectively provide comprehensive assessment of therapies for tendon repair

Aims. Femoroacetabular impingement (FAI) is a potential cause of hip osteoarthritis (OA). The purpose of this study was to investigate the expression profile of matrix metalloproteinases (MMPs) in the labral tissue with FAI pathology. Methods. In this study, labral tissues were collected from four FAI patients arthroscopically and from three normal hips of deceased donors. Proteins extracted from the FAI and normal labrums were separately applied for MMP array to screen the expression of seven MMPs and three tissue inhibitors of metalloproteinases (TIMPs). The expression of individual MMPs and TIMPs was quantified by densitometry and compared between the FAI and normal labral groups. The expression of selected MMPs and TIMPs was validated and localized in the labrum with immunohistochemistry. Results. On MMP arrays, most of the targeted MMPs and TIMPs were detected in the FAI and normal labral proteins. After data normalization, in comparison with the normal labral proteins, expression of MMP-1 and MMP-2 in the FAI group was increased and expression of TIMP-1 reduced. The histology of the FAI labrum showed disorderly cell distribution and altered composition of thick and thin collagen fibres. The labral cells expressing MMP-1 and MMP-2 were localized and their percentages were increased in the FAI labrum. Immunohistochemistry confirmed that the percentage of TIMP-1 positive cells was reduced in the FAI labrum. Conclusion. This study established an expression profile of MMPs and TIMPs in the FAI labrum. The increased expression of MMP-1 and MMP-2 and reduced expression of TIMP-1 in the FAI labrum are indicative of a pathogenic role of FAI in hip OA development. Cite this article:Bone Joint Res. 2020;9(4):173–181

Introduction: Pre-operative coronal curve flexibility assessment is of key importance in the surgical planning process for scoliosis correction. The fulcrum bending radiograph is one flexibility assessment technique which has been shown to be highly predictive of potential curve correction using posterior surgery, however little is known about the extent to which soft tissue structures govern spinal flexibility. The aim of this study was to explore how the mechanical properties of spinal ligaments and intervertebral discs affect coronal curve flexibility in the fulcrum bending test. To this end a biomechanical analysis of a scoliotic thoracolumbar spine and ribcage was carried out using a three dimensional finite element model. Methods: CT-derived spinal anatomy for a 14 year old female adolescent idiopathic scoliosis patient was used to develop the 3D finite element model. Physiological loading conditions representing the gravitational body weight forces acting on the spine when the patient lies on their side over the fulcrum bolster were simulated. Initial mechanical properties for the spinal soft tissues were derived from existing literature. In six separate analyses, the disc collagen fibre and ligament stiffness values were reduced by 10%, 25% and 40% respectively, and the effects of reduced tissue stiffness on fulcrum flexibility were assessed by comparison with the initial model. Finally, the effect of discectomy on fulcrum flexibility was simulated for thoracic levels T5 to T12. Results: Reducing disc collagen fibre stiffness resulted in a greater change in segmental rotations in the fulcrum bending test than reducing ligament stiffness. However, reductions of up to 40% in disc collagen fibre stiffness and ligament stiffness produced no clinically measurable increase in fulcrum flexibility (increase of 1.2%). By contrast, following removal of the discs, the simulated fulcrum flexibility increased by more than 80% compared to the initial case. Discussion: Disc collagen fibre and ligament stiffness both have minimal influence on scoliotic curve flexibility. However, discectomy simulation shows that the intervertebral discs are of critical importance in determining spinal flexibility

Summary Statement. Demineralised bone matrix augmented tendon-bone fixations in the animal model show less scar tissue and an enthesis morphology closer to the physiologic one which may lead to a more resistant repair construct. Introduction. Rotator cuff repair is one of the most common operative procedures in the shoulder. Yet despite its prevalence recurrent tear rates of up to 94% have been reported in the literature. High failure rates have been associated with tendon detachment from bone at the tendon – bone interface. Exogenous agents as biological strategies to augment tendon – bone healing in the shoulder represent a new area of focus to improve patient outcomes. Demineralised bone matrix (DBM) contains matrix bound proteins, exposed through acid demineralization step of DBM manufacture, and has long been recognised for its osteoinductive and osteoconductive properties. We hypothesised that DBM administered to the bone bed prior to the reattachment of the tendon, will upregulate healing and result in enhanced tissue morphology that more closely resembles that of a normal enthesis. An established ovine transosseous equivalent rotator cuff model was used. Methods. Following ethics approval, 10 adult wethers (18 months) were randomly allocated to control, n=4 (without DBM) or DBM, n=6 (DBM administered to bone bed) groups. The infraspinatus tendon was detached from its insertion and repaired in a transosseous equivalent fashion using PEEK suture anchors. In treatment animals 0.25cc of ovine DBM, previously prepared using a modified Urist protocol, was injected into two drill holes within the bony tendon footprint. Animals were culled at 4 weeks following surgery and processed for tissue histology and microcomputed tomography (μCT) endpoints. Results. No infection or tendon detachment following repair was noted in either group. 3D reconstructed images of μCT scans verified correct DBM and suture anchor placement. Histological images demonstrated distinct differences in tissue morphology between the two groups; however there was no evidence of the four – zoned structure characteristic of a healthy tendon bone insertion, in any specimens. In the control group specimens, the tendon midsubstance was highly disorganised with randomly arranged collagen fibres and diminutive areas of fibrocartilage. In the treatment group, large regions between tendon and bone were occupied by fibrocartilage. Within the fibrocartilage region, insertional collagen fibres appeared organised and chondrocytes were orientated in the direction of the insertional collagen fibres. Organised collagen fibre orientation within the tendon midsubstance was observed, though this was not consistent throughout all the specimens. DBM particles were resorbed and trabecular bone occupied the DBM holes. The PEEK anchors were all in direct contact with the ongrowing bone indicating good quality integration and fixation. Discussion. This study showed that DBM augmented tendon to bone repair leads to an upregulated cellular activity resulting in increased amounts of fibrocartilage between the repaired tendon and underlying bone. The upshot of this is an improved tissue organization which more closely resembles the morphology of the normal enthesis. Introduction of osteoinductive DBM at the tendon – bone interface during surgery may reduce failure rates associated with rotator cuff repair and improve clinical outcomes