Aims. The antidiabetic agent metformin inhibits fibrosis in various organs. This study aims to elucidate the effects of hyperglycaemia and metformin on knee joint capsule fibrosis in mice. Methods. Eight-week-old wild-type (WT) and type 2 diabetic (db/db) mice were divided into four groups without or with metformin treatment (WT met(-/+), Db met(-/+)). Mice received daily intraperitoneal administration of metformin and were killed at 12 and 14 weeks of age. Fibrosis morphology and its related genes and proteins were evaluated. Fibroblasts were extracted from the capsules of 14-week-old mice, and the expression of fibrosis-related genes in response to glucose and metformin was evaluated in vitro. Results. The expression of all fibrosis-related genes was higher in Db met(-) than in WT met(-) and was suppressed by metformin. Increased levels of fibrosis-related genes, posterior capsule thickness, and collagen density were observed in the capsules of db/db mice compared with those in WT mice; these effects were suppressed by metformin. Glucose addition increased fibrosis-related gene expression in both groups of mice in vitro. When glucose was added, metformin inhibited the expression of fibrosis-related genes other than cellular communication network factor 2 (Ccn2) in WT mouse cells. Conclusion. Hyperglycaemia promotes fibrosis in the mouse knee joint capsule, which is inhibited by metformin. These findings can help inform the development of novel strategies for treating knee joint capsule fibrosis. Cite this article: Bone Joint Res 2024;13(7):321–331

Introduction: Despite significant progress at the molecular level the etiology of aseptic loosening is still unclear. Fibrosis of the new capsule is an invariable finding at revision hip arthroplasty. Tissue fibrosis has been demonstrated in varies pathologic conditions due to elevated oxidative stress. The present retrospective study was designed to proof the hypothesis that peri-prosthetic fibrosis in aseptic loosening may be caused by elevated oxidative stress and represent an initial step in the pathomechanism of aseptic loosening. Material and methods: Levels of malondialdehyde (MDA), oxidized (GSSG) and reduced (GSH) gluthatione were assayed as markers of oxidative stress in retrieved capsules of 28 loose hips (Group I) and 12 hips revised for high rate of wear (Group II). Collagen in the periprosthetic tissues was measured as hydroxiproline content and semiquantitatively by electrophoresis. In four representative cases electron microscopy was performed. Results: MDA level as well as GSH/GSSG and GSH/ GSSG² ratios showed elevated oxidative stress in group I compared to group II and controls. SDS-PAGE electrophoresis showed higher molecular bands in 20 patients compared to controls. Hydroxiproline level in group II is significantly higher than in group I (p< 0.05). MDA, GSH and GSSG correlate significantly with hydroxiproline. A negative correlation between collagen content and osteolysis was established. Discussion and conclusion: Higher oxidative stress plays role in aseptic loosening of hip arthroplasty. The present data support the hypothesis that the process is initiated by excessive fibrosis which consequently might lead to increase of intraarticular pressure and to extension of the joint space

Aims. Dupuytren’s contracture is characterized by increased fibrosis of the palmar aponeurosis, with eventual replacement of the surrounding fatty tissue with palmar fascial fibromatosis. We hypothesized that adipocytokines produced by adipose tissue in contact with the palmar aponeurosis might promote fibrosis of the palmar aponeurosis. Methods. We compared the expression of the adipocytokines adiponectin and leptin in the adipose tissue surrounding the palmar aponeurosis of male patients with Dupuytren’s contracture, and of male patients with carpal tunnel syndrome (CTS) as the control group. We also examined the effects of adiponectin on fibrosis-related genes and proteins expressed by fibroblasts in the palmar aponeurosis of patients with Dupuytren’s contracture. Results. Adiponectin expression in the adipose tissue surrounding the palmar aponeurosis was significantly lower in patients with Dupuytren’s contracture than in those with CTS. The expression of fibrosis-related genes and proteins, such as types 1 and 3 collagen and α-smooth muscle actin, was suppressed in a concentration-dependent manner by adding AdipoRon, an adiponectin receptor agonist. The expression of fibrosis-related genes and proteins was also suppressed by AdipoRon in the in vitro model of Dupuytren’s contracture created by adding TGF-β to normal fibroblasts collected from patients with CTS. Conclusion. Fibrosis of the palmar aponeurosis in Dupuytren’s contracture in males may be associated with adiponectin expression in the adipose tissue surrounding the palmar aponeurosis. Although fibroblasts within the palmar aponeurosis are often the focus of attention when elucidating the pathogenesis of Dupuytren’s contracture, adiponectin expression in adipose tissues warrants closer attention in future research. Cite this article: Bone Joint Res 2023;12(8):486–493

Dupuytren's disease (DD) is a fibroproliferative soft tissue disease affecting the palmar fascia of the hand causing permanent and irreversible flexion contracture. Aberrant fibrosis is likely to manifest through a combination of extrinsic, intrinsic, and environmental factors, including genetics and epigenetics. However, the role of epigenetics in soft tissue fibrosis in diseases such as DD is not well established. Therefore, we conducted a comprehensive multi-omic study investigating the epigenetic profiles that influence gene expression in DD pathology. Using control (patients undergoing carpal tunnel release) and diseased fibroblasts (patients undergoing Dupuytren's fasciectomy), we conducted ATAC-seq to assess differential chromatin accessibility between control and diseased fibroblasts. Additionally, ChIP-seq mapped common histone modifications (histone H4; H3K4me3, H3K9me3, H3K27me3, H4K16Ac, H4K20Me3) associated with fibrosis. Furthermore, we extracted RNA from control and DD tissue and performed bulk RNA-seq.

ATAC-seq analysis identified 2470 accessible genomic loci significantly more accessible in diseased fibroblasts compared to control. Comparison between diseased and control cells identified numerous significantly different peaks in histone modifications (H4K20me3, H3K27me3, H3K9me3) associated with gene repression in control cells but not in diseased cells. Pathway analysis demonstrated a substantial overlap in genes being de-repressed across these histone modifications (Figure 1). Both, ATAC-seq and ChIP-seq analysis indicated pathways such as cell adhesion, differentiation, and extracellular matrix organisation were dysregulated as a result of epigenetic changes. Moreover, de novo motif enrichment analysis identified transcription factors that possibly contributed to the differential gene expression between control and diseased tissue, including HIC1, NFATC1 and TEAD2. RNA-seq analysis found that these transcription factors were upregulated in DD tissue compared to control tissue.

The current epigenetic study provides insights into the aberrant fibrotic processes associated with soft tissue diseases such as DD and indicates that epigenetic-targeted therapies may be an interesting viable treatment option in future.

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Pericytes are contractile, motile cells that surround the capillary. Recent studies have shown that pericytes promoted joint fibrosis and induced subchondral bone angiogenesis, indicating the role of pericytes in osteoarthritis (OA). However, whether pericytes are involved in regulating inflammatory and catabolic response, as well as fibrotic repair of cartilage is still unclear. Here we used 2D and 3D models to investigate the communication of pericytes and chondrocytes under inflammatory osteoarthritis conditions.

CD34-CD146+ pericytes were isolated and sorted from human bone marrow. Human OA chondrocytes were isolated from OA joints. In 2D studies, monolayer cultured chondrocytes were treated +/- pericyte conditioned media, +/- 1ng/ml IL1β for 24h. In 3D studies, pericytes and chondrocytes were cultured within fibrin gel in 3D polyurethane scaffolds, separately or combined for 7 days, followed by treatment of +/- IL1β for another 7 days (Fig 2A). The inflammatory response, catabolic activity and expression of fibrosis markers of chondrocytes and pericytes were measured by ELISA and/or q-rtPCR.

Pericytes had weak inflammatory, catabolic and fibrotic response to IL1β (data not shown). The 2D study showed that pericyte conditioned media promoted inflammation, catabolism and fibrosis markers of chondrocytes, in the absence of IL1β treatment (Figure 1). However, study in 3D showed that coculture of chondrocytes and pericytes reduced the inflammatory and catabolic response of chondrocytes to IL1β and induced fibrosis markers in chondrocytes (Figure 2).

Pericytes are involved in regulating inflammatory response, catabolic response and fibrosis of chondrocytes. The opposite results from 2D and 3D experiments indicate the variety of the regulatory role of pericytes in the interaction with chondrocytes within different culture models. The underlying mechanism is under evaluation with on-going studies.

Acknowledgements

This study was funded by SINPAIN project, from European Union's Horizon Europe research and innovation programme under Grant Agreement NO. 101057778. Funded by the European Union. Views and opinions expressed are however those of the author(s) only and do not necessarily reflect those of the European Union. Neither the European Union nor the granting authority can be held responsible for them.

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Tendinopathy is the most common form of chronic tendon disorders, accounting for up 30% of all musculoskeletal clinic visits [1]. In tendon disease, the largely avascular tendon tissue often becomes hypervascularized and fibrotic [2]. As blood vessel growth and angiogenic signaling molecules are often induced by the lack of adequate nutrients and oxygen, hypoxic signaling is speculated to be a root cause of tendon neovascularization and tendinopathy [3,4,5]. However, how the vascular switch is initiated in tendons, and how vascularization contributes to tendon pathology remains unknown. In this talk, we provide evidence that HIF-1α is implicated in tendon disease and HIF-1α stabilization in human tendon cells induces vascular recruitment of endothelial cells via VEGFa secretion. More interesting, HIF-1α stabilization in tendon cells in vivo, seems to recapitulate all main features of fibrotic human tendon disease, including vascular ingrowth, matrix disorganization, changes in tissue mechanics, cell proliferation and innervation. Surprisingly, in vivo knock-out of VEGFa rescued angiogenesis in the tendon core but it did not affect tendon mechanical properties and tissue pathophysiological changes, suggesting that blood vessels ingrowth might not be a primary cause but a consequence of HIF-1α activation.

Vascular inflammation and activation of myofibroblasts are significant contributors to the progression of fibrosis, which can severely impair tissue function. In various tissues, including tendons, Transforming growth factor beta 1 (TGF-β1) has been identified as a critical driver of adhesion and scar formation. Nevertheless, the mechanisms that underlie fibrotic peritendinous adhesions are still not well comprehended, and human microphysiological systems to help identify effective therapies remain scarce. To address this issue, we developed a novel human Tendon-on-a-Chip (hToC), comprised of an endothelialized vascular compartment harboring circulating monocytes and separated by a 5 μm/100 nm dual-scale ultrathin porous membrane from a type I/III collagen hydrogel with primary tendon fibroblasts and tissue-resident macrophages, all under defined serum-free conditions. The hToC models the crosstalk of the various cells in the system leading to the induction of inflammatory and fibrotic pathways including the activation of mTOR signaling. Consistent with phenotypes observed in vivo in mouse models and clinical human samples, we observed myofibroblast differentiation and senescence, tissue contraction, excessive extracellular matrix deposition, and monocytes’ transmigration and macrophages’ secretion of inflammatory cytokines, which were dependent on the presence of the endothelial barrier. This model offers novel insights on the role of vasculature in the pathophysiology of adhesions, which were previously underappreciated. Moreover, in testing whether the hToC could be used to evaluate efficacy of therapeutics, we were able to capture donor-specific variability in the response to Rapamycin treatment, which reduced myofibroblast activation regardless. Thus, our findings demonstrate the value of the hToC as a human microphysiological system for investigating the pathophysiology of fibrotic conditions in the context of peritendinous injury and similar fibrotic conditions, providing an alternative to animal testing.

Introduction

Initial post-operative implant instability leads to impaired osseointegration, one of the most common reasons for aseptic loosening and revision surgery. In this study, we developed a novel murine model of implant instability and demonstrated the anabolic effect of immediate and delayed intermittent Parathyroid Hormone (iPTH) treatment in the setting of instability-induced osseointegration failure.

Scar tissue formation secondary to acute muscle injury, surgical wounding and compartment syndrome can result in significant functional impairment and predispose to further injury. The source of fibroblasts, and the molecular mechanisms driving their activation and persistence in skeletal muscle fibrosis are not known. We hypothesized that cells expressing PDGFRβ become fibroblasts in response to injury and that targeting αv integrins in these cells reduces skeletal muscle fibrosis.

We used double-fluorescent reporter mice to demonstrate that cells expressing PDGFRβ become activated myofibroblasts in response to cardiotoxin (CTX) induced skeletal muscle injury. Following injury, PDGFRβ+ cells moved from perivascular locations into the interstitium in a distribution characteristic of fibroblasts, and showed marked induction of fibroblastic genes including αSMA and collagen1 (all p<0.0001). To confirm that αv integrins present on PDGFRβ cells critically regulate skeletal muscle fibrosis we used Itgavflox/flox;PDGFRβ-Cre mice (transgenic mice in which αv integrins are ‘knocked-down’ in PDGFRβ+ cells). These mice were significantly protected from CTX induced fibrosis (p<0.01). To demonstrate potential clinical utility of targeting αv integrins, we used a small molecule inhibitor of αv integrins (CWHM12). Treatment with CWHM12 significantly reduced fibrosis when delivered from the time of injury (p<0.01) and when delivered after the fibrotic response had become established (p<0.01).

We have identified a core pathway regulating fibrosis in skeletal muscle. Pharmacologic inhibition of αv integrins has potential clinical utility in the treatment and prevention of skeletal muscle fibrosis.

Tungsten has been increasing in demand for use in manufacturing and recently, medical devices, as it imparts flexibility, strength, and conductance of metal alloys. Given the surge in tungsten use, our population may be subjected to elevated exposures. For instance, embolism coils made of tungsten have been shown to degrade in some patients. In a cohort of breast cancer patients who received tungsten-based shielding for intraoperative radiotherapy, urinary tungsten levels remained over tenfold higher 20 months post-surgery. In vivo models have demonstrated that tungsten exposure increases tumor metastasis and enhances the adipogenesis of bone marrow-derived mesenchymal stem cells while inhibiting osteogenesis. We recently determined that when mice are exposed to tungsten [15 ppm] in their drinking water, it bioaccumulates in the intervertebral disc tissue and vertebrae. This study was performed to determine the toxicity of tungsten on intervertebral disc.

Bovine nucleus pulposus (bNP) and annulus fibrosus (bAF) cells were isolated from bovine caudal tails. Cells were expanded in flasks then prepared for 3D culturing in alginate beads at a density of 1×10^∧6 cells/mL. Beads were cultured in medium supplemented with increasing tungsten concentrations in the form of sodium tungstate [0, 0.5, 5, 15 ug/mL] for 12 days. A modified GAG assay was performed on the beads to determine proteoglycan content and Western blotting for type II collagen (Col II) synthesis. Cell viability was determined by counting live and dead cells in the beads following incubation with the Live/Dead Viability Assay kit (Thermo Fisher Scientific). Cell numbers in beads at the end of the incubation period was determined using Quant-iT dsDNA Assay Kit (Thermo Fisher Scientific)

Tungsten dose-dependently decreased the synthesis of proteoglycan in IVD cells, however, the effect was significant at the highest dose of 15 ug/mL. (n=3). Furthermore, although tungsten decreased the synthesis of Col II in IVD cells, it significantly increased the synthesis of Col I. Upregulation of catabolic enzymes ADAMTS4 and −5 were also observed in IVD cells treated with tungsten (n=3). Upon histological examination of spines from mice treated with tungsten [15 ug/mL] in their drinking water for 30 days, disc heights were diminished and Col I upregulation was observed (n=4). Cell viability was not markedly affected by tungsten in both bNP and bAF cells, but proliferation of bNP cells decreased at higher concentration. Surprisingly, histological examination of IVDs and gene expression analysis demonstrated upregulation of NGF expression in both NP and AF cells. In addition, endplate capillaries showed increases in CGRP and PGP9.5 expression as determined on histological sections of mouse IVDs, suggesting the development of sensory neuron invasion of the disc.

We provide evidence that prolonged tungsten exposure can induce disc fibrosis and increase the expression of markers associated with pain. Tungsten toxicity may play a role in disc degeneration disease.

Background

There are currently no effective treatments for skeletal muscle fibrosis. Myofibroblasts are the major cellular effectors of fibrosis but their origin in muscle is unknown. We report that PDGFRβ (platelet derived growth factor receptor beta) Cre inactivates genes in murine PDGFRβ+ cells and myofibroblasts in muscle with high efficiency. We used this system to delete the integrin αv subunit because of the suggested role of multiple αv integrins as central mediators of fibrosis in multiple organs.

Aims

As has been shown in larger animal models, knee immobilization can lead to arthrofibrotic phenotypes. Our study included 168 C57BL/6J female mice, with 24 serving as controls, and 144 undergoing a knee procedure to induce a contracture without osteoarthritis (OA).

Purpose of study: The objective of this retrospective clinical study was to investigate the potential benefits of using the Collagen Dural Matrix as an adhesion barrier following spine surgerin order to avoid periradicular lumbar fibrosis.

Method: Retrospective patient data was used 500patients. The study cohort (collagen matrix group) consisted of consecutive patients (250 patients) who were treated with collagen Dural matrix. Patients in the standard procedure group (250 patients in all) were operated on prior to the adoption of collagen dural matrix use by the surgeon. Patients underwent spinal surgery and completed follow-up evaluations at no less than 3 years post-operation

Study Design: Following surgery, pain was assessed at various times post operation (0 to 6 months, 6 to 12 months, and 12 to 48 months- 60 months). A post-operative MRI scan was performed on patients experiencing excessive pain to assess extent of peridural scarring and adhesions.

Summary of findings:

Post-operative Pain Assessment. Mean post-operative pain scores for the collagen matrix group at all time points were lower than those for the standard procedure group and were statistically lower at 3 to 6 months.

Comparison of Post-operative and Pre-operative MRI. Of the patients that received post-operative MRI, 43% of the standard procedure group showed adhesion formation, whereas 23% of the collagen matrix group showed adhesions.

Re-operation Rate. The difference in re-operation rate between the collagen matrix group and the standard procedure group is statistically significant (p< 0.01).

Relationship between findings and existing knowledge. Dural adhesions resulting from peridural fibrosis can cause persistent pain and may lead to re-operation. The need exists for treatments to prevent dural adhesions and minimize the impact of peridural fibrosis on post-operative outcome of spine surgery. Collagen Dural Matrix has traditionally been used for the repair of dural defects in both cranial and spinal procedures. Because of collagen dural matrix excellent clinical profile, it may be useful as an adhesion barrier.

Overall significance of findings: Findings in this study (reduced pain and lower incidence of adhesions) are consistent with the Collagen Dural Matrix acting as an effective adhesion barrier it is likely that the Collagen Dural Matrix may be used in other regions of the spine to prevent peridural fibrosis and associated sequelae. Ultimately the use of the collagen dural matrix prophylactically to prevent adhesions may result in improved patient outcomes.

1. The cases of six Chinese children affected by so-called congenital fibrosis of the vastus intermedius muscle are described. The reasons for the choice of name are discussed.

2. Reasons for early operation are put forward: in young children simple division of the tendon of the vastus intermedius is adequate.

3. With increasing age severe changes in all the joint tissues occur, notably in the articular cartilage of the patella. These changes are likely to vitiate the result after operation.

4. The importance of getting as much flexion as possible in children of Asiatic race is stressed.

Subacromial bursa fibrosis are linked to rotator cuff lesion with shoulder stiffness; however, the mechanism underlying this shoulder disorder remain elusive. MicroRNA-29s (miR-29s) are emerging fibrosis inhibitor targeting fibrogenic matrices during tissue fibrosis. This study is aimed to investigate clinical relevance and function of miR-29 signalling to subacromial bursa homeostasis in shoulder stiffness. Subacromial bursa in patients with rotator cuff lesion with or without shoulder stiffness who required open acromioplasty were harvested for assessing fibrosis histopathology using Manson's trichrome staining. Expressions of proinflammatory cytokines, fibrotic matrices, and miR-29s were quantified using RT-PCR and in situ hybridization. Range of motion and pain scores of the stiffness group were higher than those of non-stiffness group. Upregulated proinflammatory cytokines (IL-1β, IL-6, and TNF-α) and fibrotic matrices (collagen 1α1, 3α1, and 4α1) but decreased miR-29a and b expression existed in the stiffness group. Affected tissues exhibited severe fibrotic matrix accumulation, synovial hyperangiogenesis, hyperplasia, and strong miR-29a transcripts. In vitro, IL-1β rather than IL-6 and TNF-α decreased miR-29a expression of subacromial bursa fibroblasts. miR-29a knockdown escalated fibrotic matrix expression, whereas forced miR-29a expression alleviated the IL-1β-induced fibrotic matrix expression. Of interest, miR-29a transgenic mice displayed moderate responses to supraspinatus and infraspinatus tenotomy-induce fibrosis and gait irregularity of affected shoulders. Weak miR-29 signalling causes excessive fibrosis and remodelling in subacromial bursa and ultimately increases the prevalence of shoulder stiffness. This study reveals a new mechanistic underlying shoulder stiffness and highlights that sustained miR-29a potentially ameliorates the severity and function of stiff shoulder.

There are limited data on scoliosis in cystic fibrosis (CF), and the two most recent studies came to opposite conclusions. Reported prevalence ranges from 2% (within the normal range for the general population) to 15.5%. We felt that a recent study under-estimated the prevalence due to a very young population (mean age 10.9 years), since scoliosis develops most commonly in adolescents. We hypothesised that scoliosis is more prevalent in adolescents with CF compared to the general population. The aim of our study was to determine the incidence of scoliosis in adolescents with CF followed to and beyond skeletal maturity and describe the type of spinal deformity. We included all patients in our CF clinic aged >10 years, and those who have transitioned to adult services in the last 10 years. Patients with a co-existent neuromuscular condition were excluded.

We conducted a retrospective observational study. Most recent chest radiographs at end of spinal growth, or those taken at transition to adult services, were reviewed by a Consultant Radiologist and a Consultant Spine Surgeon. Scoliosis was defined as a Cobb angle of >10° in the coronal plane. Demographics and characteristics of the curves were recorded.

Our cohort included 143 CF patients (48% male) with a mean age at the time of chest radiograph of 18 years (range 15–22 years). 16 (6 male) subjects were noted to have scoliosis with a mean (range) Cobb angle of 14° (10–38°) giving a prevalence of 11%. 13 were single thoracic curves, 2 double and 1 triple. The majority were non-progressive short mid-thoracic curves, convex to the right. 5 curves were progressive, only one of which was significant and required bracing to the end of growth but no surgical treatment.

We found a prevalence of scoliosis in our adolescent CF population that is significantly greater than the general population. Only one curve was significant and progressive requiring bracing, the remainder being minor and non-progressive. A strength of our study is that all patients had achieved skeletal maturity at the time of latest X-ray and, therefore, development or further progression of scoliosis is unlikely. The negative effect of scoliosis on lung function is well-documented. With the progressive nature of CF lung disease, scoliosis may have further deleterious effects. Bone disease is increasingly recognised in CF patients, with osteopenia and osteoporosis occurring earlier and more frequently than in the general population (38% & 24% respectively in 18–32 year old CF patients). To date, studies have failed to show a correlation between scoliosis, lung function and bone mineral density. The paradigm of a radiologically significant (Cobb angle >10°) versus a clinically important scoliosis remains.

1. Twelve children are described in whom gradual limitation of knee flexion developed, without previous injury or inflammation.

2. Clinical and histological investigation showed progressive fibrous degeneration of the vastus intermedius and rectus femoris.

3. As the patella showed signs of retarded development a particular form of myodysplasia, probably congenital, may be supposed.

4. Conservative treatment always failed to stop the progress of this disease.

5. Surgical treatment (Bennett's method or an elongation of the ligamental apparatus of the intermedius muscle) was carried out in ten cases and gave good permanent results. Flexion remained possible only to the extent obtained at operation; rehabilitation gave no further improvement.

6. In the opinion of the author, these cases occur more often than is generally believed, but they are recorded under a different diagnosis and consequently are not submitted to rational treatment.

Introduction Compressive loads applied to the disc are translated into an internal hydrostatic pressure in the nucleus which is resisted by the annulus. The anisotropic, inhomogeneous, multiply, collagenous architecture of the annulus reflects the complex pattern of mainly tensile stresses developed in this region of the disc during normal function. While many previous investigators have analysed the tensile behaviour of the annulus there still remains much to be learned about the fundamental structural relationships within the disc wall and upon which normal function depends. There is also much to be learned about how alterations in these relationships might lead to disc malfunction. Both intra and inter-lamellar structural relationships will be fundamental to the maintenance of annular wall strength. The aim of this study was to use high resolution ‘live’ imaging to explore the fundamental structural relationships governing the elasticity, intrinsic strength and rupture behaviour of intra-lamellar sections.

Methods In-plane intra-lamellar sections of nominal thickness 70–90μm were cut from the outer lamellae of bovine discs using a sledging microtome. Using a micro-mechanical technique in combination with simultaneous high resolution differential interference contrast optical microscopy (DIC) structural responses both along and transverse to the primary direction of the mono-array of collagen fibres were studied.

Results and Discussion Stretching along the primary alignment direction revealed a biomechanical response consistent with the behaviour of an array of strong fibres whose strength is governed primarily by the strength of embedding in the vertebral endplates rather than from inter-fibre cohesion along their length. The mono-aligned array, even when lacerated, is highly resistant to any further tearing across the alignment direction. Although not visible in the relaxed mono-arrays, transverse stretching revealed a highly complex set of interconnecting structures embodying a series of hierarchical relationships not previously revealed. It is suggested that these structures might play an important role in the containment under pressure of the nuclear contents. The dramatic differences in rupture behaviour observed along versus across the primary fibre direction are consistent with known clinical consequences arising from varying degrees of annular wall damage, and might also explain various types of disc herniation. The lamellar architecture of the healthy disc revealed by this ‘live’ tissue investigation provides an important reference framework for exploring structural changes associated with disc trauma and degeneration.