Aims

Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear.

Stem cell therapy is an effective means to address the repair of large segmental bone defects. However, the intense inflammatory response triggered by the implants severely impairs stem cell differentiation and tissue regeneration. High-dose transforming growth factor β1 (TGF-β1), the most locally expressed cytokine in implants, inhibits osteogenic differentiation of bone marrow mesenchymal stem cells (BMSCs) and promotes tissue fibrosis, severely compromising the efficacy of stem cell therapy. Small molecule inhibitors of TGF-β1 can be used to ameliorate the osteogenic disorders caused by high concentrations of TGF-β1, but systemic inhibition of TGF-β1 function will cause strong adverse effects. How to find safe and reliable molecular targets to antagonize TGF-β1 remains to be elucidated. Orphan nuclear receptor Nr4a1, an endogenous inhibitory molecule of TGF-β1, suppresses tissue fibrosis, but its role in BMSC osteogenesis is unclear. We found that TGF-β1 inhibited Nr4a1 expression through HDAC4. Overexpression of Nr4a1 in BMSCs reversed osteogenic differentiation inhibited by high levels of TGF- β1. Mechanistically, RNA sequencing showed that Nr4a1 activated the ECM-receptor interaction and Hippo signaling pathway, which in turn promoted BMSC osteogenesis. In bone defect repair and fracture healing models, transplantation of Nr4a1-overexpressing BMSCs into C57BL/6J mice or treatment with the Nr4a1 agonist Csn-B significantly ameliorated inflammation-induced bone regeneration disorders. In summary, our findings confirm the endogenous inhibitory effect of Nr4a1 on TGF- β1 and uncover the effectiveness of Nr4a1 agonists as a therapeutic tool to improve bone regeneration, which provides a new solution strategy for the treatment of clinical bone defects and inflammatory skeletal diseases.

Aims

Lumbar spinal stenosis (LSS) is a common skeletal system disease that has been partly attributed to genetic variation. However, the correlation between genetic variation and pathological changes in LSS is insufficient, and it is difficult to provide a reference for the early diagnosis and treatment of the disease.

Critical size bone defects are frequently caused by accidental trauma, oncologic surgery, and infection. Distraction osteogenesis (DO) is a useful technique to promote the repair of critical size bone defects. However, DO is usually a lengthy treatment, therefore accompanied with increased risks of complications such as infections and delayed union.

Herein, we developed an innovative intramedullary biodegradable magnesium (Mg) nail to accelerate bone regeneration in critical size bone defect repair during DO.

We observed that Mg nail induced almost 4-fold increase of new bone formation and over 5-fold of new vessel formation at 2 weeks after distraction. Mg nail upregulated the expression of calcitonin gene-related peptide (CGRP) in the new bone as compared with the DO alone group. We further revealed that blockade of the sensory nerve by overdose capsaicin blunted Mg nail enhanced critical size bone defect repair during the DO process. Moreover, inhibitors/antagonist of CGRP receptor, FAK, and VEGF receptor blocked the Mg nail stimulated vessel and bone formation.

In summary, we revealed, for the first time, a CGRP-FAK-VEGF signaling axis linking sensory nerve and endothelial cells, which may be the main mechanism underlying Mg-enhanced critical size bone defect repair when combined with DO, suggesting a great potential of Mg implants in reducing DO treatment time for clinical applications.

Navigation in total hip arthroplasty has been shown to improve acetabular positioning and can decrease the incidence of mal-positioned acetabular components. The aim of this study was to assess two surgical guidance systems by comparing intra-operative measurements of acetabular component inclination and anteversion with a post-operative CT scan.

We prospectively collected intra-operative navigation data from 102 hips receiving conventional THA or hip resurfacing arthroplasty through either a direct anterior or posterior approach. Two guidance systems were used simultaneously: an inertial navigation system (INS) and optical navigation system (ONS). Acetabular component anteversion and inclination was measured on a post-operative CT.

The average age of the patients was 64 years (range: 24-92) and average BMI was 27 kg/m² (range 19-38). 52% had hip surgery through an anterior approach. 98% of the INS measurements and 88% of the ONS measurements were within 10° of the CT measurements. The mean (and standard deviation) of the absolute difference between the post-operative CT and the intra-operative measurements for inclination and anteversion were 3.0° (2.8) and 4.5° (3.2) respectively for the ONS, along with 2.1° (2.3) and 2.4° (2.1) respectively for the INS. There was significantly lower mean absolute difference to CT for the INS when compared to ONS in both anteversion (p<0.001) and inclination (p=0.02).

Both types of navigation produced reliable and reproducible acetabular cup positioning. It is important that patient-specific planning and navigation are used together to ensure that surgeons are targeting the optimal acetabular cup position. This assistance with cup positioning can provide benefits over free-hand techniques, especially in patients with an altered acetabular structure or extensive acetabular bone loss.

In conclusion, both ONS and INS allowed for adequate acetabular positioning as measured on postoperative CT, and thus provide reliable intraoperative feedback for optimal acetabular component placement.

Hip resurfacing arthroplasty (HRA) is a bone conserving alternative to total hip arthroplasty. We present the early 1 and 2-year clinical and radiographical follow-up of a novel ceramic-on-ceramic (CoC) HRA in a multi-centric Australian cohort.

Patient undergoing HRA between September 2018 and April 2021 were prospectively included. Patient-reported outcome measures (PROMS) in the form of the Forgotten Joint Score (FJS), HOOS Jr, WOMAC, Oxford Hip Score (OHS) and UCLA Activity Score were collected preoperatively and at 1- and 2-years post-operation. Serial radiographs were assessed for migration, component alignment, evidence of osteolysis/loosening and heterotopic ossification formation.

209 patients were identified of which 106 reached 2-year follow-up. Of these, 187 completed PROMS at 1 year and 90 at 2 years. There was significant improvement in HOOS (p< 0.001) and OHS (p< 0.001) between the pre-operative, 1-year and 2-years outcomes. Patients also reported improved pain (p<0.001), function (p<0.001) and reduced stiffness (p<0.001) as measured by the WOMAC score. Patients had improved activity scores on the UCLA Active Score (p<0.001) with 53% reporting return to impact activity at 2 years. FJS at 1 and 2-years were not significantly different (p=0.38). There was no migration, osteolysis or loosening of any of the implants. The mean acetabular cup inclination angle was 41.3° and the femoral component shaft angle was 137°. No fractures were reported over the 2-year follow-up with only 1 patient reporting a sciatic nerve palsy.

There was early return to impact activities in more than half our patients at 2 years with no early clinical or radiological complications related to the implant. Longer term follow-up with increased patient numbers are required to restore surgeon confidence in HRA and expand the use of this novel product.

In conclusion, CoC resurfacing at 2-years post-operation demonstrate promising results with satisfactory outcomes in all recorded PROMS.

Proximal humerus fractures (PHF) are common, accounting for approximately 5% of all fractures. Approximately 30% require surgical intervention which can range from open reduction with internal fixation (ORIF) to shoulder arthroplasty (including hemiarthroplasty, total shoulder arthroplasty, (TSA) or reverse total shoulder arthroplasty (RTSA)). The aim of this study was to assess trends in operative interventions for PHF in an Australian population.

Data was retrospectively collected for patients diagnosed with a PHF and requiring surgical intervention between January 2001 and December 2020. Data for patients undergoing ORIF were extracted from the Medicare database, while data for patients receiving arthroplasty for PHF were obtained from the Australian Orthopaedic Association National Joint Replacement Registry (AOANJRR).

Across the study period, ORIF was the most common surgical procedure for management of PHFs. However, since 2019, RTSA has surpassed ORIF as the most common surgical procedure to treat PHFs, accounting for 51% of operations. While the number of RTSA procedures for PHF has increased, ORIF and shoulder hemiarthroplasty has significantly reduced since 2007 (p < 0.001). TSA has remained uncommon across the follow-up period, accounting for less than 1% of all operations. Patients younger than 65 years were more likely to receive ORIF, while those aged 65 years or greater were more likely to receive hemiarthroplasty or RTSA.

While the number of ORIF procedures has increased during the period of interest, it has diminished as a proportion of overall procedure volume. RTSA is becoming increasingly popular, with decreasing utilization of hemiarthroplasty, and TSA for fracture remaining uncommon. These trends provide information that can be used to guide resource allocation and health provision in the future. A comparison to similar data from other nations would be useful.

Imageless computer navigation systems have the potential to improve acetabular cup position in total hip arthroplasty (THA), thereby reducing the risk of revision surgery. This study aimed to evaluate the accuracy of three alternate registration planes in the supine surgical position generated using imageless navigation for patients undergoing THA via the direct anterior approach (DAA).

Fifty-one participants who underwent a primary THA for osteoarthritis were assessed in the supine position using both optical and inertial sensor imageless navigation systems. Three registration planes were recorded: the anterior pelvic plane (APP) method, the anterior superior iliac spines (ASIS) functional method, and the Table Tilt (TT) functional method. Post-operative acetabular cup position was assessed using CT scans and converted to radiographic inclination and anteversion. Two repeated measures analysis of variance (ANOVA) and Bland-Altman plots were used to assess errors and agreement of the final cup position.

For inclination, the mean absolute error was lower using the TT functional method (2.4°±1.7°) than the ASIS functional method (2.8°±1.7°, ρ = .17), and the ASIS anatomic method (3.7°±2.1, ρ < .001). For anteversion, the mean absolute error was significantly lower for the TT functional method (2.4°±1.8°) than the ASIS functional method (3.9°±3.2°, ρ = .005), and the ASIS anatomic method (9.1°±6.2°, ρ < .001). All measurements were within ± 10° for the TT method, but not the ASIS functional or APP methods.

A functional registration plane is preferable to an anatomic reference plane to measure intra-operative acetabular cup inclination and anteversion accurately. Accuracy may be further improved by registering patient location using their position on the operating table rather than anatomic landmarks, particularly if a tighter target window of ± 5° is desired.

Aims

Degenerative cervical spondylosis (DCS) is a common musculoskeletal disease that encompasses a wide range of progressive degenerative changes and affects all components of the cervical spine. DCS imposes very large social and economic burdens. However, its genetic basis remains elusive.

Rheumatoid arthritis (RA) is an autoimmune disease that involves T and B cells and their reciprocal immune interactions with proinflammatory cytokines. T cells, an essential part of the immune system, play an important role in RA. T helper 1 (Th1) cells induce interferon-γ (IFN-γ), tumour necrosis factor-α (TNF-α), and interleukin (IL)-2, which are proinflammatory cytokines, leading to cartilage destruction and bone erosion. Th2 cells primarily secrete IL-4, IL-5, and IL-13, which exert anti-inflammatory and anti-osteoclastogenic effects in inflammatory arthritis models. IL-22 secreted by Th17 cells promotes the proliferation of synovial fibroblasts through induction of the chemokine C-C chemokine ligand 2 (CCL2). T follicular helper (Tfh) cells produce IL-21, which is key for B cell stimulation by the C-X-C chemokine receptor 5 (CXCR5) and coexpression with programmed cell death-1 (PD-1) and/or inducible T cell costimulator (ICOS). PD-1 inhibits T cell proliferation and cytokine production. In addition, there are many immunomodulatory agents that promote or inhibit the immunomodulatory role of T helper cells in RA to alleviate disease progression. These findings help to elucidate the aetiology and treatment of RA and point us toward the next steps.

Cite this article: Bone Joint Res 2022;11(7):426–438.

Abstract

Imageless computer navigation systems in total hip arthroplasty (THA) improve acetabular cup position, thereby reducing the risk of revision surgery for all causes as well as dislocation. We aimed to evaluate the registration accuracy of 3 alternate registration planes.

A prospective, observational study was conducted with 45 THA in the supine position using two imageless navigation systems and 3 registration planes. Patient position was registered sequentially using an optical system (Stryker OrthoMap) and an inertial sensor-based system (Navbit Sprint) with 3 planes of reference: (Plane 1) an anatomical plane using the anterior superior iliac spines (ASISs) and the pubic symphysis; (Plane 2) a functional plane parallel to the line between the ASISs and the table plane; and, (Plane 3) a functional plane that was perpendicular to the gravity vector and aligned with the longitudinal axis of the patient. The 3 measurements of acetabular cup inclination and anteversion were compared with the measurements from postoperative computed tomography (CT) scans.

For inclination, the mean absolute error was significantly lower for Plane 3 (1.80°) than for Plane 2 (2.74°), p = .038 and was lower for both functional planes than for the anatomical plane (3.75°), p < .001.

For anteversion, the mean absolute error was significantly lower for Plane 3 (2.00°) than for Plane 2 (3.69°), p = .004 and was lower for both functional planes than for the anatomical plane (8.58°), p < .001.

Patient registration using functional planes more accurately measured the acetabular cup position than registration using anatomic planes.

Introduction and Objective

Heterotopic ossification is the formation of extraskeletal mineralized tissue commonly associated with either trauma or surgery. While several mouse models have been developed to better characterize the pathologic progression of HO, no model currently exists to study HO of the hip, the most common location of acquired HO in patients. Owing to the unique biological mechanisms underpinning the formation of HO in different tissues, we sought to develop a model to study the post-surgical HO of the hip.

Osteosarcoma (OS) is the most prevalent bone tumor in children and young adults. Most tumors arise from the metaphysis of the long bones and easily metastasize to the lungs. Current therapeutic strategies of osteosarcoma are routinely surgical resection and chemotherapy, which are limited to the patients suffering from metastatic recurrence. Therefore, to investigate molecular mechanisms that contribute to osteosarcoma progression is very important and may shed light on targeted therapeutic approach to improve the survival of patients with this disease. Several miRNAs have been found expressed differentially in osteosarcoma (OS), In this study, we found that miR-144 significantly suppresses osteosarcoma cell proliferation, migration andinvasion ability in vitro, and inhibited tumor growth and metastasisin vivo. The function and molecular mechanism of miR-144 in Osteosarcoma was further investigated.

Tissue samples from fifty-one osteosarcoma patients were obtained from Shanghai Ninth People's Hospital. The in vitro function of miR-144 in Osteosarcoma was investigated by cell viability assay, wound healing assay, invasion assay, the molecular mechanism was identified by Biotin-coupled miRNA capture, Dual-luciferase reporter assays, etc. the in vivo function of miR-144 in osteosarcoma was confirmed by osteosarcoma animal model and miR-144−/− zebrafish model.

Mechanically, we demonstrated that Ras homolog family member A (RhoA) and its pivotal downstream effector Rho-associated, coiled-coil containing protein kinase 1 (ROCK1) were both identified as direct targets of miR-144. Moreover, the negative co-relation between downregulated miR-144 and upregulated ROCK1/RhoA was verified both in the osteosarcoma cell lines and clinical patients' specimens. Functionally, RhoA with or without ROCK1 co-overexpression resulted a rescue phenotype on the miR-144 inhibited cell growth, migration and invasion abilities, while individual overexpression of ROCK1 had no statistical significance compared with control in miR-144 transfected SAOS2 and U2-OS cells.

This study demonstrates that miR-144 inhibited tumor growth and metastasis in osteosarcoma via dual-suppressing of RhoA and ROCK1, which could be a new therapeutic approach for the treatment ofosteosarcoma.

Previous study reported that intra-articular injection of MgSO4 could alleviate pain related behaviors in a collagenase induced OA model in rats. It provided us a good description on the potential of Mg2+ in OA treatment. However, the specific efficiency of Mg2+ on OA needs to be further explored and confirmed. The underlying mechanisms should be elucidated as well. Increasing attention has been paid on existence of synovial fluid MSCs (SF-MSCs) (not culture expanded) which may participate in endogenous reparative capabilities of the joint. On the other hand, previous studies demonstrated that Mg2+ not only promoted the expression of integrins but also enhanced the strength of fibronectin-integrin bonds that indicated the promotive effect of Mg2+ on cell adhesion, moreover, Mg2+ was proved could enhance chondrogenic differentiation of synovial membrane derived MSCs by modulating integrins. Based on these evidence, we hypothesize herein intra-articular injection of Mg2+ can attenuate cartilage degeneration in OA rat through modulating the biological behavior of SF-MSCs.

Human and rat SF-MSCs were collected after obtaining Experimental Ethics approval. The biological behaviors of both human and rat SF-MSCs including multiple differentiation, adhesion, colony forming, proliferation, etc. were determined in vitro in presence or absence of Mg2+ (10 mmol/L). Male SD rats (body weight: 450–500 g) were used to establish anterior cruciate ligament transection and partial medial meniscectomy (ACLT+PMM) OA models. The rats received ACLT+PMM were randomly divided into saline (control) group and MgCl2 (0.5 mol/L) group (n=6 per group). Intra-articular injection was performed on week 4 post-operation, twice per week for two weeks. Knee samples were harvested on week 2, 4, 8, 12 and 16 after injection for histological analysis for assessing the progression of OA. On week 2 and 4 after injection, the rat SF-MSCs were also isolated before the rats were sacrificed for assessing the abilities of chondrogenic differentiation, colony forming and adhesion in vitro. Statistical analysis was done using Graphpad Prism 6.01. Unpaired t test was used to compare the difference between groups. Significant difference was determined at P < 0 .05.

The adhesion and chondrogenic differentiation ability of both human and rat SF-MSCs were significantly enhanced by Mg2+ (10 mmol/L) supplementation in vitro. However, no significant effects of Mg2+ (10 mmol/L) on the osteogenic and adipogenic differentiation as well as the colony forming and proliferation. In the animal study, histological analysis by Saffranin O and Toluidine Blue indicated the cartilage degeneration was significantly alleviated by intra-articular injection of Mg2+, in addition, the expression of Col2 in cartilage was also increased in MgCl2 group with respect to control group indicated by immunohistochemistry. Moreover, the OARSI scoring was decreased in MgCl2 group as well. Histological analysis and RT-qPCR indicated that the chondrogenic differentiation of SF-MSCs isolated from Mg2+ treated rats were significantly enhanced compare to control group.

In the current study, we have provided direct evidence supporting that Mg2+ attenuated the progression of OA. Except for the effect of Mg2+ on preventing cartilage degeneration had been demonstrated in this study, for the first time, we demonstrated the promoting effect of Mg2+ on adhesion and chondrogenic differentiation of endogenous SF-MSCs within knee joint that may favorite cartilage repair. We have confirmed that the anti-osteoarthritic effect of Mg2+ involves the multiple actions which refer to prevent cartilage degeneration plus enhance the adhesion and chondrogenic differentiation of SF-MSCs in knee joint to attenuate the progression of OA. These multiple actions of Mg2+ may be more advantage than traditional products. Besides, this simple, widely available and inexpensive administration of Mg2+ has the potential on reducing the massive heath economic burden of OA. However, the current data just provided a very basic concept, the exact functions and underlying mechanisms of Mg2+ on attenuating OA progression still need to be further explored both in vitro and in vivo. Formula of Mg2+ containing solution also need to be optimized, for example, a sustained and controlled release delivery system need to be developed for improving the long-term efficacy.

Bone is a connective tissue that undergoes constant remodeling. Any disturbances during this process may result in undesired pathological conditions. A single nucleotide substitution (596T-A) in exon eight which leads to a M199K mutation in human RANKL was found to cause osteoclast-poor autosomal recessive osteopetrosis (ARO). Patients with ARO cannot be cured by hematopoietic stem cell transplantation and, without proper treatments, will die in their early age. To date, how this mutation alters RANKL function has not been characterized. We thus hypothesized that hRANKL M199 residue is a structural determinant for normal RANKL-RANK interaction and osteoclast differentiation. By sharing our findings, we aim to achieve an improved clinical outcome in treating bone-related diseases such as osteoporosis, ARO and osteoarthritis.

Site-directed mutagenesis was employed to create three rat RANKL mutants, replacing the methionine 200 (human M199 equivalent residue) with either lysine (M200K), alanine (M200A) or glutamic acid (M200E). Recombinant proteins were subsequently purified through affinity chromatography and visualized by Coomassie blue staining and western blot. MTS was carried out before osteoclastogenesis assay in vitro to measure the cellular toxicity. Bone resorption pit assay, immuno-fluorescent staining, luciferase reporter assay, RT-PCR, western blot and calcium oscillation detection were also conducted to explore the biological effect of rRANKL mutants. Computational modeling, thermal Shift Assay, western blot and protein binding affinity experiments were later carried out for structural analyses.

rRANKL mutants M200K/A/E showed a drastically reduced ability to induce osteoclast formation and did not demonstrate features of competitive inhibition against wild-type rRANKL. These mutants are all incapable of supporting osteoclastic polarization and bone resorption or activating RANKL-induced osteoclast marker gene transcription. Consistently, they were unable to induce calcium flux, and also showed a diminished induction of IκBa degradation and activation of NF-kB and NFATc1 transcriptional activity. Furthermore, the transcriptional activation of the antioxidant response element (ARE) crucial in modulating oxidative stress and providing cytoprotection was also unresponsive to stimulation with rM200s. Structural analyses showed that rM200 is located in a hydrophobic pocket critical for protein folding. Thermal shift and western blot assays suggested that rM200 mutants formed unstructured proteins, with disturbed trimerisation and the loss of affinity to its intrinsic receptors RANK and OPG.

Taken together, we first demonstrates the underlying cause of M199-meidated ARO in a cellular and molecular level by establishing a phenotype in BMMs similar to observed in human samples. Further investigation hints the structural significance of a hydrophobic pocket within the TNF-like region. Combined with pharmaceutical studies on small-molecule drugs, this finding may represent a therapeutic target motif for future development of anti-resorptive treatments.

Introduction

Wear debris and metal ions originating from metal on metal hip replacements have been widely shown to recruit and activate macrophages. These cells secrete chemokines and pro-inflammatory cytokines that lead to an adverse local tissue reaction (ALTR), frequently requiring early revision. The mechanism for this response is still poorly understood. It is well documented that cobalt gives rise to apoptosis, necrosis and reactive oxygen species generation. Additionally, cobalt stimulates T cell migration, although the effect on macrophage motility remains unknown. This study tests the hypothesis that cobalt ions and nanoparticles affect macrophage migration stimulating an ALTR.

Aims

To present our experience of using a combination of intra-articular osteotomy and external fixation to treat different deformities of the knee.

The health of a synovial joint is relied on normal function and coordination of a group of tissues such as articular cartilage (AC), ligaments, tendons and muscles. Osteoarthritis (OA), which is the most common joint disease, is clinically characterised by lesion of AC. Despite this, injury of a ligament or tendon or muscle generates a joint instability, which accelerates deterioration of AC and progression of OA. Traditional histology is often used to study the pathology of biological tissues. It requires tissue biopsy, which traumatises the donor tissues. Therefore, it is not an idea method for assessing AC, ligaments and tendons as the tissues have a poor healing capability. There is a worldwide demand of an imaging technique that diagnoses the microstructural changes of chondral and connective tissues without biopsy. Confocal arthroscopy (Optiscan Pty Ltd, Australia) possesses a Ø 6.3 mm probe and offers a 0.7 µm lateral imaging resolution and 7 µm axial resolution. It has been successfully used for examining the internal microstructural disorders in rotator cuff tendons of human cadavers without tissue biopsy (WU et al., 2015). This study investigates the capability of confocal arthroscopy as optical histology for assessing the internal microstructure of AC, ligaments, tendons and muscles in a knee joint.

Four sheep keen joints were freshly donated by other research unrelated to this study. After 5 ml clinical grade fluorescein solution at 0.05 g/L was injected into the joint cavity of a knee joint, the joint was passively exercising for about 10 minutes. The joint was then open collaterally and washed thoroughly using PBS for acquiring the microstructure of AC, ligaments, tendons and muscles using the confocal arthroscopy.

Results: without biopsy, confocal arthroscopy offers an imaging resolution for onsite distinguishing the subtle microstructural difference of AC in the weight-bearing and non-weight bearing region. It also permitted visualising the hierarchical collagen structure in ligaments and tendons at a fibre level, and characterising the muscle nuclei, motor-neurons, moto-neuron synapse and striates of myofibres.

Confocal arthroscopy showed the early promise to act as optical histology for studying the microstructure of chondral and a range of connective tissues, which allows understand better the health status of a knee joint. Since a sheep knee joint is very small for operating a normal procedure of an arthroscopic examination, an open knee joint surgery was performed in this study to allow imaging the microstructure of AC and a range of connective tissues. This is accounted as a limitation in the study. Nevertheless, this study demonstrated the development of confocal arthroscopy may lead to optical histology of the internal microstructure of AC and a group of connective tissues, which offers understanding more comprehensively the healthy status of a knee joint.

A tendon is a fibrous connective tissue that acts to transmit tensile forces between muscles and bones. It mainly consists of soluble substance, collagen and small volume of elastic fibres, which are produced by tenoblasts and tenocytes. The Achilles tendon is the thickest tendon in the human body that subjects to some of the highest tensile force, thus disorders and ruptures commonly happen. As the insoluble fibrous components in Achilles tendons, the collagen fibrils and elastic fibres have unique spatial structure that plays important functional roles. Despite this, the understanding of relationship between them is still limited due to the lack of imaging evidence. Using confocal and second harmonic generation microscopy, this study aims to comprehensively investigate the spatial relationship of collagen, elastic fibres and tenocytes in hydrated tendons.

Longitudinal sections of 50 µm thick and transverse sections of 20 µm thick were cryo-sectioned respectively from the mid-portion of ten rabbit Achilles tendons. Sections were stained with 0.03g/L Acridine Orange (AO) and 1mg/ml Sulforhodamine B (SRB) solution respectively for labelling the nucleus and elastic fibres. The Leica TCS SP2 multiphoton microscopy containing second harmonic generation microscopy can image collagen without labelling. The sections were scanned by the multiphoton microscopy, and images were processed and reconstructed into 3D images to study the spatial structure of collagen, elastic fibres and cells in Achilles tendons

A rabbit Achilles tendon consists of three sub-tendons named flexor digitorum superficialis tendon, medial gastrocnemius tendon and lateral gastrocnemius tendon. Loose connective tissue connects the three sub-tendons and ensures efficient sliding between sub-tendons. The 3D network shows that the mid-portion of Achilles tendons is composed of longitudinal collagen and elastic fibres, while spindle tenocytes rest along the collagen and elastic fibres. Tenocytes appear to have a closer microstructural relationship with the elastic fibres. In comparison with the collagen, tenocytes and elastic fibres only occupy a very small volume in the 3D network. The elastic fibres exist in both tendon proper and endotenons. The tendon sheath and loose connective tissue have a higher cell density, and the cells are large and round while compared with tenocytes.

As a component of the extracellular matrix (ECM) in Achilles tendons that closely mediates with the tenocytes, the elastin may participate in the force transition and interaction between tenocytes and the ECM. The elastic fibres may also endow Achilles tendons with unique mechanical properties to stand for tensile force.