Mesenchymal stem cells (MSC) have a well recognised potential for tissue repair. This potential is two pronged: they can differentiate into the functional cell types of the damaged tissues and they can support tissue recovery by secreting trophic factors, depositing an extracellular matrix (ECM) and dampening inflammation. Three-dimensional microscopy recently shown that MSCs in the bone marrow create an intricate proteo-cellular scaffold with the ECM forming an interconnected cellular continuum whose structure is guided by the deposited ECM. This proteo-cellular scaffold controls bone marrow functions from hematopoiesis to osteogenesis. In the current study we aimed to optimise ECM production under in vitro conditions by immortalised MSCs with the view that the generated ECM can be utilised for tissue repair. With immunocytochemistry we determined the deposition of bone marrow-characteristic ECM proteins: collagen I, III, IV, V, VI, laminin and fibronectin. While primary MSCs produced slightly higher amount ECM proteins than immortalised MSCs, the relative abundancy of the ECM proteins was very similar. In order to isolate the ECM, we optimised a decellularisation method based on gentle lysis with sodium-deoxycholate and DNase digestion. Immunostaining for collagen I, III, VI and fibronectin and labelling the nuclei with Hoechst-33342 confirmed removal of all cells while retaining the ECM in its original architecture. Ideally, the decellularised ECM retains associated cytokines and chemokines, such as CXCL12, important for attracting MSCs. To test this, we seeded Molm-13 leukemia cells on decellularised ECM as MSC-produced CXCL12- and other cytokines protect leukemia cells against chemotherapeutics. We found that the decellularisation process however removed these factors and thus for therapeutic purposes, the decellularised ECM would need to be re-loaded with the essential chemo/cytokines. Overall, we developed a system for decellularised ECM production by immortalised MSCs and the results warrant further exploration of this avenue

Objectives. This study aimed to explore the role of miR-320a in the pathogenesis of osteoarthritis (OA). Methods. Human cartilage cells (C28/I2) were transfected with miR-320a or antisense oligonucleotides (ASO)-miR-320a, and treated with IL-1β. Subsequently the expression of collagen type II alpha 1 (Col2α1) and aggrecan (ACAN), and the concentrations of sulfated glycosaminoglycans (sGAG) and matrix metallopeptidase 13 (MMP-13), were assessed. Luciferase reporter assay, qRT-PCR, and Western blot were performed to explore whether pre-B-cell leukemia Homeobox 3 (PBX3) was a target of miR-320a. Furthermore, cells were co-transfected with miR-320a and PBX3 expressing vector, or cells were transfected with miR-320a and treated with a nuclear factor kappa B (NF-κB) antagonist MG132. The changes in Col2α1 and ACAN expression, and in sGAG and MMP-13 concentrations, were measured again. Statistical comparisons were made between two groups by using the two-tailed paired t-test. Results. Expression of miR-320a was elevated in OA cartilage tissues and chondrocytes, and in IL-1β-stimulated C28/I2 cells (p < 0.05 or p < 0.01). MiR-320a overexpression enhanced IL-1β-induced down-regulation of Col2α1 and ACAN and sGAG, and increased the IL-1β-induced overexpression of MMP-13 (p < 0.01). PBX3 was a direct target of miR-320a. PBX3 and MG132 co-transfection attenuated the effects of miR-320a on the expression of Col2α1, ACAN, sGAG and MMP-13(p < 0.01). Conclusion. Overexpression of miR-320a might enhance IL-1β-induced cartilage degradation factors. These effects might be via targeting PBX3 and regulating NF-κB. Cite this article: Y. Jin, X. Chen, Z. Y. Gao, K. Liu, Y. Hou, J. Zheng. The role of miR-320a and IL-1β in human chondrocyte degradation. Bone Joint Res 2017;6:–203. DOI: 10.1302/2046-3758.64.BJR-2016-0224.R1

Summary. Starting from human musculoskeletal sarcomas, we isolated a subset of cells that display cancer stem cell properties. The control of culture conditions is crucial to enhance the isolation of this cell population. Introduction. Cancer stem cells (CSCs) have emerged as the real responsible for the development, chemoresistance, and metastatic spread of different human cancers, including musculoskeletal sarcomas. However, unlike most leukemias and solid tumors, so far, data on musculoskeletal sarcomas refer to CSCs obtained from established cell lines, and only a few authors have reported on the isolation of CSCs from tissue samples [1-7]. Reasonably due to some peculiar features of mesenchymal tumors, including the lack of unique surface markers that identify tumor progenitors, there are still partial clues on the existence of a CSC population in these cancers. Here, we report the identification of putative CSCs in musculoskeletal sarcomas using the most general accepted isolation method, the sphere culture system. Accordingly to recent reports, we also analyzed the effects of reduced oxygen availability on the behavior of sarcoma CSCs. Patients & Methods. Between 2009 and 2012, we collected fresh tissue samples from 49 patients (25 males and 24 females, age 6–85 yr) with musculoskeletal sarcomas. Cells obtained from samples were cultured in anchorage-independent serum-starved conditions, in the presence of adequate growth factors, until the formation of floating spheres, here called ‘sarcospheres’. To obtain parental tumor cell cultures, single cells obtained from biopsies were in parallel seeded in anchorage-dependent conditions, in the presence of fetal bovine serum until the formation of cell monolayers. The obtained sarcospheres were characterised in terms of gene expression and in vivo tumorigenic potential. We then exposed sarcospheres obtained from a rhabdomyosarcoma model (RD cells) to a hypoxic environment (1% O. 2. ), and analyzed their growth and gene expression to that of sarcospheres grown at standard 21% O. 2. . Results. Using a sphere-forming assay, we established sphere cultures in 5 out of 49 cases (10.2 %). All sarcosphere cultures expressed consistent mRNA levels for OCT3/4, Nanog, and SOX2. CSCs from a chondrosarcoma and from a rhabdomyosarcoma also showed the ability to recapitulate the original tumor morphology in a mouse model. Finally, we observed that hypoxia induced a significant increase of the number and size of CSCs from RD. Discussion/Conclusion. Starting from human sarcoma biopsies and established cell lines, we were able to characterise the CSC subset of musculoskeletal sarcomas, that were isolated through the sphere system assay. These cells had stem-like properties, and showed in vivo tumorigenic ability. We also observed that exposure of CSCs to low oxygen conditions increased the number and size of spheres and the expression of stem cell-related markers, suggesting that the culture in hypoxic conditions could improve the yield of the isolation method here used, and that the oxygen availability is a crucial element in the physiological maintenance of CSCs of musculoskeletal sarcomas

Objectives

The role of mechanical stress and transforming growth factor beta 1 (TGF-β1) is important in the initiation and progression of osteoarthritis (OA). However, the underlying molecular mechanisms are not clearly known.

Objectives

Osteoporosis is a chronic disease. The aim of this study was to identify key genes in osteoporosis.

Objectives

This study looked to analyse the expression levels of microRNA-140-3p and microRNA-140-5p in synovial fluid, and their correlations to the severity of disease regarding knee osteoarthritis (OA).

Systemic factors are believed to be pivotal for the development of heterotopic ossification in severely-injured patients. In this study, cell cultures of putative target cells (human fibroblastic cells, osteoblastic cells (MG-63), and bone-marrow stromal cells (hBM)) were incubated with serum from ten consecutive polytraumatised patients taken from post-traumatic day 1 to day 21 and with serum from 12 healthy control subjects.

The serum from the polytraumatised patients significantly stimulated the proliferation of fibroblasts, MG-63 and of hBM cells. The activity of alkaline phosphatase in MG-63 and hBM cells was significantly decreased when exposed to the serum of the severely-injured patient. After three weeks in 3D cell cultures, matrix production and osteogenic gene expression of hBM cells were equal in the patient and control groups. However, the serum from the polytraumatised patients significantly decreased apoptosis of hBM cells compared with the control serum (4.3% vs 19.1%, p = 0.031).

Increased proliferation of osteoblastic cells and reduced apoptosis of osteoprogenitors may be responsible for increased osteogenesis in severely-injured patients.

The role of inflammatory cells and their products in tendinopathy is not completely understood. Pro-inflammatory cytokines are upregulated after oxidative and other forms of stress. Based on observations that increased cytokine expression has been demonstrated in cyclically-loaded tendon cells we hypothesised that because of their role in oxidative stress and apoptosis, pro-inflammatory cytokines may be present in rodent and human models of tendinopathy. A rat supraspinatus tendinopathy model produced by running overuse was investigated at the genetic level by custom micro-arrays. Additionally, samples of torn supraspinatus tendon and matched intact subscapularis tendon were collected from patients undergoing arthroscopic shoulder surgery for rotator-cuff tears and control samples of subscapularis tendon from ten patients with normal rotator cuffs undergoing arthroscopic stabilisation of the shoulder were also obtained. These were all evaluated using semiquantitative reverse transcription polymerase chain-reaction and immunohistochemistry.

We identified significant upregulation of pro-inflammatory cytokines and apoptotic genes in the rodent model (p = 0.005). We further confirmed significantly increased levels of cytokine and apoptotic genes in human supraspinatus and subscapularis tendon harvested from patients with rotator cuff tears (p = 0.0008).

These findings suggest that pro-inflammatory cytokines may play a role in tendinopathy and may provide a target for preventing tendinopathies.

A rat model of lumbar root constriction with an additional sympathectomy in some animals was used to assess whether the sympathetic nerves influenced radicular pain. Behavioural tests were undertaken before and after the operation.

On the 28th post-operative day, both dorsal root ganglia and the spinal roots of L4 and L5 were removed, frozen and sectioned on a cryostat (8 μm to 10 μm). Immunostaining was then performed with antibodies to tyrosine hydroxylase (TH) according to the Avidin Biotin Complex method. In order to quantify the presence of sympathetic nerve fibres, we counted TH-immunoreactive fibres in the dorsal root ganglia using a light microscope equipped with a micrometer graticule (10 x 10 squares, 500 mm x 500 mm). We counted the squares of the graticule which contained TH-immunoreactive fibres for each of five randomly-selected sections of the dorsal root ganglia.

The root constriction group showed mechanical allodynia and thermal hyperalgesia. In this group, TH-immunoreactive fibres were abundant in the ipsilateral dorsal root ganglia at L5 and L4 compared with the opposite side. In the sympathectomy group, mechanical hypersensitivity was attenuated significantly.

We consider that the sympathetic nervous system plays an important role in the generation of radicular pain.

Previous research has shown an increase in chromosomal aberrations in patients with worn implants. The type of aberration depended on the type of metal alloy in the prosthesis. We have investigated the metal-specific difference in the level of DNA damage (DNA stand breaks and alkali labile sites) induced by culturing human fibroblasts in synovial fluid retrieved at revision arthroplasty.

All six samples from revision cobalt-chromium metal-on-metal and four of six samples from cobalt-chromium metal-on-polyethylene prostheses caused DNA damage. By contrast, none of six samples from revision stainless-steel metal-on-polyethylene prostheses caused significant damage. Samples of cobalt-chromium alloy left to corrode in phosphate-buffered saline also caused DNA damage and this depended on a synergistic effect between the cobalt and chromium ions.

Our results further emphasise that epidemiological studies of orthopaedic implants should take account of the type of metal alloy used.