Ligaments which heal spontaneously have a healing process that
is similar to skin wound healing. Menopause impairs skin wound healing
and may likewise impair ligament healing. Our purpose in this study
was to investigate the effect of surgical menopause on ligament
healing in a rabbit medial collateral ligament model. Surgical menopause was induced with ovariohysterectomy surgery
in adult female rabbits. Ligament injury was created by making a
surgical gap in the midsubstance of the medial collateral ligament.
Ligaments were allowed to heal for six or 14 weeks in the presence
or absence of oestrogen before being compared with uninjured ligaments. Molecular
assessment examined the messenger ribonucleic acid levels for collagens,
proteoglycans, proteinases, hormone receptors, growth factors and
inflammatory mediators. Mechanical assessments examined ligament
laxity, total creep strain and failure stress.Objectives
Methods
Intervertebral disc (IVD) degeneration is responsible for severe clinical symptoms including chronic back pain. Galectins are a family of carbohydrate-binding proteins, some of which can induce functional disease markers in IVD cells and other musculoskeletal diseases. Galectins −4 and −8 were shown to trigger disease-promoting activity in chondrocytes but their effects on IVD cells have not been investigated yet. This study elucidates the role of galectin-4 and −8 in IVD degeneration. Immunohistochemical evidence for the presence of galectin-4 and −8 in the IVD was comparatively provided in specimens of 36 patients with spondylochondrosis, spondylolisthesis, or spinal deformity. Confocal microscopy revealed co-localization of galectin-4 and −8 in chondrocyte clusters of degenerated cartilage. The immunohistochemical presence of galectin-4 correlated with histopathological and clinical degeneration scores of patients, whereas galectin-8 did not show significant correlations. The specimens were separated into annulus fibrosus (AF), nucleus pulposus (NP) and endplate, which was confirmed histologically. Separate cell cultures of AF and NP (n=20) were established and characterized using cell type-specific markers. Potential binding sites for galectins including sialylated N-glycans and LacdiNAc structures were determined in AF and NP cells using LC/ESI-MS-MS. To assess galectin functions, cell cultures were treated with recombinant galectin-4 or −8, in comparison to IL-1β, and analyzed using RT-qPCR and In-cell Western blot. In vitro, both galectins triggered the induction of functional disease markers (CXCL8 and MMP3) on
This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs) in vitro. Our hypothesis was that irisin would improve hNPC metabolism and proliferation. hNPCs were isolated from intervertebral discs and cultured in alginate beads. hNPCs were exposed to phosphate-buffered saline (PBS) or recombinant irisin (r-irisin) at 5, 10 and 25 ng/mL (n=4). Each experiment was performed in triplicate. Cell proliferation was assessed with trypan blue staining-automated cell counting and PicoGreen assay. Glycosaminoglycan (GAG) content was measured using the DMMB assay. Metabolic activity was assessed with the MTT assay and the Griess Reagent System. Gene expression of collagen type II (COL2), matrix metalloproteinase (MMP)-13, tissue inhibitor of matrix metalloproteinase (TIMP)-1 and −3, aggrecan, interleukin (IL)-1β, a disintegrin and metalloproteinase with thrombospondin motifs (ADAMTS)-5 was measured by RT-PCR. MTT assay and ADAMTS-5, COL2, TIMP-1 and IL-1β gene expression were evaluated following incubation with 5, 10 and 25 ng/mL r-irisin for 24 hours and subsequent culture with 10 ng/ml IL-1β and vice versa (incubation for 24 hours with IL-1β and subsequent culture with r-irisin). Irisin increased hNPC proliferation (p<0.001), metabolic activity (p<0.05), GAG content (p<0.01), as well as COL2 (p<0.01), aggrecan (p<0.05), TIMP-1 and −3 (p<0.01) gene expression, while decreasing MMP-13 (p<0.05) and IL-1β (p<0.001)
Introduction. Tendinopathies represent a significant health burden, causing inflammation, pain, and reducing quality of life. The pivotal role of macrophages (Mφ) characterized by their ability to differentiate into proinflammatory (M1) or anti-inflammatory (M2) phenotypes depending on the microenvironment, has gained significant interest in tissue inflammation research. Additionally, existing literature states that the interplay between tenocytes and immune cells during inflammation involves unidentified soluble factors (SF). This study aimed to investigate the effect of extracellular vesicles (EVs) and SF derived from polarized Mφ on tendon cells to provide deeper insights of their potential therapeutic applications in the context of inflammation. Method. Human monocytes were isolated from blood donor buffy coats and differentiated into M1, M2, and hybrid M1/M2 phenotypes. Subsequently, EVs were isolated from the conditioned media from polarized Mφ and comprehensively characterized. In parallel, the elution media containing SF was collected. Furthermore, the EVs and SF were released independently onto tenocytes from human donors, previously induced with IL-1β to simulate an inflammatory environment. Finally,
Introduction:. Exercise has showed to reduce pain and improve function in patients with discogenic low back pain (LBP). Although there is currently no biologic evidence that the intervertebral disc (IVD) can respond to physical exercise in humans, a recent study has shown that chronic running exercise is associated with increased IVD hydration and hypertrophy1. Irisin, a myokine released upon muscle contraction, has demonstrated to yield anabolic effects on different cell types, including chondrocytes2. This study aimed to investigate the effect of irisin on human nucleus pulposus cells (hNPCs). Our hypothesis is that irisin may improve hNPCs metabolism and proliferation. METHODS:. The hNPCs, isolated from discectomy surgical waste material (n = 5), were expanded and encapsulated in alginate beads. The hNPCs were treated with: i) only growth medium (control); ii) medium with recombinant irisin (r-IR) at different concentrations (5, 10 and 25 ng / mL); iii) medium with Interleukin-1β (IL1β); iv) medium with IL1β for 24 h and then with IL1β and r-IR; v) medium with r-IR for 24 h and then with r-IR and IL1 β. We evaluated proliferation (trypan blue and PicoGreen), metabolic activity (MTT), nitrite concentration (Griess), and expression levels of catabolic and anabolic genes via real-time polymerase chain reaction (qPCR). Each analysis was performed in triplicate for each donor and each experiment was performed three times. Data were expressed as mean ± S.D. One-way ANOVA was used for the groups under exam. RESULTS:. Irisin increased hNPCs proliferation (p < 0.001), metabolic activity at 10 ng/mL (p < 0.05), and GAG content at concentration of 10 ng/mL and 25 ng/mL (p < 0.01; p < 0.001, respectively). The production of nitrites, used as an indicator of cellular oxidative stress, was significantly decreased (p < 0.01). Gene expression levels compared to the control group increased for COL2A1 (p < 0.01), ACAN (p < 0.05), TIMP-1 and −3 (p < 0.01), while a decrease in
Introduction and Objective. Osteonecrosis of the femoral head (ONFH) is an evolving and disabling condition that often leads to subchondral collapse in late stages. It is the underlying diagnosis for approximately 3%–12% of total hip arthroplasties (THAs) and the most frequent aetiology for young patients undergoing THA. To date, the pathophysiological mechanisms underlying ONFH remain poorly understood. In this study, we investigated whether ONFH without an obvious etiological factor is related to impaired osteoblast activities, as compared to age-matched patients with primary OA. Materials and Methods. We cultured osteoblasts isolated from trabecular bone explants taken from the femoral head of patients with ONFH and from intertrochanteric region of patients with ONFH or with OA and compared their in vitro mineralisation capacity and secretion of paracrine factors. Results. Compared to patients with OA, osteoblasts obtained from the intertrochanteric region of patients with ONFH showed reduced mineralisation capacity, which further decreased in osteoblasts from the femoral head of the same patient. Lower mineralisation of osteoblasts from patients with ONFH correlated with lower
In the differentiation of osteoclasts the differentiation factor (RANKL) interacts with the receptor activator of NF-κB (RANK) in a direct cell-to-cell contact between osteoblast and (pre)osteoclast. This is inhibited by soluble osteoprotegerin (OPG). The
Introduction. Cell-based therapy is needed to overcome the lacking intrinsic ability of cartilage to heal. Generating cartilage tissue from human bone marrow-derived stromal cells (MSC) is limited by up-regulation of COL10, ALP and other hypertrophy markers in vitro and calcifying cartilage at heterotopic sites in vivo. MSC hypertrophic differentiation reflects endochondral ossification, unable to maintain a stable hyaline stage, as observed by redifferentiation of articular chondrocytes (AC). Several transcription factors (TF), are held responsible for hypertrophic development. SOX9, the master regulator of chondrogenesis is also, alongside MEF2C, regulating hypertrophic chondrocyte maturation and COL10 expression. RUNX2/3 are terminal markers driving chondrocyte hypertrophy, and skeletogenesis. However, so far regulation of these key fate determining TFs has not been studied thoroughly on mRNA and protein level through chondrogenesis of human MSC. To fill this gap in knowledge, we aim to uncover regulation of SOX9, RUNX2/3, MEF2C and other TFs related to hypertrophy during MSC chondrogenesis in vitro and in comparison to the gold standard AC redifferentiation. Methods. Expression of SOX9, RUNX2/3 and MEF2C was compared before and during 6-week chondrogenic re-/differentiation of human MSC and AC on
Objectives. Interleukin 18 (IL-18) is a regulatory cytokine that degrades the disc matrix. Bone morphogenetic protein-2 (BMP-2) stimulates synthesis of the disc extracellular matrix. However, the combined effects of BMP-2 and IL-18 on human intervertebral disc degeneration have not previously been reported. The aim of this study was to investigate the effects of the anabolic cytokine BMP-2 and the catabolic cytokine IL-18 on human nucleus pulposus (NP) and annulus fibrosus (AF) cells and, therefore, to identify potential therapeutic and clinical benefits of recombinant human (rh)BMP-2 in intervertebral disc degeneration. Methods. Levels of IL-18 were measured in the blood of patients with intervertebral disc degenerative disease and in control patients. Human NP and AF cells were cultured in a NP cell medium and treated with IL-18 or IL-18 plus BMP-2.
Summary. Corticosteroids (CS) are commonly administered by intra-articular injection to control the symptoms of osteoarthritis; however, CSs also suppress articular chondrocyte matrix synthesis. Both triamcinolone and methylprednisolone acetate significantly suppressed BMPs −2 and −7, and TGF-b1 expression, suggesting a mechanism by which CSs suppress articular chondrocyte matrix synthesis and cartilage homeostasis. Introduction. Osteoarthritis (OA) is a common and debilitating disease that affects approximately 30% of the US population and is also a major clinical problem in companion animals. There are many drugs available to manage the symptoms of OA. Of these, intra-articular corticosteroid (CS) administration is a common and very effective anti-arthritic therapy, and is frequently administered to equine athletes. CSs exert their potent anti-inflammatory effects by blocking phospholipase A and reducing inflammatory mediator production; however, CSs also suppress matrix-biosynthetic activity of articular chondrocytes. This activity, along with ther increased joint use that symptomatic relief allows, has been linked to ‘steroid arthropathy’; a progression of arthritis driven by compromised chondrocyte homeostatic capacity. Several lines of experimental and clinical evidence emphasise the importance of TGF-b and BMP autocrine/paracrine activity in maintaining the homeostatic status of articular chondrocytes (reviewed in Oshin and Stewart 2007). This study was carried out to address the following objectives: 1) To assess the effects of CS on expression of chondro-protective TGF-β and BMP ligands in equine articular chondrocytes, and 2) To determine if exogenous BMP ligand administration can mitigate the suppressive effects of CSs on articular chondrocyte synthesis of collagen type II (Coll II) and glycosaminoglycans (sGAG). Methods. Articular cartilage was collected from clinically normal joints of adult horses, euthanased for reasons other than musculoskeletal disease. Articular chondrocytes were isolated by collagenase digestion and cultured as aggregates in serum-free medium under non-adherent conditions (Stewart et al 2000). Triamcinolone acetate (TA) or methylprednisolone acetate (MPA) was added to the articular chondrocyte cultures at 10. −10. M, 10. −7. M, and 10. −5. M; comparable to in vivo exposure concentrations. Effects on Coll II, aggrecan/sGAG, BMP and TGF-b ligand expression were assessed by QPCR, Coll II ELISAs and DMMB assays. In a separate series of experiments, exogenous BMP-2 was administered to chondrocyte cultures exposed to CS supplementation, to determine whether BMP can prevent or mitigate CS-mediated suppression of matrix synthesis. Results. BMP-2 and BMP-7
We investigated the effect of progesterone on the nerve during lengthening of the limb in rats. The sciatic nerves of rats were elongated by leg lengthening for ten days at 3 mm per day. On alternate days between the day after the operation and nerve dissection, the progesterone-treated group received subcutaneous injections of 1 mg progesterone in sesame oil and the control group received oil only. On the fifth, tenth and 17th day, the sciatic nerves were excised at the midpoint of the femur and the
Introduction. In vitro expansion of human articular chondrocytes (HACs) is required for cell-based strategies to treat cartilage defects. We have earlier shown that culturing HACs at increased osmolarity (i.e., 380 mOsm), as compared to plasma osmolarity (i.e., 280 mOsm), increases collagen type II (COL2A1) expression in vitro. Our earlier results showed that knockdown of TGF-β2, a prototypic member of the TGF-β superfamily and an accepted key regulator of chondrocyte differentiation, resulted in increased COL2A1 production. BMPs are members of the TGF-β superfamily which are known to be involved in the regulation of COL2A1 expression. In this study, we aimed to elucidate the role of BMP signaling, in the upregulation of COL2 production upon TGF-β2 knockdown (KD) under hyperosmotic culture conditions. Methods. HACs from five OA patients (passage 1) were cultured in cytokine-free medium, under 280 or 380 mOsm respectively, under standard 2D in vitro conditions. TGF-β2 knockdown (KD) by siRNA was performed in the presence or absence of the established bone morphogenetic protein (BMP) type I receptor (BMPRI) inhibitor dorsomorphin (10 μM). Expression of COL2A1 was evaluated by qRT-PCR. Results. Culturing HACs at 380 mOsm increased COL2A1 mRNA expression. Addition of dorsomorphin decreased COL2A1 mRNA expression at both 280 and 380 mOsm, but its expression was still significantly higher at 380 mOsm. In hyperosmotic 380 mOsm culture conditions, TGF-β2 KD further increased COL2A1 mRNA expression, while addition of dorsomorphin under these conditions abrogated this effect. Still, expression of COL2A1
The meniscus is a fibrocartilaginous tissue that plays an important role in controlling the complex biomechanics of the knee. Many histological and mechanical studies about meniscal attachment have been carried out, and medial meniscus (MM) root repair is recommended to prevent subsequent cartilage degeneration following MM posterior root tear. However, there are only few studies about the differences between meniscus root and horn cells. The goal of this study was to clarify the differences between these two cells. Tissue samples were obtained from the medial knee compartments of 10 patients with osteoarthritis who underwent total knee arthroplasty. Morphology, distribution, and proliferation of MM root and horn cells, as well as gene and protein expression levels of Sry-type HMG box (SOX) 9 and type II collagen (COL2A1) were determined after cyclic tensile strain (CTS) treatment. Horn cells had a triangular morphology, whereas root cells were fibroblast-like. The number of horn cells positive for SOX9 and COL2A1 was considerably higher than that of root cells. Although root and horn cells showed similar levels of proliferation after 48, 72, or 96 h of culture, more horn cells than root cells were lost following 2-h CTS (5% and 10% strain). SOX9 and COL2A1
Introduction. Cell-based therapies become more and more prominent for the treatment of intervertebral disc (IVD) injuries. Different strategies are under current development and address the restoration of either annulus fibrosus (AF) or nucleus pulposus (NP). Application of such Advanced Therapy Medicinal Products (ATMPs) is strictly regulated. One requirement is to show the identity of the cells, to make sure the cells are indeed AF or NP cells and retained their IVD cell character during manufacturing process before injection to the site of injury. Therefore, we recently identified novel marker genes that discriminate AF and NP cells on tissue level. However, expression of these AF and NP tissue markers has not been investigated in cultured cells, yet. The aim of this study was to proof the tissue marker”s specificity to discriminate cultured AF and NP cells. Furthermore, we evaluated the tissue markers robustness to different cell culture conditions. Materials & Methods. AF and NP tissue was obtained from human lumbal IVD of five donors (31–45 years) with mild to moderate degenerative changes (Pfirrmann≤3). Cells were isolated by enzymatic digestion and expanded in culture medium containing 10% human serum and 1% antibiotics. To address specificity, AF and NP cells were cultured separately. To address robustness, 1) cells were cultured up to passage P2, 2) cell culture was performed using two different cell culture media and 3) cells were cryopreserved in an optional intermediate step. Gene expression analysis was performed for 11 novel AF and NP tissue marker: LDB2, ADGRL4, EMCN, ANKRD29, OLFML2A, SPTLC3, DEFB1, DSC3, FAM132B, ARAP2, CDKN2B (patent pending). Results & Discussion. In cell culture, AF and NP cells were indistinguishable by eye. Both AF and NP cells showed same cell morphology and cell growth through monolayer expansion. For most of the tested novel AF and NP tissue marker genes no difference was seen in cultured cells AF and NP cells on
While mesenchymal stromal cells (MSCs) are a very attractive cell source for cartilage regeneration, an inherent tendency to undergo hypertrophic maturation and endochondral ossification; as well as insufficient extracellular matrix production still prevent their clinical application in cell –based cartilage repair therapies. We recently demonstrated that intermittent treatment of MSC with parathyroid hormone-related protein (PTHrP) during in vitro chondrogenesis significantly enhanced extracellular matrix deposition and concomitantly reduced hypertrophy (1) opposite to constant PTHrP treatment, which strongly suppressed chondrogenesis via the cAMP/PKA pathway (2). Since signal timing seemed to be decisive for an anabolic versus catabolic outcome of the PTHrP treatment, we here aimed to investigate the role of PTHrP pulse frequency, pulse duration and total weekly exposure time in order to unlock the full potential of PTHrP pulse application to enhance and control MSC chondrogenesis. Human bone marrow-derived MSC were subjected to in vitro chondrogenesis for six weeks. From day 7–42, cells were additionally exposed to 2.5 nM PTHrP(1–34) pulses or left untreated (control). Pulse frequency was increased from three times per week (3×6h/week) to daily, thereby maintaining either pulse duration (6h/d, total 42 h/week) or total weekly exposure time (2.6h/d, total 18 h/week). A high frequency of PTHrP-treatment (daily) was important to significantly increase extracellular matrix deposition and strongly suppress ALP activity by 87 %; independent of the pulse duration. A long pulse duration was, however, critical for the suppression of the hypertrophic marker gene IHH, while MEF2C and IBSP were significantly suppressed by all tested pulse duration and frequency protocols. COL10A1, RUNX2 and MMP13
Introduction. The ability of tendons to withstand stress generally decreases with age, often resulting in increased tissue degeneration and decreased regeneration capacity. However, the underlying molecular and cellular mechanisms of tendon senescence remain poorly characterized. Therefore, the aim of the current study was to identify genes showing an age-dependent altered expression profile in tendons. Materials and Methods. A suppression-subtractive-hybridization (SSH) screen comparing cDNA libraries generated from Achilles tendons of mature-adult (3 months) and old (18 months) female C57BL/6 mice was conducted. Subsequently, the differential expression of the identified genes was validated by RT-qPCR and selected genes were then further analysed by immunohistochemistry and Western blot. To investigate age-related structural alterations in the collagenous extracellular matrix we applied SHG-microscopy and TEM. In vitro experiments with young and old tendon derived stem/progenitor cells (TDSCs) involved wounding assays, tendon-like constructs as well as collagen gel contraction assays. Results. Among 168 identified genes, several ECM genes showed a differential expression, including Col1a1, Col3a1, fibronectin, fibromodulin, thrombospondin-1, decorin, biglycan, lysyl oxidase, and Sparc. As evidenced by RT-qPCR the
We have investigated whether cells derived from haemarthrosis caused by injury to the anterior cruciate ligament could differentiate into the osteoblast lineage in vitro. Haemarthroses associated with anterior cruciate ligament injuries were aspirated and cultured. After treatment with β-glycerophosphate, ascorbic acid and dexamethasone or 1,25 (OH). 2. D. 3. , a significant increase in the activity of alkaline phosphatase was observed. Matrix mineralisation was demonstrated after 28 days and
Introduction. Low back pain is a major public health problem in our society. Degeneration of intervertebral disc (IVD) appears to be the leading cause of chronic low-back pain [1]. Mechanical stimulations including compressive and tensional forces are directly implicated in IVD degeneration. Several studies have implicated the cytoskeleton in mechanotransduction [2, 3], which is important for communication and transport between the cells and extracellular matrix (ECM). However, the potential roles of the cytoskeletal elements in the mechanotransduction pathways in IVD are largely unknown. Methods. Outer annulus fibrosus (OAF) and nucleus pulposus (NP) cells from skeletally mature bovine IVD were either seeded onto Flexcell¯ type I collagen coated plates or seeded in 3% agarose gels, respectively. OAF cells were subjected to cyclic tensile strain (10%, 1Hz) and NP cells to cyclic compressive strain (10%, 1Hz) for 60 minutes. Post-loading, cells were processed for immunofluorescence microscopy and RNA extracted for quantitative PCR analysis. Results. F-actin reorganisation was evident in OAF and NP cells subjected to tensile and compressive strain respectively and is likely due to load-induced differential mRNA expression of actin-binding proteins. The vimentin network was also more intricately organised in loaded NP cells. Compressive strain increased type II collagen and aggrecan transcription in NP cells, whereas levels decreased in OAF cells under tension.
Summary. We demonstrate that osteoclast-like cells of GCT result from the spontaneous fusion and differentiation of CD14+ cells of the monoblastic lineage by an autocrine mechanism mediated by RANKL, rather than induced by stromal cells. This process is further enhanced by the simultaneous impairment of the negative feed-back regulation of osteoclastogenesis by interferon β. Introduction. Giant cell tumor of bone (GCT) is a benign osteolytic lesion with a complex histology, comprising prominent multinucleated osteoclast-like cells (OC), mononuclear stromal cells (SC), and monocyte-like elements. So far, most studies have focused on SC as the truly transformed elements that sustain osteoclast differentiation, while less attention has been paid on the monocyte-like cell fraction. On the contrary, we have previously shown that SC are non-transformed element that can induce osteoclastogenesis of monocytes at levels that do not exceed that of normal mesenchymal stromal cells. We therefore focused on CD14+ monocyte-like cells as an alternative key candidate for the pathogenesis of GCT. Methods. We isolated CD14+ enriched cell fraction from tumor samples by immunomagnetic separation. We analyzed CD14+ cells for ultrastructural morphology,
OBJECTIVES. Ischaemic preconditioning (IPC) is a phenomenon whereby tissues develop an increased tolerance to ischaemia and subsequent reperfusion if first subjected to sublethal periods of ischaemia. Despite extensive investigation of IPC, the molecular mechanism remains largely unknown. Our aim was to show genetic changes that occur in skeletal muscle cells in response to IPC. METHODS. Firstly, we established an in-vitro model of IPC using a human skeletal muscle cell line. Gene expression of both control and preconditioned cells at various time points was determined. The genes examined were HIF-1 alpha, EGR1, JUN, FOS, and DUSP1. HIF-1 alpha is a marker of hypoxia. EGR1, JUN, FOS and DUSP1 are early response genes and may play a role in the protective responses induced by IPC. Secondly, the expression of HSPB8 was examined in a cohort of preconditioned total knee arthroplasty patients. RESULTS. HIF-1 alpha was upregulated following 1 and 2 hours of simulated ischaemia (p = 0.076 and 0.841 respectively) verifying that hypoxic conditions were met using our model. Expression of EGR1, FOS and DUSP1 were upregulated and peaked after 1 hour of hypoxia (p = 0.001, <0.00, and 0.038 respectively). cFOS was upregulated at 2 and 3 hours of hypoxia. IPC prior to simulated hypoxia resulted in a greater level of upregulation of EGR1, JUN and FOS genes (p = <0.00, 0.047, and <0.00 respectively). HSPB8 was not significantly upregulated following IPC using the hypoxic model. It was, however, upregulated on an
