The aim of the study to analyze the circulating white blood cells including the intensity expression of surface receptors and cytoplasmic molecules in patients underwent total hip replacement, with either aseptic or septic loosening of hip prostheses in order to identify cell-surface and cytoplasmic markers that could be indicative of early loosening.
Aims. Currently, the effect of drug treatment for osteoporosis is relatively poor, and the side effects are numerous and serious. Melatonin is a potential drug to improve bone mass in postmenopausal women. Unfortunately, the mechanism by which melatonin improves bone metabolism remains unclear. The aim of this study was to further investigate the potential mechanism of melatonin in the treatment of osteoporosis. Methods. The effects of melatonin on mitochondrial apoptosis protein, bmal1 gene, and related pathway proteins of RAW264.7 (mouse mononuclear macrophage leukaemia cells) were analyzed by western blot. Cell Counting Kit-8 was used to evaluate the effect of melatonin on cell viability.
Aims. It has been established that mechanical stimulation benefits tendon-bone (T-B) healing, and macrophage phenotype can be regulated by mechanical cues; moreover, the interaction between macrophages and mesenchymal stem cells (MSCs) plays a fundamental role in tissue repair. This study aimed to investigate the role of macrophage-mediated MSC chondrogenesis in load-induced T-B healing in depth. Methods. C57BL/6 mice rotator cuff (RC) repair model was established to explore the effects of mechanical stimulation on macrophage polarization, transforming growth factor (TGF)-β1 generation, and MSC chondrogenesis within T-B enthesis by immunofluorescence and enzyme-linked immunosorbent assay (ELISA). Macrophage depletion was performed by clodronate liposomes, and T-B healing quality was evaluated by histology and biomechanics. In vitro, bone marrow-derived macrophages (BMDMs) were stretched with CELLOAD-300 load system and macrophage polarization was identified by
Aims. Circular RNA (circRNA) S-phase cyclin A-associated protein in the endoplasmic reticulum (ER) (circSCAPER, ID: hsa_circ_0104595) has been found to be highly expressed in osteoarthritis (OA) patients and has been associated with the severity of OA. Hence, the role and mechanisms underlying circSCAPER in OA were investigated in this study. Methods. In vitro cultured human normal chondrocyte C28/I2 was exposed to interleukin (IL)-1β to mimic the microenvironment of OA. The expression of circSCAPER, microRNA (miR)-140-3p, and enhancer of zeste homolog 2 (EZH2) was detected using quantitative real-time polymerase chain reaction and Western blot assays. The extracellular matrix (ECM) degradation, proliferation, and apoptosis of chondrocytes were determined using Western blot, cell counting kit-8, and
Aims. Accumulated evidence indicates that local cell origins may ingrain differences in the phenotypic activity of human osteoblasts. We hypothesized that these differences may also exist in osteoblasts harvested from the same bone type at periarticular sites, including those adjacent to the fixation sites for total joint implant components. Methods. Human osteoblasts were obtained from the acetabulum and femoral neck of seven patients undergoing total hip arthroplasty (THA) and from the femoral and tibial cuts of six patients undergoing total knee arthroplasty (TKA). Osteoblasts were extracted from the usually discarded bone via enzyme digestion, characterized by
Aims. This study investigates the effects of intra-articular injection of adipose-derived mesenchymal stem cells (AdMSCs) and platelet-rich plasma (PRP) on lameness, pain, and quality of life in osteoarthritic canine patients. Methods. With informed owner consent, adipose tissue collected from adult dogs diagnosed with degenerative joint disease was enzymatically digested and cultured to passage 1. A small portion of cells (n = 4) surplus to clinical need were characterized using
Aims. Osteosarcoma is the most common primary bone malignancy among children and adolescents. We investigated whether benzamil, an amiloride analogue and sodium-calcium exchange blocker, may exhibit therapeutic potential for osteosarcoma in vitro. Methods. MG63 and U2OS cells were treated with benzamil for 24 hours. Cell viability was evaluated with the MTS/PMS assay, colony formation assay, and
Aims. This study aimed to investigate the role and mechanism of meniscal cell lysate (MCL) in fibroblast-like synoviocytes (FLSs) and osteoarthritis (OA). Methods. Meniscus and synovial tissue were collected from 14 patients with and without OA. MCL and FLS proteins were extracted and analyzed by liquid chromatography‒mass spectrometry (LC‒MS). The roles of MCL and adenine nucleotide translocase 3 (ANT3) in FLSs were examined by enzyme-linked immunosorbent assay (ELISA),
Autografts containing bone marrow (BM) are current gold standard in the treatment of critical size bone defects, delayed union and bone nonunion defects. Although reaching unprecedented healing rates in bone reconstruction, the mode of action and cell-cell interactions of bone marrow mononuclear cell (BM-MNC) populations have not yet been described. BM-MNCs consist of a heterogeneous mixture of hematopoetic and non-hematopoetic lineage fractions. Cell culture in a 3D environment is necessary to reflect on the complex mix of these adherend and non-adherend cells in a physiologically relevant context. Therefore, the main aim of this approach was to establish conditions for a stable 3D BM-MNC culture to assess cellular responses on fracture healing strategies. BM samples were obtained from residual material after surgery with positive ethical vote and informed consent of the patients. BM-MNCs were isolated by density gradient centrifugation, and cellular composition was determined by
Aims. To explore the novel molecular mechanisms of histone deacetylase 4 (HDAC4) in chondrocytes via RNA sequencing (RNA-seq) analysis. Methods. Empty adenovirus (EP) and a HDAC4 overexpression adenovirus were transfected into cultured human chondrocytes. The cell survival rate was examined by real-time cell analysis (RTCA) and EdU and
Undifferentiated pleomorphic sarcoma (UPS) is one of the most common and aggressive adult soft tissue sarcomas (STS). Once metastatic, UPS is rapidly fatal. Most STS, including UPS, are resistant to conventional immunotherapies as these tumours have low numbers of spontaneous tumour infiltrating lymphocytes (TILs) and are densely populated with immune suppressive macrophages. Intra-tumoural activation of the STimulator of INterferon Genes (STING) pathway is a novel immunotherapeutic strategy to recruit anti-tumour TILs into the tumour microenvironment. In a murine model of UPS, we have demonstrated that intra-tumoural injection of a murine-specific STING agonist, DMXAA, results in profound immune mediated tumour clearance. Recently, molecules capable of activating both human and mouse STING pathways have been developed. In pursuit of clinically relevant therapeutic opportunities, the purpose of this study is to evaluate the anti-tumour potential of two agonists of the human and murine STING receptors: ADU-S100 and MSA-2 as monotherapies and in combination with the immune checkpoint inhibitor, anti-PD1 in a murine model of UPS. Immune competent mice were engrafted with murine UPS cells in the hindlimb muscle. Once palpable, mice in the monotherapy group were treated with a single intra-tumoural dose of 1) ADU-S100 or 2) MSA-2 or 3) DMXAA. In additional experimental groups, mice were treated with the different STING agonists and monoclonal anti-PD1. Tumour volume measurements and tumour bioluminescence were measured over time. To quantify dynamic changes in immune populations and in the tumour immune microenvironment, STING treated UPS tumours were evaluated using
Aims. The aim of this study was to evaluate blood metal ion levels, leucocyte profiles, and serum cytokines in patients with a total hip arthroplasty (THA) involving modular dual-mobility components. Patients and Methods. A total of 39 patients were recruited, with clinical follow-up of up to two years. Outcome was assessed using the Harris Hip Score (HHS, the 12-Item Short-Form Health Survey (SF-12), the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), and a visual analogue scale (VAS) for pain. Blood concentrations of cobalt (Co), chromium (Cr), and serum cytokines were measured. Subpopulations of leucocytes were analyzed by
Aims. Autologous chondrocyte implantation (ACI) is a promising treatment for articular cartilage degeneration and injury; however, it requires a large number of human hyaline chondrocytes, which often undergo dedifferentiation during in vitro expansion. This study aimed to investigate the effect of suramin on chondrocyte differentiation and its underlying mechanism. Methods. Porcine chondrocytes were treated with vehicle or various doses of suramin. The expression of collagen, type II, alpha 1 (COL2A1), aggrecan (ACAN); COL1A1; COL10A1; SRY-box transcription factor 9 (SOX9); nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX); interleukin (IL)-1β; tumour necrosis factor alpha (TNFα); IL-8; and matrix metallopeptidase 13 (MMP-13) in chondrocytes at both messenger RNA (mRNA) and protein levels was determined by quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot. In addition, the supplementation of suramin to redifferentiation medium for the culture of expanded chondrocytes in 3D pellets was evaluated. Glycosaminoglycan (GAG) and collagen production were evaluated by biochemical analyses and immunofluorescence, as well as by immunohistochemistry. The expression of reactive oxygen species (ROS) and NOX activity were assessed by luciferase reporter gene assay, immunofluorescence analysis, and
Objectives. The objective of this study was to investigate the therapeutic effect of peripheral blood mononuclear cells (PBMNCs) treated with quality and quantity control culture (QQ-culture) to expand and fortify angiogenic cells on the acceleration of fracture healing. Methods. Human PBMNCs were cultured for seven days with the QQ-culture method using a serum-free medium containing five specific cytokines and growth factors. The QQ-cultured PBMNCs (QQMNCs) obtained were counted and characterised by
Abstract. Objective. SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between mesenchymal stromal cells (MSCs) and chondrocytes by comparing SOX gene expression in their co-culture and respective monocultures. Methods. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in
SOX genes comprise a family of transcription factors characterised by a conserved HMG-box domain that confer pleiotropic effects on cell fate and differentiation through binding to the minor groove of DNA. Paracrine regulation and contact-dependant Notch signalling has been suggested to modulate the induction of SOX gene expression. The objective of this study is to investigate the crosstalk between and preconditioning of mesenchymal stem cells (MSCs) with chondrocytes through comparing SOX gene expression in their co-culture and respective monocultures. Our study adopted an in vitro autologous co-culture of p0 adipose-derived MSCs (AMSCs) and articular chondrocytes derived from Kellgren-Lawrence Grade III/IV osteoarthritic knee joints (n=7). Samples were handled according to the 2004 UK Human Tissue Act. Cells were purified and co-cultured with one AMSC for every chondrocyte at 5000 cells/cm. 2. The AMSCs were characterised by a panel of MSC surface markers in
Aims. Interleukin (IL)-1β is one of the major pathogenic regulators during the pathological development of intervertebral disc degeneration (IDD). However, effective treatment options for IDD are limited. Suramin is used to treat African sleeping sickness. This study aimed to investigate the pharmacological effects of suramin on mitigating IDD and to characterize the underlying mechanism. Methods. Porcine nucleus pulposus (NP) cells were treated with vehicle, 10 ng/ml IL-1β, 10 μM suramin, or 10 μM suramin plus IL-1β. The expression levels of catabolic and anabolic proteins, proinflammatory cytokines, mitogen-activated protein kinase (MAPK), and nuclear factor (NF)-κB-related signalling molecules were assessed by Western blotting, quantitative real-time polymerase chain reaction (qRT-PCR), and immunofluorescence analysis.
The meniscus is at the cornerstone of knee joint function, imparting stability and ensuring shock absorption, load transmission, and stress distribution within the knee joint. However, it is very vulnerable to injury and age-related degeneration. Meniscal tears are reported as the most common pathology of the knee with a mean annual incidence of 66 per 100,000. Knee osteoarthritis progresses more rapidly in the absence of a functional meniscus. Historically, tears extending to the avascular inner portion of the meniscus (white-white zone, “WW”), such as radial tears were considered as untreatable and were often resected, due to the lack of vascularity in the WW zone. Perfusion-based anatomical studies performed on cadaveric menisci in the 1980s shaped the current dogma that human meniscus has poor regenerative capacity, partly due to limited blood supply that only reaches 10 to 25% of the meniscus, commonly referred to as red-red zone (“RR”). Previous studies, including those utilizing animal models have shown mobilization of Mesenchymal Stem Cells (MSCs) upon injury into the WW zone, and successful MSC recruitment when administered externally to the injury site. We and others have recently reported positive outcomes of repaired tears in the inner zone of patients. We hypothesized that the “avascular” white-white zone of the meniscus possesses regenerative capacity due to a resident stem/progenitor cell population. Further, we sought to redefine the presence of microvessels in all meniscal zones using advanced stereology and imaging modalities. Fifteen menisci from fresh human cadaveric knees (mean age: 21.53±6.53 years) without evidence of previous injury were obtained from two tissue banks (JRF, Centennial, CO) and Biosource Medical (Lakeland, FL) and utilized for this study. The use of cadaveric specimens for research purposes was approved by the institutional review board. Tibial plateaus were dissected to harvest medial and lateral menisci along their entire length. The RR, red-white (RW) and WW zones were dissected and separated into three thirds from the inner aspect to the marginal border of the meniscus and their wet weights recorded (Fig.1A). Meniscus tissue cellular content in each zone was obtained from dissociation of meniscus tissue using 0.02% w/v pronase (Millipore) for 1h at 37oC, followed by 18h 0.02% w/v collagenase II (Worthington) at 37oC with shaking. Isolated cells were characterized immediately after harvest using
Introduction. It has been shown in vitro that human monocytes can phagocytose submicron polyethylene wear particles generated from total hip arthroplasties (THA) with highly cross-linked polyethylene inlays. The aim of our study was to detect the presence and possible phagocytosis of such particles in peripheral blood monocytes of patients with respective THA. Patients and methods. All patients were operated using the same implant, the cementless SL Plus stem; Bicon cup and a cross-linked polyethylene insert Rexpol (Smith and Nephew). Besides clinical and radiographic check-up, blood samples were collected at follow-up and analyzed by
Objectives. Cortical and cancellous bone healing processes appear to be histologically different. They also respond differently to anti-inflammatory agents. We investigated whether the leucocyte composition on days 3 and 5 after cortical and cancellous injuries to bone was different, and compared changes over time using day 3 as the baseline. Methods. Ten-week-old male C56/Bl6J mice were randomized to either cancellous injury in the proximal tibia or cortical injury in the femoral diaphysis. Regenerating tissues were analyzed with
Mesenchymal stem cells (MSC) are multipotent cells that possess regenerative functions that are of interest for in osteoarticular diseases such as osteoarthritis (OA). These functions are thought to be primarily mediated by mediators released within extracellular vesicles (EV). The aim of this study was to compare the immunomodulatory effects of two major types of EV, exosomes and microparticles, secreted by MSCs. EV subsets were isolated from murine primary MSCs by ultracentrifugation. Size and structure were evaluated by Dynamic Light Scattering and electron microscopy. Expression of membrane and endosomal markers was tested by
Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using
Aim. Periprosthetic joint infections (PJI) are severe complications after total joint arthroplasty (TJA). Up to now, a gold standard in the diagnostics of PJI is missing. Small extracellular vesicles (sEVs) are secreted by all types of cells and play a key role in immune response in presence of infection (1). In this prospective study, the diagnostic accuracy of sEVs in the synovial fluid to detect PJI of knee, hip and shoulder joints was investigated. We hypothesized increased surface markers of sEVs in PJI compared to aseptic complications (e.g. implant loosening, stress shielding related pain). Method. Synovial fluid from 48 patients with painful arthroplasty was examined. The distinction between aseptic and infectious cases was made on the basis of the 2018 Definition of Periprosthetic Hip and Knee Infection (2). 35 (72,9%) probands assigned to aseptic and 13 patients (27,1%) to PJI group. Immuno-fluorescence
As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis. Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and
Recent studies suggested that both the soluble protein of the mesenchymal stromal cell (MSC) secretome, as well as the secreted extracellular vesicles (EVs) promote bone regeneration. However, there is limited knowledge of the changes in MSC secretome vesicular fraction during aging. We therefore aimed to characterize the release profiles and cargo of EVs from MSCs of different chronological ages. Conditioned medium (CM) was collected from 13 bone marrow MSC strains (20-89 years) and from one MSC strain derived from human induced pluripotent stem cells (iPSCs). The EV-containing fraction was enriched with ultracentrifugation. The number of particles in the CM was evaluated by nanoparticle tracking analysis (NTA), and the number of EVs was evaluated by
Aims. To investigate the correlations among cytokines and regulatory T cells (T-regs) in ankylosing spondylitis (AS) patients, and their changes after anti-tumour necrosis factor-α (TNF-α) treatment. Methods. We included 72 AS patients with detailed medical records, disease activity score (Bath Ankylosing Spondylitis Disease Activity Index), functional index (Bath Ankylosing Spondylitis Functional Index), and laboratory data (interleukin (IL)-2, IL-4, IL-10, TNF-α, interferon (IFN)-γ, transforming growth factor (TGF)-β, ESR, and CRP). Their peripheral blood mononuclear cells (PBMCs) were marked with anti-CD4, anti-CD25, and anti-FoxP3 antibodies, and triple positive T cells were gated by
Background. Chronic low back pain is strongly linked to degeneration of the intervertebral disc (IVD), which currently lacks any targeted treatments. This study explores NPgel, a biomaterial combined with notochordal cells (NC), developmental precursor cells, as a potential solution. NCs, known for anti-catabolic effects on IVD cells, present a promising avenue for regenerating damaged IVD tissue. Methods. Bovine IVDs underwent enzymatic degeneration before NPgel (+/- NC) injection. Degenerated bovine IVDs were cultured under biomechanical loading for 21 days. Histology and immunohistochemistry assessed NC survival, phenotype, and matrix production. Within an in vivo sheep pilot study, NPgel (+/- NC) was injected into degenerated IVDs, blood was taken, and immune cell activation was monitored via
Meniscus is mainly composed of three different cell types; chondrocytes(Ch) situate in the superficial zone, whereas fibroblast-like cells locate in the peripheral region having long cell extensions in contact with different parts of the matrix, fibrochondrocytes(FC), is from the inner part of the meniscus and show a clear cell associated matrix. The aim of this study is to develop meniscus cell population using with mesenchymal stem cells (MSCs). For this purpose, MSCs were isolated from rabbit bone marrow and verified by
Early identification of patients at risk for impaired tendon healing and corresponding novel therapeutic approaches are urgent medical needs. This study aimed to clarify the role of CD3+ T-cells during acute Achilles tendon (AT) healing. Blood and hematoma aspirate were taken from 26 patients during AT reconstruction, and additional blood samples were obtained during clinical follow-up at 6, 26 and 52 weeks after surgery. T-cell subsets were analyzed by
Tendinopathy is the most frequent musculoskeletal disease that requires medical attention. Mechanical overload has been considered as a key driver of its pathology. However, the underline mechanism on how overload induces tendinopathy and inflammation is unclear. Extracellular mitochondria (EM) are newly identified as cell-to-cell communicators. The aim of this study is to elucidate the role of mitochondria in overload-induced inflammation. We performed three-dimensional uniaxial stretching to mouse tendon organoid in bioreactors. Cyclic strain of uniaxial loadings included underload, normal load, and overload, according to previous work. We then harvested microvesicles including EM, from the bioreactor by differential centrifugation and evaluated their characteristics by
Mesenchymal stem cells (MSCs) have been studied for the treatment of Osteoarthritis (OA), a potential mechanism of MSC therapies has been attributed to paracrine activity, in which extracellular vesicles (EVs) may play a major role. It is suggested that MSCs from younger donor compete with adult MSC in their EV production capabilities. Therefore, MSCs generated from induced pluripotent mesenchymal stem cells (iMSC) appear to provide a promising source. In this study, MSCs and iMSC during long term-expansion using a serum free clinical grade condition, were characterized for surface expression pattern, proliferation and differentiation capacity, and senescence rate. Culture media were collected continuously during cell expansion, and EVs were isolated. Nanoparticle tracking analysis (NTA), transmission electron microscopy, western blots, and
In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and
Aim. Osteomyelitis (OM) is a debilitating infection of the bone that originates from hematogenous spreading of microbes or contamination after surgery/fracture. OM is mainly caused by the opportunistic bacterium Staphylococcus aureus (SA), which can evade the host immune response, acquire antibiotic resistance and chronically colonize the musculoskeletal tissue . 1,2. , yet the underlying molecular and cellular processes are largely unclear. This study aimed to characterize the pathogenetic mechanisms of SA-OM with a focus on the long pentraxin 3 (PTX3), a soluble pattern recognition molecule and bone tissue component that is emerging as a new player in osteoimmunology . 3. and a diagnostic marker of periprosthetic joint infections, a common form of OM. 4. . Method. A murine model of OM based on intra-bone injection of SA was developed that closely mimicked surgery/trauma-related OM in humans and allowed addressing the role of PTX3 in gene-modified (Ptx3-/-) animals. Local and systemic infection and inflammation were assessed via microbiology,
Adipose-derived stem cells (ADSCs) are an effective alternative for Teno-regeneration. Despite their applications in tendon engineering, the mechanisms promoting tendon healing still need to be understood. Since there is scattered information on ovine ADSCs, this research aims to investigate in vitro their teno-differentiation for potential use in preclinical tendon regeneration models. Ovine ADSCs were isolated from the tail region according to FAT-STEM laboratories, expanded until passage six (P6), and characterized in terms of stemness, adhesion and MHC markers by
Abstract. Objectives. In relation to regenerative therapies in osteoarthritis and cartilage repair, mesenchymal stromal cells (MSCs) have immunomodulatory functions and influence macrophage behaviour. Macrophages exist as a spectrum of pro-(M1) and anti-(M2) inflammatory phenotypic subsets. In the context of cartilage repair, we investigated MSC-macrophage crosstalk, including specifically the priming of cartilage cells by macrophages to achieve a regenerative rather than fibrotic outcome. Methods. Human monocytes were isolated from blood cones and differentiated towards M1 and M2 macrophages. Monocytes (Mo), M1 and M2 macrophages were cultured directly and indirectly (trans-well system) with human bone marrow derived MSCs. MSCs were added during M1 polarisation and separately to already induced M1 cells. Outcomes (M1/M2 markers and ligands/receptors) were evaluated using RT-qPCR and
Abstract. Objectives. Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age and gender is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age and gender on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. Methods and Results. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age and gender on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory-based experiments to assess these properties. Compare the extent of the effect of age on MSC cell marker expression, proliferation and pathways. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the synovium, fat pad and bone fragments using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using
Aim. In the current study we aim to characterize the use of cationic host defense peptides (HDPs) as alternative antibacterial agents to include into novel antibacterial coatings for orthopedic implants. Staphyloccous aureus represent one the most challenging cause of infections to treat by traditional antibacterial therapies. Thanks to their lack of microbial resistance described so far, HDPs represent an attractive therapeutic alternative to antibiotics. Furthermore, HDPs have been showed to control infections via a dual function: direct antimicrobial activity and regulation of immune response. However, HDPs functions characterization and comparison is controversial, as changing test conditions or cell type used might yield different effects from the same peptide. Therefore, before moving towards the development of HDP-based coatings, we need to characterize and compare the immunomodulatory and antibacterial functions under the same conditions in vitro of 3 well-known cathelicidins: human LL-37, chicken CATH-2, and bovine-derived IDR-1018. Method. S. aureus, strain SH1000, was incubated with different concentrations of each HDP and bacterial growth was monitored overnight. Primary human monocytes were isolated from buffy coats using Ficoll-Paque density and CD14 microbeads, and differentiated for 7 days to macrophages. After 24h incubation in presence of LPS and HDPs, macrophages cytokines production was measured by ELISA. Macrophages cultured for 24h in presence of HDPs were infected with serum-opsonized S. aureus. 30 min and 24h after infection, bacterial phagocytosis and intracellular killing by macrophages were measured by
Aims. It is increasingly appreciated that coordinated regulation of angiogenesis and osteogenesis is needed for bone formation. How this regulation is achieved during peri-implant bone healing, such as osseointegration, is largely unclear. This study examined the relationship between angiogenesis and osteogenesis in a unique model of osseointegration of a mouse tibial implant by pharmacologically blocking the vascular endothelial growth factor (VEGF) pathway. Materials and Methods. An implant was inserted into the right tibia of 16-week-old female C57BL/6 mice (n = 38). Mice received anti-VEGF receptor-1 (VEGFR-1) antibody (25 mg/kg) and VEGF receptor-2 (VEGFR-2) antibody (25 mg/kg; n = 19) or an isotype control antibody (n = 19). Flow cytometric (n = 4/group) and immunofluorescent (n = 3/group) analyses were performed at two weeks post-implantation to detect the distribution and density of CD31. hi. EMCN. hi. endothelium. RNA sequencing analysis was performed using sorted CD31. hi. EMCN. hi. endothelial cells (n = 2/group). Osteoblast lineage cells expressing osterix (OSX) and osteopontin (OPN) were also detected with immunofluorescence. Mechanical pull-out testing (n = 12/group) was used at four weeks post-implantation to determine the strength of the bone-implant interface. After pull-out testing, the tissue attached to the implant surface was harvested. Whole mount immunofluorescent staining of OSX and OPN was performed to determine the amount of osteoblast lineage cells. Results.
Deriving autologous mesenchymal stem cells (MSCs) from adipose tissues without using enzymes requires sophisticated biomedical instruments. Applied pressure on tissues and cells are adjusted manually although centrifugation and filtration systems are frequently used. The number of derived MSCs therefore could differ between instruments. We compared the number of MSCs obtained from four commercially available devices and our newly designed and produced instrument (A2, B3, L3, M2 and T3). Three-hundred mL of adipose tissue was obtained from a female patient undergoing liposuction using the transillumination solution. Obtained tissue was equally distributed to each device and handled according to the producers' guides. After handling, 3 mL stromal vascular fraction (SVF) was obtained from each device. Freshly isolated SVF was characterized using multi-color
Injured skeletal muscle repairs spontaneously via regeneration, however, this process is often incomplete because of fibrotic tissue formation. In our study we wanted to show improved efficiency of regeneration process induced by antifibrotic agent decorin in a combination with Platelet Rich Plasma (PRP)-derived growth factors. A novel human myoblast cell (hMC) culture, defined as CD56 (NCAM)+ developed in our laboratory, was used for evaluation of potential bioactivity of PRP and decorin. To determine the their effect on the viability of hMC we performed a MTT assay. To perform the cell proliferation assay, hMCs were separately seeded on plates at a concentration of 30 viable cells per well. Cell growth medium prepared with different concentrations of PRP exudates (5%, 10%, and 20%) and decorin (10 ng/mL, 25 ng/mL, and 50 ng/mL) were added and incubated for 7 days. After incubation we stained the cells with crystal-violet and measured the absorbance. To study the expression of Transforming Growth Factor Beta (TGF-β) and myostatin (MSTN), two main fibrotic factors in the process of muscle regeneration we performed several ELISA assays in groups treated with all therapeutic agents (PRP, decorin and their combination). Further, we have studied the ability of these agents to influence the differential cascade of dormant myoblasts towards fully differentiated myotubes by monitoring step wise activation of single nuclear factors like MyoD and Myogenin via multicolor
Objectives. This study aimed to assess the effect of age and osteoporosis on the proliferative and differentiating capacity of bone-marrow-derived mesenchymal stem cells (MSCs) in female rats. We also discuss the role of these factors on expression and migration of cells along the C-X-C chemokine receptor type 4 (CXCR-4) / stromal derived factor 1 (SDF-1) axis. Methods. Mesenchymal stem cells were harvested from the femora of young, adult, and osteopenic Wistar rats. Cluster of differentiation (CD) marker and CXCR-4 expression was measured using
Abstract. Osteoarthritis is a common articular cartilage disorder and causes a significant global disease burden. Articular cartilage has a limited capacity of repair and there is increasing interest in the use of cell-based therapies to facilitate repair including the use of Mesenchymal Stromal Cells (MSCs). There is some evidence in the literature that suggests that advancing age is associated with declining MSC function, including reduced proliferation and differentiation potential, and greater cellular apoptosis. In our study, we first performed a systematic review of the literature to determine the effects of chronological age on the in vitro properties of MSCs, and then performed a laboratory study to investigate these properties. We initially conducted a PRISMA systematic review of the literature to review the evidence base for the effects of chronological age on the in vitro properties of MSCs including cell numbers, expansion, cell surface characterization and differentiation potential. This was followed by laboratory based experiments to assess these properties. Tissue from patients undergoing total knee replacement surgery was used to isolate MSCs from the infrapatellar fat pad using a method developed in our laboratory. The growth kinetics was determined by calculating the population doublings per day. Following expansion in culture, MSCs at P2 were characterised for a panel of cell surface markers using
Abstract. Focal articular cartilage defects do not heal and, left untreated, progress to more widespread degenerative changes. A promising new approach for the repair of articular cartilage defects is the application of cell-based regenerative therapies using mesenchymal stromal cells (MSCs). MSCs are however present in a number of tissues and studies suggest that they vary in their proliferation, cell surface characterisation and differentiation. As the phenotypic properties of MSCs vary depending on tissue source, a systematic comparison of the transcriptomic signature would allow a better understanding of these differences between tissues, and allow the identification of markers specific to a MSC source that is best suited for clinical application. Tissue was used from patients undergoing total knee replacement surgery for osteoarthritis following ethical approval and informed consent. MSCs were isolated from bone, cartilage, synovium and infrapatellar fat pad. MSC number and expansion were quantified. Following expansion in culture, MSCs were characterised using
Introduction and Objective. The early pro-inflammatory hematoma phase of bone healing is characterized by platelet activation followed by growth factor release. Bone marrow mesenchymal stromal cells (MSC) play a critical role in bone regeneration. However, the impact of the pro-inflammatory hematoma environment on the function of MSC is not fully understood. We here applied platelet-rich plasma (PRP) hydrogels to study how platelet-derived factors modulate functional properties of MSC in comparison to a non-inflammatory control environment simulated by fibrin (FBR) hydrogels. Materials and Methods. MSC were isolated from acetabular bone marrow of patients undergoing hip arthroplasty. PRP was collected from pooled apheresis thrombocyte concentrates. The phenotype of MSC was analyzed after encapsulation in hydrogels or exposure with platelet-derived factors with regards to gene expression changes, cell viability, extracellular vesicle (EV) release and immunomodulatory effects utilizing cellular and molecular,
Abstract. Objectives. Mesenchymal stromal/stem cells (MSCs) are increasingly recognized as regulators of immune cells during disease or tissue repair. During these situations, the extracellular matrix (ECM) is very dynamic and therefore, our studies aim to understand how ECM influences the activity of MSCs. Methods. Human MSCs cultured on tissue culture plastic (TCP) and encapsulated within collagen type I, fibrin, or mixed Collagen-Fibrin were exposed to low dose TNFα and IFNɣ. Transcription profiles were examined using bulk RNA sequencing (RNAseq) after 24h of treatment. ELISA, Western blot, qPCR and immunofluorescence were employed to validate RNAseq results and to investigate the significance of transcriptional changes.
Abstract. Objectives. Tendon and ligament injury poses an increasingly large burden to society. With surgical repair and grafting susceptible to high failure rates, tissue engineering provides novel avenues for treatment. This systematic review explores in vivo evidence whether mesenchymal stem cell-derived extracellular vesicles (MSC-EVs) can facilitate tendon and ligament repair in animal models. Methods. On May 26th 2021, a systematic search was performed on PubMed, Web of Science, Cochrane Library, Embase, using search terms ‘mesenchymal stem cell’ or ‘multipotent stem cell’ AND ‘extracellular vesicles’ or ‘exosomes’ AND ‘tendon’ or ‘ligament’ or ‘connective tissue’. Risk of bias was assessed using SYstematic Review Center for Laboratory animal Experimentation (SYRCLE) tool. Studies administering EVs isolated from human or animal-derived MSCs into in vivo models of tendon/ligament injury were included. In vitro, ex vivo, in silico studies were excluded, and studies without a control group were excluded. Data on isolation and characterisation of MSCs and EVs, and in vivo findings in animal models were extracted. Results. Out of 383 relevant studies, 11 case-control studies were included for data extraction, including a total of 448 animal subjects (range 10–90). Six studies utilised bone marrow-derived MSCs. All studies characterised their MSCs via
Introduction and Objective. Hyaluronic acid (HA) is an effective option for the treatment of osteoarthritis (OA) patients due to several properties such as normalization of the mechanical and rheological properties of the synovial fluid and amelioration of OA symptoms and joints function by promoting cartilage nutrition. Since OA progression is also significantly related to oxidative stress and reactive oxygen species (ROS), sodium succinate (SS) is envisioned as a promising compound for cartilage treatment by providing antioxidant defense able to normalize intracellular metabolism and tissue respiration via mitochondrial mechanism of action. The scope of this study was to investigate on an in vitro inflammatory model the efficacy of Diart. ®. product, a combination of HA and SS. Materials and Methods. Donor-matched chondrocytes and synoviocytes were obtained from KL 3–4 OA patients undergoing total knee replacement. At passage 4, inflammation was promoted with 1 ng/ml IL-1B for 48 hours in absence and presence of Diart. ®. at 1:3 dilution rate. Nitric oxide (NO) from cell culture supernatant was measured by Griess reaction. Mitochondrial and cytoplasmatic ROS evaluation was assessed by
Objectives. Mesenchymal stem cells (MSCs) are of growing interest in terms of bone regeneration. Most preclinical trials utilize bone-marrow-derived mesenchymal stem cells (bMSCs), although this is not without isolation and expansion difficulties. The aim of this study was: to compare the characteristics of bMSCs and adipose-derived mesenchymal stem cells (AdMSCs) from juvenile, adult, and ovarectomized (OVX) rats; and to assess the effect of human parathyroid hormone (hPTH) 1-34 on their osteogenic potential and migration to stromal cell-derived factor-1 (SDF-1). Methods. Cells were isolated from the adipose and bone marrow of juvenile, adult, and previously OVX Wistar rats, and were characterized with
Introduction. Tendon is prone to degeneration through ageing and injury and current therapies are largely ineffective. The recent identification of a cell population within tendon with stem cell-like characteristics holds potential for regeneration of tendon. The local stem cell environment (niche) is important for stem cell maintenance and function. This study aims to characterize extracellular matrix (ECM) components of the stem cell niche in equine tendon, which is prone to age-related degeneration and rupture. Materials and Methods. Putative tendon stem cells (TSCs) were isolated from equine superficial digital flexor tendon by low-density plating and differential adhesion to fibronectin. Cells were analysed by
Objectives. Regenerative medicine is an emerging field aimed at the repair and regeneration of various tissues. To this end, cytokines (CKs), growth factors (GFs), and stem/progenitor cells have been applied in this field. However, obtaining and preparing these candidates requires invasive, costly, and time-consuming procedures. We hypothesised that skeletal muscle could be a favorable candidate tissue for the concept of a point-of-care approach. The purpose of this study was to characterize and confirm the biological potential of skeletal muscle supernatant for use in regenerative medicine. Methods. Semitendinosus muscle was used after harvesting tendon from patients who underwent anterior cruciate ligament reconstructions. A total of 500 milligrams of stripped muscle was minced and mixed with 1 mL of saline. The collected supernatant was analysed by enzyme-linked immunosorbent assay (ELISA) and