Aims. Here we introduce a wide and complex study comparing effects of growth factors used alone and in combinations on human mesenchymal stem cell (hMSC) proliferation and osteogenic differentiation. Certain ways of cell behaviour can be triggered by specific peptides – growth factors, influencing cell fate through surface cellular receptors. Methods. In our study transforming growth factor β (TGF-β), basic fibroblast growth factor (bFGF), hepatocyte growth factor (HGF), insulin-like growth factor 1 (IGF-1), and vascular endothelial growth factor (VEGF) were used in order to induce osteogenesis and proliferation of hMSCs from bone marrow. These cells are naturally able to differentiate into various mesodermal cell lines. Effect of each factor itself is pretty well known. We designed experimental groups where two and more growth factors were combined. We supposed cumulative effect would appear when more growth factors with the same effect were combined. The cellular metabolism was evaluated using MTS assay and double-stranded DNA (dsDNA) amount using PicoGreen assay. Alkaline phosphatase (ALP) activity, as early osteogenesis marker, was observed. Phase contrast
Aims. Platelet concentrates, like platelet-rich plasma (PRP) and platelet lysate (PL), are widely used in regenerative medicine, especially in bone regeneration. However, the lack of standard procedures and controls leads to high variability in the obtained results, limiting their regular clinical use. Here, we propose the use of platelet-derived extracellular vesicles (EVs) as an off-the-shelf alternative for PRP and PL for bone regeneration. In this article, we evaluate the effect of PL-derived EVs on the biocompatibility and differentiation of mesenchymal stromal cells (MSCs). Methods. EVs were obtained first by ultracentrifugation (UC) and then by size exclusion chromatography (SEC) from non-activated PL. EVs were characterized by transmission electron
To investigate the effects of senescent osteocytes on bone homeostasis in the progress of age-related osteoporosis and explore the underlying mechanism. In a series of in vitro experiments, we used tert-Butyl hydroperoxide (TBHP) to induce senescence of MLO-Y4 cells successfully, and collected conditioned medium (CM) and senescent MLO-Y4 cell-derived exosomes, which were then applied to MC3T3-E1 cells, separately, to evaluate their effects on osteogenic differentiation. Furthermore, we identified differentially expressed microRNAs (miRNAs) between exosomes from senescent and normal MLO-Y4 cells by high-throughput RNA sequencing. Based on the key miRNAs that were discovered, the underlying mechanism by which senescent osteocytes regulate osteogenic differentiation was explored. Lastly, in the in vivo experiments, the effects of senescent MLO-Y4 cell-derived exosomes on age-related bone loss were evaluated in male SAMP6 mice, which excluded the effects of oestrogen, and the underlying mechanism was confirmed.Aims
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The effects of remnant preservation on the anterior cruciate ligament (ACL) and its relationship with the tendon graft remain unclear. We hypothesized that the co-culture of remnant cells and bone marrow stromal cells (BMSCs) decreases apoptosis and enhances the activity of the hamstring tendons and tenocytes, thus aiding ACL reconstruction. The ACL remnant, bone marrow, and hamstring tendons were surgically harvested from rabbits. The apoptosis rate, cell proliferation, and expression of types I and III collagen, transforming growth factor-β (Aims
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Tissue inhibitors of metalloproteinases (TIMPs) are the endogenous inhibitors of the zinc-dependent matrix metalloproteinases (MMP) and A disintegrin and metalloproteinases (ADAM) involved in extracellular matrix modulation. The present study aims to develop the TIMPs as biologics for osteoclast-related disorders. We examine the inhibitory effect of a high affinity, glycosyl-phosphatidylinositol-anchored TIMP variant named ‘T1PrαTACE’ on receptor activator of nuclear factor kappa-Β ligand (RANKL)-induced osteoclast differentiation.Aims
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The involvement of long non-coding RNA (lncRNA) in bone marrow mesenchymal stem cell (MSC) osteogenic differentiation during osteoporosis (OP) development has attracted much attention. In this study, we aimed to disclose how LINC01089 functions in human mesenchymal stem cell (hMSC) osteogenic differentiation, and to study the mechanism by which LINC01089 regulates MSC osteogenesis. Quantitative reverse transcription polymerase chain reaction (RT-qPCR) and western blotting were performed to analyze LINC01089, miR-1287-5p, and heat shock protein family A (HSP70) member 4 (HSPA4) expression. The osteogenic differentiation of MSCs was assessed through alkaline phosphatase (ALP) activity, alizarin red S (ARS) staining, and by measuring the levels of osteogenic gene marker expressions using commercial kits and RT-qPCR analysis. Cell proliferative capacity was evaluated via the Cell Counting Kit-8 (CCK-8) assay. The binding of miR-1287-5p with LINC01089 and HSPA4 was verified by performing dual-luciferase reporter and RNA immunoprecipitation (RIP) experiments.Aims
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Transcription factor nuclear factor kappa B (NF-κB) plays a major role in the pathogenesis of chronic inflammatory diseases in all organ systems. Despite its importance, NF-κB targeted drug therapy to mitigate chronic inflammation has had limited success in preclinical studies. We hypothesized that sex differences affect the response to NF-κB treatment during chronic inflammation in bone. This study investigated the therapeutic effects of NF-κB decoy oligodeoxynucleotides (ODN) during chronic inflammation in male and female mice. We used a murine model of chronic inflammation induced by continuous intramedullary delivery of lipopolysaccharide-contaminated polyethylene particles (cPE) using an osmotic pump. Specimens were evaluated using micro-CT and histomorphometric analyses. Sex-specific osteogenic and osteoclastic differentiation potentials were also investigated in vitro, including alkaline phosphatase, Alizarin Red, tartrate-resistant acid phosphatase staining, and gene expression using reverse transcription polymerase chain reaction (RT-PCR).Aims
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Myokine developmental endothelial locus-1 (DEL-1) has been documented to alleviate inflammation and endoplasmic reticulum (ER) stress in various cell types. However, the effects of DEL-1 on inflammation, ER stress, and apoptosis in tenocytes remain unclear. Human primary tenocytes were cultured in palmitate (400 μM) and palmitate plus DEL-1 (0 to 2 μg/ml) conditions for 24 hours. The expression levels of ER stress markers and cleaved caspase 3, as well as phosphorylated 5' adenosine monophosphate-activated protein kinase (AMPK) and autophagy markers, were assessed by Western blotting. Autophagosome formation was measured by staining with monodansylcadaverine, and apoptosis was determined by cell viability assay and caspase 3 activity assay.Aims
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Gap junction intercellular communication (GJIC) in osteocytes is impaired by oxidative stress, which is associated with age-related bone loss. Ageing is accompanied by the accumulation of advanced oxidation protein products (AOPPs). However, it is still unknown whether AOPP accumulation is involved in the impairment of osteocytes’ GJIC. This study aims to investigate the effect of AOPP accumulation on osteocytes’ GJIC in aged male mice and its mechanism. Changes in AOPP levels, expression of connexin43 (Cx43), osteocyte network, and bone mass were detected in 18-month-old and three-month-old male mice. Cx43 expression, GJIC function, mitochondria membrane potential, reactive oxygen species (ROS) levels, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation were detected in murine osteocyte-like cells (MLOY4 cells) treated with AOPPs. The Cx43 expression, osteocyte network, bone mass, and mechanical properties were detected in three-month-old mice treated with AOPPs for 12 weeks.Aims
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To develop an early implant instability murine model and explore the use of intermittent parathyroid hormone (iPTH) treatment for initially unstable implants. 3D-printed titanium implants were inserted into an oversized drill-hole in the tibiae of C57Bl/6 mice (n = 54). After implantation, the mice were randomly divided into three treatment groups (phosphate buffered saline (PBS)-control, iPTH, and delayed iPTH). Radiological analysis, micro-CT (µCT), and biomechanical pull-out testing were performed to assess implant loosening, bone formation, and osseointegration. Peri-implant tissue formation and cellular composition were evaluated by histology.Aims
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The association of auraptene (AUR), a 7-geranyloxycoumarin, on osteoporosis and its potential pathway was predicted by network pharmacology and confirmed in experimental osteoporotic mice. The network of AUR was constructed and a potential pathway predicted by Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and Gene Ontology (GO) terms enrichment. Female ovariectomized (OVX) Institute of Cancer Research mice were intraperitoneally injected with 0.01, 0.1, and 1 mM AUR for four weeks. The bone mineral density (BMD) level was measured by dual-energy X-ray absorptiometry. The bone microstructure was determined by histomorphological changes in the femora. In addition, biochemical analysis of the serum and assessment of the messenger RNA (mRNA) levels of osteoclastic markers were performed.Aims
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Tert-butylhydroquinone (tBHQ) has been identified as an inhibitor of oxidative stress-induced injury and apoptosis in human neural stem cells. However, the role of tBHQ in osteoarthritis (OA) is unclear. This study was carried out to investigate the role of tBHQ in OA. OA animal model was induced by destabilization of the medial meniscus (DMM). Different concentrations of tBHQ (25 and 50 mg/kg) were intraperitoneally injected in ten-week-old female mice. Chondrocytes were isolated from articular cartilage of mice and treated with 5 ng/ml lipopolysaccharide (LPS) or 10 ng/ml interleukin 1 beta (IL-1β) for 24 hours, and then treated with different concentrations of tBHQ (10, 20, and 40 μM) for 12 hours. The expression levels of malondialdehyde (MDA) and superoxide dismutase (SOD) in blood were measured. The expression levels of interleukin 6 (IL-6), IL-1β, and tumour necrosis factor alpha (TNF-α) leptin in plasma were measured using enzyme-linked immunoabsorbent assay (ELISA) kits. The expression of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signalling pathway proteins, and macrophage repolarization-related markers, were detected by western blot.Aims
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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. 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 flow cytometry, and cultured to passage three before measurement of metabolic activity, collagen production, alkaline phosphatase (ALP) expression, and mineralization.Aims
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LY3023414 is a novel oral phosphatidylinositol 3-kinase (PI3K)/mammalian target of rapamycin (mTOR) dual inhibitor designed for advanced cancers, for which a phase II clinical study was completed in March 2020; however, little is known about its effect on bone modelling/remodelling. In this study, we aimed to explore the function of LY3023414 in bone modelling/remodelling. The function of LY3023414 was explored in the context of osteogenesis (bone formation by osteoblasts) and osteoclastogenesis (osteoclast formation and bone resorption). Murine preosteoblast MC3T3-E1 cell line and murine bone marrow-derived macrophage cells (BMMs) were subjected to different treatments. An MTS cell proliferation assay was used to examine the cytotoxicity. Thereafter, different induction conditions were applied, such as MCSF and RANKL for osteoclastogenesis and osteogenic media for osteogenesis. Specific staining, a bone resorption assay, and quantitative real-time polymerase chain reaction (qRT-PCR) were subsequently used to evaluate the effect of LY3023414. Moreover, small interfering RNA (siRNA) was applied to knockdown Akt1 or Akt2 for further validation. Lastly, western blot was used to examine the exact mechanism of action.Aims
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Bone is a dynamic tissue with a quarter of the trabecular and a fifth of the cortical bone being replaced continuously each year in a complex process that continues throughout an individual’s lifetime. Bone has an important role in homeostasis of minerals with non-stoichiometric hydroxyapatite bone mineral forming the inorganic phase of bone. Due to its crystal structure and chemistry, hydroxyapatite (HA) and related apatites have a remarkable ability to bind molecules. This review article describes the accretion of trace elements in bone mineral giving a historical perspective. Implanted HA particles of synthetic origin have proved to be an efficient recruiting moiety for systemically circulating drugs which can locally biomodulate the material and lead to a therapeutic effect. Bone mineral and apatite however also act as a waste dump for trace elements and drugs, which significantly affects the environment and human health. Cite this article:
This study aimed to examine the effects of tumour necrosis factor-alpha (TNF-α) on osteoblasts in metal wear-induced bone loss. TNF-α immunoexpression was examined in periprosthetic tissues of patients with failed metal-on-metal hip arthroplasties and also in myeloid MM6 cells after treatment with cobalt ions. Viability and function of human osteoblast-like SaOs-2 cells treated with recombinant TNF-α were studied by immunofluorescence, terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling (TUNEL) assay, western blotting, and enzyme-linked immunosorbent assay (ELISA).Aims
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Experimental studies indicate that non-steroidal anti-inflammatory drugs (NSAIDs) may have negative effects on fracture healing. This study aimed to assess the effect of immediate and delayed short-term administration of clinically relevant parecoxib doses and timing on fracture healing using an established animal fracture model. A standardized closed tibia shaft fracture was induced and stabilized by reamed intramedullary nailing in 66 Wistar rats. A ‘parecoxib immediate’ (Pi) group received parecoxib (3.2 mg/kg bodyweight twice per day) on days 0, 1, and 2. A ‘parecoxib delayed’ (Pd) group received the same dose of parecoxib on days 3, 4, and 5. A control group received saline only. Fracture healing was evaluated by biomechanical tests, histomorphometry, and dual-energy x-ray absorptiometry (DXA) at four weeks.Objectives
Methods
Oxidative stress plays a major role in the onset and progression of involutional osteoporosis. However, classical antioxidants fail to restore osteoblast function. Interestingly, the bone anabolism of parathyroid hormone (PTH) has been shown to be associated with its ability to counteract oxidative stress in osteoblasts. The PTH counterpart in bone, which is the PTH-related protein (PTHrP), displays osteogenic actions through both its N-terminal PTH-like region and the C-terminal domain. We examined and compared the antioxidant capacity of PTHrP (1-37) with the C-terminal PTHrP domain comprising the 107-111 epitope (osteostatin) in both murine osteoblastic MC3T3-E1 cells and primary human osteoblastic cells.Objectives
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