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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Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment. Cite this article:
Aim. Biomaterial-associated infections (BAI) present a formidable clinical challenge. Bioactive glasses (BG) have proven highly successful in diverse clinical applications, especially in dentistry and orthopaedics. In this study, we aimed to determine the effect of three commonly used BG composition and particle sizes on cell and bacterial attachment and growth. Our focus is on understanding the changes in pH and osmotic pressure in the surrounding environment during glass degradation. Method. First, three different melt-derived glasses were characterized by analyzing particle size and glass network structure using Raman and NMR. The different glasses were then tested in vitro by seeding 4x 10. 4. cells/well (SaOS Cell line) in a 48 well plate. After a pre-incubation period of 72 hours, the different BGs and particle sizes were added to the cells and the pH value, ion release and live/dead staining was measured every hour. The effect of BG against bacteria (S. epidermidis) was analyzed after 24 and 72 hours of treatment by using XTT viability assay and CFU counting by plating out the treated aliquot agar to estimate the
Aim. Chemical debridement is a fundamental step during Periprosthetic joint infection (PJI) surgery. Antiseptic solutions are commonly used, but evidence on the optimal antiseptic, concentration, and irrigation time is lacking. The aim of this study is to analyze and compare the anti-biofilm capacity of povidone iodine, H. 2. 0. 2. , acetic acid and Bactisure™ after different exposure times, as well as their combinations. Method. Surgical steel discs inoculated with methicillin susceptible (MSSA) and resistant S. aureus (MRSA), P. aeruginosa, and S. epidermidis were exposed to the following antiseptic solutions: 0.3% (PI0.3) and 10% povidone iodine (PI10), H. 2. 0. 2. , 3% Acetic acid (AA3) and Bactisure™. Combinations included AA3, H. 2. 0. 2. , and PI10 in various orders. Exposure time for the antiseptics solutions was 1, 3 and 5 minutes, while combinations had a 9-minute total exposure, 3 minutes per antiseptic sequentially. All experiments were performed in triplicate and with a sterile saline control. nThe reduction in colony-forming units (CFU) was measured after sonication, and biofilm structure was analyzed via scanning electron microscopy. Results. PI showed the highest antibiofilm activity. PI0.3 eradicated bacteria on the discs after 3 and 5 minutes of exposure, but only achieved a 77.1% reduction after 1 minute. After PI10 treatment, we did not recover any bacteria regardless of exposure time. H. 2. 0. 2. , AA3, and Bactisure™ reached a significantly lower bacterial decrease at all exposure times compared to PI0.3 and PI10. AA3 was less effective against MSSA and S. epidermidis. H. 2. 0. 2. showed less activity against MRSA than PI0.3, PI10, and Bactisure™. Combinations of antiseptics starting with AA3 showed the best results in terms of CFU reduction and
Introduction. Osteoarthritis (OA) is a prevalent joint disorder characterized by cartilage degeneration, inflammation, and pain. Current treatments provide only symptomatic relief, necessitating novel molecular targets. The caspase family, known for its roles in apoptosis and inflammation regulation, may additionally influence crucial processes for cartilage homeostasis such as differentiation and proliferation. However, the specific roles of individual caspases in OA pathogenesis remain unclear. This study aims to investigate the involvement of the caspase family in OA and as potential targets for therapy, with a focus on caspase-1 and -8. Method. Chondrocytes from both healthy and OA donors were cultured in 2D and 3D culture models and stimulated with TNF-α or IL-1β. The expression and activation of caspase-1 and -8 was assessed using RT-PCR, ELISA. Transcriptome analysis of OA and healthy cartilage samples, along with Mendelian randomization (MR) analysis were conducted to explore the involvement of caspase family in OA and to assess its potential as therapeutic targets. Result. Higher expression levels of caspase-1, -8 were observed in OA cartilage compared to healthy cartilage. TNF-α stimulation increased their expression in both healthy and OA chondrocytes, while IL-1β had limited impact. Caspase-8 expression was causally associated with knee OA in MR analysis, suggesting a potential therapeutic target. The caspase-1 inhibitor VX-765 mildly reduced chondrocyte viability, with no significant effect in the presence of TNF-α. While the caspase-8 inhibitor Z-IETD-FMK exhibited slight enhancements in
Introduction. Tendon ruptures represent one of the most common acute tendon injuries in adults worldwide, affecting millions of people anually and becoming more prevalent due to longer life expectancies and sports activities. Current clinical treatments for full tears are unable to completely restore the torn tendons to their native composition, structure and mechanical properties. To address this clinical challenge, tissue-engineered substitutes will be developed to serve as functional replacements for total tendon ruptures that closely resemble the original tissue, restoring functionality. Method. Water borne polyurethanes (WBPU) containing acrylate groups, specifically polyethylene glycol methacrylate (PEGMA) or 2-hydroxyethyl methacrylate (HEMA), were combined with mouse mesenchymal stem cells (MoMSCs) and heparin sodium to formulate bioinks for the fabrication of scaffolds via extrusion-based 3D bioprinting. Result. The biocompatibility of acrylated-WBPUs was confirmed in 2D with MoMSCs using lactate dehydrogenase assay, DNA assay and live/dead assays. Cell-laden scaffolds were 3D-bioprinted by encapsulating MoMSCs at varying cell densities within the acrylated WBPUs. The resulting 3D structures support
Introduction. Supraspinatus tears comprise most rotator cuff injuries, the leading cause of shoulder pain and an increasing problem with ageing populations. Surgical repair of considerable or persistent damages is customary, although not invariably successful. Tissue engineering presents a promising alternative to generate functional tissue constructs with improved healing capacities. This study explores tendon tissue constructs’ culture in a platform providing physiological mechanical stimulation and reports on the effect of different loading regimes on the viability of human tendon cells. Method. Porcine decellularized tendon scaffolds were fixed into flexible, self-contained bioreactor chambers, seeded with human tenocytes, allocated in triplicates to either static control, low (15±0.8Newtons [N]), medium (26±0.5N), or high (49±2.1N)-force-regime groups, connected to a perfusion system and cultured under standard conditions. A humanoid robotic arm provided 30-minute adduction/abduction stimulation to chambers daily over a week. A metabolic activity assay served to assess
Introduction. Bioactive glasses (BGs) promote osteogenic differentiation of bone progenitor cells by releasing therapeutically active ions. The well-described 45S5-BG (in mol%: SiO. 2. 46.13; P. 2. O. 5. 2.60; CaO 26.91; Na. 2. O 24.35) was supplemented with CaF. 2. and NaF being added to the batch at nominal 5 (F5-BG) and 25 mol% (F25-BG), respectively. While the effect on physical and chemical properties has already been characterized, the biological properties require further studies. This study investigates the effects of fluoride-supplemented BGs on the osteogenic and angiogenic properties of human bone marrow mesenchymal stromal cells (BMSCs) in vitro. Method. BMSCs were co-cultured with melt-derived 45S5-BG, F5-BG, or F25-BG in ascending concentrations (1, 2 and 3 mg/ml). At 7 days, cell number was determined by 4,6-diamidine-2-phenylindole (DAPI) staining and
Introduction. Bereft of their optimal tissue context, cells lose their phenotype, function and therapeutic potential during in vitro culture. Despite the fact that in vivo cells are exposed simultaneously to multiple signals, traditional ex vivo cultures are monofactorial. With these in mind, herein we assessed the combined effect of surface topography, substrate rigidity, collagen type I coating and macromolecular crowding in human tenocyte, skin fibroblast and bone marrow mesenchymal stromal cell cultures. Methods. Thermal imprinted was used to pattern (groove depth: 2,000 nm, groove width: 2,000 nm, line width: 2,000 nm) polydimethylsiloxane substrates of different rigidity (50 kPa, 130 kPa, 1,000 kPa). Grooved and planar substrates were subsequently coated with collagen type I and used to culture the aforementioned cell populations without and with macromolecular crowding (100 μg/ml carrageenan). After 3, 7 and 14 days in culture,
This study aimed to demonstrate the promoting effect of elastic fixation on fracture, and further explore its mechanism at the gene and protein expression levels. A closed tibial fracture model was established using 12 male Japanese white rabbits, and divided into elastic and stiff fixation groups based on different fixation methods. Two weeks after the operation, a radiograph and pathological examination of callus tissue were used to evaluate fracture healing. Then, the differentially expressed proteins (DEPs) were examined in the callus using proteomics. Finally, in vitro cell experiments were conducted to investigate hub proteins involved in this process.Aims
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Although low-intensity pulsed ultrasound (LIPUS) combined with disinfectants has been shown to effectively eliminate portions of biofilm in vitro, its efficacy in vivo remains uncertain. Our objective was to assess the antibiofilm potential and safety of LIPUS combined with 0.35% povidone-iodine (PI) in a rat debridement, antibiotics, and implant retention (DAIR) model of periprosthetic joint infection (PJI). A total of 56 male Sprague-Dawley rats were established in acute PJI models by intra-articular injection of bacteria. The rats were divided into four groups: a Control group, a 0.35% PI group, a LIPUS and saline group, and a LIPUS and 0.35% PI group. All rats underwent DAIR, except for Control, which underwent a sham procedure. General status, serum biochemical markers, weightbearing analysis, radiographs, micro-CT analysis, scanning electron microscopy of the prostheses, microbiological analysis, macroscope, and histopathology evaluation were performed 14 days after DAIR.Aims
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In this investigation, we administered oxidative stress to nucleus pulposus cells (NPCs), recognized DNA-damage-inducible transcript 4 (DDIT4) as a component in intervertebral disc degeneration (IVDD), and devised a hydrogel capable of conveying small interfering RNA (siRNA) to IVDD. An in vitro model for oxidative stress-induced injury in NPCs was developed to elucidate the mechanisms underlying the upregulation of DDIT4 expression, activation of the reactive oxygen species (ROS)-thioredoxin-interacting protein (TXNIP)-NLRP3 signalling pathway, and nucleus pulposus pyroptosis. Furthermore, the mechanism of action of small interfering DDIT4 (siDDIT4) on NPCs in vitro was validated. A triplex hydrogel named siDDIT4@G5-P-HA was created by adsorbing siDDIT4 onto fifth-generation polyamidoamine (PAMAM) dendrimer using van der Waals interactions, and then coating it with hyaluronic acid (HA). In addition, we established a rat puncture IVDD model to decipher the hydrogel’s mechanism in IVDD.Aims
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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.
Osteoarthritis (OA) is the most common chronic pathema of human joints. The pathogenesis is complex, involving physiological and mechanical factors. In previous studies, we found that ferroptosis is intimately related to OA, while the role of Sat1 in chondrocyte ferroptosis and OA, as well as the underlying mechanism, remains unclear. In this study, interleukin-1β (IL-1β) was used to simulate inflammation and Erastin was used to simulate ferroptosis in vitro. We used small interfering RNA (siRNA) to knock down the spermidine/spermine N1-acetyltransferase 1 (Sat1) and arachidonate 15-lipoxygenase (Alox15), and examined damage-associated events including inflammation, ferroptosis, and oxidative stress of chondrocytes. In addition, a destabilization of the medial meniscus (DMM) mouse model of OA induced by surgery was established to investigate the role of Sat1 inhibition in OA progression.Aims
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Aims. Continuous local antibiotic perfusion (CLAP) has recently attracted attention as a new drug delivery system for orthopaedic infections. CLAP is a direct continuous infusion of high-concentration gentamicin (1,200 μg/ml) into the bone marrow. As it is a new system, its influence on the bone marrow is unknown. This study aimed to examine the effects of high-concentration antibiotics on human bone tissue-derived cells. Methods. Cells were isolated from the bone tissue grafts collected from six patients using the Reamer-Irrigator-Aspirator system, and exposed to different gentamicin concentrations. Live cells rate, apoptosis rate, alkaline phosphatase (ALP) activity, expression of osteoblast-related genes, mineralization potential, and restoration of
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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To investigate the efficacy of ethylenediaminetetraacetic acid-normal saline (EDTA-NS) in dispersing biofilms and reducing bacterial infections. EDTA-NS solutions were irrigated at different durations (1, 5, 10, and 30 minutes) and concentrations (1, 2, 5, 10, and 50 mM) to disrupt 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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Mechanical loading is important to maintain the homeostasis of the intervertebral disc (IVD) under physiological conditions but can also accelerate cell death and tissue breakdown in a degenerative state. Bioreactor loaded whole organ cultures are instrumental for investigating the effects of the mechanical environment on the IVD integrity and for preclinical testing of new therapies under simulated physiological conditions. Thereby the loading parameters that determine the beneficial or detrimental reactions largely depend on the IVD model and its preparation. Within this symposium we are discussing the use of bovine caudal IVD culture models to reproduce tissue inflammation or matrix degradation with or without bioreactor controlled mechanical loading. Furthermore, the outcome parameters that define the degenerative state of the whole IVD model will be outlined. Besides the disc height, matrix integrity,
In cartilage tissue engineering (TE),new solutions are needed to effectively drive chondrogenic differentiation of mesenchymal stromal cells in both normal and inflammatory milieu. Ultrasound waves represent an interesting tool to facilitate chondrogenesis. In particular, low intensity pulsed ultrasound (LIPUS)has been shown to regulate the differentiation of adipose mesenchymal stromal cells. Hydrogels are promising biomaterials capable of encapsulating MSCs by providing an instructive biomimetic environment, graphene oxide (GO) has emerged as a promising nanomaterial for cartilage TE due to its chondroinductive properties when embedded in polymeric formulations, and piezoelectric nanomaterials, such as barium titanate nanoparticles (BTNPs),can be exploited as nanoscale transducers capable of inducing cell growth/differentiation. The aim of this study was to investigate the effect of dose-controlled LIPUS in counteracting inflammation and positively committing chondrogenesis of ASCs embedded in a 3D piezoelectric hydrogel. ASCs at 2*10. 6. cells/mL were embedded in a 3D VitroGel RGD. ®. hydrogel without nanoparticles (Control) or doped with 25 µg/ml of GO nanoflakes and 50 µg/ml BTNPs.The hydrogels were exposed to basal or inflammatory milieu (+IL1β 10ng/ml)and then to LIPUS stimulation every 2 days for 10 days of culture. Hydrogels were chondrogenic differentiated and analyzed after 2,10 and 28 days. At each time point
