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Abstract Background Osteoarthritis (OA) is a degenerative musculoskeletal disease that significantly impacts the quality of life. Currently, no validated biomarkers for early detection of OA are defined. The possibility of discovering OA biomarkers is the focus of this study. Methods Human primary OA synovial cells (SVs), isolated from discarded joint tissue of patients with Kellgren & Lawrence score (KL)

Abstract Osteosarcoma (OS) is the most frequent primary malignant bone tumour, whose heterogeneity represents a major challenge for common antitumour therapies. Inflammatory cytokines are known to be necessary for OS progression. Therefore, to optimise therapy, it is important to discover reliable biomarkers by identifying the mechanism generating OS and investigating the inflammatory pathways that support the undifferentiated state. In this work, we highlight the differences of epigenetic activities of IL-1β and TNFα, and the susceptibility of TET-1 enzymatic inhibition, in tumour progression of three different OS cell lines. Investigating DNA methylation of IL-6 promoter and determining its expression, we found that TET enzymatic inhibition influences proliferation induced by inflammatory cytokines in OS cell lines. Moreover, Bobcat 339 treatment blocks IL-1β epigenetic action on IL-6 promoter, while only partially those of TNFα as well as inhibits IL-1β-dependent epithelial–mesenchymal transition (EMT) process, but only partially those of TNFα. In conclusion, this work highlights that IL-1β and TNFα have different effects on DNA demethylation in OS cell lines, making DNA methylation a potential biomarker of disease. Specifically, in IL-1β treatment, TET-1 inhibition completely blocks tumour progression, while in TNFα actions, it is only partially effective. Given that these two inflammatory pathways can be therapeutic targets for treating these tumours, knowledge of their distinct epigenetic behaviours can be useful for developing precise and specific therapeutic strategies for this disease.

Abstract Cranial reconstructions are essential for restoring both function and aesthetics in patients with craniofacial deformities or traumatic injuries. Titanium prostheses have gained popularity due to their biocompatibility, strength, and corrosion resistance. The use of Superplastic Forming (SPF) and Single Point Incremental Forming (SPIF) techniques to create titanium prostheses, specifically designed for cranial reconstructions was investigated in an ovine model through microtomographic and histomorphometric analyses. The results obtained from the explanted specimens revealed significant variations in bone volume, trabecular thickness, spacing, and number across different regions of interest (VOIs or ROIs). Those regions next to the center of the cranial defect exhibited the most immature bone, characterized by higher porosity, decreased trabecular thickness, and wider trabecular spacing. Dynamic histomorphometry demonstrated differences in the mineralizing surface to bone surface ratio (MS/BS) and mineral apposition rate (MAR) depending on the timing of fluorochrome administration. A layer of connective tissue separated the prosthesis and the bone tissue. Overall, the study provided validation for the use of cranial prostheses made using SPF and SPIF techniques, offering insights into the processes of bone formation and remodeling in the implanted ovine model.

Introduction: The development of reliable treatments for infected or potentially infected bone loss resulting from open fractures and non-unions is extremely urgent, especially to reduce the prolonged courses of antimicrobial therapy to which affected patients are subjected. Numerous bone graft substitutes have been used over the years, but there are currently no effective solutions to treat critical bone loss, especially in the presence of infection. The present study evaluated the use of the biomorphic calcium phosphate bone scaffold b. Bone™, based on a next-generation resorbable biomimetic biomaterial, in bone reconstruction surgery in cases of infection.Methods: Using an “in vitro 3D bone fracture model” to predict the behavior of this drug delivery system during critical bone loss at an infected (or potentially infected) site, the effects of scaffolds loaded with gentamicin or vancomycin on the viability and differentiation capacity of human mesenchymal stem cells (hMSCs) were evaluated.Results: This scaffold, when loaded with gentamicin or vancomycin, exhibits a typical drug release curve that determines the inhibitory effects on the growth of Staphylococcus aureus, Enterococcus faecalis, and Escherichia coli, as well as relative biofilm formation.Discussion: The study demonstrates that b.bone scaffolds can effectively address key challenges in orthopedic surgery and patient care by inhibiting bacterial growth and biofilm formation through rapid, potent antibiotic release, reducing the risk of treatment failure due to resistance, and providing a promising solution for bone infections and improved patient outcomes. Future studies could explore the combination of different antibiotics on these scaffolds for more tailored and effective treatments against post-traumatic osteomyelitis pathogens.

Soft robotics aims to replicate the structure and mechanics of skeletal muscles. The challenge lies in seamlessly integrating these muscle-inspired soft actuators with the joints they intend to actuate, resembling the natural connection between muscles and tendons (i.e., myotendinous junction). This study addresses this issue by producing electrospun bundles of aligned nanofibers using a thermoplastic polyurethane, mimicking the muscle fascicle, and nylon 6.6 for the tendon one. A novel method was developed to create electrospun bi-material bundles with two different types of myotendinous-inspired junctions, called flat and conical. Scanning electron microscopy and microtomography analyses confirmed that conical junctions mimicked natural myotendinous structures better than flat ones. Tensile mechanical tests demonstrated that bi-material junctions reached stress at failure comparable to polyurethane bundles (11 ± 2 MPa), with the conical junction showing stiffness (0.13 ± 0.02 N/mm) and net elastic modulus (153 ± 10 MPa) values closer to the natural myotendinous ones. Cyclic tests verified the mechanical stability of junctions and their ability to dampen nylon 6.6 hardening over time. Moreover, all bundles withstood cyclic loading without breaking. These findings suggest the potential of biomimetic electrospun junctions for applications in soft robotics, marking a significant step toward advancing this field.

Scaffolds are widely used devices for the treatment of osteochondral lesions of the talus (OCLT), aimed at enhancing mechanical stability and fostering chondrogenic differentiation. A systematic review and meta-analysis were performed to evaluate the safety, and clinical and radiological results of scaffolds for OCLT management. On 2 January 2024, a search was performed in four databases (PubMed, Embase, Web of Science, and Scopus), according to PRISMA guidelines. The risk of bias in the included studies was also evaluated. Thirty clinical studies were included in the qualitative analysis: 12 retrospective case series, 3 retrospective comparative studies, 9 prospective case series, 1 prospective comparative study, and 1 Randomized Controlled Trial (RCT). Natural scaffolds, such as bilayer collagen (COLL)I/III and hyaluronic scaffolds, were the most employed. Only minor adverse events were observed, even if more serious complications were shown, especially after medial malleolar osteotomy. An overall clinical and radiological improvement was observed after a mean of 36.3 months of follow-up. Patient age and Body Mass Index (BMI), lesion size, and location were correlated with the clinical outcomes, while meta-analysis revealed significant improvement in clinical scores with hyaluronic scaffolds compared to microfracture alone. This study highlights the safety and positive clinical outcomes associated with the use of scaffolds for OCLT. In the few available comparative studies, scaffolds have also demonstrated superior clinical outcomes compared to microfractures alone. Nevertheless, the analysis has shown the limitations of the current literature, characterized by an overall low quality and scarcity of RCTs.

Background: Platelets and lymphocytes levels are important in assessing systemic disorders, reflecting inflammatory and immune responses. This study investigated the relationship between blood parameters (platelet count (PLT), mean platelet volume (MPV), lymphocyte count (LINF), and platelet-to-lymphocyte ratio (PLR)) and osteoarthritis (OA) severity, considering age, sex, and body mass index (BMI). Methods: Patients aged ≥40 years were included in this cross-sectional study and divided into groups based on knee OA severity using the Kellgren–Lawrence (KL) grading system. A logistic regression model, adjusted for confounders, evaluated the ability of PLT, MPV, LINF, and PLR to categorize OA severity. Model performance in terms of accuracy, sensitivity, and specificity was assessed using ROC curves. Results: The study involved 245 OA patients (51.4% female, 48.6% male) aged 40–90 years, 35.9% with early OA (KL < 3) and 64.1% moderate/severe OA (KL ≥ 3). Most patients (60.8%) were aged ≥60 years, and BMI was 2 in 33.9%. The model showed that a 25-unit increase in PLR elevates the odds of higher OA levels by 1.30 times (1-unit OR = 1.011, 95% CI [1.004, 1.017], p < 0.005), while being ≥40 years old elevates the odds by 4.42 times (OR 4.42, 95% CI [2.46, 7.95], p < 0.0005). The model’s accuracy was 73.1%, with 84% sensitivity, 52% specificity, and an AUC of 0.74 (95% CI [0.675, 0.805]). Conclusions: Higher PLR increases the likelihood of moderate/severe OA, suggesting that monitoring these biomarkers could aid in early detection and management of OA severity. Further research is warranted to cross-validate these results in larger populations.

Regeneration of injured tendons and ligaments (T/L) is a worldwide need. In this study electrospun hierarchical scaffolds made of a poly-L (lactic) acid/collagen blend were developed reproducing all the multiscale levels of aggregation of these tissues. Scanning electron microscopy, microCT and tensile mechanical tests were carried out, including a multiscale digital volume correlation analysis to measure the full-field strain distribution of electrospun structures. The principal strains (εp1 and εp3) described the pattern of strains caused by the nanofibers rearrangement, while the deviatoric strains (εD) revealed the related internal sliding of nanofibers and bundles. The results of this study confirmed the biomimicry of such electrospun hierarchical scaffolds, paving the way to further tissue engineering and clinical applications.

Understanding the microstructure of fibrous tissues, like ligaments, is crucial due to their nonlinear stress-strain behavior from unique fiber arrangements. This study introduces a new method to analyze the relationship between the microstructure and function of anterior cruciate ligaments (ACL). We tested the procedure on two ACL samples, one from a healthy individual and one from an osteoarthritis patient, using a custom tensioning device within a micro-CT scanner. The samples were stretched and scanned at various strain levels (namely 0%, 1%, 2%, 3%, 4%, 6%, 8%) to observe the effects of mechanical stress on the microstructure. The micro-CT images were processed to identify and map fibers, assessing their orientations and volume fractions. A probabilistic mathematical model was then proposed to relate the geometric and structural characteristics of the ACL to its mechanical properties, considering fiber orientation and thickness. Our feasibility test indicated differences in mechanical behavior, fiber orientation, and volume distribution between ligaments of different origins. These indicative results align with existing literature, validating the proposed methodology. However, further research is needed to confirm these preliminary observations. Overall, our comprehensive methodology shows promise for improving ACL diagnosis and treatment and for guiding the creation of tissue-engineered grafts that mimic the natural properties and microstructure of healthy tissue, thereby enhancing integration and performance in biomedical applications.

To endow an implant surface with enhanced properties to ensure an appropriate seal with the host tissue for inflammation/infection resistance, next-generation bone implant collagen–polyphenol nanolayers were built on conventional titanium surfaces through a multilayer approach. X-ray Photoelectron Spectroscopy (XPS) analysis was performed to investigate the chemical arrangement of molecules within the surface layer and to provide an estimate of their thickness. A short-term (2 and 4 weeks) in vivo test of bone implants in a healthy rabbit model was performed to check possible side effects of the soft surface layer on early phases of osteointegration, leading to secondary stability. Results show the building up of the different nanolayers on top of titanium, resulting in a final composite collagen–polyphenol surface and a layer thickness of about 10 nm. In vivo tests performed on machined and state-of-the-art microrough titanium implants do not show significant differences between coated and uncoated samples, as the surface microroughness remains the main driver of bone-to-implant contact. These results confirm that the surface nanolayer does not interfere with the onset and progression of implant osteointegration and prompt the green light for specific investigations of the potential merits of this bioactive coating as an enhancer of the device/tissue seal.

Background: Rotator cuff tears (RCTs), resulting from degeneration or trauma of the shoulder tendons, are one of the main causes of shoulder pain. In particular, massive RCTs represent 40% of all injuries, require surgical treatment, and are characterized by poor clinical outcomes and a high rate of failure. In recent years, the use of biological decellularized patches for augmentation procedures has received great interest owing to their excellent self-integration properties, improving healing and, thus, presenting an innovative therapeutic option. However, the findings from clinical studies have emerged with conflicting viewpoints regarding the benefits of this procedure, as an excessive tension load might compromise the integrity of the tendon-to-bone connection when the patch exhibits low elasticity or insufficient strength. This could prevent the healing process, leading to unpredictable results in clinical practice.Methods: This systematic review was conducted following Preferred Reporting Items for Systematic reviews and Meta-Analyses (PRISMA) guidelines across three databases (PubMed, Scopus, and Web of Knowledge) to underline the results obtained in preclinical studies involving animal models of RCT surgeries that utilized the biological decellularized matrix augmentation technique in the last 5 years.Results: Thirteen articles were included after the screening, and the SYRCLE tools were applied to assess the risk of bias in in vivo studies. Open-surgery techniques were conducted to create tendon defects or detachment in different animal models: rat (31%), rabbit (46%), dog (15%), and sheep (8%). Patches decellularized with non-standardized protocols were used in 77% of studies, while commercially available matrices were used in 15%. Of the studies, 31% used allogenic patches, 61% used xenogenic patches, and 8% utilized both xenogenic and autologous patches.Conclusion: Overall, this review provides a comprehensive overview of the use of acellular patches and their effective therapeutic potential in rotator cuff (RC) repair at the preclinical level with the aim of expanding the strategies and matrices available for surgeons.Systematic review registration:https://www.crd.york.ac.uk/prospero/, identifier CRD42023468716.

BackgroundBone marrow aspirate (BMA), when combined with graft substitutes, has long been introduced as a promising alternative to iliac crest bone graft in spinal fusion. However, the use of BMA is limited by the absence of a standardized procedure, a structural texture, and the potential for diffusion away from the implant site. Recently, the potential use of a new formulation of BMA, named BMA clot, has been preclinically described. In this report, we present the results of a prospective pilot clinical study aimed at evaluating the safety and efficacy of autologous vertebral BMA (vBMA) clot as a three-dimensional and multifunctional bioscaffold in instrumented posterior lumbar fusion.MethodsTen consecutive patients with an indication of multilevel (≤5) posterior spinal fusion due to lumbar spine degenerative diseases were included in the study and treated with vBMA. Clinical outcomes were assessed using the Visual Analog Scale (VAS), Oswestry Disability Index (ODI), and EuroQoL-5L (EQ-5L) preoperatively and at 3 months and 12 months after spinal fusion. Bone fusion quality was evaluated at the 12-month follow-up using the Brantigan classification on radiography (XR) imaging. Bone density was measured on computed tomography (CT) scans at 6 and 12 months of follow-up visits at the intervertebral arches and intervertebral joint areas and expressed in Hounsfield unit (HU).ResultsThe results indicate a successful posterolateral fusion rate of approximately 100% (considering levels with C, D, and E grades according to the Brantigan classification) at the 12-month follow-up, along with an increase in bone density from 6 to 12 months of follow-up. An improvement in the quality of life and health status following surgery, as assessed by clinical scores (ODI, VAS, and EQ-5L), was also observed as early as 3 months postsurgery. No adverse events related to the vBMA clot were reported.ConclusionThis prospective pilot study demonstrates the effectiveness and safety profile of vBMA clot as an advanced bioscaffold capable of achieving posterior lumbar fusion in the treatment of degenerative spine diseases. This lays the groundwork for a larger randomized clinical study.

Aging comes with the loss of muscle and bone mass, leading to a condition known as osteosarcopenia. Circulating, cellular, and tissue biomarkers research for osteosarcopenia is relatively scarce and, currently, no established biomarkers exist. Here we find that osteosarcopenic patients exhibited elevated basophils and TNFα levels, along with decreased aPPT, PT/INR, IL15, alpha-Klotho, DHEA-S, and FGF-2 expression and distinctive bone and muscle tissue micro-architecture and biomarker expressions. They also displayed an increase in osteoclast precursors with a concomitant imbalance towards spontaneous osteoclastogenesis. Similarities were noted with osteopenic and sarcopenic patients, including a lower neutrophil percentage and altered cytokine expression. A linear discriminant analysis (LDA) on models based on selected biomarkers showed a classification accuracy in the range of 61–78%. Collectively, our data provide compelling evidence for novel biomarkers for osteosarcopenia that may hold potential as diagnostic tools to promote healthy aging.

In the development of bone graft substitutes, a fundamental step is the use of scaffolds with adequate composition and architecture capable of providing support in regenerative processes both on the tissue scale, where adequate resistance to mechanical stress is required, as well as at the cellular level where compliant chemical–physical and mechanical properties can promote cellular activity. In this study, based on a previous optimization study of this group, the potential of a three-dimensional construct based on polycaprolactone (PCL) and a novel biocompatible Mg- and Sr-containing glass named BGMS10 was explored. Fourier-transform infrared spectroscopy and scanning electron microscopy showed the inclusion of BGMS10 in the scaffold structure. Mesenchymal stem cells cultured on both PCL and PCL-BGMS10 showed similar tendencies in terms of osteogenic differentiation; however, no significant differences were found between the two scaffold types. This circumstance can be explained via X-ray microtomography and atomic force microscopy analyses, which correlated the spatial distribution of the BGMS10 within the bulk with the elastic properties and topography at the cell scale. In conclusion, our study highlights the importance of multidisciplinary approaches to understand the relationship between design parameters, material properties, and cellular response in polymer composites, which is crucial for the development and design of scaffolds for bone regeneration.

The damping system ensured by the osteochondral (OC) unit is essential to deploy the forces generated within load-bearing joints during locomotion, allowing furthermore low-friction sliding motion between bone segments. The OC unit is a multi-layer structure including articular cartilage, as well as subchondral and trabecular bone. The interplay between the OC tissues is essential in maintaining the joint functionality; altered loading patterns can trigger biological processes that could lead to degenerative joint diseases like osteoarthritis. Currently, no effective treatments are available to avoid degeneration beyond tissues’ recovery capabilities. A thorough comprehension on the mechanical behaviour of the OC unit is essential to (i) soundly elucidate its overall response to intra-articular loads for developing diagnostic tools capable of detecting non-physiological strain levels, (ii) properly evaluate the efficacy of innovative treatments in restoring physiological strain levels, and (iii) optimize regenerative medicine approaches as potential and less-invasive alternatives to arthroplasty when irreversible damage has occurred. Therefore, the leading aim of this review was to provide an overview of the state-of-the-art—up to 2022—about the mechanical behaviour of the OC unit. A systematic search is performed, according to PRISMA standards, by focusing on studies that experimentally assess the human lower-limb joints’ OC tissues. A multi-criteria decision-making method is proposed to quantitatively evaluate eligible studies, in order to highlight only the insights retrieved through sound and robust approaches. This review revealed that studies on human lower limbs are focusing on the knee and articular cartilage, while hip and trabecular bone studies are declining, and the ankle and subchondral bone are poorly investigated. Compression and indentation are the most common experimental techniques studying the mechanical behaviour of the OC tissues, with indentation also being able to provide information at the micro- and nanoscales. While a certain comparability among studies was highlighted, none of the identified testing protocols are currently recognised as standard for any of the OC tissues. The fibril-network-reinforced poro-viscoelastic constitutive model has become common for describing the response of the articular cartilage, while the models describing the mechanical behaviour of mineralised tissues are usually simpler (i.e., linear elastic, elasto-plastic). Most advanced studies have tested and modelled multiple tissues of the same OC unit but have done so individually rather than through integrated approaches. Therefore, efforts should be made in simultaneously evaluating the comprehensive response of the OC unit to intra-articular loads and the interplay between the OC tissues. In this regard, a multidisciplinary approach combining complementary techniques, e.g., full-field imaging, mechanical testing, and computational approaches, should be implemented and validated. Furthermore, the next challenge entails transferring this assessment to a non-invasive approach, allowing its application in vivo, in order to increase its diagnostic and prognostic potential.

Adolescent Idiopathic Scoliosis (AIS) is the most common form of three-dimensional spinal disorder in adolescents between the ages of 10 and 18 years of age, most commonly diagnosed in young women when severe disease occurs. Patients with AIS are characterized by abnormal skeletal growth and reduced bone mineral density. The etiology of AIS is thought to be multifactorial, involving both environmental and genetic factors, but to date, it is still unknown. Therefore, it is crucial to further investigate the molecular pathogenesis of AIS and to identify biomarkers useful for predicting curve progression. In this perspective, the relative abundance of a panel of microRNAs (miRNAs) was analyzed in the plasma of 20 AIS patients and 10 healthy controls (HC). The data revealed a significant group of circulating miRNAs dysregulated in AIS patients compared to HC. Further bioinformatic analyses evidenced a more restricted expression of some miRNAs exclusively in severe AIS females. These include some members of the miR-30 family, which are considered promising regulators for treating bone diseases. We demonstrated circulating extracellular vesicles (EVs) from severe AIS females contained miR-30 family members and decreased the osteogenic differentiation of mesenchymal stem cells. Proteomic analysis of EVs highlighted the expression of proteins associated with orthopedic disease. This study provides preliminary evidence of a miRNAs signature potentially associated with severe female AIS and suggests the corresponding vesicular component may affect cellular mechanisms crucial in AIS, opening the scenario for in-depth studies on prognostic differences related to gender and grade.

Poly-ε-caprolactone (PCL) has been widely used in additive manufacturing for the construction of scaffolds for bone tissue engineering. However, its use is limited by its lack of bioactivity and inability to induce cell adhesion, hence limiting bone tissue regeneration. Biomimicry is strongly influenced by the dynamics of cell–substrate interaction. Thus, characterizing scaffolds at the cell scale could help to better understand the relationship between surface mechanics and biological response. We conducted atomic force microscopy-based nanoindentation on 3D-printed PCL fibers of ~300 µm thickness and mapped the near-surface Young’s modulus at loading forces below 50 nN. In this non-disruptive regime, force mapping did not show clear patterns in the spatial distribution of moduli or a relationship with the topographic asperities within a given region. Remarkably, we found that the average modulus increased linearly with the logarithm of the strain rate. Finally, a dependence of the moduli on the history of nanoindentation was demonstrated on locations of repeated nanoindentations, likely due to creep phenomena capable of hindering viscoelasticity. Our findings can contribute to the rational design of scaffolds for bone regeneration that are capable of inducing cell adhesion and proliferation. The methodologies described are potentially applicable to various tissue-engineered biopolymers.

The objective of this review is to systematically analyze the potential correlation between gut microbiota and osteoarthritis (OA) as well as to evaluate the feasibility of microbiota-targeted therapies for treating OA. Studies conducted from October 2013 to October 2023 were identified via a search on electronic databases such as PubMed, Web of Science, and Scopus, following established PRISMA statement standards. Two reviewers independently screened, assessed, and extracted relevant data, and then they graded the studies using the ROBINS I tool for non-randomized interventions studies and SYRCLE’s risk-of-bias tool for animal studies. A search through 370 studies yielded 38 studies (24 preclinical and 14 clinical) that were included. In vivo research has predominantly concentrated on modifying the gut microbiota microenvironment, using dietary supplements, probiotics, and prebiotics to modify the OA status. Lactobacilli are the most thoroughly examined with Lactobacillus acidophilus found to effectively reduce cartilage damage, inflammatory factors, and pain. Additionally, Lactobacillus M5 inhibits the development of OA by preventing high-fat diet (HFD)-induced obesity and protecting cartilage from damage. Although there are limited clinical studies, certain compositions of intestinal microbiota may be associated with onset and progression of OA, while others are linked to pain reduction in OA patients. Based on preclinical studies, there is evidence to suggest that the gut microbiota could play a significant role in the development and progression of OA. However, due to the scarcity of clinical studies, the exact mechanism linking the gut microbiota and OA remains unclear. Further research is necessary to evaluate specific gut microbiota compositions, potential pathogens, and their corresponding signaling pathways that contribute to the onset and progression of OA. This will help to validate the potential of targeting gut microbiota for treating OA patients.

There is increasing interest in using magnesium (Mg) alloy orthopedic devices because of their mechanical properties and bioresorption potential. Concerns related to their rapid degradation have been issued by developing biodegradable micro- and nanostructured coatings to enhance corrosion resistance and limit the release of hydrogen during degradation. This systematic review based on four databases (PubMed®, Embase, Web of Science™ and ScienceDirect®) aims to present state-of-the-art strategies, approaches and materials used to address the critical factors currently impeding the utilization of Mg alloy devices. Forty studies were selected according to PRISMA guidelines and specific PECO criteria. Risk of bias assessment was conducted using OHAT and SYRCLE tools for in vitro and in vivo studies, respectively. Despite limitations associated with identified bias, the review provides a comprehensive analysis of preclinical in vitro and in vivo studies focused on manufacturing and application of Mg alloys in orthopedics. This attests to the continuous evolution of research related to Mg alloy modifications (e.g., AZ91, LAE442 and WE43) and micro- and nanocoatings (e.g., MAO and MgF2), which are developed to improve the degradation rate required for long-term mechanical resistance to loading and excellent osseointegration with bone tissue, thereby promoting functional bone regeneration. Further research is required to deeply verify the safety and efficacy of Mg alloys.

Osteoarthritis (OA) is a degenerative joint disease that is age-related and progressive. It causes the destruction of articular cartilage and underlying bone, often aggravated by inflammatory processes and oxidative stresses. This pathology impairs the quality of life of the elderly, causing pain, reduced mobility, and functional disabilities, especially in obese patients. Phytochemicals with anti-inflammatory and antioxidant activities may be used for long-term treatment of OA, either in combination with current anti-inflammatories and painkillers, or as an alternative to other products such as glucosamine and chondroitin, which improve cartilage structure and elasticity. The current systematic review provides a comprehensive understanding of the use of flavonoids. It highlights chondrocyte, cartilage, and subchondral bone activities, with a particular focus on their nutrigenomic effects. The molecular mechanisms of these molecules demonstrate how they can be used for the prevention and treatment of OA in the elderly population. However, clinical trials are still needed for effective use in clinical practice.

Musculoskeletal frailty—a common and debilitating condition linked to aging and chronic diseases—presents a major public health issue. In vivo models have become a key tool for researchers as they investigate the condition’s underlying mechanisms and develop effective interventions. This systematic review examines the current body of research on in vivo models of musculoskeletal frailty, without any time constraints. To achieve this aim, we utilized three electronic databases and incorporated a total of 11 studies. Our investigation delves into varied animal models that simulate specific features of musculoskeletal frailty, including muscle loss, bone density reduction, and functional decline. Furthermore, we examine the translational prospects of these models in augmenting our comprehension of musculoskeletal frailty and streamlining the production of groundbreaking therapeutic approaches. This review provides significant insights and guidance for healthcare researchers and practitioners who aim to combat musculoskeletal frailty, ultimately enhancing the quality of life for older adults and individuals affected by this condition.

Ageing is an irreversible and inevitable biological process and a significant risk factor for the development of various diseases, also affecting the musculoskeletal system, resulting from the accumulation of cell senescence. The aim of this systematic review was to collect the in vitro studies conducted over the past decade in which cell senescence was induced through various methods, with the purpose of evaluating the molecular and cellular mechanisms underlying senescence and to identify treatments capable of delaying senescence. Through three electronic databases, 22 in vitro studies were identified and included in this systematic review. Disc, cartilage, or muscle cells or tissues and mesenchymal stem cells were employed to set-up in vitro models of senescence. The most common technique used to induce cell senescence was the addition to the culture medium of tumor necrosis factor (TNF)α and/or interleukin (IL)1β, followed by irradiation, compression, hydrogen peroxide (H2O2), microgravity, in vitro expansion up to passage 10, and cells harvested from damaged areas of explants. Few studies evaluated possible treatments to anti-senescence effects. The included studies used in vitro models of senescence in musculoskeletal tissues, providing powerful tools to evaluate age-related changes and pathologies, also contributing to the development of new therapeutic approaches.

Prosthetic reconstruction can serve as a feasible alternative, delivering both functional and aesthetic benefits to individuals with hand and finger injuries, frequent causes of emergency room visits. Implant-related infections pose significant challenges in arthroplasty and osteosynthesis procedures, contributing to surgical failures. As a potential solution to this challenge, this study developed a new class of silver (Ag)-doped chitosan (CS) coatings via electrophoretic deposition (EPD) on osseointegrated prostheses for infection therapy. These coatings were successfully applied to additively manufactured Ti6Al4V ELI samples. In the initial phase, the feasibility of the composite coating was assessed using the Thermogravimetric Analysis (TGA) and Attenuated Total Reflection (ATR) techniques. The optimized structures exhibited impressive water uptake in the range of 300–360%. Codeposition with an antibacterial agent proved effective, and scanning electron microscopy (SEM) was used to examine the coating morphology. Biologically, CS coatings demonstrated cytocompatibility when in direct contact with a fibroblast cell line (L929) after 72 h. When exposed to the Staphylococcus epidermidis strain (ATCC 12228), these coatings inhibited bacterial growth and biofilm formation within 24 h. These findings underscore the significant potential of this approach for various applications, including endoprostheses like hip implants, internal medical devices, and transcutaneous prostheses such as osseointegrated limb prosthetics for upper and lower extremities.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen–magnesium–hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

In the context of a large animal model of early osteoarthritis (OA) treated by orthobiologics, the purpose of this study was to reveal relations between articular tissues structure/composition and cartilage viscoelasticity. Twenty-four sheep, with induced knee OA, were treated by mesenchymal stem cells in various preparations—adipose-derived mesenchymal stem cells (ADSCs), stromal vascular fraction (SVF), and amniotic endothelial cells (AECs)—and euthanized at 3 or 6 months to evaluate the (i) biochemistry of synovial fluid; (ii) histology, immunohistochemistry, and histomorphometry of articular cartilage; and (iii) viscoelasticity of articular cartilage. After performing an initial analysis to evaluate the correlation and multicollinearity between the investigated variables, this study used machine learning (ML) models—Variable Selection Using Random Forests (VSURF) and Extreme Gradient Boosting (XGB)—to classify variables according to their importance and employ them for interpretation and prediction. The experimental setup revealed a potential relation between cartilage elastic modulus and cartilage thickness (CT), synovial fluid interleukin 6 (IL6), and prostaglandin E2 (PGE2), and between cartilage relaxation time and CT and PGE2. SVF treatment was the only limit on the deleterious OA effect on cartilage viscoelastic properties. This work provides indications to future studies aiming to highlight these and other relationships and focusing on advanced regeneration targets.

Although historically, the traditional bidimensional in vitro cell system has been widely used in research, providing much fundamental information regarding cellular functions and signaling pathways as well as nuclear activities, the simplicity of this system does not fully reflect the heterogeneity and complexity of the in vivo systems. From this arises the need to use animals for experimental research and in vivo testing. Nevertheless, animal use in experimentation presents various aspects of complexity, such as ethical issues, which led Russell and Burch in 1959 to formulate the 3R (Replacement, Reduction, and Refinement) principle, underlying the urgent need to introduce non-animal-based methods in research. Considering this, three-dimensional (3D) models emerged in the scientific community as a bridge between in vitro and in vivo models, allowing for the achievement of cell differentiation and complexity while avoiding the use of animals in experimental research. The purpose of this review is to provide a general overview of the most common methods to establish 3D cell culture and to discuss their promising applications. Three-dimensional cell cultures have been employed as models to study both organ physiology and diseases; moreover, they represent a valuable tool for studying many aspects of cancer. Finally, the possibility of using 3D models for drug screening and regenerative medicine paves the way for the development of new therapeutic opportunities for many diseases.

Osteoarthritis (OA) is a joint disorder characterized by progressive degeneration of cartilage extracellular matrix (ECM), chondrocyte hypertrophy and apoptosis and inflammation. The current treatments mainly concern pain control and reduction of inflammation, but no therapeutic strategy has been identified as a disease-modifying treatment. Therefore, identifying specific biomarkers useful to prevent, treat or distinguish the stages of OA disease has become an immediate need of clinical practice. The role of microRNAs (miRNAs) in OA has been investigated in the last decade, and increasing evidence has emerged that the influence of the environment on gene expression through epigenetic processes contributes to the development, progression and aggressiveness of OA, in particular acting on the microenvironment modulations. The effects of epigenetic regulation, particularly different miRNA methylation during OA disease, were highlighted in the present systematic review. The evidence arising from this study of the literature conducted in three databases (PubMed, Scopus, Web of Science) suggested that miRNA methylation state already strongly impacts OA progression, driving chondrocytes and synoviocyte proliferation, apoptosis, inflammation and ECM deposition. However, the possibility of understanding the mechanism by which different epigenetic modifications of miRNA or pre-miRNA sequences drive the aggressiveness of OA could be the new focus of future investigations.

Recently, our group described the application of vertebral bone marrow (vBMA) clot as a cell therapy strategy for spinal fusion. Its beneficial effects were confirmed in aging-associated processes, but the influence of gender is unknown. In this study, we compared the biological properties of vBMA clots and derived vertebral mesenchymal stem cells (MSCs) from female and male patients undergoing spinal fusion procedures and treated with vBMA clot. We analyzed the expression of growth factors (GFs) in vBMA clots and MSCs as well as morphology, viability, doubling time, markers expression, clonogenicity, differentiation ability, senescence factors, Klotho expression, and HOX and TALE gene profiles from female and male donors. Our findings indicate that vBMA clots and derived MSCs from males had higher expression of GFs and greater osteogenic and chondrogenic potential compared to female patients. Additionally, vBMA-clot-derived MSCs from female and male donors exhibited distinct levels of HOX and TALE gene expression. Specifically, HOXA1, HOXB8, HOXD9, HOXA11, and PBX1 genes were upregulated in MSCs derived from clotted vBMA from male donors. These results demonstrate that vBMA clots can be effectively used for spinal fusion procedures; however, gender-related differences should be taken into consideration when utilizing vBMA-clot-based studies to optimize the design and implementation of this cell therapy strategy in clinical trials.

Curcumin has numerous biological activities and pharmaceutical applications related to its ability to inhibit reactive oxygen species. Herein, strontium-substituted monetite (SrDCPA) and strontium-substituted brushite (SrDCPD) were synthesized and further functionalized with curcumin with the aim to develop materials that combine the anti-oxidant properties of the polyphenol, the beneficial role of strontium toward bone tissue, and the bioactivity of calcium phosphates. Adsorption from hydroalcoholic solution increases with time and curcumin concentration, up to about 5–6 wt%, without affecting the crystal structure, morphology, and mechanical response of the substrates. The multi-functionalized substrates exhibit a relevant radical scavenging activity and a sustained release in phosphate buffer. Cell viability, morphology, and expression of the most representative genes were tested for osteoclast seeded in direct contact with the materials and for osteoblast/osteoclast co-cultures. The materials at relatively low curcumin content (2–3 wt%) maintain inhibitory effects on osteoclasts and support the colonization and viability of osteoblasts. The expressions of Alkaline Phosphatase (ALPL), collagen type I alpha 1 chain (COL1A1), and osteocalcin (BGLAP) suggest that curcumin reduces the osteoblast differentiation state but yields encouraging osteoprotegerin/receptor activator for the NFkB factor ligand (OPG/RANKL) ratio.

In orthopedics, titanium (Ti)-alloy implants, are often considered as the first-choice candidates for bone tissue engineering. An appropriate implant coating enhances bone matrix ingrowth and biocompatibility, improving osseointegration. Collagen I (COLL) and chitosan (CS) are largely employed in several different medical applications, for their antibacterial and osteogenic properties. This is the first in vitro study that provides a preliminary comparison between two combinations of COLL/CS coverings for Ti-alloy implants, in terms of cell adhesion, viability, and bone matrix production for probable future use as a bone implant. Through an innovative spraying technique, COLL–CS–COLL and CS–COLL–CS coverings were applied over Ti-alloy (Ti-POR) cylinders. After cytotoxicity evaluations, human bone marrow mesenchymal stem cells (hBMSCs) were seeded onto specimens for 28 days. Cell viability, gene expression, histology, and scanning electron microscopy evaluations were performed. No cytotoxic effects were observed. All cylinders were biocompatible, thus permitting hBMSCs’ proliferation. Furthermore, an initial bone matrix deposition was observed, especially in the presence of the two coatings. Neither of the coatings used interferes with the osteogenic differentiation process of hBMSCs, or with an initial deposition of new bone matrix. This study sets the stage for future, more complex, ex vivo or in vivo studies.

Osteoarthritis (OA) is a degenerative bone disease that involves the microenvironment and macroenvironment of joints. Progressive joint tissue degradation and loss of extracellular matrix elements, together with different grades of inflammation, are important hallmarks of OA disease. Therefore, the identification of specific biomarkers to distinguish the stages of disease becomes a primary necessity in clinical practice. To this aim, we investigated the role of miR203a-3p in OA progression starting from the evidence obtained by osteoblasts isolated from joint tissues of OA patients classified according to different Kellgren and Lawrence (KL) grading (KL ≤ 3 and KL > 3) and hMSCs treated with IL-1β. Through qRT-PCR analysis, it was found that osteoblasts (OBs) derived from the KL ≤ 3 group expressed high levels of miR203a-3p and low levels of ILs compared with those of OBs derived from the KL > 3 group. The stimulation with IL-1β improved the expression of miR203a-3p and the methylation of the IL-6 promoter gene, favoring an increase in relative protein expression. The gain and loss of function studies showed that the transfection with miR203a-3p inhibitor alone or in co-treatments with IL-1β was able to induce the expression of CX-43 and SP-1 and to modulate the expression of TAZ, in OBs derived from OA patients with KL ≤ 3 compared with KL > 3. These events, confirmed also by qRT-PCR analysis, Western blot, and ELISA assay performed on hMSCs stimulated with IL-1β, supported our hypothesis about the role of miR203a-3p in OA progression. The results suggested that during the early stage, miR203a-3p displayed a protective role reducing the inflammatory effects on CX-43, SP-1, and TAZ. During the OA progression the downregulation of miR203a-3p and consequently the upregulation of CX-43/SP-1 and TAZ expression improved the inflammatory response and the reorganization of the cytoskeleton. This role led to the subsequent stage of the disease, where the aberrant inflammatory and fibrotic responses determined the destruction of the joint.

Background: Osteosarcopenia, a combination of osteopenia/osteoporosis and sarcopenia, is a common condition among older adults. While numerous studies and meta-analyses have been conducted on osteoporosis biomarkers, biomarker utility in osteosarcopenia still lacks evidence. Here, we carried out a systematic review to explore and analyze the potential clinical of circulating microRNAs (miRs) shared between osteoporosis/osteopenia and sarcopenia. Methods: We performed a systematic review on PubMed, Scopus, and Embase for differentially expressed miRs (p-value < 0.05) in (i) osteoporosis and (ii) sarcopenia. Following screening for title and abstract and deduplication, 83 studies on osteoporosis and 11 on sarcopenia were identified for full-text screening. Full-text screening identified 54 studies on osteoporosis, 4 on sarcopenia, and 1 on both osteoporosis and sarcopenia. Results: A total of 69 miRs were identified for osteoporosis and 14 for sarcopenia. There were 9 shared miRs, with evidence of dysregulation (up- or down-regulation), in both osteoporosis and sarcopenia: miR-23a-3p, miR-29a, miR-93, miR-133a and b, miR-155, miR-206, miR-208, miR-222, and miR-328, with functions and targets implicated in the pathogenesis of osteosarcopenia. However, there was little agreement in the results across studies and insufficient data for miRs in sarcopenia, and only three miRs, miR-155, miR-206, and miR-328, showed the same direction of dysregulation (down-regulation) in both osteoporosis and sarcopenia. Additionally, for most identified miRs there has been no replication by more than one study, and this is particularly true for all miRs analyzed in sarcopenia. The study quality was typically rated intermediate/high risk of bias. The large heterogeneity of the studies made it impossible to perform a meta-analysis. Conclusions: The findings of this review are particularly novel, as miRs have not yet been explored in the context of osteosarcopenia. The dysregulation of miRs identified in this review may provide important clues to better understand the pathogenesis of osteosarcopenia, while also laying the foundations for further studies to lead to effective screening, monitoring, or treatment strategies.

Pivoting sports expose athletes to a high risk of knee injuries, mainly due to mechanical overloading of the joint which shatters overall tissue integrity. The present study explored the magnitude of tibiofemoral contact forces (TFCF) in high-risk dynamic tasks. A novel musculoskeletal model with modifiable frontal plane knee alignment was developed to estimate the total, medial, and lateral TFCF developed during vigorous activities. Thirty-one competitive soccer players performing deceleration and 90° sidestepping tasks were assessed via 3D motion analysis by using a marker-based optoelectronic system and TFCF were assessed via OpenSim software. Statistical parametric mapping was used to investigate the effect of frontal plane alignment, compartment laterality, and varus–valgus genu on TFCF. Further, in consideration of specific risk factors, sex influence was also assessed. A strong correlation (R = 0.71 ÷ 0.98, p < 0.001) was found between modification of compartmental forces and changes in frontal plane alignment. Medial and lateral TFCF were similar throughout most of the tasks with the exception of the initial phase, where the lateral compartment had to withstand to higher loadings (1.5 ÷ 3 BW higher, p = 0.010). Significant sex differences emerged in the late phase of the deceleration task. A comprehensive view of factors influencing the mediolateral distribution of TFCF would benefit knee injury prevention and rehabilitation in sport activities.

The increase in degenerative diseases involving articular cartilage has pushed research to focus on their pathogenesis and treatment, exploiting increasingly complex techniques. Gene expression analyses from tissue are representative of the in vivo situation, but the protocols to be applied to obtain a reliable analysis are not completely cleared through customs. Thus, RNA extraction from fresh samples and specifically from musculoskeletal tissue such as cartilage is still a challenging issue. The aim of the review is to provide an overview of the techniques described in the literature for RNA extraction, highlighting limits and possibilities. The research retrieved 65 papers suitable for the purposes. The results highlighted the great difficulty in comparing the different studies, both for the sources of tissue used and for the techniques employed, as well as the details about protocols. Few papers compared different RNA extraction methods or homogenization techniques; the case study reported by authors about RNA extraction from sheep cartilage has not found an analog in the literature, confirming the existence of a relevant blank on studies about RNA extraction from cartilage tissue. However, the state of the art depicted can be used as a starting point to improve and expand studies on this topic.

Detecting subtle changes of surface stiffness at spatial scales and forces relevant to biological processes is crucial for the characterization of biopolymer systems in view of chemical and/or physical surface modification aimed at improving bioactivity and/or mechanical strength. Here, a standard atomic force microscopy setup is operated in nanoindentation mode to quantitatively mapping the near-surface elasticity of semicrystalline polyether ether ketone (PEEK) at room temperature. Remarkably, two localized distributions of moduli at about 0.6 and 0.9 GPa are observed below the plastic threshold of the polymer, at indentation loads in the range of 120–450 nN. This finding is ascribed to the localization of the amorphous and crystalline phases on the free surface of the polymer, detected at an unprecedented level of detail. Our study provides insights to quantitatively characterize complex biopolymer systems on the nanoscale and to guide the optimal design of micro- and nanostructures for advanced biomedical applications.

Exploring innovative techniques and treatments to improve spinal fusion procedures is a global challenge. Here, we provide a scientific opinion on the ability of a vertebral bone marrow (vBM) clot to provide a local combined delivery system not only of stem cells, signaling biomolecules and anti-inflammatory factors but also of molecules and proteins endowed with antimicrobial properties. This opinion is based on the evaluation of the intrinsic basic properties of the vBM, that contains mesenchymal stem cells (MSCs), and on the coagulation process that led to the conversion of fibrinogen into fibrin fibers that enmesh cells, plasma but above all platelets, to form the clot. We emphasize that vBM clot, being a powerful source of MSCs and platelets, would allow the release of antimicrobial proteins and molecules, mainly cathelicidin LL- 37, hepcidin, kinocidins and cationic host defense peptides, that are per se gifted with direct and/or indirect antimicrobial effects. We additionally highlight that further studies are needed to deepen this knowledge and to propose vBM clot as multifunctional bioscaffold able to target all the main key challenges for spinal fusion surgery.

The reconstruction of large segmental defects still represents a critical issue in the orthopedic field. The use of functionalized scaffolds able to create a magnetic environment is a fascinating option to guide the onset of regenerative processes. In the present study, a porous hydroxyapatite scaffold, incorporating superparamagnetic Fe3O4 nanoparticles (MNPs), was implanted in a critical bone defect realized in sheep metatarsus. Superparamagnetic nanoparticles functionalized with hyperbranched poly(epsilon-Lysine) peptides and physically complexed with vascular endothelial growth factor (VEGF) where injected in situ to penetrate the magnetic scaffold. The scaffold was fixed with cylindrical permanent NdFeB magnets implanted proximally, and the magnetic forces generated by the magnets enabled the capture of the injected nanoparticles forming a VEGF gradient in its porosity. After 16 weeks, histomorphometric measurements were performed to quantify bone growth and bone-to-implant contact, while the mechanical properties of regenerated bone via an atomic force microscopy (AFM) analysis were investigated. The results showed increased bone regeneration at the magnetized interface; this regeneration was higher in the VEGF-MNP-treated group, while the nanomechanical behavior of the tissue was similar to the pattern of the magnetic field distribution. This new approach provides insights into the ability of magnetic technologies to stimulate bone formation, improving bone/scaffold interaction.

Spinal bone metastases from uterine leiomyosarcoma (LMS) are relatively uncommon and few data are present in the literature. In this study, cases of nine consecutive patients who underwent spinal surgery for metastatic uterine LMS between 2012 and 2022 at a single institution were retrospectively reviewed. The recorded demographic, operative, and postoperative factors were reviewed, and the functional outcomes were determined by changes in Frankel grade classification during follow-up. A systematic review of the literature was also performed to evaluate operative and postoperative factors and outcomes for patients with the same gynecological metastases to the spine. For our cases, the mean time between primary tumors to bone metastases diagnosis was 5.2 years, and the thoracic vertebrae were the most affected segment. Overall, median survival after diagnosis of metastatic spine lesions was 46 months. For the systematic review, the mean time between primary tumors to bone metastases was 4.9 years, with the lumbar spine as the most involved site of metastasis. Overall, median survival after diagnosis was 102 months. Once a spinal bone lesion from LMS is identified, surgical treatment can be beneficial and successful in alleviating symptoms. Further efforts will be crucial to identify prognostic markers as well as therapeutic targets to improve survival in these patients.

The existence of a tight relationship between inflammation and epigenetics that in primary breast tumor cells can lead to tumor progression and the formation of bone metastases was investigated. It was highlighted how the induction of tumor progression and bone metastasis by Interleukin-1 beta, in a non-metastatic breast cancer cell line, MCF-7, was dependent on the de-methylating actions of ten-eleven translocation proteins (TETs). In fact, the inhibition of their activity by the Bobcat339 molecule, an inhibitor of TET enzymes, determined on the one hand, the modulation of the epithelial-mesenchymal transition process, and on the other hand, the reduction in the expression of markers of bone metastasis, indicating that the epigenetic action of TETs is a prerequisite for IL-1β-dependent tumor progression and bone metastasis formation.

Osteosarcoma (OS) is the most common primary bone sarcoma affecting the life of pediatric patients. The clinical treatment faces numerous difficulties, including the adverse effects of chemotherapies, chemoresistance, and recurrences. In this study, the effects of resveratrol (RSV), a natural polyphenol, on OS cell lines were investigated to evaluate its action as an adjuvant therapy to the current chemotherapy regimens. RSV exhibited multiple tumor-suppressing activities on OS cell lines, inducing a series of critical events. We found (1) a cell growth inhibition due to an increase in cell distress, which was, in part, due to the involvement of the AKT and caspase-3 pathways, (2) an increase in cellular differentiation due to major gene expression levels of the osteoblastic differentiation genes, (3) an inhibition of IL-6 secretion due to an epigenetic effect on the IL-6 promoter, and (4) an inhibition of OS cells migration related to the decrease in IL-8 secretion levels due to an epigenetic effect on its promoter. Finally, the cotreatment of RSV with doxorubicin and cisplatin increased their cytotoxic effect on OS cells. Although further investigations are mandatory, it seems RSV might be a promising therapeutic adjuvant agent for OS cell treatment, exerting an antitumor effect when combined with chemotherapy.

Osteosarcoma (OS) is the most common primary bone tumor mainly occurring in young adults and derived from primitive bone-forming mesenchyme. OS develops in an intricate tumor microenvironment (TME) where cellular function regulated by microRNAs (miRNAs) may affect communication between OS cells and the surrounding TME. Therefore, miRNAs are considered potential therapeutic targets in cancer and one of the goals of research is to accurately define a specific signature of a miRNAs, which could reflect the phenotype of a particular tumor, such as OS. Through NGS approach, we previously found a specific molecular profile of miRNAs in OS and discovered 8 novel miRNAs. Among these, we deepen our knowledge on the fifth candidate renamed now miR-CT3. MiR-CT3 expression was low in OS cells when compared with human primary osteoblasts and healthy bone. Through TargetScan, VEGF-A was predicted as a potential biological target of miR-CT3 and luciferase assay confirmed it. We showed that enforced expression of miR-CT3 in two OS cell lines, SAOS-2 and MG-63, reduced expression of VEGF-A mRNA and protein, inhibiting tumor angiogenesis. Enforced expression of miR-CT3 also reduced OS cell migration and invasion as confirmed by soft agar colony formation assay. Interestingly, we found that miR-CT3 behaves inducing the activation of p38 MAP kinase pathway and modulating the epithelial-mesenchymal transition (EMT) proteins, in particular reducing Vimentin expression. Overall, our study highlights the novel role of miR-CT3 in regulating tumor angiogenesis and progression in OS cells, linking also to the modulation of EMT proteins.

Abstract Background Bone tissue is one of the main sites for breast metastasis; patients diagnosed with advanced breast cancer mostly develop bone metastasis characterized by severe osteolytic lesions, which heavily influence their life quality. Low Intensity Pulsed Ultrasound (LIPUS) is a form of mechanical energy able to modulate various molecular pathways both in cancer and in health cells. The purpose of the present study was to evaluate for the first time, the ability of LIPUS to modulate osteolytic capability of breast cancer cells. Methods Two different approaches were employed: a) Indirect method -conditioned medium obtained by MDA-MB-231 cell line treated or untreated with LIPUS was used to induce osteoclast differentiation of murine macrophage Raw264.7 cell line; and b) Direct method -MDA-MB-231 were co-cultured with Raw264.7 cells and treated or untreated with LIPUS. Results LIPUS treatment impaired MDA-MB-231 cell dependentosteoclast differentiation and produced a reduction of osteoclast markers such as Cathepsin K, Matrix Metalloproteinase 9 and Tartrate Resistant Acid Phosphatase, suggesting its role as an effective and safe adjuvant in bone metastasis management. Conclusion LIPUS treatment could be a good and safety therapeutic adjuvant in osteolyitic bone metastasis not only for the induction properties of bone regeneration, but also for the reduction of osteolysis.

Abstract Background Our previous study demonstrates that Citrus-limon derived nanovesicles are able to decrease colon cancer cell viability, and that this effect is associated with the downregulation of the intracellular phospholipase DDHD domain-containing protein 1 (DDHD1). While few studies are currently available on the contribution of DDHD1 in neurological disorders, there is no information on its role in cancer. This study investigates the role of DDHD1 in colon cancer. Methods DDHD1 siRNAs and an overexpression vector were transfected into colorectal cancer and normal cells to downregulate or upregulate DDHD1 expression. In vitro and in vivo assays were performed to investigate the functional role of DDHD1 in colorectal cancer cell growth. Quantitative proteomics using SWATH-MS was performed to determinate the molecular effects induced by DDHD1 silencing in colorectal cancer cells. Results The results indicate that DDHD1 supports colon cancer cell proliferation and survival, since its downregulation reduces in vitro colon cancer cell viability and increases apoptosis rate, without affecting normal cells. On the contrary, in vivo studies demonstrate that the xenograft tumors, derived from DDHD1-overexpressing cells, have a higher proliferation rate compared to control animals. Additionally, we found that functional categories, significantly affected by DDHD1 silencing, were specifically related to cancer phenotype and for the first time associated to DDHD1 activity. Conclusions In conclusion, this study provides the first evidence confirming the role of DDHD1 in cancer, providing a possibility to define a new target to design more effective therapies for colon cancer patients.

Abstract Osteosarcoma (OS) is the most common primary malignant tumor of bone, which preferentially develops lung metastasis. Although standard chemotherapy has significantly improved long-term survival over the past few decades, the outcome for patients with metastatic or recurrent OS remains dramatically poor. Novel therapies are therefore required to slow progression and eradicate the disease. Furthermore, to better understand the cellular and molecular mechanisms responsible for OS onset and progression, the development of novel predictive culture systems resembling the native three-dimensional (3D) tumor microenvironment are mandatory. ‘Tumor engineering’ approaches radically changed the previous scenario, through the development of advanced and alternative 3D cell culture in vitro models able to tightly mimic the in vivo tumor microenvironment. In this review, we will summarize the state of the art in this novel area, illustrating the different methods and techniques employed to realize 3D OS cell culture models and we report the achieved results, which highlight the efficacy of these models in reproducing the tumor milieu. Although data need to be further validated, the scientific studies reviewed here are certainly promising and give new insights into the clinical practice.

Appropriate monitoring and control of modifiable risk factors, such as the level of low-density lipoprotein cholesterol (LDL-C) and other types of dyslipidemia, have an important role in the prevention of cardiovascular diseases (CVD). Recently, various nutraceuticals with lipid-lowering effects have gained attention. In addition to the plant-derived bioactive compounds, recent studies suggested that plant cells are able to release small lipoproteic structures named extracellular vesicles (EVs). The interaction between EVs and mammalian cells could lead to beneficial effects through anti-inflammatory and antioxidant activities. The present study aimed to assess the safety of the new patented plant-based product citraVes™, containing extracellular vesicles (EVs) from Citrus limon (L.) Osbeck juice, and to investigate its ability to modulate different CV risk factors in healthy subjects. A cohort of 20 healthy volunteers was recruited in a prospective open-label study. All participants received the supplement in a spray-dried formulation at a stable dose of 1000 mg/day for 3 months. Anthropometric and hematobiochemical parameters were analyzed at the baseline and after the follow-up period of 1 and 3 months. We observed that the supplement has an effect on two key factors of cardiometabolic risk in healthy subjects. A significant change in waist circumference was found in women after 4 (85.4 [79.9, 91.0] cm, p < 0.005) and 12 (85.0 [80.0, 90.0] cm, p < 0.0005) weeks, when compared to the baseline value (87.6 [81.7, 93.6] cm). No difference was found in men (baseline: 100.3 [95.4, 105.2] cm; 4 weeks: 102.0 [95.7, 108.3] cm; 12 weeks: 100.0 [95.3, 104.7] cm). The level of LDL-C was significantly lower at 12 weeks versus 4 weeks (p = 0.0064). Our study evaluated, for the first time, the effects of a natural product containing plant-derived EVs on modifiable risk factors in healthy volunteers. The results support the use of EV extracts to manage cardiometabolic risk factors successfully.

Osteoarthritis (OA) is a degenerative bone disease that involved micro and macro-environment of joints. To date, there are no radical curative treatments for OA and novel therapies are mandatory. Recent evidence suggests the role of miRNAs in OA progression. In our previous studies, we demonstrated the role of miR-31-5p and miR-33a families in different bone regeneration signaling. Here, we investigated the role of miR-31-5p and miR-33a-5p in OA progression. A different expression of miR-31-5p and miR-33a-5p into osteoblasts and chondrocytes isolated from joint tissues of OA patients classified in based on different Kellgren and Lawrence (KL) grading was highlighted; and through a bioinformatic approach the common miRNAs target Specificity proteins (Sp1) were identified. Sp1 regulates the expression of gap junction protein Connexin43 (Cx43), which in OA drives the modification of (i) osteoblasts and chondrocytes genes expression, (ii) joint inflammation cytokines releases and (iii) cell functions. Concerning this, thanks to gain and loss of function studies, the possible role of Sp1 as a modulator of CX43 expression through miR-31-5p and miR-33a-5p action was also evaluated. Finally, we hypothesize that both miRNAs cooperate to modulate the expression of SP1 in osteoblasts and chondrocytes and interfering, consequently, with CX43 expression, and they might be further investigated as new possible biomarkers for OA.

Bone remodeling is uncoupled in the multiple myeloma (MM) bone marrow niche, resulting in enhanced osteoclastogenesis responsible of MM-related bone disease (MMBD). Several studies have disclosed the mechanisms underlying increased osteoclast formation and activity triggered by the various cellular components of the MM bone marrow microenvironment, leading to the identification of novel targets for therapeutic intervention. In this regard, recent attention has been given to non-coding RNA (ncRNA) molecules, that finely tune gene expression programs involved in bone homeostasis both in physiological and pathological settings. In this review, we will analyze major signaling pathways involved in MMBD pathophysiology, and report emerging evidence of their regulation by different classes of ncRNAs.

The purpose of this study was to verify the efficacy of a single intra-articular (i.a.) injection of a hyaluronic acid-chitlac (HY-CTL) enriched with two low dosages of triamcinolone acetonide (TA, 2.0 mg/mL and 4.5 mg/mL), in comparison with HY-CTL alone, with a clinical control (TA 40 mg/mL) and with saline solution (NaCl) in an in vivo osteoarthritis (OA) model. Seven days after chemical induction of OA, 80 Sprague Dawley male rats were grouped into five arms (n = 16) and received a single i.a. injection of: 40 mg/mL TA, HY-CTL alone, HY-CTL with 2.0 mg/mL TA (RV2), HY-CTL with 4.5 mg/mL TA (RV4.5) and 0.9% NaCl. Pain sensitivity and Catwalk were performed at baseline and at 7, 14 and 21 days after the i.a. treatments. The histopathology of the joint, meniscus and synovial reaction, type II collagen expression and aggrecan expression were assessed 21 days after treatments. RV4.5 improved the local pain sensitivity in comparison with TA and NaCl. RV4.5 and TA exerted similar beneficial effects in all gait parameters. Histopathological analyses, measured by Osteoarthritis Research Society International (OARSI) and Kumar scores and by immunohistochemistry, evidenced that RV4.5 and TA reduced OA features in the same manner and showed a stronger type II collagen and aggrecan expression; both treatments reduced synovitis, as measured by Krenn score and, at the meniscus level, RV4.5 improved degenerative signs as evaluated by Pauli score. TA or RV4.5 treatments limited the local articular cartilage deterioration in knee OA with an improvement of the physical structure of articular cartilage, gait parameters, the sensitivity to local pain and a reduction of the synovial inflammation.

Evaluating cell migration after cell-based treatment is important for several disorders, including osteoarthritis (OA), as it might influence the clinical outcome. This research explores migrating expanded-adipose stromal cells (ASCs) and adipose niches after enzymatic and mechanical processes. Bilateral anterior cruciate ligament transection induced a mild grade of OA at eight weeks in adult male New Zealand rabbits. ASCs, enzymatic stromal vascular fraction (SVF), and micro fragmented adipose tissue (MFAT) were intra-articularly injected in the knee joint. Assessments of cell viability and expression of specific markers, including CD-163 wound-healing macrophages, were done. Cell migration was explored through labelling with PKH26 dye at 7 and 30 days alongside co-localization analyses for CD-146. All cells showed good viability and high percentages of CD-90 and CD-146. CD-163 was significantly higher in MFAT compared to SVF. Distinct migratory potential and time-dependent effects were observed among cell-based treatments. At day 7, both ASCs and SVF migrated towards synovium, whereas for MFAT versus cartilage, a different migration pattern was noticed at day 30. The long-term distinct cell migration of ASCs, SVF, and MFAT open interesting clinical insights on their potential use for OA treatment. Moreover, the highest expression of CD-163 in MFAT, rather than SVF, might have an important role in directly mediating cartilage tissue repair responses.

The development of effective nanosystems for drug delivery represents a key challenge for the improvement of most current anticancer therapies. Recent progress in the understanding of structure and function of extracellular vesicles (EVs)—specialized membrane-bound nanocarriers for intercellular communication—suggests that they might also serve as optimal delivery systems of therapeutics. In addition to carrying proteins, lipids, DNA and different forms of RNAs, EVs can be engineered to deliver specific bioactive molecules to target cells. Exploitation of their molecular composition and physical properties, together with improvement in bio-techniques to modify their content are critical issues to target them to specific cells/tissues/organs. Here, we will discuss the current developments in the field of animal and plant-derived EVs toward their potential use for delivery of therapeutic agents in different pathological conditions, with a special focus on cancer.