Osteosarcoma is a highly malignant primary tumor of bone tissue. The 5-year survival rate of patients with metastasis is below 20% and this scenario is unchanged in the last two decades, despite great efforts in pre-clinical and clinical research. Traditional preclinical models of osteosarcoma do not consider the whole complexity of its microenvironment, leading to poor correlation between in vitro/in vivo results and clinical outcomes. Spheroids are a promising in vitro model to mimic osteosarcoma and perform drug-screening tests, as they (i) reproduce the microarchitecture of the tumor, (ii) are characterized by hypoxic regions and necrotic core as the in vivo tumor, (iii) and recapitulate the chemo-resistance phenomena. However, to date, the spheroid model is scarcely used in osteosarcoma research. Our aim is to develop a customized culture dish to grow and characterize spheroids and to perform advanced drug-screening tests. The resulting platform must be adapted to automated image acquisition systems, to overcome the drawbacks of commercial spheroids platforms. To this purpose, we designed and developed a micro-patterned culture dish by casting agarose on a 3D printed mold from a CAD design. We successfully obtained viable and reproducible homotypic osteosarcoma spheroids, with two different cells lines from osteosarcoma (i.e., 143b and MG-63). Using the platform, we performed viability assays and live fluorescent stainings (e.g., Calcein AM) with low reagent consumption. Moreover, the culture dish was validated as drug screening platform, administrating Doxorubicin at different doses, and evaluating its effect on OS spheroids, in terms of morphology and viability. This platform can be considered an attractive alternative to the highly expensive commercial spheroid platforms to obtain homogeneous and reproducible spheroids in a high-throughput and cost effective mode

Osteoporosis is a progressive, chronic disease of bone metabolism, characterized by decreased bone mass and mineral density, predisposing individuals to an increased risk of fractures. The use of animal models, which is the gold standard for the screening of anti-osteoporosis drugs, raises numerous ethical concerns and is highly debated because the composition and structure of animal bones is very different from human bones. In addition, there is currently a poor translation of pre-clinical efficacy in animal models to human trials, meaning that there is a need for an alternative method of screening and evaluating new therapeutics for metabolic bone disorders, in vitro. The aim of this project is to develop a 3D Bone-On-A-Chip that summarizes the spatial orientation and mutual influences of the key cellular components of bone tissue, in a citrate and hydroxyapatite-enriched 3D matrix, acting as a 3D model of osteoporosis. To this purpose, a polydimethylsiloxane microfluidic device was developed by CAD modelling, stereolithography and replica molding. The device is composed by two layers: (i) a bottom layer for a 3D culture of osteocytes embedded in an osteomimetic collagen-enriched matrigel matrix with citrate-doped hydroxyapatite nanocrystals, and (ii) a upper layer for a 2D perfused co-culture of osteoblasts and osteoclasts seeded on a microporous PET membrane. Cell vitality was evaluated via live/dead assay. Bone deposition and bone resorption was analysed respectively with ALP, Alizarin RED and TRACP staining. Osteocytes dendrite expression was evaluated via immunofluorescence. Subsequently, the model was validated as drug screening platform inducing osteocytes apoptosis and administrating standard anti-osteoporotic drugs. This device has the potential to substitute or minimize animal models in pre-clinical studies of osteoporosis, contributing to pave the way for a more precise and punctual personalized treatment

Aims

After a few passages of in vitro culture, primary human articular chondrocytes undergo senescence and loss of their phenotype. Most of the available chondrocyte cell lines have been obtained from cartilage tissues different from diarthrodial joints, and their utility for osteoarthritis (OA) research is reduced. Thus, the goal of this research was the development of immortalized chondrocyte cell lines proceeded from the articular cartilage of patients with and without OA.

Vertebral metastases are the most common type of malignant lesions of the spine. Although this tumour is still considered incurable and standard treatments are mainly palliative, the standard approach consists in surgical resection, which results in the formation of bone gaps. Hence, scaffolds, cements and/or implants are needed to fill the bone lacunae. Here, we propose a novel approach to address spinal metastases recurrence, based on the use of anti-tumour metallic-based nanostructured coatings. Moreover, for the first time, a gradient microfluidic approach is proposed for the screening of nanostructured coatings having anti-tumoral effect, to determine the optimal concentration of the metallic compound that permits selective toxicity towards tumoral cells. Coatings are based on Zinc as anti-tumour agent, which had been never explored before for treatment of bone metastases. The customized gradient generating microfluidic chip was designed by Autodesk Inventor and fabricated from a microstructured mould by using replica moulding technique. Microstructured mould were obtained by micro-milling technique. The chip is composed of a system of microfluidic channels generating a gradient of 6 concentrations of drug and a compartment with multiple arrays of cell culture chambers, one for each drug concentration. The device is suitable for dynamic cultures and in-chip biological assays. The formation of a gradient was validated using a methylene blue solution and the cell loading was successful. Preliminary biological data on 3D dynamic cultures of stromal cells (bone-marrow mesenchymal stem cells) and breast carcinoma cells (MDA-MB-231) were performed in a commercial microfluidic device. Results showed that Zn eluates had a selective cytotoxic effect for tumoral cells. Indeed, cell migration and cell replication of treated tumoral cells was inhibited. Moreover, the three-dimensionality of the model strongly affected the efficacy of Zn eluates, as 2D preliminary experiments showed a high cytotoxic effect of Zn also for stromal cells, thus confirming that traditional screening tests on 2D cultured cells usually lead to an overestimation of drug efficacy and toxicity. Based on preliminary data, the customized platform could be considered a major advancement in cancer drug screenings as it also allows the rapid and efficient screening of biomaterials having antitumor effect

The prevalence of alcohol and opioids in severely injured patients has been widely reported from 30-80%. However, despite the increasing global misuse of stimulant drugs, there is a paucity of literature regarding the presence of stimulant drugs in trauma patients. The primary aim of this study was to define the prevalence of stimulant drugs that were detected in patients who presented to Level One Trauma Centers throughout North America, and their effect on length of stay and mortality. Our triage criteria for admittance to the regional trauma centre are based on the recommendations by the American College of Surgeons Committee on Trauma, who now recommend toxicology screening on every patient. This was a retrospective analysis of data from the Trauma Quality Improvement Program, including all patients presenting emergently to participating Level 1 Trauma Centers from January 2017 to December 2018. A stimulant drug was defined as the detection of cocaine, amphetamine, or methamphetamine. Adults aged 18-64 years were included. Patient risk factors were included adjusted for in the analysis: age, sex, body mass index (BMI), alcohol screening results and smoking status. Univariate analysis was performed for all variables. Multivariable logistic regression and liner regression were used for mortality and length of stay, respectively. Of a total of 110,561 patients included in the study, 15,958 patients (14.4%) had positive screens for stimulants. The average age in the stimulants cohort was 40.8 years, with a 77.6% male preponderance, BMI of 26.9, blood alcohol content of 0.07, and ISS of 11.3. The control cohort was comparable, though 71.1% male (p<0.001) Patients who tested positive for stimulants had 1.79 times (95% CI, 1.09-2.93) the odds of dying in the emergency department as the control group (p=0.02). Following transfer from the emergency department, the odds ratio for deaths in hospital (OR=1.02, 95% CI 0.90-1.15) was comparable to the control group (p=0.78). The mean length of stay was significantly higher in the stimulant group (2.84 days) compared to the control group (1.79 days) (p<.001). In the Intensive Care Unit, length of stay was 0.64 days in the stimulant group versus 1.65 in the control (p=0.48). Stimulant misuse is a relevant issue in the trauma population, associated with a longer hospital stay and higher mortality in the emergency department. The continued routine drug screening of trauma patients may be beneficial in trauma centers, to implement preventative measures and optimise resource allocation

Abstract

Aims. Rheumatoid arthritis (RA) is a systematic autoimmune disorder, characterized by synovial inflammation, bone and cartilage destruction, and disease involvement in multiple organs. Although numerous drugs are employed in RA treatment, some respond little and suffer from severe side effects. This study aimed to screen the candidate therapeutic targets and promising drugs in a novel method. Methods. We developed a module-based and cumulatively scoring approach that is a deeper-layer application of weighted gene co-expression network (WGCNA) and connectivity map (CMap) based on the high-throughput datasets. Results. Four noteworthy RA-related modules were identified, revealing the immune- and infection-related biological processes and pathways involved in RA. HLA-DMA, HLA-DMB, HLA-DPA1, HLA-DPB1, HLA-DQB1, HLA-DRA, HLA-DRB1, BLNK, BTK, CD3D, CD4, IL2RG, INPP5D, LCK, PTPRC, RAC2, SYK, and VAV1 were recognized as the key hub genes with high connectivity in gene regulation networks and gene pathway networks. Moreover, the long noncoding RNAs (lncRNAs) in the RA-related modules, such as FAM30A and NEAT1, were identified as the indispensable interactors with the hub genes. Finally, candidate drugs were screened by developing a cumulatively scoring approach based on the selected modules. Niclosamide and the other compounds of T-type calcium channel blocker, IKK inhibitor, and PKC activator, HIF activator, and proteasome inhibitor, which harbour the similar gene signature with niclosamide, were promising drugs with high specificity and broad coverage for the RA-related modules. Conclusion. This study provides not only the promising targets and drugs for RA but also a novel methodological insight into the target and drug screening. Cite this article: Bone Joint Res 2020;9(8):501–514

The ability to edit DNA at the nucleotide level using clustered regularly interspaced short palindromic repeats (CRISPR) systems is a relatively new investigative tool that is revolutionizing the analysis of many aspects of human health and disease, including orthopaedic disease. CRISPR, adapted for mammalian cell genome editing from a bacterial defence system, has been shown to be a flexible, programmable, scalable, and easy-to-use gene editing tool. Recent improvements increase the functionality of CRISPR through the engineering of specific elements of CRISPR systems, the discovery of new, naturally occurring CRISPR molecules, and modifications that take CRISPR beyond gene editing to the regulation of gene transcription and the manipulation of RNA. Here, the basics of CRISPR genome editing will be reviewed, including a description of how it has transformed some aspects of molecular musculoskeletal research, and will conclude by speculating what the future holds for the use of CRISPR-related treatments and therapies in clinical orthopaedic practice.

Cite this article: Bone Joint Res 2020;9(7):351–359.

Bioreactors have been used in articular cartilage tissue engineering (AC-TE) to apply different mechanical stimuli in an attempt to better mimic the native AC microenvironment. However, these systems are often highly complex, costly and not very versatile. In this work, we propose a simple and customizable perfusion bioreactor fabricated by 3D-extrusion to study the effect of shear stress in human bone-marrow mesenchymal stem cells (hBMSC) cultured in 3D porous polycaprolactone (PCL) scaffolds. Prototype models were designed in a CAD-software to perfectly fit the scaffolds and computational fluid dynamics analysis was used to predict the fluid velocities and shear stress forces inside the bioreactor. For the culture studies, hBMSC-PCL constructs were cultured under static expansion for 2 weeks and then transferred to the ABS-extruded bioreactors for continuous perfusion culture (0.2mL/min) under chondrogenic induction for additional 3 weeks. Perfused constructs showed similar cell proliferation and higher sGAG production in comparison to the static counterparts (bioreactor without perfusion). Constructs exposed to shear stress stimuli presented higher expressions of chondrogenic genes (COLII/Sox9/Aggrecan) and reduced expressions of COLI and Runx2 (osteogenic) than static group. However, the higher expression of COLX in the perfused constructs suggests a shear stress role in AC hypertrophy. Both conditions (perfused/static) stained positively for GAG deposition and for the presence of collagen II and aggrecan. Overall, the results provide a proof-of-concept of our customizable extruded bioreactor envisaging applications as a platform for AC-TE repair strategies and in the development of more reliable in vitro models for disease modelling and drug screening

Human in vitro models of the neuromuscular junction (NMJ) are currently moving from embryonic stem cells to induced Pluripotent Stem Cells (iPSCs). With this, a robust model could be optimised for physiology and pathophysiology studies, as well as representing a drug screening platform. For this reason, the work presented here represents the optimisation of a human co-culture model of skeletal muscle (hSkM)/ iPSC-derived motor neurons (MNs) both in monolayer and in 3D tissue engineering collagen constructs. Firstly, human iPSC-derived motor neurons (MNs) were characterised over a period of 35 days to test their cholinergic potential. Then, primary human skeletal muscle (hSkM) and MNs were co-cultured on different substrates (gelatin and SureBond+ReadySet (Axol Bioscience)) and differentiated in various combinations of media to allow both myotube formation and neurite extension. Morphological (β-III Tubulin and Rhodamine Phalloidin) and interaction (α-Bungarotoxin and Synaptic Vesicle 2) immunofluorescent stainings were used to evaluate cell differentiation and co-localisation of pre and post-synaptic markers. Results from this study showed that the MNs presented a cholinergic phenotype up to 21 days; hSkM and MNs co-existed in culture and differentiated in neuronal Maintenance Medium (MM, Axol Bioscience); the 3D constructs allowed alignment and maturation of the muscle tissue, while providing a matrix for neurite extension and NMJ formation. This model has the potential to become a valid tool for in vitro drug screening while reducing the use of animals in research and providing the scientific community with a platform for personalised medicine

Human mesenchymal stem cells are considered the golden standard for clinical application in regenerative medicine for their multilineage differentiation potential, best candidates to treat diseases such as osteoarthritis and osteogenesis imperfecta. In the past few years several molecules have been described to induce the hMSCs differentiation into osteo cell progenitors, mainly discovered by screening of single metabolites bioactivity. However, hMSCs osteogenic differentiation potential is still poor, and the discovery of differentiation inducers with high efficiency is needed. Thanks to automated processes, High Throughput Screening (HTS) strategies shorten the metabolites bioactivity investigation timeline, allowing testing of many molecules simultaneously. In this work, reliable assays for natural products bioactivity investigation detection were developed using HTS methodologies and validated by testing 15 purified compounds derived by marine fungi and sponges. The HTS cytotoxicity investigation using HepG2 cells allowed to test in a single experiment, 15 metabolites in 4 concentrations ranging from 1 to 20µM. Low cytotoxicity was detected for metabolites concentrations from 1 to 10µM and so set as treatment concentrations to be tested in further assays. Anti-inflammatory bioactivity was tested on THP1 cells triggered by LPS. Five out of 15 metabolites showed to prevent the LPS induced THP1 inflammatory activation by lowering the TNF-α production. The metabolites pro-osteogenic potential was investigated using hMSCs: their differentiation was evaluated by calcium mineralization after 10 days differentiation. Pro-osteogenic molecules were not detected in this screening, but the method validation represents a powerful tool for future natural product and synthetic molecules libraries screenings.

We developed a 3D vascularized bone remodeling model embedding human osteoblast and osteoclast precursors and endothelial cells in a mineralized matrix. All the cells included in the model exerted their function, resulting in a vascularized system undergoing mineralized matrix remodeling. Bone remodeling is a dynamic process relying on the balance between the activity of osteoblasts and osteoclasts which are responsible for bone formation and resorption, respectively. This process is also characterized by a tight coupling between osteogenesis and angiogenesis, indicating the existence of a complex cross-talk between endothelial cells and bone cells. We have recently developed microscale in vitro hydrogel-based models, namely the 3D MiniTissue models, to obtain bone-mimicking microenvironments including a 3D microvascular network formed by endothelial cell self-assembly [1–2]. Here, we generated a vascularized 3D MiniTissue bone remodeling model through the coculture of primary human cells in a 3D collagen/fibrin (Col/Fib) matrix enriched with CaP nanoparticles (CaPn) to mimic bone mineralized matrix. Human umbilical vein endothelial cells (HUVECs), bone marrow mesenchymal stem cells (BMSCs), osteoblast (OBs) and osteoclast (OCs) precursors were cocultured in plain and CaPn-enriched Col/Fib according to the following experimental conditions: a) HUVECs-BMSCs; b) OBs-OCs; c) HUVECs-BMSCs-OBs-OCs. Undifferentiated BMSCs were used to support HUVECs in microvascular network formation. BMSCs and peripheral blood mononuclear cells were respectively pre-differentiated into OB and OC precursors through 7 days of culture in osteogenic or osteoclastogenic medium. Needle-shaped CaPn (Ø ∼20 nm, length ∼80 nm) were added to a collagen/fibrinogen solution. Cells were resuspended in a thrombin solution and then mixed with plain or CaPn-enriched collagen/fibrinogen. The cell-laden mix was injected in U-shaped PMMA masks and let to polymerize to generate constructs of 2×2×5 mm. 3. Samples were cultured for 10 days. Microvascular network formation was evaluated by confocal microscopy. OB differentiation was analyzed by quantification of Alkaline Phosphatase (ALP) and cell-mediated mineralization. OC differentiation was assessed by Tartrate-Resistant Acid Phosphatase (TRAP) and cell-mediated phosphate release quantification. HUVECs developed a robust 3D microvascular network and BMSCs differentiated into mural cells supporting vasculogenesis. The presence of CaPn enhanced OB and OC differentiation, as demonstrated by the significantly higher ALP and TRAP levels and by the superior cell-mediated mineralization and phosphate release measured in CaPn-enriched than in plain Col/Fib. The coculture of OBs and OCs with HUVECs and BMSCs further enhanced ALP and TRAP levels, indicating that the presence of HUVECs and BMSCs positively contributed to OB and OC differentiation. Remarkably, higher values of ALP and TRAP activity were measured in the tetraculture in CaPn-enriched Col/Fib compared to plain Col/Fib, indicating that also in the tetraculture the mineralized matrix stimulated OB and OC differentiation. The 3D MiniTissue bone remodeling model developed in this study is a promising platform to investigate bone cell and endothelial cell cross-talk. This system allows to minimize the use of cells and reagents and is characterized by a superior ease of use compared to other microscale systems, such as microfluidic models. Finally, it represents a suitable platform to test drugs for bone diseases and can be easily personalized with patient-derived cells further increasing its relevance as drug screening platform

SSX was initially identified as a melanoma associated tumour antigen MEL2 and in the SS18/SSX fusion gene of synovial sarcoma. It consists of a family of nine, highly homologous X chromosome genes, being SSX1, SSX2 and SSX4 the most commonly expressed in tumours. In normal tissue, SSX expression is restricted to germ cells, trophoblasts, and mesenchymal stem cells. In malignant cells, SSX expression is over-represented in sarcomas. SSX expression is epigenetically regulated by methylation and histone deacetylation. Aim. To investigate the oncogenic properties of SSX. Validate it as tumour target and identify lead compounds. Methods. RNAi system for conditional silencing of SSX expression, Protein analysis and Mass Spec, PCR arrays, tumour xenographs and drug library screening. Results. SSX expression peaks at the G1/S phase of the cell cycle where it interacts with histone H3, H4 and β-catenin. This interaction promotes the phosphorylation of β-catenin at tyrosine residues and the transactivation of β-catenin genes MMP2 and cyclin D1. The association of SSX and MMP2 expression resulted in an increased invasion capacity of tumour cells. In vivo, silencing of SSX in tumour xenographs significantly affected the tumour growth. These tumours were characterized by large necrotic areas, impaired vascularization and cytoplasmic β-catenin. In contrast, tumour xenographs expressing SSX were highly proliferative, vascularized and exibited nuclear β-catenin. We further screened a chemical library and have identified lead compounds that target SSX. Conclusion. Our results demonstrate that SSX is a tumour target for drug development. We hypothesise that drugs targeting of SSX could affect functions related with the mesenchymal cell phenotype having potential cytostatic effect in sarcomas and/or epithelial tumour cells that undergo a epithelial-to-mesenchymal transition prior to metastasis