Objectives. Osteoarthritis (OA) is characterised by articular cartilage degradation. MicroRNAs (miRNAs) have been identified in the development of OA. The purpose of our study was to explore the functional role and underlying mechanism of miR-138-5p in interleukin-1 beta (IL-1β)-induced extracellular matrix (ECM) degradation of OA cartilage. Materials and Methods. Human articular cartilage was obtained from patients with and without OA, and chondrocytes were isolated and stimulated by IL-1β. The expression levels of miR-138-5p in cartilage and chondrocytes were both determined. After transfection with miR-138-5p mimics, allele-specific oligonucleotide (ASO)-miR-138-5p, or their negative controls, the messenger RNA (mRNA) levels of aggrecan (ACAN), collagen type II and alpha 1 (COL2A1), the protein levels of glycosaminoglycans (GAGs), and both the mRNA and protein levels of matrix metalloproteinase (MMP)-13 were evaluated. Luciferase reporter assay, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blot were performed to explore whether Forkhead Box C1 (FOCX1) was a target of miR-138-5p. Further, we co-transfected OA chondrocytes with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 and then stimulated with IL-1β to determine whether miR-138-5p-mediated IL-1β-induced cartilage matrix degradation resulted from targeting FOXC1. Results. MiR-138-5p was significantly increased in OA cartilage and in chondrocytes in response to IL-1β-stimulation. Overexpression of miR-138-5p significantly increased the IL-1β-induced downregulation of COL2A1, ACAN, and GAGs, and increased the IL-1β-induced over expression of MMP-13.We found that FOXC1 is directly regulated by miR-138-5p. Additionally, co-transfection with miR-138-5p mimics and pcDNA3.1 (+)-FOXC1 resulted in higher levels of COL2A1, ACAN, and GAGs, but lower levels of MMP-13. Conclusion. miR-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes, possibly by targeting FOXC1. Cite this article: Y. Yuan, G. Q. Zhang, W. Chai,M. Ni, C. Xu, J. Y. Chen. Silencing of microRNA-138-5p promotes IL-1β-induced cartilage degradation in human chondrocytes by targeting FOXC1: miR-138 promotes cartilage degradation. Bone Joint Res 2016;5:523–530. DOI: 10.1302/2046-3758.510.BJR-2016-0074.R2

Osteoarthritis (OA) is the most common cause of joint disease and associated disability. Despite this, its pathogenesis remains incompletely understood and no specific drug exists to prevent or reverse the structural changes in OA. Basic calcium phosphate (BCP) crystals are extremely common in OA. BCP crystals consist primarily of hydroxyapatite, with smaller amounts of octacalcium phosphate, tricalcium phosphate and magnesium whitlockite. They are present in 100% of joints at the time of knee and hip joint replacement surgery. Their presence strongly correlates with radiographic severity of osteoarthitis. In mice, intra-articular BCP crystals elicit synovial inflammation and cartilage degradation. The potential mechanisms by which calcium-containing crystals may promote articular damage have been studied in the laboratory setting and in vitro properties of BCP crystals have been observed that emphasise their pathogenic potential. BCP crystals interact with articular cells such as fibroblasts and chondrocytes to induce mitogenesis with resultant cellular proliferation likely leading to synovial lining hypertrophy. BCP crystals also upregulate production of cytokines such as tumour necrosis factor alpha (TNF-α), interleukin 1 (IL-1), increase prostaglandin E2 via the cyclooxygenase pathway, stimulate matrix metalloproteinases production and increase nitrous oxide production. Therefore, BCP crystals have potent biologic effects and represent a potential therapeutic target in OA

Objectives. This study aimed to investigate the functional effects of microRNA (miR)-214-5p on osteoblastic cells, which might provide a potential role of miR-214-5p in bone fracture healing. Methods. Blood samples were obtained from patients with hand fracture or intra-articular calcaneal fracture and from healthy controls (HCs). Expression of miR-214-5p was monitored by qRT-PCR at day 7, 14 and 21 post-surgery. Mouse osteoblastic MC3T3-E1 cells were transfected with antisense oligonucleotides (ASO)-miR-214-5p, collagen type IV alpha 1 (COL4A1) vector or their controls; thereafter, cell viability, apoptotic rate, and the expression of collagen type I alpha 1 (COL1A1), type II collagen (COL-II), and type X collagen (COL-X) were determined. Luciferase reporter assay, qRT-PCR, and Western blot were performed to ascertain whether COL4A1 was a target of miR-214-5p. Results. Plasma miR-214-5p was highly expressed in patients with bone fracture compared with HCs after fracture (p < 0.05 or p < 0.01). Inhibition of miR-214-5p increased the viability of MC3T3-E1 cells and the expressions of COL1A1 and COL-X, but decreased the apoptotic rate and COL-II expression (p < 0.05 or p < 0.01). COL4A1 was a target of miR-214-5p, and was negatively regulated by miR-214-5p (p < 0.05 or p < 0.01). Overexpression of COL4A1 showed a similar impact on cell viability, apoptotic rate, and COL1A1, COL-II, and COL-X expressions inhibiting miR-214-5p (p < 0.01). Conclusion. Inhibition of miR-214-5p promotes cell survival and extracellular matrix (ECM) formation of osteoblastic MC3T3-E1 cells by targeting COL4A1. Cite this article: Q. S. Li, F. Y. Meng, Y. H. Zhao, C. L. Jin, J. Tian, X. J. Yi. Inhibition of microRNA-214-5p promotes cell survival and extracellular matrix formation by targeting collagen type IV alpha 1 in osteoblastic MC3T3-E1 cells. Bone Joint Res 2017;6:464–471. DOI: 10.1302/2046-3758.68.BJR-2016-0208.R2

We used interconnected porous calcium hydroxyapatite ceramic to bridge a rabbit ulnar defect. Two weeks after inducing the defect we percutaneously injected rabbit bone marrow-derived mesenchymal stromal cells labelled with ferumoxide. The contribution of an external magnetic targeting system to attract these cells into the ceramic and their effect on subsequent bone formation were evaluated. This technique significantly facilitated the infiltration of ferumoxide-labelled cells into ceramic and significantly contributed to the enhancement of bone formation even in the chronic phase. As such, it is potentially of clinical use to treat fractures, bone defects, delayed union and nonunion

Summary Statement

Intra-articular injection of humanised monoclonal anti-VEGF antibody (Bevacizumab, Avastin®) in a osteoarthritis rabbit model is related to positive restorative effects in terms of histopathologic evaluation.

Aseptic inflammation is the main factor causing aseptic loosening of artificial joints. Studies have shown that inflammatory cells can activate STING (stimulator of interferon genes, STING) after being stressed. This study aims to explore the specific mechanism of STING in aseptic loosening of artificial joints, and provide new strategies for disease prevention. Titanium particles with a diameter of 1.2-10 μm were prepared to stimulate macrophages (RAW 264.7) to simulate the periprosthetic microenvironment. A lentiviral vector targeting the STING gene was designed and transfected into macrophages to construct a cell line targeting STING knockdown. The expression and secretion levels of TNF-α were detected by qPCR and ELISA, the activation levels of inflammatory pathways (NF-κB, IRF3, etc.) were detected by western blot, and the nucleus translocation of P65 and IRF3 was observed by cellular immunofluorescence. After titanium particles stimulated macrophages, qPCR and ELISA showed that the transcription and secretion levels of TNF-α were significantly increased. Western blot showed that titanium particle stimulation could increase the phosphorylation levels of NF-κB and IRF3 pathways. While knockdown of STING can significantly reduce titanium particle-induced TNF production, attenuate the activation levels of NF-κB and IRF3 pathways as well as the nucleus translocation of P65 and IRF3. Conclusions: STING positively regulates the level of inflammation in macrophages induced by titanium particles, and targeted inhibition of STING can reduce inflammation, which may delay the progression of aseptic loosening of artificial joints

Low back pain affects 80% of the population with half of cases attributed to intervertebral disc (IVD) degeneration. However, the majority of treatments focus on pain management, with none targeting the underlying pathophysiological causes. PCRX-201 presents a novel gene therapy approach that addresses this issue. PCRX-201 codes for interleukin-1 receptor antagonist (IL-1Ra), the natural inhibitor of the pro-inflammatory cytokine IL-1, which orchestrates the catabolic degeneration of the IVD. Our objective here is to determine the ability of PCRX-201 to infect human nucleus pulposus (NP) cells and tissue to increase the production of IL-1Ra and assess downstream effects on catabolic protein production. Degenerate human NP cells and tissue explants were infected with PCRX-201 at 0 or 3000 multiplicities of infection (MOI) and subsequently cultured for 5 days in monolayer (n=7), 21 days in alginate beads (n=6) and 14 days in tissue explants (n=5). Cell culture supernatant was collected throughout culture duration and downstream targets associated with pain and degeneration were assessed using ELISA. IL-1Ra production was increased in NP cells and tissue infected with PCRX-201. The production of downstream catabolic proteins such as IL-1β, IL-6, MMP3, ADAMTS4 and VEGF was decreased in both 3D-cultured NP cells and tissue explants. Here, we have demonstrated that a novel gene therapy, PCRX-201, is able to infect and increase the production of IL-1Ra in degenerate NP cells and tissue in vitro. The increase of IL-1Ra also resulted in a decrease in the production of a number of pro-inflammatory and catabolic proteins, suggesting PCRX-201 enables the inhibition of IL-1-driven IVD degeneration. At present, no treatments for IVD degeneration target the underlying pathology. The ability of FX201 to elicit anti-catabolic responses is promising and warrants further investigation in vitro and in vivo, to determine the efficacy of this exciting, novel gene therapy

Tendinopathy is a disease associated with pain and tendon degeneration, leading to a decreased range of motion and an increased risk of tendon rupture. The etiology of this frequent disease is still unknown. In other musculoskeletal tissues like cartilage and intervertebral discs, transient receptor potential channels (TRP- channels) were shown to play a major role in the progression of degeneration. Due to their responsiveness to a wide range of stimuli like temperature, pH, osmolarity and mechanical load, they are potentially relevant factors in tendon degeneration as well. We therefore hypothesize that TRP- channels are expressed in tendon cells and respond to degeneration inducing stimuli. By immunohistochemistry, qRT-PCR and western blot analyses, we found three TRP channel members, belonging to the vanilloid (TRPV), and ankyrin (TRPA) subfamily, respectively, to be expressed in healthy human tendon tissue as well as in rodent tendon, with expression being located to cells within the dense tendon proper, as well as to endotenon resident cells. In vitro-inflammatory and ex vivo-mechanical stimulation led to a significant upregulation of TRPA1 expression in tendon cells, which correlates well with the fact that TRPA1 is considered as mechanosensitive channel being sensitized by inflammatory mediators. This is the first description of TRP- channels in human and rodent tendon. As these channels are pharmacologically targetable by both agonists and antagonists, they may represent a promising target for novel treatments of tendinopathy

Osteoarthritis, the most common degenerative joint disease, significantly impairs life quality and labor capability of patients. Synovial inflammation, initiated by HMGB1 (High mobility group box 1)-induced activation of macrophage, precedes other pathological changes. As an upstream regulator of NF-κB (nuclear factor-kappa B) and MAPK (mitogen-activated protein kinase) signaling pathway, TAK1 (TGF-β activated kinase 1) participates in macrophage activation, while its function in osteoarthritis remains unveiled. This study aims to investigate the role of TAK1 in the pathogenesis of osteoarthritis via both in vitro and in vivo approaches. We performed immunohistochemical staining for TAK1 in synovial tissue, both in osteoarthritis patients and healthy control. Besides, immunofluorescence staining for F4/80 as macrophage marker and TAK1 were conducted as well. TAK1 expression was examined in RAW264.7 macrophages stimulated by HMGB1 via qPCR (Quantitative polymerase chain reaction) and Western blotting, and the effect of TAK1 inhibitor (5z-7 oxozeaenol) on TNF-α production was evaluated by immunofluorescence staining. Further, we explored the influence of intra-articular shRNA (short hairpin RNA) targeting TAK1 on collagenase-induced osteoarthritis in mice. Immunohistochemical staining confirmed significant elevation of TAK1 in osteoarthritic synovium, and immunofluorescence staining suggested macrophages as predominant residence of TAK1. In HMGB1-stimulated RAW264.7 macrophages, TAK1 expression was up-regulated both in mRNA and protein level. Besides, TAK1 inhibitor significantly impairs the production of TNF-α by macrophages upon HMGB1 stimulation. Moreover, intra-articular injection of lentivirus loaded with shRNA targeting TAK1 (sh-TAK1) reduced peri-articular osteophyte formation in collagenase-induced osteoarthritis in mice. TAK1 exerts a potent role in the pathogenesis of osteoarthritis by mediating the activation of macrophages

Osteoarthritis (OA) of the equine distal interphalangeal joint (DIPJ) is a common cause of lameness. MicroRNAs (miRNAs) from biofluids such as plasma and synovial fluid make promising biomarker and therapeutic candidates. The objectives of this study are (1) Identify differentially expressed (DE) miRNAs in mild and severe equine DIPJ OA synovial fluid samples and (2) Determine the effects of DE miRNAs on equine chondrocytes in monolayer culture. Synovial fluid samples from five horses with mild and twelve horses with severe DIPJ OA were submitted for RNA-sequencing; OA diagnosis was made from MRI T2 mapping, macroscopic and histological evaluation. Transfection of equine chondrocytes (n=3) was performed using the Lipofectamine® RNAiMAX system with a negative control and a miR-92a mimic and inhibitor. qPCR was used to quantify target mRNA genes. RNA-seq showed two miRNAs (miR-16 and miR-92a) were significantly DE (p<0.05). Ingenuity Pathway Analysis (IPA) identified important downstream targets of miR-92a involved in the pathogenesis of osteoarthritis and so this miRNA was used to transfect equine chondrocytes from three donor horses diagnosed with OA. Transfection was successfully demonstrated by a 1000-20000 fold increase in miR-92a expression in the equine chondrocytes. There was a significant (p<0.05) increase in COMP, COL3A1 and Sox9 in the miR-92a mimic treatment and there was no difference in ADAMTS-5 expression between the miR-92 mimic and inhibitor treatment. RNA-seq demonstrated miR-92a was downregulated in severe OA synovial fluid samples which has not previously been reported in horses, however miR-92a is known to play a role in the pathogenesis of OA in other species. Over expression of miR-92a in equine chondrocytes led to significantly increased COMP and Sox9 expression, consistent with a chondrogenic phenotype which has been identified in human and murine chondrocytes

Osteoporosis is a common problem in postmenopausal women and the elderly. 11β-Hydroxysteroid dehydrogenase type 1 (11β-HSD1) is a bi-directional enzyme that primarily activates glucocorticoids (GCs) in vivo, which is a considerable potential target as treatment for osteoporosis. Previous studies have demonstrated its effect on osteogenesis, and our study aimed to demonstrate its effect on osteoclast activation. In vivo, we used 11β-HSD1 knock-off (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, In vivo, We used 11β-HSD1 knockoff (KO) and C57BL6/J mice to undergo the ovariectomy-induced osteoporosis (OVX). In vitro, bone marrow-derived macrophages (BMM) and bone marrow mesenchymal stem cell (BMSC) of KO and C57BL6/J mice were extracted to test their osteogenic and osteoclastic abilities. We then created osteoclastic 11β-HSD1 elimination mice (Ctsk::11β-HSD1fl/fl) and treated them with OVX. Micro-CT analysis, H&E, immunofluorescence staining, and qPCR were performed. Finally, we conducted the high-throughput sequencing to find out 11β-HSD1 and osteoclast activation related genes. We collected 6w samples after modeling. We found that KO mice were resistant to loss of bone trabeculae. The same effect was observed in osteoclastic 11β-HSD1 elimination mice. Meanwhile, BVT-2733, a classic inhibitor of 11β-HSD1, inhibited the osteoclast effect of cells without affecting osteogenic effect in vitro. High-throughput sequencing suggested that glucocorticoid receptor (GR) may play a key role in the activation of osteoclasts, which was verified by immunofluorescence staining and WB in vivo and in vitro. In the process of osteoporosis, 11β-HSD1 expression of osteoclasts is abnormally increased, which may be a new target for inhibiting osteoclast activation and treating osteoporosis

As peri-prosthetic aseptic loosening is one of the main causes of implant failure, inhibiting wear particles induced macrophages inflammation is considered as a promising therapy for AL to expand the lifespan of implant. Here, we aim at exploring the role of p110δ, a member of class IA PI3K family, and Krüppel-like factor 4 (KLF4) in titanium particles (TiPs) induced macrophages-inflammation and osteolysis. Firstly, IC87114, the inhibitor of p110δ and siRNA targeting p110δ were applied and experiments including ELISA and immunofluorescence assay were conducted to explore the role of p110δ. Sequentially, KLF4 was predicted as the transcription factor of p110δ and the relation was confirmed by dual luciferase reporter assay. Next, assays including RT-PCR, western blotting and flow cytometry were performed to ensure the specific role of KLF4. Finally, TiPs-induced mice cranial osteolysis model was established, and micro-CT scanning and immunohistochemistry assay were performed to reveal the role of p110δ and KLF4 in vivo. Here, we found that p110δ was upregulated in TiPs-stimulated macrophages. The inhibition of p110δ or knockdown of p110δ could significantly dampen the TiPs-induced secretion of TNFα and IL-6. Further mechanistic studies confirmed that p110δ was responsible for TNFα and IL-6 trafficking out of Golgi complex without affecting their expression in TiPs-treated macrophages. Additionally, we explored the upstream regulators and confirmed that Krüppel-like factor 4 (KLF4) was the transcription repressor of p110δ. Apart from that, KLF4, targeted by miR-92a, could also attenuate TiPs-induced inflammation by mediating NF-κB pathway and M1/M2 polarization. By the establishment of TiPs-induced mice cranial osteolysis model, we found that KLF4 knockdown exacerbated TiPs-induced osteolysis which was strikingly ameliorated by knockdown of p110δ. In summary, our study suggests the key role of miR-92a/KLF4/p110δ signal in TiPs-induced macrophages inflammation and osteolysis

In osteoarthritis, chondrocytes acquire a hypertrophic phenotype that contributes to matrix degradation. Inflammation is proposed as trigger for the shift to a hypertrophic phenotype. Using in vitro culture of human chondrocytes and cartilage explants we could not find evidence for a role of inflammatory signalling activation. We found, however, that tissue repair macrophages may contribute to the onset of hypertrophy (doi: 10.1177/19476035211021907) Intra-articularly injected triamcinolone acetonide to inhibit inflammation in a murine model of collagenase-induced osteoarthritis, increased synovial macrophage numbers and osteophytosis, confirming the role of macrophages in chondrocyte hypertrophy occurring in osteophyte formation (doi: 10.1111/bph.15780). In search of targets to inhibit chondrocyte hypertrophy, we combined existing microarray data of different cartilage layers of murine growth plate and murine articular cartilage after induction of collagenase-induced osteoarthritis. We identified common differentially expressed genes and selected those known to be associated to inflammation. This revealed EPHA2, a tyrosine kinase receptor, as a new target. Using in silico, in vitro and in vivo models we demonstrated that inhibition of EPHA2 might be a promising treatment for osteoarthritis. Recently, single cell RNA-seq. has revealed detailed information about different populations of chondrocytes in articular cartilage during osteoarthritis. We re-analysed a published scRNA-seq data set of healthy and osteoarthritic cartilage to obtain the differentially expressed genes in the population of hypertrophic chondrocytes compared to the other chondrocytes, applied pathway analyses and then used drug databases to search for upstream inhibitors of these pathways. This drug repurposing approach led to the selection of 6 drugs that were screened and tested using several in vitro models with human chondrocytes and cartilage explants. In this lecture I will present this sequence of studies to highlight different approaches and models that can be used in the quest for a disease modifying drug for osteoarthritis

Lumbar diseases have become a major problem affecting human health worldwide. Conservative treatment of lumbar diseases is difficult to achieve ideal results, and surgical treatment of trauma, complications, it is imperative to develop a new treatment method. This study aims to explore the regulatory mechanism of cartilage endplate ossification caused by abnormal stress, and design intervention targets for this mechanism, so as to provide theoretical reference for the prevention and treatment of lumbar degeneration. In vivo, we constructed spinal instability model in mice. In vitro, we used a mechanical tensile machine to simulate the abnormal stress conditions of the endplate cartilage cells. Through the high-throughput sequencing, we found the enrichment of Hippo signaling pathway. As YAP is a key protein in the Hippo signaling pathway, we then created cartilaginous YAP elimination mice (Col2::YAPfl/fl). The lumbar spine model was constructed again in these mice for H&E, SOFG and immunofluorescence staining. In vitro lentivirus was used to knock out YAP, immunofluorescence staining, WB and qPCR were performed. Finally, we conducted therapeutic experiments by using YAP agonist and AAV5 carrying YAP plasmids. We collected 8w samples from C57/BL6 mice after modeling. We found ossification of the endplate in mice similar to human disc degeneration. High-throughput sequencing of stretched cells demonstrated high enrichment of the Hippo signaling pathway. By immunofluorescence staining, it was confirmed that Col-II decreased and Col-X gradually increased in the endplate cartilage of mice. This was also confirmed at 7 days after an in vitro stretch of 5% and 12%. Meanwhile, we found that cartilaginous YAP elimination mice developed very severe endplate degeneration. However, the endplate was well protected by intraperitoneal injection of YAP agonist or AAV5-YAP endplate injection, and the results in vitro were consistent with that. In the process of cartilaginous ossification, abnormal stress regulates Col10a1 to promote cartilage endplate ossification through Hippo signaling pathway mediated YAP, and we expect to find potential drug targets for treatment through this mechanism

The effects of dexamethasone (dex), during in vitro human osteogenesis, are contrasting. Indeed, dex downregulates SOX9 during osteogenic differentiation of human bone marrow mesenchymal stromal cells (HBMSCs). However, dex also promotes PPARG expression, resulting in the formation of adipocyte-like cells within the osteogenic monolayers. The regulation of both SOX9 and PPARG seems to be downstream the transactivation activity of the glucocorticoid receptor (GR), thus the effect of dex on SOX9 downregulation is indirect. This study aims at determining whether PPAR-γ regulates SOX9 expression levels, as suggested by several studies. HBMSCs were isolated from bone marrow of patients with written informed consent. HBMSCs were cultured in different osteogenic induction media containing 10 or 100 nM dex. Undifferentiated cells were used as controls. Cells were treated either with a pharmacological PPAR-γ inhibitor T0070907 (donors n=4) or with a PPARG-targeting siRNA (donors n=2). Differentiation markers or PPAR-γ target genes were analysed by RT-qPCR. Mineral deposition was assessed by ARS staining. Two-way ANOVA followed by a Tukey's multiple comparison test compared the effects of treatments. At day 7, T0070907 downregulated ADIPOQ and upregulated CXCL8, respectively targets of PPAR-γ-mediated transactivation and transrepression. RUNX2 and SOX9 were also significantly downregulated in absence of dex. PPARG was successfully downregulated by siRNA. ADIPOQ expression was also inhibited, while CXCL8 did not show any significant difference between siRNA treatment groups. RUNX2 was downregulated by the PPARG-siRNA treatment in presence of 100 nM dexamethasone, while SOX9 levels were not affected. ARS showed no change in the mineralization levels when PPARG expression or activity was inhibited. Understanding how dex regulates HBMSC differentiation is of pivotal importance to refine current in vitro models. These results suggest that PPARG does not mediate SOX9 downregulation. Unexpectedly, RUNX2 expression was also unaltered or even downregulated after PPAR-γ inhibition. Acknowledgements: AO Foundation, AO Research Institute (CH) and PRIN 2017 MUR (IT) for financial support

Favoring osseointegration and avoiding bacterial contamination are the key challenges in the design of implantable devices for orthopedic applications. To meet these goals, a promising route is to tune the biointerface of the devices, that can regulate interactions with the host cells and bacteria, by using nanostructured antibacterial and bioactive coatings. Indeed, the selection of adequate metal-based coatings permits to discourage infection while avoiding the development of bacterial resistance and nanostructuring permits to tune the release of the antimicrobial compounds, allowing high efficacy and decreasing possible cytotoxic effects. In addition, metal-doped calcium phosphates-based nanostructured coatings permit to tune both composition and morphology of the biointerfaces, allowing to regulate host cells and bacteria response. To tune the biointerfaces of implantable devices, nanostructured coatings can be used, but their use is challenging when the substrate is heat-sensitive and/or porous. Here, we propose the use of Ionized Jet Deposition (IJD) to deposit metallic and ion-doped calcium phosphates materials onto different polymeric substrates, without heating and damaging the substrate morphology. 3D printed scaffolds in polylactic acid (PLA) and polyurethane (PU), and electrospun matrices in polycaprolactone (PCL) and PLA were used as substrates. Biogenic apatite (HA), ion doped (zinc, copper and iron) tricalcium phosphate (TCP) and silver (Ag) coatings were obtained on porous and custom-made polymeric substrates. Chemical analyses confirmed that coatings composition matches that of the target materials, both in terms of main phase (HA or TCP) and ion doping (presence of Cu, Zn or Fe ion). Deposition parameters, and especially its duration time, influence the coating features (morphology and thickness) and substrate damage. Indeed, SEM/EDS observations show the presence of nanostructured agglomerates on substrates surface. The dimensions of the aggregates and the thickness of the coating films increase increasing the deposition time, without affecting the substrate morphology (no porosity alteration or fibers damaging). The possible substrate damage is influenced by target and substrate material, but it can be avoided modulating deposition time. Once the parameters are optimized, the models show suitable in vitro biological efficacy for applications in bone models, regenerative medicine and infection. Indeed, HA-based coatings favor cells adhesion on printed and electrospun fibers. For antibacterial applications, the ion doped TCP coatings can reduce the bacterial growth and adhesion (E.coli and S.aureus) on electrospun matrices. To conclude, it is possible achieve different properties applying nanostructured coatings with IJD technique on polymeric substrates, modulating deposition conditions to avoid substrate damage

Decreasing the chance of local relapse or infection after surgical excision of bone metastases is a main goals in orthopedic oncology. Indeed, bone metastases have high incidence rate (up to 75%) and important cross-relations with infection and bone regeneration. Even in patients with advanced cancer, bone gaps resulting from tumor excision must be filled with bone substitutes. Functionalization of these substitutes with antitumor and antibacterial compounds could constitute a promising approach to overcome infection and tumor at one same time. Here, for the first time, we propose the use of nanostructured zinc-bone apatite coatings having antitumor and antimicrobial efficacy. The coatings are obtained by Ionized Jet Deposition from composite targets of zinc and bovine-derived bone apatite. Antibacterial and antibiofilm efficacy of the coatings is demonstrated in vitro against S. Aureus and E. Coli. Anti-tumor efficacy is investigated against MDA- MB-231 cells and biocompatibility is assessed on L929 and MSCs. A microfluidic based approach is used to select the optimal concentration of zinc to be used to obtain antitumor efficacy and avoid cytotoxicity, exploiting a custom gradient generator microfluidic device, specifically designed for the experiments. Then, coatings capable of releasing the desired amount of active compounds are manufactured. Films morphology, composition and ion-release are studies by FEG- SEM/EDS, XRD and ICP. Efficacy and biocompatibility of the coatings are verified by investigating MDA, MSCs and L929 viability and morphology by Alamar Blue, Live/Dead Assay and FEG-SEM at different timepoints. Statistical analysis is performed by SPSS/PC + Statistics TM 25.0 software, one-way ANOVA and post-hoc Sheffe? test. Data are reported as Mean ± standard Deviation at a significance level of p <0.05. Results and Discussion. Coatings have a nanostructured surface morphology and a composition mimicking the target. They permit sustained zinc release for over 14 days in medium. Thanks to these characteristics, they show high antibacterial ability (inhibition of bacteria viability and adhesion to substrate) against both the gram + and gram – strain. The gradient generator microfluidic device permits a fine selection of the concentration of zinc to be used, with many potential perspectives for the design of biomaterials. For the first time, we show that zinc and zinc-based coatings have a selective efficacy against MDA cells. Upon mixing with bone apatite, the efficacy is maintained and cytotoxicity is avoided. For the first time, new antibacterial metal-based films are proposed for addressing bone metastases and infection at one same time. At the same time, a new approach is proposed for the design of the coatings, based on a microfluidic approach. We demonstrated the efficacy of Zn against the MDA-MB-231 cells, characterized for their ability to form bone metastases in vivo, and the possibility to use nanostructured metallic coatings against bone tumors. At the same time, we show that the gradient-generator approach is promising for the design of antitumor biomaterials. Efficacy of Zn films must be verified in vivo, but the dual-efficacy coatings appear promising for orthopedic applications

Spinal surgery deals with the treatment of different pathological conditions of the spine such as tumors, deformities, degenerative disease, infections and traumas. Research in the field of vertebral surgery can be divided into two main areas: 1) research lines transversal to the different branches; 2) specific research lines for the different branches. The transversal lines of research are represented by strategies for the reduction of complications, by the development of minimally invasive surgical techniques, by the development of surgical navigation systems and by the development of increasingly reliable systems for the control of intra-operative monitoring. Instead, specific lines of research are developed within the different branches. In the field of oncological pathology, the current research concerns the development of in vitro models for the study of metastases and research for the study of targeted treatment methods such as electrochemotherapy and mesenchymal stem cells for the treatment of aneurysmal bone cysts. Research in the field of spinal deformities is focused on the development of increasingly minimally invasive methods and systems which, combined with appropriate pharmacological treatments, help reduce trauma, stress and post-operative pain. Scaffolds based on blood clots are also being developed to promote vertebral fusion, a fundamental requirement for improving the outcome of vertebral arthrodesis performed for the treatment of degenerative disc disease. To improve the management and the medical and surgical treatment of vertebral infections, research has focused on the definition of multidisciplinary strategies aimed at identifying the best possible treatment path. Thus, flow-charts have been created which allow to manage the patient suffering from vertebral infection. In addition, dedicated silver-coated surgical instrumentation and bone substitutes have been developed that simultaneously guarantee mechanical stability and reduce the risk of further local infection. In the field of vertebral traumatology, the most recent research studies have focused on the development of methods for the biostimulation of the bone growth in order to obtain, when possible, healing without surgery. Methods have also been developed that allow the minimally invasive percutaneous treatment of fractures by means of vertebral augmentation with PMMA, or more recently with the use of silicone which from a biomechanical point of view has an elastic modulus more similar to that of bone. It is clear that scientific research has changed clinical practice both in terms of medical and surgical management of patients with spinal pathologies. The results obtained stimulate the basic research to achieve even more. For this reason, new lines of research have been undertaken which, in the oncology field, aim at developing increasingly specific therapies against target receptors. Research efforts are also being multiplied to achieve regeneration of the degenerated intervertebral disc and to develop implants with characteristics increasingly similar to those of bone in order to improve mechanical stability and durability over time. Photodynamic therapies are being developed for the treatment of infections in order to reduce the use of antibiotic therapies. Finally, innovative lines of research are being launched to treat and regenerate damaged nerve structures with the goal, still far from today, of making patients with spinal cord injuries to walk

Infection in orthopedics is a challenge, since it has high incidence (rates can be up to 15-20%, also depending on the surgical procedure and on comorbidities), interferes with osseointegration and brings severe complications to the patients and high societal burden. In particular, infection rates are high in oncologic surgery, when biomedical devices are used to fill bone gaps created to remove tumors. To increase osseointegration, calcium phosphates coatings are used. To prevent infection, metal- and mainly silver-based coatings are the most diffused option. However, traditional techniques present some drawbacks, including scarce adhesion to the substrate, detachments, and/or poor control over metal ions release, all leading to cytotoxicity and/or interfering with osteointegration. Since important cross-relations exist among infection, osseointegration and tumors, solutions capable of addressing all would be a breakthrough innovation in the field and could improve clinical practice. Here, for the first time, we propose the use antimicrobial silver-based nanostructured thin films to simultaneously discourage infection and bone metastases. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture. These characteristics, in turn, allow tuning silver release and avoid delamination, thus preventing toxicity. In addition, to mitigate interference with osseointegration, here silver composites with bone apatite are explored. Indeed, capability of bone apatite coatings to promote osseointegration had been previously demonstrated in vitro and in vivo. Here, antibacterial efficacy and biocompatibility of silver-based films are tested in vitro and in vivo. Finally, for the first time, a proof-of-concept of antitumor efficacy of the silver-based films is shown in vitro. Coatings are obtained by silver and silver-bone apatite composite targets. Both standard and custom-made (porous) vertebral titanium alloy prostheses are used as substrates. Films composition and morphology depending on the deposition parameters are investigated and optimized. Antibacterial efficacy of silver films is tested in vitro against gram+ and gram- species (E. coli, P. aeruginosa, S. aureus, E. faecalis), to determine the optimal coatings characteristics, by assessing reduction of bacterial viability, adhesion to substrate and biofilm formation. Biocompatibility is tested in vitro on fibroblasts and MSCs and, in vivo on rat models. Efficacy is also tested in an in vivo rabbit model, using a multidrug resistant strain of S. aureus (MRSA, S. aureus USA 300). Absence of nanotoxicity is assessed in vivo by measuring possible presence of Ag in the blood or in target organs (ICP-MS). Then, possible antitumor effect of the films is preliminary assessed in vitro using MDA-MB-231 cells, live/dead assay and scanning electron microscopy (FEG-SEM). Statistical analysis is performed and data are reported as Mean ± standard Deviation at a significance level of p <0.05. Silver and silver-bone apatite films show high efficacy in vitro against all the tested strains (complete inhibition of planktonic growth, reduction of biofilm formation > 50%), without causing cytotoxicity. Biocompatibility is also confirmed in vivo. In vivo, Ag and Ag-bone apatite films can inhibit the MRSA strain (>99% and >86% reduction against ctr, respectively). Residual antibacterial activity is retained after explant (at 1 month). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses

Introduction and Objective. Osteoarthristis (OA) has been associated with many genes and yet the genetic basis for this disease has never formally been established. Recent realization that epigenetic changes could be the underlying pathological mechanisms has helped to explain many complex multifactorial diseases with no clear genetic cause. We therefore asked whether epigenetics could also play a role in OA. We have previously shown that the DNA epigenetic modification, specifically the hydroxymethylation on cytosine (5hmC), undergoes a fivefold increase on OA-associated genes which are activated at OA onset. In this study, we further uncovered a set of 5hmC-mediated gene targets and their mechanistic link to OA progression. Materials and Methods. We surgically induced OA on 4 to 6 months old Tet1−/− mice (Tet1tm1.1Jae, the Jackson laboratory) and wild-type littermates by performing destabilization of the medial meniscus (DMM) surgery. Joints were collected for histological assessment through blinded grading with the OARSI scoring system. Human articular chondrocytes were harvested from OA cartilage samples obtained during total knee arthroplasty or from grossly normal cartilage pieces obtained during notchplasty or debridement from patients undergoing anterior cruciate ligament (ACL) reconstruction with no history of OA symptoms, under approved Human subjects Institutional Review Board protocols. Bioinformatic analyses of RNA-sequencing and CCGG sequencing (reduced representation 5hmC profiling) were performed to identify TET1 target genes associated with OA progression. Several measurements were used to assess the effect of TET1 ablation on the phenotype of mouse cartilage tissue and human chondrocytes including, histological evaluation, and quantitative bone assessment by micro-CT imaging and multiplex cytokine analyses in the serum of mice in vivo (mouse 39-plex assay) and in the supernatant of human chondrocyte cultures (human 62-plex assay). Results. We used a mouse model with surgically induced OA and found that OA onset was accompanied by a gain of ∼40,000 differentially hydroxymethylated sites prior the notable histological onset of the disease. We additionally revealed that these changes are mediated by the ten-eleven-translocation enzyme 1 (TET1), since Tet1−/− mice lost 98% of 5hmC sites upon OA induction. Remarkably, Tet1−/− mice were protected from OA development including degeneration of the cartilage surface and osteophyte formation. Silencing of TET1 expression in human OA chondrocytes reduced the expression in a set of genes, which may represent the pathological gene targets that exacerbate OA including MMP3 and MMP13 and several inflammatory cytokines. Therefore, our study reveals the unexpected beneficial role of TET1 inhibition in blocking OA progression. In fact, intra-articular injections of a dioxygenases’ inhibitor, 2 hydroxyglutarate, on mice after surgical induction of OA stalled disease progression. Furthermore, treatment of human OA chondrocytes with the same inhibitor also phenocopied TET1 loss, implicating a therapeutic potential of TET inhibition in OA patients. Conclusions. Collectively, our study not only demonstrate the role of TET1 in OA; the 5hmC-mediated gene targets acting on multiple OA pathways were identified and can be modulated as therapeutic intervention to treat OA