Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses.

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.

Calcium phosphates-based (CaPs) nanocoatings on metallic prosthesis are widely studied in orthopedics and dentistry because they mimic the mineral component of native human bone and favor the osseointegration process. Despite the fact that different calcium phosphates have different properties (composition, crystallinity, and ion release), only stoichiometric hydroxyapatite (HA) films have been analyzed in deep. Here, we have realized films of different CaPs (HA, beta-tricalcium phosphate (β-TCP) and brushite (DCPD)) onto Ti6Al4V microrough substrates by Ionized Jet Deposition (IJD). We have implemented the heating of substrates at 400°C during deposition to see the effect on coating properties.

Different film features are evaluated: morphology and topography (FEG-SEM, AFM), physical-chemical composition (FT-IR and EDS), dissolution profile and adhesion to substrate (scratch test), with a focus on how the different CaPs and temperature changed the coating features. After coating optimization, we have studied the in vitro BM-MSC behavior, in term of viability and early adhesion.

We have obtained good transfer of fidelity in composition from target to coating for all CaPs, with nanostructured films formed by globular aggregates (~300 nm diameter), with homogeneous and uniform coverage of the substrate surface, without cracks. The heating during deposition has increased the adhesion of the films to the substrate, with higher stability in medium immersion and wettability, features that can improve the biological behavior of cells. All CaP coatings have showed excellent biocompatibility, with DCPD coating that promote higher cells viability at 14 days respect to HA and β- TCP films. About the early cell adhesion, the BM-MSC have showed switch from a globular to an elongated morphology at 6 hours in all coatings respect to the uncoated titanium, sign of better adhesion.

From these results, the fabrication of different CaP nanocoatings with IJD can be a promising for applications in orthopedics and dentistry.

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.

Prosthetic joint infections represent complications connected to the implantation of biomedical devices, they have high incidence, interfere with osseointegration, and lead to a high societal burden. The microbial biofilm, which is a complex structure of microbial cells firmly attached to a surface, is one of the main issues causing infections. Biofilm- forming bacteria are acquiring more and more resistances to common clinical treatments due to the abuse of antibiotics administration. Therefore, there is increasing need to develop alternative methods exerting antibacterial activities against multidrug-resistant biofilm-forming bacteria. In this context, metal-based coatings with antimicrobial activities have been investigated and are currently used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing their efficacy. Here, we propose the use of antimicrobial silver-based nanostructured thin films to discourage bacterial infections. Coatings are obtained by Ionized Jet Deposition, a plasma-assisted technique that permits to manufacture films of submicrometric thickness having a nanostructured surface texture, allow tuning silver release, and avoid delamination. To mitigate interference with osseointegration, here silver composites with bone apatite and hydroxyapatite were explored. The antibacterial efficacy of silver films was tested in vitro against gram- positive and gram-negative species to determine the optimal coatings characteristics by assessing reduction of bacterial viability, adhesion to substrate, and biofilm formation. Efficacy was tested in an in vivo rabbit model, using a multidrug-resistant strain of Staphylococcus aureus showing significant reduction of the bacterial load on the silver prosthesis both when coated with the metal only (>99% reduction) and when in combination with bone apatite (>86% reduction). These studies indicate that IJD films are highly tunable and can be a promising route to overcome the main challenges in orthopedic prostheses.

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.

Prosthetic joint infections represent complications connected to the implantation of biomedical devices. Bacterial biofilm is one of the main issues causing infections from contaminated orthopaedic prostheses. Biofilm is a structured community of microbial cells that are firmly attached to a surface and have unique metabolic and physiological attributes that induce improved resistance to environmental stresses including toxic compounds like antimicrobial molecules (e.g. antibiotics). Therefore, there is increasing need to develop methods/treatments exerting antibacterial activities not only against planktonic (suspended) cells but also against adherent cells of pathogenic microorganisms forming biofilms. In this context, metal-based coatings with antibacterial activities have been widely investigated and used in the clinical practice. However, traditional coatings exhibit some drawbacks related to the insufficient adhesion to the substrate, scarce uniformity and scarce control over the toxic metal release reducing the biofilm formation prevention efficacy. Additionally, standardized and systematic approaches to test antibacterial activity of newly developed coatings are still missing, while standard microbiological tests (e.g. soft-agar assays) are typically used that are limited in terms of simultaneous conditions that can be tested, potentially leading to scarce reproducibility and reliability of the results.

In this work, we combined the Calgary Biofilm Device (CBD) as a device for high-throughput screening, together with a novel plasma-assisted technique named Ionized Jet Deposition (IJD), to generate and test new generation of nanostructured silver- and zinc-based films as coatings for biomedical devices with antibacterial and antibiofilm properties. During the experiments we tested both planktonic and biofilm growth of four bacterial strains, two gram-positive and two gram-negative bacterial strains, i.e. Staphylococcus aureus ATCC 6538P, Enterococcus faecalis DP1122 and Escherichia coli ATCC 8739 and Pseudomonas aeruginosa PAO1, respectively. The use of CBD that had the only wells covered with the metal coatings while the biofilm supports (pegs) were not sheltered allowed to selectively define the toxic effect of the metal release (from the coating) against biofilm development in addition to the toxic activity exerted by contact killing mechanism (on biofilms formed on the coating). The results indicated that the antibacterial and antibiofilm effects of the metal coatings was at least partly gram staining dependent. Indeed, Gram negative bacterial strains showed high sensitivity toward silver in both planktonic growth and biofilm formation, whereas zinc coatings provided a significant inhibitory activity against Gram positive bacterial strains. Furthermore, the coatings showed the maximal activity against biofilms directly forming on them, although, Zn coating showed a strong effect against biofilms of gram-positive bacteria also formed on uncoated pegs.

We conclude that the metal-based coatings newly developed and screened in this work are efficient against bacterial growth and adherence opening possible future applications for orthopedic protheses manufacturing.

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.

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.

Fracture nonunion is a severe clinical problem for the patient, as well as for the clinician. About 5-20% of fractures does not heal properly after more than six months, with a 19% nonunion rate for tibia, 12% for femur and 13% for humerus, leading to patient morbidity, prolonged hospitalization, and high costs.

The standard treatment with iliac crest-derived autologous bone filling the nonunion site may cause pain or hematoma to the patient, as well as major complications such as infection.

The application of mesenchymal autologous cells (MSC) to improve bone formation calls for randomized, open, two-arm clinical studies to verify safety and efficacy.

The ORTHOUNION * project (ORTHOpedic randomized clinical trial with expanded bone marrow MSC and bioceramics versus autograft in long bone nonUNIONs) is a multicentric, open, randomized, comparative phase II clinical trial, approved in the framework of the H2020 funding programme, under the coordination of Enrique Gòmez Barrena of the Hospital La Paz (Madrid, Spain).

Starting from January 2017, patients with nonunion of femur, tibia or humerus have been actively enrolled in Spain, France, Germany, and Italy.

The study protocol encompasses two experimental arms, i.e., autologous bone marrow-derived mesenchymal cells after expansion (‘high dose’ or ‘low dose’ MSC) combined to ceramic granules (MBCP™, Biomatlante), and iliac crest-derived autologous trabecular bone (ICAG) as active comparator arm, with a 2-year follow-up after surgery.

Despite the COVID 19 pandemic with several lockdown periods in the four countries, the trial was continued, leading to 42 patients treated out of 51 included, with 11 receiving the bone graft (G1 arm), 15 the ‘high dose’ MSC (200x10⁶, G2a arm) and 16 the ‘low dose’ MSC (100x10⁶, G2b arm).

The Rizzoli Orthopaedic Institute has functioned as coordinator of the Italian clinical centres (Bologna, Milano, Brescia) and the Biomedical Science and Technologies and Nanobiotechnology Lab of the RIT Dept. has enrolled six patients with the collaboration of the Rizzoli’ 3rd Orthopaedic and Traumatological Clinic prevalently Oncologic.

Moreover, the IOR Lab has collected and analysed the blood samples from all the patients treated to monitor the changes of the bone turnover markers following the surgical treatment with G1, G2a or G2b protocols.

The clinical and biochemical results of the study, still under evaluation, are presented.

* ORTHOUNION Horizon 2020 GA 733288

Osteoporosis is a worldwide disease resulting in the increase of bone fragility and enhanced fracture risk in adults. In the context of osteoporotic fractures, bone tissue engineering (BTE), i.e., the use of bone substitutes combining biomaterials, cells, and bone inducers, is a potential alternative to conventional treatments. Pre-clinical testing of innovative scaffolds relies on in vitro systems where the simultaneous presence of osteoblasts (OBs) and osteoclasts (OCs) is required to mimic their crosstalk and molecular cooperation for bone remodelling. To this aim, two composite materials based on type I collagen were developed, containing either strontium-enriched mesoporous bioactive glasses or rod-like hydroxyapatite nanoparticles. Following chemical crosslinking with genipin, the nanostructured materials were tested for 2–3 weeks with an indirect co-culture of human trabecular bone-derived OBs and buffy coat-derived OC precursors. The favourable structural and biological properties of the materials proved to successfully support the viability, adhesion, and differentiation of bone cells, encouraging a further investigation of the two bioactive systems as biomaterial inks for the 3D printing of more complex scaffolds for BTE.

In the past decades, a huge amount of effort has been devoted to translate evidence based on standard preclinical models of bone tumours to effective tools for clinical applications. Although cancer is a genetic disease, hence the emphasis on -omics approaches, the complexity of cancer tissue, a mix of competing clones of transformed elements that react differently to microenvironmental stimuli, may hardly be reproduced by standard approaches. Cost, biological differences and ethical concerns are increasingly recognized as weaknessess of animal models. To overcome these limitations and provide reliable, reproducible, and affordable tools for predicting the effectiveness of treatments, environmental-controlled 3D cultures and co-cultures (spheroids, organoids) coupled with microfluidics and advanced imaging have recently being considered as effective instrument to increase knowledge on the pathophysiology of bone tumours and define effective therapeutic solutions.

Introduction and Objective

The osteocyte, recognized as a major orchestrator of osteoblast and osteoclast activity, is the most important key player during bone remodeling processes. Imbalances that occur during bone remodeling, caused by hormone perturbations or alterations in mechanical loading, can induce bone disease as osteoporosis. Due to limited understanding of the underlying mechanisms, current therapies for osteoporosis cannot adequately address this imbalance because current studies of osteocytes rely on conventional cell culture that cannot recapitulate local in vivo microenvironments for the lack of control of the spatial/temporal distribution of cells and biomolecules. Microfluidics is the science and technology of microscale fluid manipulating and sensing and can help fill this gap.

Introduction and Objective

The choice of appropriate characteristics is crucial to favor a firm bonding between orthopedic implants and the host bone and to permit bone regeneration. In particular, the morphology and composition of the biointerface plays a crucial role in orchestrating precise cellular responses. Here, to modulate the biointerface, we propose new biomimetic coatings, having multi-scale nano- to micro- morphological cues and a composition mimicking the mineral phase of bone.

Infections are among the main complications connected to implantation of biomedical devices, having high incidence rate and severe outcome. Since their treatment is challenging, prevention must be preferred. For this reason, solutions capable of exerting suitable efficacy while not causing toxicity and/or development of resistant bacterial strains are needed. To address infection, inorganic antibacterial coatings, and in particular silver coatings, have been extensively studied and used in the clinical practice, but some drawbacks have been evidenced, such as scarce adhesion to the substrate, delamination, or scarce control over silver release.

Here, antibacterial nanostructured silver-based thin films are proposed, obtained by a novel plasma-assisted technique, Ionized Jet Deposition (IJD). Coatings are obtained by deposition of metallic silver targets. Films thickness is selected based on previous results aimed at measuring extent and duration of silver release and at evaluating toxicity to host cells (fibroblasts). Here, composition (grazing incidence XRD) and morphology (SEM) of the obtained coatings are characterized for deposition onto different substrates, both metallic and polymeric. For heat sensitive substrates, possible alterations caused by coatings deposition in terms of morphology (SEM) and composition (FT-IR) is assessed. Then, a proof-of-concept study of the capability of these films to inhibit microbial biofilm formation is performed by using two different supports i.e., the Calgary Biofilm Device and the microplates. To the best of the Authors knowledge, this is the first study describing the application of specific anti-biofilm analyses to nanostructured coatings. In particular, anti-biofilm activities are tested against the following pathogenic strains: Escherichia (E.) coli NCTC12923, Staphylococcus (S.) aureus ATCC29213 and S. aureus 86. Among these, the strain 86 is not only pathogen but it also possesses several antibiotic resistance genes, allowing the evaluation of the utilization of nanostructured coatings as an alternative anti-microbial system to face the global threat of antibiotic resistance.

Results indicate that films deposited from silver targets are composed of nanosized aggregates of metallic silver, indicating a perfect transfer of composition from the deposition target to the coatings.

Results obtained here indicate that the films have significant antibacterial and antibiofilm activity. In addition, they prove that the system can be successfully applied for evaluation of coatings antibacterial efficacy for biomedical applications.

Calcium phosphates-based coatings have been widely studied to favour a firm bonding between orthopaedic implants and the host bone. To this aim, thin films (thickness below 1 μm) having high adhesion to the substrate and a nanostructured surface texture are desired, capable of boosting platelet, proteins and cells adhesion. In addition, a tunable composition is required to resemble as closely as possible the composition of mineralized tissues and/or to intentionally substitute ions having possible therapeutic functions. The authors demonstrated nanostructured films having high surface roughness and a composition perfectly resembling the deposition target one can be achieved by Ionized Jet Deposition (IJD). Highly adhesive nanostructured coatings were obtained by depositing bone-apatite like thin films by ablation of deproteinized bovine bone, capable of promoting host cells attachment, proliferation and differentiation. Here, biomimetic films are deposited by IJD, using biogenic and synthetic apatite targets. Since IJD deposition can be carried out without heating the substrate, application on heat sensitive polymeric substrate, i.e. 3D printed porous scaffolds, is investigated.

Biogenic apatite coatings are obtained by deposition of deproteinized bone (bovine, ovine, equine, porcine) and compared to ones of stoichiometry hydroxyapatite (HAp). Coatings composition (FT-IR-ATR, FT-IR microscopy, XRD, EDS) and morphology (SEM, AFM) are tested for deposition onto metallic and 3D-printed polymeric substrates (polyurethane (PU)). Different post-treatment annealing procedures for metallic substrates are compared (350–425°C), to optimize crystallinity. Then, uniformity of substrate coverage and possible damage caused to the polymeric substrate are studied by SEM, DSC and FT-IR microscopy.

Biogenic coatings are composed by carbonated HAp (XRD, FT-IR). Trace ions Na⁺ and Mg²⁺ are transferred from deposition target to coating. All coatings are nanostructured, composed by nano-sized globular aggregates, of which morphology and dimensions depend on the target characteristics. As-deposited coatings are amorphous, but crystallinity can be tuned by post-treatment annealing. A bone-like crystallinity can be achieved for heating at ≥400°C, also depending on duration. When deposited on 3D-printed PU scaffolds, coatings, owing to sub-micrometric thickness, coat them entirely, without altering their fibre shape and porosity.

Obtained biomimetic bone apatite coatings can be deposited onto a variety of metallic and polymeric biomedical devices, thus finding several perspective applications in biomedical field.

The initiation and progression of malignant tumors are supported by their microenvironment: cancer cells per se cannot explain growth and formation of the primary or metastasis, and a combination of proliferating tumor cells, cancer stem cells, immune cells, mesenchymal stromal cells and/or cancer-associated fibroblasts all contribute to the tumor bulk. The interaction between these multiple players, under different microenvironmental conditions of biochemical and physical stimuli (i.e. oxygen tension, pH, matrix mechanics), regulates the production and biological activity of several soluble factors, extracellular matrix components, and extracellular vesicles that are needed for growth, maintenance, chemoresistance and metastatization of cancer. Both in osteosarcoma and bone metastases from carcinomas this aspect has been only recently explored. In this lecture, I will discuss the role of tumor microenvironment, with a particular focus on the mesenchymal stroma, contributing to bone tumor progression through inherent. The most recent advances in the molecular cues triggered by cytokines, soluble factors, and metabolites that are partially beginning to unravel the axis between stromal elements of mesenchymal origin and bone cancer cells, under different microenvironmental conditions, will be reviewed providing insights likely to be used for novel therapeutic approaches.

Background

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. Within the REBORNE project, a multi-center orthopaedic clinical trial was focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

Background

Definitive proof is lacking on mesenchymal stem cell (MSCs) cellular therapy to regenerate bone if biological potential is insufficient. High number of MSCs after GMP expansion may solve the progenitor insufficiency at the injury but clinical trials are pending.

Cancer associated bone pain (CIBP) is a common event in patients with advanced disease with bone metastases (BM), significantly impairing their quality of life. Treatment options are limited and mainly based on the use of opioids with unacceptable side effects. Local acidosis is a well-known cause of pain since it directly stimulates nociceptors that express acid-sensing ion channels and densely innervate bone. In BM, local acidosis derives from osteoclast bone resorption activity and from the acidification by glycolytic tumor cells. Here we speculated that the pH lowering of intratumoral interstitial fluid also promotes nociceptors sensitization and hyperalgesia through the activation of cells of mesenchymal origin in BM microenvironment that might release inflammatory and nociceptive mediators.

As a model of breast cancer that can metastatise to the bone we used MDA-MB-231 (MDA), and a subclone with a higher tendency to form osteolytic BM (bmMDA). We evaluated the basal expression of proton pumps/ion transporters by Real-Time PCR (Q-RT-PCR). To evaluate the effect of extracellular acidosis on mesenchymal tumor-associated stroma, we used human osteoblast primary cultures from healthy donors and cancer-associated fibroblasts isolated with specific immunobeads from the tumor biopsies of patient with BM. We exposed the cells to pH 6.8 medium at different time points (between 3 to 24 hours). After the short-term incubation with acidosis, for the expression of and acid-sensing ion channels, inflammatory cytokines and nociceptive mediators that can produce hyperalgesia, we used both a wide screening through a deep-sequencing approach and Q-RT-PCR, and ELISA. Xenograft for osteolytic BM induced by intratibial injection of bmMDA were treated with Omeprazole and monitored for CIBP through several cognitive tests.

We found a significantly higher extracellular proton efflux and expression of proton pumps/ion transporters associated with the acid-base balance, the monocarboxylate transporter 4 (MCT4), the carbonic anhydrase (CA9), and the vacuolar ATPase (V-ATPase) V₁G₁ subunit, and V₀c subunitin bmMDA, a subclone that is prone to form BM in respect to the parental cell line MDA-MB-231. In mesenchymal stromal cells, osteoblasts and cancer-associated fibroblasts, the incubation with pH 6.8 induced the expression of the achid-sensing ion channels AISC3/ACCN3 and AICS4/ACCN4, as well as of the nociceptive modulators nerve growth factor (NGF), Brain-derived neurotrophic factor (BDNF), and of the inflammatory cytokines interleukin 6 (IL6) and 8 (IL8), and Chemokine (C-C motif) ligand 5 (CCL5). Furthermore, the targeting of V0c subunit to inhibit intratumoral acidification significantly reduced CIBP in mice model of BM.

In this study we demonstrated for the first time that, in addition to the direct acid-sensing neuronal stimulation, the acidic microenvironment of BM causes hyperalgesia through the activation of an inflammatory reaction in the tumor-associated mesenchymal stroma at the tumor site, thereby offering as a new target for palliative treatment in advanced cancer.

Maintenance of acid-base homeostasis in extracellular fluids and in the cytoplasm is essential for the physiological activities of cells and tissues [1]. However, changes in extracellular pH (pHe) occurs in a variety of physiological and pathological conditions, including hypoxia and inflammation associated with trauma and cancer. Concerning bone tissue, if abnormal acidification occurs, mineral deposition and osteoblast differentiation are inhibited, whereas osteoclast formation and activity are enhanced [2]. Indeed, acidification, that usually occurs in the early phases of fracture repair, has been suggested as a driving force for regeneration via release of growth factors that act on the stem cell fraction of repair bone [3]. However, the effect of low pHe on stemness has been insufficiently explored so far. Thus, in this study, we investigated the role of short term exposure to low pHe (6.5–6.8) on MSC stemness.

MSC derived from dental pulps (DPSC) and bone marrow (BM-MSC) were used. To perform the specific assays, culture medium at specific pH (6.5, 6.8, 7.1 and 7.4) was maintained by using different concentrations of sodium bicarbonate according to the Henderson-Hasselbach equation.

Changes in osteoblast-related gene expression (COL1A1 and ALPL), and mineral nodule formation were measured by qRT-PCR and Alizarin red staining, respectively.

The stem phenotype was analysed by measuring changes in stemness-related genes (SOX2, OCT4, KLF4, c-MYC) expression and spheres forming ability. Additionally, cell number, Ki67 index and cell cycle were analysed to monitor cell proliferation and quiescence.

We confirmed that acidic pHe inhibits the osteogenic differentiation of DPSC. Low pHe significantly but transitorily decreased the expression of osteoblast-related genes (COL1A1 and ALPL) and decreased the mineral nodule formation in vitro.

Acidic pHe conditions significantly increased the ability of DPSC and BM-MSC to form floating spheres. At acidic pHe spheres were higher but smaller when compared to spheres formed at alkaline pHe conditions. Moreover, acidic pHe increased significantly the expression of stemness-related genes. Finally, low pHe induced a significant decrease of DPSC cell number. Reduction of cell proliferation correlated with a lower number of cycling cells, as revealed by the Ki67 index that significantly decreased in a pH-dependent manner. Cell cycle analysis revealed an accumulation of cells in the G0 phase, when cultured at low pH.

In this study, we demonstrated a close relationship between acidic pHe and the regulation of MSC stemness. We therefore suggest that pHe modulation of MSC stemness is a major determinant of skeletal homeostasis and regeneration, and this finding should be considered in bone healing strategies based on cell therapy.

Osteosarcoma (OS) is an aggressive bone malignancy with a high relapse rate despite combined treatment with surgery and multiagent chemotherapy. As for other cancers, OS-associated microenvironment may contribute to tumor initiation, growth, and metastasis. We consider mesenchymal stromal cells (MSC) as a relevant cellular component of OS microenvironment, and have previously found that the interaction between MSC and tumor cells is bidirectional: tumor cells can modulate their peripheral environment that in turn becomes more favourable to tumor growth through metabolic reprogramming (1).

Stem-like cells were derived from HOS osteosarcoma cell line by using the spherogenic system (2). CSC isolated from HOS (HOS-CSC) were co-coltured with MSC isolated from bone marrow. Cell lysates and supernatants were collected for the analysis of RNA expression and of secreted cytokines, by Q-RT-PCR and specific ELISA assays, respectively.

Here, we determined the effects of MSC on OS stemness and migration, two major features associated with recurrence and chemoresistance. Recruitment of MSC to the tumor environment leads to enhanced proliferation of OS stem cells, which increase the expression levels of TGFβ1. The latter, in turn, could be responsible for the activation of NF-kB genes and IL-6 secretion by MSC. Pro-tumorigenic effects of MSC, via IL-6, including induction of HOS-CSC migration and sphere growth, can be counteracted by IL-6 neutralizing antibody. The presence of MSC is also responsible for increased expression of adhesion molecules involved in intra- or extra-vasation.

Stromal cells in combination with OS spheres exploit a vicious cycle where the presence of CSC stimulates mesenchymal cytokine secretion, which in turn increases stemness, proliferation, migration, and metastatic potential of CSC. Furthermore, for the first time we identified a novel OS stem cell marker, the Met proto-oncogene, that is frequently overexpressed and is pathogenetically relevant in OS (2 and 3). Altogether, our data corroborates the concept that a comprehensive knowledge of the interplay between tumor and stroma that also includes the stem-like fraction of tumor cells is needed to develop novel and effective anti-cancer therapies.

A complete design-manufacturing process for delivering customized foot orthoses by means of digital technologies is presented. Moreover, this feasibility study aims to combine a semi-automatic modelling approach with the use of low-cost devices for 3D scanning and 3D printing.

In clinical practice, traditional methods for manufacturing customized foot orthoses are completely manual, mainly based on plaster casting plus hand fabrication, and are widely used among practitioners. Therefore, results depend on skills and expertise of individual orthoptists and podiatrists that need considerable training and practice in order to obtain optimal functional devices.

On the other side, novel approaches for design and manufacturing customized foot orthoses by means of digital technologies (generally based on 3D scanning, 3D modelling and 3D printing) are recently reported as a valid alternative method to overcome these limitations.

This study has been carried out in an interdisciplinary approach between the staff of Design and Methods in Industrial Engineering and the staff of Podology with the aim to assess the feasibility of a novel user-friendly and cost-effective solution for delivering customized functional foot orthoses. More specifically, a Generative Design (GD) workflow has been developed to enable practitioners without enough CAD skills to easily 3D modelling and interactively customize foot orthoses. Additionally, low-cost devices for 3D scanning and 3D printing that have been acquired by the Podology Lab, were also tested and compared with the high-cost ones of the Department of Industrial Engineering.

The complete process is divided into three main steps. The first one regards the digitization of the patient's foot by means of 3D laser scanner devices. Then a user-friendly 3D modelling approach, developed for this purpose as GD workflow, allows interactively generating the customized foot orthosis, also adjusting several features and exporting the watertight mesh in STL format. Finally, the last step involves Additive Manufacturing systems to obtain the expected physical item ready to use.

First, for what concerns the digitizing step, the acquired data resulting from 3D scanning by means of the low-cost system (Sense 3D scanner) appears accurate enough for the present practical purposes.

Then, with respect to the 3D modelling step, the proposed GD workflow in Grasshopper is intuitive and allows easily and interactively customizing the final foot orthosis. Finally, regarding the Additive Manufacturing step, the low cost 3D printer (Wasp Delta 40 70) is capable to provide adequate results for the shell of the foot orthosis. Moreover, this system appears really versatile in reason of the capability to print in a wide range of different filaments. Therefore, since the market of 3D printing filaments is rapidly growing, building sessions with different materials (both flexible and rigid such, for example, PLA, AB and PETG) were completed.

This study validated, in terms of feasibility, that the use of a GD modelling approach, in combination with low-cost devices for 3D scanning and 3D printing, is a real alternative to conventional processes for providing customized foot orthosis. Moreover, the interdisciplinary approach allowed the transfer of skills and knowledge to the practitioners involved and, also, the low-cost devices Sense 3D scanner and Wasp Delta 40 70 that have been acquired by the Podology Lab, were demonstrated suitable for this kind of applications.

The glycolytic-based metabolism of cancers promotes an acidic microenvironment that is responsible for increased aggressiveness. However, the effects of acidosis on tumour metabolism have been almost unexplored, and the metabolic adaptation of cancer cells to acidosis has never been compared with the metabolic response of normal cells.

In this study, to pinpoint for the first time the different metabolic profiles between osteosarcoma (OS) cells and normal human fibroblasts (Fb) under short-term acidosis, we used capillary electrophoresis with time-of-flight mass spectrometry (CE-TOFMS). We also screened alterations of the epigenetic profiles – DNA methylation and histone acetylation – of OS cells and compared it with those of normal Fb.

Using CE-TOFMS, we observed a significant metabolic difference associated with glycolysis repression (dihydroxyacetone phosphate), increase of amino acid catabolism (phosphocreatine and glutamate) and urea cycle enhancement (arginino succinic acid) in OS cells compared with normal Fb. Noteworthy, metabolites associated with chromatin modification, like UDP-glucose and N⁸-acetylspermidine, decreased more in OS cells than in normal Fb. Further, combined bisulfite restriction analysis (COBRA) and acetyl-H3 immunoblotting indicated an epigenetic stability in OS cells than in normal Fb, and OS cells were more sensitive to an HDAC inhibitor under acidosis than under neutral condition.

Our data suggest that acidosis promotes a metabolic reprogramming that can contribute to the epigenetic maintenance under acidosis only in OS cells, and then the acidic microenvironment should be considered for future therapeutic approaches. The application of epigenetic modulators will be able to become an effectively therapeutic option to selectively target malignancies under the acidic microenvironment.

The success of biomaterials lies in the direct interaction with the host tissue. Calcium phosphates (CaP) stand as an alternative graft material for bone regeneration due to their similar composition to natural bone. Few studies have focused on the early stages of bone-like material remodeling by osteoclasts (OC), though the CaP fate is to be resorbed and then replaced by new bone. Instead, to understand how osteoclasts modify the CaP surface and initiate resorption, so as to influence subsequent osteoblast activities and bone formation, is mandatory.

Sintered hydroxyapatite (s-HA) and biomimetic hydroxyapatite with two different microstructures (b-HA-C, coarse and b-HA-F, fine) discs (1500×250 µm²) were produced from the same reagents [1]. Tissue culture polystyrene (TCPS) was used as control. Precursor human OC from buffy coats were seeded on ceramic substrates [6·10⁶cells/cm²] and supplemented with RANKL-containing osteoblast supernatant as differentiation medium over 21 days. Cell interaction with the biomaterials was investigated in terms of OC adhesion and differentiation, with gene expression, tartrate-resistant acid phosphatase (TRAP) and Hoechst staining for OC maturation. Cell culture supernatants were analyzed for ionic exchange, namely Ca and P, due to biomaterials or cells. Osteoclasts morphology was evaluated using SEM at 21 days. Innovatively, focused ion beam (FIB) was used to evaluate biomaterial structure beneath the OC to further investigate the resorption effects. To this aim, selected OC were cut cross-sectioned using a Gallium ion beam at an acceleration of 30KV, followed by a coarse milling at 10nA and a deposition of platinum to achieve a fine milling at 500pA.

Clear differences in cellular behavior were noted relative to the different substrate microstructures. Control TCPS and s-HA showed similar TRAP-positive staining and gene expression for mature OC. Several resorption pits with partial dissolution of the equiaxial grains of s-HA were noticed. b-HA substrates also showed attached and differentiated TRAP-positive OC, but gene expression resulted lower than control and s-HA. However, morphological evaluation with SEM-FIB interestingly showed early stages of osteoclast-mediated degradation on b-HA-F, i.e.an increased surface roughness in the substrate underlying cells. B-HA-C also showed attached and mature OC with a scarce degradation activity

FIB technique has been applied to cell-seeded CaP and shown as a viable method to investigate OC morphology and resorption. Though gene expression showed similarities for both biomimetic substrates, substrate morphology observed underneath OC was significantly different. b-HA-F showed early stages of OC mediated degradation underneath well spread cells similar to those seen on s-HA. No resorptive activity was found on b-HA-C even though gene expression values were similar to b-HA-F: both the acute ion exchange and the surface tortuosity on b-HA-C could explain the difficulty with the resorptive process by OC. In conclusion focused ion beam technique complements SEM imaging and may disclose changes in the inner structure of materials due to cell/material interactions.

For unrepairable nerve defects, to date autogenous nerves are considered the golden standard, but donor site morbidity, limited availability and operation time prolongation are relevant problem. Acellular nerves from cadaveric donor, introduced since more than one decade ago, represent a novel promising alternative to bridge unrepairable nerve gaps.

Aim of this study is to provide a new tool to ameliorate the assistance of the numerous patients suffering from traumatic, oncological and jatrogenic nerve lesions. For this purpose, our project is promoting a progress beyond the state of the art of nerve gaps bridging surgery by developing a new technique to obtain acellular nerve allografts (ANAs).

Several methods to examine the effect of detergents on nerve tissue morphology and protein composition have been previously reported. Most of them are too expensive and time consuming. The presented novel decellularization technique is a modification of the Michigan detergent-based organic material removal, to speed up myelin and cellular debris detachment. The previously published Hudson's method¹has been chosen as control of the decellularization process). To validate the new nerve decellularization method, in terms of histological characteristics, outcomes were estimated through morphological and immunohistochemical studies in vitro and in vivo. The in vivo study consisted of a 1 cm defect in the tibial nerve of 3 new Zealand rabbits. This nerve defect was microsurgically replaced with a “Rizzoli” acellular nerve allograft. Rabbits were sacrificed 12 weeks after surgery. Endpoints were nerve conduction studies and histology.

Histological analysis of processed acellular nerve have been performed to evaluate the preservation of the structure and almost complete clearance of donor cells and cellular debris. Immunostaining analysis confirmed absence of Schwann cells and the maintenance of basal lamina. In vivo studies showed an effective and abundant nerve regeneration through the microsurgically reconstructed nerve defects. This was histologically proven. However no electophysiological return of function was showed.

The novel method will allow the storing of acellular nerve allografts. First results obtained by morphological analysis and immunofluorescence experiments and in vivo studies indicate that the internal structure of native nerve is maintained. It is then possible to decellularize nerves with the novel technique reducing both manufacturing times and costs. The relatively inexpensive method of decellularization will facilitate the number of patients that will benefit from reconstruction of nerve defects with ANAs.

The postoperative course of median nerve decompression in the carpal tunnel syndrome may sometimes be complicated by postoperative pain, paresthesias, and other unpleasant symptoms, or be characterized by a slow recovery of nerve function due to prolonged preoperative injury causing extensive nerve damage.

The aim of this study is to explore any possible effects of alpha lipoic acid (ALA) in the postoperative period after surgical decompression of the median nerve at the wrist.

Patients were enrolled with proven carpal tunnel syndrome and randomly assigned into one of two groups: Group A: surgical decompression of the median nerve followed by ALA for 40 days. Group P: surgical decompression followed by placebo. The primary endpoint of the study was nerve conduction velocity at 3 months post surgery, Other endpoints were static 2 point discrimination, the Boston score for hand function, pillar pain and use of pain killers beyond the second postoperative day.

ALA did not show to significantly improve nerve conduction velocity or Boston score. However, a statistically significant reduction in the postoperative incidence of pillar pain was noted in Group A. In addition, static 2 point discrimination showed to be significantly improved by ALA.

Administration of ALA following decompression of the median nerve for carpal tunnel release is effective on nerve recovery, although this is not detectable through nerve conduction studies but in terms of accelerated and improved static two-point discrimination.

The use of ALA as a supplementation for nerve recovery after surgical decompression may be extended to all types of compression syndromes or conditions where a nerve is freed from a mechanical insult.

Furthermore, ALA limits post-decompression pain, including late pericicatricial pain at the base of the palm, the so called pillar pain, which seems to be associated with a reversible damage to the superfical sensitive small nerve fibers.

In conclusion postoperative administration of ALA for 40 days post-median nerve decompression was positively associated with nerve recovery, induced a lower incidence of postoperative pillar pain and was associated with a more rapid improvement of static two-point discrimination. This treatment is well tolerated and associated with high levels of satisfaction and compliance, supporting its value as a standard postoperative supplementation after carpal tunnel decompression.

Delayed bone healing and nonunion are complications of long bone fractures, with prolonged pain and disability. Regenerative therapies employing mesenchymal stromal cells (MSC) and/or bone substitutes are increasingly applied to enhance bone consolidation. The REBORNE project entailed a multi-center orthopaedic clinical trial focused on the evaluation of efficacy of expanded autologous bone marrow (BM) derived MSC combined with a CaP-biomaterial, to enhance bone healing in patients with nonunion of diaphyseal fractures. To complement the clinical and radiological examination of patients, bone turnover markers (BTM) were assayed as potential predictors of bone healing or non-union.

Peripheral blood was collected from patients at fixed time-endpoints, that is at 6,12 and 24 weeks post-surgery for implantation of expanded autologus MSC and bone-like particles. Bone-specific alkaline phosphatase (BAP), C-terminal-propeptide type I-procollagen (PICP), osteocalcin (OC), β-Cross-Laps Collagen (CTX), soluble receptor activator of NFkB (RANKL), osteoprotegerin (OPG) were measured by ELISA assays in blood samples of 22 patients at BM collection and at follow-up visits.

A significant relationship with age was found only at 6 months, with an inverse correlation for CTX, RANKL and OC, and positive for OPG. BTM levels were not related to gender. As an effect of local regenerative process, some BTM showed significant changes in comparison to the baseline value. In particular, the time course of BAP, PICP and RANKL was different in patients with a successful healing in comparison to patients with a negative outcome. The BTM profile apparently indicated a remarkable bone formation activity 12 weeks after surgery. However, the paucity of failed patients in our case series did not allow to prove statistically the role of BTM as predictors of the final outcome.

Blood markers related to bone cell function are useful to measure the efficacy of a expanded MSC-regenerative approach applied to long bone non-unions. Changes of the markers may provide a support to radiological assessment of bone healing.

The regenerative potential of bone grafts is tightly linked to the interaction of the biomaterial with the host tissue environment. Hence, strategies to confer artificial extracellular matrix (aECM) cues on the material surface are becoming a powerful tool to trigger the healing cascade and to stimulate bone regeneration. The use of glycosaminoglycans (GAGs), such as heparin, as aECM components has gained interest in the last years as a strategy to improve biological response. Calcium phosphates (CaP) are extensively used as bone grafts, however no studies have investigated the effect of GAG functionalisation on their surface. Some authors have focused on the effects of GAGs on osteoblastic cells, however, little work has been performed on the interaction with osteoclasts (OC), and still the reported effects are controversial [1]. The aim of this study was to investigate the effect of heparin on osteoclastic fate in terms of adhesion and differentiation.

Sintered CaP (β-TCP) and biomimetic CaP (calcium-deficient hydroxyapatite, CDHA) discs were synthesized at 1100 ºC and at 37ºC, respectively. Heparinisation was achieved though silane coupling (APTES) followed by amidation in the presence of EDC/NHS to covalently link heparin. The osteoclast response of heparinised (H) vsnon-heparinised substrates was studied using human monocytes as OC precursors. Tissue culture plastic (TCPS) was used as a control sample. Cell densities were 6·10⁶and 3·10⁶cells/cm²for biomaterials and TCPS, respectively. Cell cultures were supplemented every 3 days with 25% supernatant of osteoblast-like cell line as a source of RANKL, as well as other stimulating factors [2]. Tartrate-resistant acid phosphatase and Hoechst staining were used to evaluate OC adhesion, differentiation and morphology at different time points from seeding on the surfaces (14–21–28 days).

OC precursors showed adhesion on all substrates. β-TCP and β-TCP-H hosted higher number of OC precursors which might be related to the smoother sintered surface of the materials. Oppositely, the high roughness of CDHA and CDHA-H hamper the adhesion of OC, hence a lower number of cells was observed on heparin-coated and uncoated biomimetic apatites. However, the maturation of OC precursors was found to take place at earlier times (14days) on biomimetic substrates compared to sintered ones. TCPS, CDHA, CDHA-H and β-TCP-H showed clearly differentiated OC at 14 days, as revealed by TRAP positivity and multinuclearity. Interestingly, CDHA-H and β-TCP-H induced the highest multinuclearity among all differentiated OC. Both heparinised substrates point at an enhancing effect of heparin on OC maturation.

OC precursors are able to differentiate on β-TCP and CDHA substrates, a process enhanced when heparin functionalisation is performed on the materials surface. In our hands heparinisation is promoting OC differentiation at early time points, similarly to TCPS control. Interestingly, heparin substrates induced larger TRAP positive-OC and higher multinuclearity in the mature OC than TCPS control. As pointed out by Irie et al., heparin might interact through the RANKL/OPG ratio [3], thus inhibiting OPG activity and enhancing RANKL which triggers OC maturation.

Summary

We demonstrate that osteoclast-like cells of GCT result from the spontaneous fusion and differentiation of CD14+ cells of the monoblastic lineage by an autocrine mechanism mediated by RANKL, rather than induced by stromal cells. This process is further enhanced by the simultaneous impairment of the negative feed-back regulation of osteoclastogenesis by interferon β.

Summary

Starting from human musculoskeletal sarcomas, we isolated a subset of cells that display cancer stem cell properties. The control of culture conditions is crucial to enhance the isolation of this cell population.

Summary

Reciprocal metabolic reprogramming of MSCs and osteosarcoma cells influences tumor-stroma cross talk. Drugs targeting Warburg metabolism may define innovative therapeutic approaches in osteosarcoma.

Summary Statement

In this study it has been considered an alternative therapeutic approach to bone resorption diseases by using plant decoctions to improve adherence from patients to the treatment. In this context, Hemidesmus indicus represents a possible therapeutic or adjuvant natural compound.

We undertook a randomised controlled trial to compare bipolar hemiarthroplasty (HA) with a novel total hip replacement (THR) comprising a polycarbonate–urethane (PCU) acetabular component coupled with a large-diameter metal femoral head for the treatment of displaced fractures of the femoral neck in elderly patients. Functional outcome, assessed with the Harris hip score (HHS) at three months and then annually after surgery, was the primary endpoint. Rates of revision and complication were secondary endpoints.

Based on a power analysis, 96 consecutive patients aged > 70 years were randomised to receive either HA (49) or a PCU-THR (47). The mean follow-up was 30.1 months (23 to 50) and 28.6 months (22 to 52) for the HA and the PCU group, respectively.

The HHS showed no statistically significant difference between the groups at every follow-up. Higher pain was recorded in the PCU group at one and two years’ follow-up (p = 0.006 and p = 0.019, respectively). In the HA group no revision was performed. In the PCU-THR group six patients underwent revision and one patient is currently awaiting re-operation. The three-year survival rate of the PCU-THR group was 0.841 (95% confidence interval 0.680 to 0.926).

Based on our findings we do not recommend the use of the PCU acetabular component as part of the treatment of patients with fractures of the femoral neck.

Cite this article: Bone Joint J 2013;95-B:609–15.

We report a systematic review and meta-analysis of the peer-reviewed literature focusing on metal sensitivity testing in patients undergoing total joint replacement (TJR). Our purpose was to assess the risk of developing metal hypersensitivity post-operatively and its relationship with outcome and to investigate the advantages of performing hypersensitivity testing.

We undertook a comprehensive search of the citations quoted in PubMed and EMBASE: 22 articles (comprising 3634 patients) met the inclusion criteria. The frequency of positive tests increased after TJR, especially in patients with implant failure or a metal-on-metal coupling. The probability of developing a metal allergy was higher post-operatively (odds ratio (OR) 1.52 (95% confidence interval (CI) 1.06 to 2.31)), and the risk was further increased when failed implants were compared with stable TJRs (OR 2.76 (95% CI 1.14 to 6.70)).

Hypersensitivity testing was not able to discriminate between stable and failed TJRs, as its predictive value was not statistically proven. However, it is generally thought that hypersensitivity testing should be performed in patients with a history of metal allergy and in failed TJRs, especially with metal-on-metal implants and when the cause of the loosening is doubtful.

Ceramic-on-ceramic bearing is an attractive alternative to metal-on-polyethylene bearing due to the unique tri-bological advantages of alumina. However, despite the long-term satisfactory results obtained so far in the vast majority of patients, failure may occur in a few cases.

Clinical, radiographic, laboratory and microbiological data of 30 consecutive subjects with failed alumina-on-alumina total hip arthroplasties (THA) were analyzed to define if foreign body reaction to wear debris may be responsible for periprosthetic bone resorption, as in conventional metal-to-polyethylene bearings. In all cases, clinical and radiographical material was reviewed, retrieved implants were examined, and histology of periprosthetic tissues was analyzed. Massive osteolysis was never observed. Apart from 5 five patients for which revision surgery was necessary due to the occurrence of late infection, in all other cases failure had occurred due to secondary implant instability (as in the case of screwed sockets, 19 cases) or to malpositioning of the implant (5 cases). One patient suffered from chronic dislocation.

In the vast majority of cases, ceramic wear debris was absent or scarce, and did not induce any tissue reaction. In a few cases with severe wear, debris was evident in clusters of perivascular macrophages, notably in the absence of foreign body multinucleated cells, confirming the excellent biocompatibility of ceramics.

These findings indicate that wear debris and peri-prostetic bone resorption were the effect rather than the cause of failure, differently from revised metal-on-polyethylene bearings, in which foreign body cell reaction is the main pathogenetic mechanism of failure. On the contrary, mechanical problems, due to incorrect surgical technique or to inadequate prosthetic design, may cause instability of the implant, in turn resulting in wear debris production and moderate if any biological reaction.

We investigated the role of ion release in the assessment of fixation of the implant after total knee replacement and hypothesised that ion monitoring could be a useful parameter in the diagnosis of prosthetic loosening. We enrolled 59 patients with unilateral procedures and measured their serum aluminium, titanium, chromium and cobalt ion levels, blinded to the clinical and radiological outcome which was considered to be the reference standard. The cut-off levels for detection of the ions were obtained by measuring the levels in 41 healthy blood donors who had no implants. Based on the clinical and radiological evaluation the patients were divided into two groups with either stable (n = 24) or loosened (n = 35) implants.

A significant increase in the mean level of Cr ions was seen in the group with failed implants (p = 0.001). The diagnostic accuracy was 71% providing strong evidence of failure when the level of Cr ions exceeded the cut-off value. The possibility of distinguishing loosening from other causes of failure was demonstrated by the higher diagnostic accuracy of 83%, when considering only patients with failure attributable to loosening.

Measurement of the serum level of Cr ions may be of value for detecting failure due to loosening when the diagnosis is in doubt. The other metal ions studies did not have any diagnostic value.

Osteoporosis is one of the most common diseases in modern aging society. Receptor activator of nuclear factor-κB ligand (RANKL) plus macrophage colony stimulating factor (M-CSF)-mediated osteoclastogenesis has been recently implicated in the pathogenesis of this disease. Among other causes, the anticoagulant drug heparin is a notable inducer of secondary osteoporosis, although the molecular pathway underlying this process, particularly in human model, has not been clarified yet. Recently, we reported the differentiation of two subtypes of osteoclasts starting from human peripheral blood CD14-positive monocytes (Monocytes), respectively fusion regulatory protein-1 (FRP-1/CD98)-mediated osteoclasts and RANKL+M-CSF-mediated osteoclasts. We, therefore, investigated in details effects of heparin on differentiation and activation using a simple system of human osteoclastogenesis.

When Monocytes were cultured with osteoclastogenesis-relating factors and a high dose of heparin, heparin suppressed osteoclastogenesis in both pathways. However, a proper quantity of heparin enhanced tartrate-resistant acid phosphatase-positive multinucleated giant cell formation. There were significant differences in fusion indices between control osteoclasts and osteoclasts stimulated by moderate concentrations of heparin in two systems (P< 0.05). As a result of osteoclastic activity, FRP-1-mediated osteoclasts treated with a proper quantity of heparin formed larger pits on Ca plates. Moreover, lacunae on dentin surfaces induced by FRP-1-mediated osteoclasts were enhanced with moderate concentration of heparin. In contrast, heparin did not increase pit-formation area on Ca plates and on dentin surfaces by RANKL+M-CSF-mediated osteoclasts. Evaluating the relation between the concentration of heparin and the osteolytic areas on Ca plates, Pearson’s correlation coefficient of the FRP-1 and the RANKL+M-CSF were −0.973 (P< 0.05) and −0.695 (P=0.19), respectively.

In present study, although moderate doses of heparin stimulated differentiation in both systems, in osteoclastic activity, heparin promoted only to the FRP-1 system, not to RANKL+M-CSF system. Our results suggested FRP-1-induced osteoclastogenesis mainly contributes to development of heparin osteoporosis and also that the onset mechanism after long-term administration of heparin may be affected by the characteristic bone resorption ability of FRP-1osteoclasts.

Aims: Surface modification of biomaterials to be used as scaffolds in tissue engineering is a promising method to improve device multi-functionality and biological properties. Biomimetic surface treatments, such as changes in nano-structure and attachment of biomolecular signals, enhance material bioactivity and affinity for specific cells. In this study the functionalization of a titanium surface with vitronectin-derived nonapeptide(HVP) and RGD peptides was investigated. Bone forming cells were used to analyse the role of each surface modification in the initial steps of cell adhesion process and then proliferation and differentiation.

Method: Smooth titanium samples were functionalized by different chemical treatments in order to obtain varying amount of peptide adhesion. Human marrow stro-mal cells (MSC) were seeded and cultured in osteogenic medium. Cell adhesion and morphology were assessed by fluorescence microscopy after 6 hours. Viability of MSC was quantified at 7 and 14 days from plating, proliferation was measured using DNA and total protein content, and osteoblast phenotype expression was assayed using alkaline phosphatase (ALP) and calcium content.

Results: The results showed that presence of HVP and RGD peptides improves cell morphology in early adhesion on surface, compared to control (titanium without peptides). Activity of ALP and Ca²⁺ content of (1:1000)HVP and RGD samples were higher than the other experimental surfaces with or without peptides, even if they did not reach the values of control cells on tissue culture polystyrene.

Discussion: Properties acquired with chemical treatments can improve titanium surfaces. These data provide information useful to develop biomimetic cell-friendly surfaces for bone engineering.

Aims: It is well known that the success of an orthopedic implant is determined by a close apposition between bone and implant surface. The excellent physical properties and the controlled degradation of poly-ε-caprolactone (PCL) has been shown, however the suitability for bone engineering applications of a material is critically influenced by the interactions between cells and scaffold. The aim of this study was to evaluate the interaction between bone marrow cells and PCL surface. Bone marrow cells were obtained from femurs of New Zealand rabbits and seeded on PCL directly (WBMC) or after gradient centrifugation (MSC), mimicking the in vivo colonization of PCL after implantation and the pre-seeding strategy.

Methods: PCL was dissolved in chloroform (3% w/v solution) and spin coated as a thin (100nm) film onto p-doped silicon wafers. The surface wettability and roughness were analyzed by SFE measurements and AFM. Cells were seeded on PCL and adhesion/proliferation evaluated at 1, 7, 14, 21 and 28 days. Fluorescence microscopy and SEM imaging were performed at defined time endpoints.

Results: At 2 wks adherence-selected MSC had already formed confluent multilayers, whereas WBMC were still semi-confluent. At 4 wks a consistent layer of ECM was observed underneath the cell layers of both cultures.

Conclusions: PCL is a proper substrate for bone cell attachment and growth, as cell confluence was reached at 2 wks for MSC and at 3–4 wks for WBMC. To avoid any risk of bacterial contamination, the seeding of WBMC on PCL scaffold, which implies reduced handling of cells outside the body, was shown to be effective and may be recommended in the clinical practice.

Modern metal-on-metal bearings produce less wear debris and osteolysis, but have the potential adverse effect of release of ions. Improved ceramic-on-ceramic bearings have the lowest wear of all, but the corrosion process has not been analysed.

Our aim was to measure the serum ion release (ng/ml) in 23 patients having stable hip prostheses with a ceramic-on-ceramic coupling (group A) and to compare it with the release in 42 patients with a metal-on-metal bearing (group B) in the medium term. Reference values were obtained from a population of 47 healthy subjects (group C). The concentrations of chromium, cobalt, aluminium and titanium were measured.

There was a significant increase of cobalt, chromium and aluminium levels (p < 0.05) in group B compared with groups A and C. Group A did not differ significantly from the control group. Despite the apparent advantage of a metal-on-metal coupling, especially in younger patients with a long life expectancy, a major concern arises regarding the extent and duration of ion exposure. For this reason, the low corrosion level in a ceramic-on-ceramic coupling could be advantageous.

There is no diagnostic, non-invasive method for the early detection of loosening after total hip arthroplasty. In a pilot study, we have analysed two serum markers of bone remodelling, procollagen I C-terminal extension peptide (PICP) and cross-linked N-terminal telopeptide (NTx), as well as the diagnostic performance of NTx for the assessment of osteolysis. We recruited 21 patients with loosening (group I), 18 with a well-fixed prosthesis (group II) and 17 at the time of primary arthroplasty for osteoarthritis (OA) (group III). Internal normal reference ranges were obtained from 30 healthy subjects (group IV).

The serum PICP level was found to be significantly lower in patients with OA and those with loosening, when compared with those with stable implants, while the NTx level was significantly increased only in the group with loosening, suggesting that collagen degradation depended on the altered bone turnover induced by the implant. This hypothesis was reinforced by the finding that the values in the pre-surgery patients and stable subjects were comparable with the reference range of younger healthy subjects.

A high specificity and positive predictive value for NTx provided good diagnostic evidence of agreement between the test and the clinical and radiological evaluations. The NTx level could be used to indicate stability of the implant. However, further prospective, larger studies are necessary.

Aims: Different polyurethane foam matrices (PUF) loaded with hydroxyapatite (HA) or α-tricalcium phosphate were proposed as scaffold for bone regeneration [1,2]. In this work new PUFs were developed and loaded with HA or α-TCP.

Methods: PUFs were synthesized by a one-step polymerisation from a hydrophilic polyol mixture (LF 2946, Elastogran, Italy) and polymeric MDI (B141, BASF), using Fe acetyl-acetonate as catalyst and 2% water as expanding agent. The composites were prepared in the same way, by adding HA or α-TCP to the reacting mixture.

In vitro cell interactions were evaluated with human osteoblasts (HOB, 2nd passage) isolated from the trabecular bone of the femoral head of patients undergoing total hip replacement and cultured following the usual procedure. HOB cells (1x105 cells/sample) were kept in contact with the scaffolds for 7 and 14 days. At each time endpoint HOB metabolic activity, intracellular and released ALP were evaluated.

Results: By water adsorption test, newly synthesized PUFs showed a higher hydrophilicity compared to that of the previous matrices (600% vs 110% water uptake after 100h). Due to the presence of inorganic salts, composite scaffolds showed density values higher (0.131B80.200g/cm3) than those of unloaded PUFs (0.071B80.093g/cm3). Yet, open cell percentage (57–75%) and average pore size (350B8520mm) resulted similar to those of the PUFs.

HOB cells grown on scaffold samples showed an increase of metabolic activity from 7 to 14 days. The amount of intracellular ALP increased too, whereas the amount of ALP in the medium was quite low. HOB cells, after 14 days, appeared closely adherent to the scaffolds, with an elongated and flattened shape.

Conclusions: These preliminary results showed that, even if slow, the growth of HOB onto PUF scaffolds was quite good. After 14 days, PUF composites showed higher cells growth than PUF-matrices, confirming the role of the bone-like inorganic particles in improving osteoblasts functions. Long-term in vitro tests are now in progress.

Total joint arthroplasty is the most significant advance in the treatment of end-stage arthritic disease of major joints. Despite the clinical success of this surgical procedure, however, some total joint prostheses fail, and although a failed prosthesis can be replaced, the results of revision arthroplasty are not as good as the first time. Studying the failed prosthesis and the associated bone and soft tissues provides insight into the causes of failure.

Most prosthetic failures are the result of structural limitations of the implant components. Although material failure may be sudden, a much more common cause is gradual aseptic loosening of the prostheses. Aseptic loosening is caused by both mechanical (gradual loss of material by wear) and biological (osteoclastic resorption of adjacent bone) factors. Wear particles induce a foreign body reaction characterized by a pseudomembrane composed of granulomatous tissues including macrophages, fibroblasts, giant cells, and osteoclasts in addition to debris particles. The extent of this response is driven by the number, size, composition, surface area, and types of particles present. Although there are differences in the relative local toxicity of each of these particles, the end result is the same. These mechanical and biological factors are unavoidable, and the success of a total joint prosthesis depends on the rate with which they occur. Polyethylene wear particles (1–200 ?) are the primary cause of loosening. They are strongly birefringent under polarized light microscopy. Smaller particles are phagocytized by histiocytes, whereas larger particles are surrounded by foreign body giant cells. Fragmentation of PMMA may also cause particulate debris. The presence of these particles (30–100 ?) may be deduced by empty spaces into the soft tissues, often bordered by foreign body giant cells, since PMMA is dissolved by xylene during routine histological techniques. Metal oxides form on the surface of chrome-cobalt or titanium alloys due to an electrolytic process, and stresses on the surface of the metal shear the oxides into the surrounding tissues, causing a black pigmentation of the tissues. Histologically, the black deposits of oxidized metals are seen extracellularly as well as in the cytoplasm of histiocytes. In addition to oxidation, metal undergoes corrosion and, as a result, metal ions enter the soft tissues and the bloodstream. A ceramic-on-ceramic coupling generates a significantly lower amount of debris as compared to the conventional metal-on polyethylene solution. When present, ceramic debris cause a mild histiocytic reaction without giant cells and virtually no osteoclastic bone resorption. There are various secretory proteins at the interfacial membrane that can affect bone turnover, including the cytokines IL-1, IL-6, Il-10, and TNF-a. Other factors involved with bone resorption include the enzymes responsible for catabolism of the organic component of bone, such as MMPs. Prostaglandins, in particular PGE2, are also known to be important intercellular messengers in the osteolytic cascade. More recently, several mediators known to be involved in stimulation or inhibition of osteoclast differentiation and maturation, such as RANKL and osteoprotegerin, have been suggested as key factors in the development and progression of osteolysis.

Infection around a prosthesis also causes loosening in approximately 1–5% of cases. Total joint prostheses become infected by two mechanisms, wound contamination at the time of surgery by Staph. aureus or Staph.epidermidis, and late hematogenous spread of organisms (Staphylo- and Streptococci, E. Coli, Pseudomonas, and anaerobes). The following factors facilitate bacterial growth. First, reaming and sawing, as well as PMMA polymerization, cause necrosis of necrotize bone adjacent to the implant, and such nonvascularized area permits bacteria to grow, safe from circulating host defenses. Second, a highly hydrated matrix of extracellular polymeric substances (biofilm) is formed that defends bacteria from antibiotics and phagocytosis. Third, some metals, such as nickel or cobalt, may depress macrophage function. The distinguishing histologic features of an infected prosthesis is an acute inflammatory reaction: a finding of > 5 PMN or of > 50 lymphocytes/hp field are presumptive for infection. Because some low-grade infections fail to stimulate an acute inflammatory reaction, they go undiagnosed until postoperative period when microbacterial culture results are available. To date, no single routinely used clinical or laboratory test has been shown to achieve ideal sensitivity and specificity for the diagnosis of prosthetic joint infection, and in most cases the diagnosis depends on a combination of clinical features, radiographic findings, and laboratory results. Intra-operative tissue cultures may be falsely negative because of prior antimicrobial exposure, a low number of organisms, inappropriate culture media, or atypical organisms. Conversely, cultures may be falsely positive because of contamination in the operating room, during transport, or in the laboratory. If the implant is removed, the entire prosthesis can be cultured. Moreover, because prosthetic joint infection is a biofilm-mediated infection, techniques that sample bacteria in biofilm, such as sonication or enzymatic treatment, may improve the diagnosis of prosthetic joint infection. More recently, molecular techniques are being used to detect nucleic acid in samples from infected patients even when conventional techniques are negative because of unusual microbial growth requirements or failure to grow after antimicrobial exposure or due to unfavourable environmental conditions. A disadvantage of such approach is its extreme sensitivity, leading to the possibility of false positive results.

The clinical presentation of prosthetic joint infection may be indistinguishable from that of aseptic implant failure. In many cases, culture of granulomatous tissue around failed prostheses, preoperatively diagnosed as aseptically loosened, reveals the presence of bacteria that may per se significantly contribute to the recruitment, maturation and activation of osteoclasts and that superimpose to the foreign body reaction to wear debris. The presence of a smouldering infection in case of “aseptic” failure observed in revision arthroplasties. A systematic investigation on all retrieved implants is mandatory to define the precise role of each potential factor contributing to the pathogenesis of failure, in order to further improve the quality of care of patients having total joint arthroplasty.

We aimed to assess whether the immunological abnormalities which have been observed in patients with loose total hip replacements (THRs) are present in patients with a well-fixed prosthesis.

We examined blood samples from 39 healthy donors, 22 patients before THR and 41 with well-fixed THRs of different types (15 metal-on-metal, 13 metal-on-polyethylene, 13 ceramic-on-ceramic). Before THR, the patients showed a decrease in leukocytes and myeloid cells in comparison with healthy donors, and a prevalence of type-1 T lymphocytes, which was confirmed by the increase in ratio of interferon-γ to interleukin 4. Moreover, patients with metal-on-metal or metal-on-polyethylene implants showed a significant decrease in the number of T lymphocytes and a significant increase in the serum level of chromium and cobalt, although no significant correlation was observed with the immunological changes. In the ceramic-on-ceramic group, leukocytes and lymphocyte subsets were not significantly changed, but a significant increase in type-2 cytokines restored the ratio of interferon-γ to interleukin 4 to normal values.

We conclude that abnormalities of the cell-mediated immune response may be present in patients with a well-fixed THR, and that the immunological changes are more evident in those who have at least one metal component in the articular coupling.