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A stable adhesion to the cartilage is a crucial requisite for hydrogels used for cartilage regeneration. Indeed, a weak interface between the tissue and the implanted material may produce a premature detachment and thus the failure of the regeneration processes. Fibrin glue, cellulose nanofibers and catecholamines have been proposed in the state-of-the-art as primers to improve the adhesion. However, no studies focused on a systematic comparison of their performance. This work aims to evaluate the adhesion strength between ex vivo cartilage specimens and polysaccharide hydrogels (gellan gum and methacrylated gellan gum), by applying the mentioned primers as intermediate layer. Results show that the fibrin glue and the cellulose nanofibers improve the adhesion strength, while catecholamines do not guarantee reaching a clinically acceptable value. Stem cells embedded in gellan gum hydrogels reduce the adhesion strength when fibrin glue is used as a primer, being anyhow still sufficient for in vivo applications.

Objective. In this study, we aimed to verify the beneficial effects of low-intensity pulsed ultrasound (LIPUS) stimulation on two cell types: H2O2-treated RSC96 Schwann cells and THP-1 macrophages, used to model neuropathic inflammation. Approach. Using a set-up guaranteeing a fine control of the ultrasound dose at the target, different frequencies (38 kHz, 1 MHz, 5 MHz) and different intensities (20, 100, 500 mW cm−2) were screened to find the most effective experimental conditions for triggering beneficial effects on metabolic activity and release of neurotrophic cytokines (β-nerve growth factor, brain-derived neurotrophic factor, glial cell-derived neurotrophic factor) of RSC96 cells. The combination of parameters resulting the optimal one was applied to evaluate anti-inflammatory effects in terms of reactive oxygen species (ROS) and tumor necrosis factor-α (TNF-α) production, also investigating a possible anti-oxidant activity and mechanotransduction pathway for the anti-inflammatory process. The same optimal combination of parameters was then applied to THP-1 cells, differentiated into M1 and M2 phenotypes, to assess the effect on the expression and release of pro-inflammatory markers (TNF-α, interleukin (IL)-1β, IL-6, IL-8) and anti-inflammatory ones (IL-10 and CD206). Main results. 5 MHz and 500 mW cm−2 were found as the optimal stimulation parameters on RSC96 cells. Such parameters were also found to suppress ROS and TNF-α in the same cell line, thus highlighting a possible anti-inflammatory effect, involving the NF-kB pathway. An anti-oxidant effect induced by LIPUS was also observed. Finally, the same LIPUS parameters did not induce any differentiation towards the M1 phenotype of THP-1 cells, whereas they decreased TNF-α and IL-8 gene expression, reduced IL-8 cytokine release and increased IL-10 cytokine release in M1-polarized THP-1 cells. Significance. This study represents the first step towards the use of precisely controlled LIPUS for the treatment of peripheral neuropathies.

This deliverable describes the key events organized in the first year of the ADMAIORA project, as devised in the preliminary dissemination, communication and exploitation plan. In particular, the first project-related scientific workshop has been organized during the IEEE/EMBS Engineering in Medicine and Biology Conference (EMBC) in Berlin by the Project Coordinator, Prof. Leonardo Ricotti. During this event, the project strategy and preliminary results were disseminated to a broad audience of potential users, stakeholders, and scientists. A second non-technical workshop has also been organized by the Project Coordinator in collaboration with Dr. Gina Lisignoli from IOR and with the involvement of AMRER, an Italian association of rheumatic patients. Such a second event aimed at describing the project approach and preliminary results to patients affected by osteoarthritis and, in general, to elderly people affected by rheumatic problems, thus to make them aware of the technologies under development in ADMAIORA and to get a useful feedback from them. End-users concrete needs and their opinion of the different project aspects were collected and will be taken into serious consideration by the Consortium, to properly shape technical efforts towards impactful and user needs-compliant results. The deliverable describes the objectives and the details of both events, including an analysis of the patients’ feedbacks.

In this paper, a novel nanofilm type is proposed based on a blend of poly(ethylene glycol)-block-poly(ε-caprolactone) methyl ether (PEG-b-PCL) and poly(l-lactic acid), doped with zinc oxide nanoparticles (ZnO NPs) at different concentrations (0.1, 1, and 10 mg/mL). All nanofilm types were featured by a thickness value of ∼500 nm. Increasing ZnO NP concentrations implied larger roughness values (∼22 nm for the bare nanofilm and ∼67 nm for the films with 10 mg/mL of NPs), larger piezoelectricity (averaged33coefficient for the film up to ∼1.98 pm/V), and elastic modulus: the nanofilms doped with 1 and 10 mg/mL of NPs were much stiffer than the nondoped controls and nanofilms doped with 0.1 mg/mL of NPs. The ZnO NP content was also directly proportional to the material melting point and crystallinity and inversely proportional to the material degradation rate, thus highlighting the stabilization role of ZnO particles. In vitro tests were carried out with cells of the musculoskeletal apparatus (fibroblasts, osteoblasts, chondrocytes, and myoblasts). All cell types showed good adhesion and viability on all substrate formulations. Interestingly, a higher content of ZnO NPs in the matrix demonstrated higher bioactivity, boosting the metabolic activity of fibroblasts, myoblasts, and chondrocytes and enhancing the osteogenic and myogenic differentiation. These findings demonstrated the potential of these nanocomposite matrices for regenerative medicine applications, such as tissue engineering.

Background: Assessing lower limb strength in the field is problematic, as the “gold standard assessment” with isokinetic strength is cumbersome, and the device is costly and not transportable and keeps the angle of the hip at around 90°. Methods: We evaluated isometric muscle strength in a standing position with the help of an exoskeleton that holds the subject and makes the test easily repeatable. Results: The optimal device angles for hip and knee were, respectively, 20° and 80° for flexor tests and 30° and 40° for extensor tests. Test–retest reliability was very high for the right knee extensor (ICC 0.96–0.98), left knee extensor (ICC 0.96–0.97), right knee flexor (ICC 0.91–0.96), and left knee flexor (ICC 0.96–0.97). Furthermore, the typical error in percent (T.E.%) ranged from 2.50 to 5.50%, and the change in the mean in percent ranged from 0.84 to 7.72%, making it possible to determine even a slight variation in force. Conclusions: this new method could represent a valid alternative for assessing strength, due to the high reliability and the favorable joint position, particularly in football.

This systematic review is focused on the main characteristics of the hydrogels used for embedding the mesenchymal stromal cells (MSCs) in in vitro/ex vivo studies, in vivo OA models and clinical trials for favoring cartilage regeneration in osteoarthritis (OA). PubMED and Embase databases were used to select the papers that were submitted to a public reference manager Rayyan Systematic Review Screening Software. A total of 42 studies were considered eligible: 25 articles concerned in vitro studies, 2 in vitro and ex vivo ones, 5 in vitro and in vivo ones, 8 in vivo ones and 2 clinical trials. Some in vitro studies evidenced a rheological characterization of the hydrogels and description of the crosslinking methods. Only 37.5% of the studies considered at the same time chondrogenic, fibrotic and hypertrophic markers. Ex vivo studies focused on hydrogel adhesion properties and the modification of MSC-laden hydrogels subjected to compression tests. In vivo studies evidenced the effect of cell-laden hydrogels in OA animal models or defined the chondrogenic potentiality of the cells in subcutaneous implantation models. Clinical studies confirmed the positive impact of these treatments on patients with OA. To speed the translation to the clinical use of cell-laden hydrogels, further studies on hydrogel characteristics, injection modalities, chemo-attractant properties and adhesion strength are needed.

3D scaffolds for tissue engineering typically need to adopt a dynamic culture to foster cell distribution and survival throughout the scaffold. It is, therefore, crucial to know fluids' behavior inside the scaffold architecture, especially for complex porous ones. Here we report a comparison between simulated and measured permeability of a porous 3D scaffold, focusing on different modeling parameters. The scaffold features were extracted by microcomputed tomography (µCT) and representative volume elements were used for the computational fluid-dynamic analyses. The objective was to investigate the sensitivity of the model to the degree of detail of the µCT image and the elements of the mesh. These findings highlight the pros and cons of the modeling strategy adopted and the importance of such parameters in analyzing fluid behavior in 3D scaffolds.

In this work, unidirectional valves made of bare polydimethylsiloxane (PDMS) and PDMS provided with a micrometric diamond-like carbon (DLC) coating were fabricated and characterized, in terms of surface properties and opening pressure. The valve performance was also tested over 1250 repeated cycles of opening/closure in water, finding a slight decrease in the opening pressure after such cycles (10%) for the PDMS valves, while almost no variation for the PDMS + DLC ones. The valves were then immersed in urine for 126 days, evaluating the formation of encrustations and the trend of the opening pressure over time. Results showed that PDMS valves were featured by a thin layer of encrustations after 126 days, but the overall encrustation level was much smaller than the one shown by PDMS in static conditions. Furthermore, the opening pressure was almost not affected by such a thin layer of crystals. DLC-coated valves showed even less encrustations at the same time-point, with no significant loss of performance over time, although they were featured by a higher variability. These results suggest that most encrustations can be removed by the mechanical action of the valve during daily openings/closures. Such a self-cleaning behavior with respect to a static condition opens exciting scenarios for the long-term functionality of mobile devices operating in the urinary environment.

Low Intensity Pulsed Ultrasound could enable a “wireless biopsy” of gut diseases such as ulcerative colitis, by triggering the release of pathology-characterizing molecules from the intestinal wall cells. Prior to the in vivo validation, the prediction of the acoustic propagation through living tissues results fundamental to guide the design of the experimental setting and to be able to reach the target with the desired ultrasound regime. In this work we propose a simulation framework able to precisely predict the ultrasound dose at the target (i.e., mouse colon) for the above-mentioned application.

Robotic dispensing-based 3D bioprinting represents one of the most powerful technologies to develop hydrogel-based 3D constructs with enormous potential in the field of regenerative medicine. The optimization of hydrogel printing parameters, proper geometry and internal architecture of the constructs, and good cell viability during the bioprinting process are the essential requirements. In this paper, an analytical model based on the hydrogel rheological properties was developed to predict the extruded filament width in order to maximize the printed structure’s fidelity to the design. Viscosity data of two natural hydrogels were imputed to a power-law model to extrapolate the filament width. Further, the model data were validated by monitoring the obtained filament width as the output. Shear stress values occurring during the bioprinting process were also estimated. Human mesenchymal stromal cells (hMSCs) were encapsulated in the silk fibroin–gelatin (G)-based hydrogel, and a 3D bioprinting process was performed to produce cell-laden constructs. Live and dead assay allowed estimating the impact of needle shear stress on cell viability after the bioprinting process. Finally, we tested the potential of hMSCs to undergo chondrogenic differentiation by evaluating the cartilaginous extracellular matrix production through immunohistochemical analyses. Overall, the use of the proposed analytical model enables defining the optimal printing parameters to maximize the fabricated constructs’ fidelity to design parameters before the process execution, enabling to achieve more controlled and standardized products than classical trial-and-error approaches in the biofabrication of engineered constructs. Employing modeling systems exploiting the rheological properties of the hydrogels might be a valid tool in the future for guaranteeing high cell viability and for optimizing tissue engineering approaches in regenerative medicine applications.

Compared to two-dimensional cell cultures, three-dimensional ones potentially allow recreating natural tissue environments with higher accuracy. The three-dimensional approach is being investigated in the field of tissue engineering targeting the reconstruction of various tissues, among which skeletal muscle. Skeletal muscle is an electroactive tissue which strongly relies upon interactions with the extracellular matrix for internal organization and mechanical function. Studying the optimization of myogenesis in vitro implies focusing on appropriate biomimetic stimuli, as biochemical and electrical ones. Here we present a three-dimensional polyurethane-based soft porous scaffold (porosity ~ 86%) with a Young's modulus in wet conditions close to the one of natural skeletal muscle tissue (~ 9 kPa). To study the effect of external stimuli on muscle cells, we functionalized the scaffold with extracellular matrix components (laminin and fibronectin) and observed an increase in myoblast proliferation over three days. Furthermore, the combination between laminin coating and electrical stimulation resulted in more spread and thicker myotubes compared to non-stimulated samples and samples receiving the single (non-combined) inputs. These results pave the way to the development of mature muscle tissue within three-dimensional soft scaffolds, through the combination of biochemical and electrical stimuli.

This paper describes a sensorized orthosis used to objectively monitor the follow-up of pediatric patients affected by Pectus Carinatum and treated through compression therapy. The study reports an analysis made on 25 patients. The pressure exerted by the compression brace was monitored by means of a high-density sensing matrix at every outpatient check, over 150 days. The pressure maps derived from this analysis correlated with the clinical parameters indicating a successful outcome for the treatment of specific deformity types such as symmetric chondrogladiolous and rib flares. Results demonstrate that bracing is an effective tool in well-motivated patients in the previously mentioned deformity types. Pressure mapping emerged as a parameter to objectively assess treatment efficacy and may pave the way to a new generation of orthoses with high-density sensorized elements chronically embedded in their structure.

This study constitute the first attempt to systematically quantify residual limb volume changes in transfemoral amputees. The study was carried out on 24 amputees to investigate changes due to prosthesis doffing, physical activity, and testing time. A proper experimental set-up was designed, including a 3D optical scanner, to improve precision and acceptability by amputees. The first test session aimed at measuring residual limb volume at 7 time-points, with 10 minute intervals, after prosthesis doffing. This allowed for evaluating the time required for volume stabilization after prosthesis removal, for each amputee. In subsequent sessions, 16 residual limb scans in a day for each amputee were captured to evaluate volume changes due to prosthesis removal and physical activity, in two times per day (morning and afternoon). These measurements were repeated in three different days, a week apart from each other, for a total of 48 scans for each amputee. Volume changes overtime after prosthesis doffing showed a two-term decay exponential trend (R2 = 0.97), with the highest change in the initial 10 minutes and an average stabilization time of 30 minutes. A statistically significant increasing effect of both prosthesis removal and physical activity was verified. No differences were observed between measures collected in the morning and in the afternoon.

Bioinspired soft robotics aims at reproducing the complex hierarchy and architecture of biological tissues within artificial systems to achieve the typical motility and adaptability of living organisms. The development of suitable fabrication approaches to produce monolithic bodies provided with embedded variable morphological and mechanical properties, typically encountered in nature, is still a technological challenge. Here we report on a novel manufacturing approach to produce three-dimensional functionally graded hydrogels (3D-FGHs) provided with a controlled porosity gradient conferring them variable stiffness. 3D-FGHs are fabricated by means of a custom-designed liquid foam templating (LFT) technique, which relies on the inclusion of air bubbles generated by a blowing agent into the monomer-based template solution during ultraviolet-induced photopolymerization. The 3D-FGHs' apparent Young's modulus ranges from 0.37 MPa (bulky hydrogel region) to 0.09 MPa (highest porosity region). A fish-shaped soft swimmer is fabricated to demonstrate the feasibility of the LFT technique to produce bioinspired robots. Mobility tests show a significant improvement in terms of swimming speed when the robot is provided with a graded body. The proposed manufacturing approach constitutes an enabling solution for the development of macroscopic functionally graded hydrogel-based structures usable in biomimetic underwater soft robotics applications.

Creating fully implantable robots that replace or restore physiological processes is a great challenge in medical robotics. Restoring blood glucose homeostasis in patients with type 1 diabetes is particularly interesting in this sense. Intraperitoneal insulin delivery could revolutionize type 1 diabetes treatment. At present, the intraperitoneal route is little used because it relies on accessing ports connecting intraperitoneal catheters to external reservoirs. Drug-loaded pills transported across the digestive system to refill an implantable reservoir in a minimally invasive fashion could open new possibilities in intraperitoneal delivery. Here, we describe PILLSID (PILl-refiLled implanted System for Intraperitoneal Delivery), a fully implantable robotic device refillable through ingestible magnetic pills carrying drugs. Once refilled, the device acts as a programmable microinfusion system for precise intraperitoneal delivery. The robotic device is grounded on a combination of magnetic switchable components, miniaturized mechatronic elements, a wireless powering system, and a control unit to implement the refilling and control the infusion processes. In this study, we describe the PILLSID prototyping. The device key blocks are validated as single components and within the integrated device at the preclinical level. We demonstrate that the refilling mechanism works efficiently in vivo and that the blood glucose level can be safely regulated in diabetic swine. The device weights 165 grams and is 78 millimeters by 63 millimeters by 35 millimeters, comparable with commercial implantable devices yet overcoming the urgent critical issues related to reservoir refilling and powering.

Articular cartilage (AC) is a specialized connective tissue able to provide a low-friction gliding surface supporting shock-absorption, reducing stresses, and guaranteeing wear-resistance thanks to its structure and mechanical and lubrication properties. Being an avascular tissue, AC has a limited ability to heal defects. Nowadays, conventional strategies show several limitations, which results in ineffective restoration of chondral defects. Several tissue engineering approaches have been proposed to restore the AC's native properties without reproducing its mechanical and lubrication properties yet. This work reports the fabrication of a bilayered structure made of gellan gum (GG) and poly (ethylene glycol) diacrylate (PEGDA), able to mimic the mechanical and lubrication features of both AC superficial and deep zones. Through appropriate combinations of GG and PEGDA, cartilage Young's modulus is effectively mimicked for both zones. Graphene oxide is used as a dopant agent for the superficial hydrogel layer, demonstrating a lower friction than the nondoped counterpart. The bilayered hydrogel's antiwear properties are confirmed by using a knee simulator, following ISO 14243. Finally, in vitro tests with human chondrocytes confirm the absence of cytotoxicity effects. The results shown in this paper open the way to a multilayered synthetic injectable or surgically implantable filler for restoring AC defects.

This Deliverable reports the advancement in the Data Management Plan and the Innovation Management strategy. The Intermediate Data Management Plan (IDMP) includes an update of the Initial Data Management Plan submitted under D7.4 and an integration of the updates that emerged within the project, in terms of data management, curation, and preservation-related rules. The IDMP updates the general strategy that the ADMAIORA consortium adopted to fulfill the Open Access Research Data Pilot and to create a FAIR ecosystem in light of the research data already produced and the ones the Consortium is going to develop in the next steps of the project life-cycle. This part of the Deliverable confirms the strategy aimed at combining data management, dissemination activities and the overall management of the project, including the legal-ethical dimension of research data and it provides a more aware position of the Consortium towards Open Data. The Innovation Management strategy describes how the priorities and initiatives of the Consortium have been orchestrated, thus to successfully pursue exploitation, dissemination and communication initiatives, also identifying appropriate limitations to the Open Access policy drafted in the DMP. This part of the Deliverable evaluates the efficacy of the Innovation Management strategy adopted so far in the project course and if it should be maintained or not in the second part of the ADMAIORA project.

This paper aims to characterize the wear behavior of hydrogel constructs designed for human articular cartilage replacement. To this purpose, poly (ethylene glycol) diacrylate (PEGDA) 10% w/v and gellan gum (GG) 1.5% w/v were used to reproduce the superior (SUP) cartilage layer and PEGDA 15% w/v and GG 1.5% w/v were used to reproduce the deep (DEEP) cartilage layer, with or without graphene oxide (GO). These materials (SUP and DEEP) were analyzed alone and in combination to mimic the zonal architecture of human articular cartilage. The developed constructs were tested using a four-station displacement control knee joint simulator under bovine calf serum. Roughness and micro-computer tomography (&micro, CT) measurements evidenced that the hydrogels with 10% w/v of PEGDA showed a worse behavior both in terms of roughness increase and loss of uniformly distributed density than 15% w/v of PEGDA. The simultaneous presence of GO and 15% w/v PEGDA contributed to keeping the hydrogel construct&rsquo, s characteristics. The Raman spectra of the control samples showed the presence of unreacted C=C bonds in all the hydrogels. The degree of crosslinking increased along the series SUP &lt, DEEP + SUP &lt, DEEP without GO. The Raman spectra of the tested hydrogels showed the loss of diacrylate groups in all the samples, due to the washout of unreacted PEGDA in bovine calf serum aqueous environment. The loss decreased along the series SUP &gt, DEEP + SUP &gt, DEEP, further confirming that the degree of photo-crosslinking of the starting materials plays a key role in determining their wear behavior. &mu, CT and Raman spectroscopy proved to be suitable techniques to characterize the structure and composition of hydrogels.

This paper describes a biohybrid actuator consisting of a microgrooved thin film, powered by contractile, aligned skeletal muscle cells. The system was made of a thermoplastic elastomer [SBS, poly(styrene

We analyze temperature dynamics in anatomic pathology samples to identify the most efficient refrigeration method and to predict the time available for optimal sectioning before sample heating, thus getting appropriate information for a correct diagnosis by anatomopathologists. A thermal finite element (FE) analysis was carried out with comsolmultiphysics to evaluate temperature variations in paraffin-embedded tissues, i.e., muscle, bone and fat, and the corresponding thermal stresses. Experiments with different tissues and thermocouple-based measurements allowed validating the FE simulations. Simulations allowed to estimate the time needed to bring the sample at the optimal temperature for sectioning (−8 to −4 °C) in different conditions: refrigeration on a cold plate, refrigeration in a cooled environment, and refrigeration in an environment with forced convection. Among the three cooling methods tested, the forced convection at −20 °C and with an air-flow speed of 5 m/s resulted in the shortest cooling time. As compared to the other methods, thermal stresses can be modulated by varying the air-flow speed. For the different conditions, the time needed for the surface of the tissue block to exit from a temperature corresponding to an optimal cutting, when leaving the sample exposed to room temperature after refrigeration, ranged from 12 to 310 s. We quantify the time needed to adequately refrigerate paraffin-embedded tissue samples and the time available before they leave the optimal temperature window for sectioning. We also evaluate the maximum stress attained in the paraffin block during the cooling and the heating transients. This information will help optimize anatomic pathology processes.

Rare earth magnets are the elective choice when high magnetic field density is required and they are particularly intriguing for inclusion in implantable devices. A safe implantation of NdFeB magnets in muscles would enable the control of limb prostheses using a myokinetic interface

Tungsten disulfide (WS2) particles have excellent lubrication properties and low surface energy. These features are exploited for numerous applications in the biomedical field, from the development of low-friction orthodontic wires to urinary encrustation-resisting coatings. Nevertheless, the cytotoxicity of spherical WS2 particles still remains unclear. We report the biocompatibility of pristine WS2 particles in human urinary bladder cells after the exposure to a wide range of concentrations (from 0 to 100 μg/mL). Based on the cell viability assessments using trypan blue exclusion assay and the PrestoBlue Cell viability reagent after 24, 48, and 72 h exposure, it can be concluded that these particle aggregates, with a diameter of ~ 500 nm, do not show any evidence of alterations or toxicity in the cells, even with high concentrations there exist a 87% of cell viability. Furthermore, due to the predisposition to agglomerate of this material and the possible use as a coating in urinary devices, we functionalized these particles with carboxylic groups. Aggregates with a diameter of ~ 370 nm were obtained. Zeta potential measurements highlighted that carboxylation correctly occurred, showing a switch from ~ + 4 for pristine particles to ~ − 35 for COOH-functionalized ones. Cytotoxic studies with these functionalized WS2 particles also showed good biocompatibility on human urinary bladder cells, with an 86% of viability after incubation with 100 μg/mL of particles over 72 h. These results highlight a good potential of WS2 particles for a future safe application as components of implantable medical devices.

In this paper, a novel nanofilm type is proposed based on a blend of poly(ethylene glycol)

In this Deliverable, the highly controlled in vitro LIPUS stimulation prototypes are described. In section 2.1, both the low-frequency (38 kHz) and high frequency (500 kHz – 5 MHz) stimulation set-ups identified as SYSTEM 1 are presented. They will be used for ensuring the control of the US dose at the target and identifying (off-line) the optimum US parameters able to trigger beneficial bioeffects in terms of cartilage regeneration. In section 2.2, the assembled SYSTEM 2 set-up and its components is reported. It will allow investigating intracellular phenomena occurring during LIPUS stimulation in real-time. Only few details on the two systems are reported in this Deliverable, which is public, to not jeopardise possible future IP protection. Full details on the different components have been reported in Deliverable D4.1. Furthermore, all additional and confidential information on the two systems is reported in Deliverable 4.3 (which is confidential).

In this Deliverable the prototypes of PEG-fibrinogen-based hydrogel formulations developed by REGENTIS and their main features are described. In section 2, the key specifications identified for the hydrogels are reported, also with a description of the three material types developed, as well as of the methods and techniques used to optimize the hydrogel formulations based on the required specifications. Then, in section 2.1, the results of preliminary biological tests performed to assess the viability of human adipose tissue-derived stem cells (ASCs) after cell encapsulation within PEG-fibrinogen printed hydrogels are reported. These tests also served to evaluate the right amount of photoinitiator, UV intensity and exposure time to be employed to have a material respecting the specifications. After this preliminary analysis, an optimization process for PEG-based hydrogel formulations is described in the same section. This implied to vary: i) the total amount of Pluronic and ii) the ratio of non-cross-linkable and UVA-cross-linkable Pluronic. Material characterization in terms of mechanical and rheological properties, the diffusivity of proteins in the material and cell viability of human chondrocytes are also reported in section 2.1 for the different formulations. Results indicate that the formulations containing a lower amount of cross-linkable Pluronic show a higher swelling, a lower shear modulus (G') and a smaller Young modulus (E), corresponding to a few kPa. Results on protein diffusivity, obtained by testing some selected formulations, are reported in section 2.2 and suggest that the decreased amount of Pluronic increases the “permeability” of the material to nutrients available in the medium surrounding the hydrogels. However, even the formulation showing the highest diffusivity between the ones tested, does not guarantee a sufficient level of human chondrocytes cell viability, highlighting a possible relevant role played by material chemistry, with the tested cell type. In conclusion, the PEG-fibrinogen-based hydrogels do not result in good candidates usable as the matrix of the nanocomposite materials envisaged in ADMAIORA.

Micro dosing pumps are the beating heart of infusion systems. Among many technologies to inject micro quantities of fluids, peristaltic pumps show high precision and the possibility to not alter the fluid properties. However, in real drug delivery applications, the continuous release behavior of typical peristaltic pumps is not favorable. In this paper, we investigate the intermittent performance of two prototypes of peristaltic pumps, based on four and five rollers, used to occlude the tube. The pump performances are reported for different rotation speeds and lag times between consecutive infusions. The proposed pumps showed a good volumetric precision (2.88 µL for the five rollers pump and 4.11 µL for the four rollers pump) without any dependency on rotation speed and lag time.

Biohybrid actuators have the potential to overcome the limitations of traditional actuators employed in robotics, thanks to the unique features of living contractile muscle cells, which can be used to power artificial elements. This paper describes a computational approach for the estimation of the contractile capabilities of skeletal muscle cell-powered biohybrid actuators based on polymeric thin films. The proposed model grounds on the coupling between finite element modeling and smooth particle hydrodynamics. This allows describing the overall condition, including the viscous forces caused by the surrounding liquid medium, in which biohybrid systems are normally immersed. The model is calibrated by analyzing the contractile behavior of polydimethylsiloxane films coupled with skeletal muscle cells, reported in the literature as muscular thin films. Afterward, it is applied to poly (D, L-lactic acid) thin films to explore the behavior of these systems, due to myotubes cultured on them, evaluating the role of thickness, tissue maturation status, and hydrostatic pressure on the contractile performance. These results pave the way toward a novel optimization approach of biohybrid robot design relying on the simulation of all the boundary conditions, thus reducing the need for extensive trial-and-error efforts.

Urinary incontinence affects more than 300 million people worldwide. The implantation of a medical device called an artificial urinary sphincter (AUS) is the gold standard treatment when conservative and minimally invasive therapies fail. In this article, the AUSs (extra-urethral and endo-urethral sphincters) available on the market, both presented at the research level and filed as patents, are reviewed. The ability of the different solutions to effectively replace the natural sphincter are discussed, together with advantages and some possible side effects, such as tissue atrophy, overall invasiveness of the implant, and so forth. Finally, future research priorities are discussed for both endo-urethral and extra-urethral approaches considering key engineering aspects, such as materials, compression and closure mechanisms, and implantation methods, with the long-term aim of developing an effective, reliable, durable, and minimally invasive AUS capable of restoring a normal quality of life for incontinent patients.

Colorectal cancer (CRC) is one of the major causes of cancer-associated mortality worldwide. The currently approved therapeutic agents show a rather limited efficacy. We have recently demonstrated that the atypical cadherin FAT1 is a specific marker of CRC and that the FAT1-specific monoclonal antibody mAb198.3 may offer new therapeutic opportunities for CRC, being efficiently internalized by cancer cells and reducing cancer growth in colon cancer xenograft models. In this study we explored the therapeutic efficacy of mAb198.3 using two drug delivery systems (DDS) for improving the targeted treatment of CRC. The mAb198.3 was either directly bound to super-paramagnetic nanoparticles (spmNPs) or embedded into human erythrocyte-based magnetized carriers, named Erythro-Magneto-Hemagglutinin Virosomes (EMHVs) to produce two different novel mAb198.3 formulations. Both DDS were endowed with magnetic properties and were anchored in the target tumor site by means of an external permanent magnet. The antibody loading efficiency of these two magnetically driven drug delivery systems and the overall therapeutic efficacy of these two formulations were assessed both in vitro and in a proof-of-concept in vivo study. We demonstrated that mAb198.3 bound to spmNPs or embedded into EMHVs was very effective in targeting FAT1-positive colon cancer cells in vitro and accumulating in the tumor mass in vivo. Although both in vivo administered mAb198.3 formulations have approximately 200 lower antibody doses needed, these showed to achieve a relevant therapeutic effect, thus reducing cancer growth more efficiently respect to the naked antibody. These results indicate that the two proposed magnetically driven drug delivery systems have a considerable potential as platforms to improve bioavailability and pharmacodynamics of anti-FAT mAb198.3 and raise new opportunities for a targeted therapy of CRC.

ollagen-based hydrogels are an attractive option in the field of cartilage regeneration with features of high biocompatibility and low immunogenic response. Crosslinking treatments are often employed to create stable 3D gels that can support and facilitate cell embodiment. In this study, we explored the properties of JellaGel™, a novel jellyfish material extracted fromRhizostoma pulmo. In particular, we analyzed the influence of genipin, a natural crosslinker, on the formation of 3D stable JellaGel™ hydrogels embedding human chondrocytes. Three concentrations of genipin were used for this purpose (1 mM, 2.5 mM, and 5 mM). Morphological, thermal, and mechanical properties were investigated for the crosslinked materials. The metabolic activity of embedded chondrocytes was also evaluated at different time points (3, 7, and 14 days). Non-crosslinked hydrogels resulted in an unstable matrix, while genipin-crosslinked hydrogels resulted in a stable matrix, without significant changes in their properties; their collagen network revealed characteristic dimensions in the order of 20 µm, while their denaturation temperature was 57 °C. After 7 and 14 days of culture, chondrocytes showed a significantly higher metabolic activity within the hydrogels crosslinked with 1 mM genipin, compared to those crosslinked with 5 mM genipin.&nbsp

This work aims to describe the development and validation of two low-intensity pulsed ultrasound stimulation systems able to control the dose delivered to the biological target. Transducer characterization was performed in terms of pressure field shape and intensity, for a high-frequency range (500kHz to 5MHz) and for a low-frequency value (38kHz). This allowed defining the distance, on the beam axis, at which biological samples should be placed during stimulation and to exactly know the intensity at the target. Carefully designed retaining systems were developed, for hosting biological samples. Sealing tests proved their impermeability to external contaminants. The assembly/de-assembly time of the systems resulted ~3min. Time-domain acoustic simulations allowed to precisely estimate the ultrasound beam within the biological sample chamber, thus enabling the possibility to precisely control the pressure to be transmitted to the biological target, by modulating the transducer's input voltage. Biologicalin vitrotests were also carried out, demonstrating the sterility of the system and the absence of toxic and inflammatory effects on growing cells after multiple immersions in water, over seven days.

Described here is an in vitro systematic investigation of the effects on C2C12 myoblasts of exposure to finely controlled and repeatable low-intensity pulsed ultrasound of different frequencies (500 kHz, 1 MHz, 3 MHz and 5 MHz) and different intensities (250, 500 and 1000 mW/cm2). An in-house stimulation system and an ultrasound-transparent cell culture well minimized reflections and attenuations, allowing precise control of ultrasound delivery. Results indicated that a 3 MHz stimulation at 1 W/cm2 intensity maximized cell proliferation in comparison with the other exposure conditions and untreated controls. In contrast, cell differentiation and the consequent formation of multinucleated myotubes were maximized by 1 MHz stimulation at 500 mW/cm2 intensity. The highly controlled exposure conditions employed allowed precise correlation of the ultrasound delivery to the bio-effects produced, thus overcoming the inconsistency of some results available in the literature and contributing to the potential of ultrasound treatment for muscle therapy and regeneration.

Urinary incontinence is a largely spread disfunction that affects more than 300 million people worldwide. At present, no technological solutions are able to restore continence in a minimally invasive and effective way. In this article the authors report the design, fabrication, and testing of a novel artificial endourethral urinary sphincter able to fully restore continence. The device can be inserted/retracted in a minimally invasive fashion without hospital admission, does not alter the body scheme and can be applied to both women and men. The device core is a unidirectional polymeric valve and a magnetically activated system able to modulate its opening pressure. Bench tests and ex vivo tests on a human cadaver demonstrated that the device is able to fully restore continence and to allow urination when desired. Overall, the proposed system shows a high potential as a technological solution able to restore a normal daily life in patients affected by urinary incontinence.

An artificial sphincter is a device that replaces the function of the biological sphincter by occluding the relative biological lumen. The investigation of occlusion methods for artificial sphincters is crucial for a reliable and effective design of such devices. The compression induced onto the tissue by a certain pressure depends on the biomechanical and physiological features of the lumen and on the specific occlusion method. A numerical model and an experimental evaluation are presented here to assess the efficiency of different occlusion methods. Numerical models of circumferential occlusion and clamping occlusion methods to simulate the compression of the biological lumen were developed. Results revealed a relationship between the efficiency of the occlusion method and the physiological condition of the lumen. With differences related to the testing setup, this relationship was also confirmed experimentally by conducting tests on biological simulators. We analyzed the occlusion method to adopt as the physiological pressure (ie, leakage pressure values) changed. In particular, we focused on the urinary incontinence, which is a dysfunction involving the external sphincter surrounding the urethra. In this scenario, we demonstrated that a clamping occlusion is an efficient method to compress the urethra, whose physiological pressures range between 4 and 12 kPa. The clamping occlusion method resulted up to 35% more efficient in terms of sealing pressure than the circumferential one for a closing pressure varying between 2.3 and 11.5 kPa.

The ADMAIORA Management Handbook contains the instructions concerning the project procedures and other useful information to be used during the project lifetime. The Handbook includes: (1) official information about the project contract and annexes, (2) the description of the project organisation (governance structure, project coordinator, project managers, board of partners, etc.) and conflict management, (3) a summary of the tentative plan of meetings of the BoP and IPEC during the whole duration of the project, (4) a complete list of the institutions and persons involved in the ADMAIORA project, (5) a description of the project management procedures and tools used to share key documents, to provide support on financial and administrative issues, to prepare project Deliverables and periodic reports, to facilitate the communication among partners and to organise periodic meetings and videoconferences. By means of this information the Handbook is intended as an aid to all project beneficiaries, but also as a useful management tool for the coordinating institution.

The deliverable 7.1 includes complete information about the Web page of the ADMAIORA project. It has been developed with the aim to pursue effective Project dissemination and communication activities.

This deliverable consists of the Initial Data Management Plan. It describes the general strategy that the ADMAIORA consortium adopted to fulfil the Open Access Research Data Pilot and to create a FAIR ecosystem. The deliverable refers to the chosen repository features, the interplay between data management, dissemination activities and the overall management of the project, taking into account the related legal-ethical issues, where the research data are personal ones. The deliverable includes an introduction, the description of the data management strategy, the general rules to be applied to identified categories of research information, and the specific ones for each public deliverable produced in the project course.

This work aims to describe the design and development of an in vitro highly controlled ultrasonic stimulation system able to guarantee, at the same time, high usability and full sterility of the tested samples. After creating the first prototype of an ultrasound-transparent three-chambers culture well, sealing tests were conducted to prove its impermeability to external contaminants and in vitro tests were carried out to verify the usability of this system for ultrasonic stimulation of cells in vitro. No statistically significant differences were found between control and tested samples during sealing tests, thus demonstrating optimal sealing ability towards external contaminants. Furthermore, the thin polystyrene membrane used to guarantee US-transparency guaranteed a good adhesion and viability of both human fibroblasts and induced pluripotent stem cells.

Untethered magnetic microrobots hold the potential to penetrate hard-to-reach areas of the human body and to perform therapy in a controlled way. In the past decade, impressive advancements have been made in this field but the clinical adoption of magnetoresponsive microrobots is still hampered by safety issues. A tool appointed for magnetic microrobots retrieval within body fluids could enable a real paradigm change, fostering their clinical translation.By starting from the general problem to retrieve magnetic microrobots injected into the bloodstream, the authors introduce a magnetic capture model that allows to design retrieval tools for magnetic cores of different diameters (down to 10 nm) and in different environmental conditions (fluid speed up to 7 cms-1). The model robustness is demonstrated by the design and testing of a retrieval catheter. In its optimal configuration, the catheter includes 27 magnets and fits a 12 F catheter. The model provides a good prediction of capture efficiency for 250 nm magnetic particles (experimental data: 77.6%, model prediction: 65%) and a very good prediction for 500 nm particles (experimental data: 93.6%, model prediction: 94%). The results support the proposed model-based design approach, which can be extended to retrieve other magnetoresponsive agents from body compartments.

Ultrasound stimulation (US) carries the promise of a selective, reversible, and non-invasive modulation of neural activity without the need for genetic manipulation of neural structures. However, the mechanisms of US-induced generation of action potentials (APs) are still unclear. Here we address this issue by analyzing intracellularly recorded responses to controlled delivery of US of leech nociceptive neurons. US induced a depolarization linearly accumulating in time and outlasting the duration of the stimulation. Spiking activity was reliably induced for an optimal US intensity range. Moreover, we found that APs induced by US differ in smaller amplitude and faster repolarization from those induced by electrical (EL) stimulation in the same cell, but keep the same repolarization rate. These results shed light on the mechanism by which spikes are induced by US and pave the way for designing more efficient US patterns.

Skeletal muscle differentiation was proven to be influenced by changes in the substrate stiffness. However, a lack of knowledge features this field, concerning skeletal muscle tissues obtained from human induced pluripotent stem cells. Here we report the fabrication of polydimethylsiloxane-based substrates in a range of stiffness values from 3.5 to 141 kPa and the response of human induced pluripotent stem cells cultured on them for 5 days. The substrates were able to sustain cell adhesion and proliferation throughout the whole period. An inversely proportional relationship (although not significant) was found between the proliferation rate and the substrate stiffness. Initial analyses of iPSCs skeletal muscle differentiation shown no influences on markers of the early stages. These results lay the foundations for further studies on the influence of extrinsic mechanical stimuli on induced pluripotent stem cells-derived skeletal muscle tissues.

The development of small-caliber grafts still represents a challenge in the field of vascular prostheses. Among other factors, the mechanical properties mismatch between natural vessels and artificial devices limits the efficacy of state-of-the-art materials. In this paper, a novel nanocomposite graft with an internal diameter of 6 mm is proposed. The device is obtained through spray deposition using a semi-interpenetrating polymeric network combining poly(ether)urethane and polydimethilsyloxane. The inclusion of BaTiO3 nanoparticles endows the scaffold with piezoelectric properties, which may be exploited in the future to trigger beneficial biological effects. Graft characterization demonstrated a good nanoparticle dispersion and an overall porosity that was not influenced by the presence of nanoparticles. Graft mechanical properties resembled (or even ameliorated) the ones of natural vessels: both doped and non-doped samples showed a Young's modulus of ∼700 kPa in the radial direction and ∼900 kPa in the longitudinal direction, an ultimate tensile strength of ∼1 MPa, a strain to failure of ∼700%, a suture retention force of ∼1.7 N and a flexural rigidity of ∼2.5 × 10−5 N m2. The two grafts differed in terms of burst strength that resulted ∼800 kPa for the control non-doped samples and ∼1100 kPa for the doped ones. The graft doped with BaTiO3 nanoparticles showed a d33 coefficient of 1.91 pm/V, almost double than the non-doped control. The device resulted highly stable, with a mass loss smaller than 2% over 3 months and an excellent biocompatibility.

A highly crosslinked composite dextran-based scaffold (named DexFoam) was tailored to overcome specific deficiencies of polymeric and ceramic bone scaffolds and to guarantee a bone-mimicking microenvironment for the proliferation of human mesenchymal stem cells in vitro. The creep resistance for up to 90% compressive stain, the capability to regain the original shape after deformation, and the good thermal stability in both physiological and “body limit” conditions make DexFoam a valid alternative to the currently available bone scaffolds. Histopathological evaluation for host reaction and tissue colonization of DexFoam scaffold, implanted subcutaneously in mice, demonstrated its in vivo biocompatibility and biodegradability.

In the state-of-the-art, only few studies analyzed volume changes in residual lower limbs. Most of them focused on transtibial amputees, while data on transfemoral ones are largely missing. This study aims at investigating how transfemoral stumps change over time. A measurement system based on a portable 3D optical scanning technique was used to measure volume fluctuations in 6 transfemoral amputees prior to and 20 min after physical activity. Such technique allowed to measure the stump volume once outside of the prosthetic socket. Investigating stump volume changes over time after prosthesis doffing is necessary to take into account this source of error during tests. Data show that volume fluctuations range from −1.25% to +3.09%. These values were enough to cause patient discomfort. This study highlights the need to properly characterize volume changes with the aim of designing more efficient socket solutions, based on such data.

Traditional drug release systems normally rely on a passive delivery of therapeutic compounds, which can be partially programmed, prior to injection or implantation, through variations in the material composition. With this strategy, the drug release kinetics cannot be remotely modified and thus adapted to changing therapeutic needs. To overcome this issue, drug delivery systems able to respond to external stimuli are highly desirable, as they allow a high level of temporal and spatial control over drug release kinetics, in an operator-dependent fashion.On-demand drug delivery systems actually represent a frontier in this field and are attracting an increasing interest at both research and industrial level. Stimuli-responsive thin films, enabled by nanofillers, hold a tremendous potential in the field of triggerable drug delivery systems. The inclusion of responsive elements in homogeneous or heterogeneous thin film-shaped polymeric matrices strengthens and/or adds intriguing properties to conventional (bare) materials in film shape.This Expert Opinion review aims to discuss the approaches currently pursued to achieve an effective on-demand drug delivery, through nanocomposite thin films. Different triggering mechanisms allowing a fine control on drug delivery are described, together with current challenges and possible future applications in therapy and surgery.