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Calcium phosphate/Bioglass composite coatings on AISI 316L were investigated with regard to their potential role as a beneficial coating for orthopedic implants. These coatings were realized by the galvanic co-deposition of calcium phosphate compounds and Bioglass particles. A different amount of Bioglass 45S5 was used to study its effect on the performance of the composite coatings. The morphology and chemical composition of the coatings were investigated before and after their aging in simulated body fluid. The coatings uniformly covered the AISI 316L substrate and consisted of a brushite and hydroxyapatite mixture. Both phases were detected using X-ray diffraction and Raman spectroscopy. Additionally, both analyses revealed that brushite is the primary phase. The presence of Bioglass was verified through energy-dispersive X-ray spectroscopy, which showed the presence of a silicon peak. During aging in simulated body fluid, the coating was subject to a dynamic equilibrium of dissolution/reprecipitation with total conversion in only the hydroxyapatite phase. Corrosion tests performed in simulated body fluid at different aging times revealed that the coatings made with 1 g/L of Bioglass performed best. These samples have a corrosion potential of −0.068V vs. Ag/AgCl and a corrosion current density of 8.87 × 10−7 A/cm2. These values are better than those measured for bare AISI 316L (−0.187 V vs. Ag/AgCl and 2.52 × 10−6 A/cm2, respectively) and remained superior to pure steel for all 21 days of aging. This behavior indicated the good protection of the coating against corrosion phenomena, which was further confirmed by the very low concentration of Ni ions (0.076 ppm) released in the aging solution after 21 days of immersion. Furthermore, the absence of cytotoxicity, verified through cell viability assays with MC3T3-E1 osteoblastic cells, proves the biocompatibility of the coatings.

Polysaccharides are long chains of monosaccharides linked by glycosidic bonds. They are widely utilized in biomedical applications, for tissue engineering and wound management, and as excipients of pharmaceutical formulations. In cancer therapy, the development of nanoscale drug delivery systems aims at addressing issues related to the low efficacy of chemotherapeutics and protein drugs due to poor solubility and stability, and to off-target effects that severely affect patients’ body conditions. Biocompatibility, availability of functional groups, amenability of chemical derivatisation and multifunctional conjugation with drugs and targeting ligands make polysaccharide nanoparticles interesting drug delivery devices for cancer treatment. Targeting and controlling the drug release at the tumour site are promising approaches to maximise the anti-tumour effects and reduce side-effects. Xyloglucan is a highly branched, hydrophilic polysaccharide composed of a -D-glucan main chain and -D-xylose branches, partially substituted by -D-galactoxylose. Xyloglucan extracted from tamarind seeds (TS-XG) is commercially available, non-toxic, biodegradable and FDA-approved as food additive. Due to the high molecular weight and tendency to associate in ribbon-like aggregates, aqueous colloidal dispersions of xyloglucan are characterised by relatively large nanoparticles with a broad size distribution. Gamma irradiation of either the colloidal dispersion of xyloglucan (XG) or the solid powder is a very effective and clean methodology to resize the molecular weight of the polysaccharide and reduce the particle size of its colloidal dispersions. When xyloglucan is irradiated as aqueous dispersion, irradiation causes extensive chain scission, with a dramatic reduction of molecular weight already at low doses (50-500 Gy). In contrast, when it is irradiated in the solid state, higher doses are required to change the molecular weight (20-40 kGy), with moisture playing a protective role. Selected systems have been conjugated to doxorubicin (DOX), a powerful anticancer drug that is extensively applied in the clinical treatment of human malignancies, such as leukemia and cancer of the liver, ovary and breast. This drug causes severe peripheral toxicity that poses limits to the maximum dosage that can be administered, and the emergence of multidrug resistance. DOX has been bound to irradiated XG through a cleavable bond and then further loaded with DOX by self-assembly. Xyloglucan contains galactose, which can be recognised and internalised by asialoglycoprotein receptor (ASGP-R). In order to increase the targeting efficiency, XG has been also conjugated to folic acid. Drug release efficiency and tumour cells selectivity are under investigation.

Over the last decades, nanotechnology applied in medicine (nanomedicine) has sparked great interest from the scientific community, thanks to the possibility to engineer nanostructured materials, including nanoparticles (NPs), for a specific application. Their small size confers them unique properties because they are subject to physical laws in the middle between classical and quantum physics. This review is proposed to explain better how to design a specific NP and clarify the relationship between the type, size, and shape of NPs and the specific medical applications. NPs are classified into inorganic (metallic NPs, quantum dots, carbon-based nanostructures, mesoporous silica NPs) and organic (liposomes and micelles, dendrimers, and polymer NPs). Here, we report an accurate description of the potential of each NPs type focusing on their multiple areas of application, including theranostics drug delivery, imaging, tissue engineering, antimicrobial techniques, and nanovaccines. All these features make NPs a promise to revolutionize the new era of nanomedicine.

The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO32−) is the most soluble, bioavailable, and toxic Te-species. On the other hand, tellurium is a rare metalloid element whose natural supply will end shortly with possible economic and technological effects. Thus, Te-containing waste represents the source from which Te should be recycled and recovered. Among the explored strategies, the microbial TeO32− biotransformation into less toxic Te-species is the most appropriate concerning the circular economy. Actinomycetes are ideal candidates in environmental biotechnology. However, their exploration in TeO32− biotransformation is scarce due to limited knowledge regarding oxyanion microbial processing. Here, this gap was filled by investigating the cell tolerance, adaptation, and response to TeO32− of a Micromonospora strain isolated from a metal(loid)-rich environment. To this aim, an integrated biological, physical-chemical, and statistical approach combining physiological and biochemical assays with confocal or scanning electron (SEM) microscopy and Fourier-transform infrared spectroscopy in attenuated total reflectance mode (ATR-FTIR) was designed. Micromonospora cells exposed to TeO32− under different physiological states revealed a series of striking cell responses, such as cell morphology changes, extracellular polymeric substance production, cell membrane damages and modifications, oxidative stress burst, protein aggregation and phosphorylation, and superoxide dismutase induction. These results highlight this Micromonospora strain as an asset for biotechnological purposes.

The intense use of tellurium (Te) in industrial applications, along with the improper disposal of Te-derivatives, is causing their accumulation in the environment, where oxyanion tellurite (TeO

The perfusion of flow during cell culture induces cell proliferation and enhances cellular activity. Perfusion bioreactors offer a controlled dynamic environment for reliable in vitro applications in the tissue engineering field. In this work, to evaluate the effects of the operating parameters of a custom-made bioreactor, numerical simulations were performed to solve the fluid velocity profile inside the bioreactor containing multi-grid support that allows allocating of multiple seeded scaffolds at the same time. The perfusion system exhibited a uniform distribution of liquid velocities within the regions, suitable for cell growth on seeded scaffolds. The effects of the porous microstructure of scaffolds on the extracellular matrix deposition also play a crucial role during perfusion cultures. In the present study, a numerical simulation was implemented at the pore level of the scaffold for fluid flow through porous media during perfused culture. Micro-computed tomography was used to obtain the digital 3D image of the complex geometry of a PLLA scaffold, offering a detailed analysis from a volume-based methodology without simplifications of the results as for pore or Darcy's law-models. Predictions about the uniformity of the flow field through the scaffolds-bioreactor system have been assessed by quantifying the cell viability of a perfusion culture while using pre-osteoblastic cells seeded on 24 PLLA scaffolds for up to 6 days.

Over the past several decades, the development of engineered small particles as targeted and drug delivery systems (TDDS) has received great attention thanks to the possibility to overcome the limitations of classical cancer chemotherapy, including targeting incapability, nonspecific action and, consequently, systemic toxicity. Thus, this research aims at using a novel design of Poly(N-isopropylacrylamide) p(NIPAM)-based microgels to specifically target cancer cells and avoid the healthy ones, which is expected to decrease or eliminate the side effects of chemotherapeutic drugs. Smart NIPAM-based microgels were functionalized with acrylic acid and coupled to folic acid (FA), targeting the folate receptors overexpressed by cancer cells and to the chemotherapeutic drug doxorubicin (Dox). The successful conjugation of FA and Dox was demonstrated by dynamic light scattering (DLS), Fourier-transform infrared (FTIR) spectroscopy, thermogravimetric analysis (TGA), UV-VIS analysis, and differential scanning calorimetry (DSC). Furthermore, viability assay performed on cancer and healthy breast cells, suggested the microgels’ biocompatibility and the cytotoxic effect of the conjugated drug. On the other hand, the specific tumor targeting of synthetized microgels was demonstrated by a co-cultured (healthy and cancer cells) assay monitored using confocal microscopy and flow cytometry. Results suggest successful targeting of cancer cells and drug release. These data support the use of pNIPAM-based microgels as good candidates as TDDS.

Adipose derived microvascular fragments (ad-MVFs) consist of effective vascularization units able to reassemble into efficient microvascular networks. Because of their content in stem cells and related angiogenic activity, ad-MVFs represent an interesting tool for applications in regenerative medicine. Here we show that gentle dissociation of rat adipose tissue provides a mixture of ad-MVFs with a length distribution ranging from 33–955 μm that are able to maintain their original morphology. The isolated units of ad-MVFs that resulted were able to activate transcriptional switching toward angiogenesis, forming tubes, branches, and entire capillary networks when cultured in 3D collagen type-I hydrogel. The proper involvement of metalloproteases (MMP2/MMP9) and serine proteases in basal lamina and extracellular matrix ECM degradation during the angiogenesis were concurrently assessed by the evaluation of alpha-smooth muscle actin (αSMA) expression. These results suggest that collagen type-I hydrogel provides an adequate 3D environment supporting the activation of the vascularization process. As a proof of concept, we exploited 3D collagen hydrogel for the setting of ad-MVF–islet of Langerhans coculture to improve the islets vascularization. Our results suggest potential employment of the proposed in vitro system for regenerative medicine applications, such as the improving of the islet of Langerhans engraftment before transplantation.

Nanotechnology is an emerging field of modern science based on the use of nanoparticles (NPs) with a huge potential in many sectors, including nanomedicine. Their small size confers them unique properties because they are subject to physical laws that are in the middle between classical and quantum physics. In this context, NPs project plays a pivotal role because the composition, size, shape and surface proprieties need to be carefully considered for their optimal design and application. As reported in this review, NPs are classified in inorganic (metallic NPs; quantum dots; carbon-based nanostructures; mesoporous silica nanoparticles) and organic (liposomes and micelles, dendrimers and polymer nanoparticles) ones. Here, we report an accurate description of the potential of each NPs type focusing on their multiple areas of application like theranostics drug delivery, imaging, tissue engineering, antimicrobial techniques and nanovaccines, and therefore they represent a promise to revolutionize the new era of nanomedicine, especially in cancer research.

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

Chondrocyte transplantation has been successfully tested and proposed as a clinical procedure aiming to repair articular cartilage defects. However, the isolation of chondrocytes and the optimization of the enzymatic digestion process, as well as their successful in vitro expansion, remain the main challenges in cartilage tissue engineering. In order to address these issues, we investigated the performance of recombinant collagenases in tissue dissociation assays with the aim of isolating chondrocytes from bovine nasal cartilage in order to establish the optimal enzyme blend to ensure the best outcomes of the overall procedure. We show, for the first time, that collagenase H activity alone is required for effective cartilage digestion, resulting in an improvement in the yield of viable cells. The extracted chondrocytes proved able to grow and activate differentiation/dedifferentiation programs, as assessed by morphological and gene expression analyses.

An amphiphilic copolymer carrying high-dose doxorubicin (21% on a weight basis), PHEA–EDA–P,C–Doxo, was prepared by coupling doxorubicin with a biocompatible polyaminoacid through a pH-sensitive spacer. Additional derivatization with 4-pentynoic acid endows it with self-assembling properties by means of π–π stacking. These micelles can be triggered to promptly release drug in lysosomes (∼40% in 12 h) through pH-dependent micelle hydrolysis after uptake. In vitro tests on co-cultures of cancer (MDA-MB 231) and normal (HB-2) breast cells proved that the conjugate was selectively internalized into the former rather than normal cells, exploiting the caveolae-dependent endocytosis pathway, explaining the selective cytotoxic effect toward cancer cells. Intracellular trafficking study of MDA-MB 231 showed that the delivery of the endocytosed drug occurs through the direct fusion of caveosomes with late lysosomes, triggering a massive release in the cytoplasm, bringing about cell death. Dose-effectiveness and mechanistic data indicate that PHEA–EDA–P,C–Doxo is endowed with a distinctive combination of selectivity and pharmacological potency (EC50 13 μM, Emax = 77% and EC50 > 25 μM, Emax = 21% for cancer and healthy cells respectively) that makes it an excellent candidate for future preclinical studies.

Direct isolation of primary cells from tissues and organs allows for the maintenance of important cell characteristics and properties for in vitro studies and a plethora of biomedical applications. Dissociation of cells from the organ of interest is possible due to the enzymatic activity of collagenases. The choice and the dose of these enzymes is the critical step to obtain the maximum number of cells with intact structure and function. In this contest, Abiel collagenases class I (Col G) and class II (Col H) were synthesised using recombinant DNA technologies and their ability to degrade collagen in cell isolation from different tissues was tested. Examples of cells isolated with these enzymes include Langerhans islets, hepatocytes, chondrocytes, osteoblasts, fibroblasts, and stem and retinal cells that be used for in vitro studies. In this contest, primary cardiomyocytes represent a perfect murine model to investigate numerous heart diseases. Herein, a protocol of cardiomyocyte extraction from rat heart, using a combination of Abiel collagenases supplemented with thermolysin protease, is proposed. The structure and the viability of isolated cells were tested over time by optical and fluorescence microscopy and viability assays. Further cellular structure characterisation was performed by western blot analysis, using specific cardiomyocytes markers. Isolation of viable primary heart cells with unaltered properties and functionality can potentially provide in vitro models to study heart function, arrhythmias, long Q-T syndrome and cardiotoxicity.

The development of various nanotechnologies have provided a new field of research, which allows the manipulation of molecular components of matter and covers a vast array of nanodevices. The “smart” multifunctional nanostructures should work as customizable, targeted drug-delivery vehicles capable of carrying large doses of therapeutic agents into malignant cells. Some nanomedical approaches are based on the use of functionalized nanoparticles (NPs), not only to reduce toxicity and side effects of drugs but, also in potential the biological barriers crossing on, such as: the blood–brain barrier, different cellular compartments, including the nucleus. Currently, many materials are used for nanoparticle preparation, several of biological derivation, such as albumin, gelatin, phospholipids, etc.; others of chemical nature, such as various polymers, hydrogels, solid metals. Covalent and noncovalent chemical linking using different molecules have been reported for NPs surface functionalization, confer them specific properties, such as targeting ability. Based on the strategy chosen to control release (pH or redox or temperature sensitive NPs), different drugs are linked to NPs by adsorption, incorporation, or chemical binding. Use of smart nanocarriers can be a successful approach to overcome the limits of drug delivery.

(1) Background: A new family of nanosystems able to discern between normal and tumor cells and to release a therapeutic agent in controlled way were synthetized by e-beam irradiation. This technique permits to obtain biocompatible, sterile, carboxyl-functionalized polyvinylpyrrolidone (PVP-co-acrylic acid) nanogels (NGs); (2) Methods: Here, we performed a targeting strategy based on the recognition of over-expressed proteins on tumor cells, like the folate receptor. The selective targeting was demonstrated by co-culture studies and flow cytometry analysis, using folate conjugated NGs. Moreover, nanoparticles were conjugated to a chemotherapeutic drug or to a pro-apoptotic siRNA through a glutathione sensitive spacer, in order to obtain a controlled release mechanism, specific for cancer cells. The drug efficiency was tested on tumor and healthy cells by flow cytometric analysis, confocal and epifluorescence microscopy and cytotoxicity assay; the siRNA effect was investigated by RNAi experiment; (3) Results: The data obtained showed that the use of NGs permits a faster cargo release in cancer cells, in response to high cytosolic glutathione level, also improving their efficacy; (4) Conclusion: The possibility of releasing biological molecules in a controlled way and to recognize a specific tumor target allows overcoming the typical limits of the classic cancer therapy.

In this work the sterilization with dense carbon dioxide of poly( l -lactic acid) (PLLA) porous scaffolds intended for tissue engineering applications was investigated with the main objective of confirming the three-dimensional efficacy of the treatment and of analysing the scaffold properties after CO2 treatment. For this purpose the scaffold was contaminated with a conventional bacterium (Escherichia coli) and with spores (Streptomyces coelicolor), a species more fascinating and difficult to inactivate. Contamination was performed in such a way to soak the whole matrix with bacteria and spores. The effect of pressure and treatment time on the efficacy of the sterilization was evaluated. The E. coli was eradicated from the whole matrix after just 5 min of exposure to supercritical CO2 at 10 MPa and 40 °C. The spores required more severe conditions due to their different and very resistant structures since complete inactivation was obtained after 360 min of exposure at 30 MPa and 40 °C or at 30 MPa and 30 °C after the addition of 200 ppm of H2O2 to near critical CO2. The treatment with dense CO2 did not alter the biocompatibility and the structure of the scaffold as demonstrated by biological culture tests and calorimetric and SEM analyses. Collected data suggest that dense CO2 is a promising alternative to conventional sterilization techniques to sterilize biodegradable PLLA scaffolds.

Biological structures control cell behavior via physical, chemical, electrical, and mechanical cues. Approaches that allow us to build devices that mimic these cues in a combinatorial way are lacking due to there being no suitable instructive materials and limited manufacturing procedures. This challenge is addressed by developing a new conductive composite material, allowing for the fabrication of 3D biomimetic structures in a single manufacturing method based on two?photon polymerization. The approach induces a combinatorial biostimulative input that can be tailored to a specific application. Development of the 3D architecture is performed with a chemically actuating photocurable acrylate previously identified for cardiomyocyte attachment. The material is made conductive by impregnation with multiwalled carbon nanotubes. The bioinstructive effect of 3D nano? and microtopography is combined with electrical stimulation, incorporating biochemical, and electromechanical cues to stimulate cells in serum?free media. The manufactured architecture is combined with cardiomyocytes derived from human pluripotent stem cells. It is demonstrated that by mimicking biological occurring cues, cardiomyocyte behavior can be modulated. This represents a step change in the ability to manufacture 3D multifunctional biomimetic modulatory architectures. This platform technology has implications in areas spanning regenerative medicine, tissue engineering to biosensing, and may lead to more accurate models for predicting toxicity.