Improvement of Osteogenic Differentiation of Mouse Pre-Osteoblastic MC3T3-E1 Cells on Core-Shell Polylactic Acid/Chitosan Electrospun Scaffolds for Bone Defect Repair.

Electrospun hybrid scaffolds composed of synthetic and natural polymers have gained increasing interest in tissue engineering applications over the last decade. In this work, scaffolds composed of polylactic acid electrospun fibers, either treated (P-PLA) or non-treated (PLA) with air-plasma, were coated with high molecular weight chitosan to create a core-shell microfibrous structure. The effective thickness control of the chitosan layer was confirmed by gravimetric, spectroscopic (FTIR-ATR) and morphological (SEM) investigations. The chitosan coating increased the fiber diameter of the microfibrous scaffolds while the tensile mechanical tests, conducted in dry and wet environments, showed a reinforcing action of the coating layer on the scaffolds, in particular when deposited on P-PLA samples. The stability of the Chi coating on both PLA and P-PLA substrates was confirmed by gravimetric analysis, while their mineralization capacity was evaluated though scanning electron microscopy (SEM) and energy-dispersive spectroscopy (EDS) after immersing the scaffolds in simulated body fluids (SBF) at 37 °C for 1 week. Sample biocompatibility was investigated through cell viability assay and SEM analysis on mouse pre-osteoblastic MC3T3-E1 cells grown on scaffolds at different times (1, 7, 14 and 21 days). Finally, Alizarin Red assay and qPCR analysis suggested that the combination of plasma treatment and chitosan coating on PLA electrospun scaffolds influences the osteoblastic differentiation of MC3T3-E1 cells, thus demonstrating the great potential of P-PLA/chitosan hybrid scaffolds for bone tissue engineering applications.

Collagen-Based Scaffolds for Chronic Skin Wound Treatment.

Chronic wounds, commonly known as ulcers, represent a significant challenge to public health, impacting millions of individuals every year and imposing a significant financial burden on the global health system. Chronic wounds result from the interruption of the natural wound-healing process due to internal and/or external factors, resulting in slow or nonexistent recovery. Conventional medical approaches are often inadequate to deal with chronic wounds, necessitating the exploration of new methods to facilitate rapid and effective healing. In recent years, regenerative medicine and tissue engineering have emerged as promising avenues to encourage tissue regeneration. These approaches aim to achieve anatomical and functional restoration of the affected area through polymeric components, such as scaffolds or hydrogels. This review explores collagen-based biomaterials as potential therapeutic interventions for skin chronic wounds, specifically focusing on infective and diabetic ulcers. Hence, the different approaches described are classified on an action-mechanism basis. Understanding the issues preventing chronic wound healing and identifying effective therapeutic alternatives could indicate the best way to optimize therapeutic units and to promote more direct and efficient healing.

Nanoformulations based on collagenases loaded into halloysite/Veegum® clay minerals for potential pharmaceutical applications.

The design and development of nanomaterials capable of penetrate cancer cells is fundamental when anticancer therapy is involved. The use of collagenase (Col) is useful since this enzyme can degrade collagen, mainly present in the tumor extracellular matrix. However, its use is often limited since collagenase suffers from inactivation and short half-life. Use of recombinant ultrapure collagenase or carrier systems for their delivery are among the strategies adopted to increase the enzyme stability. Herein, based on the more stability showed by recombinant enzymes and the possibility to use them in anticancer therapy, we propose a novel strategy to further increase their stability by using halloysite nanotubes (HNTs) as carrier. ColG and ColH were supramolecularly loaded onto HNTs and used as fillers for Veegum gels. The systems could be used for potential local administration of collagenases for solid tumor treatment. All techniques adopted for characterization showed that halloysite interacts with collagenases in different ways depending with the Col considered. Furthermore, the hydrogels showed a very slow release of the collagenases within 24 h. Finally, biological assays were performed by studying the digestion of a type-I collagen matrix highlighting that once released the Col still possessed some activity. Thus we developed carrier systems that could further increase the high recombinant collagenases stability, preventing their inactivation in future in vivo applications for potential local tumor treatment.

Recovery of Bioactive Compounds from Marine Organisms: Focus on the Future Perspectives for Pharmacological, Biomedical and Regenerative Medicine Applications of Marine Collagen.

Marine environments cover more than 70% of the Earth's surface and are among the richest and most complex ecosystems. In terms of biodiversity, the ocean represents an important source, still not widely exploited, of bioactive products derived from species of bacteria, plants, and animals. However, global warming, in combination with multiple anthropogenic practices, represents a serious environmental problem that has led to an increase in gelatinous zooplankton, a phenomenon referred to as jellyfish bloom. In recent years, the idea of "sustainable development" has emerged as one of the essential elements of green-economy initiatives; therefore, the marine environment has been re-evaluated and considered an important biological resource. Several bioactive compounds of marine origin are being studied, and among these, marine collagen represents one of the most attractive bio-resources, given its use in various disciplines, such as clinical applications, cosmetics, the food sector, and many other industrial applications. This review aims to provide a current overview of marine collagen applications in the pharmacological and biomedical fields, regenerative medicine, and cell therapy.

Tolerance, Adaptation, and Cell Response Elicited by Micromonospora sp. Facing Tellurite Toxicity: A Biological and Physical-Chemical Characterization.

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.

Functionalized Poly(N-isopropylacrylamide)-Based Microgels in Tumor Targeting and Drug Delivery.

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.

Core-shell PLA/Kef hybrid scaffolds for skin tissue engineering applications prepared by direct kefiran coating on PLA electrospun fibers optimized via air-plasma treatment.

Over the recent years, there is a growing interest in electrospun hybrid scaffolds composed of synthetic and natural polymers that can support cell attachment and proliferation. In this work, the physical and biological properties of polylactic acid (PLA) electrospun mats coated with kefiran (Kef) were evaluated. Gravimetric, spectroscopic (FTIR-ATR) and morphological investigations via scanning electron microscopy confirmed the effective formation of a thin kefiran layer wrapped on the PLA fibers with an easy-tunable thickness. Air plasma pre-treatment carried out on PLA (P-PLA) affected both the morphology and the crystallinity of Kef coating as confirmed by differential scanning calorimetry and X-ray diffraction analyses. Scaffolds were mechanically characterized with tensile tests to evaluate the reinforcing action of the Kef coating. The water resistance of Kefiran coating in distilled water at 37 °C evaluated on both PLA/Kef and P-PLA/Kef was carried out by gravimetric and morphological analyses. Finally, cell culture assays with embryonic fibroblast cells were conducted on selected hybrid scaffolds to compare the cell proliferation, morphology, and collagen production with PLA and P-PLA electrospun scaffolds. Based on the results, we can demonstrate that direct coating of PLA from Kef/water solutions is an effective approach to prepare hybrid scaffolds with tunable properties and that the plasma pre-treatment enhances the affinity between PLA and Kefiran. In vitro tests demonstrated the great potential of PLA/Kef hybrid scaffolds for skin tissue engineering.

3D Collagen Hydrogel Promotes In Vitro Langerhans Islets Vascularization through ad-MVFs Angiogenic Activity.

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.

Neural Crest-Derived Chondrocytes Isolation for Tissue Engineering in Regenerative Medicine.

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.

Multi-Functional Nanogels for Tumor Targeting and Redox-Sensitive Drug and siRNA 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.