An integrated design strategy coupling additive manufacturing and matrix-assisted pulsed laser evaporation (MAPLE) towards the development of a new concept 3D scaffold with improved properties for tissue regeneration.

Bioinspired strategies for scaffold design and optimization were improved by the introduction of Additive Manufacturing (AM), thus allowing for replicating and reproducing complex shapes and structures in a reliable manner, adopting different kinds of polymeric and nanocomposite materials properly combined according to the features of the natural host tissues. Benefiting from recent findings in AM, a Matrix-Assisted Pulsed Laser Evaporation (MAPLE) technique was employed for obtaining graphene-like material (GL) uniform coatings on 3D scaffolds for tissue repair strategies, towards the development of a new concept 3D scaffold with controlled morphological/architectural and surface features and mechanical and biological properties. The effect of the material-design combination through an integrated technological approach (i.e., MAPLE deposition of GL on 3D AM PCL scaffolds) was assessed through scanning electron microscopy, atomic force microscopy, contact angle measurements, mechanical measurements and biological analyses (cell viability assay and alkaline phosphatase activity) in conjunction with confocal laser scanning microscopy. The differentiation of hMSCs towards the osteoblast phenotype was also investigated analysing the gene expression profile. The obtained findings provided a further insight into the development of improved strategies for the functionalization or combination of GL with other materials and 3D structures in a hybrid fashion for ensuring a tighter adhesion onto the substrates, improving cell fate over time, without negatively altering the mechanical properties and behaviour of the neat constructs. In particular, the results provided interesting information, making 3D AM GL-coated scaffolds potential candidates for bone tissue engineering.

Development of Two-Layer Hybrid Scaffolds Based on Oxidized Polyvinyl Alcohol and Bioactivated Chitosan Sponges for Tissue Engineering Purposes.

Oxidized polyvinyl alcohol (OxPVA) is a new polymer for the fabrication of nerve conduits (NCs). Looking for OxPVA device optimization and coupling it with a natural sheath may boost bioactivity. Thus, OxPVA/chitosan sponges (ChS) as hybrid scaffolds were investigated to predict in the vivo behaviour of two-layered NCs. To encourage interaction with cells, ChS were functionalized with the self-assembling-peptide (SAP) EAK, without/with the laminin-derived sequences -IKVAV/-YIGSR. Thus, ChS and the hybrid scaffolds were characterized for mechanical properties, ultrastructure (Scanning Electron Microscopy, SEM), bioactivity, and biocompatibility. Regarding mechanical analysis, the peptide-free ChS showed the highest values of compressive modulus and maximum stress. However, among +EAK groups, ChS+EAK showed a significantly higher maximum stress than that found for ChS+EAK-IKVAV and ChS+EAK-YIGSR. Considering ultrastructure, microporous interconnections were tighter in both the OxPVA/ChS and +EAK groups than in the others; all the scaffolds induced SH-SY5Y cells' adhesion/proliferation, with significant differences from day 7 and a higher total cell number for OxPVA/ChS+EAK scaffolds, in accordance with SEM. The scaffolds elicited only a slight inflammation after 14 days of subcutaneous implantation in Balb/c mice, proving biocompatibility. ChS porosity, EAK 3D features and neuro-friendly attitude (shared with IKVAV/YIGSR motifs) may confer to OxPVA certain bioactivity, laying the basis for future appealing NCs.

New Aß(1-42) ligands from anti-amyloid antibodies: Design, synthesis, and structural interaction.

Alzheimer's disease (AD), is the most common neurodegenerative disorder of the aging population resulting in progressive cognitive and functional decline. Accumulation of amyloid plaques around neuronal cells is considered a critical pathogenetic event and, in most cases, a hallmark of the pathology. In the attempt to identify anti-AD drug candidates, hundreds of molecules targeting Aß peptides have been screened. Peptide molecules have been widely explored, appreciating chemical stability, biocompatibility, and low production cost. More recently, many anti-Aß(1-42) monoclonal antibodies have been developed, given the excellent potential of immunotherapy for treating or preventing AD. Antibodies are versatile ligands that bind a large variety of molecules with high affinity and specificity; however, their extensive therapeutic application is complex and requires huge economic investments. Novel approaches to identify alternative antibody formats are considered with great interest. In this context, taking advantage of the favorable peptide properties and the availability of Aß-antibodies structural data, we followed an innovative research approach to identify short peptide sequences on the model of the binding sites of Aß(1-42)/antibodies. WAibH and SYSTPGK were designed as mimics of solanezumab and aducanumab, respectively. Circular dichroism and nuclear magnetic resonance analysis reveal that the antibody-derived peptides interact with Aß(1-42) in the soluble monomeric form. Moreover, AFM microscopy imaging shows that WAibH and SYSTPGK are capable of controlling the Aß(1-42) aggregation. The strategy to identify WAibH and SYSTPGK is innovative and can be widely applied for new anti-Aß antibody mimicking peptides.

PEOT/PBT Polymeric Pastes to Fabricate Additive Manufactured Scaffolds for Tissue Engineering.

The advantages of additive manufactured scaffolds, as custom-shaped structures with a completely interconnected and accessible pore network from the micro- to the macroscale, are nowadays well established in tissue engineering. Pore volume and architecture can be designed in a controlled fashion, resulting in a modulation of scaffold's mechanical properties and in an optimal nutrient perfusion determinant for cell survival. However, the success of an engineered tissue architecture is often linked to its surface properties as well. The aim of this study was to create a family of polymeric pastes comprised of poly(ethylene oxide therephthalate)/poly(butylene terephthalate) (PEOT/PBT) microspheres and of a second biocompatible polymeric phase acting as a binder. By combining microspheres with additive manufacturing technologies, we produced 3D scaffolds possessing a tailorable surface roughness, which resulted in improved cell adhesion and increased metabolic activity. Furthermore, these scaffolds may offer the potential to act as drug delivery systems to steer tissue regeneration.

Covalent functionalization of decellularized tissues accelerates endothelialization.

In the field of tissue regeneration, the lack of a stable endothelial lining may affect the hemocompatibility of both synthetic and biological replacements. These drawbacks might be prevented by specific biomaterial functionalization to induce selective endothelial cell (EC) adhesion. Decellularized bovine pericardia and porcine aortas were selectively functionalized with a REDV tetrapeptide at 10-5 M and 10-6 M working concentrations. The scaffold-bound peptide was quantified and REDV potential EC adhesion enhancement was evaluated in vitro by static seeding of human umbilical vein ECs. The viable cells and MTS production were statistically higher in functionalized tissues than in control. Scaffold histoarchitecture, geometrical features, and mechanical properties were unaffected by peptide anchoring. The selective immobilization of REDV was effective in accelerating ECs adhesion while promoting proliferation in functionalized decellularized tissues intended for blood-contacting applications.

Erratum to "Design of Additively Manufactured Lattice Structures for Biomedical Applications".

[This corrects the article DOI: 10.1155/2020/2707560.].

Nipple Sparing Mastectomy as a Risk-Reducing Procedure for BRCA-Mutated Patients.

Growing numbers of asymptomatic women who become aware of carrying a breast cancer gene mutation (BRCA) mutation are choosing to undergo risk-reducing bilateral mastectomies with immediate breast reconstruction. We reviewed the literature with the aim of assessing the oncological safety of nipple-sparing mastectomy (NSM) as a risk-reduction procedure in BRCA-mutated patients. Nine studies reporting on the incidence of primary breast cancer post NSM in asymptomatic BRCA mutated patients undergoing risk-reducing bilateral procedures met the inclusion criteria. NSM appears to be a safe option for BRCA mutation carriers from an oncological point of view, with low reported rates of new breast cancers, low rates of postoperative complications, and high levels of satisfaction and postoperative quality of life. However, larger multi-institutional studies with longer follow-up are needed to establish this procedure as the best surgical option in this setting.

A 3D Printed Composite Scaffold Loaded with Clodronate to Regenerate Osteoporotic Bone: In Vitro Characterization.

Additive manufacturing (AM) is changing our current approach to the clinical treatment of bone diseases, providing new opportunities to fabricate customized, complex 3D structures with bioactive materials. Among several AM techniques, the BioCell Printing is an advanced, integrated system for material manufacture, sterilization, direct cell seeding and growth, which allows for the production of high-resolution micro-architectures. This work proposes the use of the BioCell Printing to fabricate polymer-based scaffolds reinforced with ceramics and loaded with bisphosphonates for the treatment of osteoporotic bone fractures. In particular, biodegradable poly(ε-caprolactone) was blended with hydroxyapatite particles and clodronate, a bisphosphonate with known efficacy against several bone diseases. The scaffolds' morphology was investigated by means of Scanning Electron Microscopy (SEM) and micro-Computed Tomography (micro-CT) while Energy Dispersive X-ray Spectroscopy (EDX) and X-ray Photoelectron Spectroscopy (XPS) revealed the scaffolds' elemental composition. A thermal characterization of the composites was accomplished by Thermogravimetric analyses (TGA). The mechanical performance of printed scaffolds was investigated under static compression and compared against that of native human bone. The designed 3D scaffolds promoted the attachment and proliferation of human MSCs. In addition, the presence of clodronate supported cell differentiation, as demonstrated by the normalized alkaline phosphatase activity. The obtained results show that the BioCell Printing can easily be employed to generate 3D constructs with pre-defined internal/external shapes capable of acting as a temporary physical template for regeneration of cancellous bone tissues.

Design of 3D Additively Manufactured Hybrid Structures for Cranioplasty.

A wide range of materials has been considered to repair cranial defects. In the field of cranioplasty, poly(methyl methacrylate) (PMMA)-based bone cements and modifications through the inclusion of copper doped tricalcium phosphate (Cu-TCP) particles have been already investigated. On the other hand, aliphatic polyesters such as poly(ε-caprolactone) (PCL) and polylactic acid (PLA) have been frequently investigated to make scaffolds for cranial bone regeneration. Accordingly, the aim of the current research was to design and fabricate customized hybrid devices for the repair of large cranial defects integrating the reverse engineering approach with additive manufacturing, The hybrid device consisted of a 3D additive manufactured polyester porous structures infiltrated with PMMA/Cu-TCP (97.5/2.5 w/w) bone cement. Temperature profiles were first evaluated for 3D hybrid devices (PCL/PMMA, PLA/PMMA, PCL/PMMA/Cu-TCP and PLA/PMMA/Cu-TCP). Peak temperatures recorded for hybrid PCL/PMMA and PCL/PMMA/Cu-TCP were significantly lower than those found for the PLA-based ones. Virtual and physical models of customized devices for large cranial defect were developed to assess the feasibility of the proposed technical solutions. A theoretical analysis was preliminarily performed on the entire head model trying to simulate severe impact conditions for people with the customized hybrid device (PCL/PMMA/Cu-TCP) (i.e., a rigid sphere impacting the implant region of the head). Results from finite element analysis (FEA) provided information on the different components of the model.

Impact of Magnetic Stimulation on Periodontal Ligament Stem Cells.

The aim of this study was to evaluate the effect of a time-dependent magnetic field on the biological performance of periodontal ligament stem cells (PDLSCs). A Western blot analysis and Alamar Blue assay were performed to investigate the proliferative capacity of magnetically stimulated PDLSCs (PDLSCs MAG) through the study of the MAPK cascade (p-ERK1/2). The observation of ALP levels allowed the evaluation of the effect of the magnetic field on osteogenic differentiation. Metabolomics data, such as oxygen consumption rate (OCR), extracellular acidification rate (ECAR) and ATP production provided an overview of the PDLSCs MAG metabolic state. Moreover, the mitochondrial state was investigated through confocal laser scanning microscopy. Results showed a good viability for PDLSCs MAG. Magnetic stimulation can activate the ERK phosphorylation more than the FGF factor alone by promoting a better cell proliferation. Osteogenic differentiation was more effectively induced by magnetic stimulation. The metabolic panel indicated significant changes in the mitochondrial cellular respiration of PDLSCs MAG. The results suggested that periodontal ligament stem cells (PDLSCs) can respond to biophysical stimuli such as a time-dependent magnetic field, which is able to induce changes in cell proliferation and differentiation. Moreover, the magnetic stimulation also produced an effect on the cell metabolic profile. Therefore, the current study demonstrated that a time-dependent magnetic stimulation may improve the regenerative properties of PDLSCs.

Breast Cancer Cell Cultures on Electrospun Poly(ε-Caprolactone) as a Potential Tool for Preclinical Studies on Anticancer Treatments.

During anticancer drug development, most compounds selected by in vitro screening are ineffective in in vivo studies and clinical trials due to the unreliability of two-dimensional (2D) in vitro cultures that are unable to mimic the cancer microenvironment. Herein, HCC1954 cell cultures on electrospun polycaprolactone (PCL) were characterized by morphological analysis, cell viability assays, histochemical staining, immunofluorescence, and RT-PCR. Our data showed that electrospun PCL allows the in vitro formation of cultures characterized by mucopolysaccharide production and increased cancer stem cell population. Moreover, PCL-based cultures were less sensitive to doxorubicin and electroporation/bleomycin than those grown on polystyrene plates. Collectively, our data indicate that PCL-based cultures may be promising tools for preclinical studies.

Stress Distributions for Hybrid Composite Endodontic Post Designs with and without a Ferrule: FEA Study.

The aim of the current work was to analyze the influence of the ferrule effect for hybrid composite endodontic post designs consisting of carbon (C) and glass (G) fiber-reinforced polyetherimide (PEI), in upper canine teeth. Starting from theoretical designs of C-G/PEI hybrid composite posts with different Young's moduli (Post A-57.7 GPa, Post B-31.6 GPa, Post C-graduated from 57.7 to 9.0 GPa in the coronal-apical direction) in endodontically treated anterior teeth, the influence of the ferrule effect was determined through finite element analysis (FEA). On the surface of the crown, a load of 50 N was applied at 45° to the longitudinal axis of the tooth. Maximum principal stresses were evaluated along the C-G/PEI post as well as at the interface between the surrounding tooth structure and the post. Maximum stress values were lower than those obtained for the corresponding models without a ferrule. The presence of a ferrule led to a marked decrease of stress and gradients especially for posts A and B. A less marked effect was globally found for Post C, except in a cervical margin section along a specific direction, where a significant decrease of the stress was probably due to local geometric features, compared to the model without a ferrule. The presence of a ferrule did not generally provide a marked benefit in the case of the graduated Post C, in comparison to other C-G/PEI posts. The outcomes suggest how such a hybrid composite post alone should be sufficient to optimize the stress distribution, dissipating stress from the coronal to the apical end.

A Preliminary Evaluation of the Pro-Chondrogenic Potential of 3D-Bioprinted Poly(ester Urea) Scaffolds.

Degeneration of articular cartilage (AC) is a common healthcare issue that can result in significantly impaired function and mobility for affected patients. The avascular nature of the tissue strongly burdens its regenerative capacity contributing to the development of more serious conditions such as osteoarthritis. Recent advances in bioprinting have prompted the development of alternative tissue engineering therapies for the generation of AC. Particular interest has been dedicated to scaffold-based strategies where 3D substrates are used to guide cellular function and tissue ingrowth. Despite its extensive use in bioprinting, the application of polycaprolactone (PCL) in AC is, however, restricted by properties that inhibit pro-chondrogenic cell phenotypes. This study proposes the use of a new bioprintable poly(ester urea) (PEU) material as an alternative to PCL for the generation of an in vitro model of early chondrogenesis. The polymer was successfully printed into 3D constructs displaying adequate substrate stiffness and increased hydrophilicity compared to PCL. Human chondrocytes cultured on the scaffolds exhibited higher cell viability and improved chondrogenic phenotype with upregulation of genes associated with type II collagen and aggrecan synthesis. Bioprinted PEU scaffolds could, therefore, provide a potential platform for the fabrication of bespoke, pro-chondrogenic tissue engineering constructs.

Combination Design of Time-Dependent Magnetic Field and Magnetic Nanocomposites to Guide Cell Behavior.

The concept of magnetic guidance is still challenging and has opened a wide range of perspectives in the field of tissue engineering. In this context, magnetic nanocomposites consisting of a poly(ε-caprolactone) (PCL) matrix and iron oxide (Fe3O4) nanoparticles were designed and manufactured for bone tissue engineering. The mechanical properties of PCL/Fe3O4 (80/20 w/w) nanocomposites were first assessed through small punch tests. The inclusion of Fe3O4 nanoparticles improved the punching properties as the values of peak load were higher than those obtained for the neat PCL without significantly affecting the work to failure. The effect of a time-dependent magnetic field on the adhesion, proliferation, and differentiation of human mesenchymal stem cells (hMSCs) was analyzed. The Alamar Blue assay, confocal laser scanning microscopy, and image analysis (i.e., shape factor) provided information on cell adhesion and viability over time, whereas the normalized alkaline phosphatase activity (ALP/DNA) demonstrated that the combination of a time-dependent field with magnetic nanocomposites (PCL/Fe3O4 Mag) influenced cell differentiation. Furthermore, in terms of extracellular signal-regulated kinase (ERK)1/2 phosphorylation, an insight into the role of the magnetic stimulation was reported, also demonstrating a strong effect due the combination of the magnetic field with PCL/Fe3O4 nanocomposites (PCL/Fe3O4 Mag).

NMR Structure of the FIV gp36 C-Terminal Heptad Repeat and Membrane-Proximal External Region.

Feline immunodeficiency virus (FIV), a lentivirus causing an immunodeficiency syndrome in cats, represents a relevant model of pre-screening therapies for human immunodeficiency virus (HIV). The envelope glycoproteins gp36 in FIV and gp41 in HIV mediate the fusion of the virus with the host cell membrane. They have a common structural framework in the C-terminal region that includes a Trp-rich membrane-proximal external region (MPER) and a C-terminal heptad repeat (CHR). MPER is essential for the correct positioning of gp36 on the lipid membrane, whereas CHR is essential for the stabilization of the low-energy six-helical bundle (6HB) that is necessary for the fusion of the virus envelope with the cell membrane. Conformational data for gp36 are missing, and several aspects of the MPER structure of different lentiviruses are still debated. In the present work, we report the structural investigation of a gp36 construct that includes the MPER and part of the CHR domain (737-786gp36 CHR-MPER). Using 2D and 3D homo and heteronuclear NMR spectra on 15N and 13C double-labelled samples, we solved the NMR structure in micelles composed of dodecyl phosphocholine (DPC) and sodium dodecyl sulfate (SDS) 90/10 M: M. The structure of 737-786gp36 CHR-MPER is characterized by a helix-turn-helix motif, with a regular α-helix and a moderately flexible 310 helix, characterizing the CHR and the MPER domains, respectively. The two helices are linked by a flexible loop regulating their orientation at a ~43° angle. We investigated the positioning of 737-786gp36 CHR-MPER on the lipid membrane using spin label-enhanced NMR and ESR spectroscopies. On a different scale, using confocal microscopy imaging, we studied the effect of 737-786gp36 CHR-MPER on 1,2-dioleoyl-sn-glycero-3-phosphocholine/1,2-dioleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (DOPC/DOPG) multilamellar vesicles (MLVs). This effect results in membrane budding and tubulation that is reminiscent of a membrane-plasticizing role that is typical of MPER domains during the event in which the virus envelope merges with the host cell membrane.

Synthesis and Characterization of Sintered Sr/Fe-Modified Hydroxyapatite Bioceramics for Bone Tissue Engineering Applications.

In the current study, Sr/Fe co-substituted hydroxyapatite (HAp) bioceramics were prepared by the sonication-assisted aqueous chemical precipitation method followed by sintering at 1100 °C for bone tissue regeneration applications. The sintered bioceramics were analyzed for various structural and chemical properties through X-ray diffraction, scanning electron microscopy, and Fourier transform infrared spectroscopy, which confirmed the phase purity of HAp and Sr/Fe co-substitution into its lattice. The Vickers hardness measurement, high blood compatibility (less than 5% hemolysis), and ability to support the adhesion, proliferation, and osteogenic differentiation of human mesenchymal stem cells suggest the suitability of Sr/Fe:HAp bioceramics for bone implant applications. The physicochemical analysis revealed that the developed Sr/Fe:HAp bioceramics exhibited a polyphasic nature (HAp and ßTCP) with almost identical structural morphology having a particle size less than 0.8 µm. The dielectric constant (ε') and dielectric loss (ε″) were potentially affected by the incorporated foreign ions together with the polyphasic nature of the material. The Sr/Fe co-substituted samples demonstrated extended drug (5-fluorouracil and amoxicillin) release profiles at the pH of physiological medium. The multifunctional properties of the developed HAp bioceramics enabled them to be an auspicious candidate for potential biomedical applications, including targeted drug-delivery applications, heating mediator in hyperthermia, and bone tissue repair implants.

EAK Hydrogels Cross-Linked by Disulfide Bonds: Cys Number and Position Are Matched to Performances.

Hydrogels produced by self-assembling peptides are intrinsically biocompatible and thus appropriate for many biomedical purposes. Their application field may be even made wider by reducing the softness and improving the hydrogel mechanical properties through cross-linking treatments. To this aim, modifications of EAK16-II sequence by including Cys residues in its sequence were here investigated in order to obtain hydrogels cross-linkable through a disulfide bridge. Two sequences, namely, C-EAK and C-EAK-C, that contain Cys residues at the N-terminus or at both ends were characterized. Fiber-forming abilities and biological and dynamic mechanical properties were explored before and after the oxidative treatment. In particular, the oxidized version of C-EAK presents a good cell viability and sustains osteoblast proliferation. Furthermore, molecular dynamics (MD) simulations on monomeric and assembled forms of the peptides were performed. MD simulations explained how a specific Cys functionalization was better than the other one. In particular, the results suggested that EAK16-II functionalization with a single Cys residue, instead of two, together with biocompatible cross-linking may be considered an intriguing strategy to obtain a support with better dynamic mechanical properties and biological performances.

Modification of PMMA Cements for Cranioplasty with Bioactive Glass and Copper Doped Tricalcium Phosphate Particles.

Cranioplasty represents the surgical repair of bone defects or deformities in the cranium arising from traumatic skull bone fracture, cranial bone deformities, bone cancer, and infections. The actual gold standard in surgery procedures for cranioplasty involves the use of biocompatible materials, and repair or regeneration of large cranial defects is particularly challenging from both a functional and aesthetic point of view. PMMA-based bone cement are the most widely biomaterials adopted in the field, with at least four different surgical approaches. Modifications for improving biological and mechanical functions of PMMA-based bone cement have been suggested. To this aim, the inclusion of antibiotics to prevent infection has been shown to provide a reduction of mechanical properties in bending. Therefore, the development of novel antibacterial active agents to overcome issues related to mechanical properties and bacterial resistance to antibiotics is still encouraged. In this context, mechanical, biological, and antibacterial feature against P. aeruginosa and S. aureus bacterial strains of surgical PMMA cement modified with BG and recently developed Cu-TCP bioactive particles have been highlighted.

A Further Analysis on Ti6Al4V Lattice Structures Manufactured by Selective Laser Melting.

Mechanical and architectural features play an important role in designing biomedical devices. The use of materials (i.e., Ti6Al4V) with Young's modulus higher than those of natural tissues generally cause stress shielding effects, bone atrophy, and implant loosening. However, porous devices may be designed to reduce the implant stiffness and, consequently, to improve its stability by promoting tissue ingrowth. If porosity increases, mass transport properties, which are crucial for cell behavior and tissue ingrowth, increase, whereas mechanical properties decrease. As reported in the literature, it is always possible to tailor mass transport and mechanical properties of additively manufactured structures by varying the architectural features, as well as pore shape and size. Even though many studies have already been made on different porous structures with controlled morphology, the aim of current study was to provide only a further analysis on Ti6Al4V lattice structures manufactured by selective laser melting. Experimental and theoretical analyses also demonstrated the possibility to vary the architectural features, pore size, and geometry, without dramatically altering the mechanical performance of the structure.