In Vitro Studies on 3D-Printed PLA/HA/GNP Structures for Bone Tissue Regeneration.

The successful regeneration of large-size bone defects remains one of the most critical challenges faced in orthopaedics. Recently, 3D printing technology has been widely used to fabricate reliable, reproducible and economically affordable scaffolds with specifically designed shapes and porosity, capable of providing sufficient biomimetic cues for a desired cellular behaviour. Natural or synthetic polymers reinforced with active bioceramics and/or graphene derivatives have demonstrated adequate mechanical properties and a proper cellular response, attracting the attention of researchers in the bone regeneration field. In the present work, 3D-printed graphene nanoplatelet (GNP)-reinforced polylactic acid (PLA)/hydroxyapatite (HA) composite scaffolds were fabricated using the fused deposition modelling (FDM) technique. The in vitro response of the MC3T3-E1 pre-osteoblasts and RAW 264.7 macrophages revealed that these newly designed scaffolds exhibited various survival rates and a sustained proliferation. Moreover, as expected, the addition of HA into the PLA matrix contributed to mimicking a bone extracellular matrix, leading to positive effects on the pre-osteoblast osteogenic differentiation. In addition, a limited inflammatory response was also observed. Overall, the results suggest the great potential of the newly developed 3D-printed composite materials as suitable candidates for bone tissue engineering applications.

Synthesis, Physicochemical Characteristics, and Biocompatibility of Multi-Component Collagen-Based Hydrogels Developed by E-Beam Irradiation.

Herein, three different recipes of multi-component hydrogels were synthesized by e-beam irradiation. These hydrogels were obtained from aqueous polymer mixtures in which different proportions of bovine collagen gel, sodium carboxymethylcellulose (CMC), poly(vinylpyrrolidone), chitosan, and poly(ethylene oxide) were used. The cross-linking reaction was carried out exclusively by e-beam cross-linking at 25 kGy, a dose of irradiation sufficient both to complete the cross-linking reaction and effective for hydrogel sterilization. The hydrogels developed in this study were tested in terms of physical and chemical stability, mechanical, structural, morphological, and biological properties. They are transparent, maintain their structure, are non-adhesive when handling, and most importantly, especially from the application point of view, have an elastic structure. Likewise, these hydrogels possessed different swelling degrees and expressed rheological behavior characteristic of soft solids with permanent macromolecular network. Morphologically, collagen- and CMC based-hydrogels showed porous structures with homogeneously distributed pores assuring a good loading capacity with drugs. These hydrogels were investigated by indirect and direct contact studies with Vero cell line (CCL-81™, ATCC), demonstrating that they are well tolerated by normal cells and, therefore, showed promising potential for further use in the development of drug delivery systems based on hydrogels.

Selection Route of Precursor Materials in 3D Printing Composite Filament Development for Biomedical Applications.

Additive manufacturing or 3D printing technologies might advance the fabrication sector of personalised biomaterials with high-tech precision. The selection of optimal precursor materials is considered the first key-step for the development of new printable filaments destined for the fabrication of products with diverse orthopaedic/dental applications. The selection route of precursor materials proposed in this study targeted two categories of materials: prime materials, for the polymeric matrix (acrylonitrile butadiene styrene (ABS), polylactic acid (PLA)); and reinforcement materials (natural hydroxyapatite (HA) and graphene nanoplatelets (GNP) of different dimensions). HA was isolated from bovine bones (HA particles size < 40 µm, <100 µm, and >125 µm) through a reproducible synthesis technology. The structural (FTIR-ATR, Raman spectroscopy), morphological (SEM), and, most importantly, in vitro (indirect and direct contact studies) features of all precursor materials were comparatively evaluated. The polymeric materials were also prepared in the form of thin plates, for an advanced cell viability assessment (direct contact studies). The overall results confirmed once again the reproducibility of the HA synthesis method. Moreover, the biological cytotoxicity assays established the safe selection of PLA as a future polymeric matrix, with GNP of grade M as a reinforcement and HA as a bioceramic. Therefore, the obtained results pinpointed these materials as optimal for future composite filament synthesis and the 3D printing of implantable structures.

Metal Oxide Nanoparticles: Review of Synthesis, Characterization and Biological Effects.

In the last few years, the progress made in the field of nanotechnology has allowed researchers to develop and synthesize nanosized materials with unique physicochemical characteristics, suitable for various biomedical applications. Amongst these nanomaterials, metal oxide nanoparticles (MONPs) have gained increasing interest due to their excellent properties, which to a great extent differ from their bulk counterpart. However, despite such positive advantages, a substantial body of literature reports on their cytotoxic effects, which are directly correlated to the nanoparticles' physicochemical properties, therefore, better control over the synthetic parameters will not only lead to favorable surface characteristics but may also increase biocompatibility and consequently lower cytotoxicity. Taking into consideration the enormous biomedical potential of MONPs, the present review will discuss the most recent developments in this field referring mainly to synthesis methods, physical and chemical characterization and biological effects, including the pro-regenerative and antitumor potentials as well as antibacterial activity. Moreover, the last section of the review will tackle the pressing issue of the toxic effects of MONPs on various tissues/organs and cell lines.

TiO2 Nanotubes Functionalized with Icariin for an Attenuated In Vitro Immune Response and Improved In Vivo Osseointegration.

Due to their superior mechanical and chemical properties, titanium (Ti) and its alloys have been widely used as orthopedic implantable devices. However, their bioinertness represents a limitation, which can be overcome by employing various surface modifications, such as TiO2 nanotube (TNT) fabrication via electrochemical anodization. Anodic TNTs present tunable dimensions and unique structures, turning them into feasible drug delivery platforms. In the present work, TNTs were loaded with icariin (Ica) through an adhesive intermediate layer of polydopamine (DP), and their in vitro and in vivo biological performance was evaluated. The successful fabrication of the modified surfaces was verified by scanning electron microscopy (SEM), atomic force microscopy (AFM), Fourier transform infrared spectroscopy (FTIR), and contact angle measurements (CA), while the in vitro release of Ica was evaluated via UV-VIS spectrophotometry. In terms of in vitro behaviour, comparative studies on RAW 264.7 macrophages demonstrated that the TNT substrates, especially TNT-DP-Ica, elicited a lower inflammatory response compared to the Ti support. Moreover, the in vivo implantation studies evinced generation of a reduced fibrotic capsule around this implant and increased thickness of the newly formed bone tissue at 1 month and 3 months post-implantation, respectively. Overall, our results indicate that the controlled release of Ica from TNT surfaces could result in an improved osseointegration process.

Macrophage-like Cells Are Responsive to Titania Nanotube Intertube Spacing-An In Vitro Study.

With the introduction of a new interdisciplinary field, osteoimmunology, today, it is well acknowledged that biomaterial-induced inflammation is modulated by immune cells, primarily macrophages, and can be controlled by nanotopographical cues. Recent studies have investigated the effect of surface properties in modulating the immune reaction, and literature data indicate that various surface cues can dictate both the immune response and bone tissue repair. In this context, the purpose of the present study was to investigate the effects of titanium dioxide nanotube (TNT) interspacing on the response of the macrophage-like cell line RAW 264.7. The cells were maintained in contact with the surfaces of flat titanium (Ti) and anodic TNTs with an intertube spacing of 20 nm (TNT20) and 80 nm (TNT80), under standard or pro-inflammatory conditions. The results revealed that nanotube interspacing can influence macrophage response in terms of cell survival and proliferation, cellular morphology and polarization, cytokine/chemokine expression, and foreign body reaction. While the nanostructured topography did not tune the macrophages' differentiation into osteoclasts, this behavior was significantly reduced as compared to flat Ti surface. Overall, this study provides a new insight into how nanotubes' morphological features, particularly intertube spacing, could affect macrophage behavior.

The State of the Art and Prospects for Osteoimmunomodulatory Biomaterials.

The critical role of the immune system in host defense against foreign bodies and pathogens has been long recognized. With the introduction of a new field of research called osteoimmunology, the crosstalk between the immune and bone-forming cells has been studied more thoroughly, leading to the conclusion that the two systems are intimately connected through various cytokines, signaling molecules, transcription factors and receptors. The host immune reaction triggered by biomaterial implantation determines the in vivo fate of the implant, either in new bone formation or in fibrous tissue encapsulation. The traditional biomaterial design consisted in fabricating inert biomaterials capable of stimulating osteogenesis; however, inconsistencies between the in vitro and in vivo results were reported. This led to a shift in the development of biomaterials towards implants with osteoimmunomodulatory properties. By endowing the orthopedic biomaterials with favorable osteoimmunomodulatory properties, a desired immune response can be triggered in order to obtain a proper bone regeneration process. In this context, various approaches, such as the modification of chemical/structural characteristics or the incorporation of bioactive molecules, have been employed in order to modulate the crosstalk with the immune cells. The current review provides an overview of recent developments in such applied strategies.

In Vitro Macrophage Immunomodulation by Poly(ε-caprolactone) Based-Coated AZ31 Mg Alloy.

Due to its excellent bone-like mechanical properties and non-toxicity, magnesium (Mg) and its alloys have attracted great interest as biomaterials for orthopaedic applications. However, their fast degradation rate in physiological environments leads to an acute inflammatory response, restricting their use as biodegradable metallic implants. Endowing Mg-based biomaterials with immunomodulatory properties can help trigger a desired immune response capable of supporting a favorable healing process. In this study, electrospun poly(ε-caprolactone) (PCL) fibers loaded with coumarin (CM) and/or zinc oxide nanoparticles (ZnO) were used to coat the commercial AZ31 Mg alloy as single and combined formulas, and their effects on the macrophage inflammatory response and osteoclastogenic process were investigated by indirect contact studies. Likewise, the capacity of the analyzed samples to generate reactive oxygen species (ROS) has been investigated. The data obtained by attenuated total reflection Fourier-transform infrared (FTIR-ATR) and X-ray photoelectron spectroscopy (XPS) analyses indicate that AZ31 alloy was perfectly coated with the PCL fibers loaded with CM and ZnO, which had an important influence on tuning the release of the active ingredient. Furthermore, in terms of degradation in phosphate-buffered saline (PBS) solution, the PCL-ZnO- and secondary PCL-CM-ZnO-coated samples exhibited the best corrosion behaviour. The in vitro results showed the PCL-CM-ZnO and, to a lower extent, PCL-ZnO coated sample exhibited the best behaviour in terms of inflammatory response and receptor activator of nuclear factor kappa-B ligand (RANKL)-mediated differentiation of RAW 264.7 macrophages into osteoclasts. Altogether, the results obtained suggest that the coating of Mg alloys with fibrous PCL containing CM and/or ZnO can constitute a feasible strategy for biomedical applications.