Collagen/κ-Carrageenan-Based Scaffolds as Biomimetic Constructs for In Vitro Bone Mineralization Studies.

Tissue engineering offers attractive strategies to develop three-dimensional scaffolds mimicking the complex hierarchical structure of the native bone. The bone is formed by cells incorporated in a molecularly organized extracellular matrix made of an inorganic phase, called biological apatite, and an organic phase mainly made of collagen and noncollagenous macromolecules. Although many strategies have been developed to replicate the complexity of bone at the nanoscale in vitro, a critical challenge has been to control the orchestrated process of mineralization promoted by bone cells in vivo and replicate the anatomical and biological properties of native bone. In this study, we used type I collagen to fabricate mineralized scaffolds mimicking the microenvironment of the native bone. The sulfated polysaccharide κ-carrageenan was added to the scaffolds to fulfill the role of noncollagenous macromolecules in the organization and mineralization of the bone matrix and cell adhesion. Scanning electron microscopy images of the surface of the collagen/κ-carrageenan scaffolds showed the presence of a dense and uniform network of intertwined fibrils, while images of the scaffolds' lateral sides showed the presence of collagen fibrils with a parallel alignment, which is characteristic of dense connective tissues. MC3T3-E1 osteoblasts were cultured in the collagen scaffolds and were viable after up to 7 days of culture, both in the absence and in the presence of κ-carrageenan. The presence of κ-carrageenan in the collagen scaffolds stimulated the maturation of the cells to a mineralizing phenotype, as suggested by the increased expression of key genes related to bone mineralization, including alkaline phosphatase (Alp), bone sialoprotein (Bsp), osteocalcin (Oc), and osteopontin (Opn), as well as the ability to mineralize the extracellular matrix after 14 and 21 days of culture. Taken together, the results described in this study shed light on the potential use of collagen/κ-carrageenan scaffolds to study the role of the structural organization of bone-mimetic synthetic matrices in cell function.

Influence of Er:YAG laser irradiation on surface properties of Ti-6Al-4V machined and hydroxyapatite coated.

Surface treatment by laser irradiation can change the topography of titanium; however, little is known about the changes it causes when applied to other coatings. This study aimed to evaluate the influence of Er:YAG laser irradiation on the surface properties of titanium-aluminum-vanadium (Ti-6Al-4V) discs. Four Ti-6Al-4V surfaces were evaluated (n = 10): CON-control, machined without surface treatment; LT-machined + laser treatment; HA-hydroxyapatite coating; and LT-HA-hydroxyapatite coating + laser treatment. For the laser treatment, an Er:YAG laser with a wavelength of 2940 nm, a frequency of 10 Hz, and an energy density of 12.8 J/cm2 was used. The morphology of the coating was investigated by scanning electron microscopy and the surface composition by energy-dispersive X-ray spectroscopy. The influence of laser irradiation treatment on roughness and wettability was also evaluated. The Er:YAG laser promoted a significant reduction in the roughness Sa (p < 0.05) and in the contact angle (p = 0.002) of the LT surface compared to the CON surface. On the LT-HA surface, a significant decrease in roughness was observed only for the Rz parameter (p = 0.015) and an increase in the contact angle (p < 0.001) compared to the HA surface. The use of the Er:YAG laser with the evaluated parameters decreased the surface roughness and improved the wetting capacity of machined without surface treatment. In the group with hydroxyapatite coating, the laser influenced the surface roughness only for the parameter Rz and reduced their wetting capacity.

Ultrasensitive Diamond Microelectrode Application in the Detection of Ca2+ Transport by AnnexinA5-Containing Nanostructured Liposomes.

This report describes the innovative application of high sensitivity Boron-doped nanocrystalline diamond microelectrodes for tracking small changes in Ca2+ concentration due to binding to Annexin-A5 inserted into the lipid bilayer of liposomes (proteoliposomes), which could not be assessed using common Ca2+ selective electrodes. Dispensing proteoliposomes to an electrolyte containing 1 mM Ca2+ resulted in a potential jump that decreased with time, reaching the baseline level after ~300 s, suggesting that Ca2+ ions were incorporated into the vesicle compartment and were no longer detected by the microelectrode. This behavior was not observed when liposomes (vesicles without AnxA5) were dispensed in the presence of Ca2+. The ion transport appears Ca2+-selective, since dispensing proteoliposomes in the presence of Mg2+ did not result in potential drop. The experimental conditions were adjusted to ensure an excess of Ca2+, thus confirming that the potential reduction was not only due to the binding of Ca2+ to AnxA5 but to the transfer of ions to the lumen of the proteoliposomes. Ca2+ uptake stopped immediately after the addition of EDTA. Therefore, our data provide evidence of selective Ca2+ transport into the proteoliposomes and support the possible function of AnxA5 as a hydrophilic pore once incorporated into lipid membrane, mediating the mineralization initiation process occurring in matrix vesicles.

Curcumin-loaded carrageenan nanoparticles: Fabrication, characterization, and assessment of the effects on osteoblasts mineralization.

The use of Curcumin (CR) as a bioactive molecule to prevent and treat inflammation- related diseases is widespread. However, the high hydrophobicity hinders the in vivo bioavailability of CR, reducing its therapeutic index. In the present study, we described the use of nanoparticles (NPs) made of kappa-carrageenan (κ-Carr), a sulphated polysaccharide, as cost-effective, biodegradable and biocompatible CR carriers. CR-loaded κ-Carr nanoparticles (CR@Carr NPs) were prepared by mixing a κ-Carr aqueous solution with a CR ethanolic solution. The final suspension was centrifuged and re-suspended in phosphate buffer solution. The NPs' size was tuned by changing the concentration of the polysaccharide. CR@CarrNPs displayed high CR incorporation efficiency (~80 wt%) and a double-exponential curve of CR release at physiological conditions (37 °C and pH 7.4) with a cumulative drug release of 32 wt% after 24 h for the smaller NP. Our results also showed that CR@CarrNPs were not cytotoxic to osteoblasts at concentrations up to 1 µM. Confocal microscopy images revealed the internalization of CR by the cells guided by the NPs. Cells treated with CR@CarrNPs exhibited higher activity of alkaline phosphatase and higher expression of the main osteogenic genes (Sp7, Col1 and Runx2), and mineralized the extracellular matrix in a higher extent compared to the cells cultivated in absence of the NPs. We posited that these effects were related to the NP-driven internalization of CR by osteoblasts. Our study sheds light on the possible use of CR@CarrNPs as efficient and safe therapeutic tools for the treatment of bone-related diseases.

Fabrication and characterization of a bioactive polymethylmethacrylate-based porous cement loaded with strontium/calcium apatite nanoparticles.

Polymethylmethacrylate (PMMA)-based cements are used for bone reparation due to their biocompatibility, suitable mechanical properties, and mouldability. However, these materials suffer from high exothermic polymerization and poor bioactivity, which can cause the formation of fibrous tissue around the implant and aseptic loosening. Herein, we tackled these problems by adding Sr2+ -substituted hydroxyapatite nanoparticles (NPs) and a porogenic compound to the formulations, thus creating a microenvironment suitable for the proliferation of osteoblasts. The NPs resembled the structure of the bone's apatite and enabled the controlled release of Sr2+ . Trends in the X-ray patterns and infrared spectra confirmed that Sr2+ replaced Ca2+ in the whole composition range of the NPs. The inclusion of an effervescent additive reduced the polymerization temperature and lead to the formation of highly porous cement exhibiting mechanical properties comparable to the trabecular bone. The formation of an opened and interconnected matrix allowed osteoblasts to penetrate the cement structure. Most importantly, the gas formation confined the NPs at the surface of the pores, guaranteeing the controlled delivery of Sr2+ within a concentration sufficient to maintain osteoblast viability. Additionally, the cement was able to form apatite when immersed into simulated body fluids, further increasing its bioactivity. Therefore, we offer a formulation of PMMA cement with improved in vitro performance supported by enhanced bioactivity, increased osteoblast viability and deposition of mineralized matrix assigned to the loading with Sr2+ -substituted hydroxyapatite NPs and the creation of an interconnected porous structure. Altogether, our results hold promise for enhanced bone reparation guided by PMMA cements.