A New Dermal Substitute Containing Polyvinyl Alcohol with Silver Nanoparticles and Collagen with Hyaluronic Acid: In Vitro and In Vivo Approaches.

The experimental use of poly (alcohol-vinyl) (PVA) as a skin curative is increasing widely. However, the use of this hydrogel is challenging due to its favorable properties for microbiota growth. The association with silver nanoparticles (AgNPs) as an antimicrobial agent turns the match for PVA as a dressing, as it focuses on creating a physical barrier to avoid wound dehydration. When associated with extracellular components, such as the collagen matrix, the device obtained can create the desired biological conditions to act as a skin substitute. This study aimed to analyze the anti-microbiological activity and the in vitro and in vivo responses of a bilaminar device of PVA containing AgNPs associated with a membrane of collagen-hyaluronic acid (col-HA). Additionally, mesenchymal stem cells were cultured in the device to evaluate in vitro responses and in vivo immunomodulatory and healing behavior. The device morphology revealed a porous pattern that favored water retention and in vitro cell adhesion. Controlled wounds in the dorsal back of rat skins revealed a striking skin remodeling with new epidermis fulfilling all previously injured areas after 14 and 28 days. No infections or significant inflammations were observed, despite increased angiogenesis, and no fibrosis-markers were identified as compared to controls. Although few antibacterial activities were obtained, the addition of AgNPs prevented fungal growth. All results demonstrated that the combination of the components used here as a dermal device, chosen according to previous miscellany studies of low/mid-cost biomaterials, can promote skin protection avoiding infections and dehydration, minimize the typical wound inflammatory responses, and favor the cellular healing responses, features that give rise to further clinical trials of the device here developed.

The hypertrophic cartilage induction influences the building-block capacity of human adipose stem/stromal cell spheroids for biofabrication.

As an alternative to the classical tissue engineering approach, bottom-up tissue engineering emerges using building blocks in bioassembly technologies. Spheroids can be used as building blocks to reach a highly complex ordered tissue by their fusion (bioassembly), representing the foundation of biofabrication. In this study, we analyzed the biomechanical properties and the fusion capacity of human adipose stem/stromal cell (ASC) we spheroids during an in vitro model of hypertrophic cartilage established by our research group. Hypertrophic induced-ASC spheroids showed a statistically significant higher Young's modulus at weeks 2 (P < .001) and 3 (P < .0005) compared with non-induced. After fusion, non-induced and induced-ASC spheroids increased the contact area and decreased their pairs' total length. At weeks 3 and 5, induced-ASC spheroids did not fuse completely, and the cells migrate preferentially in the fusion contact region. Alizarin red O staining showed the highest intensity of staining in the fused induced-ASC spheroids at week 5, together with intense staining for collagen type I and osteocalcin. Transmission electron microscopy and element content analysis (X-ray Energy Dispersive Spectroscopy) revealed in the fused quartet at week 3 a crystal-like structure. Hypertrophic induction interferes with the intrinsic capacity of spheroids to fuse. The measurements of contact between spheroids during the fusion process, together with the change in viscoelastic profile to the plastic, will impact the establishment of bioassembly protocols using hypertrophic induced-ASC spheroids as building blocks in biofabrication.

Maternal Low-Protein Diet Deregulates DNA Repair and DNA Replication Pathways in Female Offspring Mammary Gland Leading to Increased Chemically Induced Rat Carcinogenesis in Adulthood.

Studies have shown that maternal malnutrition, especially a low-protein diet (LPD), plays a key role in the developmental mechanisms underlying mammary cancer programming in female offspring. However, the molecular pathways associated with this higher susceptibility are still poorly understood. Thus, this study investigated the adverse effects of gestational and lactational low protein intake on gene expression of key pathways involved in mammary tumor initiation after a single dose of N-methyl-N-nitrosourea (MNU) in female offspring rats. Pregnant Sprague-Dawley rats were fed a normal-protein diet (NPD) (17% protein) or LPD (6% protein) from gestational day 1 to postnatal day (PND) 21. After weaning (PND 21), female offspring (n = 5, each diet) were euthanized for histological analysis or received NPD (n = 56 each diet). At PND 28 or 35, female offspring received a single dose of MNU (25 mg/kg body weight) (n = 28 each diet/timepoint). After 24 h, some females (n = 10 each diet/timepoint) were euthanized for histological, immunohistochemical, and molecular analyses at PDN 29 or 36. The remaining animals (n = 18 each diet/timepoint) were euthanized when tumors reached ≥2 cm or at PND 250. Besides the mammary gland development delay observed in LPD 21 and 28 groups, the gene expression profile demonstrated that maternal LPD deregulated 21 genes related to DNA repair and DNA replication pathways in the mammary gland of LPD 35 group after MNU. We further confirmed an increased γ-H2AX (DNA damage biomarker) and in ER-α immunoreactivity in mammary epithelial cells in the LPD group at PND 36. Furthermore, these early postnatal events were followed by significantly higher mammary carcinogenesis susceptibility in offspring at adulthood. Thus, the results indicate that maternal LPD influenced the programming of chemically induced mammary carcinogenesis in female offspring through increase in DNA damage and deregulation of DNA repair and DNA replication pathways. Also, Cidea upregulation gene in the LPD 35 group may suggest that maternal LPD could deregulate genes possibly leading to increased risk of mammary cancer development and/or poor prognosis. These findings increase the body of evidence of early-transcriptional mammary gland changes influenced by maternal LPD, resulting in differential response to breast tumor initiation and susceptibility and may raise discussions about lifelong prevention of breast cancer risk.

Scaffold- and serum-free hypertrophic cartilage tissue engineering as an alternative approach for bone repair.

Human adipose stem/stromal cell (ASC) spheroids were used as a serum-free in vitro model to recapitulate the molecular events and extracellular matrix organization that orchestrate a hypertrophic cartilage phenotype. Induced-ASC spheroids (ø = 450 µm) showed high cell viability throughout the period of culture. The expression of collagen type X alpha 1 chain (COLXA1) and matrix metallopeptidase 13 (MMP-13) was upregulated at week 2 in induced-ASC spheroids compared with week 5 (P < .001) evaluated by quantitative real-time PCR. In accordance, secreted levels of IL-6 (P < .0001), IL-8 (P < .0001), IL-10 (P < .0001), bFGF (P < .001), VEGF (P < .0001), and RANTES (P < .0001) were the highest at week 2. Strong in situ staining for collagen type X and low staining for TSP-1 was associated with the increase of hypertrophic genes expression at week 2 in induced-ASC spheroids. Collagen type I, osteocalcin, biglycan, and tenascin C were detected at week 5 by in situ staining, in accordance with the highest expression of alkaline phosphatase (ALPL) gene and the presence of calcium deposits as evaluated by Alizarin Red O staining. Induced-ASC spheroids showed a higher force required to compression at week 2 (P < .0001). The human ASC spheroids under serum-free inducer medium and normoxic culture conditions were induced to a hypertrophic cartilage phenotype, opening a new perspective to recapitulate endochondral ossification in vivo.

In vitro and in vivo evaluations of nanocrystalline Zn-doped carbonated hydroxyapatite/alginate microspheres: zinc and calcium bioavailability and bone regeneration.

Background: Zinc-doped hydroxyapatite has been proposed as a graft biomaterial for bone regeneration. However, the effect of zinc on osteoconductivity is still controversial, since the release and resorption of calcium, phosphorus, and zinc in graft-implanted defects have rarely been studied. Methods: Microspheres containing alginate and either non-doped carbonated hydroxyapatite (cHA) or nanocrystalline 3.2 wt% zinc-doped cHA (Zn-cHA) were implanted in critical-sized calvarial defects in Wistar rats for 1, 3, and 6 months. Histological and histomorphometric analyses were performed to evaluate the volume density of newly formed bone, residual biomaterial, and connective tissue formation. Biomaterial degradation was characterized by transmission electron microscopy (TEM) and synchrotron radiation-based X-ray microfluorescence (SR-µXRF), which enabled the elemental mapping of calcium, phosphorus, and zinc on the microsphere-implanted defects at 6 months post-implantation. Results: The bone repair was limited to regions close to the preexistent bone, whereas connective tissue occupied the major part of the defect. Moreover, no significant difference in the amount of new bone formed was found between the two microsphere groups. TEM analysis revealed the degradation of the outer microsphere surface with detachment of the nanoparticle aggregates. According to SR-µXRF, both types of microspheres released high amounts of calcium, phosphorus, and zinc, distributed throughout the defective region. The cHA microsphere surface strongly adsorbed the zinc from organic constituents of the biological fluid, and phosphorus was resorbed more quickly than calcium. In the Zn-cHA group, zinc and calcium had similar release profiles, indicating a stoichiometric dissolution of these elements and non-preferential zinc resorption. Conclusions: The nanometric size of cHA and Zn-cHA was a decisive factor in accelerating the in vivo availability of calcium and zinc. The high calcium and zinc accumulation in the defect, which was not cleared by the biological medium, played a critical role in inhibiting osteoconduction and thus impairing bone repair.

Improved tribocorrosion performance of bio-functionalized TiO2 nanotubes under two-cycle sliding actions in artificial saliva.

After insertion into bone, dental implants may be subjected to tribocorrosive conditions resulting in the release of metallic ions and solid wear debris, which can induce to peri-implant inflammatory reactions accompanied by bone loss, and ultimately implant loosening. Despite the promising ability of TiO2 nanotubes (NTs) to improve osseointegration and avoid infection-related failures, the understanding of their degradation under the simultaneous action of wear and corrosion (tribocorrosion) is still very limited. This study aims, for the first time, to study the tribocorrosion behavior of bio-functionalized TiO2 NTs submitted to two-cycle sliding actions, and compare it with conventional TiO2 NTs. TiO2 NTs grown by anodization were doped with bioactive elements, namely calcium (Ca), phosphorous (P), and zinc (Zn), through reverse polarization anodization treatments. Characterization techniques such as scanning electron microscopy (SEM), energy dispersive X-ray spectroscopy (EDS) and scanning transmission electron microscopy (STEM), were used to characterize the films. Tribocorrosion tests were carried out in artificial saliva (AS) by applying two cycles of reciprocating sliding actions. The open circuit potential (OCP) was monitored before, during, and after both cycles of sliding, during which the coefficient of friction (COF) was calculated. The resulting wear scars were analyzed by SEM and EDS, and wear volume measurements were performed by 2D profilometry. Finally, the mechanical features of TiO2 NTs were accessed by nanoindentation. The results show that bio-functionalized TiO2 NTs display an enhanced tribocorrosion performance, ascribed to the growth of a nano-thick oxide film at Ti/TiO2 NTs interface, which significantly increased their adhesion strength to the substrate and consequently their hardness. Furthermore, it was discovered that during tribo-electrochemical solicitations, the formation of a P-rich tribofilm takes place, which grants both electrochemical protection and resistance to mechanical wear. This study provides fundamental and new insights for the development of multifunctional TiO2 NTs with long-term biomechanical stability and improved clinical outcomes.

Intracellular pathway and subsequent transformation of hydroxyapatite nanoparticles in the SAOS-2 osteoblast cell line.

Internalization of hydroxyapatite nanoparticles in SAOS-2 osteoblasts for 2 and 24 h was investigated in vitro using 5 and 50 µg/mL nanoparticles in culture medium. No cytotoxic effects were observed in a PrestoBlue viability assay. Focused ion beam-scanning electron microscopy and transmission electron microscopy were used to study nanoparticle trafficking inside cells and to characterize the physicochemical properties of the remodeled nanoparticles. Nanoparticles were actively internalized by cells and maintained in intracellular membrane-bound compartments. Dissolution of hydroxyapatite nanoparticles was observed inside phagolysosome in all samples. After 24 h of internalization in cell culture assays, reprecipitation of calcium phosphate minerals was observed in membrane-bound compartments in 5 and 50 µg/mL samples. Compared to the original nanoparticles, the reprecipitated calcium phosphate phase presented a different morphology, structure, and chemical composition. Two sample preparation methods were used and confirmed that reprecipitation of the calcium phosphate crystallites occurred in the intracellular environment and not during electron microscopy sample preparation. Reprecipitation of calcium phosphate prevented the release of large amounts of calcium and phosphate ions inside the cells. This phenomenon may be linked to physiological processes in the cell that control the concentration and trafficking of intracellular calcium ions, which are highly controlled by cells. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 428-439, 2018.

Shape Tailored Magnetic Nanorings for Intracellular Hyperthermia Cancer Therapy.

ABSTARCT: This work explores a new class of vortex/magnetite/iron oxide nanoparticles designed for magnetic hyperthermia applications. These nanoparticles, named Vortex Iron oxide Particles (VIPs), are an alternative to the traditional Superparamagnetic Iron Oxide Nanoparticles (SPIONs), since VIPs present superior heating power while fulfilling the main requirements for biomedical applications (low cytotoxicity and nonremanent state). In addition, the present work demonstrates that the synthesized VIPs also promote an internalization and aggregation of the particles inside the cell, resulting in a highly localized hyperthermia in the presence of an alternating magnetic field. Thereby, we demonstrate a new and efficient magnetic hyperthermia strategy in which a small, but well localized, concentration of VIPs can promote an intracellular hyperthermia process.

The effects of strontium on bone mineral: A review on current knowledge and microanalytical approaches.

The interest in effects of strontium (Sr) on bone has greatly increased in the last decade due to the development of the promising drug strontium ranelate. This drug is used for treating osteoporosis, a major bone disease affecting hundreds of millions of people worldwide, especially postmenopausal women. The novelty of strontium ranelate compared to other treatments for osteoporosis is its unique effect on bone: it simultaneously promotes bone formation by osteoblasts and inhibits bone resorption by osteoclasts. Besides affecting bone cells, treatment with strontium ranelate also has a direct effect on the mineralized bone matrix. Due to the chemical similarities between Sr and Ca, a topic that has long been of particular interest is the incorporation of Sr into bones replacing Ca from the mineral phase, which is composed by carbonated hydroxyapatite nanocrystals. Several groups have analyzed the mineral produced during treatment; however, most analysis were done with relatively large samples containing numerous nanocrystals, resulting thus on data that represents an average of many crystalline domains. The nanoscale analysis of the bone apatite crystals containing Sr has only been described in a few studies. In this study, we review the current knowledge on the effects of Sr on bone mineral and discuss the methodological approaches that have been used in the field. In particular, we focus on the great potential that advanced microscopy and microanalytical techniques may have on the detailed analysis of the nanostructure and composition of bone apatite nanocrystals produced during treatment with strontium ranelate.

Chlorhexidine-loaded hydroxyapatite microspheres as an antimicrobial delivery system and its effect on in vivo osteo-conductive properties.

Hydroxyapatite (HA) has been investigated as a delivery system for antimicrobial and antibacterial agents to simultaneously stimulate bone regeneration and prevent infection. Despite evidence supporting the bactericidal efficiency of these HA carriers, few studies have focused on the effect of this association on bone regeneration. In this work, we evaluated the physico-chemical properties of hydroxyapatite microspheres loaded with chlorhexidine (CHX) at two different concentrations, 0.9 and 9.1 µgCHX/cm2 HA, and characterized their effects on in vitro osteoblast viability and bone regeneration. Ultraviolet-visible spectroscopy, scanning and transmission electron microscopy associated with energy-dispersive X-ray spectroscopy and electron energy loss spectroscopy were used to characterize the association of CHX and HA nanoparticles. The high CHX loading dose induced formation of organic CHX plate-like aggregates on the HA surface, whereas a Langmuir film was formed at the low CHX surface concentration. Quantitative evaluation of murine osteoblast viability parameters, including adhesion, mitochondrial activity and membrane integrity of cells exposed to HA/CHX extracts, revealed a cytotoxic effect for both loading concentrations. Histomorphological analysis upon implantation into the dorsal connective tissues and calvaria of rats for 7 and 42 days showed that the high CHX concentration induced the infiltration of inflammatory cells, resulting in retarded bone growth. Despite a strong decrease in in vitro cell viability, the low CHX loading dose did not impair the biocompatibility and osteoconductivity of HA during bone repair. These results indicate that high antimicrobial doses may activate a strong local inflammatory response and disrupt the long-term osteoconductive properties of CHX-HA delivery systems.

Long-range crystalline order in spicules from the calcareous sponge Paraleucilla magna (Porifera, Calcarea).

We investigated the ultrastructure and crystallographic orientation of spicules from the calcareous sponge Paraleucilla magna (subclass Calcaronea) by transmission and scanning electron microscopy using two different methods of sample preparation: ultramicrotomy and focused ion beam (FIB). It was found that the unpaired actine from the spicules was oriented in the [211] zone axis. The plane that contains the unpaired actine and divides symmetrically the paired actines is the (-120). This plane is a mirror plane of the hexagonal lattice system. All the spicule types analyzed presented the same crystallographic orientation. Electron nanodiffraction maps from 4µm×4µm regions prepared by FIB showed disorientation of <2° between diffraction patterns obtained from neighbor regions, indicating the presence of a unique, highly aligned calcite crystalline phase. Among the eight FIB sections obtained, four presented high pore density. In one section perpendicular to the actine axis pores were observed only in the center of the spicule aligned in a circular pattern and surrounded by a faint circular contour with a larger radius. The presence of amorphous carbon representative of organic molecules detected by electron energy loss spectroscopy was correlated neither with porosity nor with specific lattice planes.

Effect of strontium ranelate on bone mineral: Analysis of nanoscale compositional changes.

Strontium ranelate has been used to prevent bone loss and stimulate bone regeneration. Although strontium may integrate into the bone crystal lattice, the chemical and structural modifications of the bone when strontium interacts with the mineral phase are not completely understood. The objective of this study was to evaluate apatite from the mandibles of rats treated with strontium ranelate in the drinking water and compare its characteristics with those from untreated rats and synthetic apatites with and without strontium. Electron energy loss near edge structures from phosphorus, carbon, calcium and strontium were obtained by electron energy loss spectroscopy in a transmission electron microscope. The strontium signal was detected in the biological and synthetic samples containing strontium. The relative quantification of carbon by analyzing the CK edge at an energy loss of ΔE = 284 eV showed an increase in the number of carbonate groups in the bone mineral of treated rats. A synthetic strontium-containing sample used as control did not exhibit a carbon signal. This study showed physicochemical modifications in the bone mineral at the nanoscale caused by the systemic administration of strontium ranelate.

Growth of crystalline hydroxyapatite thin films at room temperature by tuning the energy of the RF-magnetron sputtering plasma.

Right angle radio frequency magnetron sputtering technique (RAMS) was redesigned to favor the production of high-quality hydroxyapatite (HA) thin coatings for biomedical applications. Stoichiometric HA films with controlled crystallinity, thickness varying from 254 to 540 nm, crystallite mean size of 73 nm, and RMS roughness of 1.7 ± 0.9 nm, were obtained at room temperature by tuning the thermodynamic properties of the plasma sheath energy. The plasma energies were adjusted by using a suitable high magnetic field confinement of 143 mT (1430 G) and a substrate floating potential of 2 V at the substrate-to-magnetron distance of Z = 10 mm and by varying the sputtering geometry, substrate-to-magnetron distance from Z = 5 mm to Z = 18 mm, forwarded RF power and reactive gas pressure. Measurements that were taken with a Langmuir probe showed that the adjusted RAMS geometry generated a plasma with an adequate effective temperature of Teff ≈ 11.8 eV and electron density of 2.0 × 10(15) m(-3) to nucleate nanoclusters and to further crystallize the nanodomains of stoichiometric HA. The deposition mechanism in the RAMS geometry was described by the formation of building units of amorphous calcium phosphate clusters (ACP), the conversion into HA nanodomains and the crystallization of the grain domains with a preferential orientation along the HA [002] direction.

Synthesis of hydroxyapatite nanotubes for biomedical applications.

Single phase, stoichiometrically pure, hollow nanotubes of hydroxyapatite have been synthesized and single-particle analysis has been performed to successfully prove the sole formation of Ca10(PO4)6(OH)2 phase. The facile synthesis involves a sol-gel process under neutral conditions in the presence of a sacrifical anodic alumina template. The structures formed are hollow nanotubes that have been characterized by XRD, SEM, TEM, SAED, EELS, EDS and BET measurements. The diameter of the resulting tubes is in the range of 140-350 nm, length is on the order of a few microns and the wall thickness of the tubes was found to be ca. 30 nm. Moreover these tubes had a large BET surface area of 115 m(2)/g and were found to be biocompatible. They displayed inertness in the presence of NIH 3T3 mouse fibroblast cells as dictated by an MTT assay.

Ultrastructure of regenerated bone mineral surrounding hydroxyapatite-alginate composite and sintered hydroxyapatite.

We report the ultrastructure of regenerated bone surrounding two types of biomaterials: hydroxyapatite-alginate composite and sintered hydroxyapatite. Critical defects in the calvaria of Wistar rats were filled with micrometer-sized spherical biomaterials and analyzed after 90 and 120 days of implantation by high-resolution transmission electron microscopy and Fourier transform infrared attenuated total reflectance microscopy, respectively. Infrared spectroscopy showed that hydroxyapatite of both biomaterials became more disordered after implantation in the rat calvaria, indicating that the biological environment induced modifications in biomaterials structure. We observed that the regenerated bone surrounding both biomaterials had a lamellar structure with type I collagen fibers alternating in adjacent lamella with angles of approximately 90°. In each lamella, plate-like apatite crystals were aligned in the c-axis direction, although a rotation around the c-axis could be present. Bone plate-like crystal dimensions were similar in regenerated bone around biomaterials and pre-existing bone in the rat calvaria. No epitaxial growth was observed around any of the biomaterials. A distinct mineralized layer was observed between new bone and hydroxyapatite-alginate biomaterial. This region presented a particular ultrastructure with crystallites smaller than those of the bulk of the biomaterial, and was possibly formed during the synthesis of alginate-containing composite or in the biological environment after implantation. Round nanoparticles were observed in regions of newly formed bone. The findings of this work contribute to a better understanding of the role of hydroxyapatite based biomaterials in bone regeneration processes at the nanoscale.