Modified Titanium Surface with Nano Amorphous Calcium Phosphate@Chitosan Oligolactate as Ion Loading Platform with Multifunctional Properties for Potential Biomedical Application.

Titanium (Ti) is widely used in medical and dental implants. Calcium phosphate (CPs) coatings enhance Ti implants' osteoinductive properties, and additives further improve these coatings. Recently, a nano amorphous calcium phosphate (nACP) coating decorated with chitosan oligolactate (ChOL) and selenium (Se) showed immunomodulatory effects. This study investigates the surface morphology, composition, bioactivity, mechanical properties, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates. Amorphous calcium phosphate (ACP) was synthesized, and the nACP@ChOL-Se hybrid coating was deposited on Ti substrates using in situ anaphoretic deposition. Physico-chemical characterization was used to analyze the surface of the coating (scanning electron microscopy (SEM), X-ray diffraction (XRD), and Fourier Transform Infrared Spectroscopy). The distribution of Se within the coating was examined with energy-dispersive X-ray spectroscopy (EDS). Bioactivity was evaluated in simulated body fluid (SBF), and adhesion was tested using a scratch test method. In vitro testing determined the release mechanism of Se. SEM images illustrated the surface morphology, while AFM provided a detailed analysis of surface roughness. XRD analysis revealed structural and phase composition, and EDS confirmed Se distribution within the coating. The coating exhibited bioactivity in SBF and showed good adhesion according to the scratch test. In vitro testing uncovered the release mechanism of Se from the coating. This study successfully characterized the surface morphology, composition, bioactivity, and Se-release mechanism of the nACP@ChOL-Se hybrid coating on Ti substrates, offering insights for developing immunomodulatory coatings for medical and dental applications.

Antimicrobial and Osteogenic Effects of Collagen Membrane Decorated with Chitosan-Nano-Hydroxyapatite.

Collagen membranes are routinely used in oral surgery for bone regeneration. Despite their numerous advantages, such as stimulating bone growth, bacterial contamination still remains one of the disadvantages of membrane use. Thus, we assessed the biocompatibility and osteogenic and antibacterial properties of a collagen membrane (OsteoBiol) modified with chitosan (CHI) and hydroxyapatite nanoparticles (HApNPs). Attenuated total reflectance-Fourier transform infrared spectroscopy (ATR FT-IR), X-ray powder diffraction (XRD), and field emission scanning electron microscopy (FE-SEM) were performed for membrane characterization. Biocompatibility was assessed on dental pulp stem cells (DPSCs) by an MTT assay, while the osteogenic effect was assessed by an ALP activity assay and qPCR analysis of osteogenic markers (BMP4, ALP, RUNX2, and OCN). Antimicrobial properties were investigated by counting colony-forming units (CFUs) of Streptococcus mitis, Porphyromonas gingivalis, and Fusobaterium nucleatum on membranes and in the surrounding medium. Membranes showed no cytotoxicity. ALP activity was higher and ALP, BMP4, and OCN genes were up-regulated in DPSCs on modified membranes compared to unmodified membranes. The CFUs were reduced on modified membranes and in the medium. Modified membranes showed great biocompatibility and a high osteoinductive effect. Additionally, they showed antimicrobial and antibiofilm effects against periopathogens. It can be concluded that the incorporation of CHI and hydroxyapatite nanoparticles in collagen membranes may be advantageous to promote osteogenesis and reduce bacterial adhesion.

Immunomodulatory Effects Mediated by Nano Amorphous Calcium Phosphate/Chitosan Oligosaccharide Lactate Coatings Decorated with Selenium on Titanium Implants.

The aim of this work is in situ anodization/anaphoretic deposition of a nano amorphous calcium phosphate (ACP)/chitosan oligosaccharide lactate (ChOL) multifunctional hybrid coating decorated with selenium (Se) on a titanium substrate and in vivo investigation of its immunomodulatory and anti-inflammatory effect. Investigating phenomena at the implant-tissue interface of interest for controlled inflammation and immunomodulation was also the aim of the research. In our earlier research, we designed coatings based on ACP and ChOL on titanium with anticorrosive, antibacterial and biocompatible properties, while in the presented results we show that selenium addition makes this coating an immunomodulator. The immunomodulatory effect of the novel hybrid coating is characterized by the examination of the functional aspects in the tissue around the implant (in vivo): proinflammatory cytokines' gene expression, M1 (iNOS) and M2 (Arg1) macrophages, fibrous capsule formation (TGF-ß) and vascularization (VEGF). The EDS, FTIR and XRD analyses prove the formation of a ACP/ChOL/Se multifunctional hybrid coating on Ti and the presence of Se. A higher M2/M1 macrophage ratio in the ACP/ChOL/Se-coated implants compared to pure titanium implants (a higher level of Arg1 expression) is noted at all time points examined (after 7, 14 and 28 days). Lower inflammation measured by gene expression of proinflammatory cytokines IL-1ß and TNF, lower expression of TGF-ß in the surrounding tissue and higher IL-6 expression (solely at day 7 post-implantation) is noted in presence of the ACP/ChOL/Se-coated implants.

Effect of argon plasma pre-treatment of healing abutments on peri-implant microbiome and soft tissue integration: a proof-of-concept randomized study.

PURPOSE: Biofilm-free implant surface is ultimate prerequisite for successful soft and bone tissue integration. Objective of the study was to estimate the effects of argon plasma healing abutment pre-treatment (PT) on peri-implant soft-tissue phenotype (PiSP), inflammation, plaque accumulation and the microbiome (PiM) between non-treated (NPT) and treated (PT) abutments following 3-months healing period. The hypothesis was that cell-conductive and antimicrobial properties of PT would yield optimal conditions for soft tissue integration. MATERIAL AND METHODS: Two months following second-phase surgery, microbiological and clinical parameters were assessed around thirty-six healing abutments with two types of microtopography, smooth surface (MACHINED) and ultrathin threaded microsurface (ROUGH). A two level randomization schema was used to achieve equal distribution and abutments were randomly divided into rough and machined groups, and then divided into PT and NPT groups. PiM was assessed using next-generation DNA sequencing. RESULTS: PiM bacterial composition was highly diverse already two months post-implantation, consisting of key-stone pathogens, early and late colonizers, while the mycobiome was less diverse. PT was associated with lower plaque accumulation and inflammation without significant impact on PiSP, while in NPT clinical parameters were increased and associated with periopathogens. NPT mostly harbored late colonizers, while PT exerted higher abundance of early colonizers suggesting less advanced plaque formation. Interaction analysis in PT demonstrated S. mitis co-occurrence with pro-healthy Rothia dentocariosa and co-exclusion with Parvimonas micra, Porphyromonas endodontalis and Prevotella oris. PiSP parameters were generally similar between the groups, but significant association between PiM and keratinized mucosa width was observed in both groups, with remarkably more expressed diversity in NPT compared to PT. PT resulted in significantly lower BOP and PI around rough and machined abutments, respectively, without specific effect on PiM and PiSP. CONCLUSIONS: PT contributed to significantly the less advanced biofilm accumulation and inflammation without specific effects on PiSP.

Temperature Sensing Properties of Biocompatible Yb/Er-Doped GdF3 and YF3 Mesocrystals.

Y0.8-xGdxF3:Yb/Er mesocrystals with a biocompatible surface and diverse morphological characteristics were successfully synthesized using chitosan-assisted solvothermal processing. Their structural properties, studied using X-ray powder diffraction, Fourier transform infrared spectroscopy, scanning and transmission electron microscopy and energy dispersive X-ray analysis, were further correlated with the up-conversion emission (λexc = 976 nm) recorded in function of temperature. Based on the change in the visible green emissions originating from the thermally coupled 2H11/2 and 4S3/2 levels of Er3+, the corresponding LIR was acquired in the physiologically relevant range of temperatures (25-50 °C). The detected absolute sensitivity of about 0.0042 °C-1, along with the low cytotoxicity toward both normal human lung fibroblasts (MRC-5) and cancerous lung epithelial (A549) cells, indicate a potential for use in temperature sensing in biomedicine. Additionally, their enhanced internalization in cells, without suppression of cell viability, enabled in vitro labeling of cancer and healthy cells upon 976 nm laser irradiation.

Hormone receptor binding, selectivity and cytotoxicity of steroid D-homo lactone loaded chitosan nanoparticles for the treatment of breast and prostate cancer cells.

Chemically modified steroids have a long history as anti-neoplastic drugs. Incorporation of a lactone moiety in the steroid nucleus, as in previously obtained 3ß-acetoxy-17-oxa-17a-homoandrost-5-en-16-one (A) and 3ß-hidroxy-17-oxa-17a-homoandrost-5-en-16-one (B), often results in enhanced anticancer properties. In this work, chitosan-based (Ch) nanoparticles were created and loaded with potent anticancer steroidal compounds, A and B. Changes to hormone receptor binding and cytotoxicity were then measured. In agreement with our previous results for A and B, A- and B-loaded Ch displayed cytotoxic properties against cancer cell lines. Both A-Ch and B-Ch showed activity toward estrogen negative breast cancer (MDA-MB-231) and androgen negative prostate cancer cell lines (PC-3). Greater selectivity toward cancer cells versus healthy lung fibroblast (MRC-5) was observed for B-Ch particles. Cell viability and cytotoxicity measurements after a recovery period indicate more robust recovery of healthy cells versus malignant cells. Compounds A and B or their Ch-encapsulated forms were shown to have negligible affinity for the ligand binding domain of estrogen receptor ß or the androgen receptor in a fluorescent yeast screen, suggesting a lack of estrogenicity and androgenicity. Steroid-loaded chitosan nanoparticles display strong cytotoxicity towards MDA-MB-231 and PC-3 with a lack of hormone activity, indicating their safety and efficacy.

Synthesis and Biological Properties of Alanine-Grafted Hydroxyapatite Nanoparticles.

Hydroxyapatite attracts great attention as hard tissues implant material for bones and teeth. Its application in reconstructive medicine depends on its biocompatibility, which is in a function of composition and surface properties. The insertion of a protein element in the composition of implants can improve the cell adhesion and the osseointegration. Having this in mind, the proposal of this work was to develop L-alanine-grafted hydroxyapatite nanoparticles and to study their biocompatibility. Two L-alanine sources and three grafting methods were used for hydroxyapatite surface functionalization. The efficiency of grafting was determined based on X-ray powder diffraction, Fourier-transform infrared spectroscopy, thermal analyses, and field-emission scanning electron microscopy. The results indicated the formation of hydroxyapatite with 8-25 wt% of organic content, depending on the grafting method. Protein adsorption, cell adhesion, and viability studies were carried out to evaluate biological properties of grafted materials. The viability of MG-63 human osteoblastic cells following 24 h incubation with the alanine-grafted hydroxyapatite samples is well preserved, being in all cases above the viability of cells incubated with hydroxyapatite. The alanine-grafted hydroxyapatite prepared in situ and by simple mixture showed higher protein adsorption and cell adhesion, respectively, indicating their potential toward use in regenerative medicine.

Anodizing/Anaphoretic Electrodeposition of Nano-Calcium Phosphate/Chitosan Lactate Multifunctional Coatings on Titanium with Advanced Corrosion Resistance, Bioactivity, and Antibacterial Properties.

The aim of this work was to investigate corrosion resistivity, bioactivity, and antibacterial activity of novel nano-amorphous calcium phosphate (ACP) potentially multifunctional composite coatings with and without chitosan oligosaccharide lactate (ChOL), ACP + ChOL/TiO2 and ACP/TiO2 ACP + ChOL/TiO2, respectively, on the titanium substrate. The coatings were obtained by new single-step in situ anodization of the substrate to generate TiO2 and the anaphoretic electrodeposition process of ACP and ChOL. The obtained coatings were around 300 ± 15 µm thick and consisted of two phases, namely, TiO2 and hybrid composite phases. Both ACP/TiO2 and ACP + ChOL/TiO2 have improved corrosion stability, whereas the ACP + ChOL/TiO2 coating showed better corrosion stability. It was shown that at the very start of the deposition process, the formation of the ChOL/TiO2 layer takes place predominantly, which is followed by the inclusion of ChOL into ACP with simultaneous growth of TiO2. This deposition mechanism resulted in the formation of strongly bonded uniform stable coating with high corrosion resistance. In vitro bioactivity was investigated by immersion of the samples in simulated body fluid (SBF). There is in-bone-like apatite formation on both ACP/TiO2 and ACP + ChOL/TiO2 surfaces upon immersion into SBF, which was proven by X-ray diffraction and Fourier transform infrared spectroscopy. While ACP/TiO2 shows no antibacterial activity, ACP + ChOL/TiO2 samples exhibited three- to fourfold decreases in the number of Staphylococcus aureus and Pseudomonas aeruginosa, respectively, after 420 min. The probable mechanism is binding ChOL with the bacterial cell wall, inhibiting its growth, altering the permeability of the cell membrane, and leading to bacterial death.

Thermal crystallization of amorphous calcium phosphate combined with citrate and fluoride doping: a novel route to produce hydroxyapatite bioceramics.

Amorphous calcium phosphate (ACP) is a material of high interest for dentistry, orthopedics, and other biomedical sectors. Being intrinsically metastable, the process of transformation of ACP into a crystalline phase upon heating is of high relevance for the development of innovative bioceramics. Here we have first studied the thermal behavior of a citrate-stabilized ACP (Cit-ACP) also doped with fluoride ions (Cit-FACP) prepared at three different nominal Cit/Ca ratios (i.e. 4, 2, 1) by differential thermal analysis. Next, the physico-chemical features of the crystalline products as well as the in vitro cell response to the materials were investigated. A citrate and fluoride free ACP sample was also tested as the blank. We have found that the activation energy of crystallization of Cit-(F)ACP samples is lower in comparison to the blank ACP and this is influenced by the nominal Cit/Ca molar ratio. Interestingly, we have discovered that the thermal treatment of Cit-(F)ACP at 800 °C yields hydroxyapatite (HA) or fluorapatite (FHA) as the main products differently from blank ACP that, like most of the ACPs reported in the literature, yields ß-tricalcium phosphate. This was attributed to the Ca/P ratio of Cit-(F)ACP, which is similar to HA. A study of the crystalline products has revealed that all the (F)HA samples were non-cytotoxic, and retained carbonate ions in the crystal structure despite the heat treatment that should have induced decarbonation. The morphology of the products is influenced by the nominal Cit/Ca ratio and the presence of fluoride, ranging from spherical nanoparticles to micrometric hexagonal rods. Overall, our results prove that the thermal crystallization of Cit-(F)ACP is markedly different from classic ACP based materials and the thermal treatment of Cit-(F)ACP represents an attractive route for producing pure bioactive HA ceramics.

Chitosan nanobeads loaded with Biginelli hybrids as cell-selective toxicity systems with a homogeneous distribution of the cell cycle in cancer treatment.

Tetrahydropyrimidines are a class of azaheterocycles, also called Biginelli hybrids (obtained from the Biginelli reaction), that have attracted an enormous interest in the medicinal chemistry community in recent years, due to a broad biological activity, such as anticancer, antiviral, anti-inflammatory, antidiabetic, antituberculosis activities, etc. According to SciFinder®, more than 70 000 different Biginelli-like compounds have been covered in publications. However, although the Biginelli reaction can yield a large number of compounds with a broad range of activities, none of them have been captured in a carrier. In this study, chitosan-based (Ch) nanoparticles (NPs) containing three different molecules (Biginelli hybrids) were developed and tested for the first time as simple and promising vehicles for anticancer Biginelli-based drugs. The key features of NPs, such as size, surface morphology, drug encapsulation efficiency, and in vitro release were systematically investigated. Rather weak cell selectivity of pure Biginelli hybrids (A-C) to selected cancer cell lines has improved and this has been accompanied with two-to-four times stronger cytotoxic effect of A-C loaded Ch NPs, with a triple reduction in toxicity to healthy cells (MRC-5). It has been observed that the examined NPs induce apoptosis. The cell cycle analysis has confirmed the influence of A-loaded Ch (A-Ch), B-loaded Ch (B-Ch), and C-loaded Ch (C-Ch) on the cell cycle distribution, which was homogenously affected. This is the difference with regard to the effect of A, B, and C on the cell cycle. It has been established that the increased selectivity and antitumor activity of NPs are related to the presence of the carrier.

Rare-earth (Gd3+,Yb3+/Tm3+, Eu3+) co-doped hydroxyapatite as magnetic, up-conversion and down-conversion materials for multimodal imaging.

Taking advantage of the flexibility of the apatite structure, nano- and micro-particles of hydroxyapatite (HAp) were doped with different combinations of rare earth ions (RE3+ = Gd, Eu, Yb, Tm) to achieve a synergy among their magnetic and optical properties and to enable their application in preventive medicine, particularly diagnostics based on multimodal imaging. All powders were synthesized through hydrothermal processing at T ≤ 200 °C. An X-ray powder diffraction analysis showed that all powders crystallized in P63/m space group of the hexagonal crystal structure. The refined unit-cell parameters reflected a decrease in the unit cell volume as a result of the partial substitution of Ca2+ with smaller RE3+ ions at both cation positions. The FTIR analysis additionally suggested that a synergy may exist solely in the triply doped system, where the lattice symmetry and vibration modes become more coherent than in the singly or doubly doped systems. HAp:RE3+ optical characterization revealed a change in the energy band gap and the appearance of a weak blue luminescence (λex = 370 nm) due to an increased concentration of defects. The "up"- and the "down"-conversion spectra of HAp:Gd/Yb/Tm and HAp:Gd/Eu powders showed characteristic transitions of Tm3+ and Eu3+, respectively. Furthermore, in contrast to diamagnetic HAp, all HAp:RE3+ powders exhibited paramagnetic behavior. Cell viability tests of HAp:Gd/Yb/Tm and HAp:Gd/Eu powders in human dental pulp stem cell cultures indicated their good biocompatibility.

Effects of hydroxyapatite@poly-lactide-co-glycolide nanoparticles combined with Pb and Cd on liver and kidney parenchyma after the reconstruction of mandibular bone defects.

Reconstruction of bone defects with the use of biomaterials based on hydroxyapatite (HAp) has been a popular approach in medicine and dentistry. Most often the process of new bone formation is analyzed with the focus only on the region of the reconstructed defect. The effects of the therapy on distant organs have been rarely reported in the literature, especially not in synergy with the exposure to other bioactive chemicals. In this study, reconstruction of the mandibular bone in vivo using poly-lactide-co-glycolide-coated HAp (HAp/PLGA) nanoparticles was monitored with a simultaneous histopathological analysis of distant organs, specifically kidney and liver parenchyma. Heavy metals are among the most prominent environmental pollutants and have a high affinity for the crystal lattice of HAp, where they get incorporated by replacing calcium ions. Lead (Pb) and cadmium (Cd) are two such metals that can be found in food, water and air, but are most commonly present in cigarette smoke, the frequent contaminant of hospital settings in the developing world. The influence of their presence in the repaired bone on the content of calcium (Ca) in the reconstructed bone defect was analyzed, along with the histopathological changes in liver and kidneys. A study performed on 24 female Wistar rats demonstrated that the reconstruction of mandibular bone defects using HAp/PLGA particles induced an increase in the content of Ca in the newly created bone without causing any pathological changes to the liver and the kidneys. The presence of Pb and Cd in the defects reconstructed with HAp/PLGA nanoparticles impeded the regenerative process and led to a severe and irreversible damage to the liver and kidney parenchyma.

Insights into the kinetics of thermally induced crystallization of amorphous calcium phosphate.

Transformations between amorphous and crystalline apatite mechanistically govern some of the most essential processes in bone metabolism, including biomineralization and bone remodeling. Fundamental understanding of this phase transition can help us gain control over the formation and dissolution of boney tissues in vivo and utilize that knowledge for various therapeutic ends. Crystallization of hydroxyapatite (HAp) and two tricalcium phosphate (TCP) polymorphs from the metastable precursor, amorphous calcium phosphate (ACP) was here studied kinetically and mechanistically using thermal analyses, X-ray diffraction and Fourier-transform infrared spectroscopy. Crystallization was detected in the differential thermal analysis as the exothermic peak at 639.5 °C at the slowest heating regimen of 5 °C min-1, while a combination of different kinetics models, including Augis-Bennett, Borchardt-Daniels, Johnson-Mehl-Avrami, Kissinger, Ozawa and Piloyan, yielded activation energies in the 435-450 kJ mol-1 range. Dehydrated ACP required a significant energy input to transform to HAp, thus indirectly proving the key role that structural water plays in this process in a biological setting. The phase transformation at high temperatures involved preformed nuclei and was solely due to their 3D growth, contrasting the edge-controlled nucleation derived earlier as the mechanism of growth in the solution. Crystallization was in both cases accompanied by the formation of needle-shape crystals of HAp through aggregation of ultrafine spherical units of ACP. Relationship between crystallinity and the heating rate was detected only for the initially amorphous structure, indicating a more intense and coherent lattice ordering process in annealed ACP than in HAp. Despite that, crystallization disobeyed the rule of inverse proportionality between the thermal energy required for the relaxation of defects and the level of strain, as the recovery rate of the initially poorly crystalline HAp was higher than that of ACP.

The effect of the androstane lung cancer inhibitor content on the cell-selective toxicity of hydroxyapatite-chitosan-PLGA nanocomposites.

An androstane (17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (derivative A)) cancer inhibitor was successfully captured in a carrier made of nano-sized hydroxyapatite (HAp) coated with chitosan-PLGA polymer blends (Ch-PLGA). In our previous studies, we demonstrated that it was convenient to use spherical HAp/Ch-PLGA carriers as vehicles to target the lungs following intravenous administration. In this study, we used emulsification and subsequent freeze-drying to load the spherical HAp/Ch-PLGA carriers with varying contents of the derivative A, in order to examine the selective toxicity towards cancerous/healthy lung cells. The XRD and FT-IR techniques confirmed the drug loading process, and the content of the poorly water soluble derivative A was estimated directly via the DSC technique. The particles were spherical in shape with the d50 distribution varying between 167 and 231 nm, whereas the content of the derivative A ranged from 6.5 to 19.3 wt%. Cell-selective cytotoxicity was examined simultaneously on two cell lines: human lung carcinoma (A549 ATCC CCL 185) and human lung fibroblasts (MRC-5 ATCC CCL 171). All particles exhibited nearly three times larger cytotoxicity towards cancer cells (A549) than towards healthy cells (MRC5), where the particles with the derivative A content of 6.5 wt% allowed for the viability of healthy cells >80%. Ninety-six hours after the treatment of cells with particles with different contents of derivative A (after incubation and recovery), recovery was faster in damaged healthy cells than in cancerous cells.

Chitosan Oligosaccharide Lactate Coated Hydroxyapatite Nanoparticles as a Vehicle for the Delivery of Steroid Drugs and the Targeting of Breast Cancer Cells.

Low targeting efficiency and fast metabolism of antineoplastic drugs are hindrances to effective chemotherapies and there is an ongoing search for better drugs, but also better carriers. Steroid derivatives, 3ß-hydroxy-16-hydroxymino-androst-5-en-17-one (A) and 3ß,17ß-dihydroxy-16-hydroxymino-androst-5-ene (B) as cancer growth inhibitors were chemically synthesized and captured in a carrier composed of hydroxyapatite (HAp) nanoparticles coated with chitosan oligosaccharide lactate (ChOLS). The only difference between the two derivatives is that A has a carbonyl group at the C17 position of the five-membered ring and B has a hydroxyl. This small difference in the structure resulted not only in different physicochemical properties of the A- and B-loaded HAp/ChOSL, but also in different biological activities. The morphology of drug-loaded HAp/ChOSL particles was spherical, but the size depended on the drug identity: d50=138 nm for A-loaded HAp/ChOSL and d50=223 nm for B-loaded HAp/ChOSL. Cell-selective toxicity was tested against human breast carcinoma (MCF7 and MDA-MB-231), human lung carcinoma (A549) and human lung fibroblasts (MRC-5). The small selectivity of pure derivatives A and B toward breast cancer cells became drastically increased when they were delivered using HAp/ChOSL particles. Whereas the ratio of the cytotoxicity imposed onto breast cancer cells and the cytotoxicity imposed onto healthy MRC-5 fibroblasts ranged from 1.5 to 1.7 for pure A and from 1.5 to 2.3 for pure derivative B depending on the concentration, it increased to 5.4 for A-loaded HAp/ChOSL and 5.1 for B-loaded HAp/ChOSL. FACS analysis demonstrated poor uptake of HAp/ChOSL particles by MCF7 cells, suggesting that the drug release occurs extracellularly. The augmented activity of the drugs was most likely due to sustained release, although the favorable positive charge of the carrier, allowing it to adhere to the negatively charged plasma membrane and release the drugs steadily and directly to the hydrophobic cell membrane milieu, was delineated as a possible complementary mechanism.

Instrumental methods and techniques for structural and physicochemical characterization of biomaterials and bone tissue: A review.

A review of recent advances in instrumental methods and techniques for structural and physicochemical characterization of biomaterials and bone tissue is presented in this paper. In recent years, biomaterials attracted great attention primarily because of the wide range of biomedical applications. This paper focuses on the practical aspects of instrumental methods and techniques that were most often applied (X-ray methods, vibrational spectroscopy (IR and Raman), magnetic-resonance spectroscopy (NMR and ESR), mass spectrometry (MS), atomic absorption spectrometry (AAS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES), thermogravimetry (TG), differential thermal analysis (DTA) and differential scanning calorimetry (DSC), scanning electron microscopy (SEM), transmission electron microscopy (TEM)) in the structural investigation and physicochemical characterization of biomaterials and bone tissue. The application of some other physicochemical methods was also discussed. Hands-on information is provided about these valuable research tools, emphasizing practical aspects such as typical measurement conditions, their limitations and advantages, interpretation of results and practical applications.

Selective anticancer activity of hydroxyapatite/chitosan-poly(d,l)-lactide-co-glycolide particles loaded with an androstane-based cancer inhibitor.

In an earlier study we demonstrated that hydroxyapatite nanoparticles coated with chitosan-poly(d,l)-lactide-co-glycolide (HAp/Ch-PLGA) target lungs following their intravenous injection into mice. In this study we utilize an emulsification process and freeze drying to load the composite HAp/Ch-PLGA particles with 17ß-hydroxy-17α-picolyl-androst-5-en-3ß-yl-acetate (A), a chemotherapeutic derivative of androstane and a novel compound with a selective anticancer activity against lung cancer cells. 1H NMR and 13C NMR techniques confirmed the intact structure of the derivative A following its entrapment within HAp/Ch-PLGA particles. The thermogravimetric and differential thermal analyses coupled with mass spectrometry were used to assess the thermal degradation products and properties of A-loaded HAp/Ch-PLGA. The loading efficiency, as indicated by the comparison of enthalpies of phase transitions in pure A and A-loaded HAp/Ch-PLGA, equaled 7.47wt.%. The release of A from HAp/Ch-PLGA was sustained, neither exhibiting a burst release nor plateauing after three weeks. Atomic force microscopy and particle size distribution analyses were used to confirm that the particles were spherical with a uniform size distribution of d50=168nm. In vitro cytotoxicity testing of A-loaded HAp/Ch-PLGA using MTT and trypan blue dye exclusion assays demonstrated that the particles were cytotoxic to the A549 human lung carcinoma cell line (46±2%), while simultaneously preserving high viability (83±3%) of regular MRC5 human lung fibroblasts and causing no harm to primary mouse lung fibroblasts. In conclusion, composite A-loaded HAp/Ch-PLGA particles could be seen as promising drug delivery platforms for selective cancer therapies, targeting malignant cells for destruction, while having a significantly lesser cytotoxic effect on the healthy cells.

Hydrothermally processed 1D hydroxyapatite: Mechanism of formation and biocompatibility studies.

Recent developments in bone tissue engineering have led to an increased interest in one-dimensional (1D) hydroxyapatite (HA) nano- and micro-structures such as wires, ribbons and tubes. They have been proposed for use as cell substrates, reinforcing phases in composites and carriers for biologically active substances. Here we demonstrate the synthesis of 1D HA structures using an optimized, urea-assisted, high-yield hydrothermal batch process. The one-pot process, yielding HA structures composed of bundles of ribbons and wires, was typified by the simultaneous occurrence of a multitude of intermediate reactions, failing to meet the uniformity criteria over particle morphology and size. To overcome these issues, the preparation procedure was divided to two stages: dicalcium phosphate platelets synthesized in the first step were used as a precursor for the synthesis of 1D HA in the second stage. Despite the elongated particle morphologies, both the precursor and the final product exhibited excellent biocompatibility and caused no reduction of viability when tested against osteoblastic MC3T3-E1 cells in 2D culture up to the concentration of 2.6mg/cm(2). X-ray powder diffraction combined with a range of electron microscopies and laser diffraction analyses was used to elucidate the formation mechanism and the microstructure of the final particles. The two-step synthesis involved a more direct transformation of DCP to 1D HA with the average diameter of 37nm and the aspect ratio exceeding 100:1. The comparison of crystalline domain sizes along different crystallographic directions showed no signs of significant anisotropy, while indicating that individual nanowires are ordered in bundles in the b crystallographic direction of the P63/m space group of HA. Intermediate processes, e.g., dehydration of dicalcium phosphate, are critical for the formation of 1D HA alongside other key aspects of this phase transformation, it must be investigated in more detail in the continuous design of smart HA micro- and nano-structures with advanced therapeutic potentials.

In Vitro Evaluation of Nanoscale Hydroxyapatite-Based Bone Reconstructive Materials with Antimicrobial Properties.

In the field of oral implantology the loss of bone tissue prevents adequate patient care, and calls for the use of synthetic biomaterials with properties that resemble natural bone. Special attention is paid to the risk of infection after the implantation of these materials. Studies have suggested that some nanocontructs containing metal ions have antimicrobial properties. The aim of this study was to examine the antimicrobial and hemolytic activity of cobalt-substituted hydroxyapatite nanoparticles, compared to hydroxyapatite and hydroxyapatite/poly-lactide-co-glycolide. The antibacterial effects of these powders were tested against two pathogenic bacterial strains: Escherichia coi (ATCC 25922) and Staphylococcus aureus (ATCC 25923), using the disc diffusion method and the quantitative antimicrobial test in a liquid medium. The quantitative antimicrobial test showed that all of the tested biomaterials have some antibacterial properties. The effects of both tests were more prominent in case of S. aureus than in E coli. A higher percentage of cobalt in the crystal structure of cobalt-substituted hydroxyapatite nanoparticles led to an increased antimicrobial activity. All of the presented biomaterial samples were found to be non-hemolytic. Having in mind that the tested of cobalt-substituted hydroxyapatite (Ca/Co-HAp) material in given concentrations shows good hemocompatibility and antimicrobial effects, along with its previously studied biological properties, the conclusion can be reached that it is a potential candidate that could substitute calcium hydroxyapatite as the material of choice for use in bone tissue engineering and clinical practices in orthopedic, oral and maxillofacial surgery.

Chitosan-PLGA polymer blends as coatings for hydroxyapatite nanoparticles and their effect on antimicrobial properties, osteoconductivity and regeneration of osseous tissues.

Composite biomaterials comprising nanostructured hydroxyapatite (HAp) have an enormous potential for natural bone tissue reparation, filling and augmentation. Chitosan (Ch) as a naturally derived polymer has many physicochemical and biological properties that make it an attractive material for use in bone tissue engineering. On the other hand, poly-D,L-lactide-co-glycolide (PLGA) is a synthetic polymer with a long history of use in sustained drug delivery and tissue engineering. However, while chitosan can disrupt the cell membrane integrity and may induce blood thrombosis, PLGA releases acidic byproducts that may cause tissue inflammation and interfere with the healing process. One of the strategies to improve the biocompatibility of Ch and PLGA is to combine them with compounds that exhibit complementary properties. In this study we present the synthesis and characterization, as well as in vitro and in vivo analyses of a nanoparticulate form of HAp coated with two different polymeric systems: (a) Ch and (b) a Ch-PLGA polymer blend. Solvent/non-solvent precipitation and freeze-drying were used for synthesis and processing, respectively, whereas thermogravimetry coupled with mass spectrometry was used for phase identification purposes in the coating process. HAp/Ch composite particles exhibited the highest antimicrobial activity against all four microbial strains tested in this work, but after the reconstruction of the bone defect they also caused inflammatory reactions in the newly formed tissue where the defect had lain. Coating HAp with a polymeric blend composed of Ch and PLGA led to a decrease in the reactivity and antimicrobial activity of the composite particles, but also to an increase in the quality of the newly formed bone tissue in the reconstructed defect area.