In Vitro Studies Regarding the Effect of Cellulose Acetate-Based Composite Coatings on the Functional Properties of the Biodegradable Mg3Nd Alloys.

Magnesium (Mg) alloys are adequate materials for orthopedic and maxilo-facial implants due to their biocompatibility, good mechanical properties closely related to the hard tissues, and processability. Their main drawbacks are the high-speed corrosion process and hydrogen release. In order to improve corrosion and mechanical properties, the Mg matrix can be strengthened through alloying elements with high temperature-dependent solubility materials. Rare earth elements (RE) contribute to mechanical properties and degradation improvement. Another possibility to reduce the corrosion rate of Mg-based alloys was demonstrated to be the different types of coatings (bioceramics, polymers, and composites) applied on their surface. The present investigation is related to the coating of two Mg-based alloys from the system Mg3Nd (Mg-Nd-Y-Zr-Zn) with polymeric-based composite coatings made from cellulose acetate (CA) combined with two fillers, respectively hydroxyapatite (HAp) and Mg particles. The main functions of the coatings are to reduce the biodegradation rate and to modify the surface properties in order to increase osteointegration. Firstly, the microstructural features of the experimental Mg3Nd alloys were revealed by optical microscopy and scanning electron microscopy (SEM) coupled with energy-dispersive spectroscopy. Apart from the surface morphology revealed by SEM, the roughness and wettability of all experimental samples were evaluated. The corrosion behavior of the uncoated and coated samples of both Mg3Nd alloys was investigated by immersion testing and electrochemical testing using Simulated Body Fluid as the medium. The complex in vitro research performed highlights that the composite coating based on CA with HAp particles exhibited the best protective effect for both Mg3Nd alloys.

Ion-Doped Calcium Phosphate-Based Coatings with Antibacterial Properties.

Ion-substituted calcium phosphate (CP) coatings have been extensively studied as promising materials for biomedical implants due to their ability to enhance biocompatibility, osteoconductivity, and bone formation. This systematic review aims to provide a comprehensive analysis of the current state of the art in ion-doped CP-based coatings for orthopaedic and dental implant applications. Specifically, this review evaluates the effects of ion addition on the physicochemical, mechanical, and biological properties of CP coatings. The review also identifies the contribution and additional effects (in a separate or a synergistic way) of different components used together with ion-doped CP for advanced composite coatings. In the final part, the effects of antibacterial coatings on specific bacteria strains are reported. The present review could be of interest to researchers, clinicians, and industry professionals involved in the development and application of CP coatings for orthopaedic and dental implants.

An in situ study of thermal crystallization of amorphous calcium phosphates.

The heat-induced crystallization of amorphous calcium phosphate (ACP) is an intriguing process not yet well comprehended. This is because most of the works on this topic are based on ex situ studies where the materials are characterized after the heat and cooldown cycles, thus missing transient structural changes. Here, we used time-resolved energy dispersive X-ray diffraction and infrared spectroscopy to study, for the first time, the thermal crystallization of ACP in situ. The thermal crystallization of two kinds of citrate-stabilized carbonated ACP was studied, as they are promising materials for the preparation of advanced bioceramics. The behavior of these samples was compared to that of two citrate-free ACPs, either doped or non-doped with carbonate ions. Our results evinced that several phenomena occur during ACP thermal annealing. Before crystallization, all ACP samples undergo a decrease in the short-range order process, followed by several internal reorganizations. We have assessed that differently from carbonate-free ACP, carbonated ACPs with and without citrate directly crystallize into a biomimetic poorly crystalline carbonated hydroxyapatite. Citrate-stabilized ACPs in comparison to citrate-free ACPs have a faster hydroxyapatite formation kinetics, which is due to their higher specific surface area. This work reveals the necessity and the potentialities of using in situ techniques to effectively probe complex processes such as the heat-induced crystallization of ACPs.

Effect of the Antimicrobial Agents Peppermint Essential Oil and Silver Nanoparticles on Bone Cement Properties.

The main problems directly linked with the use of PMMA bone cements in orthopedic surgery are the improper mechanical bond between cement and bone and the absence of antimicrobial properties. Recently, more research has been devoted to new bone cement with antimicrobial properties using mainly antibiotics or other innovative materials with antimicrobial properties. In this paper, we developed modified PMMA bone cement with antimicrobial properties proposing some experimental antimicrobial agents consisting of silver nanoparticles incorporated in ceramic glass and hydroxyapatite impregnated with peppermint oil. The impact of the addition of antimicrobial agents on the structure, mechanical properties, and biocompatibility of new PMMA bone cements was quantified. It has been shown that the addition of antimicrobial agents improves the flexural strength of the traditional PMMA bone cement, while the yield strength values show a decrease, most likely because this agent acts as a discontinuity inside the material rather than as a reinforcing agent. In the case of all samples, the addition of antimicrobial agents had no significant influence on the thermal stability. The new PMMA bone cement showed good biocompatibility and the possibility of osteoblast proliferation (MTT test) along with a low level of cytotoxicity (LDH test).

Raman Spectroscopy in Skeletal Tissue Disorders and Tissue Engineering: Present and Prospective.

Musculoskeletal disorders are the most common reason of chronic pain and disability, representing an enormous socioeconomic burden worldwide. In this review, new biomedical application fields for Raman spectroscopy (RS) technique related to skeletal tissues are discussed, showing that it can provide a comprehensive profile of tissue composition in situ, in a rapid, label-free, and nondestructive manner. RS can be used as a tool to study tissue alterations associated to aging, pathologies, and disease treatments. The main advantage with respect to currently applied methods in clinics is its ability to provide specific information on molecular composition, which goes beyond other diagnostic tools. Being compatible with water, RS can be performed without pretreatment on unfixed, hydrated tissue samples, without any labeling and chemical fixation used in histochemical methods. This review first provides the description of the basic principles of RS as a biotechnology tool and is introduced into the field of currently available RS-based techniques, developed to enhance Raman signals. The main spectral processing, statistical tools, fingerprint identification, and available databases are mentioned. The recent literature has been analyzed for such applications of RS as tendon and ligaments, cartilage, bone, and tissue engineered constructs for regenerative medicine. Several cases of proof-of-concept preclinical studies have been described. Finally, advantages, limitations, future perspectives, and challenges for the translation of RS into clinical practice have been also discussed. Impact statement Raman spectroscopy (RS) is a powerful noninvasive tool giving access to molecular vibrations and characteristics of samples in a wavelength window of 600 to 3200 cm-1, thus giving access to a molecular fingerprint of biological samples in a nondestructive way. RS could not only be used in clinical diagnostics, but also be used for quality control of tissues and tissue-engineered constructs, reducing number of samples, time, and the variety of analysis required in the quality control chain before implantation.

Factors influencing the drug release from calcium phosphate cements.

Thanks to their biocompatibility, biodegradability, injectability and self-setting properties, calcium phosphate cements (CPCs) have been the most economical and effective biomaterials of choice for use as bone void fillers. They have also been extensively used as drug delivery carriers owing to their ability to provide for a steady release of various organic molecules aiding the regeneration of defective bone, including primarily antibiotics and growth factors. This review provides a systematic compilation of studies that reported on the controlled release of drugs from CPCs in the last 25 years. The chemical, compositional and microstructural characteristics of these systems through which the control of the release rates and mechanisms could be achieved have been discussed. In doing so, the effects of (i) the chemistry of the matrix, (ii) porosity, (iii) additives, (iv) drug types, (v) drug concentrations, (vi) drug loading methods and (vii) release media have been distinguished and discussed individually. Kinetic specificities of in vivo release of drugs from CPCs have been reviewed, too. Understanding the kinetic and mechanistic correlations between the CPC properties and the drug release is a prerequisite for the design of bone void fillers with drug release profiles precisely tailored to the application area and the clinical picture. The goal of this review has been to shed light on these fundamental correlations.

Quality control methods in musculoskeletal tissue engineering: from imaging to biosensors.

Tissue engineering is rapidly progressing toward clinical application. In the musculoskeletal field, there has been an increasing necessity for bone and cartilage replacement. Despite the promising translational potential of tissue engineering approaches, careful attention should be given to the quality of developed constructs to increase the real applicability to patients. After a general introduction to musculoskeletal tissue engineering, this narrative review aims to offer an overview of methods, starting from classical techniques, such as gene expression analysis and histology, to less common methods, such as Raman spectroscopy, microcomputed tomography, and biosensors, that can be employed to assess the quality of constructs in terms of viability, morphology, or matrix deposition. A particular emphasis is given to standards and good practices (GXP), which can be applicable in different sectors. Moreover, a classification of the methods into destructive, noninvasive, or conservative based on the possible further development of a preimplant quality monitoring system is proposed. Biosensors in musculoskeletal tissue engineering have not yet been used but have been proposed as a novel technology that can be exploited with numerous advantages, including minimal invasiveness, making them suitable for the development of preimplant quality control systems.

Antibacterial and cell-friendly copper-substituted tricalcium phosphate ceramics for biomedical implant applications.

The development of new materials with antibacterial properties and the scope to decrease or eliminate the excessive antibiotic use is an urgent priority due to the growing antibiotic resistance-related mortalities. New bone substitute materials with intrinsic antibacterial characteristics are highly requested for various clinical applications. In this study, the choice of copper ions as substitutes for calcium in tricalcium phosphate (TCP) has been justified by their pronounced broad-spectrum antibacterial properties. Copper-substituted TCP (Cu-TCP) ceramics with the copper content of 1.4 and 0.1 wt% were synthesized by mechano-chemical activation. X-ray diffraction (XRD) analyses established that both pure and copper-containing compounds adopted the structure of whitlockite (ß-TCP). XRD and electron paramagnetic resonance (EPR) spectroscopy revealed the partial isovalent substitution of calcium ions with copper ions in the ß-TCP lattice. With the use of infrared and EPR spectroscopies, it was detected that carbonate ions got incorporated into the ß-TCP structure during the synthesis procedure. By releasing the tension in the M(5)O6 octahedron consequential to the lower CaO bond length than the corresponding sum of ionic radii, the substitution of calcium with smaller copper ions stabilizes the structure of ß-TCP. As concluded form the thermal analyses, the introduction of Cu prevented the polymorphic transformation of ß- to α-TCP. At the same time, the introduction of Cu to the ß-TCP structure enhanced the crystal growth and porosity of the ceramics, which had a positive effect on the cytocompatibility of the material. The MTT colorimetric assay showed that the metabolic activity of the mouse fibroblast NCTC L929 cell line during 24 h of incubation with 3-day extracts from Cu-TCP (1.4 wt%) and ß-TCP pellets in the cell culture medium was similar to the negative control, indicating the absence of any inhibitory effects on cells. The seeding and the growth of human dental pulp stem cells on the surface of Cu-TCP (1.4 wt%) and ß-TCP ceramics also showed the absence of any signs of cytotoxicity. Finally, microbiological assays demonstrated the antibacterial activity of Cu-TCP ceramics against Escherichia coli and Salmonella enteritidis, whereas ß-TCP did not exhibit such an activity. Overall, the addition of Cu ions to ß-TCP improves its antibacterial properties without diminishing the biocompatibility of the material, thus making it more attractive than pure ß-TCP for clinical applications such as synthetic bone grafts and orthopaedic implant coatings.

High-Fat Diet Impairs Mouse Median Eminence: A Study by Transmission and Scanning Electron Microscopy Coupled with Raman Spectroscopy.

Hypothalamic dysfunction is an initial event following diet-induced obesity, primarily involving areas regulating energy balance such as arcuate nucleus (Arc) and median eminence (ME). To gain insights into the early hypothalamic diet-induced alterations, adult CD1 mice fed a high-fat diet (HFD) for 6 weeks were studied and compared with normo-fed controls. Transmission and scanning electron microscopy and histological staining were employed for morphological studies of the ME, while Raman spectroscopy was applied for the biochemical analysis of the Arc-ME complex. In HFD mice, ME ß2-tanycytes, glial cells dedicated to blood-liquor crosstalk, exhibited remarkable ultrastructural anomalies, including altered alignment, reduced junctions, degenerating organelles, and higher content of lipid droplets, lysosomes, and autophagosomes. Degenerating tanycytes also displayed an electron transparent cytoplasm filled with numerous vesicles, and they were surrounded by dilated extracellular spaces extending up to the subependymal layer. Consistently, Raman spectroscopy analysis of the Arc-ME complex revealed higher glycogen, collagen, and lipid bands in HFD mice compared with controls, and there was also a higher band corresponding to the cyanide group in the former compared to the last. Collectively, these data show that ME ß2-tanycytes exhibit early structural and chemical alterations due to HFD and reveal for the first-time hypothalamic cyanide presence following high dietary lipids consumption, which is a novel aspect with potential implications in the field of obesity.

Improved cytocompatibility and antibacterial properties of zinc-substituted brushite bone cement based on ß-tricalcium phosphate.

For bone replacement materials, osteoconductive, osteoinductive, and osteogenic properties are desired. The bacterial resistance and the need for new antibacterial strategies stand among the most challenging tasks of the modern medicine. In this work, brushite cements based on powders of Zinc (Zn) (1.4 wt%) substituted tricalcium phosphate (ß-TCP) and non-substituted ß-TCP were prepared and investigated. Their initial and final phase composition, time of setting, morphology, pH evolution, and compressive strength are reported. After soaking for 60 days in physiological solution, the cements transformed into a mixture of brushite and hydroxyapatite. Antibacterial activity of the cements against Enterococcus faecium, Escherichia coli, and Pseudomonas aeruginosa bacteria strains was attested. The absence of cytotoxicity of cements was proved for murine fibroblast NCTC L929 cells. Moreover, the cell viability on the ß-TCP cement containing Zn2+ ions was 10% higher compared to the ß-TCP cement without zinc. The developed cements are perspective for applications in orthopedics and traumatology.

Ionized jet deposition of antimicrobial and stem cell friendly silver-substituted tricalcium phosphate nanocoatings on titanium alloy.

Orthopedic infections pose severe societal and economic burden and interfere with the capability of the implanted devices to integrate in the host bone, thus significantly increasing implants failure rate. To address infection and promote integration, here nanostructured antibacterial and bioactive thin films are proposed, obtained, for the first time, by Ionized Jet Deposition (IJD) of silver-substituted tricalcium phosphate (Ag-TCP) targets on titanium. Coatings morphology, composition and mechanical properties are characterized and proof-of-concept of biocompatibility is shown. Antimicrobial efficacy is investigated against four Gram positive and Gram negative bacterial strains and against C. albicans fungus, by investigating the modifications in planktonic bacterial growth in the absence and presence of silver. Then, for all bacterial strains, the capability of the film to inhibit bacterial adhesion is also tested. Results indicate that IJD permits a fine control over films composition and morphology and deposition of films with suitable mechanical properties. Biological studies show a good efficacy against Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, Enterococcus faecalis and against fungus Candida albicans, with evidences of efficacy against planktonic growth and significant reduction of bacterial cell adhesion. No cytotoxic effects are evidenced for equine adipose tissue derived mesenchymal stem cells (ADMSCs), as no reductions are caused to cells viability and no interference is assessed in cells differentiation towards osteogenic lineage, in the presence of silver. Instead, thanks to nanostructuration and biomimetic composition, tricalcium phosphate (TCP) coatings favor cells viability, also when silver-substituted. These findings show that silver-substituted nanostructured coatings are promising for orthopedic implant applications.

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.

In vitro characterization of novel nanostructured collagen-hydroxyapatite composite scaffolds doped with magnesium with improved biodegradation rate for hard tissue regeneration.

New materials are required for bone healing in regenerative medicine able to temporarily substitute damaged bone and to be subsequently resorbed and replaced by endogenous tissues. Taking inspiration from basic composition of the mammalian bones, composed of collagen, apatite and a number of substitution ions, among them magnesium (Mg2+), in this work, novel composite scaffolds composed of collagen(10%)-hydroxyapatite (HAp)(90%) and collagen(10%)-HAp(80%)-Mg(10%) were developed. The lyophilization was used for composites preparation. An insight into the nanostructural nature of the developed scaffolds was performed by Scanning Electron Microscopy coupled with Energy Dispersive X-Ray and Transmission Electron Microscopy coupled with Energy Dispersive X-Ray. The HAp nanocrystallite clusters and Mg nanoparticles were homogeneously distributed within the scaffolds and adherent to the collagen fibrils. The samples were tested for degradation in Simulated Body Fluid (SBF) solution by soaking for up to 28 days. The release of Mg from collagen(10%)-HAp(80%)-Mg(10%) composite during the period of up to 21 days was attested, this composite being characterized by a decreased degradation rate with respect to the composite without Mg. The developed composite materials are promising for applications as bone substitute materials favouring bone healing and regeneration.

In Vitro Properties of Manganese-Substituted Tricalcium Phosphate Coatings for Titanium Biomedical Implants Deposited by Arc Plasma.

Bioactive manganese (Mn)-doped ceramic coatings for intraosseous titanium (Ti) implants are developed. Arc plasma deposition procedure is used for coatings preparation. X-ray Diffraction, Scanning Electron Microscopy-Energy Dispersive X-ray Spectroscopy, and Electron Paramagnetic Resonance (EPR) methods are applied for coatings characterization. The coatings are homogeneous, composed of the main phase α-tricalcium phosphate (α-TCP) (about 67%) and the minor phase hydroxyapatite (about 33%), and the Mn content is 2.3 wt%. EPR spectroscopy demonstrates that the Mn ions are incorporated in the TCP structure and are present in the coating in Mn2+ and Mn3+ oxidation states, being aggregated in clusters. The wetting contact angle of the deposited coatings is suitable for cells' adhesion and proliferation. In vitro soaking in physiological solution for 90 days leads to a drastic change in phase composition; the transformation into calcium carbonate and octacalcium phosphate takes place, and no more Mn is present. The absence of antibacterial activity against Escherichia coli, Enterococcus faecalis, and Pseudomonas aeruginosa bacteria strains is observed. A study of the metabolic activity of mouse fibroblasts of the NCTC L929 cell line on the coatings using the MTT (dye compound 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) test demonstrates that there is no toxic effect on the cell culture. Moreover, the coating material supports the adhesion and proliferation of the cells. A good adhesion, spreading, and proliferative activity of the human tooth postnatal dental pulp stem cells (DPSC) is demonstrated. The developed coatings are promising for implant application in orthopedics and dentistry.

Tricalcium phosphate cement supplemented with boron nitride nanotubes with enhanced biological properties.

A self-setting bone cement containing ß-tricalcium phosphate (TCP) supplemented with boron nitride nanotubes (BNNTs, 1 wt%) was synthesized and analyzed in situ for its kinetics of hardening and selected physicochemical and biological properties. Moderately delayed due to the presence of BNNTs, the hardening reaction involved the transformation of the TCP precursor to the dicalcium phosphate (DCPD) product. In spite of the short-lived chemical transformations in the cement upon its hardening, the structural changes in it were extended. As a result, the compressive strength increased from day 1 to day 7 of the hardening reaction and the presence of BNNTs further increased it by ~25%. Fitting of the time-resolved energy-dispersive diffractometric data to the Johnson-Mehl-Avrami-Kolmogorov crystallization kinetics model conformed to the one-dimensional nucleation at a variable rate during the growth of elongated DCPD crystals from round TCP grains. For the first seven days of growth of human mesenchymal stem cells (hMSCs) on the cement, no difference in their proliferation was observed compared to the control. However, between the 7th and the 21st day, the cell proliferation decreased compared to the control because of the ongoing stem cell differentiation toward the osteoblast phenotype. This differentiation was accompanied by the higher expression of alkaline phosphatase, an early marker of hMSC differentiation into a pre-osteoblast phenotype. The TCP cement supplemented with BNNTs was able to thwart the production of reactive oxygen species (ROS) in hMSCs treated with H2O2/Fe2+ and bring the ROS levels down to the concentrations detected in the control cells, indicating the good capability of the material to protect the cells against the ROS-associated damage. Simultaneously, the cement increased the expression of mediators of inflammation in a co-culture of osteoblasts and macrophages, thus attesting to the direct reciprocity between the degrees of inflammation and stimulated new bone production.

Raman spectroscopy discriminates malignant follicular lymphoma from benign follicular hyperplasia and from tumour metastasis.

Raman spectroscopy is a non-destructive label-free technique providing biochemical tissue fingerprint. The objective of the present work was to test if Raman spectroscopy is a suitable tool to differentiate lymph nodes affected by different conditions, such as reactive follicular hyperplasia (benign), follicular lymphoma (low grade primary tumour), diffuse large B cell lymphoma (high grade primary tumour) and tumour metastasis (secondary tumours). Moreover, we tested its ability to discriminate follicular lymphomas by the tumour grade and the BCL2 protein expression. Lymph nodes collected from 20 patients, who underwent surgery for suspected malignancy, were investigated. Imaging of tissue areas from about 400 µm2 up to 2 mm2 was performed collecting Raman maps containing thousands of spectra. Partial least squares discriminant analysis (PLS-DA) - a bilinear classification method - was used to calculate lymph node classification models, in order to discriminate at first between benign and malignant tissues and successively among cancer types, grades and the BCL2 protein expression. This proof-of-concept study paves the way for the development of clinical optical biopsy tools for lymph node cancer diagnosis, complementary to histopathological assessment.

Gold is for the mistress, silver for the maid: Enhanced mechanical properties, osteoinduction and antibacterial activity due to iron doping of tricalcium phosphate bone cements.

Self-hardening calcium phosphate cements present ideal bone tissue substitutes from the standpoints of bioactivity and biocompatibility, yet they suffer from (a) weak mechanical properties, (b) negligible osteoinduction without the use of exogenous growth factors, and (c) a lack of intrinsic antibacterial activity. Here we attempt to improve on these deficiencies by studying the properties of self-setting Fe-doped bone-integrative cements containing two different concentrations of the dopant: 0.49 and 1.09 wt% Fe. The hardening process, which involved the transformation of Fe-doped ß-tricalcium phosphate (Fe-TCP) to nanocrystalline brushite, was investigated in situ by continuously monitoring the cements using the Energy Dispersive X-Ray Diffraction technique. The setting time was 20 min and the hardening time 2 h, but it took 50 h for the cement to completely stabilize compositionally and mechanically. Still, compared to other similar systems, the phase transformation during hardening was relatively fast and it also followed a relatively simple reaction path, virtually free of complex intermediates and noisy background. Mössbauer spectrometry demonstrated that 57Fe atoms in Fe-TCP were located in two non-equivalent crystallographic sites and distributed over positions with a strong crystal distortion. The pronounced presence of ultrafine crystals in the final, brushite phase contributed to the reduction of the porosity and thereby to the enhancement of the mechanical properties. The compressive strength of the hardened TCP cements increased by more than twofold when Fe was added as a dopant, i.e., from 11.5 ±â€¯0.5 to 24.5 ±â€¯2.0 MPa. The amount of iron released from the cements in physiological media steadied after 10 days and was by an order of magnitude lower than the clinical threshold that triggers the toxic response. The cements exhibited osteoinductive activity, as observed from the elevated levels of expression of genes encoding for osteocalcin and Runx2 in both undifferentiated and differentiated MC3T3-E1 cells challenged with the cements. The osteoinductive effect was inversely proportional to the content of Fe ions in the cements, indicating that an excessive amount of iron can have a detrimental effect on the induction of bone growth by osteoblasts in contact with the cement. In contrast, the antibacterial activity of the cement in the agar assay increased against all four bacterial species analysed (E. coli, S. enteritidis, P. aeruginosa, S. aureus) in direct proportion with the concentration of Fe ions in it, indicating their key effect on the promotion of the antibacterial effect in this material. This effect was less pronounced in broth assays. Experiments involving co-incubation of cements with cells in an alternate magnetic radiofrequency field for 30 min demonstrated a good potential for the use of these magnetic cements in hyperthermia cancer therapies. Specifically, the population of human glioblastoma cells decreased six-fold at the 24 h time point following the end of the magnetic field treatment, while the population of the bone cancer cells dropped approximately twofold. The analysis of the MC3T3-E1 cell/cement interaction reiterated the effects of iron in the cement on the bone growth marker expression by showing signs of adverse effects on the cell morphology and proliferation only for the cement containing the higher concentration of Fe ions (1.09 wt%). Biological testing concluded that the effects of iron are beneficial from the perspective of a magnetic hyperthermia therapy and antibacterial prophylaxis, but its concentration in the material must be carefully optimized to avoid the adverse effects induced above a certain level of iron concentrations.

Raman Spectroscopy Applied to Parathyroid Tissues: A New Diagnostic Tool to Discriminate Normal Tissue from Adenoma.

Primary hyperparathyroidism is an endocrine disorder characterized by autonomous production of parathyroid hormone. Patients with the symptomatic disease should be referred for parathyroidectomy. However, the distinction between the pathological condition and the benign one is very challenging in the surgical setting; therefore, accurate recognition is important to ensure success during minimally invasive surgery. At present, all intraoperative techniques significantly increase surgical time and, consequently, cost. In this proof-of-concept study, Raman microscopy was used to differentiate between healthy parathyroid tissue and parathyroid adenoma from 18 patients. The data showed different spectroscopic features for the two main tissue types of healthy and adenoma. Moreover, the parathyroid adenoma subtypes (chief cells and oxyphil cells) were characterized by their own Raman spectra. The partial least-squares discriminant analysis (PLS-DA) model built to discriminate healthy from adenomatous parathyroid tissue was able to correctly classify all samples in the calibration and validation data sets, providing 100% prediction accuracy. The PLS-DA model built to discriminate chief cell adenoma from oxyphil cell adenoma allowed us to correctly classify >99% of the spectra during calibration and cross-validation and to correctly predict 100% of oxyphil and 99.8% of chief cells in the external validation data set. The results clearly demonstrate the great potential of Raman spectroscopy. The final goal would be development of a Raman portable fiber probe device for intraoperative optical biopsy, both to improve the surgical success rate and reduce surgical cost.

Proof-of-concept Raman spectroscopy study aimed to differentiate thyroid follicular patterned lesions.

Inter-observer variability and cancer over-diagnosis are emerging clinical problems, especially for follicular patterned thyroid lesions. This challenge strongly calls for a new clinical tool to reliably identify neoplastic lesions and to improve the efficiency of differentiation between benign and malignant neoplasms, especially considering the increased diagnosis of small carcinomas and the growing number of thyroid nodules. In this study, we employed a Raman spectroscopy (RS) microscope to investigate frozen thyroid tissues from fourteen patients with thyroid nodules. To generate tissue classification models, a supervised statistical analysis of the Raman spectra was performed. The results obtained demonstrate an accuracy of 78% for RS based diagnosis to discriminate between normal parenchyma and follicular patterned thyroid nodules, and 89% accuracy - for very challenging follicular lesions (carcinoma versus adenoma). RS translation into intraoperative diagnosis of frozen sections and in preoperative analysis of biopsies can be very helpful to reduce unnecessary surgery in patients with indeterminate cytological reports.