In Vitro Viability Tests of New Ecofriendly Nanosystems Incorporating Essential Oils for Long-Lasting Conservation of Stone Artworks.

The study explores the application of natural biocides (oregano essential oil and eugenol, directly applied in solutions or encapsulated within silica nanocapsules) for safeguarding stone cultural heritage from biodeterioration, using green algae (Chlorococcum sp.) and cyanobacteria (Leptolyngbya sp.) as common pioneer biodeteriogens. Core-shell nanocontainers were built for a controlled release of microbicidal agents, a safe application of chemicals and a prolonged efficacy. The qualitative and quantitative evaluations of biocide efficiency at different doses were periodically performed in vitro, after six scheduled intervals of time (until 100 days). The release kinetics of composite biocide-embedding silica nanocapsules were characterized by the UV-Vis spectroscopy technique. Data showed both promising potential and some limitations. The comparative tests of different biocidal systems shed light on their variable efficacy against microorganisms, highlighting how encapsulation influences the release dynamics and the overall effectiveness. Both the essential oils showed a potential efficacy in protective antifouling coatings for stone artifacts. Ensuring compatibility with materials, understanding their differences in biocidal activity and their release rates becomes essential in tailoring gel, microemulsion or coating products for direct on-site application.

Bio-Functionalized Chitosan for Bone Tissue Engineering.

Hybrid biomaterials allow for the improvement of the biological properties of materials and have been successfully used for implantology in medical applications. The covalent and selective functionalization of materials with bioactive peptides provides favorable results in tissue engineering by supporting cell attachment to the biomaterial through biochemical cues and interaction with membrane receptors. Since the functionalization with bioactive peptides may alter the chemical and physical properties of the biomaterials, in this study we characterized the biological responses of differently functionalized chitosan analogs. Chitosan analogs were produced through the reaction of GRGDSPK (RGD) or FRHRNRKGY (HVP) sequences, both carrying an aldehyde-terminal group, to chitosan. The bio-functionalized polysaccharides, pure or "diluted" with chitosan, were chemically characterized in depth and evaluated for their antimicrobial activities and biocompatibility toward human primary osteoblast cells. The results obtained indicate that the bio-functionalization of chitosan increases human-osteoblast adhesion (p < 0.005) and proliferation (p < 0.005) as compared with chitosan. Overall, the 1:1 mixture of HVP functionalized-chitosan:chitosan is the best compromise between preserving the antibacterial properties of the material and supporting osteoblast differentiation and calcium deposition (p < 0.005 vs. RGD). In conclusion, our results reported that a selected concentration of HVP supported the biomimetic potential of functionalized chitosan better than RGD and preserved the antibacterial properties of chitosan.

Hydroxyapatite Surfaces Functionalized with a Self-Assembling Peptide: XPS, RAIRS and NEXAFS Study.

Hydroxyapatite (HAP) coatings can improve the biocompatibility and bioactivity of titanium alloys, such as Ti6Al4V, commonly used as material for orthopedic prostheses. In this framework, we have studied the surface of HAP coatings enriched with Mg and either Si or Ti deposited by RF magnetron sputtering on Ti6Al4V. HAP coatings have been furtherly functionalized by adsorption of a self-assembling peptide (SAP) on the HAP surface, with the aim of increasing the material bioactivity. The selected SAP (peptide sequence AbuEAbuEAbuKAbuKAbuEAbuEAbuKAbuK) is a self-complementary oligopeptide able to generate extended ordered structures by self-assembling in watery solutions. Samples were prepared by incubation of the HAP coatings in SAP solutions and subsequently analyzed by X-Ray Photoelectron Spectroscopy (XPS), Fourier Transform Infrared (FTIR) and Near Edge X-Ray Absorption Fine Structure (NEXAFS) spectroscopies, in order to determine the amount of adsorbed peptide, the peptide stability and the structure of the peptide overlayer on the HAP coatings as a function of the HAP substrate and of the pH of the mother SAP solution. Experimental data yielded evidence of SAP adsorption on the HAP surface, and peptide overlayers showed ordered structure and molecular orientation. The thickness of the SAP overlayer depends on the composition of the HAP coating.

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.

The Bone Building Blues: Self-hardening copper-doped calcium phosphate cement and its in vitro assessment against mammalian cells and bacteria.

A blue calcium phosphate cement with optimal self-hardening properties was synthesized by doping whitlockite (ß-TCP) with copper ions. The mechanism and the kinetics of the cement solidification process were studied using energy dispersive X-ray diffraction and it was found out that hardening was accompanied by the phase transition from TCP to brushite. Reduced lattice parameters in all crystallographic directions resulting from the rather low (1:180) substitution rate of copper for calcium was consistent with the higher ionic radius of the latter. The lower cationic hydration resulting from the partial Ca→Cu substitution facilitated the release of constitutive hydroxyls and lowered the energy of formation of TCP from the apatite precursor at elevated temperatures. Addition of copper thus effectively inhibited the formation of apatite as the secondary phase. The copper-doped cement exhibited an antibacterial effect, though exclusively against Gram-negative bacteria, including E. coli, P. aeruginosa and S. enteritidis. This antibacterial effect was due to copper ions, as demonstrated by an almost negligible antibacterial effect of the pure, copper-free cement. Also, the antibacterial activity of the copper-containing cement was significantly higher than that of its precursor powder. Since there was no significant difference between the kinetics of the release of copper from the precursor TCP powder and from the final, brushite phase of the hardened cement, this has suggested that the antibacterial effect was not solely due to copper ions, but due to the synergy between cationic copper and a particular phase and aggregation state of calcium phosphate. Though inhibitory to bacteria, the copper-doped cement increased the viability of human glial E297 cells, murine osteoblastic K7M2 cells and especially human primary lung fibroblasts. That this effect was also due to copper ions was evidenced by the null effect on viability increase exhibited by the copper-free cements. The difference in the mechanism of protection of dehydratases in prokaryotes and eukaryotes was used as a rationale for explaining the hereby evidenced selectivity in biological response. It presents the basis for the consideration of copper as a dually effective ion when synergized with calcium phosphates: toxic for bacteria and beneficial for the healthy cells.

RAMAN spectroscopy imaging improves the diagnosis of papillary thyroid carcinoma.

Recent investigations strongly suggest that Raman spectroscopy (RS) can be used as a clinical tool in cancer diagnosis to improve diagnostic accuracy. In this study, we evaluated the efficiency of Raman imaging microscopy to discriminate between healthy and neoplastic thyroid tissue, by analyzing main variants of Papillary Thyroid Carcinoma (PTC), the most common type of thyroid cancer. We performed Raman imaging of large tissue areas (from 100 × 100 µm2 up to 1 × 1 mm2), collecting 38 maps containing about 9000 Raman spectra. Multivariate statistical methods, including Linear Discriminant Analysis (LDA), were applied to translate Raman spectra differences between healthy and PTC tissues into diagnostically useful information for a reliable tissue classification. Our study is the first demonstration of specific biochemical features of the PTC profile, characterized by significant presence of carotenoids with respect to the healthy tissue. Moreover, this is the first evidence of Raman spectra differentiation between classical and follicular variant of PTC, discriminated by LDA with high efficiency. The combined histological and Raman microscopy analyses allow clear-cut integration of morphological and biochemical observations, with dramatic improvement of efficiency and reliability in the differential diagnosis of neoplastic thyroid nodules, paving the way to integrative findings for tumorigenesis and novel therapeutic strategies.