Marco Fosca

Real-time monitoring of the mechanism of poorly crystalline apatite cement conversion in the presence of chitosan, simulated body fluid and human blood.

In this study, the real-time monitoring of structural changes, occurring upon poorly crystalline apatite bone cement hardening in the presence of chitosan, simulated body fluid and human blood, was performed. Strong experimental evidence of octacalcium phosphate intermediate phase is provided. The energy dispersive X-ray diffraction was applied in situ to monitor the structural changes upon the transformation process, while the Fourier transform infrared spectroscopy and the scanning electron microscopy supplied information on the vibrational and morphological properties of the system. The cooperative action of chitosan and simulated body fluid induces the formation of a preferentially oriented hydroxyapatite phase, this process being similar to the oriented self-assembling process in collagen-apatite matrix in human plasma, occurring upon in vivo biomineralization. Conversely, the presence of blood does not induce any significant change in hardening kinetics and the final structure of the investigated cement.

Bioactive, nanostructured Si-substituted hydroxyapatite coatings on titanium prepared by pulsed laser deposition

AIMS The aim of this work was to deposit silicon-substituted hydroxyapatite (Si-HAp) coatings on titanium for biomedical applications, since it is known that Si-HAp is able to promote osteoblastic cells activity, resulting in the enhanced bone ingrowth. MATERIALS AND METHODS Pulsed laser deposition (PLD) method was used for coatings preparation. For depositions, Si-HAp targets (1.4 wt % of Si), made up from nanopowders synthesized by wet method, were used. RESULTS Microstructural and mechanical properties of the produced coatings, as a function of substrate temperature, were investigated by scanning electron and atomic force microscopies, X-ray diffraction, Fourier transform infrared spectroscopy, and Vickers microhardness. In the temperature range of 400-600°C, 1.4-1.5 µm thick Si-HAp films, presenting composition similar to that of the used target, were deposited. The prepared coatings were dense, crystalline, and nanostructured, characterized by nanotopography of surface and enhanced hardness. Whereas the substrate temperature of 750°C was too high and led to the HAp decomposition. Moreover, the bioactivity of coatings was evaluated by in vitro tests in an osteoblastic/osteoclastic culture medium (α-Modified Eagles Medium). CONCLUSIONS The prepared bioactive Si-HAp coatings could be considered for applications in orthopedics and dentistry to improve the osteointegration of bone implants.

Glass-ceramic coated Mg-Ca alloys for biomedical implant applications.

Biodegradable metals and alloys are promising candidates for biomedical bone implant applications. However, due to the high rate of their biodegradation in human body environment, they should be coated with less reactive materials, such, for example, as bioactive glasses or glass-ceramics. Fort this scope, RKKP composition glass-ceramic coatings have been deposited on Mg-Ca(1.4wt%) alloy substrates by Pulsed Laser Deposition method, and their properties have been characterized by a number of techniques. The prepared coatings consist of hydroxyapatite and wollastonite phases, having composition close to that of the bulk target material used for depositions. The 100μm thick films are characterized by dense, compact and rough morphology. They are composed of a glassy matrix with various size (from micro- to nano-) granular inclusions. The average surface roughness is about 295±30nm due to the contribution of micrometric aggregates, while the roughness of the fine-texture particulates is approximately 47±4nm. The results of the electrochemical corrosion evaluation tests evidence that the RKKP coating improves the corrosion resistance of the Mg-Ca (1.4wt%) alloy in Simulated Body Fluid.

Structural study of octacalcium phosphate bone cement conversion in vitro.

The nature of precursor phase during the biomineralization process of bone tissue formation is still controversial. Several phases were hypothesized, among them octacalcium phosphate. In this study, an in situ monitoring of structural changes, taking place upon the octacalcium phosphate bone cement hardening, was carried out in the presence of biopolymer chitosan and simulated body fluid (SBF). Several systems with different combinations of components were studied. The energy dispersive X-ray diffraction was applied to study the structural changes in real time, while morphological properties of the systems were investigated by the scanning electron microscopy. The obtained results evidence that final hydroxyapatite phase is formed only in the presence of chitosan and/or SBF, providing new insights into the in vivo biomineralization mechanism and, consequently, favoring the development of new approaches in biomaterials technology.

Phase Development During Setting and Hardening of a Bone Cement Based on α-Tricalcium and Octacalcium Phosphates

In this study, the phase development in the cement system α-TCP–OCP with phosphoric acid as a setting liquid was studied. The most promising formulation of α-TCP (60 wt%) and OCP (40 wt%) is proposed. This cement has the following characteristics: setting time 10 min, pH = 6.7, the compressive strength about 30 MPa, and high dissolution rate in an isotonic solution; the final wt% composition of α-TCP/DCPD/HA/OCP equals 27/38/20/15. Energy dispersive X-ray diffraction techniques were used for in situ monitoring of the processes taking place in the cement in real time.

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 (β-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.

Silver-Doped Calcium Phosphate Bone Cements with Antibacterial Properties.

Calcium phosphate bone cements (CPCs) with antibacterial properties are demanded for clinical applications. In this study, we demonstrated the use of a relatively simple processing route based on preparation of silver-doped CPCs (CPCs-Ag) through the preparation of solid dispersed active powder phase. Real-time monitoring of structural transformations and kinetics of several CPCs-Ag formulations (Ag = 0 wt %, 0.6 wt % and 1.0 wt %) was performed by the Energy Dispersive X-ray Diffraction technique. The partial conversion of β-tricalcium phosphate (TCP) phase into the dicalcium phosphate dihydrate (DCPD) took place in all the investigated cement systems. In the pristine cement powders, Ag in its metallic form was found, whereas for CPC-Ag 0.6 wt % and CPC-Ag 1.0 wt % cements, CaAg(PO3)3 was detected and Ag (met.) was no longer present. The CPC-Ag 0 wt % cement exhibited a compressive strength of 6.5 ± 1.0 MPa, whereas for the doped cements (CPC-Ag 0.6 wt % and CPC-Ag 1.0 wt %) the reduced values of the compressive strength 4.0 ± 1.0 and 1.5 ± 1.0 MPa, respectively, were detected. Silver-ion release from CPC-Ag 0.6 wt % and CPC-Ag 1.0 wt % cements, measured by the Atomic Emission Spectroscopy, corresponds to the average values of 25 µg/L and 43 µg/L, respectively, rising a plateau after 15 days. The results of the antibacterial test proved the inhibitory effect towards pathogenic Escherichia coli for both CPC-Ag 0.6 wt % and CPC-Ag 1.0 wt % cements, better performances being observed for the cement with a higher Ag-content.

In Situ Time-Resolved Energy Dispersive X-Ray Diffraction Studies of Calcium Phosphate Based Bone Cements

In situ monitoring of structural changes, taking place upon calcium phosphate bone cements hardening process was carried out by means of the Energy Dispersive X-Ray Diffraction method. Two different cement systems were studied, one of them based on the octacalcium phosphate and another - on the dicalcium phosphate dehydrate. Both systems contained natural biopolymer chitosan and were soaked in Simulated Body Fluid. The obtained experimental results evidence that during the hardening of the cement containing octacalcium phosphate its partial transformation into hydroxyapatite takes place, whereas no significant changes were detected during the hardening process of cement containing the dicalcium phosphate dehydrate.

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.

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.