Fani Anagnostou

Osteoblastic differentiation of human mesenchymal stem cells with platelet lysate.

Culture of expanded mesenchymal stem cells (MSCs) seeded on biomaterials may represent a clinical alternative to autologous bone graft in bone regeneration. Foetal bovine serum (FBS) is currently used for MSC expansion, despite risks of infectious disease transmission and immunological reaction due to its xenogenic origin. This study aimed to compare the osteogenic capacities of clinical-grade human MSCs cultured with FBS or allogenic human platelet lysate (PL). In vitro, MSCs cultured in PL both accelerate the expansion rate over serial passages and spontaneously induce osteoblastic gene expression such as alkaline phosphatase (ALP), bone sialoprotein (BSP), osteopontin (Op) and bone morphogenetic protein-2 (BMP-2). In vivo, ectopic bone formation is only observed on ceramics seeded with MSCs grown in PL medium implanted under the skin of immunodeficient mice for 7 weeks. In conclusion, allogenic human PL accelerates MSC proliferation and enhances MSC osteogenic differentiation.

Bioactive polymer grafting onto titanium alloy surfaces

Bioactive polymers bearing sulfonate (styrene sodium sulfonate, NaSS) and carboxylate (methylacrylic acid, MA) groups were grafted onto Ti6Al4V alloy surfaces by a two-step procedure. The Ti alloy surfaces were first chemically oxidized in a piranha solution and then directly subjected to radical polymerization at 70 degrees C in the absence of oxygen. The grafted surfaces were characterized by X-ray photoelectron spectroscopy (XPS), time-of-flight secondary ion mass spectrometry (ToF-SIMS) and the toluidine blue colorimetric method. Toluidine blue results showed 1-5microgcm(-2) of polymer was grafted onto the oxidized Ti surfaces. Grafting resulted in a decrease in the XPS Ti and O signals from the underlying Ti substrate and a corresponding increase in the XPS C and S signals from the polymer layer. The ToF-SIMS intensities of the S(-) and SO(-) ions correlated linearly with the XPS atomic percent S concentrations and the ToF-SIMS intensity of the TiO(3)H(2)(-) ion correlated linearly with the XPS atomic per cent Ti concentration. Thus, the ToF-SIMS S(-), SO(-) and TiO(3)H(2)(-) intensities can be used to quantify the composition and amount of grafted polymer. ToF-SIMS also detected ions that were more characteristic of the polymer molecular structure (C(6)H(4)SO(3)(-) and C(8)H(7)SO(3)(-) from NaSS, C(4)H(5)O(2)(-) from MA), but the intensity of these peaks depended on the polymer thickness and composition. An in vitro cell culture test was carried out with human osteoblast-like cells to assess the influence of the grafted polymers on cell response. Cell adhesion after 30min of incubation showed significant differences between the grafted and ungrafted surfaces. The NaSS grafted surfaces showed the highest degree of cell adhesion while the MA-NaSS grafted surfaces showed the lowest degree of cell adhesion. After 4 weeks in vivo in rabbit femoral bones, bone was observed to be in direct contact with all implants. The percentage of mineralized tissue around the implants was similar for NaSS grafted and non-grafted implants (59% and 57%). The MA-NaSS grafted implant exhibited a lower amount of mineralized tissue (47%).

Use of the induced membrane technique for bone tissue engineering purposes: animal studies.

Animal experiments using the induced membrane procedure for bone tissue engineering purposes have provided evidence that the membrane has structural characteristics and biologic properties that may be used for bone tissue engineering purposes. Clinically relevant animal models have demonstrated that standardized particulate bone constructs can be used to repair large bone defects using the procedure and that the osteogenic ability of these constructs partially approaches that of bone autografts.

Ultrasonic Evaluation of Dental Implant Biomechanical Stability: An In Vitro Study

Dental implants are widely used for oral rehabilitation. However, there remain risks of failure that are difficult to anticipate. The objective of this ex vivo study is to investigate the potentiality of quantitative ultrasound (QUS) to assess the amount of bone in contact with titanium prototype cylindrical implants. Four groups of 10 rabbit femurs each are considered, corresponding to different amounts of bone in contact with the implant. The 10 MHz ultrasonic response of the implant is processed to derive a quantitative indicator I, based on the temporal variation of the signal amplitude. Analysis of variance (ANOVA) (p < 10(-5)) tests revealed a statistical distribution of I significantly correlated with the amount of bone in contact with the cylinders. An analytical model considering the propagation of lateral waves allows the understanding of the physical origin of the echoes. QUS technique may be used to investigate the amount of bone in contact with a cylinder implant.

Micro-Brillouin Scattering Measurements in Mature and Newly Formed Bone Tissue Surrounding an Implant

The evolution of implant stability in bone tissue remains difficult to assess because remodeling phenomena at the bone-implant interface are still poorly understood. The characterization of the biomechanical properties of newly formed bone tissue in the vicinity of implants at the microscopic scale is of importance in order to better understand the osseointegration process. The objective of this study is to investigate the potentiality of micro-Brillouin scattering techniques to differentiate mature and newly formed bone elastic properties following a multimodality approach using histological analysis. Coin-shaped Ti-6Al-4V implants were placed in vivo at a distance of 200 μm from rabbit tibia leveled cortical bone surface, leading to an initially empty cavity of 200 μm×4.4 mm. After 7 weeks of implantation, the bone samples were removed, fixed, dehydrated, embedded in methyl methacrylate, and sliced into 190 μm thick sections. Ultrasonic velocity measurements were performed using a micro-Brillouin scattering device within regions of interest (ROIs) of 10 μm diameter. The ROIs were located in newly formed bone tissue (within the 200 μm gap) and in mature bone tissue (in the cortical layer of the bone sample). The same section was then stained for histological analysis of the mineral content of the bone sample. The mean values of the ultrasonic velocities were equal to 4.97×10(-3) m/s in newly formed bone tissue and 5.31×10(-3) m/s in mature bone. Analysis of variance (p=2.42×10(-4)) tests revealed significant differences between the two groups of measurements. The standard deviation of the velocities was significantly higher in newly formed bone than in mature bone. Histological observations allow to confirm the accurate locations of the velocity measurements and showed a lower degree of mineralization in newly formed bone than in the mature cortical bone. The higher ultrasonic velocity measured in newly formed bone tissue compared with mature bone might be explained by the higher mineral content in mature bone, which was confirmed by histology. The heterogeneity of biomechanical properties of newly formed bone at the micrometer scale may explain the higher standard deviation of velocity measurements in newly formed bone compared with mature bone. The results demonstrate the feasibility of micro-Brillouin scattering technique to investigate the elastic properties of newly formed bone tissue.

Numerical simulation of ultrasonic wave propagation for the evaluation of dental implant biomechanical stability

Osseointegration of dental implants remains poorly understood. The objective of this numerical study is to understand the propagation phenomena of ultrasonic waves in prototypes cylindrically shaped implants and to investigate the sensitivity of their ultrasonic response to the surrounding bone biomechanical properties. The 10 MHz ultrasonic response of the implant was calculated using a finite difference numerical simulation tool and was compared to rf signals taken from a recent experimental study by Mathieu et al. [Ultrasound Med. Biol. 37, 262-270 (2011a)]. Reflection and mode conversion phenomena were analyzed to understand the origin of the different echoes and the importance of lateral wave propagation was evidenced. The sensitivity of the ultrasonic response of the implant to changes of (i) amount of bone in contact with the implant, (ii) cortical bone thickness, and (iii) surrounding bone material properties, was compared to the reproducibility of the measurements. The results show that, either a change of 1 mm of bone in contact with the implant, or 1.1 mm of cortical thickness or 12% of trabecular bone mass density should be detectable. This study paves the way for the investigation of the use of quantitative ultrasound techniques for the evaluation of bone-implant interface properties and implant stability.

Ecto-alkaline phosphatase considered as levamisole-sensitive phosphohydrolase at physiological pH range during mineralization in cultured fetal calvaria cells

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p‐nitrophenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6–8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP were close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole was uncompetitive (Ki = 45 μM). Phosphatidylinositol‐specific phospholipase C (PI‐PLC) produced the release into the medium of a p‐nitrophenyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The released activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto‐pNPPase activity. After 75 min of PI‐PLC treatment the ecto‐pNPPase activity remained unchanged despite the 30% decrease in Nonidet P‐40‐extractable ALP activity. High levels of 45Ca incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in accordance with ecto‐pNPPase activity variations. These results evidenced the levamisole‐sensitive activity of a glycosylphosphatidylinositol‐anchored pNPPase consistent with ALP acting as an ecto‐enzyme whose functioning under physiological conditions was correlated to 45Ca incorporation and permit the prediction of the physiological importance of the enzyme dynamic equilibrium at the cell surface in cultured fetal calvaria cells.

Ultrasonic evaluation of dental implant osseointegration

Dental implants are widely used for oral rehabilitation. However, there remain risks of failure which are difficult to anticipate and depend on the implant osseointegration. The objective of this in vivo study is to determine the variation of the echographic ultrasonic response of a dental implant to bone healing around the implant interface. Twenty one dental implants were inserted in the femur of seven New Zealand white rabbits. Two animals were sacrificed after a healing duration of two weeks, three animals after six weeks and six animals after eleven weeks. The 10 MHz ultrasonic response of the implant was measured just after the implantation using a dedicated device positioned at the emerging surface of each dental implant. The measurements were realized again before the sacrifice with the same device. An indicator I˜ was derived based on the amplitude of the rf signal obtained for each configuration. The bone-Implant Contact (BIC) ratio was determined by histological analyses. The average value of the relative variation of the indicator I˜ obtained after initial surgery and after the corresponding healing period varies between 7% and 40%. A Kruskal-Wallis test (p<0.01) revealed a significant decrease of the value of the indicator I˜ as function of healing time. The indicator I˜ was significantly correlated (R(2)=0.45) with the BIC ratio. The results show that the ultrasonic response of a dental implant varies significantly as a function of healing time, which paves the way for the development of a new quantitative ultrasound (QUS) method in oral implantology.

Role of β-GP-derived pi in mineralization via ecto-alkaline phosphatase in cultured fetal calvaria cells

The permissive effect of β‐GP on mineralization in cultured rat fetal calvaria cells was investigated in relationship with phosphohydrolase activity of ecto‐ALP at physiological pH range. β‐GP present in the culture medium for 8 days exerted a stimulatory effect on 45Ca incorporation into matrix cell layers while the ecto‐ALP activity level measured on intact cells with a saturating concentration of p cells grown either in the presence or absence of β‐GP. In both types of cultures, β‐GP addition inhibited pNPP hydrolysis in a competitive and reversible manner and increased Pi concentration in the medium. The dose dependency of the effect of β‐GP on 45Ca incorporation and generation of Pi was similar (kϕ = 3 mM). Levamisole, but not dexamisole, inhibited both pNPP and β‐GP hydrolyses, which were likely catalyzed by the same ecto‐enzyme. The rate of 45Ca incorporation into matrix cell layers, which was high (0.90 μmol/4h/mg cell protein) in cells grown in the absence of β‐GP, was inhibited by 50% by levamisole. In cells grown in the absence of β‐GP, the 45Ca incorporation rate increased progressively after β‐GP addition, reaching after 12 h the value of cultures grown in the presence of β‐GP, the increase being totally inhibited by levamisole. In both types of cells, addition of exogenous Pi at concentrations corresponding to medium levels of β‐GP‐derived Pi rapidly led to high 45Ca incorporation rate which was unaffected by levamisole. β‐GP removal from cultures grown in its presence reduced by 50% the 45Ca incorporation rate which recovered the initial value after exogenous Pi addition independently of levamisole presence. Thus, mineral deposition did not affect the level and catalytic efficiency of ecto‐ALP to hydrolyze β‐GP in cultured fetal calvaria cells, yet it influenced the β‐GP‐stimulatory effect on mineralization so as to render this process not sensitive to high medium Pi levels.

Influence of healing time on the ultrasonic response of the bone-implant interface

The aim of the present study is to investigate the effect of bone healing on the ultrasonic response of coin-shaped titanium implants inserted in rabbit tibiae. The ultrasound response of the interface was measured in vitro at 15 MHz after 7 and 13 weeks of healing time. The average value of the ratio r between the amplitudes of the echo of the bone-implant interface and of the water-implant interface was determined. The bone-implant contact (BIC) was measured by histomorphometry and the degree of mineralisation of bone was estimated qualitatively by histologic staining. The significant decrease of the ultrasonic quantitative indicator r (p = 2.10⁻⁴) vs. healing time (from r = 0.53 to r = 0.49) is explained by (1) the increase of the BIC (from 27% to 69%) and (2) the increase of mineralization of newly formed bone tissue, both phenomena inducing a decrease of the gap of acoustical impedance.