Harnessing Wharton's jelly stem cell differentiation into bone-like nodule on calcium phosphate substrate without osteoinductive factors.

An important aim of bone regenerative medicine is to design biomaterials with controlled chemical and topographical features to guide stem cell fate towards osteoblasts without addition of specific osteogenic factors. Herein, we find that sprayed bioactive and biocompatible calcium phosphate substrates (CaP) with controlled topography induce, in a well-orchestrated manner, Wharton's jelly stem cells (WJ-SCs) differentiation into osteoblastic lineage without any osteogenic supplements. The resulting WJ-SCs commitment exhibits features of native bone, through the formation of three-dimensional bone-like nodule with osteocyte-like cells embedded into a mineralized type I collagen. To our knowledge, these results present the first observation of a whole differentiation process from stem cell to osteocytes-like on a synthetic material. This suggests a great potential of sprayed CaP and WJ-SCs in bone tissue engineering. These unique features may facilitate the transition from bench to bedside and the development of successful engineered bone. STATEMENT OF SIGNIFICANCE: Designing materials to direct stem cell fate has a relevant impact on stem cell biology and provides insights facilitating their clinical application in regenerative medicine. Inspired by natural bone compositions, a friendly automated spray-assisted system was used to build calcium phosphate substrate (CaP). Sprayed biomimetic solutions using mild conditions led to the formation of CaP with controlled physical properties, good bioactivity and biocompatibility. Herein, we show that via optimization of physical properties, CaP substrate induce osteogenic differentiation of Wharton's jelly stem cells (WJ-SCs) without adding osteogenic supplement factors. These results suggest a great potential of sprayed CaP and WJ-SCs in bone tissue engineering and may facilitate the transition from bench to beside and the development of clinically successful engineered bone.

An optimized device for staining electron microscopy grids.

Proper staining of grids is critical for transmission electron microscopy (TEM). Staining must be done as quickly as possible using minimal reagents and with consideration for the environment. We developed a new device for efficient staining of multiple TEM grids. We studied reagent evaporation, rinsing volume, flow rate and re-use of uranyl acetate, and provide here a procedure for efficient staining using the new device. Our device permits TEM grids to be stained with less reagent than alternative staining apparatuses; staining requires a total volume of 260 µl for five grids. Reagent evaporation is less than 6% even if used at 37° C. Moreover, our staining apparatus reduces chemical waste and shortens experiment time by staining several grids simultaneously. Our staining device is a compromise between time-consuming single grid processing and expensive commercial devices that consume large amounts of reagents.

Homozygous and compound heterozygous MMP20 mutations in amelogenesis imperfecta.

In this article, we focus on hypomaturation autosomal-recessive-type amelogenesis imperfecta (type IIA2) and describe 2 new causal Matrix metalloproteinase 20 (MMP20) mutations validated in two unrelated families: a missense mutation p.T130I at the expected homozygous state, and a compound heterozygous mutation having the same mutation combined with a nucleotide deletion, leading to a premature stop codon (p.N120fz*2). We characterized the enamel structure of the latter case using scanning electron microscopy analysis and microanalysis (Energy-dispersive X-ray Spectroscopy, EDX) and confirmed the hypomaturation-type amelogenesis imperfecta as identified in the clinical diagnosis. The mineralized content was slightly decreased, with magnesium substituting for calcium in the crystal structure. The anomalies affected enamel with minimal inter-rod enamel present and apatite crystals perpendicular to the enamel prisms, suggesting a possible new role for MMP20 in enamel formation.

Polysaccharide films built by simultaneous or alternate spray: a rapid way to engineer biomaterial surfaces.

We investigated polysaccharide films obtained by simultaneous and alternate spraying of a chitosan (CHI) solution as polycation and hyaluronic acid (HA), alginate (ALG), and chondroitin sulfate (CS) solutions as polyanions. For simultaneous spraying, the film thickness increases linearly with the cumulative spraying time and passes through a maximum for polyanion/CHI molar charge ratios lying between 0.6 and 1.2. The size of polyanion/CHI complexes formed in solution was compared with the simultaneously sprayed film growth rate as a function of the polyanion/CHI molar charge ratio. A good correlation was found. This suggests the importance of polyanion/polycation complexation in the simultaneous spraying process. Depending on the system, the film topography is either liquid-like or granular. Film biocompatibility was evaluated using human gingival fibroblasts. A small or no difference is observed in cell viability and adhesion between the two deposition processes. The CHI/HA system appears to be the best for cell adhesion inducing the clustering of CD44, a cell surface HA receptor, at the membrane of cells. Simultaneous or alternate spraying of CHI/HA appears thus to be a convenient and fast procedure for biomaterial surface modifications.

Simultaneous spray coating of interacting species: general rules governing the poly(styrene sulfonate)/poly(allylamine) system.

Simultaneous spraying of two solutions of interacting species onto a substrate held vertically leads to the formation of nanometer-sized coatings. Here we investigate the simultaneous spraying of poly(styrene sulfonate) (PSS) and poly(allylamine hydrochloride) (PAH) solutions leading to the formation of a film composed of PSS/PAH complexes. The thickness of this film increases linearly with the cumulative spraying time. For a given spraying rate of PAH (respectively PSS), the growth rate of the film depends strongly upon the PSS/PAH ratio and passes through a maximum for a PSS/PAH ratio lying between 0.55 and 0.8. For a PSS/PAH ratio that is maintained constant, the growth speed of the film increases linearly with the spraying rate of polyelectrolyte of both solutions. Using X-ray photoelectron spectroscopy, we find that the film composition is almost independent of the PSS/PAH (spayed) ratio, with composition very close to 1:1 in PSS:PAH film. The 1:1 PSS:PAH composition is explained by the fact that the simultaneous spraying experiments are carried out with salt-free solutions; thus, electroneutrality in the film requires exact matching of the charges carried by the polyanions and the polycations. Zeta potential measurements reveal that, depending on whether the PSS/PAH spraying rate ratio lies below or above the optimal spraying rate ratio, the film acquires a positive or a negative excess charge. We also find that the overall film morphology, investigated by AFM, is independent of the spraying rate ratio and appears to be composed of nanometer-sized grains which are typically in the 100 nm range.

Ultrastructural assessments of the melanosome distribution patterns and pigmentation features in human epidermal cells after UV irradiation and kojic acid treatment.

It is well-known that skin pigmentation depends, among others, on number, aggregation and distribution of melanosomes in the epidermis. Here we describe a correlative microscopy-based ultrastructural approach that investigates the spatial distribution and pigmentation features of the melanosomes within melanocytes and keratinocytes. Data obtained from control skin, ultraviolet (UV)-stimulated tissue and kojic acid-treated UV-irradiated explants are compared. We introduce original parameters for the evaluation of the aggregation and pigmentation features of the melanosomes: the aggregation and pigmentation indexes. The aggregation index evaluates the presence of clustered melanosomes when the pigmentation index expresses the electron-density level of the pigment granules. The present study demonstrates that the last parameters clearly express histological effects induced by UVB irradiation. Results indicate that UV light did not change the number of melanosomes within either melanocytes or keratinocytes, but it definitely modified the distribution patterns of the pigment granules in both cell types. It also enhanced the pigmentation state of the epidermal cells. Moreover, statistical analysis concerning keratinocytes discloses a significant decrease in the mean pigmentation index when explants exposed to UV light were treated with kojic acid. Obviously, the present numerical findings point out the relevance of the introduced parameters to characterize the pigmentation state of skin.

Changes in silicon elastomeric surface properties under stretching induced by three surface treatments.

Poly(dimethylsiloxane) (PDMS) substrates are used in many applications where the substrates need to be elongated and various treatments are used to regulate their surface properties. In this article, we compare the effect of three of such treatments, namely, UV irradiation, water plasma, and plasma polymerization, both from a molecular and from a macroscopic point of view. We focus our attention in particular on the behavior of the treated surfaces under mechanical stretching. UV irradiation induces the substitution of methyl groups by hydroxyl and acid groups, water plasma leads to a silicate-like layer, and plasma polymerization causes the formation of an organic thin film with a major content of anhydride and acid groups. Stretching induces cracks on the surface both for silicate-like layers and for plasma polymer thin coatings. This is not the case for the UV irradiated PDMS substrates. We then analyzed the chemical composition of these cracks. In the case of water plasma, the cracks reveal native PDMS. In the case of plasma polymerization, the cracks reveal modified PDMS. The contact angles of plasma polymer and UV treated surfaces vary only very slightly under stretching, whereas large variations are observed for water plasma treatments. The small variation in the contact angle values observed on the plasma polymer thin film under stretching even when cracks appear on the surface are explained by the specific chemistry of the PDMS in the cracks. We find that it is very different from native PDMS and that its structure is somewhere between Si(O2) and Si(O3). This is, to our knowledge, the first study where different surface treatments of PDMS are compared for films under stretching.

Mechanically responsive films of variable hydrophobicity made of polyelectrolyte multilayers.

Mechanically responsive surfaces that allow to switch reversibly from a hydrophobic to a hydrophilic substrate are reported. The surfaces are constituted of polyelectrolyte multilayers deposited on modified charged silicone sheets. n bilayers of poly(allylamine)-Nafion (PAH-Naf) and m bilayers of poly(allylamine)-poly(acrylic acid) (PAH-PAA) composed the multilayers. A (PAH-Naf)(n) film possesses a water contact angle of around 105 degrees, whereas the contact angle of a (PAH-Naf)(4)-(PAH-PAA)(m) multilayer is around 50 degrees. When such a film with m < 5 and terminated by PAA is stretched out, its water contact angle increases up to around 100 degrees. Successive elongation/retraction cycles allow the water contact angle to alternate reversibly between 100 and 57 degrees indicating the reversible mechanical responsive nature of the film.

Modeling of the detachment of a molecule from a surface: illustration of the "Bell-Evans effect".

This article deals with the modeling of the detachment of a molecule initially adsorbed on a surface and submitted to an external force whose strength increases with time. By means of an atomic force microscope (AFM), it is possible to measure the force when the molecule separates from the substrate. However, it is known that this force depends to a large extend on the rate at which the pulling force is applied ("Bell-Evans effect"). Two models are described to illustrate this behavior. First, a random walk approach is suggested to reveal the fundamental principle of the escape over a time-dependent energy barrier. Second, a multi bead-and-spring model is proposed to mimic the AFM experiment and numerical simulations, based on Brownian dynamics, are performed.

Odontoblast dysfunction in osteogenesis imperfecta: an LM, SEM, and ultrastructural study.

The inherited dentin defect dentinogenesis imperfecta (DI), while clinically obvious in osteogenesis imperfecta (OI) Types IB and IC, II, III, and IVB, is now thought to be present in all children with OI, in a continuum from minimal to severe dentin pathology. This collaborative study further clarifies the structural and ultrastructural dentin changes in the teeth of OI children with clinically obvious DI, and attempts to explain these in terms of odontoblast dysfunction. Collaborative studies were carried out in Melbourne, Australia, and Strasbourg, France, using light and polarized-light microscopy, scanning and transmission electron microscopy (SEM, TEM), selected-area diffraction (SAD), and x-ray spectroscopy (EDX). These showed structurally normal enamel (but containing long and broad lamellae) and a normally scalloped dentino-enamel junction (DEJ), but severe pathologic changes in the dentin. An initial narrow band of normal-appearing dentin tubules (including the mantle layer) ceased abruptly and was replaced by a wavelike laminar zone parallel to the DEJ with occluded tubules. Multiple parallel channels of 5-10 microns diameter were present at right angles to the DEJ indenting this zone, some terminating in retro-curved "processes." The abnormal dentin containing these channels almost completely occluded the pulp chamber. The structural and ultrastructural changes seen can be explained on the basis of the collagen defect in OI resulting in odontoblast dysfunction, which produces a distinct phenotype and one that is different from that in bone.

Semi-automatized processing of AFM force-spectroscopy data.

Atomic force microscopy operated in the force-spectroscopy mode is now a widespread technique, often used to investigate ligand-receptor interactions with the goal of measuring forces at the individual molecule level. However, in an experiment, the simultaneous interaction of several ligand/receptor pairs cannot be excluded. This may produce complicated force curves, although unambiguous ruptures are sometimes observed. In the case of the non-specific adhesion of molecules, such as fibrinogen, to a surface, it is usually difficult to identify the real events on the force curves. This can render the application of fixed rules uneasy and in addition can introduce some degree of arbitrariness if the analysis has to be performed by hand. In the present paper a computer algorithm, aimed at speeding up the processing, and at applying selection rules in a reproducible manner, is proposed. It is applied to force recordings performed at various retraction velocities, thus various loading rates. The influence on the evaluation of the rupture forces of the different parameters that can be set by the operator is discussed.

Unbinding process of adsorbed proteins under external stress studied by atomic force microscopy spectroscopy.

We report the study of the dynamics of the unbinding process under a force load f of adsorbed proteins (fibrinogen) on a solid surface (hydrophilic silica) by means of atomic force microscopy spectroscopy. By varying the loading rate r(f), defined by f = r(f) t, t being the time, we find that, as for specific interactions, the mean rupture force increases with r(f). This unbinding process is analyzed in the framework of the widely used Bell model. The typical dissociation rate at zero force entering in the model lies between 0. 02 and 0.6 s(-1). Each measured rupture is characterized by a force f(0), which appears to be quantized in integer multiples of 180-200 pN.

Characterization, at the bone crystal level, of the titanium-coating/bone interfacial zone.

Well integrated and clinically functional titanium dental implants retrieved in humans after 14 and 40 months of settlement were used for the characterization of the interfacial area between the plasma-sprayed coating and the surrounding mineralized bone. Electron microscopic studies were performed from undecalcified intact coating/bone interfaces. The concomitant presence of direct bone tissue apposition on the titanium coating, as well as the interposition of amorphous material along the same interfacial zones emphasizes the dynamic biologic aspect of the osseointegration process. A very striking finding of these ultrastructural and microanalytical investigations was the presence of tiny titanium grains ranging from 5 to 50 nm in diameter at the surface of bulky plasma-sprayed coating particles. High-resolution transmission electron microscopy revealed a continuity between the lattice planes of the coating material and those of the minute titanium grains; thus indicating a chemical binding. The thickness of the granular titanium layer interposed between coating particles and mineralized bone tissue ranged up to 600 nm. The observation of calcium-phosphate needle-like crystallites within the porous layer made of titanium grains indicates a bone ingrowth process, suggesting a bone-binding mechanism to the outer surface of the coated titanium implant.

Influence of applied quantity, water-immersion and air-drying on covering and microstructure of physical sunscreen films.

The features of topically applied suncream films depend on the applied quantities and could be modified by water immersion and air drying. The aim of this study was to investigate the effects of the aforementioned factors on physical sunscreen films and to establish the correlation between the in vitro determined Sun Protection Factor (SPF) and the microstructure of the mineral coating. The assessments were conducted by using UV spectroscopy and electron microscopy on sunscreen films applied on both synthetic membranes and human skin. The results emphasize the paramount role played by the applied quantity to produce a continuous and protecting sunscreen film. The microscopic findings show that water immersion induces mainly a compactness of the sunscreen films, whereas no significant alterations were noted after air drying. Lastly, the SPF values, measured before and after water immersion, disclose the satisfactory water resistance of the broad-spectrum physical sunscreen considered.

Direct observation of the anchoring process during the adsorption of fibrinogen on a solid surface by force-spectroscopy mode atomic force microscopy.

Atomic force microscopy in a force-spectroscopy mode has been used to investigate the kinetics of the adsorption process of fibrinogen molecules on a silica surface. An original "approach/retraction" cycle of the tip/surface was used for this purpose. Fibrinogen molecules were adsorbed on the atomic force microscopy tip and were brought into contact with the silica surface for different interaction times varying from 5 to 2,000 ms. Multiple consecutive ruptures were observed. The mean number of ruptures nr per cycle increases steadily with the interaction time as well as the mean strength fr which varies from 300 pN for 5 ms to 1,400 pN for 2,000 ms. The minimal interaction time for a fibrinogen molecule to bind strongly to a silica surface during an adsorption process appears to lie between 50 and 200 ms. The histograms of the distances between two consecutive ruptures in one cycle exhibit maxima around 20-25 nm. This length is comparable to the characteristic distance between D and E globules of one fibrinogen molecule and suggests that fibrinogen molecules mainly adsorb through their D and E globules.

Characterization and histological analyses of a coral-collagen composite used for bone-replacement graft material: a report of clinical cases.

Several studies, devoted to the osteogenic potentialities of natural CaCO3 have already been reported. However, it seems questionable if the data obtained from natural calcium carbonates can be extrapolated to a composite biomaterial incorporating coralline material. For these reasons, in the present investigations the structural and crystallographic features of the biomaterial (Biocoral gel) were thoroughly analyzed prior to implantation, with the aid of X-ray diffraction and electron microscopy. Then, biopsied samples, taken from Biocoral gel-filled sites, respectively after 7, 8, 9, 12 and 29 mon implantation, were studied with optical and electron microscopy. It could be concluded from the histological analyses of the biopsies, that mineral still remained after long implantation periods. This composite biomaterial may thus be considered for uses in clinical situations where neither incorporation nor dissolution of the implanted biomaterial are essential, i.e. maintenance of edentulous ridge volume.

Ultrastructural features of the bone response to a plasma-sprayed hydroxyapatite coating in sheep.

The intentions of this study were to characterize the macroscopic, microscopic, and structural aspects of a plasma-sprayed implant and to thoroughly investigate bone tissue response after its implantation in sheep. Therefore, we used scanning electron microscopy, transmission electron microscopy (TEM), high-resolution TEM, X-ray diffraction, and energy-dispersive X-ray analyses. Assessment of the biomaterial prior to implantation showed a coating with irregular outlines and varying thickness, mainly consisting of hydroxyapatite (HA) covering a rough metallic implant core. Six months after insertion of the HA-coated Ti-6A1-4V implant, neither mechanical failure of the coating-substrate interface nor a significant loss of coating thickness was evident. However, an occasional lack of HA coating and phagocytosis of HA particles were noted. More generally, the implant was surrounded by well-mineralized bone investing the smallest cavities of the plasma-sprayed layer. Newly formed microcrystals with size, shape, and structure similar to those of bone apatite crystals were growing directly at the coating surface. These results suggest that the bone-bonding behavior of the considered grooved implant should provide satisfactory osseointegration and be suitable for fixed prostheses.

Characterization of the mineral content of a physical sunscreen emulsion and its distribution onto human stratum corneum.

When using physical sunscreens, protection and aesthetic aspects are directly related to particle size and mineral distribution onto skin. These investigations aimed to characterize the different mineral raw materials entering into the composition of a specific sunscreen emulsion and to assess the distribution of the sunscreeen agents at the surface of human stratum corneum. X-ray diffractometry revealed the crystallographic features of the TiO2 and ZnO crystals. Frequency distributions of crystal length and aspect ratio were determined for the zinc oxide which presented the largest particle size and varying shapes. Scanning and transmission electron microscopy disclosed structural information. The results reported in this study specify the characteristics of the oxide particles involved in the formulation and show a satisfactory mineral distribution along the irregular topography of the skin. No intracellular penetration was noted.

HRTEM study of biological crystal growth mechanisms in the vicinity of implanted synthetic hydroxyapatite crystals.

Calcium phosphates are widely used as biomaterials. Ultrastructural assessments are of the utmost importance in our understanding of interfacial phenomena. The aim of this study was to learn more about the newly formed crystal growth mechanisms. The interfaces between implanted synthetic hydroxyapatite crystals (HAS) and newly formed crystallites were thoroughly examined on a molecular level. The bone-grafting material (HAS) was implanted into two adult patients, and small biopsies were recovered 6 months after implantation. The raw biomaterial was analyzed by x-ray diffraction and high-resolution transmission electron microscopy (HRTEM). Six months after their implantation, the HAS aggregates were surrounded by a mineralized bone matrix. Tiny crystallites also filled the spaces between the HAS crystals within the aggregates. These newly formed crystallites growing at the surfaces of the implanted HAS crystals appeared to be apatitic. The crystallographic investigations of the nucleation and growing mechanisms of the newly formed crystallites were performed by HRTEM in association with computer simulation and mathematical processing of digitized images. A relationship was noted between the orientation axes of crystallites growing nearby and the zone axes of the implanted HAS, thus strongly suggesting a guiding or substratum role of the HAS particles.

Transformation of hydroxyapatite to fluorapatite by irradiation with high-energy CO2 laser.

High-energy laser irradiation has been shown to cause crystalline transformations in apatites, which may lead to the formation of tricalcium phosphates with a resulting decrease in acid resistance. Depending on the nature and energy density of laser irradiation used, however, an increase of acid resistance of dental enamel has also been reported after laser irradiation. The aim of the present study was to investigate the phase transformation of hydroxyapatite (HA) to fluorapatite (FA) in a model system that incorporates sodium fluoride (NaF) into apatite structure by using laser irradiation. A CO2 laser was used at energy densities ranging from 21 to 500 J/cm2. Synthetic HA mixed with NaF (10:1) was the target of laser irradiation. The crystalline structures were then investigated using X-ray diffraction analysis. The results showed that a phase transformation of HA to FA could be realized, and that the threshold energy density needed was 38 J/cm2. Not only is the finding crystallographically important, but it also opens new perspectives for future research regarding the development of laser technology for clinical purposes.