Preparation of collagen fibrils from mineralized tissues and evaluation by atomic force microscopy.

Mineralized tissues like bone and dentin are materials that support the distribution of mechanical loads through the body of humans and other animals. While their organic content plays a critical role on the structural behavior of these materials, investigations that quantify the structural properties of collagen fibrils in mineralized tissues at the nanoscale are rather limited. We report a new experimental methodology to prepare samples of dentinal collagen fibrils for evaluation by atomic force microscopy and characterize their mechanical behavior. Specifically, a Dynamic Mechanical Analysis (DMA) of the collagen fibrils was performed to study their viscoelastic behavior. The capacity for viscous dampening in the fibrils was characterized in terms of measures of the energy dissipation, phase angle and loss modulus in both the peak and trough regions of the fibrils. According to the phase angle and the loss modulus, the peak regions of the fibrils exhibit significantly greater stiffness and capacity for dampening than the trough regions. This new approach will help in exploring the role of collagen fibrils in the mechanical behavior of dentin and other mineralized tissues as well as help to understand the potential effects from changes in fibril confirmation with tissue treatments, aging or that result from chronic disease.

Engineered Escherichia coli Biofilms Produce Adhesive Nanomaterials Shaped by a Patterned 43 kDa Barnacle Cement Protein.

Barnacles integrate multiple protein components into distinct amyloid-like nanofibers arranged as a bulk material network for their permanent underwater attachment. The design principle for how chemistry is displayed using adhesive nanomaterials, and fragments of proteins that are responsible for their formation, remains a challenge to assess and is yet to be established. Here, we use engineered bacterial biofilms to display a library of amyloid materials outside of the cell using full-length and subdomain sequences from a major component of the barnacle adhesive. A staggered charged pattern is found throughout the full-length sequence of a 43 kDa cement protein (AACP43), establishing a conserved sequence design evolved by barnacles to make adhesive nanomaterials. AACP43 domain deletions vary in their propensity to aggregate and form fibers, as exported extracellular materials are characterized through staining, immunoblotting, scanning electron microscopy, and atomic force microscopy. Full-length AACP43 and its domains have a propensity to aggregate into nanofibers independent of all other barnacle glue components, shedding light on its function in the barnacle adhesive. Curliated Escherichia coli biofilms are a compatible system for heterologous expression and the study of foreign functional amyloid adhesive materials, used here to identify the c-terminal portion of AACP43 as critical in material formation. This approach allows us to establish a common sequence pattern between two otherwise dissimilar families of cement proteins, laying the foundation to elucidate adhesive chemistries by one of the most tenacious marine fouling organisms in the ocean.

Adhesion of acorn barnacles on surface-active borate glasses.

Concerns about the bioaccumulation of toxic antifouling compounds have necessitated the search for alternative strategies to combat marine biofouling. Because many biologically essential minerals have deleterious effects on organisms at high concentration, one approach to preventing the settlement of marine foulers is increasing the local concentration of ions that are naturally present in seawater. Here, we used surface-active borate glasses as a platform to directly deliver ions (Na+, Mg2+ and BO43-) to the adhesive interface under acorn barnacles (Amphibalanus (=Balanus) amphitrite). Additionally, surface-active glasses formed reaction layers at the glass-water interface, presenting another challenge to fouling organisms. Proteomics analysis showed that cement deposited on the gelatinous reaction layers is more soluble than cement deposited on insoluble glasses, indicating the reaction layer and/or released ions disrupted adhesion processes. Laboratory experiments showed that the majority (greater than 79%) of adult barnacles re-attached to silica-free borate glasses for 14 days could be released and, more importantly, barnacle larvae did not settle on the glasses. The formation of microbial biofilms in field tests diminished the performance of the materials. While periodic water jetting (120 psi) did not prevent the formation of biofilms, weekly cleaning did dramatically reduce macrofouling on magnesium aluminoborate glass to levels below a commercial foul-release coating. This article is part of the theme issue 'Transdisciplinary approaches to the study of adhesion and adhesives in biological systems'.

Below the Hall-Petch Limit in Nanocrystalline Ceramics.

Reducing the grain size of metals and ceramics can significantly increase strength and hardness, a phenomenon described by the Hall-Petch relationship. The many studies on the Hall-Petch relationship in metals reveal that when the grain size is reduced to tens of nanometers, this relationship breaks down. However, experimental data for nanocrystalline ceramics are scarce, and the existence of a breakdown is controversial. Here we show the Hall-Petch breakdown in nanocrystalline ceramics by performing indentation studies on fully dense nanocrystalline ceramics fabricated with grain sizes ranging from 3.6 to 37.5 nm. A maximum hardness occurs at a grain size of 18.4 nm, and a negative (or inverse) Hall-Petch relationship reduces the hardness as the grain size is decreased to around 5 nm. At the smallest grain sizes, the hardness plateaus and becomes insensitive to grain size change. Strain rate studies show that the primary mechanism behind the breakdown, negative, and plateau behavior is not diffusion-based. We find that a decrease in density and an increase in dissipative energy below the breakdown correlate with increasing grain boundary volume fraction as the grain size is reduced. The behavior below the breakdown is consistent with structural changes, such as increasing triple-junction volume fraction. Grain- and indent-size-dependent fracture behavior further supports local structural changes that corroborate current theories of nanocrack formation at triple junctions. The synergistic grain size dependencies of hardness, elasticity, energy dissipation, and nanostructure of nanocrystalline ceramics point to an opportunity to use the grain size to tune the strength and dissipative properties.

On the stiffness of demineralized dentin matrices.

UNLABELLED: Resin bonding to dentin requires the use of self-etching primers or acid etching to decalcify the surface and expose a layer of collagen fibrils of the dentin matrix. Acid-etching reduces the stiffness of demineralized dentin from approximately 19 GPa-1 MPa, requiring that it floats in water to prevent it from collapsing during bonding procedures. Several publications show that crosslinking agents like gluteraladehyde, carbodiimide or grape seed extract can stiffen collagen and improve resin-dentin bond strength. OBJECTIVE: The objective was to assess a new approach for evaluating the changes in stiffness of decalcified dentin by polar solvents and a collagen cross-linker. METHODS: Fully demineralized dentin beams and sections of etched coronal dentin were subjected to indentation loading using a cylindrical flat indenter in water, and after treatment with ethanol or ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC). The stiffness was measured as a function of strain and as a function of loading rate from 1 to 50 µm/s. RESULTS: At a strain of 0.25% the elastic modulus of the fully demineralized dentin was approximately 0.20 MPa. It increased to over 0.90 MPa at strains of 1%. Exposure to ethanol caused an increase in elastic modulus of up to four times. Increasing the loading rate from 1 to 50 µm/s caused an increase in the apparent modulus of up to three times in both water and ethanol. EDC treatment caused increases in the stiffness in fully demineralized samples and in acid-etched demineralized dentin surfaces in situ. SIGNIFICANCE: Changes in the mechanical behavior of demineralized collagen matrices can be measured effectively under hydration via indentation with cylindrical flat indenters. This approach can be used for quantifying the effects of bonding treatments on the properties of decalcified dentin after acid etching, as well as to follow the loss of stiffness over time due to enzymatic degradation.

Importance of age on the dynamic mechanical behavior of intertubular and peritubular dentin.

An experimental evaluation of human coronal dentin was performed using nanoscopic dynamic mechanical analysis (nanoDMA). The primary objectives were to quantify any unique changes in mechanical behavior of intertubular and peritubular dentin with age, and to evaluate the microstructure and mechanical behavior of the mineral deposited within the lumens. Specimens of coronal dentin were evaluated by nanoDMA using single indents and in scanning mode via scanning probe microscopy. Results showed that there were no significant differences in the storage modulus or complex modulus between the two age groups (18-25 versus 54-83 yrs) for either the intertubular or peritubular tissue. However, there were significant differences in the dampening behavior between the young and old dentin, as represented in the loss modulus and tanδ responses. For both the intertubular and peritubular components, the capacity for dampening was significantly lower in the old group. Scanning based nanoDMA showed that the tubules of old dentin exhibit a gradient in elastic behavior, with decrease in elastic modulus from the cuff to the center of tubules filled with newly deposited mineral.

A characterization of the mechanical behavior of resin-infiltrated dentin using nanoscopic Dynamic Mechanical Analysis.

UNLABELLED: This study explored the spatial variations in mechanical behavior of resin-infiltrated dentin using nanoscopic Dynamic Mechanical Analysis (DMA). OBJECTIVE: The objectives were to: (1) evaluate the mechanical behavior of resin-infiltrated dentin using a scanning-based approach to nanoindentation, (2) identify contributions of the collagen matrix to time-dependent deformation of the hybrid layer, and (3) assess the importance of specimen hydration on the nanoDMA response. METHODS: Specimens of completely demineralized dentin infiltrated with commercial resin adhesive and control samples of resin adhesive were evaluated using a nanoindenter in scanning mode. The load and displacement responses were used to perform DMA and to estimate the complex (E*), storage (E') and loss (E″) moduli over selected regions of evaluation. The importance of hydration on the mechanical behavior was also examined from a comparison of responses in the hydrated and dehydrated conditions. RESULTS: In the hydrated state the apparent complex, storage and loss moduli for the resin-infiltrated dentin samples were 3.5±0.3GPa, 3.4±0.2GPa and 0.9±0.3GPa, respectively. Those values for the resin adhesive control were 2.7±0.3GPa, 2.7±0.3GPa and 0.2±0.02GPa, respectively. Viscoelastic deformation of the resin-infiltrated collagen exceeded that occurring in regions of uniform resin adhesive. Though dehydration resulted in a significant increase in both the complex and storage moduli of the macro hybrid layer, the largest changes occurred to the resin adhesive. SIGNIFICANCE: The microstructure and hydration play critical roles on the mechanical behavior of the hybrid layer and nanoDMA provides a potent measurement tool for identifying the spatial variations.

Biomimetic silicification of demineralized hierarchical collagenous tissues.

Unlike man-made composite materials, natural biominerals containing composites usually demonstrate different levels of sophisticated hierarchical structures which are responsible for their mechanical properties and other metabolic functions. However, the complex spatial organizations of the organic-inorganic phases are far beyond what they achieved by contemporary engineering techniques. Here, we demonstrate that carbonated apatite present in collagen matrices derived from fish scale and bovine bone may be replaced by amorphous silica, using an approach that simulates what is utilized by phylogenetically ancient glass sponges. The structural hierarchy of these collagen-based biomaterials is replicated by the infiltration and condensation of fluidic polymer-stabilized silicic acid precursors within the intrafibrillar milieu of type I collagen fibrils. This facile biomimetic silicification strategy may be used for fabricating silica-based, three-dimensional functional materials with specific morphological and hierarchical requirements.

Fatigue of the resin-enamel bonded interface and the mechanisms of failure.

The durability of adhesive bonds to enamel and dentin and the mechanisms of degradation caused by cyclic loading are important to the survival of composite restorations. In this study a novel method of evaluation was used to determine the strength of resin-enamel bonded interfaces under oth static and cyclic loading, and to identify the mechanisms of failure. Specimens with twin interfaces of enamel bonded to commercial resin composite were loaded in monotonic and cyclic 4-point flexure to failure within a hydrated environment. Results for the resin-enamel interface were compared with those for the resin composite (control) and values reported for resin-dentin adhesive bonds. Under both modes of loading the strength of the resin-enamel interface was significantly (p≤0.0001) lower than that of the resin composite and the resin-dentin bonded interface. Fatigue failure of the interface occurred predominantly by fracture of enamel, adjacent to the interface, and not due to adhesive failures. In the absence of water aging or acid production of biofilms, the durability of adhesive bonds to enamel is lower than that achieved in dentin bonding.

Fatigue of the resin-dentin interface: a new approach for evaluating the durability of dentin bonds.

UNLABELLED: There are concerns regarding the longevity of resin composite restorations and the clinical relevance of in vitro bond strength testing to the durability of dentin bonds in vivo. OBJECTIVE: The objectives of this investigation were to: (1) develop a new method of experimental evaluation for quantifying the durability of dentin bonds, (2) apply this method to characterize the interfacial strength of a selected commercial system under both monotonic and cyclic loading, and (3) distinguish mechanisms contributing to the interface degradation and failure. METHODS: A new method for fatigue testing the resin-dentin interface was developed based on a four-point flexure arrangement that includes two identical bonded interfaces. Cyclic loading of specimens comprised of coronal dentin bonded to a commercial resin composite and controls of resin composite was performed to failure within a hydrated environment. Scanning electron microscopy and nanoscopic dynamic mechanical analysis were used to evaluate failure mechanisms. RESULTS: The fatigue strength of the resin-dentin interface was significantly lower (p≤0.0001) than that of the resin composite and reported for dentin over the entire finite life regime. Defined at 1×10(7) cycles, the apparent endurance limit of the resin-dentin interface was 13MPa, in comparison to 48MPa and 44MPa for the resin composite and dentin, respectively. The ratio of fully reversed endurance limit to ultimate strength of the interface (0.26) was the lowest of the three materials. SIGNIFICANCE: The proposed approach for characterizing the fatigue strength of resin-dentin bonds may offer new insights concerning durability of the bonded interface.

On the fatigue behavior of resin-dentin bonds after degradation by biofilm.

The durability of resin-dentin bonds is a growing concern in the placement of composite restorations. Most reported evaluations concerning the mechanical behavior of the bonded interface are conducted using static loading to failure only. They also do not account for the acid production of biofilms, which is one of the most common contributors to interfacial failures in vivo. In this investigation resin-dentin bonded interface specimens were exposed to S. mutans for 14 days and then subjected to quasi-static or cyclic four-point flexure to failure. Control specimens (without biofilm) were evaluated after aging for one and fourteen days. While no significant difference in flexure strength resulted from the duration of water aging (66.2 MPa vs. 56.9 MPa), biofilm exposure caused a significant reduction in strength (29.3 MPa; p ≤ 0.000). After water aging for one and fourteen days the apparent endurance limits were 13.0 MPa and 13.1 MPa, respectively. Biofilm treatment caused a significant (p ≤ 0.001) reduction in fatigue resistance of the interface, and the endurance limit was reduced to 9.9 MPa. Fatigue failure of the control specimens initiated within the resin composite adjacent to the interface, whereas failure of the biofilm treated specimens initiated within the hybrid layer and appeared attributed to the localized demineralization of dentin. Biofilm degradation is an important consideration in assessing the durability of resin-dentin bonds.

Quaternary ammonium silane-functionalized, methacrylate resin composition with antimicrobial activities and self-repair potential.

The design of antimicrobial polymers to address healthcare issues and minimize environmental problems is an important endeavor with both fundamental and practical implications. Quaternary ammonium silane-functionalized methacrylate (QAMS) represents an example of antimicrobial macromonomers synthesized by a sol-gel chemical route; these compounds possess flexible Si-O-Si bonds. In present work, a partially hydrolyzed QAMS co-polymerized with 2,2-[4(2-hydroxy 3-methacryloxypropoxy)-phenyl]propane is introduced. This methacrylate resin was shown to possess desirable mechanical properties with both a high degree of conversion and minimal polymerization shrinkage. The kill-on-contact microbiocidal activities of this resin were demonstrated using single-species biofilms of Streptococcus mutans (ATCC 36558), Actinomyces naeslundii (ATCC 12104) and Candida albicans (ATCC 90028). Improved mechanical properties after hydration provided the proof-of-concept that QAMS-incorporated resin exhibits self-repair potential via water-induced condensation of organic modified silicate (ormosil) phases within the polymerized resin matrix.

Nanoscopic dynamic mechanical properties of intertubular and peritubular dentin.

An experimental evaluation of intertubular and peritubular dentin was performed using nanoindentation and Dynamic Mechanical Analysis (DMA). The objective of the investigation was to evaluate the differences in dynamic mechanical behavior of these two constituents and to assess whether their response is frequency dependent. Specimens of hydrated coronal dentin were evaluated by DMA using single indents over a range of parametric conditions and using scanning probe microscopy. The complex (E∗), storage (E') and loss moduli (E″) of the intertubular and peritubular dentin were evaluated as a function of the dynamic loading frequency and static load in the fully hydrated condition. The mean complex E∗ (19.6 GPa) and storage E' (19.2 GPa) moduli of the intertubular dentin were significantly lower than those for peritubular dentin (E∗ = 31.1 GPa, p < 0.05; E' = 30.3 GPa, p < 0.05). There was no significant influence of dynamic loading frequency on these measures. Although there was no significant difference in the loss modulus (E″) between the two materials (p > 0.05), both constituents exhibited a significant increase in E″ with dynamic load frequency and reduction in the quasi-static component of indentation load. The largest difference in dynamic behavior of the two tissues was noted at small quasi-static indentation loads and the highest frequency.

Subtleties of biomineralisation revealed by manipulation of the eggshell membrane.

Biocalcification of collagen matrices with calcium phosphate and biosilicification of diatom frustules with amorphous silica are two discrete processes that have intrigued biologists and materials scientists for decades. Recent advancements in the understanding of the mechanisms involved in these two biomineralisation processes have resulted in the use of biomimetic strategies to replicate these processes separately using polyanionic, polycationic or zwitterionic analogues of extracellular matrix proteins to stabilise amorphous mineral precursor phases. To date, there is a lack of a universal model that enables the subtleties of these two apparently dissimilar biomineralisation processes to be studied together. Here, we utilise the eggshell membrane as a universal model for differential biomimetic calcification and silicification. By manipulating the eggshell membrane to render it permeable to stabilised mineral precursors, it is possible to introduce nanostructured calcium phosphate or silica into eggshell membrane fibre cores or mantles. We provide a model for infiltrating the two compartmental niches of a biopolymer membrane with different intrafibre minerals to obtain materials with potentially improved structure-property relationships.