Fabrication and in vitro characterization of three-dimensional organic/inorganic scaffolds by robocasting.

A key issue for the fabrication of scaffolds for tissue engineering is the development of processing techniques flexible enough to produce materials with a wide spectrum of solubility (bioresorption rates) and mechanical properties matching those of calcified tissues. These techniques must also have the capability of generating adequate porosity to further serve as a framework for cell penetration, new bone formation, and subsequent remodeling. In this study we show how hybrid organic/inorganic scaffolds with controlled microstructures can be built using robotic assisted deposition at room temperature. Polylactide or polycaprolactone scaffolds with pore sizes ranging between 200-500 microm and hydroxyapatite contents up to 70 wt % were fabricated. Compressive tests revealed an anisotropic behavior of the scaffolds, strongly dependant on their chemical composition. The inclusion of an inorganic component increased their stiffness but they were not brittle and could be easily machined even for ceramic contents up to 70 wt %. The mechanical properties of hybrid scaffolds did not degrade significantly after 20 days in simulated body fluid. However, the stiffness of pure polylactide scaffolds increased drastically due to polymer densification. Scaffolds containing bioactive glasses were also printed. After 20 days in simulated body fluid they developed an apatite layer on their surface.

Role of alcohol in the fracture resistance of teeth.

Healthy dentin, the mineralized tissue that makes up the bulk of the tooth, is naturally hydrated in vivo; however, it is known that various chemical reagents, including acetone and ethanol, can induce dehydration and thereby affect its properties. Here, we sought to investigate this in light of the effect of alcohol on the mechanical properties of dentin, specifically by measuring the stiffness, strength, and toughness of dentin in simulated body fluid and Scotch whisky. Results indicated that chemical dehydration induced by the whisky had a significant beneficial effect on the elastic modulus, strength, and fracture toughness of dentin. Although this made teeth more resistant to fracture, the change in properties was fully reversible upon rehydration. This effect is considered to be associated with increased cross-linking of the collagen molecules from intermolecular hydrogen-bonding, where water is replaced with weaker hydrogen-bond-forming solvents such as alcohol.

Propagation of surface fatigue cracks in human cortical bone.

An understanding of how fatigue cracks grow in bone is of importance as fatigue is thought to be the main cause of clinical stress fractures. This study presents new results on the fatigue-crack growth behavior of small surface cracks (approximately 75-1000 microm in size) in human cortical bone, and compares their growth rates with data from other published studies on the behavior of both surface cracks and many millimeter, through-thickness large cracks. Results are obtained with a cyclically loaded cantilever-beam geometry using optical microscopy to examine for crack growth after every 100-500 cycles. Based on the current and previous results, small fatigue cracks appear to become more resistant to fatigue-crack growth with crack extension, analogous to the way the fracture resistance of cortical bone increases with crack growth. Mechanistically, a theory attributing such behavior to the development of bridges in the wake of the crack with crack growth is presented. The existence of such bridges is directly confirmed using optical microscopy.

Re-evaluating the toughness of human cortical bone.

Data for fracture in human humeral cortical bone are re-analyzed to assess the validity for this material of linear-elastic fracture mechanics (LEFM), which is the standard method of analyzing toughness and one basis for analyzing clinical data relating to bone quality. A nonlinear fracture model, which is based on representing the damage zone in the bone by a cohesive model, is calibrated against a number of sets of test data for normal (not diseased or aged) human cortical bone taken from cadavers. The data consist of load vs. load-point displacement measurements from standard compact-tension fracture tests. Conventional LEFM is unable to account for the shape of the load-displacement curves, but the nonlinear model overcomes this deficiency. Calibration of the nonlinear model against one data curve leads to predictions of the peak load and the displacement to peak load for two other data curves that are, for this limited test set, more accurate than those made using LEFM. Furthermore, prior observations of damage mechanisms in bone are incompatible with the modeling assumption of LEFM that all nonlinearity is confined to a zone much smaller than the specimen and the crack length. The predictions of the cohesive model and the prior observations concur that the length of the nonlinear zone in human cortical bone varies in the range 3-10 mm, which is comparable to or larger than naturally-occurring bones and the specimens used to test them. We infer that LEFM is not an accurate model for cortical bone. The fracture toughness of bone deduced via LEFM from test data will not generally be a material constant, but will take different values for different crack lengths and test configurations. The accuracy of using LEFM or single-parameter fracture toughness for analyzing the significance of data from clinical studies is called into question. The nonlinear cohesive zone model is proposed to be a more accurate model of bone and the traction-displacement or cohesive law is hypothesized to be a material property. The cohesive law contains a more complete representation of the mechanics of material failure than the single-parameter fracture toughness and may therefore provide a superior measure of bone quality, e.g., for assessing the efficacy of therapy for osteoporosis.

Fabrication and mechanical properties of PLA/HA composites: A study of in vitro degradation.

The adverse effects of stress shielding from the use of high-modulus metallic alloy bio-implant materials has led to increased research into developing polymer-ceramic composite materials that match the elastic modulus of human bone. Of particular interest are poly-l-lactic acid- hydroxyapatite (PLA/HA)-based composites which are fully resorbable in vivo. However, their bioresorbability has a deleterious effect on the mechanical properties of the implant. The purpose of this study is to investigate, from a micromechanistic perspective, the in vitro degradation behavior of such composites manufactured using a simple hot-pressing route for two different hydroxyapatite particles: a fine-grained (average particle size ∼5 µm) commercial powder or coarser whiskers (∼25-30 µm long, ∼5 µm in diameter). We observed that composites with ceramic contents ranging between 70 and 85 wt.% have mechanical properties that match reasonably those of human cortical bone. However, the properties deteriorate with immersion in Hanks' Balanced Salt Solution due to the degradation of the polymer phase. The degradation is more pronounced in samples with larger ceramic content due to the dissolution of the smaller amount of polymer between the ceramic particles.

Deep-ultraviolet Raman spectroscopy study of the effect of aging on human cortical bone.

The age-related deterioration in bone quality and consequent increase in fracture incidence is an obvious health concern that is becoming increasingly significant as the population ages. Raman spectroscopy with deep-ultraviolet excitation (244 nm) is used to measure vibrational spectra from human cortical bone obtained from donors over a wide age range (34-99 years). The UV Raman technique avoids the fluorescence background usually found with visible and near-infrared excitation and, due to resonance Raman effects, is particularly sensitive to the organic component of bone. Spectral changes in the amide I band at 1640 cm(-1) are found to correlate with both donor age and with previously reported fracture toughness data obtained from the same specimens. These results are discussed in the context of possible changes in collagen cross-linking chemistry as a function of age, and are deemed important to further our understanding of the changes in the organic component of the bone matrix with aging.

Ultrastructural examination of dentin using focused ion-beam cross-sectioning and transmission electron microscopy.

Focused ion-beam (FIB) milling is a commonly used technique for transmission electron microscopy (TEM) sample preparation of inorganic materials. In this study, we seek to evaluate the FIB as a TEM preparation tool for human dentin. Two particular problems involving dentin, a structural analog of bone that makes up the bulk of the human tooth, are examined. Firstly, the process of aging is studied through an investigation of the mineralization in 'transparent' dentin, which is formed naturally due to the filling up of dentinal tubules with large mineral crystals. Next, the process of fracture is examined to evaluate incipient events that occur at the collagen fiber level. For both these cases, FIB-milling was able to generate high-quality specimens that could be used for subsequent TEM examination. The changes in the mineralization suggested a simple mechanism of mineral 'dissolution and reprecipitation', while examination of the collagen revealed incipient damage in the form of voids within the collagen fibers. These studies help shed light on the process of aging and fracture of mineralized tissues and are useful steps in developing a framework for understanding such processes.

Fracture in human cortical bone: local fracture criteria and toughening mechanisms.

Micromechanical models for fracture initiation that incorporate local failure criteria have been widely developed for metallic and ceramic materials; however, few such micromechanical models have been developed for the fracture of bone. In fact, although the fracture event in "hard" mineralized tissues such as bone is commonly believed to be locally strain-controlled, only recently has there been experimental evidence (using double-notched four-point bend testing) to support this widely held belief. In the present study, we seek to shed further light on the nature of the local cracking events that precede catastrophic fracture in human cortical bone, and to define their relationship to the microstructure. Specifically, numerical computations are reported that demonstrate that the stress and strain states ahead of such a notch are qualitatively similar irrespective of the deformation mechanism (pressure-insensitive plasticity vs. pressure-sensitive microcracking). Furthermore, we use the double-notched test to examine crack-microstructure interactions from a perspective of determining the salient toughening mechanisms in bone and to characterize how these may affect the anisotropy in fracture properties. Based on preliminary micromechanical models of these processes, the relative contributions of various toughening mechanisms are established. In particular, crack deflection and uncracked-ligament bridging are identified as the major mechanisms of toughening in cortical bone.

Dentin erosion simulation by cantilever beam fatigue and pH change.

Exposed root surfaces frequently exhibit non-carious notches representing material loss by abrasion, erosion, and/or abfraction. Although a contribution from mechanical stress is often mentioned, no definitive proof exists of a cause-effect relationship. To address this, we examined dimensional changes in dentin subjected to cyclic fatigue in two different pH environments. Human dentin cantilever-beams were fatigued under load control in pH = 6 (n = 13) or pH = 7 (n = 13) buffer, with a load ratio (R = minimum load/maximum load) of 0.1 and frequency of 2 Hz, and stresses between 5.5 and 55 MPa. Material loss was measured at high- and low-stress locations before and after cycling. Of the 23 beams, 7 withstood 1,000,000 cycles; others cracked earlier. Mean material loss in high-stress areas was greater than in low-stress areas, and losses were greater at pH = 6 than at pH = 7, suggesting that mechanical stress and lower pH both accelerate erosion of dentin surfaces.

Effects of polar solvents on the fracture resistance of dentin: role of water hydration.

Although healthy dentin is invariably hydrated in vivo, from a perspective of examining the mechanisms of fracture in dentin, it is interesting to consider the role of water hydration. Furthermore, it is feasible that exposure to certain polar solvents, e.g., those found in clinical adhesives, can induce dehydration. In the present study, in vitro deformation and fracture experiments, the latter involving a resistance-curve (R-curve) approach (i.e., toughness evolution with crack extension), were conducted in order to assess changes in the constitutive and fracture behavior induced by three common solvents-acetone, ethanol and methanol. In addition, nanoindentation-based experiments were performed to evaluate the deformation behavior at the level of individual collagen fibers and ultraviolet Raman spectroscopy to evaluate changes in bonding. The results indicate a reversible effect of chemical dehydration, with increased fracture resistance, strength, and stiffness associated with lower hydrogen bonding ability of the solvent. These results are analyzed both in terms of intrinsic and extrinsic toughening phenomena to further understand the micromechanisms of fracture in dentin and the specific role of water hydration.

Mechanistic aspects of fracture and R-curve behavior in human cortical bone.

An understanding of the evolution of toughness is essential for the mechanistic interpretation of the fracture of cortical bone. In the present study, in vitro fracture experiments were conducted on human cortical bone in order to identify and quantitatively assess the salient toughening mechanisms. The fracture toughness was found to rise linearly with crack extension (i.e., rising resistance- or R-curve behavior) with a mean crack-initiation toughness, K0 of approximately 2 MPa square root m for crack growth in the proximal-distal direction. Uncracked ligament bridging, which was observed in the wake of the crack, was identified as the dominant toughening mechanism responsible for the observed R-curve behavior. The extent and nature of the bridging zone was examined quantitatively using multi-cutting compliance experiments in order to assess the bridging zone length and estimate the bridging stress distribution. Additionally, time-dependent cracking behavior was observed at stress intensities well below those required for overload fracture; specifically, slow crack growth occurred at growth rates of approximately 2 x 10(-9) m/s at stress intensities approximately 35% below the crack-initiation toughness. In an attempt to measure slower growth rates, it was found that the behavior switched to a regime dominated by time-dependent crack blunting, similar to that reported for dentin; however, such blunting was apparent over much slower time scales in bone, which permitted subcritical crack growth to readily take place at higher stress intensities.

Mechanistic aspects of in vitro fatigue-crack growth in dentin.

Although the propagation of fatigue cracks has been recognized as a problem of clinical significance in dentin, there have been few fracture mechanics-based studies that have investigated this issue. In the present study, in vitro cyclic fatigue experiments were conducted over a range of cyclic frequencies (1-50 Hz) on elephant dentin in order to quantify fatigue-crack growth behavior from the perspective of understanding the mechanism of fatigue in dentin. Specifically, results obtained for crack extension rates along a direction parallel to the dentinal tubules were found to be well described by the stress-intensity range, DeltaK, using a simple Paris power-law approach with exponents ranging from 12 to 32. Furthermore, a frequency dependence was observed for the crack-growth rates, with higher growth rates associated with lower frequencies. By using crack-growth experiments involving alternate cyclic and static loading, such fatigue-crack propagation was mechanistically determined to be the result of a "true" cyclic fatigue mechanism, and not simply a succession of static fracture events. Furthermore, based on the observed frequency dependence of fatigue-crack growth in dentin and observations of time-dependent crack blunting, a cyclic fatigue mechanism involving crack-tip blunting and re-sharpening is proposed. These results are deemed to be of importance for an improved understanding of fatigue-related failures in teeth.

Aspects of in vitro fatigue in human cortical bone: time and cycle dependent crack growth.

Although fatigue damage in bone induced by cyclic loading has been recognized as a problem of clinical significance, few fracture mechanics based studies have investigated how incipient cracks grow by fatigue in this material. In the present study, in vitro cyclic fatigue experiments were performed in order to quantify fatigue-crack growth behavior in human cortical bone. Crack-growth rates spanning five orders of magnitude were obtained for the extension of macroscopic cracks in the proximal-distal direction; growth-rate data could be well characterized by the linear-elastic stress-intensity range, using a simple (Paris) power law with exponents ranging from 4.4 to 9.5. Mechanistically, to discern whether such behavior results from "true" cyclic fatigue damage or is simply associated with a succession of quasi-static fracture events, cyclic crack-growth rates were compared to those measured under sustained (non-cyclic) loading. Measured fatigue-crack growth rates were found to exceed those "predicted" from the sustained load data at low growth rates ( approximately 3 x 10(-10) to 5 x 10(-7) m/cycle), suggesting that a "true" cyclic fatigue mechanism, such as alternating blunting and re-sharpening of the crack tip, is active in bone. Conversely, at higher growth rates ( approximately 5 x 10(-7) to 3 x 10(-5) m/cycle), the crack-growth data under sustained loads integrated over the loading cycle reasonably predicts the cyclic fatigue data, indicating that quasi-static fracture mechanisms predominate. The results are discussed in light of the occurrence of fatigue-related stress fractures in cortical bone.

Age-related transparent root dentin: mineral concentration, crystallite size, and mechanical properties.

Many fractures occur in teeth that have been altered, for example restored or endodontically repaired. It is therefore essential to evaluate the structure and mechanical properties of these altered dentins. One such altered form of dentin is transparent (sometimes called sclerotic) dentin, which forms gradually with aging. The present study focuses on differences in the structure and mechanical properties of normal versus transparent dentin. The mineral concentration, as measured by X-ray computed microtomography, was significantly higher in transparent dentin, the elevated concentration being consistent with the closure of the tubule lumens. Crystallite size, as measured by small angle X-ray scattering, was slightly smaller in transparent dentin, although the importance of this finding requires further study. The elastic properties were unchanged by transparency; however, transparent dentin, unlike normal dentin, exhibited almost no yielding before failure. In addition, the fracture toughness was lowered by roughly 20% while the fatigue lifetime was deleteriously affected at high stress levels. These results are discussed in terms of the altered microstructure of transparent dentin.

Effect of aging on the toughness of human cortical bone: evaluation by R-curves.

Age-related deterioration of the fracture properties of bone, coupled with increased life expectancy, is responsible for increasing incidence of bone fracture in the elderly, and hence, an understanding of how its fracture properties degrade with age is essential. The present study describes ex vivo fracture experiments to quantitatively assess the effect of aging on the fracture toughness properties of human cortical bone in the longitudinal direction. Because cortical bone exhibits rising crack-growth resistance with crack extension, unlike most previous studies, the toughness is evaluated in terms of resistance-curve (R-curve) behavior, measured for bone taken from wide range of age groups (34-99 years). Using this approach, both the ex vivo crack-initiation and crack-growth toughness are determined and are found to deteriorate with age; the initiation toughness decreases some 40% over 6 decades from 40 to 100 years, while the growth toughness is effectively eliminated over the same age range. The reduction in crack-growth toughness is considered to be associated primarily with a degradation in the degree of extrinsic toughening, in particular, involving crack bridging in the wake of the crack.

On the origin of the toughness of mineralized tissue: microcracking or crack bridging?

Two major mechanisms that could potentially be responsible for toughening in mineralized tissues, such as bone and dentin, have been identified-microcracking and crack bridging. While evidence has been reported for both mechanisms, there has been no consensus thus far on which mechanism plays the dominant role in toughening these materials. In the present study, we seek to present definitive experimental evidence supporting crack bridging, rather than microcracking, as the most significant mechanism of toughening in cortical bone and dentin. In vitro fracture toughness experiments were conducted to measure the variation of the fracture resistance with crack extension [resistance-curve (R-curve) behavior] for both materials with special attention paid to changes in the sample compliance. Because these two toughening mechanisms induce opposite effects on the sample compliance, such experiments allow for the definitive determination of the dominant toughening mechanism, which in the present study was found to be crack bridging for microstructurally large crack sizes. The results of this work are of relevance from the perspective of developing a micromechanistic framework for understanding fracture behavior of mineralized tissue and in predicting failure in vivo.

On the in vitro fatigue behavior of human dentin: effect of mean stress.

Human dentin is susceptible to failure under repetitive cyclic-fatigue loading. This investigation seeks to address the paucity of data that reliably quantify this phenomenon. Specifically, the effect of alternating vs. mean stresses, characterized by the stress- or load-ratio R (ratio of minimum-to-maximum stress), was investigated for three R values (-1, 0.1, and 0.5). Dentin was observed to be prone to fatigue failure under cyclic stresses, with susceptibility varying, depending upon the stress level. The "stress-life" (S/N) data obtained are discussed in the context of constant-life diagrams for fatigue failure. The results provide the first fatigue data for human dentin under tension-compression loading and serve to map out safe and unsafe regimes for failure over a wide range of in vitro fatigue lives (< 10(3) to > 10(6) cycles).

On the fracture of human dentin: is it stress- or strain-controlled?

Despite substantial clinical interest in the fracture resistance of human dentin, there is little mechanistic information in archival literature that can be usefully used to model such fracture. In fact, although the fracture event in dentin, akin to other mineralized tissues like bone, is widely believed to be locally strain-controlled, there has never been any scientific proof to support this belief. The present study seeks to address this issue through the use of a novel set of in vitro experiments in Hanks' balanced salt solution involving a double-notched bend test geometry, which is designed to discern whether the critical failure events involved in the onset of fracture are locally stress- or strain-controlled. Such experiments are further used to characterize the notion of "plasticity" in dentin and the interaction of cracks with the salient microstructural features. It is observed that fracture in dentin is indeed locally strain-controlled and that the presence of dentinal tubules does not substantially affect this process of crack initiation and growth. The results presented are believed to be critical steps in the development of a micromechanical model for the fracture of human dentin that takes into consideration the influence of both the microstructure and the local failure mode.

Crack blunting, crack bridging and resistance-curve fracture mechanics in dentin: effect of hydration.

Few studies have focused on a description of the fracture toughness properties of dentin in terms of resistance-curve (R-curve) behavior, i.e., fracture resistance increasing with crack extension, particularly in light of the relevant toughening mechanisms involved. Accordingly, in the present study, fracture mechanics based experiments were conducted on elephant dentin in order to determine such R-curves, to identify the salient toughening mechanisms and to discern how hydration may affect their potency. Crack bridging by uncracked ligaments, observed directly by microscopy and X-ray tomography, was identified as a major toughening mechanism, with further experimental evidence provided by compliance-based experiments. In addition, with hydration, dentin was observed to display significant crack blunting leading to a higher overall fracture resistance than in the dehydrated material. The results of this work are deemed to be of importance from the perspective of modeling the fracture behavior of dentin and in predicting its failure in vivo.

In vitro fracture toughness of human dentin.

The in vitro fracture toughness of human dentin has been reported to be of the order of 3 MPa (square root)m. This result, however, is based on a single study for a single orientation, and furthermore involves notched, rather than fatigue precracked, test samples. The present study seeks to obtain an improved, lower-bound, value of the toughness, and to show that previously reported values may be erroneously high because of the absence of a sharp crack as the stress concentrator. Specifically, the average measured critical stress intensity, K(c), for the onset of unstable fracture along an orientation perpendicular to the long axis of the tubules in dentin is found to be 1.8 MPa (square root)m in simulated body fluid (Hanks' balanced salt solution), when tested in a three-point bending specimen containing a (nominally) atomically sharp precrack generated during prior fatigue cycling. This is to be compared with a value of 2.7 MPa (square root)m measured under identical experimental conditions except that the bend specimen contained a sharp machined notch (of root radius 30-50 microm). The effect of acuity of the precrack on the fracture toughness of human dentin is discussed in the context of these data.