Alix C. Deymier-Black

Synchrotron X-ray diffraction study of load partitioning during elastic deformation of bovine dentin.

The elastic properties of dentin, a biological composite consisting of stiff hydroxyapatite (HAP) nano-platelets within a compliant collagen matrix, are determined by the volume fraction of these two phases and the load transfer between them. We have measured the elastic strains in situ within the HAP phase of bovine dentine by high energy X-ray diffraction for a series of static compressive stresses at ambient temperature. The apparent HAP elastic modulus (ratio of applied stress to elastic HAP strain) was found to be 18+/-2GPa. This value is significantly lower than the value of 44GPa predicted by the lower bound load transfer Voigt model, using HAP and collagen volume fractions determined by thermo-gravimetric analysis. This discrepancy is explained by (i) a reduction in the intrinsic Youngs modulus of the nano-size HAP platelets due to the high fraction of interfacial volume and (ii) an increase in local stresses due to stress concentration around the dentin tubules.

Bone cell-independent benefits of raloxifene on the skeleton: a novel mechanism for improving bone material properties.

Raloxifene is an FDA approved agent used to treat bone loss and decrease fracture risk. In clinical trials and animal studies, raloxifene reduces fracture risk and improves bone mechanical properties, but the mechanisms of action remain unclear because these benefits occur largely independent of changes to bone mass. Using a novel experimental approach, machined bone beams, both from mature male canine and human male donors, were depleted of living cells and then exposed to raloxifene ex vivo. Our data show that ex vivo exposure of non-viable bone to raloxifene improves intrinsic toughness, both in canine and human cortical bone beams tested by 4-point bending. These effects are cell-independent and appear to be mediated by an increase in matrix bound water, assessed using basic gravimetric weighing and sophisticated ultrashort echo time magnetic resonance imaging. The hydroxyl groups (OH) on raloxifene were shown to be important in both the water and toughness increases. Wide and small angle X-ray scattering patterns during 4-pt bending show that raloxifene alters the transfer of load between the collagen matrix and the mineral crystals, placing lower strains on the mineral, and allowing greater overall deformation prior to failure. Collectively, these findings provide a possible mechanistic explanation for the therapeutic effect of raloxifene and more importantly identify a cell-independent mechanism that can be utilized for novel pharmacological approaches for enhancing bone strength.

Effect of high-energy X-ray doses on bone elastic properties and residual strains.

Bone X-ray irradiation occurs during medical treatments, sterilization of allografts, space travel and in vitro studies. High doses are known to affect the post-yield properties of bone, but their effect on the bone elastic properties is unclear. The effect of such doses on the mineral-organic interface has also not been adequately addressed. Here, the evolution of elastic properties and residual strains with increasing synchrotron X-ray dose (5-3880 kGy) is examined on bovine cortical bone. It is found that these doses affect neither the degree of nanometer-level load transfer between the hydroxyapatite (HAP) platelets and the collagen up to stresses of -60 MPa nor the microscopic modulus of collagen fibrils (both measured by synchrotron X-ray scattering during repeated in situ loading and unloading). However, the residual elastic strains in the HAP phase decrease markedly with increased irradiation, indicating damage at the HAP-collagen interface. The HAP residual strain also decreases after repeated loading/unloading cycles. These observations can be explained by temporary de-bonding at the HAP/collagen interface (thus reducing the residual strain), followed by rapid re-bonding (so that load transfer capability is not affected).

Evolution of load transfer between hydroxyapatite and collagen during creep deformation of bone

While the matrix/reinforcement load-transfer occurring at the micro- and nanoscale in nonbiological composites subjected to creep deformation is well understood, this topic has been little studied in biological composites such as bone. Here, for the first time in bone, the mechanisms of time-dependent load transfer occurring at the nanoscale between the collagen phase and the hydroxyapatite (HAP) platelets are studied. Bovine cortical bone samples are subjected to synchrotron X-ray diffraction to measure in situ the evolution of elastic strains in the crystalline HAP phase and the evolution of viscoelastic strains accumulating in the mineralized collagen fibrils under creep conditions at body temperature. For a constant compressive stress, both types of strains increase linearly with time. This suggests that bone, as it deforms macroscopically, is behaving as a traditional composite, shedding load from the more compliant, viscoelastic collagen matrix to the reinforcing elastic HAP platelets. This behavior is modeled by finite-element simulation carried out at the fibrillar level.

Variability in the elastic properties of bovine dentin at multiple length scales

Various methods are used to investigate the variability in elastic properties across a population of deciduous bovine incisor root dentin samples spanning different animals, incisor types, and locations within teeth. First, measurements of elastic strains by high-energy synchrotron X-ray scattering during compressive loading of dentin specimens provided the effective modulus--the ratio of applied stress to elastic phase strain--for the two main phases of dentin (hydroxyapatite crystals and mineralized collagen fibrils), shedding light on load transfer operating at the nanoscale between collagen and mineral phases. Second, Youngs moduli were measured at the macroscale by ultrasonic time-of-flight measurements. Third, thermogravimetry quantified the volume fractions of hydroxyapatite, protein and water at the macroscale. Finally, micro-Computed Tomography determined spatial variations of the mineral at the sub-millimeter scale. Statistical comparison of the above properties reveals: (i) no significant differences for dentin samples taken from different animals or different incisor types but (ii) significant differences for samples taken from the cervical or apical root sections as well as from different locations between buccal and lingual edges.

Stochastic Interdigitation as a Toughening Mechanism at the Interface between Tendon and Bone

Reattachment and healing of tendon to bone poses a persistent clinical challenge and often results in poor outcomes, in part because the mechanisms that imbue the uninjured tendon-to-bone attachment with toughness are not known. One feature of typical tendon-to-bone surgical repairs is direct attachment of tendon to smooth bone. The native tendon-to-bone attachment, however, presents a rough mineralized interface that might serve an important role in stress transfer between tendon and bone. In this study, we examined the effects of interfacial roughness and interdigital stochasticity on the strength and toughness of a bimaterial interface. Closed form linear approximations of the amplification of stresses at the rough interface were derived and applied in a two-dimensional unit-cell model. Results demonstrated that roughness may serve to increase the toughness of the tendon-to-bone insertion site at the expense of its strength. Results further suggested that the natural tendon-to-bone attachment presents roughness for which the gain in toughness outweighs the loss in strength. More generally, our results suggest a pathway for stochasticity to improve surgical reattachment strategies and structural engineering attachments.

Allometry of the Tendon Enthesis: Mechanisms of Load Transfer Between Tendon and Bone

Several features of the tendon-to-bone attachment were examined allometrically to determine load transfer mechanisms. The humeral head diameter increased geometrically with animal mass. Area of the attachment site exhibited a near isometric increase with muscle physiological cross section. In contrast, the interfacial roughness as well as the mineral gradient width demonstrated a hypoallometric relationship with physiologic cross-sectional area (PCSA). The isometric increase in attachment area indicates that as muscle forces increase, the attachment area increases accordingly, thus maintaining a constant interfacial stress. Due to the presence of constant stresses at the attachment, the micrometer-scale features may not need to vary with increasing load.

Effect of high-energy X-ray irradiation on creep mechanisms in bone and dentin

Under long-term loading creep conditions, mineralized biological tissues like bone are expected to behave in a similar manner to synthetic composites where the creeping matrix sheds load to the elastic reinforcement as creep deformation progresses. To study this mechanism in biological composites, creep experiments were performed at 37 °C on bovine compact bone and dentin. Static compressive stresses were applied to the samples, while wide- and small-angle scattering patterns from high energy synchrotron X-rays were used to determine, respectively, the elastic strain in the hydroxyapatite (HAP) platelets and the strain in the mineralized collagen fibril, as a function of creep time. In these highly irradiated biological composites, the reinforcing hydroxyapatite platelets progressively transfer some of their stress back to the softer protein matrix during creep. While such behavior can be explained by damage at the interface between the two phases, it is not consistent with measurements of the apparent moduli--the ratio of applied stress to elastic HAP strain measured throughout the creep experiments by elastic unload/load segments--which remained constant throughout the experiment and thus indicated good HAP/protein bonding. A possible explanation is a combination of X-ray and load induced interfacial damage explaining the shedding of load from the HAP during long term creep, coupled with interfacial re-bonding of the load-disrupted reversible bonds upon unloading, explaining the unaffected elastic load partitioning during unload/load segments. This hypothesis is further supported by finite element modeling which shows results mirroring the experimental strain measurements when considering interfacial delamination and a compliant interstitial space at the ends of the HAP platelets.

Bovine and equine peritubular and intertubular dentin.

Dentin contains 1-2μm diameter tubules extending from the pulp cavity to near the junction with enamel. Peritubular dentin (PTD) borders the tubule lumens and is surrounded by intertubular dentin (ITD). Differences in PTD and ITD composition and microstructure remain poorly understood. Here, a (∼200nm)(2), 10.1keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15-30μm thick bovine and equine specimens. Increased Ca fluorescence surrounding tubule lumens confirms that PTD is present, and the relative intensities in PTD and ITD correspond to carbonated apatite (cAp) volume fraction of ∼0.8 in PTD vs. 0.65 assumed for ITD. In the PTD near the lumen edges, Zn intensity is strongly peaked, corresponding to a Zn content of ∼0.9mgg(-1) for an assumed concentration of ∼0.4mgg(-1) for ITD. In the equine specimen, the Zn K-edge position indicates that Zn(2+) is present, similar to bovine dentin (Deymier-Black et al., 2013), and the above edge structure is consistent with spectra from macromolecules related to biomineralization. Transmission X-ray diffraction shows only cAp, and the 00.2 diffraction peak (Miller-Bravais indices) width is constant from ITD to the lumen edge. The cAp 00.2 average preferred orientation is axisymmetric (about the tubule axis) in both bovine and equine dentin, and the axisymmetric preferred orientation continues from ITD through the PTD to the tubule lumen. These data indicate that cAp structure does not vary from PTD to ITD.

Effect of stress and temperature on the micromechanics of creep in highly irradiated bone and dentin

Synchrotron X-ray diffraction is used to study in situ the evolution of phase strains during compressive creep deformation in bovine bone and dentin for a range of compressive stresses and irradiation rates, at ambient and body temperatures. In all cases, compressive strains in the collagen phase increase with increasing creep time (and concomitant irradiation), reflecting macroscopic deformation of the sample. By contrast, compressive elastic strains in the hydroxyapatite (HAP) phase, created upon initial application of compressive load on the sample, decrease with increasing time (and irradiation) for all conditions; this load shedding behavior is consistent with damage at the HAP-collagen interface due to the high irradiation doses (from ~100 to ~9,000 kGy). Both the HAP and fibril strain rates increase with applied compressive stress, temperature and irradiation rate, which is indicative of greater collagen molecular sliding at the HAP-collagen interface and greater intermolecular sliding (i.e., plastic deformation) within the collagen network. The temperature sensitivity confirms that testing at body temperature, rather than ambient temperature, is necessary to assess the in vivo behavior of bone and teeth. The characteristic pattern of HAP strain evolution with time differs quantitatively between bone and dentin, and may reflect their different structural organization.