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Apatitic mineral of dentin forms within the collagenous matrix (intertubular dentin, ITD) secreted from the odontoblastic processes (OP). Highly calcified mineral (peritubular dentin, PTD) is deposited at the interface between the ITD and each process membrane, creating a tubular system penetrating the dentin that extends from the dentino-enamel junction to the predentin-dentin junction. We focus on determining the composition of the PTD both with regard to its organic matrix and the inorganic phase. A laser capture technique has been adapted for the isolation of the mineralized PTD free from the ITD, and for the analysis of the PTD by SEM, TEM, and energy dispersive spectrometry (EDS), these data were subsequently compared with similar analyses of intact dentin slices containing ITD bounded-PTD annuli. Elemental line scans reveal clearly marked boundaries between ITD, PTD, and OP components, and illustrate the differences in composition, and topographical surface roughness. The organic matrix of the PTD was shown to be sulfur rich, and further antibody labeling showed the sulfated organic component to be chondroitin sulfate [corrected]. In this PTD organic matrix the S/Ca and Ca/P ratios were distinctly higher than in the ITD, indicating that polysaccharide bound S supplies the anionic counterion facilitating the formation of the apatitic PTD mineral.

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.

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.

Stabilization of biological materials by freezing is widespread in the fields of medicine and biomaterials research and yet, in the case of hard biomaterials such as dentin, there is not a good understanding of how such treatments might affect the mechanical properties. The freezing and thawing may have a number of different effects on dentin including formation of cracks in the microstructure and denaturation of the collagen. Using high-energy synchrotron X-ray diffraction, the apparent moduli of bovine dentin samples were measured before and after various numbers of freeze–thaw cycles. It was determined that repeated freezing and thawing has no measurable effect on the hydroxyapatite or fibrillar apparent moduli up to 10 cycles. This confirms that the use of low temperature storage for stabilization of dentin is reasonable in cases where stiffness is a property of importance.

Copper artifacts from Cahokia Mounds, Illinois were analyzed from a materials science perspective to shed light on techniques used by Mississippian copper workers to deform nuggets of native copper into thin sheets. Eight small copper pieces from a copper-working site at Cahokia’s Mound 34 were subjected to metallographic examination. Replication experiments thereafter recreated features of the artifacts under controlled conditions. It is concluded that copper sheets were thinned through repeated cycles of hammering and annealing performed at temperatures achievable in an open wood fire. The welding of sheets to create multilayered objects was not observed in any artifacts and could not be accomplished experimentally. Additionally, a possible cutting method used on some artifacts was identified.

We present a previously unrecognized effect of sound waves on gap-junction-based intercellular signaling such as in biological tissues composed of endothelial cells. We suggest that sound irradiation may, through temporal and spatial modulation of cell-to-cell conductance, create intercellular calcium waves with unidirectional signal propagation associated with nonconventional topologies. Nonreciprocity in calcium wave propagation induced by sound wave irradiation is demonstrated in the case of a linear and a nonlinear reaction-diffusion model. This demonstration should be applicable to other types of gap-junction-based intercellular signals, and it is thought that it should be of help in interpreting a broad range of biological phenomena associated with the beneficial therapeutic effects of sound irradiation and possibly the harmful effects of sound waves on health.

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.

Apatitic mineral of dentin forms within the collagenous matrix (intertubular dentin, ITD) secreted from the odontoblastic processes (OP). Highly calcified mineral (peritubular dentin, PTD) is deposited at the interface between the ITD and each process membrane, creating a tubular system penetrating the dentin that extends from the dentino-enamel junction to the predentin-dentin junction. We focus on determining the composition of the PTD both with regard to its organic matrix and the inorganic phase. A laser capture technique has been adapted for isolation of the mineralized PTD free of the ITD, and for the analysis of the PTD by SEM, TEM, and energy dispersive spectrometry (EDS), and comparison with similar analyses of intact dentin slices containing ITD bounded-PTD annuli. Elemental line scans clearly marked the boundaries between ITD, PTD, and OP components, and revealed differences in composition, and topographical surface roughness. The organic matrix of the PTD was shown to be sulfur rich, and further antibody labeling showed the sulfated organic component to be chondroitin sulfate B. In this organic matrix the Ca/P ratio was distinctly higher than in the ITD and intact PTD, indicating that polysaccharide bound S supplies the anionic counterion facilitating the formation of the apatitic PTD mineral.

We demonstrate the capability and technique to perform microdissection and isolation of select regions of untreated, mineralized dentin using laser capture. Dentin is a complex, non-homogeneous tissue comprised of a mineralized collagenous matrix (intertubular dentin [ITD]), odontoblastic processes (ODPs), a void space (tubules) that forms within the ITD left behind by the retraction of ODPs during dentin maturation, and a highly mineralized non-collagenous component that exists at the interface between the tubules and ITD known as peritubular dentin (PTD). PTD forms as the dentin matures. The ODPs retract toward the direction of the pulp; leaving very little PTD at either the DEJ or near the pulp. Statistical analysis of thin cross-sections of coronal bovine dentin imaged by light microscopy reveal that the area occupied by PTD >50%. To examine the nature of PTD and its relation to both the tubules and ITD, we devised a series of steps to carefully prepare sections of coronal bovine dentin so that areas of the dentin tissue could be cut and isolated for further analysis. We demonstrate that it is possible to selectively isolate targeted regions of dentin for analysis and that high resolution analysis of such sections can be performed using electron microscopy. Results show that the mineralized PTD has a different texture than mineralized ITD and that there is a distinct boundary between the PTD and the ITD. Selective isolation of mineralized tissue components for further analytical study opens the door for the investigation of similar enigmatic mineralized structures.

Dentin contains 1–2 μm 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 (∼200 nm)2, 10.1 keV synchrotron X-ray beam maps X-ray fluorescence and X-ray diffraction simultaneously around tubules in 15–30 μm 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 ∼0.9 mg g−1 for an assumed concentration of ∼0.4 mg g−1 for ITD. In the equine specimen, the Zn K-edge position indicates that Zn2+ 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.

The scattering of elastic waves in a one dimensional phononic (PnC) crystal composed of alternate collagen and hydroxy-apatite constituent layers is studied. These superlattices are metaphors for mineralized tissues present in bones and teeth. The collagen is treated as an open system elastic medium with water content which can vary depending on the level of stress applied. The open system nature of the collagen-water system leads to a non-linear stress-strain response. The finite difference time domain method is employed to investigate the propagation of non-linear mechanical waves through the superlattice. The spectral energy density method enables the calculation of the non-linear vibrational wave band structure. The non-linearity in the mechanical response of the collagen-water system enables a variety of multi-phonon scattering processes resulting in an increase in the number of channels for the dissipation of elastic waves and therefore for the dissipation of mechanical energy. These results provide an explanation for the relationship between bone fragility and decreased hydration.

Tough, graded interfaces are a broadly conserved feature of biological attachment of two dissimilar materials, but the mechanisms by which toughening occurs are not fully known. In biological systems, graded structures are commonly found in the attachments of materials with very different properties such as bone, which is very stiff, and tendon, which is compliant 1. Previous research has shown that the interface between these two materials is composed mostly of collagen, proteoglycans, and carbonated apatite crystals (mineral). Our working hypothesis is that gradients in mineral content and collagen orientation combine to provide a compliant energy absorbing region between the tendon and bone 1.

The relationship between the mineralization of peritubular dentin (PTD) and intertubular dentin (ITD) is not well understood. Tubules are quite small, diameter ∼2 µm, and this makes the near-tubule region of dentin difficult to study. Here, advanced characterization techniques are applied in a novel way to examine what organic or nanostructural signatures may indicate the end of ITD or the beginning of PTD mineralization. X-ray fluorescence intensity (Ca, P, and Zn) and X-ray diffraction patterns from carbonated apatite (cAp) were mapped around dentintubules at resolutions ten times smaller than the feature size (200 nm pixels), representing a 36% increase in resolution over earlier work. In the near tubule volumes of near-pulp, root dentin, Zn intensity was higher than in ITD remote from the tubules. This increase in Zn(2+), as determined by X-ray absorption near edge structure analysis, may indicate the presence of metalloenzymes or transcription factors important to ITD or PTD mineralization. The profiles of the cAp 00.2 X-ray diffraction rings were fitted with a pseudo-Voigt function, and the spatial and azimuthal distribution of these rings' integrated intensities indicated that the cAp platelets were arranged with their c-axes aligned tangential to the edge of the tubule lumen. This texture was continuous throughout the dentin indicating a lack of structural difference between in the Zn rich near-tubular region and the remote ITD.

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.

Both the load partitioning between hydroxyapatite (HAP) and collagen during compressive creep deformation of bone and the HAP residual strain in unloaded bone have been shown in previous synchrotron X-ray diffraction studies to be affected by the X-ray irradiation dose. Here, through detailed analysis of the X-ray diffraction patterns of bovine bone, the effect of X-ray dose on (i) the rate of HAP elastic strain accumulation/shedding under creep conditions and (ii) the HAP lattice spacing and average root mean square (RMS) strain under load-free conditions are examined. These strain measurements exhibit three stages in response to increasing X-ray dose. Up to ∼75 kGy (stage I) no effect of dose is observed, indicating a threshold behavior. Between ∼75 and ∼300 kGy (stage II) in unloaded bone the HAP d-spacing increases and the RMS strain decreases with dose, indicating strain relaxation of HAP. Furthermore, under constant compressive load creep conditions, the rate of compressive elastic strain accumulation in HAP decreases with increasing dose until, at ∼115 kGy, it changes sign, indicating that the HAP phase is shedding load during creep deformation. These stage II behaviors are consistent with HAP-collagen interfacial damage, which allows the HAP elastic strain to relax within both the loaded and unloaded samples. Finally, for doses in excess of ∼300 kGy (stage III, measured up to 7771 kGy) the HAP lattice spacing and RMS strain for load-free samples and the rate of HAP elastic strain shedding for crept samples remain independent of dose, suggesting a saturation of damage and/or stiffening of the collagen matrix due to intermolecular cross-linking.

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.

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).

Bones are important natural materials which compose the skeleton of the animals. The basic and fundamental function of bone is mechanical – to provide a frame to keep the body supported and moved, to protect internal organs, while they also serve with other important biological functions, such as blood production, mineral storage, etc. Such multifunctionality makes bone with a very complex hierarchical structure, while the millions of years’ evolution makes bone have outstanding characteristics, in mechanical aspect of view, bone has a high strength and high toughness while keeping lightweight. These natural exceptional mechanical properties of bone are much better than that of the conventional artificial materials are inspiring the materials engineering community to design the novel biomimetic materials. However, on the other hand, as a natural material which lies in the body of organism, numbers of diseases infects bones and severely weakens their instinct properties, therefore, to cure the patients and recover basic functions of bone, many researches are needed to find the biological substitute of bone.

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 ± 2 GPa. This value is significantly lower than the value of 44 GPa 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 Young’s 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 and dentin are highly complex, hierarchical composite materials with exceptional properties due to their unique composition and structure. They are essentially the same material with varied structural organization. They are three phase composites made up of a ceramic component, hydroxyapatite (HAP), a polymeric or proteinaceous component, collagen, and fluid filled porosity. A number of macroscopic studies have shown that both dentin [1-6] and bone [7-9] undergo visco-elastic, creep deformation and stress-relaxation behaviors. The problem with these bulk experiments is that they do not give information about which phase is contributing to the macroscopic creep or how. Some of these inquiries have suggested that the collagen is not responsible [9] and that creep in hard biological materials is primarily due to dislocations in the HAP mineral. On the other hand, others have said that collagen is completely responsible for the creep [8, 10]. These uncertainties make it essential to use techniques that allow for the study of the behavior of these very different components simultaneously during loading, determining their participation in creep. One such technique is synchrotron diffraction.