Bone mineral properties and 3D orientation of human lamellar bone around cement lines and the Haversian system.

Bone is a complex, biological tissue made up primarily of collagen fibrils and biomineral nanoparticles. The importance of hierarchical organization in bone was realized early on, but the actual interplay between structural features and the properties on the nanostructural and crystallographic level is still a matter of intense discussion. Bone is the only mineralized tissue that can be remodeled and, at the start of the formation of new bone during this process, a structure called a cement line is formed on which regular bone grows. Here, the orientational relationship of nanostructural and crystallographic constituents as well as the structural properties of both nanostructural and crystallographic constituents around cement lines and the Haversian system in human lamellar bone are investigated. A combination of small- and wide-angle X-ray scattering tensor tomography is employed together with diffraction tomography and synchrotron computed tomography to generate a multi-modal image of the sample. This work shows that the mineral properties vary as a function of the distance to the Haversian canal and, importantly, shows that the cement line has differing mineral properties from the surrounding lamellar bone, in particular with respect to crystallite size and degree of orientation. Cement lines make up a significant portion of the bone matrix despite their small size, hence the reported findings on an altered mineral structure, together with the spatial modulation around the Haversian canal, have implications for the formation and mechanics of bone.

Osteon Myospalacem Baileyi attenuates osteoclast differentiation through RANKL induced NFAT pathways.

ETHNOPHARMACOLOGICAL RELEVANCE: Osteon Myospalacem Baileyi, known as Sai long gu (Tibetan language, means "blind rat bone"), is the whole skeleton of Tibet plateau rodentia animal Myospalacem Baileyi. Osteon Myospalacem Baileyi had been widely used in the Tibet region as an anti-osteoporosis drug and since 1991 Osteon Myospalacem Baileyi has been listed in the Pharmacopoeia of People's Republic of China as the first-class animal new medical material. However, the mechanism of its anti-osteoporosis activities is still unclear. It is very desirable to solve this problem for further study. MATERIALS AND METHODS: in this study, preparative chromatography was employed to produce the active fraction ET4 from Osteon Myospalacem Baileyi crude. Flow cytometry and MTT assay were used to evaluate the toxicities of ET4. BMM cells were separated from mouse bone marrow to test the inhibition effects of ET4 on osteoclastogenesis. Western blot was used to find out the pathways, through which ET4 could act on osteoclastogenesis. Q-PCR was used to test the osteoclastogenesis marker genes. At last, immunofluorescence confocal microscopy was used to test the osteoclastogenesis master protein NFATc1 nuclei translocation. RESULTS: In this study we report that ET4, at the dose of 60µg/mL, significantly inhibited the formation of osteoclasts. Notably, ET4 did not affect the BMM viability at that dose. In addition, Osteon Myospalacem Baileyi could inhibit the expression of osteoclast marker genes, including cathepsin K (CTSK), nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAP, Acp5) dendrite cell-specific transmembrane protein (DC-STAMP), calcitonin receptor (CTR), osteoclast associated and immunoglobulin-like receptor (OSCAR). Mechanistically, ET4 dose- and time-dependently blocked the RANKL-induced activation of ERK and c-Fos as well as the induction of NFATc1 which is essential for OC formation. CONCLUSIONS: These data suggest that ET4 might be a useful alternative therapy in preventing or treating osteolytic diseases.

Radiopacity of alloplastic bone grafts measured with cone beam computed tomography: An analysis in rabbit calvaria.

Availability of adequate bone structure for dental implants is still a problem in dentistry. Alloplastic grafts, which promote bone regeneration, are used as bone substitutes in orthopedic and oral surgical procedures. The aim of this study was to evaluate the radiopacity of three different synthetic bone grafts in rabbit calvaria, over 3 months, using cone beam computed tomography (CBCT). Four critical-size defects were made on the calvaria of 11 rabbits. The lesions were classified into three groups according to the alloplastic grafts they received: Osteon® 70/30, Osteon collagen®, and Osteon II® groups. The fourth group received blood clot, and served as a control. The bone samples were collected and analyzed with CBCT after the 1st, 2nd, and 3rd month. One month after surgery, the lesions that received Osteon® 70/30 and Osteon collagen® grafts showed the highest radiopacity compared to the lesions with Osteon II® and blood clot. After the 2nd month, the radiopacity values between the three groups that received the grafts were more similar compared to the group with blood clot. After the 3rd month, the lesions with Osteon® 70/30 graft showed the highest radiopacity values, followed by Osteon collagen® and Osteon II® groups. The group that received blood clot showed the lowest radiopacity values. In conclusion, the grafts used in this study had higher radiopacity values compared to blood clot. Among the grafts used, the Osteon® 70/30 graft showed the highest radiopacity values in the 3-month period.

Photo-cross-linkable methacrylated gelatin and hydroxyapatite hybrid hydrogel for modularly engineering biomimetic osteon.

Modular tissue engineering holds great potential in regenerating natural complex tissues by engineering three-dimensional modular scaffolds with predefined geometry and biological characters. In modular tissue-like construction, a scaffold with an appropriate mechanical rigidity for assembling fabrication and high biocompatibility for cell survival is the key to the successful bioconstruction. In this work, a series of composite hydrogels (GH0, GH1, GH2, and GH3) based on a combination of methacrylated gelatin (GelMA) and hydroxyapatite (HA) was exploited to enhance hydrogel mechanical rigidity and promote cell functional expression for osteon biofabrication. These composite hydrogels presented a lower swelling ratio, higher mechanical moduli, and better biocompatibility when compared to the pure GelMA hydrogel. Furthermore, on the basis of the composite hydrogel and photolithograph technology, we successfully constructed an osteon-like concentric double-ring structure in which the inner ring encapsulating human umbilical vascular endothelial cells (HUVECs) was designed to imitate blood vessel tubule while the outer ring encapsulating human osteoblast-like cells (MG63s) acts as part of bone. During the coculture period, MG63s and HUVECs exhibited not only satisfying growth status but also the enhanced genic expression of osteogenesis-related and angiogenesis-related differentiations. These results demonstrate this GelMA-HA composite hydrogel system is promising for modular tissue engineering.

Novel Diketopiperazine Dihydroorotate Dehydrogenase Inhibitors Purified from Traditional Tibetan Animal Medicine Osteon Myospalacem Baileyi.

Traditional Tibetan medicine provides an abundant source of knowledge on human ailments and their treatment. As such, it is necessary to explore their active single compounds used to treat these ailments to discover lead compounds with good pharmacologic properties. In this present work, animal medicine, Osteon Myospalacem Baileyi extracts have been separated using a two-dimensional preparative chromatographic method to obtain single compounds with high purity as part of the following pharmacological research. Five high-purity cyclic dipeptides from chromatography work were studied for their dihydroorotate dehydrogenase inhibitory activity on recombinant human dihydroorotate dehydrogenase enzyme and compound Fr. 1-4 was found to contain satisfying inhibition activity. The molecular modeling study suggests that the active compound Fr. 1-4 may have a teriflunomide-like binding mode. Then, the energy decomposition study suggests that the hydrogen bond between Fr. 1-4 and Arg136 can improve the binding mode to indirectly increase the van der Waals binding energy. All the results above together come to the conclusion that the 2, 5-diketopiperazine structure group can interact with the polar residues well in the active pocket using electrostatic power. If some proper hydrophobic groups can be added to the sides of the 2, 5-diketopiperazine group, it is believed that better 2, 5-diketopiperazine dihydroorotate dehydrogenase inhibitors will be found in the future.

A novel two-dimensional preparative chromatography method designed for the separation of traditional animal Tibetan medicine Osteon Myospalacem Baileyi.

Animal medicine is an important part in traditional Tibetan medicine. However, information about the chemical composition of animal medicine is very limited, and there is a lack of comprehensive chromatographic purification methods. In the present work, animal medicine Osteon Myospalacem Baileyi was taken as an example and a novel two-dimensional preparative chromatographic method was established for the preparation of single compounds with high purity from the extract of Osteon Myospalacem Baileyi. The first-dimension preparation was carried on a DAISO Silica prep column, and ten fractions were obtained from the 112.3 g crude sample within 12 injections. A diol prep column used in nonaqueous mobile phase was selected for the second-dimension preparation. The purity of the compounds isolated from the crude extract was >98%, which indicated that the method built in this work was efficient to manufacture single compounds of high purity from the extract of Osteon Myospalacem Baileyi. Additionally, this method showed great potential in the purification of weakly polar chemicals and it could act as a good example in the purification of other traditional animal medicines.

Chemical etching in processing cortical bone specimens for scanning electron microscopy.

Transverse and longitudinal sectioning of undecalcified cortical bone is a commonly employed technique for investigating the lamellar structure of the osteons. Since a flat surface is required, the specimen has to be grinded and then polished. Whereas the smear of debris and inorganic/organic deposits left by these treatments cannot be removed by ultrasonication alone, a chemical treatment of the specimen surface with either a basic or an acid etching solution is currently employed. A further effect of the latter can be the enhancement of the lamellar bone pattern. The kind of etching solution, its pH, the concentration of etchants, and the contact time significantly affect the sectioned surface when it is observed with scanning electron microscopy (SEM). The etching procedures can severely influence the obtained images. Homogeneous cortical bone specimens were sampled from the first metatarsal of two fresh human subjects. One or two cut surfaces were exposed to different acid and basic solutions in bonded conditions. Considering the type of chemical agents, the solution pH, and the exposure time of the specimens, the effects of several etching media have been investigated and compared. Strong etching, either acid or basic produced surface decalcification and severe damage of the collagen matrix, compromising any morphological or morphometric analysis. Weak acid etching (for example citric and acetic acid), even though causing distinctive alteration of the sample, enhanced the visibility of the lamellar pattern, while the polyphosphate treatment of the surface decalcified a thin layer matrix, ensuring a good visibility of fibrils and avoiding rough distortions.

Mapping dynamical mechanical properties of osteonal bone by scanning acoustic microscopy in time-of-flight mode.

An important determinant of mechanical properties of bone is Young's modulus and its variation in individual osteons of cortical bone tissue. Its mechanical behavior also depends on deformation rate owing to its visco- or poroelastic properties. We developed a method to measure dynamical mechanical properties of bulk bone tissue at osteonal level based on scanning acoustic microscopy (SAM) using time-of-flight (TOF) measurements in combination with quantitative backscattered electron imaging (qBEI). SAM-TOF yields local sound velocities and qBEI corresponding material densities together providing elastic properties. Osteons (n=55) were measured in three human femoral diaphyseal ground bone sections (∼ 30 µm in thickness). In addition, subchondral bone and mineralized articular cartilage were investigated. The mean mineral contents, the mean sound velocities, and the mean elastic modulus of the osteons ranged from 20 to 26 wt%, from 3,819 to 5,260 m/s, and from 21 to 44 GPa, respectively. There was a strong positive correlation between material density and sound velocity (Pearson's r=0.701; p<0.0001) of the osteons. Sound velocities between cartilage and bone was similar, though material density was higher in cartilage (+4.46%, p<0.0001). These results demonstrate the power of SAM-TOF to estimate dynamic mechanical properties of the bone materials at the osteonal level.

Osteo-radio-necrosis (ORN) and bisphosphonate-related osteonecrosis of the jaws (BRONJ): the histopathological differences under the clinical similarities.

OBJECTIVES: Both osteoradionecrosis (ORN) and bisphosphonate associated osteonecrosis of the jaws (BRONJ) present clinically as regions of exposed necrotic bone. The study aimed to demonstrate the histopathological differences behind the observed clinical similarities. STUDY DESIGN: Ten ORN specimens and ten BRONJ specimens were used, as well as ten samples of normal mandibular bone as control. Two bone-specific stainings were used, i.e. Sirius Red for the study of the relative presence of collagen types I and III and toluidine blue for the study the osteon density. RESULTS: The Red Green Blue (RGB)-analysis of the specimens stained with Sirius Red identified significant differences between the chromatic patterns observed in bone preparations of patients suffering from ORN when compared to both BRONJ and control samples. Moreover, the osteon density of the BRONJ samples was significantly lower when compared to ORN and normal bone samples. CONCLUSIONS: The demonstrated differences in the bone architecture and in the bone collagen content between the two pathological conditions most likely reflect underlying pathophysiological differences.

The effects of freezing, boiling and degreasing on the microstructure of bone.

The histology of bone has been a useful tool in research. It is commonly used to estimate the age of an individual at death, to assess if the bone is of human or non-human origin and in trauma analysis. Factors that affect the histology of bone include age, sex, population affinity and burning to name but a few. Other factors expected to affect bone histology are freezing, boiling and degreasing but very little information is available for freezing and the effect thereof, and it is unknown if boiling and degreasing affects bone histology. The aim of this study was to assess the effects of freezing, freezing and boiling, and freezing, boiling and degreasing on the histological structure of compact bone. Five cadaver tibiae were frozen at -20°C for 21 days followed by segments being boiled in water for three days and degreased in trichloroethylene at 82°C for three days. Anterior midshaft sections were prepared as ground sections and for Scanning Electron Microscopy (SEM). Quantitatively, there were no significant differences between freezing, boiling and degreasing; however, qualitative differences were observed using SEM. After being frozen the bone displayed cracks and after boiling the bones displayed erosion pits on the surface. It is suggested that further research, using different durations and temperatures for boiling and freezing be undertaken on bone samples representing different ages and various skeletal elements.

A quantitative collagen fibers orientation assessment using birefringence measurements: calibration and application to human osteons.

Even though mechanical properties depend strongly on the arrangement of collagen fibers in mineralized tissues, it is not yet well resolved. Only a few semi-quantitative evaluations of the fiber arrangement in bone, like spectroscopic techniques or circularly polarized light microscopy methods are available. In this study the out-of-plane collagen arrangement angle was calibrated to the linear birefringence of a longitudinally fibered mineralized turkey leg tendon cut at variety of angles to the main axis. The calibration curve was applied to human cortical bone osteons to quantify the out-of-plane collagen fibers arrangement. The proposed calibration curve is normalized to sample thickness and wavelength of the probing light to enable a universally applicable quantitative assessment. This approach may improve our understanding of the fibrillar structure of bone and its implications on mechanical properties.

Biochemical characterization of major bone-matrix proteins using nanoscale-size bone samples and proteomics methodology.

There is growing evidence supporting the need for a broad scale investigation of the proteins and protein modifications in the organic matrix of bone and the use of these measures to predict fragility fractures. However, limitations in sample availability and high heterogeneity of bone tissue cause unique experimental and/or diagnostic problems. We addressed these by an innovative combination of laser capture microscopy with our newly developed liquid chromatography separation methods, followed by gel electrophoresis and mass spectrometry analysis. Our strategy allows in-depth analysis of very limited amounts of bone material, and thus, can be important to medical sciences, biology, forensic, anthropology, and archaeology. The developed strategy permitted unprecedented biochemical analyses of bone-matrix proteins, including collagen modifications, using nearly nanoscale amounts of exceptionally homogenous bone tissue. Dissection of fully mineralized bone-tissue at such degree of homogeneity has not been achieved before. Application of our strategy established that: (1) collagen in older interstitial bone contains higher levels of an advanced glycation end product pentosidine then younger osteonal tissue, an observation contrary to the published data; (2) the levels of two enzymatic crosslinks (pyridinoline and deoxypiridinoline) were higher in osteonal than interstitial tissue and agreed with data reported by others; (3) younger osteonal bone has higher amount of osteopontin and osteocalcin then older interstitial bone and this has not been shown before. Taken together, these data show that the level of fluorescent crosslinks in collagen and the amount of two major noncollagenous bone matrix proteins differ at the level of osteonal and interstitial tissue. We propose that this may have important implications for bone remodeling processes and bone microdamage formation.

Microstructural mechanical study of a transverse osteon under compressive loading: The role of fiber reinforcement and explanation of some geometrical and mechanical microscopic properties.

This Finite Element study aims at understanding the transverse osteon as a composite microstructure, and at differentiating the actions of each of its main components and their interactions. Three components of the osteon have been distinguished: the lamellae mineral-collagen matrix, the lamellae mineral-collagen reinforcement fibers and the Haversian canal content made of intracortical fluid and soft tissues. Numerical compression experiments have been performed, varying the microstructure properties. Our results show that fiber reinforcement of transverse osteons is only efficient at resisting dynamic compressive loadings, but that the improvement of the static compressive properties is very poor. Furthermore, the modeled stress distribution within the matrix and reinforcement fibers may explain why transverse osteons are often limited to a small number of lamellae (<8) and why internal lamellae could be stiffer than external ones.

The sealed osteons of cortical diaphyseal bone. Early observations revisited with scanning electron microscopy.

The frequency, structure, mode of formation and significance of sealed osteons remain unsettled. Sealed osteons have been reported as an unusual finding in the cortical bone of experimental animals: we extended the observation to human cortical bone studied with SEM. Tibial bone specimens from three patients who sustained a traumatic below-the-knee amputation were used in the study. The observed total mean density of osteons was 19.25/mm(2) and the percentage of sealed and partially sealed osteons was 4.2% and 1.7% respectively. The material sealing the central canal showed an X-ray microanalysis spectrum with the same Ca/P ratio as the peripheral lamellae and a lower carbon signal. The morphology suggested a reactivation of bone apposition triggered by exclusion of the occluded canal from blood flow rather than a physiological evolution of the closing process of secondary osteons. This presupposes collapse and degeneration of the central vessel before the osteoblasts resting on the inner surface of the canal could start to lay down new bone matrix. This explanation is consistent with a dynamic model of intracortical blood flow.

New method for Raman investigation of the orientation of collagen fibrils and crystallites in the Haversian system of bone.

Knowledge of the organization of the components of bone is of primary importance in understanding how this tissue responds to stresses and provides a starting point for the design and development of biomaterials. Bone structure has been the subject of numerous studies. The mineralized fiber arrangement in cortical bone is either a twisted or orthogonal plywood structure. Both mineral models coexist in compact bone. Raman polarized spectroscopy offers definite advantages in the study of biological samples, enabling the simultaneous analysis of mineral and organic components and the determination of molecular orientation through the polarization properties of the Raman scattering. In this study, we used the Raman polarization approach to simultaneously investigate the orientation of collagen fibrils and apatite crystals in human cortical bone. Raman bands ratios were monitored as a function of sample orientation. Specific ratios were chosen--such as nu(3) PO(4)/nu(1) PO(4), amide III (1271 cm(-1))/amide III (1243 cm(-1)), and amide I/amide III (1243 cm(-1))--due to their sensitivity to apatite-crystal and collagen-fibril orientation. Based on this original approach, spatial changes were monitored as a function of distance from the Haversian canal. The results revealed simultaneous tilting in intra-lamellar collagen-fibril and mineral crystal orientations. These results are consistent with a twisted plywood organization in the Haversian bone structure at the lamellar level. But at molecular level, the co-alignment of the collagen fibrils and the apatite crystal is observed in the innermost lamellae and becomes gradually less ordered as the distance from the Haversian canal increases. This work highlights the interest of Raman spectroscopy for the multiscale investigation of bone structure.

Technical note: PCR analysis of minimum target amount of ancient DNA.

The study of ancient DNA plays an important role in archaeological and palaeontological research as well as in pathology and forensics. Here, we present a new tool for ancient DNA analysis, which overcomes contamination problems, DNA degradation, and the negative effects of PCR inhibitors while reducing the amount of starting target material in the picogram range. Ancient bone samples from four Egyptian mummies were examined by combining laser microdissection, conventional DNA extraction, and low-volume PCR. Initially, several bone particles (osteons) in the micrometer range were extracted by laser microdissection. Subsequently, ancient DNA amplification was performed to verify our extraction method. Amelogenin and beta-actin gene specific fragments were amplified via low-volume PCR in a total reaction volume of 1 microl. Results of microdissected mummy DNA samples were compared to mummy DNA, which was extracted using a standard DNA extraction method based on pulverization of bone material. Our results highlight the combination of laser microdissection and low-volume PCR as a promising new technique in ancient DNA analysis.

Orientation of collagen at the osteocyte lacunae in human secondary osteons.

This work characterizes an aspect of human bone micro-structure, pertinent to fracture initiation and arrest. It addresses how the orientation of elementary components proximate to osteocyte lacunae influences secondary osteon micro-biomechanics. New data at the perilacunar region concerning orientation of collagen-apatite, and prior data on collagen orientation outside the perilacunar region, are incorporated in a novel simulation of osteons to investigate how orientation relates to strains and stresses during mechanical testing. The perilacunar region was observed by confocal microscopy within single lamellar specimens, isolated from osteons. The specimens were separated by extinct or bright appearance in transverse section under circularly polarizing light. This is because synchrotron diffraction and confocal microscopy had established that each type, away from the perilacunar region, corresponds to specific dominant collagen orientation (extinct lamellae's dominant collagen forming small angles with the original osteon axis, while the bright lamellae's forms larger angles). Morphometry of serial confocal images of each perilacunar region showed collagen orientation generally following the orientation of canaliculi, circumambiently-perpendicular to the lacuna. The lacunae tilted relative to the lamellar walls were more numerous in extinct than in bright lamella. Their apices were less likely in extinct than bright lamella to show collagen following the canalicular orientation. The simulation of osteocyte lacunae in osteons, under tension or compression loading, supports the hypothesis that collagen orientation affects strains and stresses at the equatorial perilacunar region in conjunction with the presence of the lacuna. We further conjecture that collagen orientation diverts propagation of micro-cracks initiating from apices.

In situ examination of the time-course for secondary mineralization of Haversian bone using synchrotron Fourier transform infrared microspectroscopy.

At the tissue level it is well established that the rate of remodeling is related to the degree of mineralization. However, it is unknown how long it takes for an individual bone structural unit (BSU) to become fully mineralized during secondary mineralization. Using synchrotron Fourier transform infrared microspectroscopy (FTIRM) we examined the time required for newly formed bone matrix to reach a physiological mineralization limit. Twenty-six, four-month old female New Zealand white rabbits were administered up to four different fluorochrome labels at specific time points to evaluate the chemical composition of labeled osteons from the tibial diaphysis that had mineralized for 1, 8, 18, 35, 70, 105, 140, 175, 210, 245, 280, 315, 350, and 385 days. Interstitial bone from 505 day old rabbits was used as a reference value for the physiological limit to which bone mineralizes. Using synchrotron FTIRM, area integrations were carried out on protein (Amide I: 1688-1623 cm(-1)), carbonate (v(2)CO(3)(2-): 905-825 cm(-1)), and phosphate (v(4)PO(4)(3-): 650-500 cm(-1)) IR bands. IR spectral data are presented as ratios of phosphate/protein (overall matrix mineralization) and carbonate/protein. The rate of mineralization of osteonal bone proceeded rapidly between day 1 and 18, reaching 67% of interstitial bone levels. This was followed by a slower, more progressive accumulation of mineral up to day 350. By 350 days the rate of increase plateaued. The ratio of carbonate/protein also increased rapidly during the first 18 days, reaching 73% of interstitial bone levels. The ratio of carbonate/protein plateaued by day 315, reaching levels not significantly different to interstitial bone levels. In conclusion, our data demonstrate that bone accumulates mineral rapidly during the first 18 days (primary mineralization), followed by a more gradual increase in the accumulation of mineral (secondary mineralization) which we found to be completed in 350 days.

Some considerations on biomaterials and bone.

Osteoinduction is a property not traditionally attributed to Calcium Phosphate ceramics. Histologic, SEM and X-ray microanalyses of a biopsy of pulmonary alveolar microlithiasis allow to discredit this opinion. Bone, even lamellar type, was ectopically formed on microliths undergoing osteoclastic erosion. The SEM and X-ray microanalyses of coral granules implanted in humans indicate an osteoconductive property for both Calcium and Phosphorus. Analysis of in vitro allows to propose an enhancement of the osteocapability of coral. Lamellar bone formation in the near absence of loads undermines the opinion which sees a correlation between lamellar bone and mechanical loads. Analysis of the bone surrounding an uncemented titanium hip prosthesis highlights that both remodeled and newly formed bone have lamellae oriented parallel to prosthesis surfaces, i.e. orthogonal to loads, as opposed to that of lamellar bone of osteons which are oriented parallel to loads. Analysis of longitudinal sections of cortical bone under polarized light points out that lamellae are displaced parallel to the cement line surface both in the conic end of osteons and in Volkman's canals with thick wall, i.e. undergoing sloped load directions. In conclusion, there may be a relationship between lamellae formation and gravity.