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1.  Computational and experimental methodology for site-matched investigations of the influence of mineral mass fraction and collagen orientation on the axial indentation modulus of lamellar bone☆ 
Relationships between mineralization, collagen orientation and indentation modulus were investigated in bone structural units from the mid-shaft of human femora using a site-matched design. Mineral mass fraction, collagen fibril angle and indentation moduli were measured in registered anatomical sites using backscattered electron imaging, polarized light microscopy and nano-indentation, respectively. Theoretical indentation moduli were calculated with a homogenization model from the quantified mineral densities and mean collagen fibril orientations. The average indentation moduli predicted based on local mineralization and collagen fibers arrangement were not significantly different from the average measured experimentally with nanoindentation (p=0.9). Surprisingly, no substantial correlation of the measured indentation moduli with tissue mineralization and/or collagen fiber arrangement was found. Nano-porosity, micro-damage, collagen cross-links, non-collagenous proteins or other parameters affect the indentation measurements. Additional testing/simulation methods need to be considered to properly understand the variability of indentation moduli, beyond the mineralization and collagen arrangement in bone structural units.
Highlights
•Site-matched assessment of nanoindentation modulus, mineral mass fraction and collagen fibers orientation in human cortical bone sections.•Comparison of experimental nanoindentation modulus with its computed equivalent based on the site-matched morphological data.•While mean experimental and computed nanoindentation moduli match well, their variations exhibit very weak correlations.•Considering factors like nano-porosity and damage may be necessary to understand variability of lamellar stiffness of bone structural units.•This is not in conflict with the well known anisotropy associated with the rotated plywood model at the sublamellar scale.
doi:10.1016/j.jmbbm.2013.07.004
PMCID: PMC3843116  PMID: 23994944
Collagen fibril orientation; Nanoindentation; Homogenization; Quantitative polarized light microscopy (qPLM); Mineralization; Site-matching
2.  Elastic anisotropy of uniaxial mineralized collagen fibers measured using two-directional indentation. Effects of hydration state and indentation depth 
Mineralized turkey leg tendon (MTLT) is an attractive model of mineralized collagen fibers, which are also present in bone. Its longitudinal structure is advantageous for the relative simplicity in modeling, yet its anisotropic elastic properties remain unknown. The aim of this study was to quantify the extent of elastic anisotropy of mineralized collagen fibers by using nano- and microindentation to probe a number on MTLT samples in two orthogonal directions. The large dataset allowed the quantification of the extent of anisotropy, depending on the final indentation depth and on the hydration state of the sample.
Anisotropy was observed to increase with the sample re-hydration process. Artifacts of indentation in a transverse direction to the main axis of the mineralized tendons in re-hydrated condition were observed. The indentation size effect, that is, the increase of the measured elastic properties with decreasing sampling volume, reported previously on variety of materials, was also observed in MTLT. Indentation work was quantified for both directions of indentation in dried and re-hydrated conditions.
As hypothesized, MTLT showed a higher extent of anisotropy compared to cortical and trabecular bone, presumably due to the alignment of mineralized collagen fibers in this tissue.
Graphical abstract
Highlights
► Assessment of the elastic anisotropy extent in mineralized collagen fibers. ► Two-directional nano- and microindentation of mineralized tissue. ► Effects of hydration state and indentation depth on the stiffness measured with two-directional indentation.
doi:10.1016/j.jmbbm.2012.03.004
PMCID: PMC3424420  PMID: 22664658
Mineralized turkey leg tendon (MTLT); Uniaxial mineralized collagen fibers; Anisotropy; Microindentation; Nanoindentation; Re-hydration; Indentation modulus; Indentation work
3.  COL1 C-propeptide Cleavage Site Mutations Cause High Bone Mass Osteogenesis Imperfecta 
Human mutation  2011;32(6):598-609.
Osteogenesis imperfecta (OI) is most often caused by mutations in the type I procollagen genes (COL1A1/COL1A2). We identified two children with substitutions in the type I procollagen C-propeptide cleavage site, which disrupt a unique processing step in collagen maturation and define a novel phenotype within OI. The patients have mild OI caused by mutations in COL1A1 (Patient 1: p.Asp1219Asn) or COL1A2 (Patient 2: p.Ala1119Thr), respectively. Patient 1 L1-L4 DXA z-score was +3.9 and pQCT vBMD was +3.1; Patient 2 had L1-L4 DXA z-score of 0.0 and pQCT vBMD of −1.8. Patient BMD contrasts with radiographic osteopenia and histomorphometry without osteosclerosis. Mutant procollagen processing is impaired in pericellular and in vitro assays. Patient dermal collagen fibrils have irregular borders. Incorporation of pC-collagen into matrix leads to increased bone mineralization. FT-IR imaging confirms elevated mineral/matrix ratios in both patients, along with increased collagen maturation in trabecular bone, compared to normal or OI controls. Bone mineralization density distribution revealed a marked shift toward increased mineralization density for both patients. Patient 1 has areas of higher and lower bone mineralization than controls; Patient 2’s bone matrix has a mineral content exceeding even classical OI bone. These patients define a new phenotype of high BMD OI and demonstrate that procollagen C-propeptide cleavage is crucial to normal bone mineralization.
doi:10.1002/humu.21475
PMCID: PMC3103631  PMID: 21344539
Osteogenesis imperfecta; C-propeptide; collagen; C-proteinase; mineralization; high bone mass
4.  Microcracks and Osteoclast Resorption Activity In Vitro 
Calcified Tissue International  2012;90(3):230-238.
During bone remodeling osteoclasts resorb bone, thus removing material, e.g., damaged by microcracks, which arises as a result of physiological loading and could reduce bone strength. Such a process needs targeted bone resorption exactly at damaged sites. Osteocytic signaling plays a key role in this process, but it is not excluded that osteoclasts per se may possess toposensitivity to recognize and resorb damaged bone since it has been shown that resorption spaces are associated with microcracks. To address this question, we used an in vitro setup of a pure osteoclast culture and mineralized substrates with artificially introduced microcracks and microscratches. Histomorphometric analyses and statistical evaluation clearly showed that these defects had no effect on osteoclast resorption behavior. Osteoclasts did not resorb along microcracks, even when resorption started right beside these damages. Furthermore, quantification of resorption on three different mineralized substrates, cortical bone, bleached bone (bone after partial removal of the organic matrix), and dentin, revealed lowest resorption on bone, significantly higher resorption on bleached bone, and highest resorption on dentin. The difference between native and bleached bone may be interpreted as an inhibitory impact of the organic matrix. However, the collagen-based matrix could not be the responsible part as resorption was highest on dentin, which contains collagen. It seems that osteocytic proteins, stored in bone but not present in dentin, affect osteoclastic action. This demonstrates that osteoclasts per se do not possess a toposensitivity to remove microcracks but may be influenced by components of the organic bone matrix.
doi:10.1007/s00223-011-9568-z
PMCID: PMC3282896  PMID: 22271249
Osteoclast; Microcrack; Mineralized tissue; Toposensitivity
5.  Assessment of chemical species of lead accumulated in tidemarks of human articular cartilage by X-ray absorption near-edge structure analysis 
Journal of Synchrotron Radiation  2011;18(Pt 2):238-244.
Lead is a toxic trace element that shows a highly specific accumulation in the transition zone between calcified and non-calcified articular cartilage, the so-called ‘tidemark’. Excellent agreement has been found between XANES spectra of synthetic Pb-doped carbonated hydroxyapatite and spectra obtained in the tidemark region and trabecular bone of normal human samples, confirming that in both tissues Pb is incorporated into the hydroxyapatite crystal structure of bone. During this study the µ-XANES set-up at the SUL-X beamline at ANKA was tested and has proven to be well suited for speciation of lead in human mineralized tissue samples.
A highly specific accumulation of the toxic element lead was recently measured in the transition zone between non-calcified and calcified normal human articular cartilage. This transition zone, the so-called ‘tidemark’, is considered to be an active calcification front of great clinical importance. However, little is known about the mechanisms of accumulation and the chemical form of Pb in calcified cartilage and bone. Using spatially resolved X-ray absorption near-edge structure analysis (µ-XANES) at the Pb L 3-edge, the chemical state of Pb in the osteochondral region was investigated. The feasibility of the µ-XANES set-up at the SUL-X beamline (ANKA synchrotron light source) was tested and confirmed by comparing XANES spectra of bulk Pb-reference compounds recorded at both the XAS and the SUL-X beamline at ANKA. The µ-XANES set-up was then used to investigate the tidemark region of human bone (two patella samples and one femoral head sample). The spectra recorded at the tidemark and at the trabecular bone were found to be highly correlated with the spectra of synthetic Pb-doped carbonated hydroxyapatite, suggesting that in both of these very different tissues Pb is incorporated into the hydroxyapatite structure.
doi:10.1107/S0909049510052040
PMCID: PMC3042330  PMID: 21335911
X-ray absorption spectroscopy; Pb L3-edge XANES; human bone; tidemark; trabecular bone

Results 1-5 (5)