Ex-vivo observation of calcification process in chick tibia slice: Augmented calcification along collagen fiber orientation in specimens subjected to static stretch

Bone formation through matrix synthesis and calcification in response to mechanical loading is an essential process of the maturation in immature animals, although how mechanical loading applied to the tissue increases the calcification and improves mechanical properties, and which directions the calcification progresses within the tissue are largely unknown. To address these issues, we investigated the calcification of immature chick bone under static tensile stretch using a newly developed real-time observation bioreactor system. Bone slices perpendicular to the longitudinal axis obtained from the tibia in 2- to 4-day-old chick legs were cultured in the system mounted on a microscope, and their calcification was observed up to 24 h while they were stretched in the direction parallel to the slice. Increase in the calcified area, traveling distance and the direction of the calcification and collagen fiber orientation in the newly calcified region were analyzed. There was a significant increase in calcified area in the bone explant subjected to tensile strain over ∼3%, which corresponds to the threshold strain for collagen fibers showing alignment in the direction of stretch, indicating that the fiber alignment may enhance tissue calcification. The calcification progressed to a greater distance to the stretching direction in the presence of the loading. Moreover, collagen fiber orientation in the calcified area in the loaded samples was coincided with the progression angle of the calcification. These results clearly show that the application of static tensile strain enhanced tissue calcification, which progresses along collagen fibers aligned to the loading direction.     

Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone tissue

Summary The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl₂MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl₂MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed.Using discriminant analysis the significant differences between all three studied groups of animals were found. The widest differences were found between the Cl₂MBP and HEBP treated animals. They were larger than those between each of these two groups and the control one. In control as well as in HEBP treated animals thicker bundles of collagen fibers were more frequently observed than in the Cl₂MBP treated group, while in the latter thin bundles, nondetected in the former two groups were found. Generally, the radial distribution of light intensity in diffraction patterns of the HEBP treated animals resembles more that in the control group than in the Cl₂MBP treated one. The different effects of the two analysed bisphosphonates (BPs) on the organic bone matrix of heterotopically induced bone is interpreted as differences in their influence on osteogenic cells and/or as differences in their direct influence on extracellular collagen fiber bundles formation.Dedicated to Professor Dr. T.H. Schiebler on the occasion of his 65th birthday     

Effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone tissue

The effect of 1-hydroxyethylidene-1,1-bisphosphonate (HEBP) and dichloromethylidene-bisphosphonate (Cl2MBP) on the structure of the organic matrix of heterotopically induced bone in guinea pig was studied. Heterotopic bone formation was induced by transplantation of allogenic urinary bladder epithelium. Starting from the day of transplantation the animals were treated subcutaneously with HEBP and Cl2MBP with a dose of 12.5 mg P/kg/day during 35 days. The control group was injected with 0.9% NaCl solution. The advantage of heterotopic bone induction as an experimental model is the fact that the applied drugs act on de novo bone formation. Collagen fibers were treated as markers of bone because their size and spatial arrangement reflect the structure and maturity of organic matrix of this tissue. Decalcified histological sections of induced bone, taken 35 days after implantation of inductor, were stained by the picrosirius method. This staining enhances the natural birefringency of collagen fibers and allows for better and specific visualization of collagen fibers bundles under polarizing microscope. In this way the amount of information in the analysed image is increased. Thirty five microphotographs were analysed from each of the investigated groups with the use of optical diffractometry. The radial distribution of light intensity in diffraction patterns was analysed what allowed to evaluate spatial frequencies connected with the width of collagen bundles in induced bone tissue. Since the spatial arrangement of collagen fibers in newly formed bone is random, analysis of angular distribution of light intensity in diffractograms was not performed. Using discriminant analysis the significant differences between all three studied groups of animals were found.(ABSTRACT TRUNCATED AT 250 WORDS)     

Immunolocalization of collagen types I and III, tenascin, and fibronectin in intramembranous bone.

Structural components of the organic bone matrix were located by immunohistochemical techniques in fresh-frozen sections of normal and dysplastic bone. Fine and coarse birefringent fibers were identified as separate and distinctive features in the extracellular matrix by antibodies raised against human collagen Type III. The glycoprotein tenascin was located on a proportion of the fibers in a characteristic beaded pattern, which was absent in dysplastic bone. The fibers originated in the periosteum or in the fibrous stroma of the marrow cavity and were oriented with regard to both the spatial and the lamellar organization of the bone. The disposition and composition of the fibers suggests that they form a preliminary framework on which intramembranous bone modeling proceeds, and that the specific location of tenascin on the fibers in normal developing membrane bone may be important in determining the alignment of the bone tissue. Epitopes recognized by the collagen Type I and fibronectin antibodies were demonstrated throughout the mineralized matrix, but their incorporation into the collagen "Type III" fibers was evident only outside the mineralized matrix.     

Tensile properties and fiber alignment of human supraspinatus tendon in the transverse direction demonstrate inhomogeneity,nonlinearity, and regional isotropy

A recent study (Lake et al., 2009); reported the properties of human supraspinatus tendon (SST) tested along the predominant fiber direction. The SST was found to have a relatively disperse distribution of collagen fibers, which may represent an adaptation to multiaxial loads imposed by the complex loading environment of the rotator cuff. However, the multiaxial mechanical properties of human SST remain unknown. The objective of this study, therefore, was to evaluate the mechanical properties, fiber alignment, change in alignment with applied load, and structure–function relationships of SST in transverse testing. Samples from six SST locations were tested in uniaxial tension with samples oriented transverse to the tendon long-axis. Polarized light imaging was used to quantify collagen fiber alignment and change in alignment under applied load. The mechanical properties of samples taken near the tendon–bone insertion were much greater on the bursal surface compared to the joint surface (e.g., bursal moduli 15–30 times greater than joint; p<0.001). In fact, the transverse moduli values of the bursal samples were very similar to values obtained from samples tested along the tendon long-axis (Lake et al., 2009). This key and unexpected finding suggests planar mechanical isotropy for bursal surface samples near the insertion, which may be due to complex in vivo loading. Organizationally, fiber distributions became less aligned along the tendon long-axis in the toe-region of the stress–strain response. Alignment changes occurred to a slightly lesser degree in the linear-region, suggesting that movement of collagen fibers may play a role in mechanical nonlinearity. Transverse mechanical properties were significantly correlated with fiber alignment (e.g., for linear-region modulus r_s=0.74, p<0.0001), demonstrating strong structure–function relationships. These results greatly enhance current understanding of the properties of human SST and provide clinicians and scientists with vital information in attempting to treat or replace this complex tissue.     

Monitoring dynamic collagen reorganization during skin stretching with fast polarization‐resolved second harmonic generation imaging

The mechanical properties of biological tissues are strongly correlated to the specific distribution of their collagen fibers. Monitoring the dynamic reorganization of the collagen network during mechanical stretching is however a technical challenge, because it requires mapping orientation of collagen fibers in a thick and deforming sample. In this work, a fast polarization‐resolved second harmonic generation microscope is implemented to map collagen orientation during mechanical assays. This system is based on line‐to‐line switching of polarization using an electro‐optical modulator and works in epi‐detection geometry. After proper calibration, it successfully highlights the collagen dynamic alignment along the traction direction in ex vivo murine skin dermis. This microstructure reorganization is quantified by the entropy of the collagen orientation distribution as a function of the stretch ratio. It exhibits a linear behavior, whose slope is measured with a good accuracy. This approach can be generalized to probe a variety of dynamic processes in thick tissues.      

The susceptibility of pure tubulin to high magnetic fields: a magnetic birefringence and x-ray fiber diffraction study.

The orientational behavior of microtubules assembled in strong magnetic fields has been studied. It is shown that when microtubules are assembled in a magnetic field, they align with their long axis parallel to the magnetic field. The effect of several parameters known to affect the microtubule assembly are investigated with respect to their effect on the final degree of alignment. Aligned samples of hydrated microtubules suitable for low-resolution x-ray fiber diffraction experiments have been produced, and the results obtained from the fiber diffraction experiments have been compared with the magnetic birefringence experiments. Comparisons with earlier fiber diffraction work and small-angle x-ray solution scattering experiments have been made.     

Quick Shear-Flow Alignment of Biological Filaments for X-ray Fiber Diffraction Facilitated by Methylcellulose

X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s⁻¹) to the medium and that methylcellulose contained in the medium (∼1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5°). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.     

Biomaterial-loaded allograft threaded cage for the treatment of femoral head osteonecrosis in a goat model

The purpose of this study was to evaluate the efficacy of core decompression with a biomaterial-loaded allograft threaded cage (ATC) for the treatment of femoral head osteonecrosis in an established goat model. First, bilateral early-stage osteonecrosis was induced. After core decompression, the remaining goats were randomly divided into three groups: Group A, the goats were left without any treatment; Group B, the goats were treated with implanting a composite of autologous bone and decalcified bone matrix (DBM); Group C, the goats were treated using insertion of ATC loaded with DBM and autogenous bone graft. Then radiographic, histological and biomechanical analysis were taken in each group at 5, 10, and 20 weeks postoperation. In Group A, the classical signs of osteonecrosis of the femoral head were identified 10 weeks after the induction. Twenty weeks later, the density, surface and biomechanical stability of the femoral head were normal in Group C, while an irregular surface and an inhomogeneous microstructure or variation of density/hardness were identified in Group B. The specimens revealed a continuous trabaecular bone structure throughout the cage and extensive bone ingrowth and remodeling in Group C, while fibrous tissue was evident in Group B. Core decompression with a biomaterial loaded ATC almost uniformly delays or arrests the progression of the disease before articular collapse, and it could help to get the balance between bone resorption and new bone formation, strengthen structural mechanics of the femoral head, provide structure support of articular cartilage.     

Hydroxyapatite-Sheet Parallel Microstructure of Shinbone

Bone is a natural biomaterial. It behaves favorable strength, stiffness and fracture toughness, which are closely related to its eximious microstructure. Scanning Electron Microscope (SEM) observation on a shinbone showed that the bone is a bioceramic composite consisting of laminated hydroxyapatite and collagen matrix. The hydroxyapatite layers are parallel with the surface of the bone and consist of long and thin hydroxyapatite sheets. The observation also showed that the hydroxyapatite sheets in different hydroxyapatite layers also parallel with each other, which composes a hydroxyapatite-sheet parallel microstructure. The maximum pullout energy of the parallel microstructure was investigated based on its representative model. It was shown that the long and thin shape of the hydroxyapatite sheets in the parallel microstructure is profitable to increase the maximum pullout energy and enhance the fracture toughness of the bone.     

Rapid Quantification of 3D Collagen Fiber Alignment and Fiber Intersection Correlations with High Sensitivity

Metastatic cancers aggressively reorganize collagen in their microenvironment. For example, radially orientated collagen fibers have been observed surrounding tumor cell clusters in vivo. The degree of fiber alignment, as a consequence of this remodeling, has often been difficult to quantify. In this paper, we present an easy to implement algorithm for accurate detection of collagen fiber orientation in a rapid pixel-wise manner. This algorithm quantifies the alignment of both computer generated and actual collagen fiber networks of varying degrees of alignment within 5°°. We also present an alternative easy method to calculate the alignment index directly from the standard deviation of fiber orientation. Using this quantitative method for determining collagen alignment, we demonstrate that the number of collagen fiber intersections has a negative correlation with the degree of fiber alignment. This decrease in intersections of aligned fibers could explain why cells move more rapidly along aligned fibers than unaligned fibers, as previously reported. Overall, our paper provides an easier, more quantitative and quicker way to quantify fiber orientation and alignment, and presents a platform in studying effects of matrix and cellular properties on fiber alignment in complex 3D environments.     

Long-Range Force Transmission in Fibrous Matrices Enabled by Tension-Driven Alignment of Fibers

Cells can sense and respond to mechanical signals over relatively long distances across fibrous extracellular matrices. Recently proposed models suggest that long-range force transmission can be attributed to the nonlinear elasticity or fibrous nature of collagen matrices, yet the mechanism whereby fibers align remains unknown. Moreover, cell shape and anisotropy of cellular contraction are not considered in existing models, although recent experiments have shown that they play crucial roles. Here, we explore all of the key factors that influence long-range force transmission in cell-populated collagen matrices: alignment of collagen fibers, responses to applied force, strain stiffening properties of the aligned fibers, aspect ratios of the cells, and the polarization of cellular contraction. A constitutive law accounting for mechanically driven collagen fiber reorientation is proposed. We systematically investigate the range of collagen-fiber alignment using both finite-element simulations and analytical calculations. Our results show that tension-driven collagen-fiber alignment plays a crucial role in force transmission. Small critical stretch for fiber alignment, large fiber stiffness and fiber strain-hardening behavior enable long-range interaction. Furthermore, the range of collagen-fiber alignment for elliptical cells with polarized contraction is much larger than that for spherical cells with diagonal contraction. A phase diagram showing the range of force transmission as a function of cell shape and polarization and matrix properties is presented. Our results are in good agreement with recent experiments, and highlight the factors that influence long-range force transmission, in particular tension-driven alignment of fibers. Our work has important relevance to biological processes including development, cancer metastasis, and wound healing, suggesting conditions whereby cells communicate over long distances.     

A comparative study of the effect of cholesterol on bicelle model membranes using X-band and Q-band EPR spectroscopy

X-band and Q-band electron paramagnetic resonance (EPR) spectroscopic techniques were used to investigate the structure and dynamics of cholesterol containing phospholipid bicelles based upon molecular order parameters (S_mol), orientational dependent hyperfine splittings and line shape analysis of the corresponding EPR spectra. The nitroxide spin-label 3-β-doxyl-5-α-cholestane (cholestane) was incorporated into DMPC/DHPC bicelles to report the alignment of bicelles in the static magnetic field. The influence of cholesterol on aligned phospholipid bicelles in terms of ordering, the ease of alignment, phase transition temperature have been studied comparatively at X-band and Q-band. At a magnetic field of 1.25 T (Q-band), bicelles with 20 mol% cholesterol aligned at a much lower temperature (313 K), when compared to 318 K at a 0.35 T field strength for X-band, showed better hyperfine splitting values (18.29 G at X-band vs. 18.55 G at Q-band for perpendicular alignment and 8.25 G at X-band vs. 7.83 G at Q-band for the parallel alignment at 318 K) and have greater molecular order parameters (0.76 at X-band vs. 0.86 at Q-band at 318 K). Increasing cholesterol content increased the bicelle ordering, the bicelle-alignment temperature and the gel to liquid crystalline phase transition temperature. We observed that Q-band is more effective than X-band for studying aligned bicelles, because it yielded a higher ordered bicelle system for EPR spectroscopic studies.     

Imaging of Scleral Collagen Deformation Using Combined Confocal Raman Microspectroscopy and Polarized Light Microscopy Techniques

This work presents an optospectroscopic characterization technique for soft tissue microstructure using site-matched confocal Raman microspectroscopy and polarized light microscopy. Using the technique, the microstructure of soft tissue samples is directly observed by polarized light microscopy during loading while spatially correlated spectroscopic information is extracted from the same plane, verifying the orientation and arrangement of the collagen fibers. Results show the response and orientation of the collagen fiber arrangement in its native state as well as during tensile and compressive loadings in a porcine sclera model. An example is also given showing how the data can be used with a finite element program to estimate the strain in individual collagen fibers. The measurements demonstrate features that indicate microstructural reorganization and damage of the sclera’s collagen fiber arrangement under loading. The site-matched confocal Raman microspectroscopic characterization of the tissue provides a qualitative measure to relate the change in fibrillar arrangement with possible chemical damage to the collagen microstructure. Tests and analyses presented here can potentially be used to determine the stress-strain behavior, and fiber reorganization of the collagen microstructure in soft tissue during viscoelastic response.     

Influence of osteocalcin and collagen I on the mechanical and biological properties of Biocement D

In order to generate a calcium-phosphate bone cement as a transient replacement for bone defects, we modified Biocement D (Merck Biomaterial GmbH) containing mineralised collagen with osteocalcin, the most abundant non-collageneous protein of bone. Osteocalcin was added to the cement paste during setting in order to control the crystallisation kinetics of hydroxyapatite (HAP) as well as to stimulate the interaction of osteoblasts and osteoclasts with the bone replacement material. Analysis by SEM and AFM shows, that the addition of osteocalcin causes a nanosize microstructure of the calcium cement, which can be explained by inhibited growth of HAP crystals. The fracture strength of the material decreased by incorporation of osteocalcin, pointing onto a higher defect concentration of the crystalline structure. The impact of osteocalcin onto the interaction of bone cells with HAP-Collagen I-cements was studied in a cell culture system using the human osteosarcoma cell line SAOS-2. Results suggest, that osteocalcin might possibly improve the initial adherence of osteoblast-like cells, whereas proliferation of the cells is not effected.     

Collagen fiber orientation at the tendon to bone insertion and its influence on stress concentrations

The tendon to bone insertion serves the mechanical role of transferring loads from a relatively compliant tendon to a relatively rigid bone. The details of the mechanism of load transfer are of great importance, since current surgical procedures for tendon reattachment have high failure rates. We hypothesized that the microscopic structure of the insertion is optimized to minimize stress concentrations associated with this load transfer. To explore this, collagen fiber orientation distributions were measured in the supraspinatus tendons of rats. The angular deviation of fibers was fairly uniform across the insertion, and the mean angles of the local distributions deviated mildly from the tendon axis. To explore how these observed property distributions could influence load transfer, these distributions were used to derive material properties for an idealized two-dimensional mechanical model of an insertion. Comparison between stress concentrations in this idealized model and those in three comparison models suggests that the microstructure serves to (1) simultaneously reduce stress concentrations and material mass, and (2) shield the insertion's outward splay from the highest stresses.     

Elastic properties of woven bone: effect of mineral content and collagen fibrils orientation

Woven bone is a type of tissue that forms mainly during fracture healing or fetal bone development. Its microstructure can be modeled as a composite with a matrix of mineral (hydroxyapatite) and inclusions of collagen fibrils with a more or less random orientation. In the present study, its elastic properties were estimated as a function of composition (degree of mineralization) and fibril orientation. A self-consistent homogenization scheme considering randomness of inclusions’ orientation was used for this purpose. Lacuno-canalicular porosity in the form of periodically distributed void inclusions was also considered. Assuming collagen fibrils to be uniformly oriented in all directions led to an isotropic tissue with a Young’s modulus \(E = 1.90\) GPa, which is of the same order of magnitude as that of woven bone in fracture calluses. By contrast, assuming fibrils to have a preferential orientation resulted in a Young’s modulus in the preferential direction of 9–16 GPa depending on the mineral content of the tissue. These results are consistent with experimental evidence for woven bone in foetuses, where collagen fibrils are aligned to a certain extent.     

Microstructural densification and alignment by aspiration-ejection influence cancer cell interactions with three-dimensional collagen networks

Extracellular matrix microstructure and mechanics are crucial to breast cancer progression and invasion into surrounding tissues. The peritumor collagen network is often dense and aligned, features which in vitro models lack. Aspiration of collagen hydrogels led to densification and alignment of microstructure surrounding embedded cancer cells. Two metastasis-derived breast cancer cell lines, MDA-MB-231 and MCF-7, were cultured in initially 4 mg/ml collagen gels for 3 days after aspiration, as well as in unaspirated control hydrogels. Videomicroscopy during aspiration, and at 0, 1, and 3 days after aspiration, epifluorescence microscopy of phalloidin-stained F-actin cytoskeleton, histological sections, and soluble metabolic byproducts from constructs were collected to characterize effects on the embedded cell morphology, the collagen network microstructure, and proliferation. Breast cancer cells remained viable after aspiration-ejection, proliferating slightly less than in unaspirated gels. Furthermore, MDA-MB-231 cells appear to partially relax the collagen network and lose alignment 3 days after aspiration. Aspiration-ejection generated aligned, compact collagen network microstructure with immediate cell co-orientation and higher cell number density apparently through purely physical means, though cell-collagen contact guidance and network remodeling influence cell organization and collagen network microstructure during subsequent culture. This study establishes a platform to determine the effects of collagen density and alignment on cancer cell behavior, with translational potential for anticancer drug screening in a biomimetic three-dimensional matrix microenvironment, or implantation in preclinical models.     

Multiscale,Converging Defects of Macro-Porosity,Microstructure and Matrix Mineralization Impact Long Bone Fragility in NF1

Bone fragility due to osteopenia, osteoporosis or debilitating focal skeletal dysplasias is a frequent observation in the Mendelian disease Neurofibromatosis type 1 (NF1). To determine the mechanisms underlying bone fragility in NF1 we analyzed two conditional mouse models, Nf1Prx1 (limb knock-out) and Nf1Col1 (osteoblast specific knock-out), as well as cortical bone samples from individuals with NF1. We examined mouse bone tissue with micro-computed tomography, qualitative and quantitative histology, mechanical tensile analysis, small-angle X-ray scattering (SAXS), energy dispersive X-ray spectroscopy (EDX), and scanning acoustic microscopy (SAM). In cortical bone of Nf1Prx1 mice we detected ectopic blood vessels that were associated with diaphyseal mineralization defects. Defective mineral binding in the proximity of blood vessels was most likely due to impaired bone collagen formation, as these areas were completely devoid of acidic matrix proteins and contained thin collagen fibers. Additionally, we found significantly reduced mechanical strength of the bone material, which was partially caused by increased osteocyte volume. Consistent with these observations, bone samples from individuals with NF1 and tibial dysplasia showed increased osteocyte lacuna volume. Reduced mechanical properties were associated with diminished matrix stiffness, as determined by SAM. In line with these observations, bone tissue from individuals with NF1 and tibial dysplasia showed heterogeneous mineralization and reduced collagen fiber thickness and packaging. Collectively, the data indicate that bone fragility in NF1 tibial dysplasia is partly due to an increased osteocyte-related micro-porosity, hypomineralization, a generalized defect of organic matrix formation, exacerbated in the regions of tensional and bending force integration, and finally persistence of ectopic blood vessels associated with localized macro-porotic bone lesions.     

Dietary pseudopurpurin improves bone geometry architecture and metabolism in red-bone Guishan goats

Red-colored bones were found initially in some Guishan goats in the 1980s, and they were designated red-boned goats. However, it is not understood what causes the red color in the bone, or whether the red material changes the bone geometry, architecture, and metabolism of red-boned goats. Pseudopurpurin was identified in the red-colored material of the bone in red-boned goats by high-performance liquid chromatography-electrospray ionization-mass spetrometry and nuclear magnetic resonance analysis. Pseudopurpurin is one of the main constituents of Rubia cordifolia L, which is eaten by the goats. The assessment of the mechanical properties and micro-computed tomography showed that the red-boned goats displayed an increase in the trabecular volume fraction, trabecular thickness, and the number of trabeculae in the distal femur. The mean thickness, inner perimeter, outer perimeter, and area of the femoral diaphysis were also increased. In addition, the trabecular separation and structure model index of the distal femur were decreased, but the bone mineral density of the whole femur and the mechanical properties of the femoral diaphysis were enhanced in the red-boned goats. Meanwhile, expression of alkaline phosphatase and osteocalcin mRNA was higher, and the ratio of the receptor activator of the nuclear factor kappa B ligand to osteoprotegerin was markedly lower in the bone marrow of the red-boned goats compared with common goats. To confirm further the effect of pseudopurpurin on bone geometry, architecture, and metabolism, Wistar rats were fed diets to which pseudopurpurin was added for 5 months. Similar changes were observed in the femurs of the treated rats. The above results demonstrate that pseudopurpurin has a close affinity with the mineral salts of bone, and consequently a high level of mineral salts in the bone cause an improvement in bone strength and an enhancement in the structure and metabolic functions of the bone.