Quantitative Ultrasound Assessment of Cortical Bone Properties Beyond Bone Mineral Density

The development of quantitative ultrasound (QUS) technologies to measure bone is motivated by the need to overcome the limitations of X-ray based methods, measuring bone mineral density (BMD) which is the gold standard to date for the diagnosis of osteoporosis. Because it uses mechanical waves, the ultrasound modality is a particularly relevant means to probe bone mechanical resistance. The vast majority of QUS technologies commercialized to date merely aim to provide surrogate markers for BMD. During the past decade, innovative QUS approaches have emerged to assess bone beyond BMD. This may be achieved by (1) specifically assessing the cortical bone compartment, independently of trabecular bone, and (2) providing intrinsic bone properties such as cortical bone thickness and material properties. One specific motivation is to estimate intracortical porosity, a quantity reflected in material properties. This article aims at an overview of recent QUS developments to measure cortical bone properties. We also draw a picture of the current knowledge on bone material properties of interest for bone QUS. We discuss the potential of ultrasound to provide novel biomarkers of bone health through the assessment of material properties.     

Comparative Mechanical Properties and Histology of Bone

Different bone tissues differ in their amounts of porosity,mineralization,reconstruction, and preferred orientation. Allthese have important effects on mechanical properties. Veryporous, cancellous bone is always weaker and morecompliant thancompact bone on a weight for weight basis, yet it occurs inplaceswhere its energyabsorbing ability, or its low density,is advantageous. Bonevaries considerably in its mineralization,and such variations have quite disproportionate effects on mechanicalproperties. These variations can be shown to be adaptive. Inparticular, there must always be a compromise between stiffnessandresistanceto fracture; these two properties run contrary to each other.The reason for secondary remodeling is an unresolved problem,though in a few places the role of such remodeling in changingthe grain of the bone is clearly mechanically adaptive. Themechanical properties of non-mammalian bone are obscure, andas the histology of such bone is often quite different fromthat of mammalian bone, we are no doubt in for some surpriseswhen the mechanical properties ofnonmammalian bone are discovered.     

Non-invasive Predictors of Human Cortical Bone Mechanical Properties: T2-Discriminated 1H NMR Compared with High Resolution X-ray

Recent advancements in magnetic resonance imaging (MRI) have enabled clinical imaging of human cortical bone, providing a potentially powerful new means for assessing bone health with molecular-scale sensitivities unavailable to conventional X-ray-based diagnostics. To this end, ¹H nuclear magnetic resonance (NMR) and high-resolution X-ray signals from human cortical bone samples were correlated with mechanical properties of bone. Results showed that ¹H NMR signals were better predictors of yield stress, peak stress, and pre-yield toughness than were the X-ray derived signals. These ¹H NMR signals can, in principle, be extracted from clinical MRI, thus offering the potential for improved clinical assessment of fracture risk.     

Collagenase-Released Non-Collagenous Proteins of Cortical Bone Matrix

Two distinct groups of non-collagenous components were isolated from rat cortical bone gelatin which had previously been digested with purified bacterial col-lagenase. One component was disulfide-bonded, strongly acidic, trypsin-labile glycoprotein aggregate with a molecular mass of more than 100, 000 daltons. When reduced with β-mercaptoethanol this protein disaggregated into subunits with a molecular mass of about 60, 000 daltons. The other components consisted of a group of polypeptides with a molecular mass of about 5, 000 daltons. The latter group was present in collagenase digests prepared from normal bone gelatin but was hardly detectable or absent in digests of gelatin prepared from either autolyzed, trypsinized or lathyritic bone, or from the residue of neutral salt extracted rat tail tendon.

A recently discovered group of non-collagenous proteins is tightly bound to the highly crosslinked insoluble structure of collagen fibrils of bone and dentin. Dische et al ¹ predicted the existence of these proteins in bone by analyzing the products of KOH hydrolysis of the EDTA-insoluble residues of cortical bone collagen. Comparable products were separated following digestion of bone matrix with collagenase by Herring², and oxidation of dentin matrix by alkaline sodium metaperiodate by Shuttleworth and Veis.³ Leaver et al ⁴ proposed the name collagenase-released proteins (CRP) for non-collagenous proteins obtained from the EDTA-insoluble residue of bone and dentin. There are at least two limitations in information about CRP in the above-cited reports. The first is that the collagenases were not certified to be protease-free. The second is that EDTA-insoluble residues of cortical bone were only partly digestable by bacterial collagenase. We report here isolation and partial characterization of CRP from a special preparation of bone matrix gelatin which is quantitatively digested by a protease-free bacterial collagenase-gelatinase purified by the method of Peterkofsky and Diegelmann.^{5, 6}     

Changes in the Mechanical Properties and Composition of Bone during Microdamage Repair

Under normal conditions, loading activities result in microdamage in the living skeleton, which is repaired by bone remodeling. However, microdamage accumulation can affect the mechanical properties of bone and increase the risk of fracture. This study aimed to determine the effect of microdamage on the mechanical properties and composition of bone. Fourteen male goats aged 28 months were used in the present study. Cortical bone screws were placed in the tibiae to induce microdamage around the implant. The goats were euthanized, and 3 bone segments with the screws in each goat were removed at 0 days, 21 days, 4 months, and 8 months after implantation. The bone segments were used for observing microdamage and bone remodeling, as well as nanoindentation and bone composition, separately. Two regions were measured: the first region (R1), located 1.5 mm from the interface between the screw hole and bone; and the second region (R2), located>1.5 mm from the bone-screw interface. Both diffuse and linear microdamage decreased significantly with increasing time after surgery, with the diffuse microdamage disappearing after 8 months. Thus, screw implantation results in increased bone remodeling either in the proximal or distal cortical bone, which repairs the microdamage. Moreover, bone hardness and elastic modulus decreased with microdamage repair, especially in the proximal bone tissue. Bone composition changed greatly during the production and repair of microdamage, especially for the C (Carbon) and Ca (Calcium) in the R1 region. In conclusion, the presence of mechanical microdamage accelerates bone remodeling either in the proximal or distal cortical bone. The bone hardness and elastic modulus decreased with microdamage repair, with the micromechanical properties being restored on complete repair of the microdamage. Changes in bone composition may contribute to changes in bone mechanical properties.     

Effects of Hydrogen Peroxide Cleaning Procedures on Bone Graft Osteoinductivity and Mechanical Properties

Bone allografts are frequently used during orthopaedic trauma cases or other reconstructive procedures. Most allografts are processed and cleaned before use. Our goals were to determine if an improved cleaning procedure compromises the strength or osteoinductivity of a graft. We compared our improved cleaning procedure to our standard cleaning procedure on cortical bone allograft. The cleaning procedures are generally composed of a series of chemical steps with nonionic detergents, hydrogen peroxide, and alcohol under time and temperature control, subjected to ultrasonic agitation. We tested the compressive strength, impact strength, and shear strength following the standard and improved cleaning procedures. Osteoinductivity was tested in 4 groups, using the improved cleaning procedure with four different hydrogen peroxide cleaning times: 0, 1, 3, and 5 h. Osteoinductivity was evaluated in vivo, using a 28-day implant in the hamstring muscle of an athymic, nude mouse. Results demonstrated that osteoinductivity is maintained with cleaning in hydrogen peroxide for up to 1 h, and that compressive strength, impact strength, and shear strength were all unaffected by the improved cleaning procedure. The improved cleaning procedure therefore did not compromise the strength or osteoinductivity of cortical bone allografts in comparison to the standard procedure.     

Influence of Nano-HA Coated Bone Collagen to Acrylic (Polymethylmethacrylate) Bone Cement on Mechanical Properties and Bioactivity

Objective

This research investigated the mechanical properties and bioactivity of polymethylmethacrylate (PMMA) bone cement after addition of the nano-hydroxyapatite(HA) coated bone collagen (mineralized collagen, MC).

Materials & Methods

The MC in different proportions were added to the PMMA bone cement to detect the compressive strength, compression modulus, coagulation properties and biosafety. The MC-PMMA was embedded into rabbits and co-cultured with MG 63 cells to exam bone tissue compatibility and gene expression of osteogenesis.

Results

15.0%(wt) impregnated MC-PMMA significantly lowered compressive modulus while little affected compressive strength and solidification. MC-PMMA bone cement was biologically safe and indicated excellent bone tissue compatibility. The bone-cement interface crosslinking was significantly higher in MC-PMMA than control after 6 months implantation in the femur of rabbits. The genes of osteogenesis exhibited significantly higher expression level in MC-PMMA.

Conclusions

MC-PMMA presented perfect mechanical properties, good biosafety and excellent biocompatibility with bone tissues, which has profoundly clinical values.     

Quantitative Evaluation of Collagen Crosslinks and Corresponding Tensile Mechanical Properties in Mouse Cervical Tissue during Normal Pregnancy

The changes in the mechanical integrity of the cervix during pregnancy have implications for a successful delivery. Cervical collagens are known to remodel extensively in mice with progressing gestation leading to a soft cervix at term. During this process, mature crosslinked collagens are hypothesized to be replaced with immature less crosslinked collagens to facilitate cervical softening and ripening. To determine the mechanical role of collagen crosslinks during normal mouse cervical remodeling, tensile load-to-break tests were conducted for the following time points: nonpregnant (NP), gestation day (d) 6, 12, 15, 18 and 24 hr postpartum (PP) of the 19-day gestation period. Immature crosslinks (HLNL and DHLNL) and mature crosslinks (DPD and PYD) were measured using ultra performance liquid chromatography-electrospray ionization tandem mass spectrometry (UPLC-ESI-MS/MS). There were no significant changes in the total immature crosslink density (HLNL+DHLNL mol per collagen mol) throughout normal mouse gestation (range: 0.31–0.49). Total mature crosslink density (PYD+DPD mol per collagen mol) decreased significantly in early softening from d6 to d15 (d6: 0.17, d12: 0.097, d15: 0.026) and did not decrease with further gestation. The maturity ratio (total mature to total immature crosslinks) significantly decreased in early softening from d6 to d15 (d6: 0.2, d15: 0.074). All of the measured crosslinks correlated significantly with a measure of tissue stiffness and strength, with the exception of the immature crosslink HLNL. This data provides quantitative evidence to support the hypothesis that as mature crosslinked collagens decline, they are replaced by immature collagens to facilitate increased tissue compliance in the early softening period from d6 to d15.     

A Three-Dimensional Finite Element Scheme to Investigate the Apparent Mechanical Properties of Trabecular Bone

A computational technique is described for investigating the apparent mechanical properties of trabecular bone based on tissue geometry obtained from the marching cubes volume rendering scheme. Using this scheme, a 3D representation of the trabecular bone was extracted from two-dimensional cross-sections of the tissue originating from a quantitative serial sectioning procedure. Surface information consists of node coordinates and polygon connectivity in a 3D space. A custom, adaptive mesh generation technique using a normal offset was used to prepare 3D finite element volume meshes (4-node tetrahedral elements) of variable mesh density from the extracted surface geometry. Nine target mesh resolutions (32 μm to 107 μm) were examined for a (1.5 mmx 1.5 mmx 2 mm) volume of trabecular bone. A mesh density of 50,000 elements/mm(3) of bone tissue was found to be adequate for convergence of apparent (bulk) modulus for 1% uniaxial compression. For this convergent case, the maximum local normal compressive tissue stress was 400 MPa which was six hundred-fold greater than the computed apparent stress. Variation in the apparent modulus was less than 5% when Poisson's ratio values were varied between 0.1 and 0.4. Poisson's ratio values greater than 0.4 had a more marked effect on the apparent modulus. Based upon these results, approximately 1 million, 4-node tetrahedral elements are required to analyze a continuum scale model of trabecular bone (5 mm cube).     

The Changing Pore Structure of Aging Human Mandibular Bone1

The spatial structure of human mandibular bone has been studied using microradiography, scanning electron microscopy, and methacrylate replication. Microradiographs have demonstrated lamellar organization and mineralization. Methacrylate replicas have been examined by scanning electron microscopy which revealed the complex structure and inter-connection of the vascular, lacunar, and canalicular spaces. The spatial replicas, representing the pore volume, have been dispersed ultrasonically and separated by sedimentation in a fluid column. The harvested methacrylate replicas of the three types of space have been weighed and knowing the density of methacrylate (1.18g/ml) their volumes determined by calculation. Pore volume increases with age. The vascular space makes the greatest contribution to this increasing porosity with age. Although lacunar volume increases with age, their numbers may fall. Canalicular volume appears to fall with age. This method has provided more information about the contribution the three different spatial compartments make to the increasing porosity of aging bone. There is no difference in the volume of these compartments in alveolar and basal mandibular bone but their distribution indicates that some of the smaller channels are more readily obstructed in alveolar bone.     

Potential of Magnetic Nanofiber Scaffolds with Mechanical and Biological Properties Applicable for Bone Regeneration

Magnetic nanofibrous scaffolds of poly(caprolactone) (PCL) incorporating magnetic nanoparticles (MNP) were produced, and their effects on physico-chemical, mechanical and biological properties were extensively addressed to find efficacy for bone regeneration purpose. MNPs 12 nm in diameter were citrated and evenly distributed in PCL solutions up to 20% and then were electrospun into nonwoven nanofibrous webs. Incorporation of MNPs greatly improved the hydrophilicity of the nanofibers. Tensile mechanical properties of the nanofibers (tensile strength, yield strength, elastic modulus and elongation) were significantly enhanced with the addition of MNPs up to 15%. In particular, the tensile strength increase was as high as ∼25 MPa at 15% MNPs vs. ∼10 MPa in pure PCL. PCL-MNP nanofibers exhibited magnetic behaviors, with a high saturation point and hysteresis loop area, which increased gradually with MNP content. The incorporation of MNPs substantially increased the degradation of the nanofibers, with a weight loss of ∼20% in pure PCL, ∼45% in 10% MNPs and ∼60% in 20% MNPs. Apatite forming ability of the nanofibers tested in vitro in simulated body fluid confirmed the substantial improvement gained by the addition of MNPs. Osteoblastic cells favored the MNPs-incorporated nanofibers with significantly improved initial cell adhesion and subsequent penetration through the nanofibers, compared to pure PCL. Alkaline phosphatase activity and expression of genes associated with bone (collagen I, osteopontin and bone sialoprotein) were significantly up-regulated in cells cultured on PCL-MNP nanofibers than those on pure PCL. PCL-MNP nanofibers subcutaneously implanted in rats exhibited minimal adverse tissue reactions, while inducing substantial neoblood vessel formation, which however, greatly limited in pure PCL. In vivo study in radial segmental defects also signified the bone regeneration ability of the PCL-MNP nanofibrous scaffolds. The magnetic, bone-bioactive, mechanical, cellular and tissue attributes of MNP-incorporated PCL nanofibers make them promising candidate scaffolds for bone regeneration.     

Interfacial Strength of Cement Lines in Human Cortical Bone

In human cortical bone, cement lines (or reversal lines) separate osteons from the interstitial bone tissue, which consists of remnants of primary lamellar bone or fragments of remodeled osteons. There have been experimental evidences of the cement line involvement in the failure process of bone such as fatigue and damage. However, there are almost no experimental data on interfacial properties of cement lines in human cortical bone. The objective of this study is to design and assemble a precision and computer controlled osteon pushout microtesting system, and to experimentally determine the interfacial strength of cement lines in human cortical bone by performing osteon pushout tests. Thirty specimens were prepared from humeral diaphyses of four human subjects. Twenty specimens were tested under the condition of a small hole in the supporting plate, in which the cement line debonding occurred. The cement line interfacial strength ranged from 5.38 MPa to 10.85 MPa with an average of 7.31±1.73 MPa. On the other hand, ten specimens were tested under the condition of a large hole in the supporting plate, in which the shear failure inside osteons was observed. The specimens tested under the condition of the large hole resulted in an average shear strength of 73.71±15.06 MPa, ranging from 45.97 MPa to 93.74 MPa. Therefore, our results suggest that the cement line interface between osteon and interstitial bone tissue is weaker than that between bone tissue lamellae.     

Bone Balance within a Cortical BMU: Local Controls of Bone Resorption and Formation

Maintaining bone volume during bone turnover by a BMU is known as bone balance. Balance is required to maintain structural integrity of the bone and is often dysregulated in disease. Consequently, understanding how a BMU controls bone balance is of considerable interest. This paper develops a methodology for identifying potential balance controls within a single cortical BMU. The theoretical framework developed offers the possibility of a directed search for biological processes compatible with the constraints of balance control. We first derive general control constraint equations and then introduce constitutive equations to identify potential control processes that link key variables that describe the state of the BMU. The paper describes specific local bone volume balance controls that may be associated with bone resorption and bone formation. Because bone resorption and formation both involve averaging over time, short-term fluctuations in the environment are removed, leaving the control systems to manage deviations in longer-term trends back towards their desired values. The length of time for averaging is much greater for bone formation than for bone resorption, which enables more filtering of variability in the bone formation environment. Remarkably, the duration for averaging of bone formation may also grow to control deviations in long-term trends of bone formation. Providing there is sufficient bone formation capacity by osteoblasts, this leads to an extraordinarily robust control mechanism that is independent of either osteoblast number or the cellular osteoid formation rate. A complex picture begins to emerge for the control of bone volume. Different control relationships may achieve the same objective, and the 'integration of information' occurring within a BMU may be interpreted as different sets of BMU control systems coming to the fore as different information is supplied to the BMU, which in turn leads to different observable BMU behaviors.     

Cement Line Staining in Undecalcified Thin Sections of Cortical Bone

A technique for demonstrating cement lines in thin, undecalcified transverse sections of cortical bone has been developed. Cortical bone samples are processed and embedded undecalcified in methyl methacrylate plastic. After sectioning at 3-5 μm, cross-sections are transferred to a glass slide and flattened for 10 min. Sections of cortical bone are stained for 20 sec free-floating in a fresh solution of 1% toluidine blue dissolved in 0.1% formic acid. The section is dehydrated in t-butyl alcohol, cleared in xylene, and mounted with Eukitt's medium. Reversal lines appear as thin, scalloped, dark blue lines against a light blue matrix, whereas bone formation arrest lines are thicker with a smooth contour. With this technique cellular detail, osteoid differentiation, and fluorochrome labels are retained. Results demonstrate the applicability of a one-step staining method for cement lines which will facilitate the assessment of bone remodeling activity in thin sections of undecalcified cortical bone.     

The Effect of Naturally Occurring Chronic Kidney Disease on the Micro-Structural and Mechanical Properties of Bone

Chronic kidney disease (CKD) is a growing public health concern worldwide, and is associated with marked increase of bone fragility. Previous studies assessing the effect of CKD on bone quality were based on biopsies from human patients or on laboratory animal models. Such studies provide information of limited relevance due to the small size of the samples (biopsies) or the non-physiologic CKD syndrome studied (rodent models with artificially induced CKD). Furthermore, the type, architecture, structure and biology of the bone of rodents are remarkably different from human bones; therefore similar clinicopathologic circumstances may affect their bones differently. We describe the effects of naturally occurring CKD with features resembling human CKD on the skeleton of cats, whose bone biology, structure and composition are remarkably similar to those of humans. We show that CKD causes significant increase of resorption cavity density compared with healthy controls, as well as significantly lower cortical mineral density, cortical cross-sectional area and cortical cross-sectional thickness. Young''s modulus, yield stress, and ultimate stress of the cortical bone material were all significantly decreased in the skeleton of CKD cats. Cancellous bone was also affected, having significantly lower trabecular thickness and bone volume over total volume in CKD cats compared with controls. This study shows that naturally occurring CKD has deleterious effects on bone quality and strength. Since many similarities exist between human and feline CKD patients, including the clinicopathologic features of the syndrome and bone microarchitecture and biology, these results contribute to better understanding of bone abnormalities associated with CKD.     

Acellularization-Induced Changes in Tensile Properties Are Organ Specific - An In-Vitro Mechanical and Structural Analysis of Porcine Soft Tissues

Introduction

Though xenogeneic acellular scaffolds are frequently used for surgical reconstruction, knowledge of their mechanical properties is lacking. This study compared the mechanical, histological and ultrastructural properties of various native and acellular specimens.

Materials and Methods

Porcine esophagi, ureters and skin were tested mechanically in a native or acellular condition, focusing on the elastic modulus, ultimate tensile stress and maximum strain. The testing protocol for soft tissues was standardized, including the adaption of the tissue’s water content and partial plastination to minimize material slippage as well as templates for normed sample dimensions and precise cross-section measurements. The native and acellular tissues were compared at the microscopic and ultrastructural level with a focus on type I collagens.

Results

Increased elastic modulus and ultimate tensile stress values were quantified in acellular esophagi and ureters compared to the native condition. In contrast, these values were strongly decreased in the skin after acellularization. Acellularization-related decreases in maximum strain were found in all tissues. Type I collagens were well-preserved in these samples; however, clotting and a loss of cross-linking type I collagens was observed ultrastructurally. Elastins and fibronectins were preserved in the esophagi and ureters. A loss of the epidermal layer and decreased fibronectin content was present in the skin.

Discussion

Acellularization induces changes in the tensile properties of soft tissues. Some of these changes appear to be organ specific. Loss of cross-linking type I collagen may indicate increased mechanical strength due to decreasing transverse forces acting upon the scaffolds, whereas fibronectin loss may be related to decreased load-bearing capacity. Potentially, the alterations in tissue mechanics are linked to organ function and to the interplay of cells and the extracellular matrix, which is different in hollow organs when compared to skin.     

Effects of Decreased Occlusal Loading during Growth on the Mandibular Bone Characteristics

Background

Bone mass and mineralization are largely influenced by loading. The purpose of this study was to evaluate the reaction of the entire mandibular bone in response to decreased load during growth. It is hypothesized that decreased muscular loading will lead to bone changes as seen during disuse, i.e. loss of bone mass.

Methods and Findings

Ten 21-day-old Wistar strain male rats were divided into two groups (each n=5) and fed on either a hard- or soft-diet for 11 weeks. Micro-computed tomography was used for the investigation of bone mineralization, bone volume, bone volume fraction (BV/TV) and morphological analysis. Mandibular mineralization patterns were very consistent, showing a lower degree of mineralization in the ramus than in the corpus. In the soft-diet group, mineralization below the molars was significantly increased (p<0.05) compared to the hard diet group. Also, bone volume and BV/TV of the condyle and the masseter attachment were decreased in the soft-diet group (p<0.05). Morphological analysis showed inhibited growth of the ramus in the soft-diet group (p<0.05).

Conclusion

Decreased loading by a soft diet causes significant changes in the mandible. However, these changes are very region-specific, probably depending on the alterations in the local loading regime. The results suggest that muscle activity during growth is very important for bone quality and morphology.