Virginia L. Ferguson

Changes in Bone Structure and Mass With Advancing Age in the Male C57BL/6J Mouse

To determine whether the mouse loses bone with aging and whether the changes mimic those observed in human aging, we examined the changes in the tibial metaphysis and diaphysis in the male C57BL/6J mouse over its life span using microcomputed tomography (μCT). Cancellous bone volume fraction (BV/TV) decreased 60% between 6 weeks and 24 months of age. Loss was characterized by decreased trabecular number (Tb.N), increased trabecular spacing (Tb.Sp), and decreased connectivity. Anisotropy decreased while the structure model index increased with age. Cortical bone thickness increased between 6 weeks and 6 months of age and then decreased continuously to 24 months (−12%). Cortical bone area (Ct.Ar) remained constant between 6 and 24 months. Fat‐free weight reached a peak at 12 months and gradually declined to 24 months. Total mass lost between 12 and 24 months reached 10%. Overall, the age‐related changes in skeletal mass and architecture in the mouse were remarkably similar to those seen in human aging. Furthermore, the rapid early loss of cancellous bone suggests that bone loss is not just associated with old age in the mouse but rather occurs as a continuum from early growth. We conclude that the C57BL/6J male mouse maybe a useful model to study at least some aspects of age‐related bone loss in humans.

Bone development and age-related bone loss in male C57BL/6J mice

The objective of this study was to examine changes in the long bones of male C57BL/6J mice with growth and aging, and to consider the applicability of this animal for use in studying Type II osteoporosis. Male C57BL/6J mice were aged in our colony between 4 and 104 weeks (n=9-15/group). The right femur and humeri were measured for length and subjected to mechanical testing (3-point flexure) and compositional analysis. The left femurs were embedded and thick slices at the mid-diaphysis were assessed for morphology, formation indices, and bone structure. In young mice, rapid growth was marked by substantial increases in bone size, mineral mass, and mechanical properties. Maturity occurred between 12 and 42 weeks of age with the maintenance of bone mass and mechanical properties. From peak levels, mice aged for 104 weeks experienced decreased whole femur mass (12.1 and 18.6% for dry and ash mass, respectively), percentage mineralization (7.4%), diminished whole bone stiffness (29.2%), energy to fracture (51.8%), and decreased cortical thickness (20.1%). Indices of surface-based formation decreased rapidly from the onset of the study. However, the periosteal perimeter and, consequently, the cross-sectional moments of inertia continued to increase through 104 weeks, thus maintaining structural properties. This compensated for cortical thinning and increased brittleness due to decreased mineralization and stiffness. The shape of the mid-diaphysis became increasingly less elliptical in aged mice, and endocortical resorption and evidence of subsequent formation were present in 20-50% of femurs aged > or =78 weeks. This, combined with the appearance of excessive endocortical resorption after 52 weeks, indicated a shift in normal mechanisms regulating bone shape and location, and was suggestive of remodeling. The pattern of bone loss at the femoral mid-diaphysis in this study is markedly similar to that seen in cortical bone in the human femoral neck in Type II osteoporosis. This study has thus demonstrated that the male C57BL/6J mouse is a novel and appropriate model for use in studying endogenous, aging-related osteopenia and may be a useful model for the study of Type II osteoporosis.

Nanomechanical properties and mineral concentration in articular calcified cartilage and subchondral bone

We studied articular calcified cartilage (ACC) and the immediately subchondral bone (SCB) in normal and osteoarthritic human femoral heads. Thick slices of human normal reference post mortem (PM) and osteoarthritic (OA) femoral heads (age 55–89 years) were embedded in PMMA, micromilled, carbon coated and studied using quantitative backscattered electron (qBSE) imaging to determine variations in degree of mineralization. With exact microanatomical location, nanoindentation was performed on the same block faces in representative superior (more highly loaded) and medial regions of the joint surface. Using a partial unloading method, elastic modulus as a function of indenter penetration depth was determined using a spherical tipped diamond indenter. A pointed indenter was used to determine the tissue hardness in selected locations. The relationship between mineralization and indentation modulus was more distinct in ACC than in SCB, the latter having a higher matrix concentration with variable collagen orientation. In OA, the bulk of the measurements were coincident with those in the PM samples, although there was a greater range in the levels of mineralization and modulus in ACC. In OA, extremely hypermineralized ACC was found in ACC proper, especially in superior regions, and translocated into SCB and hyaline cartilage. The very highly mineralized cartilage fragments may function as a hard grinding abrasive, accelerating wear rates whether attached to or fragmented from the eburnated surfaces of OA ACC. Highly mineralized regions would also alter loading patterns and thereby contribute to further destruction of the joint tissues.

Spaceflight effects on T lymphocyte distribution, function and gene expression

The immune system is highly sensitive to stressors present during spaceflight. The major emphasis of this study was on the T lymphocytes in C57BL/6NTac mice after return from a 13-day space shuttle mission (STS-118). Spleens and thymuses from flight animals (FLT) and ground controls similarly housed in animal enclosure modules (AEM) were evaluated within 3-6 h after landing. Phytohemagglutinin-induced splenocyte DNA synthesis was significantly reduced in FLT mice when based on both counts per minute and stimulation indexes (P < 0.05). Flow cytometry showed that CD3(+) T and CD19(+) B cell counts were low in spleens from the FLT group, whereas the number of NK1.1(+) natural killer (NK) cells was increased (P < 0.01 for all three populations vs. AEM). The numerical changes resulted in a low percentage of T cells and high percentage of NK cells in FLT animals (P < 0.05). After activation of spleen cells with anti-CD3 monoclonal antibody, interleukin-2 (IL-2) was decreased, but IL-10, interferon-gamma, and macrophage inflammatory protein-1alpha were increased in FLT mice (P < 0.05). Analysis of cancer-related genes in the thymus showed that the expression of 30 of 84 genes was significantly affected by flight (P < 0.05). Genes that differed from AEM controls by at least 1.5-fold were Birc5, Figf, Grb2, and Tert (upregulated) and Fos, Ifnb1, Itgb3, Mmp9, Myc, Pdgfb, S100a4, Thbs, and Tnf (downregulated). Collectively, the data show that T cell distribution, function, and gene expression are significantly modified shortly after return from the spaceflight environment.

Effects of spaceflight on innate immune function and antioxidant gene expression

Spaceflight conditions have a significant impact on a number of physiological functions due to psychological stress, radiation, and reduced gravity. To explore the effect of the flight environment on immunity, C57BL/6NTac mice were flown on a 13-day space shuttle mission (STS-118). In response to flight, animals had a reduction in liver, spleen, and thymus masses compared with ground (GRD) controls (P < 0.005). Splenic lymphocyte, monocyte/macrophage, and granulocyte counts were significantly reduced in the flight (FLT) mice (P < 0.05). Although spontaneous blastogenesis of splenocytes in FLT mice was increased, response to lipopolysaccharide (LPS), a B-cell mitogen derived from Escherichia coli, was decreased compared with GRD mice (P < 0.05). Secretion of IL-6 and IL-10, but not TNF-alpha, by LPS-stimulated splenocytes was increased in FLT mice (P < 0.05). Finally, many of the genes responsible for scavenging reactive oxygen species were upregulated after flight. These data indicate that exposure to the spaceflight environment can increase anti-inflammatory mechanisms and change the ex vivo response to LPS, a bacterial product associated with septic shock and a prominent Th1 response.

Composite bounds on the elastic modulus of bone

Advances in diagnosis and treatment of some bone disorders can be made by understanding the linkage between mineral content and mechanical function. Bone is approximately half by volume a hydrated protein network, and the remainder is a biomineral analogue of hydroxyapatite. In the current work, paired measurements of mechanical properties, using nanoindentation, and of bone mineral volume fraction, computed from quantitative back-scattered electron imaging, were made on six different types of normal and outlier bone samples. Local elastic modulus was plotted against mineral fraction and compared with predictions of engineering bounds for a two-phase composite material. Experimental data spanning the composite bounds showed no one-to-one relationship between mechanical stiffness and bone composition, excluding the possibility of any single, simple composites model for bone at nanometer length-scales.

Osteoprotegerin Mitigates Tail Suspension-Induced Osteopenia

Osteoprotegerin (OPG) is a recently discovered protein related to the tumor necrosis factor receptor family. It has been shown to inhibit ovariectomy (ovx)-induced resorption in rats and increase bone mineral density in young mice. Tail suspension is a procedure that inhibits bone formation in maturing rodents. This study was designed to quantify OPGs effect on cortical bone formation. Fifty-four mice were assigned to one of five groups (n = 10-11/group). A baseline control group was killed on day 0 of the 10 day study. The remaining groups were: vivarium housed (nonsuspended) control mice receiving 0.3 mg/kg per day OPG; vivarium control mice receiving daily placebo injections; tail-suspended mice receiving 0. 3 mg/kg per day OPG; and tail-suspended mice receiving placebo injections. Tetracycline was administered on days 0 and 8. OPG treatment of tail-suspended mice produced mechanical properties similar to those of placebo-treated, vivarium-housed mice: structural stiffness (8.5%, 20.7%) and elastic (13.9%, 10.1%) and maximum (4.7%, 8.1%) force were increased compared with placebo controls (vivarium, suspended groups). Percent mineral composition was highly significantly greater (p < 0.001 for all comparisons) for OPG-treated mice in the femur, tibia, and humerus, relative to placebo treatment. Matrix mass was also significantly increased in the femur, although not to the same degree as mineral mass. OPG decreased the amount of femoral endocortical resorption compared with the placebo-treated groups for both vivarium (27%) and suspended (24%) mice. Administration of OPG significantly decreased endocortical formation of the tibia. Periosteal bone formation rates were not altered by OPG. OPG-mitigated tail suspension induced osteopenia not by returning bone formation to normal levels, but by inhibiting resorption and increasing percent mineral composition.

Effects of Weak Static Magnetic Fields on Endothelial Cells

Pulsed electromagnetic fields (PEMFs) have been used extensively in bone fracture repairs and wound healing. It is accepted that the induced electric field is the dose metric. The mechanisms of interaction between weak magnetic fields and biological systems present more ambiguity than that of PEMFs since weak electric currents induced by PEMFs are believed to mediate the healing process, which are absent in magnetic fields. The present study examines the response of human umbilical vein endothelial cells to weak static magnetic fields. We investigated proliferation, viability, and the expression of functional parameters such as eNOS, NO, and also gene expression of VEGF under the influence of different doses of weak magnetic fields. Applications of weak magnetic fields in tissue engineering are also discussed. Static magnetic fields may open new venues of research in the field of vascular therapies by promoting endothelial cell growth and by enhancing the healing response of the endothelium.

Comparative study of the viscoelastic mechanical behavior of agarose and poly(ethylene glycol) hydrogels.

This study presents a comparative investigation into differences in the mechanical properties between two hydrogels commonly used in cartilage tissue engineering [agarose vs. poly(ethylene glycol) (PEG)], but which are formed through distinctly different crosslinking mechanisms (physical vs. covalent, respectively). The effects of hydrogel chemistry, precursor concentration, platen type (nonporous vs. porous) used in compression bioreactors, and degradation (for PEG) on the swelling properties and static and dynamic mechanical properties were examined. An increase in precursor concentration resulted in decreased equilibrium mass swelling ratios but increased equilibrium moduli and storage moduli for both hydrogels (p < 0.05). Agarose displayed large stress relaxations and a frequency dependence indicating its viscoelastic properties. Contrarily, PEG hydrogels displayed largely elastic behavior with minimal stress relaxation and frequency dependence. In biodegradable PEG hydrogels, the largely elastic behavior was retained during degradation. The type of platen did not affect static mechanical properties, but porous platens led to a reduced storage modulus for both hydrogels implicating fluid flow. In summary, agarose and PEG exhibit vastly different mechanical behaviors; a finding largely attributed to differences in their chemistries and fluid movement. Taken together, these design choices (hydrogel chemistry/structure, loading conditions) will likely have a profound effect on the tissue engineering outcome.

Bioengineering aspects of the umbilical cord

The umbilical cord and its constituent tissues: an outer layer of amnion, porous Whartons jelly, two umbilical arteries, and one umbilical vein, are designed to protect blood flow to the fetus during a term pregnancy. The outer amnion layer may regulate fluid pressure within the umbilical cord. The porous, fluid filled Whartons jelly likely acts to prevent compression of the vessels. Blood flow is regulated by smooth muscle surrounding the arteries that is intermingled with a collagen based extracellular matrix (ECM). Doppler ultrasound measurements of blood flow within the umbilical cord, and at specific sites within the developing fetus, provide evidence of impaired blood flow in conditions such as preeclampsia. Mechanosensory communication between cells and the extracellular matrix (ECM) may likely result in cords possessing abnormal physical dimensions, impaired hemodynamics, and altered composition within the umbilical cord tissues. Few studies have explored the biomechanics of the intact umbilical cord, with its constituent tissues, from normal pregnancies or abnormal pregnancies, maternal or fetal complications. Here, alterations in the umbilical cord are reviewed concerning anatomical abnormalities, disease, or chromosomal alterations using sonography, Doppler ultrasound, histology, and biomolecular and biochemical analyses. This paper considers how current knowledge of the umbilical cord and its constituent tissues can be used to infer biomechanical function. In addition, the mechanical consequences of structural abnormalities and altered tissue structure or composition are discussed with a specific focus on preeclampsia.