Daniel P. Nicolella

Dendritic processes of osteocytes are mechanotransducers that induce the opening of hemichannels

Osteocytes with long dendritic processes are known to sense mechanical loading, which is essential for bone remodeling. There has been a long-standing debate with regard to which part(s) of osteocyte, the cell body versus the dendritic process, acts as a mechanical sensor. To address this question experimentally, we used a transwell filter system that differentiates the cell body from the dendritic processes. Mechanical loading was applied to either the cell body or the dendrites, and the osteocyte’s response was observed through connexin 43 hemichannel opening. The hemichannels located on the cell body were induced to open when mechanical loading was applied to either the dendritic processes or the cell body. However, no significant hemichannel activity in the dendrites was detected when either part of the cell was mechanically stimulated. Disruption of the glycocalyx by hyaluronidase on the dendrite side alone is sufficient to diminish a dendrite’s ability to induce the opening of hemichannels on the cell body, while hyaluronidase has no such effect when applied to the cell body. Importantly, hyaluronidase treatment to the dendrite side resulted in formation of poor integrin attachments with the reduced ability of the dendrites to form integrin attachments on the underside of the transwell filter. Together, our study suggests that the glycocalyx of the osteocyte dendritic process is required for forming strong integrin attachments. These integrin attachments probably serve as the mechanotransducers that transmit the mechanical signals to the cell body leading to the opening of hemichannels, which permits rapid exchange of factors important for bone remodeling.

Mechanism by which MLO-A5 Late Osteoblasts/Early Osteocytes Mineralize in Culture: Similarities with Mineralization of Lamellar Bone

The mechanisms whereby bone mineralizes are unclear. To study this process, we used a cell line, MLO-A5, which has highly elevated expression of markers of the late osteoblast such as alkaline phosphatase, bone sialoprotein, parathyroid hormone type 1 receptor, and osteocalcin and will mineralize in sheets, not nodules. In culture, markers of osteocytes and dendricity increase with time, features of differentiation from a late osteoblast to an early osteocyte. Mineral formation was examined using transmission electron microscopy, scanning electron microscopy with energy-dispersive X-ray analysis, and atomic force microscopy. At 3–4 days of culture, spheres of approximately 20–50 nm containing calcium and phosphorus were observed budding from and associated with developing cellular projections. By 5–6 days, these calcified spheres were associated with collagen fibrils, where over time they continued to enlarge and to engulf the collagen network. Coalescence of these mineralized spheres and collagen-mediated mineralization were responsible for the mineralization of the matrix. Similar calcified spheres were observed in cultured fetal rat calvarial cells and in murine lamellar bone. We propose that osteoid-osteocytes generate spherical structures that calcify during the budding process and are fully mineralized on their developing cellular processes. As the cellular process narrows in diameter, these mineralized structures become associated with and initiate collagen-mediated mineralization.

Machine vision photogrammetry: a technique for measurement of microstructural strain in cortical bone

Understanding local microstructural deformations and strains in cortical bone may lead to a better understanding of cortical bone damage development, fracture, and remodeling. Traditional experimental techniques for measuring deformation and strain do not allow characterization of these quantities at the microstructural level in cortical bone. This study describes a technique based on digital stereoimaging used to measure the microstructural strain fields in cortical bone. The technique allows the measurement of material surface displacements and strains by comparing images acquired from a specimen at two distinct stress states. The accuracy of the system is investigated by analyzing an undeformed image set; the test image is identical to the reference image but translated by a known pixel amount. An increase in the correlation sub-image train parameter results in an increase in displacement measurement accuracy from 0.049 to 0.012 pixels. Errors in strain calculated from the measured displacement field were between 39 and 564 microstrain depending upon the sub-image train size and applied image displacement. The presence of a microcrack in cortical bone results in local strain at the crack tip reaching 0.030 (30,000 microstrain) and 0.010 (10,000 microstrain) near osteocyte lacunae. It is expected that the use of this technique will allow a greater understanding of bone strength and fracture as well as bone mechanotransduction.

Statistical shape modeling describes variation in tibia and femur surface geometry between Control and Incidence groups from the Osteoarthritis Initiative database

We hypothesize that variability in knee subchondral bone surface geometry will differentiate between patients at risk and those not at risk for developing osteoarthritis (OA) and suggest that statistical shape modeling (SSM) methods form the basis for developing a diagnostic tool for predicting the onset of OA. Using a subset of clinical knee MRI data from the osteoarthritis initiative (OAI), the objectives of this study were to (1) utilize SSM to compactly and efficiently describe variability in knee subchondral bone surface geometry and (2) determine the efficacy of SSM and rigid body transformations to distinguish between patients who are not expected to develop osteoarthritis (i.e. Control group) and those with clinical risk factors for OA (i.e. Incidence group). Quantitative differences in femur and tibia surface geometry were demonstrated between groups, although differences in knee joint alignment measures were not statistically significant, suggesting that variability in individual bone geometry may play a greater role in determining joint space geometry and mechanics. SSM provides a means of explicitly describing complete articular surface geometry and allows the complex spatial variation in joint surface geometry and joint congruence between healthy subjects and those with clinical risk of developing or existing signs of OA to be statistically demonstrated.

Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice

We investigated whether polycystin‐1 is a bone mechanosensor. We conditionally deleted Pkd1 in mature osteoblasts/osteocytes by crossing Dmp1‐Cre with Pkd1flox/m1Bei mice, in which the m1Bei allele is nonfunctional. We assessed in wild‐type and Pkd1‐deficient mice the response to mechanical loading in vivo by ulna loading and ex vivo by measuring the response of isolated osteoblasts to fluid shear stress. We found that conditional Pkd1 heterozygotes (Dmp1‐Cre;Pkd1flox/+) and null mice (Pkd1Dmp1‐cKO) exhibited a ~40 and ~90% decrease, respectively, in functional Pkd1 transcripts in bone. Femoral bone mineral density (12 vs. 27%), trabecular bone volume (32 vs. 48%), and cortical thickness (6 vs. 17%) were reduced proportionate to the reduction of Pkd1 gene dose, as were mineral apposition rate (MAR) and expression of Runx2‐II, Osteocalcin, Dmp1, and Phex. Anabolic load‐induced periosteal lamellar MAR (0.58±0.14; Pkd1Dmp1‐cKO vs. 1.68±0.34 μm/d; control) and increases in Cox‐2, c‐Jun, Wnt10b, Axin2, and Runx2‐II gene expression were significantly attenuated in Pkd1Dmp1‐cKO mice compared with controls. Application of fluid shear stress to immortalized osteoblasts from Pkd1null/null and Pkd1m1Bei/m1Bei‐derived osteoblasts failed to elicit the increments in cytosolic calcium observed in wild‐type controls. These data indicate that polycystin‐1 is essential for the anabolic response to skeletal loading in osteoblasts/osteocytes.—Xiao, Z., Dallas, M., Qiu, N., Nicolella, D., Cao, L., Johnson, M., Bonewald, L., Quarles, L. D. Conditional deletion of Pkd1 in osteocytes disrupts skeletal mechanosensing in mice. FASEB J. 25, 2418–2432 (2011). www.fasebj.org

Measurement of microstructural strain in cortical bone.

It is well known that mechanical factors affect bone remodeling such that increased mechanical demand results in net bone formation, whereas decreased demand results in net bone resorption. Current theories suggest that bone modeling and remodeling is controlled at the cellular level through signals mediated by osteocytes. The objective of this study was to investigate how macroscopically applied bone strains similar in magnitude to those that occur in vivo are manifest at the microscopic level in the bone matrix. Using a digital image correlation strain measurement technique, experimentally determined bone matrix strains around osteocyte lacuna resulting from macroscopic strains of approximately 2,000 microstrain (0.2%) reach levels of over 30,000 microstrain (3%) over fifteen times greater than the applied macroscopic strain. Strain patterns were highly heterogeneous and in some locations similar to observed microdamage around osteocyte lacuna indicating the resulting strains may represent the precursors to microdamage. This information may lead to a better understanding of how bone cells are affected by whole bone functional loading.

Fracture risk assessment

Having traditionally relied on measurements of bone mineral density, it is now established that the consideration of other risk variables improves the categorisation of fracture risk. Whereas several models are available, the FRAX models are the most extensively used. The approach uses easily obtained clinical risk factors to estimate 10 year fracture probability, with or without femoral neck bone mineral density (BMD), to enhance fracture risk prediction. It has been constructed and validated using primary data from population based cohorts around the world, including centres from North America, Europe, Asia and Australia. The FRAX® tool should not be considered as a gold standard, but rather as a platform technology on which to build as new validated risk indicators become available. Notwithstanding, the present models provide an aid to enhance patient assessment by the integration of clinical risk factors alone and/or in combination with BMD.

Assessment of water distribution changes in human cortical bone by nuclear magnetic resonance

A NMR spin–spin (T2) relaxation technique has been described for determining water distribution changes in human cortical bone tissue. The advantages of using NMR T2 relaxation techniques for bone water distribution are illustrated. The CPMG T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to a pore size distribution with the longer relaxation times corresponding to larger pores. The FID T2 relaxation data can be inverted to T2 relaxation distribution and this distribution then can be transformed to bound- and mobile-water distribution with the longest relaxation time corresponding to mobile water and the middle relaxation time corresponding to bound water. The technique is applied to quantify apparent changes in porosity, bound and mobile water in cortical bone. Overall bone porosity is determined using the calibrated NMR fluid volume from the proton relaxation data divided by overall bone volume. The NMR bound and mobile water changes were determined from cortical bone specimens obtained from male and female donors of different ages. Differences in water distribution were found between specimens from male and female donors. Furthermore, the distribution of water within a single specimen was found to be non-homogeneous. Our results show that the ratio of the average bound to mobile water in bone from male donors is higher than in bone from female donors when the bone porosities are similar between male and female groups. We also show that the average bone porosity multiplied by the ratio of bound to mobile water is constant for both male and female bone groups. This parameter may be used as a measure of bone quality describing both porosity and water content, both of which may be important determinants of bone strength and fracture resistance.

Platelet-derived growth-factor-releasing aligned collagen-nanoparticle fibers promote the proliferation and tenogenic differentiation of adipose-derived stem cells.

In order to enhance the healing potential of an injured tendon, we have prepared a novel biomimetic aligned collagen-nanoparticle (NP) composite fiber using an electrochemical process. The aligned collagen-NP composite fiber is designed to affect the cellular activity of adipose-derived stem cells (ADSCs) through two different ways: (i) topographic cues from the alignment of collagen fibril and (ii) controlled release of platelet-derived growth factors (PDGFs) from the NPs. PDGF released from collagen-NP fibers significantly enhanced the proliferation of ADSCs when tested for up to 7 days. Moreover, compared to random collagen fibers with PDGFs, aligned collagen-NP fibers significantly promoted the desirable tenogenic differentiation of ADSCs, as evidenced by an increased level of tendon markers such as tenomodulin and scleraxis. On the other hand, no undesirable osteogenic differentiation, as measured by the unchanged level of alkaline phosphatase and osteocalcin, was observed. Together, these results indicate that the aligned collagen-NP composite fiber induced the tenogenic differentiation of ADSCs through both a topographic cue (aligned collagen fibril) and a chemical cue (PDGF released from NPs). Thus, our novel aligned collagen-NP composite fiber has a significant potential to be used for tendon tissue engineering and regeneration.

Baboons as a Model to Study Genetics and Epigenetics of Human Disease

A major challenge for understanding susceptibility to common human diseases is determining genetic and environmental factors that influence mechanisms underlying variation in disease-related traits. The most common diseases afflicting the US population are complex diseases that develop as a result of defects in multiple genetically controlled systems in response to environmental challenges. Unraveling the etiology of these diseases is exceedingly difficult because of the many genetic and environmental factors involved. Studies of complex disease genetics in humans are challenging because it is not possible to control pedigree structure and often not practical to control environmental conditions over an extended period of time. Furthermore, access to tissues relevant to many diseases from healthy individuals is quite limited. The baboon is a well-established research model for the study of a wide array of common complex diseases, including dyslipidemia, hypertension, obesity, and osteoporosis. It is possible to acquire tissues from healthy, genetically characterized baboons that have been exposed to defined environmental stimuli. In this review, we describe the genetic and physiologic similarity of baboons with humans, the ability and usefulness of controlling environment and breeding, and current genetic and genomic resources. We discuss studies on genetics of heart disease, obesity, diabetes, metabolic syndrome, hypertension, osteoporosis, osteoarthritis, and intrauterine growth restriction using the baboon as a model for human disease. We also summarize new studies and resources under development, providing examples of potential translational studies for targeted interventions and therapies for human disease.