Amber Rath Stern

Isolation and culture of primary osteocytes from the long bones of skeletally mature and aged mice

The purpose of this work was to establish a methodology to enable the isolation and study of osteocytes from skeletally mature young (4-month-old) and old (22-month-old) mice. The location of osteocytes deep within bone is ideal for their function as mechanosensors. However, this location makes the observation and study of osteocytes in vivo technically difficult. Osteocytes were isolated from murine long bones through a process of extended collagenase digestions combined with EDTA-based decalcification. A tissue homogenizer was used to reduce the remaining bone fragments to a suspension of bone particles, which were placed in culture to yield an outgrowth of osteocyte-like cells. All of the cells obtained from this outgrowth that displayed an osteocyte-like morphology stained positive for the osteocyte marker E11/GP38. The osteocyte phenotype was further confirmed by a lack of staining for alkaline phosphatase and the absence of collagen1a1 expression. The outgrowth of osteocytes also expressed additional osteocyte-specific genes such as Sost and Mepe. This technique facilitates the isolation of osteocytes from skeletally mature bone. This novel enabling methodology should prove useful in advancing our understanding of the roles mature osteocytes play in bone health and disease.

Deletion of a Single β-Catenin Allele in Osteocytes Abolishes the Bone Anabolic Response to Loading

The Wnt/β‐catenin signaling pathway is essential for bone cell viability and function and for skeletal integrity. To determine if β‐catenin in osteocytes plays a role in the bone anabolic response to mechanical loading, 18‐ to 24‐week‐old osteocyte β‐catenin haploinsufficient mice (Dmp1‐Cre × β‐catenin fl/ + ; HET cKO) were compared with their β‐catenin fl/fl (control) littermates. Trabecular bone volume (BV/TV) was significantly less (58.3%) in HET cKO females versus controls, whereas male HET cKO and control mice were not significantly different. Trabecular number was significantly less in HET cKO mice compared with controls for both genders, and trabecular separation was greater in female HET cKO mice. Osteoclast surface was significantly greater in female HET cKO mice. Cortical bone parameters in males and females showed subtle or no differences between HET cKO and controls. The right ulnas were loaded in vivo at 100 cycles, 2 Hz, 2500 µϵ, 3 days per week for 3 weeks, and the left ulnas served as nonloaded controls. Calcein and alizarin complexone dihydrate were injected 10 days and 3 days before euthanization, respectively. Micro‐computed tomography (µCT) analysis detected an 8.7% and 7.1% increase in cortical thickness in the loaded right ulnas of male and female control mice, respectively, compared with their nonloaded left ulnas. No significant increase in new cortical bone formation was observed in the HET cKO mice. Histomorphometric analysis of control mice showed a significant increase in endocortical and periosteal mineral apposition rate (MAR), bone‐formation rate/bone surface (BFR/BS), BFR/BV, and BFR/TV in response to loading, but no significant increases were detected in the loaded HET cKO mice. These data show that deleting a single copy of β‐catenin in osteocytes abolishes the anabolic response to loading, that trabecular bone in females is more severely affected and suggest that a critical threshold of β‐catenin is required for bone formation in response to mechanical loading.

Measurement and estimation of osteocyte mechanical strain

Osteocytes are the most abundant cell type in bone and are responsible for sensing mechanical strain and signaling bone (re)modeling, making them the primary mechanosensors within bone. Under aging and osteoporotic conditions, bone is known to be less responsive to loading (exercise), but it is unclear why. Perhaps, the levels of mechanical strain required to initiate these biological events are not perceived by the osteocytes embedded within the bone tissue. In this review we examine the methods used to measure and estimate the strains experienced by osteocytes in vivo as well as the results of related published experiments. Although the physiological levels of strain experienced by osteocytes in vivo are still under investigation, through computational modeling and laboratory experiments, it has been shown that there is significant amplification of average bone strain at the level of the osteocyte lacunae. It has also been proposed that the material properties of the perilacunar region surrounding the osteocyte can have significant effects of the strain perceived by the embedded osteocyte. These facts have profound implications for studies involving osteoporotic bone where the material properties are known to become stiffer.

Deletion of Mbtps1 (Pcsk8, S1p, Ski-1) Gene in Osteocytes Stimulates Soleus Muscle Regeneration and Increased Size and Contractile Force with Age.

Conditional deletion of Mbtps1 (cKO) protease in bone osteocytes leads to an age-related increase in mass (12%) and in contractile force (30%) in adult slow twitch soleus muscles (SOL) with no effect on fast twitch extensor digitorum longus muscles. Surprisingly, bone from 10–12-month-old cKO animals was indistinguishable from controls in size, density, and morphology except for a 25% increase in stiffness. cKO SOL exhibited increased expression of Pax7, Myog, Myod1, Notch, and Myh3 and 6-fold more centralized nuclei, characteristics of postnatal regenerating muscle, but only in type I myosin heavy chain-expressing cells. Increased expression of gene pathways mediating EGF receptor signaling, circadian exercise, striated muscle contraction, and lipid and carbohydrate oxidative metabolism were also observed in cKO SOL. This muscle phenotype was not observed in 3-month-old mice. Although Mbtps1 mRNA and protein expression was reduced in cKO bone osteocytes, no differences in Mbtps1 or cre recombinase expression were observed in cKO SOL, explaining this age-related phenotype. Understanding bone-muscle cross-talk may provide a fresh and novel approach to prevention and treatment of age-related muscle loss.

Blunt eye trauma: Empirical histopathologic paintball impact thresholds in fresh mounted porcine eyes

PURPOSE Ballistic studies were conducted using gelatin-embedded abattoir-fresh porcine eyes suspended within clear acrylic orbits to discern the energy required to produce specific ocular injuries. Paintball impact provides a robust ballistic model for isolating and quantifying the role of direct blunt force in ocular trauma. METHODS Fifty-nine porcine orbital preparations received direct blows from 0.68 caliber (16-18 mm diameter/3.8 g) paintballs fired at impact velocities ranging from 26 to 97 meters per second (2-13.5 J). Five additional eyes not subjected to ballistic impact were also evaluated as controls. Impact energies were correlated with histopathologic damage. RESULTS Minimum impact energies consistently producing damage in experimental eyes unobserved in control specimens were: 2 joules--posterior lens dislocation, zonulysis, capsular rupture, and choroidal detachment; 3.5 joules--moderate angle recession; 4 joules--anterior lens dislocation; 4.8 joules--peripapillary retinal detachment; 7 joules--severe angle recession, iridodialysis, and cyclodialysis; 7.5 joules--corneal stromal distraction; 9.3 joules--choroidal segmentation; and 10 joules--globe rupture. CONCLUSIONS Impact thresholds correlating traumatic ocular pathology with impact energy followed a positive stepwise progression in severity with impact energies between 2 and 10 joules. Moderate angle recession commensurate with typical clinical traumatic glaucoma was not observed among control eyes, but occurred at relatively low impact energy of 3.5 joules among test eyes. Extensive disruption in and around the angle (iridodialysis/cyclodialysis) consistently occurred at energies >7 joules. Globe rupture required a minimum energy of 10 joules.

Osteoblast-specific deletion of Pkd2 leads to low-turnover osteopenia and reduced bone marrow adiposity

Polycystin-1 (Pkd1) interacts with polycystin-2 (Pkd2) to form an interdependent signaling complex. Selective deletion of Pkd1 in the osteoblast lineage reciprocally regulates osteoblastogenesis and adipogenesis. The role of Pkd2 in skeletal development has not been defined. To this end, we conditionally inactivated Pkd2 in mature osteoblasts by crossing Osteocalcin (Oc)-Cre;Pkd2 +/null mice with floxed Pkd2 (Pkd2 flox/flox) mice. Oc-Cre;Pkd2 flox/null (Pkd2 Oc-cKO) mice exhibited decreased bone mineral density, trabecular bone volume, cortical thickness, mineral apposition rate and impaired biomechanical properties of bone. Pkd2 deficiency resulted in diminished Runt-related transcription factor 2 (Runx2) expressions in bone and impaired osteoblastic differentiation ex vivo. Expression of osteoblast-related genes, including, Osteocalcin, Osteopontin, Bone sialoprotein (Bsp), Phosphate-regulating gene with homologies to endopeptidases on the X chromosome (Phex), Dentin matrix protein 1 (Dmp1), Sclerostin (Sost), and Fibroblast growth factor 23 (FGF23) were reduced proportionate to the reduction of Pkd2 gene dose in bone of Oc-Cre;Pkd2 flox/+ and Oc-Cre;Pkd2 flox/null mice. Loss of Pkd2 also resulted in diminished peroxisome proliferator-activated receptor γ (PPARγ) expression and reduced bone marrow fat in vivo and reduced adipogenesis in osteoblast culture ex vivo. Transcriptional co-activator with PDZ-binding motif (TAZ) and Yes-associated protein (YAP), reciprocally acting as co-activators and co-repressors of Runx2 and PPARγ, were decreased in bone of Oc-Cre;Pkd2 flox/null mice. Thus, Pkd1 and Pkd2 have coordinate effects on osteoblast differentiation and opposite effects on adipogenesis, suggesting that Pkd1 and Pkd2 signaling pathways can have independent effects on mesenchymal lineage commitment in bone.

Paintball Trauma and Mechanisms of Optic Nerve Injury: Rotational Avulsion and Rebound Evulsion

PURPOSE Ballistic impact studies and supercomputer modeling were performed to elicit the mechanisms of optic nerve rupture that may accompany blunt ocular trauma. METHODS Paintball ocular impact responses were studied with abattoir-fresh porcine eyes. Physics-based numerical code CTH was used to produce robust geometric and constitutive models of the eye and orbit, providing a comparative 3-D finite volume model to help determine the mechanisms underlying empirical ballistic observations. RESULTS Among 59 porcine eye specimens submitted to paintball impact in the 1- to 13-J range, 10 (17%) disengaged completely from the orbital mount. In each instance the paintball penetrated the orbit adjacent to the globe, producing rotation and eventual globe repulsion, dramatically evident on high-speed film images. Supercomputer modeling yielded similar globe-expulsive results when orbital constraints were in place, but not when these were removed. In these models, tangential (grazing) impact sheared the nerve flush with the globe via a strain rate effect within 260 μs, with minimal posterior displacement and just 5° of globe rotation. Midperipheral impact produced compressive globe distortion and posterior displacement, followed by rebound and tractional nerve avulsion 10 mm behind the lamina after 700 μs and 20° of globe rotation. CONCLUSIONS Constitutive modeling studies suggest at least two trajectory-dependent mechanisms for optic nerve rupture with paintball impact on the eye. Tangential glancing blows produce strain-rate rotational avulsion, abscising the optic nerve with minimal internal globe disruption, whereas off-center direct impact produces slower rotational-rebound evulsion, traumatizing the globe and breaching the nerve posteriorly. The latter mechanism would be expected to arise more commonly and would most likely be clinically masked by accompanying intraocular injury.

Osteocyte isolation and culture methods

The aim of this paper is to present several popular methods for in vitro culture of osteocytes and osteocyte cell lines. Osteocytes are located extremely suitably within the calcified bone matrix to sense mechanical signals, and are equipped with a multitude of molecular features that allow mechanosensing. However, osteocytes are more than specialized mechanosensing cells. Several signaling molecules are preferentially produced by osteocytes, and osteocytes hold a tight reign over osteoblast and osteoclast formation and activity, but also have a role as endocrine cell, communicating with muscles or organs as remote as the kidneys. In order to facilitate further research into this fascinating cell type, three protocols will be provided in this paper. The first protocol will be on the culture of mouse (early) osteocyte cell lines, the second on the isolation and culture of primary mouse bone cells, and the third on the culture of fully embedded human osteocytes within their own three-dimensional bone matrix.

Isolation of osteocytes from mature and aged murine bone

Osteocytes are thought to be the mechanosensors of bone by sensing mechanical loads imposed upon the bone and transmitting these signals to the other bone cells to initiate bone modeling and remodeling. The location of osteocytes deep within bone is ideal for their function. However, this location makes the study of osteocytes in vivo technically difficult. There are several methods for obtaining and culturing primary osteocytes for in vitro experiments and ex vivo observation. In this chapter, several proven methods are discussed including the isolation of avian osteocytes from chicks and osteocytes from calvaria and long bones of young mice. A detailed protocol for the isolation of osteocytes from hypermineralized bone of mature and aged animals is provided.

TRANSDUCTION OF STRAIN TO CELLS SEEDED ONTO SCAFFOLDS EXPOSED TO UNIAXIAL STRETCHING: A THREE DIMENSIONAL FINITE ELEMENT STUDY

When preparing tissue engineering and regenerative medicine constructs, a commonly encountered problem is the failure of seeded cells to infiltrate the scaffold. In an increasing number of cases, constructs are being mechanically preconditioned with the expectation that preconditioning will enhance the constructs maturation and effectiveness by pre-exposing seeded cells to stimuli the tissue of interest experiences in vivo. However, whether or not mechanostimulation of a scaffold actually results in transmission of stimuli to the seeded cells remains poorly understood. The purpose of this research was to develop a model that quantifies how strain is transmitted to cells layered on a scaffolds surface compared to cells embedded within a scaffold. Three-dimensional finite element models representative of these conditions were created. When 10% strain was applied to the construct, embedded cells received the full imposed strain. However, cells growing on top of the scaffold received 5% strain within the fi...