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
and
. 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.
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 (Pkd2flox/flox) mice. Oc-Cre;Pkd2flox/null (Pkd2Oc-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;Pkd2flox/+ and Oc-Cre;Pkd2flox/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;Pkd2flox/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.
Though osteoporosis is a significant cause of disability worldwide, treatment with pharmacologic agents decreases risk of fragility fracture. Though these treatments act through the bone remodeling ...system to improve bone mass, it is unclear if they alter the response of bone to mechanical loading at the level of the osteocyte. This pre-clinical study determined the relationship between microstructural bone tissue properties and osteocyte lacunar size and density to strain around osteocytes with standard osteoporosis treatment or sequential therapies. Six-month-old female ovariectomized (OVX) Sprague-Dawley rats were cycled through various sequences of pharmacological treatments alendronate (Aln), raloxifene (Ral) and human parathyroid hormone-1,34 (PTH) for three month intervals, over nine months. Linear nanoindentation mapping was used to determine Young's modulus in perilacunar and bone matrix regions around cortical bone osteocyte lacunae. Measurements of lacunar diameter and density were completed. Treatment-related differences in Young's modulus in the perilacunar and bone matrix regions were not observed. We confirmed previous data that showed that the bone matrix region was stiffer than the perilacunar matrix region. Whole bone material properties were correlated to perilacunar matrix stiffness. Finite element models predicted a range of mechanical strain amplification factors estimated at the osteocyte across treatment groups. In summary, though the perilacunar matrix near cortical osteocyte lacuna is not as stiff as bone matrix further away, osteoporosis treatment agents do not affect the stiffness of bone tissue near osteocyte lacunae.
•Monotherapy with osteoporosis treatment agents does not affect the stiffness of bone tissue around osteocyte lacunae.•Sequential use of osteoporosis treatment agents does not affect bone tissue stiffness around osteocyte lacunae.•Perilacunar cortical bone tissue is not as stiff as bone matrix further from osteocyte lacunae.•Whole bone material properties are negatively correlated to the stiffness of perilacunar bone tissue.
Isolation of Murine and Human Osteocytes Prideaux, Matthew; Stern, Amber Rath; Bonewald, Lynda F
Methods in molecular biology (Clifton, N.J.),
2021, Letnik:
2221
Journal Article
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. In addition, a modified version of this protocol that can be used to isolate osteocytes from human trabecular bone is described.
Abstract 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. This article is part of a Special Issue entitled "The Osteocyte".
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.
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