Activation of the P2Y2 receptor regulates bone cell function by enhancing ATP release

Bone cells constitutively release ATP into the extracellular environment where it acts locally via P2 receptors to regulate bone cell function. Whilst P2Y2 receptor stimulation regulates bone mineralisation, the functional effects of this receptor in osteoclasts remain unknown. This investigation used the P2Y2 receptor knockout (P2Y2R−/−) mouse model to investigate the role of this receptor in bone. MicroCT analysis of P2Y2R−/− mice demonstrated age-related increases in trabecular bone volume (≤48%), number (≤30%) and thickness (≤17%). In vitro P2Y2R−/− osteoblasts displayed a 3-fold increase in bone formation and alkaline phosphatase activity, whilst P2Y2R−/− osteoclasts exhibited a 65% reduction in resorptive activity. Serum cross-linked C-telopeptide levels (CTX, resorption marker) were also decreased (≤35%). The resorption defect in P2Y2R−/− osteoclasts was rescued by the addition of exogenous ATP, suggesting that an ATP deficit could be a key factor in the reduced function of these cells. In agreement, we found that basal ATP release was reduced up to 53% in P2Y2R−/− osteoclasts. The P2Y2 receptor agonists, UTP and 2-thioUTP, increased osteoclast activity and ATP release in wild-type but not in P2Y2R−/− cells. This indicates that the P2Y2 receptor may regulate osteoclast function indirectly by promoting ATP release. UTP and 2-thioUTP also stimulate ATP release from osteoblasts suggesting that the P2Y2 receptor exerts a similar function in these cells. Taken together, our findings are consistent with the notion that the primary action of P2Y2 receptor signalling in bone is to regulate extracellular ATP levels.

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Review on material parameters to enhance bone cell function in vitro and in vivo

Biochemical Society Transactions ◽

10.1042/bst20200210 ◽

2020 ◽

Vol 48 (5) ◽

pp. 2039-2050

Author(s):

Eric Madsen ◽

Merjem Mededovic ◽

David H. Kohn

Keyword(s):

Cell Function ◽

Bone Cells ◽

Bone Cell ◽

Bone Augmentation ◽

Material Parameters ◽

Specific Material ◽

Resorbable Implants ◽

Resorbable Materials

Bone plays critical roles in support, protection, movement, and metabolism. Although bone has an innate capacity for regeneration, this capacity is limited, and many bone injuries and diseases require intervention. Biomaterials are a critical component of many treatments to restore bone function and include non-resorbable implants to augment bone and resorbable materials to guide regeneration. Biomaterials can vary considerably in their biocompatibility and bioactivity, which are functions of specific material parameters. The success of biomaterials in bone augmentation and regeneration is based on their effects on the function of bone cells. Such functions include adhesion, migration, inflammation, proliferation, communication, differentiation, resorption, and vascularization. This review will focus on how different material parameters can enhance bone cell function both in vitro and in vivo.

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Regulation of Bone Mass and Bone Turnover by Neuronal Nitric Oxide Synthase

Endocrinology ◽

10.1210/en.2004-0205 ◽

2004 ◽

Vol 145 (11) ◽

pp. 5068-5074 ◽

Cited By ~ 64

Author(s):

Robert J. van’t Hof ◽

Jeny MacPhee ◽

Helene Libouban ◽

Miep H. Helfrich ◽

Stuart H. Ralston

Keyword(s):

Nitric Oxide ◽

Bone Turnover ◽

Cell Function ◽

Bone Cells ◽

Physiological Role ◽

Bone Cell ◽

Nodule Formation ◽

Mineral Density

Abstract Nitric oxide (NO) is produced by NO synthase (NOS) and plays an important role in the regulation of bone cell function. The endothelial NOS isoform is essential for normal osteoblast function, whereas the inducible NOS isoform acts as a mediator of cytokine effects in bone. The role of the neuronal isoform of NOS (nNOS) in bone has been studied little thus far. Therefore, we investigated the role of nNOS in bone metabolism by studying mice with targeted inactivation of the nNOS gene. Bone mineral density (BMD) was significantly higher in nNOS knockout (KO) mice compared with wild-type controls, particularly the trabecular BMD (P < 0.01). The difference in BMD between nNOS KO and control mice was confirmed by histomorphometric analysis, which showed a 67% increase in trabecular bone volume in nNOS KO mice when compared with controls (P < 0.001). This was accompanied by reduced bone remodeling, with a significant reduction in osteoblast numbers and bone formation surfaces and a reduction in osteoclast numbers and bone resorption surfaces. Osteoblasts from nNOS KO mice, however, showed increased levels of alkaline phosphatase and no defects in proliferation or bone nodule formation in vitro, whereas osteoclastogenesis was increased in nNOS KO bone marrow cultures. These studies indicate that nNOS plays a hitherto unrecognized but important physiological role as a stimulator of bone turnover. The low level of nNOS expression in bone and the in vitro behavior of nNOS KO bone cells indicate that these actions are indirect and possibly mediated by a neurogenic relay.

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From Mouse to Man: Redefining the Role of Insulin-Like Growth Factor-I in the Acquisition of Bone Mass

Experimental Biology and Medicine ◽

10.1177/153537020322800302 ◽

2003 ◽

Vol 228 (3) ◽

pp. 245-252 ◽

Cited By ~ 72

Author(s):

Shoshana Yakar ◽

Clifford J. Rosen

Keyword(s):

Growth Factor ◽

Cell Function ◽

Bone Cells ◽

Bone Cell ◽

Insulin Like Growth Factor ◽

In Vivo Models ◽

Igf I

The insulin-like growth factor system (IGF) has been linked to the process of bone acquisition through epidemiologic analyses of large cohorts and in vitro studies of bone cells. But the exact relationship between expression of IGF-I in bone and skeletal homeostasis or pathologic conditions, such as osteoporosis, remains poorly defined. Recent advances in genomic engineering have resulted in the development of better in vivo models to test the role of IGF-I during development and maintenance of the adult skeleton. It is now established that skeletal expression of IGF-I is critical for differentiative bone cell function. It may also be essential for the full anabolic effects of parathyroid hormone on trabecular bone and for some component of biomineralization. Evidence from conditional mutagenesis studies suggests that serum IGF-I may represent more than a storage depot or permissive factor during the final phase of skeletal acquisition. This work re-examines the original tenets of the “somatomedin hypothesis” in light of these newer mouse models and their remarkable skeletal phenotypes. The implications are far reaching and suggest that newer approaches for manipulating the IGF regulatory system may one day be useful as therapeutic adjuncts for the treatment of osteoporosis.

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Megakaryocyte-driven changes in bone health: lessons from mouse models of myelofibrosis and related disorders

AJP Cell Physiology ◽

10.1152/ajpcell.00328.2021 ◽

2021 ◽

Author(s):

Mariya Stavnichuk ◽

Svetlana V. Komarova

Keyword(s):

Bone Marrow ◽

Mouse Models ◽

Cell Function ◽

Bone Cells ◽

Bone Marrow Cells ◽

Bone Cell ◽

Bone Architecture ◽

Bone Homeostasis

Over the years, numerous studies demonstrated reciprocal communications between processes of bone marrow hematopoiesis and bone remodeling. Megakaryocytes, rare bone marrow cells responsible for platelet production, were demonstrated to be involved in bone homeostasis. Myelofibrosis, characterized by an increase in pleomorphic megakaryocytes in the bone marrow, commonly leads to the development of osteosclerosis. In vivo, an increase in megakaryocyte number was shown to result in osteosclerosis in GATA-1low, NF-E2-/-, TPOhigh, Mpllf/f;PF4cre, Lnk-/-, Mpig6b-/-, Mpig6bfl/fl;Gp1ba-Cr+/KI, Pt-vWD mouse models. In vitro, megakaryocytes stimulate osteoblast proliferation and have variable effects on osteoclast proliferation and activity through soluble factors and direct cell-cell communications. Intriguingly, new studies revealed that the ability of megakaryocytes to communicate with bone cells is affected by the age and sex of animals. This mini-review summarises changes seen in bone architecture and bone cell function in mouse models with an elevated number of megakaryocytes and the effects megakaryocytes have on osteoblasts and osteoclasts in vitro, and discusses potential molecular players that can mediate these effects.

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Molecular Basis of Bone Aging

International Journal of Molecular Sciences ◽

10.3390/ijms21103679 ◽

2020 ◽

Vol 21 (10) ◽

pp. 3679 ◽

Cited By ~ 2

Author(s):

Addolorata Corrado ◽

Daniela Cici ◽

Cinzia Rotondo ◽

Nicola Maruotti ◽

Francesco Paolo Cantatore

Keyword(s):

Animal Studies ◽

Molecular Mechanisms ◽

Bone Cells ◽

Current Knowledge ◽

Cellular Tissue ◽

Bone Cell ◽

Age Related ◽

Bone Cell Differentiation ◽

Bone Changes

A decline in bone mass leading to an increased fracture risk is a common feature of age-related bone changes. The mechanisms underlying bone senescence are very complex and implicate systemic and local factors and are the result of the combination of several changes occurring at the cellular, tissue and structural levels; they include alterations of bone cell differentiation and activity, oxidative stress, genetic damage and the altered responses of bone cells to various biological signals and to mechanical loading. The molecular mechanisms responsible for these changes remain greatly unclear and many data derived from in vitro or animal studies appear to be conflicting and heterogeneous, probably due to the different experimental approaches; nevertheless, understanding the main physio-pathological processes that cause bone senescence is essential for the development of new potential therapeutic options for treating age-related bone loss. This article reviews the current knowledge concerning the molecular mechanisms underlying the pathogenesis of age-related bone changes.

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Modulation of Endocannabinoid Tone in Osteoblastic Differentiation of MC3T3-E1 Cells and in Mouse Bone Tissue over Time

Cells ◽

10.3390/cells10051199 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1199

Author(s):

Magdalena Kostrzewa ◽

Ali Mokhtar Mahmoud ◽

Roberta Verde ◽

Federica Scotto di Carlo ◽

Fernando Gianfrancesco ◽

...

Keyword(s):

Osteoblast Differentiation ◽

Cannabinoid Receptors ◽

Bone Growth ◽

Cell Function ◽

Bone Cell ◽

Mature Adult ◽

Age Related ◽

Over Time

Bone is a highly complex and metabolically active tissue undergoing a continuous remodeling process, which endures throughout life. A complex cell-signaling system that plays role in regulating different physiological processes, including bone remodeling, is the endocannabinoid system (ECS). Bone mass expresses CB1 and CB2 cannabinoid receptors and enzymatic machinery responsible for the metabolism of their endogenous ligands, endocannabinoids (AEA and 2-AG). Exogenous AEA is reported to increase the early phase of human osteoblast differentiation in vitro. However, regarding this cell context little is known about how endocannabinoids and endocannabinoid-related N-acylethanolamines like PEA and OEA are modulated, in vitro, during cell differentiation and, in vivo, over time up to adulthood. Here we characterized the endocannabinoid tone during the different phases of the osteoblast differentiation process in MC3T3-E1 cells, and we measured endocannabinoid levels in mouse femurs at life cycle stages characterized by highly active bone growth (i.e., of juvenile, young adult, and mature adult bone). Endocannabinoid tone was significantly altered during osteoblast differentiation, with substantial OEA increment, decline in 2-AG and AEA, and consistent modulation of their metabolic enzymes in maturing and mineralized MC3T3-E1 cells. Similarly, in femurs, we found substantial, age-related, decline in 2-AG, OEA, and PEA. These findings can expand existing knowledge underlying physiological bone cell function and contribute to therapeutic strategies for preventing bone-related metabolic changes accruing through lifespan.

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Protocol of Co-Culture of Human Osteoblasts and Osteoclasts to Test Biomaterials for Bone Tissue Engineering

Methods and Protocols ◽

10.3390/mps5010008 ◽

2022 ◽

Vol 5 (1) ◽

pp. 8

Author(s):

Giorgia Borciani ◽

Giorgia Montalbano ◽

Nicola Baldini ◽

Chiara Vitale-Brovarone ◽

Gabriela Ciapetti

Keyword(s):

Tissue Engineering ◽

Bone Tissue Engineering ◽

Bone Tissue ◽

Bone Cells ◽

Bone Cell ◽

Seeding Density ◽

Bone Homeostasis ◽

Bone Microenvironment

New biomaterials and scaffolds for bone tissue engineering (BTE) applications require to be tested in a bone microenvironment reliable model. On this assumption, the in vitro laboratory protocols with bone cells represent worthy experimental systems improving our knowledge about bone homeostasis, reducing the costs of experimentation. To this day, several models of the bone microenvironment are reported in the literature, but few delineate a protocol for testing new biomaterials using bone cells. Herein we propose a clear protocol to set up an indirect co-culture system of human-derived osteoblasts and osteoclast precursors, providing well-defined criteria such as the cell seeding density, cell:cell ratio, the culture medium, and the proofs of differentiation. The material to be tested may be easily introduced in the system and the cell response analyzed. The physical separation of osteoblasts and osteoclasts allows distinguishing the effects of the material onto the two cell types and to evaluate the correlation between material and cell behavior, cell morphology, and adhesion. The whole protocol requires about 4 to 6 weeks with an intermediate level of expertise. The system is an in vitro model of the bone remodeling system useful in testing innovative materials for bone regeneration, and potentially exploitable in different application fields. The use of human primary cells represents a close replica of the bone cell cooperation in vivo and may be employed as a feasible system to test materials and scaffolds for bone substitution and regeneration.

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Control of Oriented Extracellular Matrix Similar to Anisotropic Bone Microstructure

Materials Science Forum ◽

10.4028/www.scientific.net/msf.783-786.72 ◽

2014 ◽

Vol 783-786 ◽

pp. 72-77 ◽

Cited By ~ 2

Author(s):

Takayoshi Nakano ◽

Aira Matsugaki ◽

Takuya Ishimoto ◽

Mitsuharu Todai ◽

Ai Serizawa ◽

...

Keyword(s):

Extracellular Matrix ◽

Bone Tissue ◽

Bone Cells ◽

Crystal Surface ◽

Early Stage ◽

Bone Microstructure ◽

Bone Cell ◽

Collagen Fibers

Bone microstructure is dominantly composed of anisotropic extracellular matrix (ECM) in which collagen fibers and epitaxially-oriented biological apatite (BAp) crystals are preferentially aligned depending on the bone anatomical position, resulting in exerting appropriate mechanical function. The regenerative bone in bony defects is however produced without the preferential alignment of collagen fibers and the c-axis of BAp crystals, and subsequently reproduced to recover toward intact alignment. Thus, it is necessary to produce the anisotropic bone-mimetic tissue for the quick recovery of original bone tissue and the related mechanical ability in the early stage of bone regeneration. Our group is focusing on the methodology for regulating the arrangement of bone cells, the following secretion of collagen and the self-assembled mineralization by oriented BAp crystallites. Cyclic stretching in vitro to bone cells, principal-stress loading in vivo on scaffolds, step formation by slip traces on Ti single crystal, surface modification by laser induced periodic surface structure (LIPSS), anisotropic collagen substrate with the different degree of orientation, etc. can dominate bone cell arrangement and lead to the construction of the oriented ECM similar to the bone tissue architecture. This suggests that stress/strain loading, surface topography and chemical anisotropy are useful to produce bone-like microstructure in order to promote the regeneration of anisotropic bone tissue and to understand the controlling parameters for anisotropic osteogenesis induction.

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