The Impairment of Osteogenesis in Bone Sialoprotein (BSP) Knockout Calvaria Cell Cultures Is Cell Density Dependent

Bouet, Guénaëlle; Bouleftour, Wafa; Juignet, Laura; Linossier, Marie‐Thérèse; Thomas, Mireille; Vanden‐Bossche, Arnaud; Aubin, Jane E.; Vico, Laurence; Marchat, David; Malaval, Luc

doi:10.1371/journal.pone.0117402

Cited by 34 publications

(34 citation statements)

References 47 publications

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“…Seeding of scaffolds led to a starting cell density (≈ 10,000 cells/cm 2 ) for this specific cell type, which is close to standard plating densities on plastic (e.g. 5,000 cells/cm², (Bouet et al, 2015a)). Mouse calvaria cells grown in 2D at this cell density form osteoblast colonies expressing bone markers and laying down an osteoid matrix which mineralises as HA (bone nodules (Bellows et al, 1998).…”

Section: G Bouet Et Alsupporting

confidence: 66%

“…These values are similar to what has been reported for human trabecular bone tissue (Guo, 2000), where the macroscopic elastic modulus varies between 1 and 5 GPa depending on the anatomical site and age, while the microscopic modulus (obtained by nanoindentation) ranges from 11 GPa to 18 GPa (Guo, 2000). It is well known that the mechanical properties of scaffolds affect cellular behaviour and fate (Bouet et al, 2015b). Indeed, cells have the ability to sense and probe the stiffness of their surroundings as they adhere and interact with the extracellular matrix (ECM) (Discher et al, 2005).…”

Section: G Bouet Et Almentioning

confidence: 99%

“…cell seeding, matrix deposition and mineralisation) and its supporting environment (e.g. nutrient supply, waste removal), which necessitates optimisation of the scaffold and the bioreactor (Bouet et al, 2015b).…”

Section: Introductionmentioning

confidence: 99%

“…compressive strength). Therefore, we decided to work with synthetic calcium phosphates (CaP) macroporous ceramics which present most of the qualities required for in vitro bone applications, such as controllable chemistry and architecture, biocompatibility, tuneable biodegradability, osteoconductivity, stiffness similar to bone, and adequate chemical surface functional groups (Bose et al, 2013;Bouet et al, 2015b;Shepherd and Best, 2011;Shepherd et al, 2012). The brittle nature of these ceramics can be partly compensated by architectural optimisation (Bouet et al, 2015b).…”

Section: Introductionmentioning

confidence: 99%

“…Therefore, we decided to work with synthetic calcium phosphates (CaP) macroporous ceramics which present most of the qualities required for in vitro bone applications, such as controllable chemistry and architecture, biocompatibility, tuneable biodegradability, osteoconductivity, stiffness similar to bone, and adequate chemical surface functional groups (Bose et al, 2013;Bouet et al, 2015b;Shepherd and Best, 2011;Shepherd et al, 2012). The brittle nature of these ceramics can be partly compensated by architectural optimisation (Bouet et al, 2015b). Among existing tools, perfusion-based bioreactors represent the most efficient culture system to provide uniform nutrient transport, waste elimination as well as cell distribution, growth and osteoblastic differentiation in the interior of the scaffold (Goldstein et al, 2001;Szpalski et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

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Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

Bouet¹,

Cruel²,

Laurent³

et al. 2015

eCM

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An engineered three dimensional (3D) in vitro cell culture system was designed with the goal of inducing and controlling in vitro osteogenesis in a reproducible manner under conditions more similar to the in vivo bone microenvironment than traditional two-dimensional (2D) models. This bioreactor allows efficient mechanical loading and perfusion of an original cubic calcium phosphate bioceramic of highly controlled composition and structure. This bioceramic comprises an internal portion containing homogeneously interconnected macropores surrounded by a dense layer, which minimises fluid flow bypass around the scaffold. This dense and flat layer permits the application of a homogeneous loading on the bioceramic while also enhancing its mechanical strength. Numerical modelling of constraints shows that the system provides direct mechanical stimulation of cells within the scaffold. Experimental results establish that under perfusion at a steady flow of 2 µL/min, corresponding to 3 ≤ Medium velocity ≤ 23 µm/s, mouse calvarial cells grow and differentiate as osteoblasts in a reproducible manner, and lay down a mineralised matrix. Moreover, cells respond to mechanical loading by increasing C-fos expression, which demonstrates the effective mechanical stimulation of the culture within the scaffold. In summary, we provide a "proof-of-concept" for osteoblastic cell culture in a controlled 3D culture system under perfusion and mechanical loading. This model will be a tool to analyse bone cell functions in vivo, and will provide a bench testing system for the clinical assessment of bioactive bone-targeting molecules under load.

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Section: G Bouet Et Alsupporting

confidence: 66%

Section: G Bouet Et Almentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

Bouet¹,

Cruel²,

Laurent³

et al. 2015

eCM

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Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP‐/‐ Mice, Along With Faster Hematopoietic Recolonization

Bouleftour

Granito

Vanden‐Bossche

et al. 2017

Journal Cellular Physiology

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The bone organ integrates the activity of bone tissue, bone marrow, and blood vessels and the factors ensuring this coordination remain ill defined. Bone sialoprotein (BSP) is with osteopontin (OPN) a member of the small integrin binding ligand N-linked glycoprotein (SIBLING) family, involved in bone formation, hematopoiesis and angiogenesis. In rodents, bone marrow ablation induces a rapid formation of medullary bone which peaks by ∼8 days (d8) and is blunted in BSP-/- mice. We investigated the coordinate hematopoietic and vascular recolonization of the bone shaft after marrow ablation of 2 month old BSP+/+ and BSP-/- mice. At d3, the ablated area in BSP-/- femurs showed higher vessel density (×4) and vascular volume (×7) than BSP+/+. Vessel numbers in the shaft of ablated BSP+/+ mice reached BSP-/- values only by d8, but with a vascular volume which was twice the value in BSP-/-, reflecting smaller vessel size in ablated mutants. At d6, a much higher number of Lin (×3) as well as LSK (Lin IL-7Rα Sca-1 c-Kit , ×2) and hematopoietic stem cells (HSC: Flt3 LSK, ×2) were counted in BSP-/- marrow, indicating a faster recolonization. However, the proportion of LSK and HSC within the Lin was lower in BSP-/- and more differentiated stages were more abundant, as also observed in unablated bone, suggesting that hematopoietic differentiation is favored in the absence of BSP. Interestingly, unablated BSP-/- femur marrow also contains more blood vessels than BSP+/+, and in both intact and ablated shafts expression of VEGF and OPN are higher, and DMP1 lower in the mutants. In conclusion, bone marrow ablation in BSP-/- mice is followed by a faster vascular and hematopoietic recolonization, along with lower medullary bone formation. Thus, lack of BSP affects the interplay between hematopoiesis, angiogenesis, and osteogenesis, maybe in part through higher expression of VEGF and the angiogenic SIBLING, OPN. J. Cell. Physiol. 232: 2528-2537, 2017. © 2016 Wiley Periodicals, Inc.

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The role of Meteorin‐like in skeletal development and bone fracture healing

Ron

Balu

Molitoris

et al. 2022

Journal Orthopaedic Research

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Meteorin‐like protein (Metrnl), homologous to the initially identified neurotrophic factor Meteorin, is a secreted, multifunctional protein. Here we used mouse models to investigate Metrnl's role in skeletal development and bone fracture healing. During development Metrnl was expressed in the perichondrium and primary ossification center. In neonates, single cell RNA‐seq of diaphyseal bone demonstrated strongest expression of Metrnl transcript by osteoblasts. In vitro, Metrnl was osteoinductive, increasing osteoblast differentiation and mineralization in tissue culture models. In vivo, loss of Metrnl expression resulted in no change in skeletal metrics in utero, at birth, or during postnatal growth. Six‐week‐old Metrnl‐null mice displayed similar body length, body weight, tibial length, femoral length, BV/TV, trabecular number, trabecular thickness, and cortical thickness as littermate controls. In 4‐month‐old mice, lack of Metrnl expression did not change structural stiffness, ultimate force, or energy to fracture of femora under 3‐point‐bending. Last, we investigated the role of Metrnl in bone fracture healing. Metrnl expression increased in response to tibial injury, however, loss of Metrnl expression did not affect the amount of bone deposited within the healing tissue nor did it change the structural parameters of healing tissue. This work identifies Metrnl as a dispensable molecule for skeletal development. However, the osteoinductive capabilities of Metrnl may be utilized to modulate osteoblast differentiation in cell‐based orthopedic therapies.

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The Impairment of Osteogenesis in Bone Sialoprotein (BSP) Knockout Calvaria Cell Cultures Is Cell Density Dependent

Cited by 34 publications

References 47 publications

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

Validation of an in vitro 3D bone culture model with perfused and mechanically stressed ceramic scaffold

Bone Shaft Revascularization After Marrow Ablation Is Dramatically Accelerated in BSP‐/‐ Mice, Along With Faster Hematopoietic Recolonization

The role of Meteorin‐like in skeletal development and bone fracture healing

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