19 research outputs found
Développement de modèles pour l'étude de la formation osseuse en culture tridimensionnelle et en ingénierie tissulaire osseuse
La culture tridimensionnelle (3D) et l'ingénierie du tissu osseux sont deux thématiques basées sur l'utilisation de matrices permettant de véhiculer des cellules ostéogéniques dans le but d'obtenir une formation osseuse in vitro et in vivo respectivement. La culture 3D est un enjeu important en biologie car elle permet de restaurer certaines propriétés tissulaires perdues en culture bidimensionnelle (2D) sur plastique. De nombreux travaux sont actuellement dédiés à la mise au point de matrices utilisables comme support de culture 3D des cellules osseuses. Sur la base d'une matrice constituée de particules de phosphate de calcium biphasé (BCP) j'ai mis au point un modèle original de culture 3D qui permet le développement d un tissu ostéoïde et la différenciation spontanée d'ostéoblastes humains en ostéocytes. Ce modèle 3D ouvre une nouvelle voie d étude des ostéocytes qui sont les cellules majoritaires du tissu osseux mais les plus mal connues du fait de leur accessibilité difficile et du manque de modèles d'étude disponibles. L'ingénierie tissulaire osseuse a pour but de reconstruire le stock osseux grâce à l'association de matrices, de facteurs ostéoinducteurs et/ou de cellules ostéogéniques. La majorité des travaux menés actuellement dans ce domaine préconisent l utilisation de cellules stromales mésenchymateuses (MSC) pour améliorer les performances de ces matrices. Cependant le mécanisme d action de ces cellules est encore peu documenté. Basé sur l'utilisation des mêmes particules de BCP, j'ai participé à la mise au point d'un nouveau biomatériau développé et breveté au laboratoire et à son utilisation comme véhicule de MSC de souris pour l'étude de la formation osseuse en site ectopique. La mise au point d'une méthode de suivi quantitatif de la survie des cellules implantées a permis de montrer que ces MSC disparaissaient très rapidement, laissant la place aux cellules de l'hôte qui sont à l'origine du tissu osseux. Nous avons conclu que, dans ce modèle, les MSC implantées jouent très probablement un rôle chimiotactique pour les cellules de l'organisme receveur. Une étude préliminaire des molécules impliquées dans ce rôle chimiotactique à été effectuée, permettant de proposer une nouvelle approche pour l ingénierie tissulaire osseuse.Three-dimensional culture (3D) of bone cells and bone tissue engineering are both based on the use of scaffolds to convey osteogenic cells and obtain in vitro and in vivo bone formation respectively. 3D culture is an important field in cell biology, dedicated to reduce the gap between two-dimensional culture and complex tissue architecture. Many works have described various scaffolds as support for the 3D culture of bone cells but in two studies only the presence of osteocyte-like cells have been detected after very long periods of culture. I have engineered an original model of 3D culture in which human primary osteoblasts are seeded within the interspace of calibrated biphasic calcium phosphate particles (BCP). This system results, after one week, in the development of an osteoid matrix and the spontaneous differentiation of the osteoblasts in osteocytes. This model of primary osteocyte differentiation in 3D is a new tool to gain insights into the biology of osteocytes, which compose over 90-95% of bone cells but are difficult to study due to their accessibility and the very rare models available in vitro. The aim of bone tissue engineering is to regenerate the bone stock through a combination of scaffolds, osteogenic factors and / or osteogenic cells. The majority of the studied in this field advocates the use of mesenchymal stromal cells (MSC) but the mechanism of action of these cells is still poorly documented. Based on the use of BCP particles, I have participated to the development of a new bone substitute, which has been patented in our laboratory. I have used this new biomaterial as a vehicle for mouse MSC in a model of ectopic bone formation. Using a method of quantitative tracking of the implanted cells, I have shown that the implanted MSC disappeared very quickly from the implants whereas host cells were progressively recruited suggesting that host cells are responsible for the bone formation. We have concluded that, in this model, MSC play a chemotactic function towards host cells. A preliminary study of the putative molecules involved in this phenomenon was performed with the aim of proposing a newNICE-BU Sciences (060882101) / SudocSudocFranceF
Adaptive Immune Response Inhibits Ectopic Mature Bone Formation Induced by BMSCs/BCP/Plasma Composite in Immune-Competent Mice
International audienceA combination of autologous bone marrow stromal cells (BMSCs) and biomaterials is a strategy largely developed in bone tissue engineering, and subcutaneous implantation in rodents or large animals is often a first step to evaluate the potential of new biomaterials. This study aimed at investigating the influence of the immune status of the recipient animal on BMSCs-induced bone formation. BMSCs prepared from C57BL/6 mice, composed of a mixture of mesenchymal stromal and monocytic cells, were combined with a biomaterial that consisted of biphasic calcium phosphate (BCP) particles and plasma clot. This composite was implanted subcutaneously either in syngenic C57BL/6 immune-competent mice or in T-lymphocyte-deficient Nude (Nude) mice. Using histology, immunohistochemistry, and histomorphometry, we show here that this BMSC/BCP/plasma clot composite implanted in Nude mice induces the formation of mature lamellar bone associated to hematopoietic areas and numerous vessels. Comparatively, implantation in C57BL/6 results in the formation of woven bone without hematopoietic tissue, a lower number of new vessels, and numerous multinucleated giant cells (MNGCs). In situ hybridization, which enabled to follow the fate of the BMSCs, revealed that BMSCs implanted in Nude mice survived longer than BMSCs implanted in C57BL/6 mice. Quantitative expression analysis of 280 genes in the implants indicated that the differences between C57BL/6 and Nude implants corresponded almost exclusively to genes related to the immune response. Gene expression profile in C57BL/6 implants was consistent with a mild chronic inflammation reaction characterized by Th1, Th2, and cytotoxic T-lymphocyte activation. In the implants retrieved from T-deficient Nude mice, Mmp14, Il6st, and Tgfbr3 genes were over-expressed, suggesting their putative role in bone regeneration and hematopoiesis. In conclusion, we show here that the T-mediated inflammatory microenvironment is detrimental to BMSCs-induced bone formation and shortens the survival of implanted cells. Conversely, the lack of T-lymphocyte reaction in T-deficient animals is beneficial to BMSCs-induced mature bone formation. This should be taken into account when evaluating cell/biomaterial composites in these models
Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells
International audienceWe have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate-hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function
Ectopic bone formation using an injectable biphasic calcium phosphate/Si-HPMC hydrogel composite loaded with undifferentiated bone marrow stromal cells.
We have used a new synthetic injectable composite constituted of hydroxyapatite/tricalcium phosphate (HA/TCP) particles in suspension in a self-hardening Si-hydroxypropylmethylcellulose (HPMC) hydrogel. The aim of this study was to evaluate in vivo the biocompatibility and the new bone formation efficacy of this scaffold loaded with undifferentiated bone marrow stromal cells (BMSCs). This biomaterial was mixed extemporaneously with BMSCs prepared from C57BL/6 mice, injected in subcutaneous and intramuscular sites and retrieved 4 and 8 weeks after implantation. Dissection of the implants revealed a hard consistency and the absence of a fibrous capsule reflecting a good integration into the host tissues. Histological analysis showed mineralized woven bone in the granule inter-space with numerous active osteoclasts attached to the particles as assessed by the presence of multinucleated cells positively stained for TRAP activity and for the a3 subunit of the V-ATPase. Small vessels were homogenously distributed in the whole implants. Similar results were obtained in SC and IM sites and no bone formation was observed in the control groups when cell-free and particle-free transplants were injected. These results indicate that this injectable biphasic calcium phosphate-hydrogel composite mixed with undifferentiated BMSCs is a new promising osteoinductive bone substitute. It also provides with an original in vivo model of osteoclast differentiation and function
Monocytes differentiation upon treatment with a peptide corresponding to the C-terminus of activated T cell-expressed Tirc7 protein
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Fate of bone marrow stromal cells in a syngenic model of bone formation.
International audienceBone marrow stromal cells (BMSCs) have been demonstrated to induce bone formation when associated to osteoconductive biomaterials and implanted in vivo. Nevertheless, their role in bone reconstruction is not fully understood and rare studies have been conducted to follow their destiny after implantation in syngenic models. The aim of the present work was to use sensitive and quantitative methods to track donor and recipient cells after implantation of BMSCs in a syngenic model of ectopic bone formation. Using polymerase chain reaction (PCR) amplification of the Sex determining Region Y (Sry) gene and in situ hybridization of the Y chromosome in parallel to histological analysis, we have quantified within the implants the survival of the donor cells and the colonization by the recipient cells. The putative migration of the BMSCs in peripheral organs was also analyzed. We show here that grafted cells do not survive more than 3 weeks after implantation and might migrate in peripheral lymphoid organs. These cells are responsible for the attraction of host cells within the implants, leading to the centripetal colonization of the biomaterial by new bone
In vivo resorption of injectable apatitic calcium phosphate cements: Critical role of the intergranular microstructure
International audienceThe in vivo resorption rate of two injectable apatitic calcium phosphate cements used in clinics (Graftys® HBS and NORIAN®) was compared, using a GLP study based on an animal model of critical-sized bone defect. To rationalize the markedly different biological properties observed for both cements, key physical features were investigated, including permeability and water-accessible porosity, total porosity measured by mercury intrusion and gravimetry, and microstructure. Due to a different concept for creating porosity between the two cements investigated in this study, a markedly different microstructural arrangement of apatite crystals was observed in the intergranular space, which was found to significantly influence both the mechanical strength and in vivo degradation of the two CPCs
Calcium supplementation decreases BCP-induced inflammatory processes in blood cells through the NLRP3 inflammasome down-regulation
International audienceInteraction of host blood with biomaterials is the first event occurring after implantation in a bone defect. This study aimed at investigating the cellular and molecular consequences arising at the interface between whole blood and biphasic calcium phosphate (BCP) particles. We observed that, due to calcium capture, BCP inhibited blood coagulation, and that this inhibition was reversed by calcium supplementation. Therefore, we studied the impact of calcium supplementation on BCP effects on blood cells. Comparative analysis of BCP and calcium supplemented-BCP (BCP/Ca) effects on blood cells showed that BCP as well as BCP/Ca induced monocyte proliferation, as well as a weak but significant hemolysis. Our data showed for the first time that calcium supplementation of BCP microparticles had anti-inflammatory properties compared to BCP alone that induced an inflammatory response in blood cells. Our results strongly suggest that the anti-inflammatory property of calcium supplemented-BCP results from its down-modulating effect on P2X7R gene expression and its capacity to inhibit ATP/P2X7R interactions, decreasing the NLRP3 inflammasome activation. Considering that monocytes have a vast regenerative potential, and since the excessive inflammation often observed after bone substitutes implantation limits their performance, our results might have great implications in terms of understanding the mechanisms leading to an efficient bone reconstruction
Human Primary Osteocyte Differentiation in a Three-Dimensional Culture System.
International audienceAbstract Introduction: Investigations on primary osteocytes, which compose over 90-95% of bone cells, embedded throughout the mineralized matrix, is a major challenge due to their difficult accessibility and the very rare models available in vitro. We engineered a three-dimensional (3D) culture method of primary human osteoblast differentiation into osteocytes. These 3D-differentiated osteocytes were compared with 2D-cultured cells and with human microdissected cortical osteocytes obtained from bone cryosections. Materials and Methods: Human primary osteoblasts were seeded either within the interspace of calibrated biphasic calcium phosphate particles or on plastic culture dishes and cultured for four weeks in the absence of differentiation factors. Osteocyte differentiation was assessed by histological and immunohistological analysis after paraffin embedding of culture after various times as well as by quantitative RT-PCR analysis of a panel of osteoblast and osteocyte markers after nucleic acid extraction. Results: Histological analysis revealed, after only one week, the presence of an osteoid matrix including many lacunae in which the cells were individually embedded, exhibiting characteristics of osteocyte-like cells. Real time PCR expression of a set of bone-related genes confirmed their osteocyte phenotype. Comparison with plastic-cultured cells and mature osteocytes microdissected from human cortical bone allowed to assess their maturation stage as osteoid-osteocytes. Conclusions: This model of primary osteocyte differentiation is a new tool to gain insights into the biology of osteocytes. It should be a suitable method to study the osteoblast-osteocyte differentiation pathway, the osteocyte interaction with the other bone cells and orchestration of bone remodeling transmitted by mechanical loading and shear stress. It should be used in important cancer research areas such as the crosstalk of osteocytes with tumor cells in bone metastasis since it has been recently shown that gene expression in osteocytes is strongly affected by cancer cells of different origin. It could be also a very efficient tool for drug testing and bone tissue engineering applications