Publications by authors named "Jean-Michel Bouler"

49 Publications

Proteomic analysis identified LBP and CD14 as key proteins in blood/biphasic calcium phosphate microparticle interactions.

Acta Biomater 2021 06 6;127:298-312. Epub 2021 Apr 6.

Université Côte d'Azur, CNRS (UMR 7277), Inserm (U1091), iBV, Nice, France. Electronic address:

Immediately upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to the biomaterial surface and the protein layer affects both blood cell functions and biomaterial bioactivity. Previously, we reported that 80-200 µm biphasic calcium phosphate (BCP) microparticles embedded in a blood clot, induce ectopic woven bone formation in mice, when 200-500 µm BCP particles induce mainly fibrous tissue. Here, in a LC-MS/MS proteomic study we compared the differentially expressed blood proteins (plasma and blood cell proteins) and the deregulated signaling pathways of these osteogenic and fibrogenic blood composites. We showed that blood/BCP-induced osteogenesis is associated with a higher expression of fibrinogen (FGN) and an upregulation of the Myd88- and NF-κB-dependent TLR4 signaling cascade. We also highlighted the key role of the LBP/CD14 proteins in the TLR4 activation of blood cells by BCP particles. As FGN is an endogenous ligand of TLR4, able to modulate blood composite stiffness, we propose that different FGN concentrations modify the blood clot mechanical properties, which in turn modulate BCP/blood composite osteoactivity through TLR4 signaling. The present findings provide an insight at the protein level, into the mechanisms leading to an efficient bone reconstruction by blood/BCP composites. STATEMENT OF SIGNIFICANCE: Upon implantation, scaffolds for bone repair are exposed to the patient's blood. Blood proteins adhere to bone substitute surface and this protein layer affects both biomaterial bioactivity and bone healing. Therefore, for the best outcome for patients, it is crucial to understand the molecular interactions between blood and bone scaffolds. Biphasic calcium phosphate (BCP) ceramics are considered as the gold standard in bone reconstruction surgery. Here, using proteomic analyses we showed that the osteogenic properties of 80-200 µm BCP particles embedded in a blood clot is associated with a higher expression of fibrinogen. Fibrinogen upregulates the Myd88- and NF-κB-dependent TLR4 pathway in blood cells and, BCP-induced TLR4 activation is mediated by the LBP and CD14 proteins.
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http://dx.doi.org/10.1016/j.actbio.2021.03.070DOI Listing
June 2021

Combination of biocompatible hydrogel precursors to apatitic calcium phosphate cements (CPCs): Influence of the in situ hydrogel reticulation on the CPC properties.

J Biomed Mater Res B Appl Biomater 2021 Jan 23;109(1):102-116. Epub 2020 Jul 23.

Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, France.

In the field of bone regenerative medicine, injectable calcium phosphate cements (CPCs) are used for decades in clinics, as bone void fillers. Most often preformed polymers (e.g., hyaluronic acid, collagen, chitosan, cellulose ethers…) are introduced in the CPC formulation to make it injectable and improve its cohesion. Once the cement has hardened, the polymer is simply trapped in the CPC structure and no organic subnetwork is present. By contrast, in this work a CPC was combined with organic monomers that reticulated in situ so that a continuous biocompatible 3D polymeric subnetwork was formed in the CPC microstructure, resulting in a higher permeability of the CPC, which might allow to accelerate its in vivo degradation. Two options were investigated depending on whether the polymer was formed before the apatitic inorganic network or concomitantly. In the former case, conditions were found to reach a suitable rheology for easy injection of the composite. In addition, the in situ formed polymer was shown to strongly affect the size, density, and arrangement of the apatite crystals formed during the setting reaction, thereby offering an original route to modulate the microstructure and porosity of apatitic cements.
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http://dx.doi.org/10.1002/jbm.b.34685DOI Listing
January 2021

In vivo resorption of injectable apatitic calcium phosphate cements: Critical role of the intergranular microstructure.

J Biomed Mater Res B Appl Biomater 2020 02 29;108(2):367-376. Epub 2019 Apr 29.

CEISAM, Université de Nantes, CNRS, UMR 6230, Nantes, France.

The in vivo resorption rate of two injectable apatitic calcium phosphate cements used in clinics (Graftys® HBS and NORIAN®) was compared, using a good laboratory practice (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 calcium phosphate cements.
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http://dx.doi.org/10.1002/jbm.b.34395DOI Listing
February 2020

Delivery systems of local anesthetics in bone surgery: are they efficient and safe?

Drug Discov Today 2018 11 26;23(11):1897-1903. Epub 2018 Jun 26.

CEISAM, CNRS UMR 6230, University of Nantes, Nantes, France; Faculty of Pharmaceutical Sciences, University of Nantes, Nantes, France. Electronic address:

Management of postoperative pain following bone surgery includes administration of local anesthetics (LAs). Smart delivery systems, including triggered systems, have been designed to provide a continuous release of LA in situ. However, these systems can provide a high level of LA locally. This review will examine the state-of-the-art regarding the LA delivery systems optimized for management of postoperative pain in bone surgery and will discuss the potential adverse effects of LAs on the overall pathways of bone healing, including the inflammation response phase, hemostasis phase, tissue repair phase and remodeling phase. There is a clinical need to document these effects and the potential impacts on the clinical outcome of the patient.
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http://dx.doi.org/10.1016/j.drudis.2018.06.019DOI Listing
November 2018

Pain Management After Bone Reconstruction Surgery Using an Analgesic Bone Cement: A Functional Noninvasive In Vivo Study Using Gait Analysis.

J Pain 2018 10 15;19(10):1169-1180. Epub 2018 May 15.

CEISAM, CNRS UMR 6230, University of Nantes, Nantes, France.

Postoperative pain after bone reconstruction is a serious complication that could jeopardize the global success of a surgery. This pain must be controlled and minimized during the first 3 to 4 postoperative days to prevent it from becoming chronic. In this study, a critical-size bone defect was created at the femoral distal end of rats and filled by an injectable calcium phosphate cement (CPC) loaded or not with local anesthetics (bupivacaine or ropivacaine). A functional evaluation of the gait was performed using the CatWalk system to compare the postoperative pain relief enhanced by the different CPCs after such a bone filling surgery. The results demonstrated significant pain relief during the short-term postoperative period, as shown by the print area and intensity parameters of the operated paw. At 24hours, the print area decreased by 65%, 42%, and 24%, and the intensity decreased by 25%, 9%, and 1% for unloaded, ropivacaine-loaded, and bupivacaine-loaded CPCs, respectively, compared with the preoperative values. Bupivacaine-loaded CPC provided an earlier return to full functional recovery than ropivacaine-loaded CPC. Moreover, the CPCs retained their biologic and mechanical properties. For all these reasons, anesthetic-loaded CPCs could be part of the global pain management protocol after bone reconstruction surgery such as iliac crest bone grafting procedures.

Perspective: Bupivacaine-loaded CPC provided an earlier return to full gait function than ropivacaine-loaded CPC, with preserved bone filling properties. Such analgesic CPCs deserve further in vivo investigation and may be part of the global pain management protocol after bone reconstruction or bone augmentation surgery such as iliac crest bone grafting.
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http://dx.doi.org/10.1016/j.jpain.2018.04.014DOI Listing
October 2018

Combination of blood and biphasic calcium phosphate microparticles for the reconstruction of large bone defects in dog: A pilot study.

J Biomed Mater Res A 2018 07 24;106(7):1842-1850. Epub 2018 Mar 24.

Université Côte d'Azur, CNRS, Inserm, iBV, France.

We previously reported that biphasic calcium phosphate (BCP) microparticles embedded in a blood clot induces ectopic bone formation in mice and repairs a critical femoral defect in rat. The present pilot study aimed to evaluate in dog and in two models of large defects the efficacy of this composite named "blood for reconstruction of bone" (BRB). We show here that BRB is a cohesive biomaterial easy to prepare from dog autologous blood and to mold to fill large bone defects. First in a model of cylindrical femoral condyle defect, the BRB was compared with BCP particles alone. After 8 weeks, this revealed that the amount of mature bone was slightly and significantly higher with BRB than with BCP particles. Second, in a model consisting in a 2 cm-long critical interruptive defect of the ulna, the BRB was compared with autologous bone. After 6 months, we observed that implantation of BRB can induce the complete reconstruction of the defect and that newly formed bone exhibits high regenerative potential. Comparison with the results obtained with autologous bone grafting strongly suggests that the BRB might be an efficient biomaterial to repair large bone defects, as an alternative or in addition to autologous bone. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1842-1850, 2018.
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http://dx.doi.org/10.1002/jbm.a.36384DOI Listing
July 2018

Design and properties of a novel radiopaque injectable apatitic calcium phosphate cement, suitable for image-guided implantation.

J Biomed Mater Res B Appl Biomater 2018 11 11;106(8):2786-2795. Epub 2017 Dec 11.

Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, BP 92208, 44322, NANTES Cedex 3, France.

An injectable purely apatitic calcium phosphate cement (CPC) was successfully combined to a water-soluble radiopaque agent (i.e., Xenetix ), to result in an optimized composition that was found to be as satisfactory as poly(methyl methacrylate) (PMMA) formulations used for vertebroplasty, in terms of radiopacity, texture and injectability. For that purpose, the Xenetix dosage in the cement paste was optimized by injection of the radiopaque CPC in human cadaveric vertebrae under classical PMMA vertebroplasty conditions, performed by interventional radiologists familiar with this surgical procedure. When present in the cement paste up to 70 mg I mL , Xenetix did not influence the injectability, cohesion, and setting time of the resulting composite. After hardening of the material, the same observation was made regarding the microstructure, mechanical strength and alpha-tricalcium phosphate to calcium deficient apatite transformation rate. Upon implantation in bone in a small animal model (rat), the biocompatibility of the Xenetix-containing CPC was evidenced. Moreover, an almost quantitative release of the contrast agent was found to occur rapidly, on the basis of in vitro static and dynamic quantitative studies simulating in vivo implantation. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 2786-2795, 2018.
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http://dx.doi.org/10.1002/jbm.b.34059DOI Listing
November 2018

A straightforward approach to enhance the textural, mechanical and biological properties of injectable calcium phosphate apatitic cements (CPCs): CPC/blood composites, a comprehensive study.

Acta Biomater 2017 10 30;62:328-339. Epub 2017 Aug 30.

Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, 2, rue de la Houssinière, BP 92208, 44322 NANTES Cedex 3, France. Electronic address:

Two commercial formulations of apatitic calcium phosphate cements (CPCs), Graftys® Quickset (QS) and Graftys® HBS (HBS), similar in composition but with different initial setting time (7 and 15min, respectively), were combined to ovine whole blood. Surprisingly, although a very cohesive paste was obtained after a few minutes, the setting time of the HBS/blood composite dramatically delayed when compared to its QS analogue and the two blood-free references. Using solid state NMR, scanning electron microscopy and high frequency impedance measurements, it was shown that, in the particular case of the HBS/blood composite, formation of a reticulated and porous organic network occurred in the intergranular space, prior to the precipitation of apatite crystals driven by the cement setting process. The resulting microstructure conferred unique biological properties to this material upon implantation in bone defects, since its degradation rate after 4 and 12weeks was more than twice that for the three other CPCs, with a significant replacement by newly formed bone.

Statement Of Significance: A major challenge in the design of bone graft substitutes is the development of injectable, cohesive, resorbable and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with initial mechanical properties as close as bone ones. Thus, we describe specific conditions in CPC-blood composites where the formation of a 3D clot-like network can interact with the precipitated apatite crystals formed during the cement setting process. The resulting microstructure appears more ductile at short-term and more sensitive to biological degradation which finally promotes new bone formation. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology.
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http://dx.doi.org/10.1016/j.actbio.2017.08.040DOI Listing
October 2017

Solid-state P and H chemical MR micro-imaging of hard tissues and biomaterials with magic angle spinning at very high magnetic field.

Sci Rep 2017 08 15;7(1):8224. Epub 2017 Aug 15.

CNRS, CEMHTI UPR3079, Université d'Orléans, F-45071, Orléans, France.

In this work, we show that it is possible to overcome the limitations of solid-state MRI for rigid tissues due to large line broadening and short dephasing times by combining Magic Angle Spinning (MAS) with rotating pulsed field gradients. This allows recording ex vivo P 3D and 2D slice-selected images of rigid tissues and related biomaterials at very high magnetic field, with greatly improved signal to noise ratio and spatial resolution when compared to static conditions. Cross-polarization is employed to enhance contrast and to further depict spatially localized chemical variations in reduced experimental time. In these materials, very high magnetic field and moderate MAS spinning rate directly provide high spectral resolution and enable the use of frequency selective excitation schemes for chemically selective imaging. These new possibilities are exemplified with experiments probing selectively the 3D spatial distribution of apatitic hydroxyl protons inside a mouse tooth with attached jaw bone with a nominal isotropic resolution nearing 100 µm.
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http://dx.doi.org/10.1038/s41598-017-08458-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5557955PMC
August 2017

Calcium supplementation decreases BCP-induced inflammatory processes in blood cells through the NLRP3 inflammasome down-regulation.

Acta Biomater 2017 07 17;57:462-471. Epub 2017 May 17.

Université Côte d'Azur, CNRS, Inserm, iBV, France.

Interaction 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.

Statement Of Significance: Although scaffolds and biomaterials unavoidably come into direct contact with blood during bone defect filling, whole blood-biomaterials interactions have been poorly explored. By studying in 3D the interactions between biphasic calcium phosphate (BCP) in microparticulate form and blood, we showed for the first time that calcium supplementation of BCP microparticles (BCP/Ca) has anti-inflammatory properties compared to BCP-induced inflammation in whole blood cells and provided information related to the molecular mechanisms involved. The present study also showed that BCP, as well as BCP/Ca particles stimulate monocyte proliferation. As monocytes represent a powerful target for regenerative therapies and as an excessive inflammation limits the performance of biomaterials in bone tissue engineering, our results might have great implications to improve bone reconstruction.
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http://dx.doi.org/10.1016/j.actbio.2017.05.039DOI Listing
July 2017

An analysis model for investigating the staining effect of various chlorhexidine-based mouthwashes.

J Clin Exp Dent 2017 Mar 1;9(3):e410-e416. Epub 2017 Mar 1.

Prof, PhD, UFR d'odontologie. 1, Place A. Ricordeau, 44082, Nantes cedex2.

Background: There are different mouthwashes containing chlorhexidine in different concentrations, as well as various excipients. Chlorhexidine induce stains or discoloration in teeth and mucous membranes. The aim of this work was to design a model to reproduce staining associated with the use of different mouthwashes containing chlorhexidine.

Material And Methods: We used as substrates of natural teeth and elephant ivory slices. Different incubation baths were conducted over 21 days in culture dishes at 37°C. At the beginning of experiment before incubation (D0) and after 21 days (D21) of incubation with different mouthwashes, pictures of substrates were taken in a standardized manner and an image analysis software was used to analyse and quantify the staining under the various conditions by using the 3 main colours (Red, Green, Blue, RGB).

Results: The results of this work demonstrate a very good reproducibility of the protocol, and secondly, a different expression statistically significant of the primary blue colour. We suggest that for a given concentration of chlorhexidine, the staining effects may vary depending on the excipients used.

Conclusions: This replicable model, easy to implement over a relatively short duration, can be used for evaluation of existing mouthwashes, and to test the excipients anti discoloration proposed by manufacturers. In vitro, chlorhexidine, mouthwashes, dental stain, tooth discoloration.
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http://dx.doi.org/10.4317/jced.53375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5347291PMC
March 2017

Gallium enhances reconstructive properties of a calcium phosphate bone biomaterial.

J Tissue Eng Regen Med 2018 02 23;12(2):e854-e866. Epub 2017 May 23.

Université Nice Sophia Antipolis, CNRS, Inserm, iBV, Nice, France.

Calcium phosphate (CaP)-based biomaterials are commonly used in bone reconstructive surgery to replace the damaged tissue, and can also serve as vectors for local drug delivery. Due to its inhibitory action on osteoclasts, the semi-metallic element gallium (Ga) is used for the systemic treatment of disorders associated with accelerated bone resorption. As it was demonstrated that Ga could be incorporated in the structure of CaP biomaterials, we investigated the biological properties of Ga-loaded CaP biomaterials. Culturing bone cells on Ga-CaP, we observed a decrease in osteoclast number and a downregulation of late osteoclastic markers expression, while Ga-CaP upregulated the expression of osteoblastic marker genes involved in the maturation of bone matrix. We next investigated in vivo bone reconstructive properties of different Ga-loaded biomaterials using a murine bone defect healing model. All implanted biomaterials showed a good osseointegration into the surrounding host tissue, accompanied by a successful bone ingrowth and bone marrow reconstruction, as evidenced by histological analysis. Moreover, quantitative micro-computed tomography analysis of implants revealed that Ga enhanced total defect filling. Lastly, we took advantage for the first time of a particular mode of non-linear microscopy (second harmonic generation) to quantify in vivo bone tissue reconstruction within a CaP bone substitute. By doing so, we showed that Ga exerted a positive impact on mature organized collagen synthesis. As a whole, our data support the hypothesis that Ga represents an attractive additive to CaP biomaterials for bone reconstructive surgery. Copyright © 2017 John Wiley & Sons, Ltd.
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http://dx.doi.org/10.1002/term.2396DOI Listing
February 2018

Gallium, a promising candidate to disrupt the vicious cycle driving osteolytic metastases.

Biochem Pharmacol 2016 Sep 1;116:11-21. Epub 2016 Jul 1.

INSERM, U791, LIOAD, Nantes F-44042, France. Electronic address:

Bone metastases of breast cancer typically lead to a severe osteolysis due to an excessive osteoclastic activity. On the other hand, the semi-metallic element gallium (Ga) displays an inhibitory action on osteoclasts, and therefore on bone resorption, as well as antitumour properties. Thus, we explored in vitro Ga effects on osteoclastogenesis in an aggressive bone metastatic environment based on the culture of pre-osteoclast RAW 264.7 cells with conditioned medium from metastatic breast tumour cells, i.e. the breast tumour cell line model MDA-MB-231 and its bone-seeking clone MDA-231BO. We first observed that Ga dose-dependently inhibited the tumour cells-induced osteoclastic differentiation of RAW 264.7 cells. To mimic a more aggressive environment where pro-tumourigenic factors are released from bone matrix due to osteoclastic resorption, metastatic breast tumour cells were stimulated with TGF-β, a mayor cytokine in bone metastasis vicious cycle. In these conditions, we observed that Ga still inhibited cancer cells-driven osteoclastogenesis. Lastly, we evidenced that Ga affected directly and strongly the proliferation/viability of both cancer cell lines, as well as the expression of major osteolytic factors in MDA-231BO cells. With the exception of two small scale clinical studies from 1980s, this is the first time that antitumour properties of Ga have been specifically studied in the context of bone metastases. Our data strongly suggest that, through its action against the vicious cycle involving bone cells and tumour cells, Ga represents a relevant and promising candidate for the local treatment of bone metastases in patients with breast cancer.
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http://dx.doi.org/10.1016/j.bcp.2016.06.020DOI Listing
September 2016

A simple and effective approach to prepare injectable macroporous calcium phosphate cement for bone repair: Syringe-foaming using a viscous hydrophilic polymeric solution.

Acta Biomater 2016 Feb 26;31:326-338. Epub 2015 Nov 26.

Université de Nantes, INSERM UMRS 791, Laboratoire d'Ingénierie Ostéo-Articulaire et Dentaire, 1 place Alexis Ricordeau, BP 84215, 44042 Nantes Cedex 1, France; CHU de Nantes, Nantes University Hospital, PHU 4 OTONN, 1 Pl A. Ricordeau Nantes, France. Electronic address:

Unlabelled: In this study, we propose a simple and effective strategy to prepare injectable macroporous calcium phosphate cements (CPCs) by syringe-foaming via hydrophilic viscous polymeric solution, such as using silanized-hydroxypropyl methylcellulose (Si-HPMC) as a foaming agent. The Si-HPMC foamed CPCs demonstrate excellent handling properties such as injectability and cohesion. After hardening the foamed CPCs possess hierarchical macropores and their mechanical properties (Young's modulus and compressive strength) are comparable to those of cancellous bone. Moreover, a preliminary in vivo study in the distal femoral sites of rabbits was conducted to evaluate the biofunctionality of this injectable macroporous CPC. The evidence of newly formed bone in the central zone of implantation site indicates the feasibility and effectiveness of this foaming strategy that will have to be optimized by further extensive animal experiments.

Statement Of Significance: A major challenge in the design of biomaterial-based injectable bone substitutes is the development of cohesive, macroporous and self-setting calcium phosphate cement (CPC) that enables rapid cell invasion with adequate initial mechanical properties without the use of complex processing and additives. Thus, we propose a simple and effective strategy to prepare injectable macroporous CPCs through syringe-foaming using a hydrophilic viscous polymeric solution (silanized-hydroxypropyl methylcellulose, Si-HPMC) as a foaming agent, that simultaneously meets all the aforementioned aims. Evidence from our in vivo studies shows the existence of newly formed bone within the implantation site, indicating the feasibility and effectiveness of this foaming strategy, which could be used in various CPC systems using other hydrophilic viscous polymeric solutions.
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http://dx.doi.org/10.1016/j.actbio.2015.11.055DOI Listing
February 2016

Design and properties of novel gallium-doped injectable apatitic cements.

Acta Biomater 2015 Sep 12;24:322-32. Epub 2015 Jun 12.

Université de Nantes, CNRS, UMR 6230, CEISAM, UFR Sciences et Techniques, 44322 NANTES Cedex 3, France.

Unlabelled: Different possible options were investigated to combine an apatitic calcium phosphate cement with gallium ions, known as bone resorption inhibitors. Gallium can be either chemisorbed onto calcium-deficient apatite or inserted in the structure of β-tricalcium phosphate, and addition of these gallium-doped components into the cement formulation did not significantly affect the main properties of the biomaterial, in terms of injectability and setting time. Under in vitro conditions, the amount of gallium released from the resulting cement pellets was found to be low, but increased in the presence of osteoclastic cells. When implanted in rabbit bone critical defects, a remodeling process of the gallium-doped implant started and an excellent bone interface was observed.

Statement Of Significance: The integration of drugs and materials is a growing force in the medical industry. The incorporation of pharmaceutical products not only promises to expand the therapeutic scope of biomaterials technology but to design a new generation of true combination products whose therapeutic value stem equally from both the structural attributes of the material and the intrinsic therapy of the drug. In this context, for the first time an injectable calcium phosphate cement containing gallium was designed with properties suitable for practical application as a local delivery system, implantable by minimally invasive surgery. This important and original paper reports the design and in-depth chemical and physical characterization of this groundbreaking technology.
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http://dx.doi.org/10.1016/j.actbio.2015.05.027DOI Listing
September 2015

Vertebroplasty using bisphosphonate-loaded calcium phosphate cement in a standardized vertebral body bone defect in an osteoporotic sheep model.

Acta Biomater 2014 Nov 19;10(11):4887-4895. Epub 2014 Jul 19.

Université de Nantes, INSERM, UMR 791, LIOAD, Faculté de Chirurgie Dentaire, BP 84215, 44042 Nantes Cedex 1, France.

In the context of bone regeneration in an osteoporotic environment, the present study describes the development of an approach based on the use of calcium phosphate (CaP) bone substitutes that can promote new bone formation and locally deliver in situ bisphosphonate (BP) directly at the implantation site. The formulation of a CaP material has been optimized by designing an injectable apatitic cement that (i) hardens in situ despite the presence of BP and (ii) provides immediate mechanical properties adapted to clinical applications in an osteoporotic environment. We developed a large animal model for simulating lumbar vertebroplasty through a two-level lateral corpectomy on L3 and L4 vertebrae presenting a standardized osteopenic bone defect that was filled with cements. Both 2-D and 3-D analysis of microarchitectural parameters demonstrated that implantation of BP-loaded cement in such vertebral defects positively influenced the microarchitecture of the adjacent trabecular bone. This biological effect was dependent on the distance from the implant, emphasizing the in situ effect of the BP and its release from the cement. As a drug device combination, this BP-containing apatitic cement shows good promise as a local approach for the prevention of osteoporotic vertebral fractures through percutaneous vertebroplasty procedures.
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http://dx.doi.org/10.1016/j.actbio.2014.07.012DOI Listing
November 2014

Therapeutic strategies for treating osteolytic bone metastases.

Drug Discov Today 2014 Sep 14;19(9):1419-26. Epub 2014 Apr 14.

INSERM U791, Université de Nantes, Faculté de Chirurgie Dentaire, 44042 Nantes, France.

The recent progress in oncologic management of patients with localized cancer or metastatic disease has permitted a significant improvement in life expectancy. Nevertheless, bone metastases and their consequent skeletal-related events (SREs) are still associated with unfavorable prognosis and greatly affect quality of life. Global management of these bone metastases includes traditional local approaches (surgery, radiotherapy, etc.) and systemic administration of chemotherapeutic agents. This review focuses on treatments specific for bone metastases and, in particular, on inhibitors of bone resorption that are effective for preventing and delaying the development of SREs.
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http://dx.doi.org/10.1016/j.drudis.2014.04.004DOI Listing
September 2014

A novel injectable, cohesive and toughened Si-HPMC (silanized-hydroxypropyl methylcellulose) composite calcium phosphate cement for bone substitution.

Acta Biomater 2014 Jul 20;10(7):3335-45. Epub 2014 Mar 20.

Université de Nantes, INSERM, UMR 791, LIOAD, Faculté de Chirurgie Dentaire, BP 84215, 44042 Nantes Cedex 1, France; Centre Hospitalier Universitaire de Nantes, 1 place Alexis Ricordeau, 44093 Nantes Cedex 1, France.

This study reports on the incorporation of the self-setting polysaccharide derivative hydrogel (silanized-hydroxypropyl methylcellulose, Si-HPMC) into the formulation of calcium phosphate cements (CPCs) to develop a novel injectable material for bone substitution. The effects of Si-HPMC on the handling properties (injectability, cohesion and setting time) and mechanical properties (Young's modulus, fracture toughness, flexural and compressive strength) of CPCs were systematically studied. It was found that Si-HPMC could endow composite CPC pastes with an appealing rheological behavior at the early stage of setting, promoting its application in open bone cavities. Moreover, Si-HPMC gave the composite CPC good injectability and cohesion, and reduced the setting time. Si-HPMC increased the porosity of CPCs after hardening, especially the macroporosity as a result of entrapped air bubbles; however, it improved, rather than compromised, the mechanical properties of composite CPCs, which demonstrates a strong toughening and strengthening effect. In view of the above, the Si-HPMC composite CPC may be particularly promising as bone substitute material for clinic application.
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http://dx.doi.org/10.1016/j.actbio.2014.03.009DOI Listing
July 2014

High Frequency Impedance Measurement as a Relevant Tool for Monitoring the Apatitic Cement Setting Reaction.

Acta Biomater 2014 02 25;10(2):940-950. Epub 2013 Oct 25.

LCPME, Université de Lorraine CNRS UMR 7564, 405 rue de Vandoeuvre 54600 Villers Lès Nancy, France.

This work reports the development of a relevant and general method based on high frequency impedance measurements, for the in situ monitoring of the alpha-tricalcium phosphate (α-TCP) to calcium-deficient hydroxyapatite (CDA) transformation which is the driving force of the hardening processes of some calcium phosphate cements (CPC) used as bone substitutes. The three main steps of the setting reaction are identified in a non invasive way through the variation of dielectric permittivity and dielectric losses. The method is also likely to characterize the effect of the incorporation of additives (i.e, antiosteoporotic bisphosphonate drugs such as Alendronate) in the CPC formulation on the hydration process. It allows not only to confirm the retarding effect of bisphosphonate by an accurate determination of setting times, but also to assess the phenomena taking place whether alendronate is added in the liquid phase or combined to the solid phase of the cement composition. Compared to the conventional Gillmore needle test, the present method offers the advantage of accurate, user-independent, in situ and real-time determination of the initial and final times of the chemical hardening process, which are important parameters when considering surgical applications.
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http://dx.doi.org/10.1016/j.actbio.2013.10.019DOI Listing
February 2014

Calcium phosphate cements for bone substitution: chemistry, handling and mechanical properties.

Acta Biomater 2014 Mar 11;10(3):1035-49. Epub 2013 Nov 11.

Université de Nantes, INSERM UMRS 791, Laboratoire d'Ingénierie Ostéo-Articulaire et Dentaire, 1 place Alexis Ricordeau, BP 84215, 44042 Nantes Cedex 1, France. Electronic address:

Since their initial formulation in the 1980s, calcium phosphate cements (CPCs) have been increasingly used as bone substitutes. This article provides an overview on the chemistry, kinetics of setting and handling properties (setting time, cohesion and injectability) of CPCs for bone substitution, with a focus on their mechanical properties. Many processing parameters, such as particle size, composition of cement reactants and additives, can be adjusted to control the setting process of CPCs, concomitantly influencing their handling and mechanical performance. Moreover, this review shows that, although the mechanical strength of CPCs is generally low, it is not a critical issue for their application for bone repair--an observation not often realized by researchers and clinicians. CPCs with compressive strengths comparable to those of cortical bones can be produced through densification and/or homogenization of the cement matrix. The real limitation for CPCs appears to be their low fracture toughness and poor mechanical reliability (Weibull modulus), which have so far been only rarely studied.
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http://dx.doi.org/10.1016/j.actbio.2013.11.001DOI Listing
March 2014

Polarized infrared reflectance spectra of brushite (CaHPO4·2H2O) crystal investigation of the phosphate stretching modes.

Spectrochim Acta A Mol Biomol Spectrosc 2013 Jul 23;111:7-13. Epub 2013 Mar 23.

CNRS, Institut des Matériaux Jean-Rouxel (IMN) - UMR 6502, Université de Nantes, 2 rue de la Houssinière, B.P. 32229, 44322 Nantes Cedex 3, France.

Polarized infrared (IR) reflectance measurements at near-normal incidence were recorded from the ac-plane of a monoclinic brushite (CaHPO4·2H2O) crystal in the 800-1200 cm(-1) spectral range (P-O stretching modes). The adjustment of these data, on the basis of a dispersion analysis (DA) model for monoclinic case, allowed the determination of oscillators parameters for the four P-O stretching observed modes of the phosphate group.
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http://dx.doi.org/10.1016/j.saa.2013.03.047DOI Listing
July 2013

Biomaterial porosity determined by fractal dimensions, succolarity and lacunarity on microcomputed tomographic images.

Mater Sci Eng C Mater Biol Appl 2013 May 17;33(4):2025-30. Epub 2013 Jan 17.

LUNAM Université, GEROM Groupe Etudes Remodelage Osseux et bioMatériaux-LHEA, IRIS-IBS Institut de Biologie en Santé, CHU d'Angers, 49933 ANGERS Cedex, France.

Porous structures are becoming more and more important in biology and material science because they help in reducing the density of the grafted material. For biomaterials, porosity also increases the accessibility of cells and vessels inside the grafted area. However, descriptors of porosity are scanty. We have used a series of biomaterials with different types of porosity (created by various porogens: fibers, beads …). Blocks were studied by microcomputed tomography for the measurement of 3D porosity. 2D sections were re-sliced to analyze the microarchitecture of the pores and were transferred to image analysis programs: star volumes, interconnectivity index, Minkowski-Bouligand and Kolmogorov fractal dimensions were determined. Lacunarity and succolarity, two recently described fractal dimensions, were also computed. These parameters provided a precise description of porosity and pores' characteristics. Non-linear relationships were found between several descriptors e.g. succolarity and star volume of the material. A linear correlation was found between lacunarity and succolarity. These techniques appear suitable in the study of biomaterials usable as bone substitutes.
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http://dx.doi.org/10.1016/j.msec.2013.01.020DOI Listing
May 2013

The influence of different cellulose ethers on both the handling and mechanical properties of calcium phosphate cements for bone substitution.

Acta Biomater 2013 Mar 28;9(3):5740-50. Epub 2012 Nov 28.

L'UNAM, Université de Nantes, Polytech Nantes, Institut des Matériaux Jean Rouxel, Rue Christian Pauc, BP 50609, 44306 Nantes Cedex 3, France.

The influence of cellulose ether additives (CEAs) on the performance of final calcium phosphate cement (CPC) products is thoroughly investigated. Four CEAs were added into the liquid phase of apatitic CPCs based on the hydrolysis of α-tricalcium phosphate, to investigate the influence of both molecular weight and degree of substitution on the CPCs' properties, including handling (e.g. injectability, cohesion, washout resistance and setting time), microstructure (e.g. porosity and micromorphology) and mechanical properties (e.g. fracture toughness and compressive strength). The results showed that even a small amount of CEAs modified most of these CPCs' features, depending on the structural parameters of the CEAs. The CEAs dramatically improved the injectability, cohesion and washout resistance of the pastes, prolonged the final setting time and increased the porosity of CPCs. Moreover, the CEAs had an evident toughening effect on CPCs, and this effect become more significant with increasing molecular weight and mass fraction of CEAs, inducing a significant tolerance to damage. Overall, the molecular weight of CEAs played a major role compared to their degree of substitution in CPCs' performances.
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http://dx.doi.org/10.1016/j.actbio.2012.11.020DOI Listing
March 2013

Effects of citrate and NaCl on size, morphology, crystallinity and microstructure of calcium phosphates obtained from aqueous solutions at acidic or near-neutral pH.

J Dairy Res 2012 May;79(2):238-48

INRA/Agrocampus Ouest, UMR1253 Science et Technologie du Lait et de l'Oeuf, 65 rue de Saint Brieuc, F-35042 Rennes, France.

Precipitation of calcium phosphates occurs in dairy products and depending on pH and ionic environment, several salts with different crystallinity can form. The present study aimed to investigate the effects of NaCl and citrate on the characteristics of precipitates obtained from model solutions of calcium phosphate at pH 6·70 maintained constant or left to drift. The ion speciation calculations showed that all the starting solutions were supersaturated with respect to dicalcium phosphate dihydrate (DCPD), octacalcium phosphate (OCP) and hydroxyapatite (HAP) in the order HAP>OCP>DCPD. X-ray diffraction (XRD) and Fourier transform infrared (FTIR) analyses of the precipitates showed that DCPD was formed at drifting pH (acidic final pH) whereas poor crystallised calcium deficient apatite was mainly formed at constant pH (6·70). Laser light scattering measurements and electron microscopy observations showed that citrate had a pronounced inhibitory effect on the crystallisation of calcium phosphates both at drifting and constant pH. This resulted in the decrease of the particle sizes and the modification of the morphology and the microstructure of the precipitates. The inhibitory effect of citrate mainly acted by the adsorption of the citrate molecules onto the surfaces of newly formed nuclei of calcium phosphate, thereby changing the morphology of the growing particles. These findings are relevant for the understanding of calcium phosphate precipitation from dairy byproducts that contain large amounts of NaCl and citrate.
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http://dx.doi.org/10.1017/S0022029912000076DOI Listing
May 2012

Assay of in vitro osteoclast activity on dentine, and synthetic calcium phosphate bone substitutes.

J Mater Sci Mater Med 2012 Mar 22;23(3):797-803. Epub 2011 Dec 22.

Osteo-Articular and Dental Tissue Engineering Laboratory LIOAD, Department of Periodontology, School of Dental Surgery, INSERM U791, Nantes, France.

Resorption of synthetic bone substitute materials is essential for the integration of these materials into the natural bone remodeling process. Osteoclast behavior in the presence of calcium phosphate bioceramics (CaPB) is partially understood, and a better understanding of the underlying mechanisms is expected to facilitate the development of new synthetic bone substitutes to improve bone regeneration. In the present study, our aim was to investigate osteoclastic resorption of various synthetic CaPB. We used neonatal total rabbit bone cells to generate osteoclasts. Osteoclast-generated resorption on dentine and multiple CaPB was investigated by quantifying the surface resorbed and measuring tartrate resistant acid phosphatase (TRAP) enzyme activity. In this study, we observed that osteoclastic cells responded in a different way to each substrate. Both dentine and CaPB were resorbed but the quantitative results for the surface resorbed and TRAP activity showed a specific response to each substrate and that increased mineral density seemed to inhibit osteoclast activity.
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http://dx.doi.org/10.1007/s10856-011-4534-xDOI Listing
March 2012

Characterization and properties of novel gallium-doped calcium phosphate ceramics.

Inorg Chem 2011 Sep 27;50(17):8252-60. Epub 2011 Jul 27.

Graftys SA, Eiffel Park, Bâtiment D, 415 Rue Claude Nicolas Ledoux, Pôle d'activités d'Aix en Provence, 13854 Aix en Provence CEDEX 3, France.

Addition of a gallium (Ga) precursor in the typical reaction protocols used for the preparation of β-tricalcium phosphate (β-TCP) led to novel Ga-doped β-TCP ceramics with rhombohedral structures (R3c space group). From the refinement of their X-ray diffraction patterns, it was found that the incorporation of Ga in the β-TCP network occurs by substitution of one of the five calcium (Ca) sites, while occupation of another Ca site decreases in inverse proportion to the Ga content in the structure. The Ga local environment and the modification of the phosphorus environments due to the Ga/Ca substitution in Ga-doped β-TCP compounds are probed using (31)P and (71)Ga magic-angle spinning NMR. A decrease of the unit cell volume is observed with increasing Ga content, together with improved mechanical properties. Indeed, the compressive strength of these new bioceramics is enhanced in direct proportion of the Ga content, up to a 2.6-fold increase as compared to pure β-TCP.
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http://dx.doi.org/10.1021/ic2007777DOI Listing
September 2011

Fate of bone marrow stromal cells in a syngenic model of bone formation.

Tissue Eng Part A 2011 Sep 14;17(17-18):2267-78. Epub 2011 Jun 14.

GEPITOS, Université Nice Sophia-Antipolis, CNRS, UFR de Médecine, Nice, France.

Bone 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.
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http://dx.doi.org/10.1089/ten.TEA.2010.0461DOI Listing
September 2011

Na-doped β-tricalcium phosphate: physico-chemical and in vitro biological properties.

J Mater Sci Mater Med 2011 Mar 8;22(3):593-600. Epub 2011 Jan 8.

INSERM, UMR 791, LIOAD, Faculté de Chirurgie Dentaire, Université de Nantes, BP 84215, 44042, Nantes Cedex 1, France.

Synthetic calcium phosphate ceramics as β-tricalcium phosphate (Ca(3)(PO(4))(2); β-TCP) are currently successfully used in human bone surgery. The aim of this work was to evaluate the influence of the presence of sodium ion in β-TCP on its mechanical and biological properties. Five Na-doped-β-TCP [Ca(10.5-x/2)Na(x)(PO(4))(7), 0 ≤ x ≤ 1] microporous pellets were prepared via solid phase synthesis, and their physico-chemical data (lattice compacity, density, porosity, compressive strength, infrared spectra) denote an increase of the mechanical properties and a decrease of the solubility when the sodium content is raised. On the other hand, the in vitro study of MC3T3-E1 cell activity (morphology, MTS assay and ALP activity) shows that the incorporation of sodium does not modify the bioactivity of the β-TCP. These results strongly suggest that Na-doped-β-TCP appear to be good candidates for their use as bone substitutes.
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http://dx.doi.org/10.1007/s10856-010-4219-xDOI Listing
March 2011

Osteoclastic differentiation of mouse and human monocytes in a plasma clot/biphasic calcium phosphate microparticles composite.

Eur Cell Mater 2010 Dec 10;20:379-92. Epub 2010 Dec 10.

GéPITOs, UMR6235 UFR de Médecine, 28 Avenue de Valombrose, F-06107 NICE, Cedex 2, France.

We recently demonstrated that blood clotted around biphasic calcium phosphate (BCP) microparticles constituted a composite biomaterial that could be used for bone defect filling. In addition, we showed that mononuclear cells, i.e. monocytes and lymphocytes, play a central role in the osteogenic effect of this biomaterial. Hypothesizing that osteoclast progenitors could participate to the pro-osteogenic effect of mononuclear cells we observed previously, we focus on this population through the study of mouse monocyte/macrophage cells (RAW264.7 cell line), as well as human pre-osteoclastic cells derived from mononuclear hematopoietic progenitor cells (monocytes-enriched fraction from peripheral blood). Using monocyte-derived osteoclast progenitors cultured within plasma clot/BCP microparticles composite, we aimed in the present report at the elucidation of transcriptional profiles of genes related to osteoclastogenesis and to bone remodelling. For both human and mouse monocytes, real-time PCR experiments demonstrated that plasma clot/BCP scaffold potentiated the expression of marker genes of the osteoclast differentiation such as Nfactc1, Jdp2, Fra2, Tracp and Ctsk. By contrast, Mmp9 was induced in mouse but not in human cells, and Ctr expression was down regulated for both species. In addition, for both mouse and human precursors, osteoclastic differentiation was associated with a strong stimulation of VegfC and Sdf1 genes expression. At last, using field-emission scanning electron microscopy analysis, we observed the interactions between human monocytes and BCP microparticles. As a whole, we demonstrated that plasma clot/BCP microparticles composite provided monocytes with a suitable microenvironment allowing their osteoclastic differentiation, together with the production of pro-angiogenic and chemoattractant factors.
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http://dx.doi.org/10.22203/ecm.v020a31DOI Listing
December 2010