Effect of a composite matrix containing various ratios of hydroxyapatite doped with strontium on tissue mineralization formed in ectopic site
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1
University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, France
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2
University ParisDiderot, University Paris 13, Inserm U1148, LVTS, France
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3
University of Bordeaux, ICMCB, France
Introduction: Worldwide 500,000 cases of maxillofacial cancer are diagnosed each year. After carcinologic surgery, the reconstruction of large bone defect is often required. The technique of reconstruction described by Masquelet with the induced membranes is one of the strategies, but still exhibits limitations in an oncological context, i.e. the use of bone autografts or bone morphogenetic proteins (BMP) for stimulating bone reconstruction. The main objective is to develop an injectable osteoinductive and osteoconductive composite matrix composed of doped strontium (Sr) hydroxyapatite (HA) particules dispersed within a polysaccharide-based scaffold to fill the induced membrane and to replace autologous bone and BMP2.
Materials and Methods: HA particles and strontium-doped HA particles (Sr-doped HA) were synthesized by wet chemical precipitation at 90°C with different ratios of Sr (8 and 50 % w/w). X-ray diffraction (XRD), Inductively Coupled Plasma (ICP), Transmission Electronic Microscopy (TEM) and particle size analysis using Nanosizer™, were used to characterize these HA particles. HA and Sr-doped HA particles were dispersed at different ratios (2.8%; 5%; 10% and 25% v/v) within the pullulan-dextran based matrices, already used by our group for bone reconstruction [1]. In vitro assays were performed using hMSC according to our previous work [2]. Live/Dead assay was used to evaluate the viability of hMSC cultured within the composite matrix for two weeks. Expression of bone markers (runx2, ALP, OCN) was quantified by quantitative polymerase chain reaction. Matrices containing the HA and Sr-doped HA particles were then implanted subcutaneously in Balb/c mice. After 2 and 4 weeks, implants were collected and analyzed by Micro-Computed Tomography (micro-CT). Fixed samples were then decalcified, dehydrated and paraffin-embedded. Sections were stained with Masson’s Trichrome.
Results and Discussion: Phase identification by X-ray diffraction (XRD) analysis of the HA material (Diffract-Plus Software, Bruker) was compatible with a carbonated hydroxyapatite. X-Ray diffraction patterns of Sr-doped HA are consistent of the influence of Sr substitution on HA structure. Morphological evaluation by TEM and Nanosizer™ analyses show that HA and Sr-doped HA particles mainly form globular agglomerates with a size of 150 nm to 4 mm.
Environmental Scanning Electron Microscopy (ESEM) of matrices composed with different ratios of HA or Sr-doped HA, shows an homogenous distribution of the particles within the matrices, whatever the conditions of dispersion of the particules. In vitro studies revealed that the presence of Sr-doped HA particles within the matrix stimulates the expression of osteoblastic markers, compared to non-doped HA matrices.
Finally, subcutaneous implantation of the matrices containing different ratios of HA and Sr-doped HA particles, demonstrated the formation of a mineralized tissue visualized by micro-CT, up to 2 weeks of implantation and well evidenced at 4 weeks (Fig 1). BV/TV data show that the mineralization of the implants is dependent of the amount of HA particles dispersed within the polysaccharide matrices (Fig 2) with an optimal ratio of HA particles within the scaffold of 5 %. Moreover, the use of 50% Sr-doped HA dispersed at 5% within the matrix increases significantly the tissue mineralization after two and four weeks of implantation compared to non-doped HA particles (Fig 2).
Conclusion: These osteoinductive matrices doped with Sr could represent an alternative to the autografts supplemented with BMP2 used for the regeneration of large bone defects in an oncological context, where vascularization of the host irradiated tissue is altered, and where an osteoinductive material deprived of growth factors is required for promoting bone formation.
The authors would like to thank the “Fondation pour la Recherche Médicale” for providing financial support to this project as well as Inserm, University of Bordeaux, University Paris Diderot and Paris 13.
References:
[1] Fricain JC. et al., J. Biomaterials, 2013, 34:2947-59.
[2] Guerrerro et al, Tissue Eng Part A. 2015;21(5-6):861-74.
Keywords:
Bone Regeneration,
Cell Differentiation,
3D scaffold,
in vivo tissue engineering
Conference:
10th World Biomaterials Congress, Montréal, Canada, 17 May - 22 May, 2016.
Presentation Type:
Poster
Topic:
Biomaterials in constructing tissue substitutes
Citation:
Ehret
C,
Aid
R,
Kalisky
J,
Sagerdoy
T,
Siadous
R,
Bareille
R,
Pechev
S,
Etienne
L,
Fricain
J,
Letourneur
D and
Amedee
J
(2016). Effect of a composite matrix containing various ratios of hydroxyapatite doped with strontium on tissue mineralization formed in ectopic site.
Front. Bioeng. Biotechnol.
Conference Abstract:
10th World Biomaterials Congress.
doi: 10.3389/conf.FBIOE.2016.01.01938
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Received:
27 Mar 2016;
Published Online:
30 Mar 2016.
*
Correspondence:
Dr. Camille Ehret, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, camille.erhet@inserm.fr
Dr. Rachida Aid, University ParisDiderot, University Paris 13, Inserm U1148, LVTS, Paris, France, rachida.aid@gmail.com
Dr. Jerome Kalisky, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, jerome.kalisky@inserm.fr
Dr. Thomas Sagerdoy, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, thomas.sagerdoy@gmail.com
Dr. Robin Siadous, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, robin.siadous@inserm.fr
Dr. Reine Bareille, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, reine.bareille@inserm.fr
Dr. Stanislav Pechev, University of Bordeaux, ICMCB, Talence, France, pechev@icmcb-bordeaux.cnrs.fr
Dr. Laetitia Etienne, University of Bordeaux, ICMCB, Talence, France, etiennel@icmcb-bordeaux.cnrs.fr
Dr. Jean Christophe Fricain, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, jean-christophe.fricain@inserm.fr
Dr. Didier Letourneur, University ParisDiderot, University Paris 13, Inserm U1148, LVTS, Paris, France, didier.letourneur@inserm.fr
Dr. Joëlle Amedee, University of Bordeaux, Inserm U1026, Tissue Bio-Engineering, Bordeaux, France, Email1