In-vitro and in-vivo design and validation of an injectable polysaccharide-hydroxyapatite composite material for sinus floor augmentation

Objective: Polysaccharide-based composite matrices consisting of natural polysaccharides, pullulan and dextran supplemented with hydroxyapatite (Matrix-HA) have recently been developed. The principal objective of this study was to evaluate the capacities of this composite material to promote new bone formation in a sinus lift model in the sheep. Secondary objectives were to evaluate in vitro properties of the material regarding cell adhesion and proliferation.Methods: In this report, once such composite matrix was prepared as injectable beads after dispersion in a physiological buffer, and evaluated using a large animal model (sheep) for a sinus lift procedure.Results: In vitro studies revealed that these microbeads (250-550μm in diameter) allow vascular cell adhesion and proliferation of Endothelial Cells (EC) after 1 and 7 days of culture. In vivo studies were performed in 12 adult sheep, and newly formed tissue was analyzed by Cone Beam Computed Tomography (CBCT scanning electron microscopy (SEM) and by histology 3 and 6 months post-implantation. CBCT analyses at the implantation time revealed the radiolucent properties of these matrices. Quantitative analysis showed an increase of a dense mineralized tissue in the Matrix-HA group up to 3 months of implantation. The mineralized volume over total volume after 6 months reached comparable values to those obtained for Bio-Oss® used as positive control. Histological examination confirmed that the Matrix-HA did not induce any long term inflammatory events, and promoted direct contact between the osteoid tissue and lamellar bone structures and beads. After 6 months, we observed a dense network of osteocytes surrounding both biomaterials as well as a newly vascularized formed tissue in close contact to the biomaterials.Significance: In conclusion, the absence of animal components in Matrix-HA, the osteoconductive property of Matrix-HA in sheep, resulting in a dense bone and vascularized tissue, and the initial radiolucent property to follow graft integration offer great promises of this composite material for clinical use

mRNA expression of early and late osteoblastic gene in MSCs cultured on the three different matrices.

<p>Runx 2 (A) and OPN (B) expression were quantified by qPCR after 1, 3 and 7 days of hMSCs cultured on Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA. Three separate experiments were performed and each assay was done in duplicate. Data were expressed in relative expression normalized to P0 expression, compared to day 1. Runx2 (A) and OPN (B) gene expression were set as “1.0” at Day 1 for the three scaffolds. Results were expressed as average ± SD. The symbol * denotes <i>p</i>< 0.05; ** denotes <i>p</i><0.01 and *** indicates a significant difference with <i>p</i>< 0.001.</p

Structural characterization of the synthesized samples of hydroxyapatite.

<p>(A) XRD patterns of non-substituted HA samples (HA); HA powders with 8% of Sr-substitution (8Sr-HA); HA powders with 50% of Sr-substitution (50Sr-HA). (B) FTIR spectra of non-substituted HA samples (HA); HA powders with 8% of Sr-substitution (8Sr-HA); HA powders with 50% of Sr-substitution (50Sr-HA). (C) Representative images of HA powders (a: HA, b: 8Sr-HA, c: 50Sr-HA) by Transmission Electron Microscopy (TEM). Scale bars = 100nm.</p

Micro-CT analysis of Matrix HA containing different ratios of HA particles, implanted subcutaneously in mice.

<p>(A) Representative Micro-CT images of Matrix-HA supplemented with three different amounts of HA (dispersion 1 (D1), dispersion 2 (D2), and dispersion 3 (D3)), before implantation (Time 0), after 2 weeks (W2) and 4 weeks (W4) of subcutaneous implantation. (B) Quantification of the mineralized volume / total volume (MV/TV) after 2 weeks (W2) and 4 weeks (W4) of subcutaneous implantation. Six samples were evaluated for each condition at each time point (2 and 4weeks). Results were expressed as average ± SD. The symbol ** denotes p<0.01.</p

mRNA expression of Runx2 and osteopontin (OPN) gene in MSCs cultured on the three different matrices.

<p>Runx 2 and OPN expression were quantified by qPCR after 3 and 7 days of MSCs cultured on Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA. Three separate experiments were performed and each assay was done in duplicate. Data were expressed in relative expression normalized to P0 expression, compared to Matrix-HA for each time. Gene expression were set as “1.0” for each day (Day 3 and Day 7) for Matrix-HA. Results were expressed as average ± SD.</p

Histological and immunochemistry analysis of the newly formed tissue within the Matrix-HA supplemented with strontium, implanted subcutaneously in mice.

<p>(A) Masson’s Trichrome staining of the newly tissue formed within the three matrices after 2 and 4 weeks of subcutaneous implantation. (B) Quantitative analysis: images were analysed using the Nikon software. The whole surface as well as the newly bone surface were quantified in mm². Stained slides from 2 samples per condition were processed for histological analysis, 3 sections were fully imaged and analysed per sample and per condition. Results were expressed as average ± SD per group of matrix, with time of implantation (2 and 4 weeks: W2, W4). The symbols *; **; and *** denote <i>p</i><0.05; <i>p</i><0.01 or <i>p</i><0.001, respectively. (C) CD31 immunostaining of the newly formed tissue within the three matrices, Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA. (D) Quantification of the number of vessels within the tissue was performed by using NDP view software. The whole surface as well as the number of vessels were quantitated in mm². Stained slides from 2 samples per condition were processed for immunostaining analysis, and 3 sections were fully imaged and analysed per sample and per group of matrix. Results were expressed as average ± SD per group of matrix, with time of implantation (2 and 4weeks: W2, W4). The symbols *; **; and *** indicate <i>p</i><0.05; <i>p</i><0.01 or <i>p</i><0.001, respectively.</p

Average agglomerate sizes of HA particles as determined by Dynamic light scattering (DLS).

<p>Values shown (in μm) represent the average ± SD of <i>n</i> = 3 samples for each condition.</p

Peak assignments for the different HA powders.

<p>Summary of vibrational frequencies observed by FTIR for standard carbonated HA as described by Cox <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184663#pone.0184663.ref017" target="_blank">17</a>], for non-substituted HA produced by wet precipitation according to Catros <i>et al</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0184663#pone.0184663.ref014" target="_blank">14</a>], or measured for non-substituted HA, 8Sr-HA and 50Sr-HA synthesized in this work.</p