Strontium-doped hydroxyapatite polysaccharide materials effect on ectopic bone formation

Abstract

Previous studies performed using polysaccharide-based matrices supplemented with hydroxyapatite (HA) particles showed their ability to form in subcutaneous and intramuscular sites a mineralized and osteoid tissue. Our objectives are to optimize the HA content in the matrix and to test the combination of HA with strontium (Sr-HA) to increase the matrix bioactivity. First, non-doped Sr-HA powders were combined to the matrix at three different ratios and were implanted subcutaneously for 2 and 4 weeks. Interestingly, matrices showed radiolucent properties before implantation. Quantitative analysis of micro-CT data evidenced a significant increase of mineralized tissue formed ectopically with time of implantation and allowed us to select the best ratio of HA to polysaccharides of 30% (w/w). Then, two Sr-substitution of 8% and 50% were incorporated in the HA powders (8Sr-HA and 50Sr-HA). Both Sr-HA were chemically characterized and dispersed in matrices. In vitro studies performed with human mesenchymal stem cells (MSCs) demonstrated the absence of cytotoxicity of the Sr-doped matrices whatever the amount of incorporated Sr. They also supported osteoblastic differentiation and activated the expression of one late osteoblastic marker involved in the mineralization process i.e. osteopontin. In vivo, subcutaneous implantation of these Sr-doped matrices induced osteoid tissue and blood vessels formation.

Fig 1. Structural characterization of the synthesized samples of hydroxyapatite.

(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.

Fig 2. Micro-CT analysis of Matrix HA containing different ratios of HA particles, implanted subcutaneously in mice.

(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.

Fig 3. Characterization of the Matrix-HA.

(A) Environmental scanning electron microscopy (ESEM) and back scattering electron microscopy (ESEM-BSE) images showing the presence of the macroporous structure of the 3D matrices and the presence of numerous HA particles (indicated by white arrows). Sale bars = 0.5 mm and 100 μm for ESEM; and 5 μm for ESEM-BSE (B) Energy Dispersive X-ray spectroscopy (EDX) spectra of Matrix-HA, Matrix-8Sr-HA, and Matrix-50Sr-HA.

Fig 4. Effect of the various Matrix-HA doped or not with strontium on MSCs viability.

(A) LIVE/DEAD^® assays after 3 and 7 days of culture of MSCs on the three different matrices (Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA). Scale bars = 50 μm. (B) Alamar Blue assay after 3 and 7 days of culture of MSCs on the three different matrices (Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA). Data were expressed in % of metabolic activity normalized to day 1 (100%) for each condition (average ± SD). The symbol * denotes p<0.05 and ns, “non-significant”.

Fig 5. mRNA expression of early and late osteoblastic gene in MSCs cultured on the three different matrices.

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 p< 0.05; ** denotes p<0.01 and *** indicates a significant difference with p< 0.001.

Fig 6. In vivo evaluation of the Matrix-HA supplemented with strontium implanted subcutaneously in mice.

(A) Representative micro-CT images of Matrix-HA, Matrix-8Sr-HA and Matrix-50Sr-HA, before the implantation (Time 0), and after 2 (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 points 2 weeks and 4weeks (W2, W4). Results were expressed as average ± SD. The symbol ** denotes p<0.01.

Fig 7. Histological and immunochemistry analysis of the newly formed tissue within the Matrix-HA supplemented with strontium, implanted subcutaneously in mice.

(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 p<0.05; p<0.01 or p<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 p<0.05; p<0.01 or p<0.001, respectively.