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. 2017 Aug 2;7(1):7110.
doi: 10.1038/s41598-017-07672-0.

Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse

Giovanna Calabrese  1   2 , Raffaella Giuffrida  1 , Stefano Forte  1 , Claudia Fabbi  3 , Elisa Figallo  3 , Lucia Salvatorelli  4 , Lorenzo Memeo  1   5 , Rosalba Parenti  2 , Massimo Gulisano  2 , Rosario Gulino  6   7
Affiliations

Human adipose-derived mesenchymal stem cells seeded into a collagen-hydroxyapatite scaffold promote bone augmentation after implantation in the mouse

Giovanna Calabrese et al. Sci Rep. .

Abstract

Traumatic injury or surgical excision of diseased bone tissue usually require the reconstruction of large bone defects unable to heal spontaneously, especially in older individuals. This is a big challenge requiring the development of biomaterials mimicking the bone structure and capable of inducing the right commitment of cells seeded within the scaffold. In particular, given their properties and large availability, the human adipose-derived stem cells are considered as the better candidate for autologous cell transplantation. In order to evaluate the regenerative potential of these cells along with an osteoinductive biomaterial, we have used collagen/hydroxyapatite scaffolds to test ectopic bone formation after subcutaneous implantation in mice. The process was analysed both in vivo, by Fluorescent Molecular Tomography (FMT), and ex vivo, to evaluate the formation of bone and vascular structures. The results have shown that the biomaterial could itself be able of promoting differentiation of host cells and bone formation, probably by means of its intrinsic chemical and structural properties, namely the microenvironment. However, when charged with human mesenchymal stem cells, the ectopic bone formation within the scaffold was increased. We believe that these results represent an important advancement in the field of bone physiology, as well as in regenerative medicine.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Figure 1
Figure 1
SEM images of the scaffold internal structure at two different magnifications. At higher magnification, an open and interconnected porosity is visible within the bone-like scaffold and Mg-HA particles are visible within and on the surface of the collagen fibers (B). Magnification: 30× in A; 300× in B. Scale bars: 1000 µm in (A) 100 µm in (B).
Figure 2
Figure 2
Phenotypic characterization of hADSCs by immunofluorescence and flow cytometry. Different samples were analyzed and, as expected, they didn’t exhibit any expression of CD31 (A,G), CD34 (B,H) or CD45 (C,I), whereas a strong expression of CD73 (D,J), CD105 (E,K) and CD90 (F,L) was found consistently. Scale bar in A for (A–F) 100 µm.
Figure 3
Figure 3
FMT images and in vivo quantification of osteogenesis within the implanted scaffolds. The color scale indicates the mean fluorescent probe concentrations within the ROI, and the graph (A) shows the mean values of probe content (in pmol) calculated within groups and reported as percent of the mean value of the 2 wk group with cell-free scaffold. It is evident that a higher concentration of probe (OsteoSense 750) is present when the scaffolds contain hADSCs, at all time-points (B–D) when compared with cell-free scaffolds (E–G). In the graph A, the number into the circle indicates significant difference (P < 0.05) from the corresponding time-point, in weeks (same treatment group). The asterisk indicates significant difference (P < 0.05) from the other treatment group (same time-point).
Figure 4
Figure 4
FMT images and in vivo quantification of angiogenesis within the implanted scaffolds. The color scale indicates the mean fluorescent probe concentrations within the ROI, and the graph (A) shows the mean values of probe content (in pmol) calculated within groups and reported as percent of the mean value of the 2 wk group with cell-free scaffold. A higher concentration of AngioSense 680 is present when the scaffolds contain hADSCs, especially at 2 and 8 weeks (B,D) when compared with cell-free scaffolds (E–G). In the graph A, the numbers into the circles indicate significant differences (P < 0.05) from the corresponding time-points, in weeks (same treatment groups), while the asterisk indicates significant difference (P < 0.05) from the other treatment group (same time-point).
Figure 5
Figure 5
Alizarin Red S staining showing the time-course of mineralization within the scaffolds implanted either charged with hADSCs (A–C) or cell-free (D–F) and explanted at 2, 4 or 8 weeks after surgery. It is evident that the mineralization increased overtime in both treatment groups, but the addition of hADSCs determined a strong increase of this process. The graph (G) shows the quantitative evaluation of Alizarin Red staining by measuring the optical density (OD). The numbers into the circles indicate significant differences (P < 0.05) from the corresponding time-points, in weeks (same treatment groups), while the asterisk indicates significant difference (P < 0.05) from the other treatment group (same time-point). Scale bar: 100 μm.
Figure 6
Figure 6
Time-course of tissue modifications observed by Hematoxylin and Eosin staining in the scaffolds explanted at 2 (A,D), 4 (B,E) or 8 (C,F) weeks after subcutaneous implantation, either with (A–C) or without (D–F) the addition of hADSCs. It is evident that the material was wrapped, and then invaded, by fibroblast-like cells (asterisks in G), and this process was more robust in scaffolds seeded with hADSCs (compare A,B with D,E). Along the observed time-points, the material undergone a visible mineralization that, again, appeared dramatically increased in scaffolds seeded with hADSCs prior to implantation (compare A–C with D–F). In particular, the scaffold appeared almost completely mineralized at 8 weeks (C). The mineralization is evident also comparing the scaffolds sections at low magnification in H (2 weeks, without hADSCs) and I (8 weeks, with hADSCs). The formation of vascular elements is evident in G (arrow) and at higher magnification in J (arrows). Scale bar in A for A-F: 100 μm; in G: 200 μm; in H for H, I: 1 mm; in J: 50 μm.
Figure 7
Figure 7
Panel of fluorescence microscope images showing the time-course of expression of osteogenic and angiogenic markers in scaffolds with or without hADSCs (red fluorescence). It is evident that the expression of some osteogenic markers in scaffolds plus hADSCs, especially ALPL (A,F,K), Osteocalcin (B,G,L) and Osteonectin (C,H,M) is increasing with time, whereas the expression of Osterix was constant along time-points (D,I,N) and CD31 appeared higher at 2-4 weeks post-implantation (E,J) and then decreased at the longer time-point (O). A similar pattern of time-course expression appears in scaffolds without human cells. In particular, the expression of ALPL (A’,F’,K’) and Osteonectin (C’,H’,M’) is increasing with time, whereas the expression of CD31 appeared higher at 2-4 weeks post-implantation (E’,J’) and then decreased at the longer time-point (O’). It is also evident that the expression of all analysed markers seems to be lower than that observed in scaffolds containing hADSCs. Nuclei have been stained with DAPI (blue). Scale bars: 100 μm.
Figure 8
Figure 8
Panel of fluorescence microscope images showing typical examples of ALPL (A), Osteocalcin (B), Osteonectin (C), Osterix (D) and CD31 (E) expression (red fluorescence). Arrows indicates some positive cells. A negative control obtained by omitting the primary antibody shows the absence of any fluorescent signal. Nuclei have been stained with DAPI (blue). Scale bar: 40 μm.
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