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. 2019 Jan;8(1):22-34.
doi: 10.1002/sctm.18-0085. Epub 2018 Sep 5.

Mesenchymal Stromal Cell-Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl-Deficient Osteopetrosis

Ciro Menale  1   2 , Elisabetta Campodoni  3 , Eleonora Palagano  2   4 , Stefano Mantero  1   2 , Marco Erreni  2 , Antonio Inforzato  2   4 , Elena Fontana  1   2 , Francesca Schena  5 , Rob Van't Hof  6 , Monica Sandri  3 , Anna Tampieri  3 , Anna Villa  1   2 , Cristina Sobacchi  1   2
Affiliations

Mesenchymal Stromal Cell-Seeded Biomimetic Scaffolds as a Factory of Soluble RANKL in Rankl-Deficient Osteopetrosis

Ciro Menale et al. Stem Cells Transl Med. 2019 Jan.

Abstract

Biomimetic scaffolds are extremely versatile in terms of chemical composition and physical properties, which can be defined to accomplish specific applications. One property that can be added is the production/release of bioactive soluble factors, either directly from the biomaterial, or from cells embedded within the biomaterial. We reasoned that pursuing this strategy would be appropriate to setup a cell-based therapy for RANKL-deficient autosomal recessive osteopetrosis, a very rare skeletal genetic disease in which lack of the essential osteoclastogenic factor RANKL impedes osteoclast formation. The exogenously administered RANKL cytokine is effective in achieving osteoclast formation and function in vitro and in vivo, thus, we produced murine Rankl-/- mesenchymal stromal cells (MSCs) overexpressing human soluble RANKL (hsRL) following lentiviral transduction (LVhsRL). Here, we described a three-dimensional (3D) culture system based on a magnesium-doped hydroxyapatite/collagen I (MgHA/Col) biocompatible scaffold closely reproducing bone physicochemical properties. MgHA/Col-seeded murine MSCs showed improved properties, as compared to two-dimensional (2D) culture, in terms of proliferation and hsRL production, with respect to LVhsRL-transduced cells. When implanted subcutaneously in Rankl-/- mice, these cell constructs were well tolerated, colonized by host cells, and intensely vascularized. Of note, in the bone of Rankl-/- mice that carried scaffolds with either WT or LVhsRL-transduced Rankl-/- MSCs, we specifically observed formation of TRAP+ cells, likely due to sRL released from the scaffolds into circulation. Thus, our strategy proved to have the potential to elicit an effect on the bone; further work is required to maximize these benefits and achieve improvements of the skeletal pathology in the treated Rankl-/- mice. Stem Cells Translational Medicine 2019;8:22-34.

Keywords: Biomimetic scaffold; Cell therapy; Gene therapy; Mesenchymal stromal cell; Osteopetrosis; RANKL.

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Figures

Figure 1
Figure 1
Validation of a lentiviral vector expressing human soluble RANKL (hsRL). (A) Schematic representation of the lentiviral vector for the expression of hsRL. (B) Quantization of HEK293T cell transduction with the LVhsRL and mock vector at increasing MOI, expressed as percentage of GFP+ cells (left) and as GFP mean fluorescence intensity (MFI; right). (C) Evaluation of cell growth rate over time of untransduced and transduced HEK293T cells, by MTT assay. (D) Quantization of hsRL production over time by untransduced and transduced HEK293T cells, by ELISA assay on the corresponding supernatants. Only statistics between different time points of the same MOI is indicated. (E) Quantization of hsRL in protein extracts (left) and in supernatants (right) of untransduced and transduced HEK293T cells 2 weeks after transduction. * p < .05; ** p < .01, *** p < .001.
Figure 2
Figure 2
Functional evaluation of hsRL produced by transduced HEK293T cells. Osteoclastogenesis and resorption assays from (A) human PBMCs and (B) murine WT and Rankl −/− splenocytes in the presence of M‐CSF and ×50 concentrated conditioned medium from mock‐transduced and LVhsRL‐transduced HEK293T cells (left and right plots, respectively). Mature osteoclasts were identified by TRAP staining, while resorption pits were visualized by staining dentin discs with toluidine blue. Scale bars: 200 μm (A); 400 μm (B).
Figure 3
Figure 3
Characterization of 3D biomimetic MgHA/Col scaffold. (A) Scanning Electron Microscopy micrographs at different magnifications, showing scaffold porosity and micro‐architectures of the hybrid composite. (B) Porosity and chemical composition of the hybrid MgHA/Col scaffold. (C) TGA, (D) FTIR, and (E) XRD analysis. In plots D and E, the solid line indicates the MgHA/Col, while the dashed one the HA. (F) Evaluation of the swelling ratio and (G) percentage of degradation of MgHA/Col scaffolds overtime.
Figure 4
Figure 4
Characterization of MSC culture on MgHA/Col scaffolds. (A) Confocal analysis of LVhsRL‐transduced Rankl −/− MSC‐seeded scaffolds at different time points; blue color corresponds to collagen fibers, green to MSCs. Scale bar: 200 μm (B) Assessment of WT, mock‐transduced and LVhsRL‐transduced Rankl −/− MSC proliferation in 2D versus 3D culture overtime by MTT assay. Cell growth is expressed as fold increase compared to time 0. Statistics is referred to comparison of 2D versus 3D for each group. Cell growth fold increase data ± SEM are reported in Supporting Information Table 1. (C) Evaluation of hsRL concentration in the supernatant of LVhsRL‐transduced Rankl −/− MSCs in 2D versus 3D culture by ELISA assay. * p < .05; ** p < .01.
Figure 5
Figure 5
Histological analysis of MSC‐seeded MgHA/Col scaffolds after 2 months implantation in Rankl −/− mice. (A) Hematoxylin–Eosin staining and CD31 immunostaining on serial sections of decalcified scaffolds from the different groups of treatment. Scale bar: 100 μm. (B) GFP immunostaining on sections of decalcified scaffolds from the different groups of treatment. Scale bar: 50 μm.
Figure 6
Figure 6
Evaluation of the effect of cell‐constructs implantation on the bone phenotype of Rankl −/− mice. (A) TRAP staining on long bone of Rankl −/− mice belonging to the different treatment groups. Scale bars: 50 μm (left); 20 μm (right). (B) Number of TRAP+ cells per bone section of treated Rankl −/− mice. nd: not detected. (C) Measurement of serum total calcium concentration in treated Rankl −/− mice. The red dashed line indicates the average concentration in age matched wild type mice. * p < .05.
Figure 7
Figure 7
Histological analysis of bone after 2 months MSC‐seeded MgHA/Col scaffolds implantation in Rankl −/− mice. H&E staining on sections of decalcified femurs from the different groups of treatment. Scale bar: 500 μm.
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