Abstract
Summary Statement
This study reports that hMSC can be manipulated in order to engineer a bone organ, characterised by mature osseous and vascular components and capable to recruit, host and maintain functional HSCs.
Introduction
Bone tissue engineering strategies are typically based on methods involving adult human Mesenchymal Stromal Cells (hMSC) in a process resembling intramembranous ossification. However, most bones develop and repair through endochondral ossification. In addition, endochondral ossification presents several advantages for regenerative purposes such as osteogenic activity, capability to drive formation of the Hematopoietic Stem Cell (HSC) niche, resistance to hypoxia, intrinsic vasculogenic potential and, consequently, efficiency of engraftment. In this study, we aimed at developing an endochondral bone organ model characterised by functional osseous and hematopoietic compartments by using hMSC.
Materials & Methods
Expanded hMSC were seeded onto 8 mm diameter, 2 mm thick collagen sponges (UltrafoamTM, Davol Inc.), cultured for vitro under defined chondrogenic (3 weeks) and hypertrophic (2 weeks) conditions and then implanted ectopically in subcutaneous pouches in nude mice. Consistently with the normal process of bone regeneration, which requires an inflammatory environment, we added IL-1β to the hypertrophic medium and assessed its effect on in vitro mineralization, hypertrophy, extracellular matrix processing and in vivo remodeling/bone formation. Samples were analyzed by histology, IHC, Luminex® assays, ISH for human Alu repeats and µCT. Bone marrow cells, extracted after 12 weeks from the implanted samples were analyzed by flow cytometry and transplanted into lethally irradiated congenic animals to asses functionality of the engrafted bone marrow.
Results
In vitro, samples showed a mineralised collar, rich in Collagen I and BSP, and a hypertrophic core, rich in proteoglycans and Collagen X. In vivo, extensive remodeling occurred, with mature vessel ingrowth (CD31+, NG2+, α-SMA+) and osteoclast (TRAP+ and MMP9+ multinucleated cells). Bone formation displayed a peculiar topography: at the periphery of the samples, perichondral bone was formed, corresponding to the in vitro pre-mineralised outer ring; in the core of the samples, endochondral bone was formed, corresponding to the in vitro non-mineralised cartilaginous areas. Human cells could be still detected after 12 weeks in vivo, mainly in the bone in the core of the samples. IL-1β resulted in (i) enhanced MMP13 endogenous activity; (ii) enhanced osteoclasts activity by increased M-CSF levels and RANKL/OPG ratio; (iii) faster vascularization; (iv) larger regions of bone marrow, possibly because of an increased synthesis of SDF1, IL-8, M-CSF and MCP-1. Murine bone marrow cells in the newly generated bone included phenotypically and functionally defined HSC at a comparable frequency than normal bones of the same mice.
Discussion/Conclusion
We reported the generation of an ectopic “bone organ” with a size, structure and functionality comparable to native bones by appropriately primed hMSC. The use of hMSC and IL-1β makes this model closer to bone regeneration than to bone development. The work, provides a model useful for fundamental and translational studies on bone development and regeneration, as well as for the modeling of normal and malignant hematopoiesis.
