Abstract
Introduction
The use of mesenchymal stem cells (MSCs) loaded on osteoconductive scaffolds has emerged as a potential new treatment of large bone defects but has generated marginally successful results in terms of new bone formation. It is supposed that MSC massive death post implantation is a major obstacle for the exhibition of their osteogenic potential. Yet, the very few studies conducted using primary human MSCs derived from bone marrow (hMSCs), a clinically pertinent cell source, did not demonstrate that cell survival is required for new bone formation. In order to elucidate whether cell survival is needed for hMSC to express their osteogenic potential, the present study examined in an ectopic mouse model the relationship between cell survival and osteogenic potential of hMSCs loaded onto osteoconductive scaffold.
Materials and Methods
hMSCs (106) were seeded on 40-mg calcium carbonate (Biocoral) particles (size: 610–1000 µm), wrapped in fibrin gel (Baxter), and implanted subcutaneously into immunodeficient (nu/nu) mice (n=8/group). The fate of implanted cells was analysed using the bioluminescence and immunohistochemistry. For this, hMSCs were transduced with Luc-GFP (Luciferase-Green fluorescent protein) lentiviral vectors prior to experimentation. Bone formation was analysed 8 weeks post implantation on both non-decalcified and decalcified samples.
Results
Bone formation was observed in all cell-containing scaffolds as soon as 4 weeks post implantation. The number of viable Luc-expressing cells decreased progressively post implantation: while at most 4 % of the initially seeded cells survived at 2 weeks, it was less than 0.3% at 8 weeks. In addition, cell survival was not improved when either (i) hMSCs were seeded on calcium carbonate particles of other sizes such as 45–80 µm, 80–200 µm, 300–450 µm, and 3 mm or (ii) hMSCs seeded on particles were led to deposit extracellular matrix and differentiate towards the osteoblastic phenotype prior implantation. The results of cell surviving analysis were further supported by immunohistochemistry data. Only few cells of human origin (i.e., GFP positive cells) were detected in explanted specimens at 15, 30 days, but not at 60 days. Surviving human cells were located either in the vicinity of blood vessels or at the interface between the fibrous tissue and the scaffold surfaces. However, no GFP-positive either osteoblasts or osteocytes were detected in the newly-formed bone.
Conclusion and Discussion
In the present ectopic mouse model, massive death of implanted hMSCs that occurred after implantation did not prevent new bone formation. According to the fact that neither osteoblasts nor osteocytes were found to be of human origin, it may be suggested that hMSCs have an osteogenic-inductive potential. These results are in agreement with the theory that implanted MSCs contribute indirectly (through secretion of cytokines and growth factors) to new bone formation.
