Authors:
Froilan Granero-Molto, PhD, Timothy J Myers, PhD, Jared A Weis, Lara Longobardi, PhD, Tieshi Li, PhD, Yun Yan, MD, Natasha Case, PhD, Janet Rubin, MD, and Anna Spagnoli, MD
Summary:
Failures of fracture repair (nonunions) occur in 10% of the fractures. Clinical and animal studies have indicated that the use of mesemchymal stem cells (MSC) in tissue regeneration is safe and effective. The use of MSC expressing IGF-I enhanced such effects increasing callus bone formation. Objectives: To determine, 1) the intracellular pathway mediating osteoblastic differentiation of MSC by IGF-I and 2) whether IGF-I expressed by MSC, through autocrine effects, improved the fracture healing promoting new bone formation in a model of fracture non-union with a defective IGF signaling. Methods: 1) MSC were induced to differentiate into osteoblasts in serum free conditions. The effect of IGF-I on the differentiation process was assayed by quantification of the RNA expression levels of osteogenic markers. The contribution of the intracellular signaling pathways initiated by IGF-I was characterized using shRNA mediated gene silencing. 2) MSC isolated from WT mice were engineered to express human IGF-I (MSCIGF). A stabilized tibia fracture was produced in adult Irs1 knockout females (Irs1KO). Mice were transplanted with 106 MSC or MSCIGF by IV injection; untransplanted mice were used as controls. Fractured tibias were analyzed 14 days postfracture by µCT analysis. Calluses were subjected to histological studies to determine the distribution of bone and cartilage. Results: We determined that IGF-I induced osteoblastic differentiation of MSC in vitro. IGF-I induced significant expression of the osteoblast specific genes Osterix and Osteocalcin and a significant accumulation of calcium deposits. We characterized the intracellular pathway mediating the differentiation process and found that is dependent of an intact IRS1-PI3K pathway. Using Irs1KO as nonunion model of fracture healing through altered IGF-I signaling, we demonstrated, by µCT analysis, that, through autocrine properties, MSCIGF improved the fracture healing process inducing the formation of a callus that bridged the gap. Histological analysis confirmed these observations. In addition, MSCIGF transplant induced a significant increase of the callus new bone and soft tissue content when compared with control group (bone: 1.747±0.518 mm3, n=2 vs 1.063±0.037 mm3, n=3, p<0.05; soft tissue: 1.207±0.396 mm3; n=2 vs 0.689±0.078 mm3, n=3; p<0.05). Conclusions: We provided evidence of the effects and mechanism of MSCIGF in fracture repair and showed their therapeutic potential to treat fracture non-unions.
Source:
Endocrine Reviews; Vol. 32, OR12-5 (06/11)