Co-culture of the bone and bone marrow: a novel way to obtain mesenchymal stem cells with enhanced osteogenic ability for fracture healing in SD rats.

Abstract

Mesenchymal stem cells (MSCs) have great potential for the repair and regeneration of bone fracture, but their optimal origins remain controversial. Bone marrow-MSCs (BM-MSCs) and bone-bone marrow-MSCs (B-BM-MSCs) were isolated from 12 SD rats, and the morphology, MSC-associated markers, and proliferative capacity of these cells were compared using an inverted microscope, flow cytometry, and CCK-8 assays, respectively. After 14 days of osteoblastic induction, osteoblast phenotypes were detected by ALP and calcium nodule staining, and the expression of BMP-2 and TGF-β1 was observed by western blotting. Then, the rat tibia fracture model was established with 3 groups (n = 6 per group), the control, BM-MSC, and B-BM-MSC groups. Computed tomography (CT) imaging was performed to evaluate fracture healing at weeks 2, 4, and 6. Finally, the fractured bones were removed at weeks 4 and 6, and HE staining was performed to evaluate fracture healing. Although the 2 types of MSCs shared the same cellular morphology and MSC-associated markers, B-BM-MSCs had a higher proliferative rate than BM-MSCs from day 9 to day 12 (p < 0.05), and the expression levels of ALP and calcium were obviously higher in B-BM-MSCs than in BM-MSCs after osteogenic induction (p < 0.01 and p < 0.001, respectively). Western blot results showed that the expression levels of BMP-2 and TGF-β1 in B-BM-MSCs were higher than in BM-MSCs before and after osteogenic induction (p < 0.01). In the animal experiments, CT imaging and gross observation showed that B-BM-MSCs had a greater capacity than BM-MSCs to promote fracture healing, as the Lane-Sandhu scores of B-BM-MSCs at weeks 4 and 6 after operation (3.00 ± 0.81 and 9.67 ± 0.94, respectively) were higher than those of BM-MSCs (1.33 ± 0.47 and 6.67 ± 1.25, respectively; both p < 0.05). The HE staining results further supported this conclusion. Taken together, our study results proved that MSCs obtained by co-culturing the bone and bone marrow from SD rats had better proliferative, osteogenic differentiation, and fracture healing capacities than BM-MSCs, perhaps suggesting a novel way to obtain MSCs for bone tissue repair.