Osteogenic potential of mandibular vs. long-bone marrow stromal cells

Abstract

Although fundamentally similar to other bones, the jaws demonstrate discrete responses to developmental, mechanical, and homeostatic regulatory signals. Here, we hypothesized that rat mandible vs. long-bone marrow-derived cells possess different osteogenic potential. We established a protocol for rat mandible and long-bone marrow stromal cell (BMSC) isolation and culture. Mandible BMSC cultures formed more colonies, suggesting an increased CFU-F population. Both mandible and long-bone BMSCs differentiated into osteoblasts. However, mandible BMSCs demonstrated augmented alkaline phosphatase activity, mineralization, and osteoblast gene expression. Importantly, upon implantation into nude mice, mandible BMSCs formed 70% larger bone nodules containing three-fold more mineralized bone compared with long-bone BMSCs. Analysis of these data demonstrates an increased osteogenic potential and augmented capacity of mandible BMSCs to induce bone formation in vitro and in vivo. Our findings support differences in the mechanisms underlying mandible homeostasis and the pathophysiology of diseases unique to the jaws.

Figure 1.

Isolation and culture of mandible BMSCs. (A) Radiograph of hemimandible showing the final position of the needle in the alveolar ridge superior to the incisor. The third molar has been extracted. (B) DSPP expression of long-bone (LB) BMSCs, mandible (MB) BMSCs, and pulp tissue by qPCR representative of more than 6 independent experiments. (C) Quantification of total, ALP-positive, and ALP-negative colonies formed by long-bone (LB) vs. mandible (MB) BMSC cultures (average of 3 independent experiments). *p < 0.05; error bars represent standard error of the mean.

Figure 2.

In vitro characterization of mandible vs. long-bone BMSCs. (A) Representative of 5 independent experiments of ALP staining at 3-, 7-, 14-, and 21-day cultures of long-bone (LB) vs. mandible (MB) BMSCs in osteogenic media. (B) Representative of 4 independent experiments of von Kossa staining at 14- and 21-day cultures of long-bone (LB) vs. mandible (MB) BMSCs in osteogenic media. (C) ALP activity assay at 3- and 7-day cultures of long-bone (LB) vs. mandible (MB) BMSCs in osteogenic media (average of 3 independent experiments). (D) ^[45]Ca assay at 21 days’ culture of long-bone (LB) vs. mandible (MB) BMSCs in osteogenic media (average of 3 independent experiments). (E) ALP and (F) OCN mRNA expression determined by qPCR of long-bone (LB) vs. mandible (MB) BMSCs cultured in osteogenic media for 3 and 7 days (average of 4 independent experiments). *p < 0.05; error bars represent standard error of the mean.

Figure 3.

3D-reconstructed (A,B) microCT images of representative gelatin sponges seeded with (A) long-bone- or (B) mandible-derived marrow cells. (C) Tissue volume (TV) and (D) bone volume (BV) of long-bone (LB) vs. mandible (MB) marrow cell-seeded sponges, quantified by µCT (average of 8 individual transplants from 2 independent experiments). *p < 0.05; error bars represent standard error of the mean.

Figure 4.

H&E sections at 2X (A,D), 10X (B,E), and 20X (C,F) of sponges seeded with long-bone marrow cells (A,B,C) or sponges seeded with mandible marrow cells (D,E,F) and implanted at the intrascapular area of nude mice.