Putative heterotopic ossification progenitor cells derived from traumatized muscle

Abstract

Heterotopic ossification (HO) is a frequent complication following combat-related trauma, but the pathogenesis of traumatic HO is poorly understood. Building on our recent identification of mesenchymal progenitor cells (MPCs) in traumatically injured muscle, the goal of this study was to evaluate the osteogenic potential of the MPCs in order to assess the role of these cells in HO formation. Compared to bone marrow-derived mesenchymal stem cells (MSCs), a well-characterized population of osteoprogenitor cells, the MPCs exhibited several significant differences during osteogenic differentiation and in the expression of genes related to osteogenesis. Upon osteogenic induction, MPCs showed increased alkaline phosphatase activity, production of a mineralized matrix, and up-regulated expression of the osteoblast-associated genes CBFA1 and alkaline phosphatase. However, MPCs did not appear to reach terminal differentiation as the expression of osteocalcin was not substantially up-regulated. With the exception of a few genes, the osteogenic gene expression profile of traumatized muscle-derived MPCs was comparable to that of the MSCs after osteogenic induction. These findings indicate that traumatized muscle-derived MPCs have the potential to function as osteoprogenitor cells when exposed to the appropriate biochemical environment and are the putative osteoprogenitor cells that initiate ectopic bone formation in HO.

Figure 1

Morphology of traumatized muscle-derived MPCs. (A,B) Phase contrast microscopy 24 h after initial plating. Bar = 100 µm (A) or 50 µm (B). (C,D) After 21 days, the MPCs formed ALP-positive colonies stained with fast blue BB. (C) Whole mount, bar = 25 mm. (D) Bright-field view, bar = 100 µm.

Figure 2

ALP activity of MPCs and MSCs during osteogenic differentiation. (A) ALP staining with fast blue BB. MPCs were cultured in growth medium (GM) or osteogenic induction medium (OM) and compared to bone marrow MSCs cultured in OM. Bar = 5 mm. (B) Quantitative ALP activity profile. The ALP activity levels in each condition are normalized to the day 7 values from cells cultured with GM and also expressed as specific activities (per µg DNA).Groups that are not labeled with the same letter were significantly different (p < 0.05, ANOVA with multiple comparisons and n = 3).

Figure 3

Matrix mineralization of osteogenic MPC and MSC cultures. Alizarin red staining of mineralized matrix in cultures of MPCs cultured in growth medium (GM) or osteogenic induction medium (OM) and compared to bone marrow MSCs cultured in OM. Low magnification: bar = 5 mm. High magnification: phase contrast, bar = 100 µm.

Figure 4

Expression of osteogenic genes during osteogenic differentiation of MPCs and MSCs. (A) RT-PCR analysis of MPCs and MSC cultured in growth medium (GM) or osteogenic induction medium (OM). (B) Relative gene expression in MPCs and MSCs measured using quantitative real-time RT-PCR (*p < 0.01, Student’s t-test with n = 3).

Figure 5

Osteogenic gene expression profile of MPCs and MSCs. (A) The differential gene expression of 84 genes related to osteogenesis between MPCs and MSCs maintained in growth medium (GM). The table lists all genes that are differentially expressed more than fourfold. Genes differentially expressed with significance p < 0.018 (Student’s t-test with n = 3) are drawn inside a box in the plot and are written in bold in the table. (B,C) The differential expression of genes related to osteogenesis in (B) MPCs and (C) MSCs cultured in either osteogenic induction medium (OM) or growth medium (GM). Circles (●) represent CBFA1, squares (■) represent ALP, and diamonds (♦) represent BGLAP (osteocalcin) expression.