Although HO is a known sequela of traumatic injury, and occurs frequently in patients following combat-related extremity injuries and traumatic amputation,1
little is known about the etiology of this pathological wound repair process. In this study, we have rigorously characterized the osteogenic potential of a population of MPCs isolated from within the traumatized muscle tissue.5
The morphology and cell surface epitope profile of the MPCs are similar to those of bone marrow-derived MSCs,6
osteoprogenitor cells resident within the bone marrow. We have also demonstrated that the traumatized muscle-derived MPCs are capable of forming ALP-positive colonies, which is consistent with the mesenchymal osteoprogenitor phenotype.11
Upon osteogenic induction, the MPCs increase their ALP activity and begin to generate a mineralized matrix, although unlike the bone marrow-derived MSCs, significant cell proliferation continues in the MPC population while being cultured in the osteoinduction medium. The osteogenic gene expression profile of the MPCs is characteristic of early differentiation into osteoblasts, although some significant differences are apparent between the MPCs and bone marrow-derived MSCs. Taken together, these findings provide insight into the possible involvement of the traumatized muscle-derived MPCs in the formation of HO following orthopedic trauma.
Several aspects of our study lend strength to the relevance of the findings. First, the muscle samples in this study were obtained from a patient population that has a documented predisposition to HO formation. Therefore, the MPC populations were derived from tissues that were likely to undergo HO, and our findings are directly applicable to HO formation in humans following traumatic injury. Second, we have compared the osteogenic differentiation of MPCs to bone marrow-derived MSCs, a well-characterized population of osteoprogenitor cells8–10
that served as a valid positive control. Finally, the differentiation of the MPCs into osteoblasts was evaluated based on a number of criteria, including histology, histochemistry, and gene expression analyses, with each assay performed using a different set of patients. The corroborative evidence is taken together to accurately assess the osteogenic potential of the traumatized muscle-derived MPCs.
However, there are two caveats to this study that should be noted. First, the bone marrow-derived MSCs were obtained from patients undergoing elective total hip arthroplasties, and thus were not age or sex matched to the substantially younger population of soldiers from whom we obtained the traumatized muscle samples. Although the MSCs are an adequate population to use as a positive control, any observed differences between the cell populations must take into account age-related changes12,13
and sexual dimorphism14
in the osteogenic potential of bone marrow-derived MSCs. Second, a modified alpha (α = 0.018) was used to assess statistical significance in the RT-PCR array assay and limit the false discovery rate. While comparisons of the osteogenic gene expression profile as a whole may be made between the two cell populations, the differential expression of any specific genes in the array will need to be independently verified before beginning further investigation to determine their role in osteogenic differentiation of the MPCs or ectopic bone formation in traumatized muscle.
Despite these caveats, significant differences were noted between the traumatized muscle-derived MPCs and bone marrow-derived MSCs. First, the MPCs continued to proliferate while being induced to differentiate into osteoblasts. It is not known whether the entire population is slow to shift from the proliferative state to differentiation, or if a subset of the population continues to proliferate while a second subset differentiates. There is evidence supporting the former, because histological evidence of differentiation appears homogeneous throughout the MPC cultures undergoing osteogenesis. These cells also express lower levels of osteocalcin, an osteoblastic gene that is expressed during later stages of osteogenic differentiation. Second, there are differences in the osteogenic gene expression profile between the MPCs and MSCs, which may reflect the tissue of origin for both cell types. MPCs express higher levels of COL15A1
, a gene associated with muscle tissue development,15
, shown to be a negative regulator of osteogenesis,16
whereas the bone marrow-derived MSCs express higher levels of genes associated with bone physiology and maintenance: VEGF-A
These differences may also reflect the fact that traumatized muscle-derived MPCs are harvested from an active wound bed, where they likely participate in the process of muscle tissue repair. During osteogenic differentiation, COL15A1
are substantially, albeit nonsignificantly, down-regulated, while VEGF-A, VCAM-1
, and IGF-2
are similarly up-regulated, suggesting that the MPCs can assume the role of osteoprogenitors under the appropriate biological environment, in a manner similar to other populations of MSCs.20
The MPCs used in this study were harvested from traumatized muscle tissue, and when the tissue was obtained, it was not possible to determine whether the patient would eventually develop HO. However, the osteogenic potential of the MPCs does not appear to be patient specific. Using the limited clinical outcomes data, we could not make any correlation between the ability of MPCs to undergo osteogenic differentiation and the incidence of HO in our patient population. Given this apparent homogeneity of the MPCs harvested from different patients, it is reasonable to assume that the potential to differentiate into osteoblasts is an inherent property of all the MPCs in the traumatized tissue. Multipotent stem cells have previously been isolated from untraumatized muscle using immuno-selective techniques,21
and the plastic-adherent MPCs studied here may be the descendants of these stem cells that have been activated within their niche by the injury and have begun to proliferate in the tissue. It has also been hypothesized that pericytes, which occupy a perivascular niche in vivo, are the cells that exhibit an MSC phenotype in vitro22
; thus the MPCs might also be activated pericytes that have entered the wound bed. These scenarios imply that migration of MPCs into the traumatized muscle is a part of the normal wound healing response, and HO occurs when pathological factors are present in the environment to encourage the expression of their osteogenic phenotype. The traumatized muscle tissue is an active wound bed, with intense inflammatory and wound healing responses. We are currently studying the cytokine expression profiles of the traumatized tissue to determine which factors are sufficient to trigger osteogenic differentiation of the MPCs and initiate the formation of ectopic bone.
In summary, the findings of this study indicate that traumatized muscle-derived MPCs are the putative osteoprogenitor cells responsible for HO following traumatic injury. Although there are notable differences between the MPCs and MSCs, which likely reflect their tissue of origin and in vivo function, both cell types demonstrate the ability to adopt the osteogenic differentiation pathway under the appropriate induction conditions. It is unclear what pathological signaling events occur in vivo to initiate the differentiation of traumatized muscle-derived MPCs into osteoblasts, and the identification of this triggering mechanism is an active area of investigation in our laboratory. Furthermore, the MPCs appear to differentiate more slowly than the bone marrow-derived MSCs, which suggests that an additional, intermediate step may be involved to encourage their terminal differentiation into osteoblasts, such as the endochondral ossification of the fibroproliferative lesions. This investigation will be followed up using an in vivo model to study the mechanism of osteogenic differentiation of MPCs that leads to ectopic bone formation in a physiological system. Nevertheless, our findings have shed substantial light on the mechanism of HO by identifying the MPCs in traumatized muscle as the osteoprogenitor cells that are likely to initiate bone formation, and may be used in future investigations to further elucidate the signaling mechanism involved in this process.