A more recent line of thinking among orthopaedic surgeons, however, states that the majority of nonunions occur due to mechanical instability, and consequently, high strain at the fracture site. If this is so, it is argued that the nonunion will heal if the mechanical environment is corrected by surgery, with no need to excise the nonunion tissue or add autografts. This study is based on the concept that the tissue which is formed in the fracture site maintains its biological functions, despite the failed healing process.
Indeed, previous studies have shown that the nonunion tissue contained cells with similar characteristics as BMSC,[13,16,17,28,29] from which emerged the idea that nonunion stromal cells could be reactivated in vivo to act as an adjuvant to stimulate bone formation. Nevertheless, none of these previous reports effectively evaluated whether nonunion cells were really capable of making bone in vivo. As nonunion cells experienced failed bone healing conditions, one could wonder that their proliferation and osteogenic capacities might have been irreversibly lost. To evaluate this hypothesis—the main objective of this study—we isolated the cells from atrophic nonunion tissues, to evaluate cell activity exempt of the confounding factors from the fibrous tissue environment.
Here we showed that, in vitro, NUSC had proliferative and senescence rates comparable to BMSC and osteoblasts, and homogeneously expressed the markers CD90 and CD73. However, the expression of CD105 and CD146 in NUSC was more closely related to that of osteoblasts, and significantly inferior in comparison to BMSC. In spite of this, NUSC differentiated along the osteogenic and adipogenic pathways in vitro, and when transplanted in vivo, formed ossicles displaying hematopoietic marrow, with the ability to host and support hematopoiesis.
Comparing the in vitro properties of NUSC versus BMSC isolated from healthy donors undergoing spinal fusion surgery, Bajada and co-workers reported that NUSC had a doubling time—a measure of how long a given cell culture takes to double in number in vitro—of 12 to 16 days and that the percentage of senescent cells in the cultures increased during cell culture expansion, in a way independent of patient's age. In this study, however, NUSC doubling time was similar to that of BMSC and osteoblasts. Also, NUSC did not senesce over time. We attribute these differences to the fact that Bajada and collaborators compared NUSC to BMSC isolated from healthy donors, while we compared NUSC to BMSC and osteoblasts also isolated from nonunion patients. Evidence indicates that nonunion patients have polymorphisms[4,5,33–36] in genes that regulate cell proliferation, such as FGFR1. Although a polymorphism does not directly dictate a loss of function, we cannot rule out the possibility that cells from nonunion patients could have a slower proliferation rate as compared to cells isolated from healthy subjects. In agreement with our findings, Takahara and colleagues showed that cells isolated from synovial pseudoarthrosis, defined as an end-stage nonunion, could be expanded in vitro for 10 passages with minimal decline in their initial proliferative capacity. Therefore, we concluded that NUSC was not a senescence-prone population and was able to proliferate, under its own intrinsic rate, when appropriate signaling conditions were provided.
Regarding cell surface protein expression, 2 reports showed that NUSC expressed the BMSC-related markers CD29, CD44, CD166, and CD105.[13,29] However, none of these markers are specific of osteoprogenitors.[26,32,37] Here we chose to characterize the immunophenotypic profile of nonunion cells using the BMSC-related markers recommended by the International Society of Cellular Therapy. We also included CD146, which was shown to be expressed by the multipotent subpopulation that reside within the total BMSC fraction.[26,39] CD146 expression had not been previously evaluated in NUSC populations. We observed that along with CD90 and CD73, both known to be homogeneously expressed by cells of the osteoblastic lineage,[26,38,40,41] only a small fraction (<10%) of NUSC was CD105+/CD146+. Considering that CD146 expression decreases as multipotent cells progress down the differentiation cascade toward osteoblasts[26,42] —what was indeed confirmed in our BMSC and osteoblast cultures — we concluded that NUSC mostly contained cells that already progressed on the line of differentiation and were closer to the mature osteoblastic stage. This raised the hypothesis that the signaling disfunction that lead to the interruption of the healing process might have occurred after osteoprogenitors had been recruited to the fracture site and had initiated differentiation. Further transcriptomic profiling of NUSC would certainly contribute to confirm this question.
This observation brings important new perspectives both in the clinical scenario and in nonunion etiology research. First, it supports the thesis of some clinicians that the mechanical environment is the strongest determinant of nonunion, and should, therefore, base the treatment.[18,19] In other words, according to this notion, management of nonunion should rely on minimally invasive mechanical restabilization of the fracture, in order to reduce the level of local strain, with no need to remove the nonunion tissue. Bone grafts or any biological adjuvants would only be applied to those cases where significant bone loss occurred, as in this context, the biological vector of the healing process is also severely compromised.
Finally, from the biological point of view, the knowledge that NUSC are able to form bone indicates that future studies should focus on the identification of specific signaling cues that could endogenously reactivate, that is, stimulate the proliferation and/or differentiation of the in situ NUSC, marrow, and periosteal osteoprogenitors. Besides the mechanotransduction signaling provided by fixation revision, this reactivation could be achieved by providing specific growth factors and/or cytokines involved in bone formation. But unfortunately, a specific (or a combination of) signaling factor that can endogenous promote bone formation in a controllable and safe way is, at present, not known. To a certain extent, this has already been achieved with the percutaneous injection of bone marrow concentrates in the nonunion site.[51–55] However, marrow preparations have the drawback of not having a homogeneous and reproducible composition of either signaling factors or cell types. Therefore, this strategy does not allow the predictability of results. Finding a strategy that combines the critical mechanical and biological healing-restarting components will be the next fundamental challenge for the development of less invasive and more effective treatments for nonunion.
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