These data demonstrate unequivocally a population of multipotent mesenchymal progenitor cells within the human ACL. The recent identification of such progenitor cells by Cheng et al
that were isolated by collagenase digestion of anterior and posterior cruciate ligaments supports this conclusion. The present study, however, focuses on the repair relevant outgrowth ACL cell populations (ACLOUT
) and offers a unique perspective on ACL progenitors by examining their origins within the tissue and by comparing them directly to ACL populations released by collagenase digestion (ACLDIG
) and BMSCs from human bone marrow. Both ACL populations are very similar to human BMSCs, but differ in certain details. All cell types examined show the ability of colony formation () and revealed almost similar osteogenic () and adipogenic () capacities. However, the ACL populations show less cell proliferation () as well as chondrogenic capacity (), and do not express CD106 or CD146 as highly or STRO-1 as consistently, but they express higher levels of CD97 ( and ). CD146 in particular was less expressed in the ACLOUT
compared to the ACLDIG
, although levels of significance were not reached (). Expression of STRO-1 by MSCs declines with cell passage,35
which may explain the marginal staining for this epitope on those cells examined by FACS (). This would be consistent with the considerable staining for STRO-1 noted by immunocytochemistry, and immunohistochemistry, where less subculture is involved (). However, this study is limited to the examination of only a finite number of characteristic cell surface markers, and analyses of, for example, whole transcriptomes might be a suitable approach, to further resolve differences between the cell types.
Although cells with the general properties of MSCs have been recovered from a number of connective tissues, there are small phenotypic variations depending on the tissue source.14,16–24,26,36–39
The MSCs that are released from ACL tissues fit this pattern, but differ in subtle ways from the BMSCs used as a reference population. However, the three cell types could not be age- and patient-matched, so these variables could also account for the differences.
Progenitor cells were isolated from ligaments recovered 4–16 weeks after injury, at the time of surgical reconstruction. Given the evidence that MSCs migrate to sites of injury,40
there is the possibility that they are not normally present in the ACL. Indeed, a recent article by Morito et al
suggests that MSCs enter the synovial fluid after rupture of the ACL. The surface immunophenotype of the synovial fluid cells is similar to that reported here for ACL cells, except for a much higher expression of STRO-1.29
Despite the presence of these cells in the synovial fluid, our immunohistochemical findings argue against them being the source of the MSCs within the ACL. Cells positive for CD44, CD90, CD105, and STRO-1 were noted deep within the body of the ACL, closely aligned with the collagen fibers, as well as surrounding small blood vessels. The latter may be pericytes, long suspected of being a source of mesenchymal progenitors within vascularized tissues.39
The former, however, are morphologically indistinguishable from what are normally considered to be ligament fibroblasts, lying adjacent to the collagen bundles of this tissue. Further, we confirmed our results by using ACL outgrowth and digest populations from intact ACLs, derived from OA patients undergoing knee arthroplasty surgery, and noted an almost identical surface antigen expression and trilineage differentiation potential (not shown), compared to the data from cells recovered from torn ACLs (–), supporting the concept of a tissue-inherent MSC population and confirming findings in the tendon.24
The capacity of self-renewal is also an important criterion of a stem cell that is met by the ACLOUT
population as evidenced by an in vitro
assay using BMP12 adenovirus and three-dimensional culture in a collagen hydrogel for ligamentogenic induction (). Using a similar approach, we were recently able to delineate the ligamentogenic potential of the ACLDIG
and BMSC populations,34
which were used as comparative controls here (not shown). Our study, however, is limited to its in vitro
nature, and only in vivo
experiments can provide the necessary evidence for final validation of ACL regenerative approaches using inherent stem cells. The strong regenerative response of the BMP12 adenovirus, a strong ligamentogenic inductor of stem cells,41
in a rat model of Achilles tendon healing supports this concept.42
The results of our study differ in some ways to the data of Cheng and colleagues,43
who recently compared the cell proliferation and differentiation capacities of BMSCs to that of collagenase-digested ACL cells. In contrast to their study, where ACL cells revealed higher proliferation rates compared to BMSC,43
we found the BMSCs to elicit higher rates of cell proliferation compared to both ACLOUT
populations at all time points (). Further, we found the BMSC to exhibit a higher chondrogenic potential and similar osteogenic potential compared to both ACL cells types (–), whereas, in contrast, Cheng and colleagues found the BMSCs to have higher osteogenic capacity and similar chondrogenic capacity compared to ACL digest cells.43
We attribute the discrepancies in both studies to the different protocols that were used for cell isolation, maintenance, and differentiation as well as in differences in growth factor and FBS supplementation used to the respective cultures. However, the data in both studies correspond in that the adipogenic and ligamentogenic potential in all cell types examined were at comparable levels,43
undermining the relevance of ligament inherent stem cell populations and their implications over a possible use of this technology toward biological ACL repair. The fact that ACL outgrowth cells, the primary cell source that mediates any direct biological repair of the ACL,44
share these features further adds to this conclusion.
Putative stem/progenitor cells have recently been identified in similar locations of the tendon, suggesting that this may be a general phenomenon within fibrous tissues and several extracellular matrix components are important organizers of the stem cell niche.24
Not all fibroblasts within the ACL stained positively for MSC surface epitopes (), suggesting the presence of a mixed population of MSCs and ligament fibroblasts within the tissue. The heterogeneity of the cell population may be reflected in the nature of the cartilaginous pellets formed during chondrogenesis assays, where the inner core was much less cartilaginous than the outer component ().
While the information gained in this study provided insight into the cellular characteristics of ACL outgrowth cell populations, the study is limited to its in vitro nature, and further in vivo work is required to determine its relevance to ligament repair including evaluation for structural and mechanical properties of the repair tissue.
The presence of MSCs within the ACL opens new opportunities for biological repair of this ligament. As noted above, biological repair has been largely ignored because of the belief that the ACL lacks regenerative capacity. Our data suggest, on the contrary, that the ACL houses a rich population of progenitors that are rapidly mobilized in response to injury. These cells migrate from the injured ligament, and can colonize a suitable, adjacent scaffold to synthesize repair tissue.9,10
Recent work by Cheng and colleagues revealed a vivid response of collagenase released ACL progenitors to several growth factors with respect to cell proliferation and differentiation.45
In animal models, the repair of tendons and ligaments can be enhanced by various morphogens and growth factors,38
such as BMP12,46
growth and differentiation factor (GDF)5,48
and platelet derived growth factor (PDGF).49
Gene transfer is an attractive technology for delivering these factors, especially if the vectors are associated with the matrix into which the ACL outgrowth cells migrate. Under these conditions, selective gene transfer to the ACL outgrowth cells occurs very efficiently in situ
This suggests one way in which these newly identified cells can be manipulated to improve healing of the ACL.