Abstract| Volume 25, SUPPLEMENT 1, S157-S158, April 2017

Endogenous PRRX1+ progenitors are present after full thickness cartilage injury but do not directly contribute to fibrocartilage or subchondral bone tissues during the repair process

      Purpose: While it is known that full thickness cartilage defects (FTCD) in mice and humans repair with a fibrocartilage patch rather than native articular cartilage (AC), it remains unknown what cell type(s) are responsible for producing the fibrocartilage patch. It has long been assumed clinically that mesenchymal stem cells (MSCs) present in the bone marrow are required for this fibrocartilage repair (e.g. rationale behind micro-fracture procedures), but to our knowledge this has never been demonstrated in vivo with endogenous cells. MSCs are unique in their ability to self-renew or differentiate into chondrocytes (cartilage cells), however, we have recently shown that exogenously delivered MSCs (GFP+) from C57BL/6 mice (C57) do not migrate to AC defects after injury, nor contribute to the resulting fibrocartilage patch. Furthermore, many published studies have shown that MSCs rarely directly contribute to formation of new tissues after injury, but rather they direct other cell types to promote repair. Therefore, in this study we have examined the role of endogenous Prrx1+ cells after FTCD injury in C57 mice. The MSC/progenitor marker, Paired Related Homeobox 1 (Prrx1), was employed in this study, as Prrx1+ cells can give rise to bone, fat and cartilage (cartilaginous callus) in vivo and these cells are present in the bone marrow, periosteum, fat pad and synovium within the joint.
      Methods: Prrx1CreERT2-GFPR26RTdTomato; C57BL/6 (C57) 8 week old mice were used for all experiments. FTCD (∼0.4mm) were induced in the trochlear groove (Fig. 1 A-D). Fluorescence imaging was used to identify the location of undifferentiated Prrx1+ MSCs (GFP+;TdTomato-) and their differentiated progeny (GFP-;TdTomato+) before and after cartilage injury. Immunohistochemistry was used on serial sections to identify actively proliferating cells (Ki-67+). Data was collected 1 day or 1, 2 and 4 weeks after FTCD injury.
      Results: Neither Prrx1+ cells (GFP+) or their differentiated progeny (GFP-TdTomato+) were observed inside AC defects in C57 mice at any time point (Fig. 1E-H).
      However, Prrx1+ cells and their progeny were observed directly adjacent to AC defects at 1 week post-injury (Fig. 1 F), and were also observed in the subchondral bone (SCB), patella/tendon, periosteum, and blood vessels within the bone marrow at all time points after injury (Fig. 1 E-H). While Prrx+ cells and their progeny were observed adjacent to the defect, no GFP or TdTomato staining was observed within the resulting fibrocartilage or remodeled SCB at any time point post-injury. Actively proliferating cells (Ki-67+) were observed at all time points in the AC injury site (Fig 1I-L). Specifically, Ki-67+ cells were located adjacent to, and also appeared to fill AC defects 1 week post-injury (Fig. 1J), but were also found within the underlying SCB as early as 1 day post-injury (Fig. 1 I-L). Interestingly, in a number of instances, Ki-67+ cells were observed in ‘chains’ between the SCB and AC injury, and were also found on the surface of the AC in and around the defect area (Fig. 1 I-K), however, Ki-67+ cells did not co-localize with GFP or TdTomato staining.
      Conclusions: These results support the hypothesis that endogenous MSCs (Prrx1+) do not directly contribute to the formation of new tissues (e.g. fibrocartilage or SCB) after FTCD in C57 mice. These results can neither confirm nor refute that Prrx1+ cells in the injury area are directing repair through trophic effects, but is interesting that Prrx1+ cells are observed at the margins of the defect area, but not within the defect itself. Furthermore, the resultant fibrocartilage and underlying remodeled SCB are derived from a replicating cell population likely originating from the bone marrow. Additionally, since Ki-67+ staining rarely co-localized with GFP+/TdTomato+ staining at the time points examined post-injury, this suggests that Prrx1+ cells do not proliferate once they have reached the defect area, but instead must proliferate and then migrate to the defect margins. Since we observed GFP+/TdTomato- cells in proximity to each other after 1 week after injury, this suggests that new Prrx1+ cells were produced from another cell type and able to migrate within this time period in response to injury. Future studies utilizing other progenitor/MSC lineage tracking mice such as GDF-5 or leptin-receptor may help elucidate the composition of the fibrocartilage patch observed during the healing response, however, from this study it is clear that Prrx1+ cells generate neither the fibrocartilage nor bone observed after FTCD in mice.