The results of this study supported the hypothesis that MSCs from either source enhance the migration, proliferation, and collagen gene expression of the ACL fibroblasts in vitro, but that they do so in different ways. While ADSCs had a greater effect on stimulating ACL fibroblast proliferation and procollagen production, PBMCs were more effective in stimulating ACL fibroblast migration. Both ADSCs and PBMCs significantly accelerated gene expression for both type I and type III collagen for the ACL fibroblasts. This suggests that ADSCs and PBMCs may contribute to wound healing not just through their own production of collagen but also by stimulating the in situ ACL fibroblasts. These findings are consistent with previous work with dermal fibroblasts, where Kim et al. [4
] and Wang et al. [27
] reported that media conditioned by culture of ADSCs [4
] and plastic adherent PBMCs [27
] (named fibrocytes by the authors) significantly increased the collagen gene expression of those cells as well as their migration in vitro. Our article builds on those findings by comparing ADSCs and PBMCs and also using co-culture of the three cell types and FACS separation after culture to begin to differentiate potential roles of the MSCs in influencing ACL fibroblast behavior. Even though the MSC cells tend to disappear over time in vivo, their effects in the early weeks of wound healing may be profound—a 100% or 300% increase in migration (seen in our migration assay for the co-culture with ADSCs or PBMCs, respectively) of fibroblasts to the wound site or the 20% increase in early procollagen production in the ACL fibroblast/ADSC co-culture—findings that can be critical to stabilization of the provisional scaffold during the healing of the primarily repaired ACL.
ACL fibroblasts and ADSC monocultures show a similar proliferation rate in monoculture and both cell types are significantly faster proliferating than the PBMC monoculture. However, the FACS analysis indicates a decrease of the ADSCs/ACL and PBMC/ACL ratio over time. There are several possible reasons for the observed changing ratio of ACL cells to ADSCs and PBMCs in co-culture. For the PBMCs, the significantly lower basic proliferation rate compared to the ACL fibroblasts could be the reason for a proportional decrease over time, since the ACL fibroblasts will divide at a higher rate until confluence is reached. This simple explanation fails in case of the ADSCs, which should proliferate at the same rate as the ACL fibroblasts. The higher proliferation rate of the ACL fibroblasts in co-culture with the ADSCs could be caused by the ADSCs' function as trophic mediators, inducing a higher proliferation rate in the ACL fibroblasts by excreting growth factors [28
]. The same characteristic is known for the PBMCs [27
] and might even have boosted the proportional difference in cell number between the PBMCs and the ACL fibroblasts during cell culture. Additionally, the stemness of the ADSCs and PBMCs could result in greater stimulation of fibroblast proliferation than ADSC or PBMC proliferation [12
], which enabled them to undergo differentiation while in contact with the ACL fibroblasts. Another possibility is that the PBMCs and ADSCs begin to undergo apoptosis when co-cultured with the ACL fibroblasts. This would lead to an increased rate of cell death for these particular cells, and thus a change in the ratio of ACL/ADSC or ACL/PBMC in the culture. This would be supported by prior work by Jones et al. showed in their research that fibroblasts could have an antiproliferative effect on PBMCs [30
]. Additional experiments are planned to investigate these hypotheses.
Both ADSC and PBMC co-culture increased the collagen gene expression of the ACL fibroblasts above the rate of that seen in the ACL fibroblasts cultured alone. This positive effect on collagen synthesis of fibroblasts is also consistent with previous reports where an indirect effect of media conditioned by exposure to MSCs stimulated collagen gene expression of fibroblasts [4
]. The increase in mRNA expression by the ACL fibroblasts here is consistent with previous reports in the literature [4
], but a new finding in this report is that the co-culture also resulted in an upregulation of the collagen mRNAs in the ADSCs and plastic adherent PBMCs themselves, as shown in the qPCR results after FACS sorting of the co-cultures. This supports our second hypothesis that MSC differentiation can be influenced by the surrounding microenvironment as suggested by Ball [31
] and Karaoz [32
]. Our findings suggest that MSC-like cells derived from various tissues might be able to adapt and differentiate depending on the surrounding tissue. This seems to be true for the fat pad-derived ADSCs, since they significantly altered their collagen mRNA levels when they were co-cultured with the ACL fibroblasts. However, our data did not support the same finding for the PBMCs.
In addition, we were also able to show that cells from the ACL, fat pad, and the peripheral blood would show MSC-like properties, a finding consistent with prior reports [7
]. The cells used in this study were third or fourth passage following isolation as expansion was required both before and after lentiviral infection. Although no further experiments were conducted to test the influence of multiple passages on the MSCs, studies indicate that proliferation or differentiation capability seems not to be influenced until greater than the fifth passage [33
We were able to confirm the findings of Steinert et al. [14
], Jürgens et al. [25
], Cao et al. [9
], Faast et al. [7
], and Chong et al. [10
], who were able to differentiate ACL fibroblasts, fat pad-derived cells, and plastic adherent peripheral blood mononuclear cells along the three pathways. In our experiments, ADSCs showed the greatest ability to differentiate down all three pathways. Their ability to differentiate is well documented in the literature [12
] and recent findings suggest a strong similarity of ADSCs and bone marrow-derived MSCs in their molecular signature [34
], growth kinetics and differentiation potential [11
], as well as the expression of different growth factor genes related to their trophic potential [34
], suggesting that the fat pad may be a good source of multipotent MSCs. The superior differentiation and trophic potential could have had a positive influence on the ACL fibroblasts in co-culture as well, inducing a higher proliferation rate, upregulation of collagen mRNA, and stimulation of the collagen production compared to the PBMC co-culture.
PBMCs were found to have almost similar molecular expression patterns [9
] as bone marrow-derived MSCs or ADSCs. Nonetheless, there are varying results in the literature about their stemness and differentiation potential. Cao et al. [9
] were able to differentiate PBMCs along all three pathways, and Chong et al. [10
] showed similar chondrogenic potential for human peripheral blood and bone marrow-derived MSCs. However, our findings are more similar to prior reports for plastic adherent PBMCs from horse peripheral blood, which showed a no chondrogenic differentiation in the PBMC culture [35
]. Also, in healthy donors, the number of mesenchymal progenitor cells as well as their trophic potential seems to be lower in the peripheral blood compared to the bone marrow or adipose tissue [36
], but proliferation as well as growth factor excretion reached levels comparable to bone marrow-derived MSCs after inflammation, injury, or hypoxia [37
]. Since healthy donor animals were used in our study, this might have contributed to the lower proliferative, differentiation, and trophic potential of the PBMC culture in our results. The lower trophic and differentiation potential of the PBMCs compared to the ADSCs might also have impaired the stimulatory effect of the PBMCs on the ACL fibroblasts in co-culture, ultimately leading to the observed inferior performance compared to the ADSC/ACL fibroblast co-culture.
In summary, our results suggest that ACL fibroblasts, ligament healing, and therefore ACL-repair, in general, might be enhanced by the addition of MSCs from the fat pad and the peripheral blood. The fat pad-derived cells showed not only a significant stimulatory effect on the ACL fibroblasts but were able to be coaxed into differentiating along the fibroblast pathway as evidenced by upregulation of collagen gene expression. On the other hand, cells obtained from the peripheral blood, PBMCs, showed a stimulatory effect on ACL fibroblasts but their potential to differentiate along a fibroblast pathway appeared more limited. Nonetheless, they might play an important role in the regulation of ACL healing because of their strong influence on the migration of the ACL fibroblasts, which could ultimately lead to a faster re-colonization of the ACL defect. This migratory stimulus could also potentially enhance the migration of ADSCs from the retro-patellar fat-pad into the ACL defect and bolster the healing process further. It might also be beneficial to combine ADSCs and PBMCs in vivo to have a synergistic effect of faster migration of the resident ACL fibroblasts into the defect promoted by the PBMCs and an enhanced proliferation and collagen production stimulated by the ADSCs.
Future studies evaluating the signaling mechanisms behind these primary observations and in vivo studies of the effects of implanting these cells in an ACL wound are planned.