**4.2 In vivo evidence**

Several in vivo studies have investigated the effect of MSC treatment on tendon ECM composition and structure, as well as on tendon biomechanical parameters. In most of these studies, including an equine large animal study with a follow-up of 45 weeks, the ECM composition was improved by BMSC and ASC treatment, with higher expression of collagen I on gene and/or protein level [106, 114, 120, 122, 140]. Collagen III expression was found to be decreased after ASC implantation [110, 125, 126] but increased after BMSC implantation [106, 122]. Tenascin-C and decorin were found to be increased following BMSC and ASC treatment [112, 114, 140], and glycosaminoglycans were decreased after BMSC treatment [141]. Based on these data, MSCs appear to increase collagen I deposition in healing tendons. Furthermore, as an increase of human-specific collagen I and tenascin-C was demonstrated in a rat model after human ASC implantation, there is also some evidence that MSCs actively contribute to the synthesis of new ECM [114]. The contribution of collagen III, tenascin-C, and decorin synthesis/modulation to tendon healing is to be considered controversially, as illustrated above, and certainly depends on its balance with regard to other ECM components. Yet, beyond mere collagen I synthesis, BMSC and ASC have also repeatedly been shown to improve the structural organization of healing tendons, again including the study with a 45-week follow-up, as well as an experimental trial in horses with naturally occurring tendinopathy [108, 115, 121, 140, 141]. In conjunction with the synthesis and protection of desired ECM components such as collagen I, this could be due to active ECM remodeling and the contribution of synthesized small ECM molecules to collagen fibrillogenesis. Still, it should be acknowledged that some studies in the equine model could demonstrate only few compositional or structural improvements 5 months after ASC treatment [133, 137]. Moreover, despite generally improved ECM structure and collagen I synthesis, collagen II deposits and areas staining positive for

**79**

research.

*Mechanisms of Action of Multipotent Mesenchymal Stromal Cells in Tendon Disease*

alizarin red were found in BMSC-treated tendons [106], suggesting that erroneous MSC differentiation toward the chondrogenic and osteogenic lineage had occurred. Nevertheless, functional testing of BMSC- and ASC-treated tendons indicated an improvement of functional parameters in the majority of studies [107, 108, 112–115, 117, 119, 121, 122], which represents a beneficial effect that

So far, very few in vivo studies have investigated the effect of MSC on the presence and activation of matrix-remodeling enzymes and their endogenous inhibitors. In the equine model, MMP-13 activity was decreased 6 months after BMSC treatment [141], and MMP-3 gene expression was upregulated in the healing tendons 45 weeks after BMSC treatment [140]. Together, these results might suggest that collagen degradation could be inhibited while degradation of small ECM components is promoted. However, there is much overlap regarding MMP substrates [167], and other studies found no significant differences in MMP and TIMP expression due to ASC treatment [112]. Further studies have to substantiate

When MSCs were combined with tenogenic growth factors, conflicting results were reported. Treatment with ASC and GDF-5 decreased MMP-2 and TIMP-2 expression and resulted in inferior biomechanical properties compared to ASC treatment alone [112]. Treatment with ASC and BMP-12 promoted ECM degradation, which was interpreted as a side effect of the fibrin-based delivery vehicle [124], but improved tendon ECM regeneration when delivered as cell sheets without fibrin [123]. Interestingly, the latter study showed that this may have been mediated by modulating the ECM remodeling activity of macrophages [123]. A further study from the same group demonstrated beneficial effects of combined ASC and CTGF treatment, although not evaluating effects of ASC alone [125]. A different study showed that predifferentiated BMSC sheets, induced by stepwise stimulation with TGF-β1 and CTGF, resulted in superior tendon regeneration, including improved biomechanical properties than BMSC alone [54]. However, in this study, again, fibrin was used for delivery of noninduced cells, which may have contributed to the differences observed. Thus, although some data suggest that the additional use of growth factors potentiates the beneficial effects of MSC on ECM regeneration, more evidence supporting this hypothesis is required. It should also be acknowledged that growth factor supplementation might impair other regenera-

There is a substantial body of evidence that demonstrates the immunomodulatory potential of MSC. While not all underlying mechanisms have been elucidated in detail yet, it is well-understood that MSCs suppress T cell proliferation and promote the modulatory M2 macrophage phenotype [168]. Furthermore, small ECM molecules synthesized by the MSC, such as tenascin-C and decorin, could contribute to immunomodulation [163, 169]. Therefore, it is likely that immunomodulation plays an important role in MSC-based tendon therapies. Against that background, it appears surprising that relatively few studies have addressed the interplay between MSC and the immune system in the context of tendon disease. This may be due to the long-existing perception that inflammation is absent during most stages of tendon disease, which, however, has been changing [5, 170]. While so far existing findings are summarized in the following, immunomodulation in the context of tendon disease will remain a promising field of future

*DOI: http://dx.doi.org/10.5772/intechopen.83745*

can be attributed to ECM regeneration [3].

tive mechanisms of MSC at the same time [93].

**5. Immunomodulation**

this hypothesis.

### *Mechanisms of Action of Multipotent Mesenchymal Stromal Cells in Tendon Disease DOI: http://dx.doi.org/10.5772/intechopen.83745*

alizarin red were found in BMSC-treated tendons [106], suggesting that erroneous MSC differentiation toward the chondrogenic and osteogenic lineage had occurred. Nevertheless, functional testing of BMSC- and ASC-treated tendons indicated an improvement of functional parameters in the majority of studies [107, 108, 112–115, 117, 119, 121, 122], which represents a beneficial effect that can be attributed to ECM regeneration [3].

So far, very few in vivo studies have investigated the effect of MSC on the presence and activation of matrix-remodeling enzymes and their endogenous inhibitors. In the equine model, MMP-13 activity was decreased 6 months after BMSC treatment [141], and MMP-3 gene expression was upregulated in the healing tendons 45 weeks after BMSC treatment [140]. Together, these results might suggest that collagen degradation could be inhibited while degradation of small ECM components is promoted. However, there is much overlap regarding MMP substrates [167], and other studies found no significant differences in MMP and TIMP expression due to ASC treatment [112]. Further studies have to substantiate this hypothesis.

When MSCs were combined with tenogenic growth factors, conflicting results were reported. Treatment with ASC and GDF-5 decreased MMP-2 and TIMP-2 expression and resulted in inferior biomechanical properties compared to ASC treatment alone [112]. Treatment with ASC and BMP-12 promoted ECM degradation, which was interpreted as a side effect of the fibrin-based delivery vehicle [124], but improved tendon ECM regeneration when delivered as cell sheets without fibrin [123]. Interestingly, the latter study showed that this may have been mediated by modulating the ECM remodeling activity of macrophages [123]. A further study from the same group demonstrated beneficial effects of combined ASC and CTGF treatment, although not evaluating effects of ASC alone [125]. A different study showed that predifferentiated BMSC sheets, induced by stepwise stimulation with TGF-β1 and CTGF, resulted in superior tendon regeneration, including improved biomechanical properties than BMSC alone [54]. However, in this study, again, fibrin was used for delivery of noninduced cells, which may have contributed to the differences observed. Thus, although some data suggest that the additional use of growth factors potentiates the beneficial effects of MSC on ECM regeneration, more evidence supporting this hypothesis is required. It should also be acknowledged that growth factor supplementation might impair other regenerative mechanisms of MSC at the same time [93].
