**5. Immunomodulation**

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 research.

*Tendons*

in future studies.

**4.2 In vivo evidence**

Some studies also provide first insight into the interplay of MSC and tenocytes/ tendon ECM in matrix remodeling and will therefore be addressed in more detail. In direct co-cultures of ASC and tenocytes, a different temporal regulation of MMP and ECM components was observed compared to tenocytes alone [105]. This included the upregulation of collagen I and tenascin-C gene expression at day 7 and downregulation of tenascin-C and collagen III at later time points (14 and 21 days, respectively) and a higher collagen I to collagen III ratio on protein level at day 7. MMP-1, -2 and -3, as well as TIMP-1 gene expression, increased over time in tenocytes alone but showed a different temporal regulation pattern in the cocultures with a significantly increased MMP-3 expression at day 7 [105]. A different study from the same group investigated the indirect co-culture of ASC and tendon explants [104]. Here, total protease activity was increased in the co-cultures at day 3, as were the collagenases (putatively MMP-1 and -14) but not the stromelysins MMP-3 and -10. Furthermore, collagen III and tenascin-C deposition by ASC were reduced at day 7. Histology also suggested that ASCs had protective effects on the explant structure, but this was not consistent between donors [104]. However, seemingly in contrast to these findings, MMP-8, -9, and -13 expression by ASC in collagen scaffolds was lower upon stimulation with tendon ECM extract [46], and microvesicles from amniotic membrane mesenchymal cells induced a downregulation of MMP-1, -9, and -13 in tenocytes [101]. Thus, while it can be assumed that MSC actively contribute to and/or modulate tendon ECM remodeling, the exact temporal regulation and context-sensitivity of this mechanism need to be addressed

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

**78**

### **5.1 In vitro evidence**

In vitro evidence for MSC immunomodulation in tendon disease is scarce. The most comprehensive study investigated whether ASCs influence the effects of differently polarized macrophages on tenocytes in a tri-culture system [98]. In co-cultures of M1 macrophages and tenocytes, release of inflammatory mediators, such as PGE2 and IL-1β, was increased compared to M1 macrophage cultures alone or compared to co-cultures with M0 or M2 macrophages, suggesting inflammatory tenocyte activation. When ASCs were directly co-cultured with the macrophages for 5 days, with the tenocytes added for the last 24 h, tenocyte activation was decreased, with significantly lower release of TNF-α and IL-1β in tri-cultures with M1 macrophages. At the same time, the presence of ASC had increased CD206 expression in M0 and M1 macrophage populations, indicating a switch toward the anti-inflammatory M2 macrophage phenotype and providing insight into the suppressive mechanism. However, ASCs did not effectively counteract inflammatory activation of tenocytes by IL-1β, even when ASCs had been primed with IFN-γ [98].

Interestingly, it has also been shown that tenogenic differentiation of BMSC induced by GDF-5 involves arachidonic acid production and signaling pathways [67], suggesting a link between differentiation and inflammatory processes. In this line, addition of BMP-12 increased IL-6 secretion by ASC and attenuated the suppressive effect of ASC in a mixed lymphocyte reaction [93]. Microvesicles from amniotic membrane mesenchymal cells downregulated TNF-α expression in tenocytes but in contrast to conditioned medium, they had no effect on peripheral blood mononuclear cell proliferation [101, 171]. These studies provide preliminary insight into the modulation of inflammatory tenocyte activation by MSC, while they also suggest that their immunomodulatory potential may be higher when not tenogenically differentiated. Yet, MSC immunomodulation is highly contextspecific and influenced by a variety of factors including three-dimensional culture environments as well as inflammatory priming/licensing [172, 173]. Therefore, it remains crucial to perform further studies specifically mimicking aspects of tendon pathophysiology.

### **5.2 In vivo evidence**

The most insightful studies were performed by the same group, shedding light on ASC-mediated immunomodulation in tendon healing in the canine model [123–126]. Corresponding to the group's in vitro findings, ASC alone, delivered via cell sheets, stimulated the anti-inflammatory M2 macrophage phenotype in healing tendons and reduced total mononuclear cell infiltration. The M2 macrophage markers CD163, MRC1, and CD204 were increased on mRNA and/or protein level, as well as IL-4, prostaglandin reductase-1, and VEGF [123, 126]. Combined administration of ASC and BMP-12 promoted these effects, particularly with respect to IL-4 expression [123]. Furthermore, combined treatment with ASC and CTGF decreased IL-1β, IL-6, and IFN-γ and increased IL-4 expression [125]. These latter findings challenge the hypothesis that tenogenic differentiation decreases the MSC immunomodulatory potential. However, when the inflammatory reaction at the tendon repair site was promoted by a fibrin-based delivery vehicle, ASC and BMP-12 further fostered these unwanted effects [124]. This might indicate that strong inflammation alters the MSC immunomodulatory properties toward a proinflammatory phenotype. In contrast, priming with TNF-α increased the anti-inflammatory effects of BMSC: While nonprimed as well as primed BMSC increased IL-10 and reduced IL-1α, primed BMSC also reduced IL-12 and the numbers of M1 macrophages and increased IL-4 and the numbers of M2 macrophages in

**81**

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

BMSC in tendon healing was demonstrated in a rat model, in which TNFα, IFN-γ, and IL-1β were reduced, along with an increase of IL-2 and growth factors, including VEGF [111]. Apparently in contrast to most of these findings, however, we observed that clinical signs of inflammation were increased by ASC treatment in the equine model, although this effect was transient [137]. This again illustrates that MSCs can also adopt a pro-inflammatory phenotype and raises questions as to how and whether this should be controlled. When addressing this issue, it should be acknowledged that a certain extent of inflammation is required to drive resolution. In this respect, macrophages and their M2 polarization driven by MSC

rat Achilles tendon defects [118]. Further evidence of anti-inflammatory effects of

In addition to the direct effects of MSC on ECM composition and immune cells, trophic support and protection of resident cells are likely to contribute to beneficial effects of MSC in tendon healing. Tenocytes and tendon stem cells rescued by the MSC may be enabled to promote ECM regeneration and counteract inflammation. Furthermore, a MSC-mediated increase in vascularity may be beneficial at least in some stages of tendon healing, as it would improve energy and oxygen supply, as well as disposal of metabolites, thus reduce oxidative and metabolic stress. The presence of vascular endothelial cells, as well as the combination of tenogenic growth factors with VEGF, has also been shown to promote tenogenic differentiation [60, 74]. However, increased vascularity is also associated with tendinopathy pathogenesis and may foster neurogenic inflammation [6], thus this issue is dis-

Trophic effects on tenocytes were demonstrated in vitro, when ASC and BMSC, as well as BMSC-conditioned medium, promoted the proliferation of tenocytes [94, 102, 103]. Furthermore, ASC as well as BMSC-conditioned medium promoted tenocyte migration [102, 103], and ASC promoted healing in a microwound model [92]. In vivo, results are inconsistent as to whether BMSC and ASC decrease [137, 141] or increase [117] cellularity within healing tendons. However, the rate of apoptosis was lower following BMSC treatment [107], suggesting protective effects of the MSC. Moreover, ASC combined with CTGF locally increased the numbers of CD146-positive tendon stem cells, suggesting an activation and possible rescue of

Pro-angiogenetic effects were observed in small, as well as large animal studies, which demonstrated that BMSC and ASC implantation increased vascularity [106, 129, 131], likely mediated by an increase in VEGF (see below). Yet, the opposite effect was observed in horses suffering from naturally occurring tendinopathy

With respect to possible growth factor signaling, in vitro, higher TGF-β bioactivity was found in the BMSC secretome compared to tenocytes [100]. Upon tenogenic differentiation of ASC using BMP-12, VEGF secretion was significantly increased, although no effect on TGF-β was observed [93]. First in vivo evidence regarding the contribution of growth factors in tendon healing following BMSC or ASC implantation was obtained in rat models, in which VEGF, TGF-β, and hepatocyte growth factor expression were increased in the MSC treatment groups [106, 111, 112]. Yet, these studies did not comprehensively reveal whether these factors

The brevity of this subsection illustrates that the insight into trophic and protective mechanisms, as well as growth factor release by MSC, in the context of tendon

were released by the MSC or other cells within the tendon lesion.

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

may play a particularly important role.

cussed controversially.

this endogenous cell population [125].

following implantation of BMSC [141].

**6. Trophic support and pro-angiogenetic effects**

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

rat Achilles tendon defects [118]. Further evidence of anti-inflammatory effects of BMSC in tendon healing was demonstrated in a rat model, in which TNFα, IFN-γ, and IL-1β were reduced, along with an increase of IL-2 and growth factors, including VEGF [111]. Apparently in contrast to most of these findings, however, we observed that clinical signs of inflammation were increased by ASC treatment in the equine model, although this effect was transient [137]. This again illustrates that MSCs can also adopt a pro-inflammatory phenotype and raises questions as to how and whether this should be controlled. When addressing this issue, it should be acknowledged that a certain extent of inflammation is required to drive resolution. In this respect, macrophages and their M2 polarization driven by MSC may play a particularly important role.

### **6. Trophic support and pro-angiogenetic effects**

In addition to the direct effects of MSC on ECM composition and immune cells, trophic support and protection of resident cells are likely to contribute to beneficial effects of MSC in tendon healing. Tenocytes and tendon stem cells rescued by the MSC may be enabled to promote ECM regeneration and counteract inflammation. Furthermore, a MSC-mediated increase in vascularity may be beneficial at least in some stages of tendon healing, as it would improve energy and oxygen supply, as well as disposal of metabolites, thus reduce oxidative and metabolic stress. The presence of vascular endothelial cells, as well as the combination of tenogenic growth factors with VEGF, has also been shown to promote tenogenic differentiation [60, 74]. However, increased vascularity is also associated with tendinopathy pathogenesis and may foster neurogenic inflammation [6], thus this issue is discussed controversially.

Trophic effects on tenocytes were demonstrated in vitro, when ASC and BMSC, as well as BMSC-conditioned medium, promoted the proliferation of tenocytes [94, 102, 103]. Furthermore, ASC as well as BMSC-conditioned medium promoted tenocyte migration [102, 103], and ASC promoted healing in a microwound model [92]. In vivo, results are inconsistent as to whether BMSC and ASC decrease [137, 141] or increase [117] cellularity within healing tendons. However, the rate of apoptosis was lower following BMSC treatment [107], suggesting protective effects of the MSC. Moreover, ASC combined with CTGF locally increased the numbers of CD146-positive tendon stem cells, suggesting an activation and possible rescue of this endogenous cell population [125].

Pro-angiogenetic effects were observed in small, as well as large animal studies, which demonstrated that BMSC and ASC implantation increased vascularity [106, 129, 131], likely mediated by an increase in VEGF (see below). Yet, the opposite effect was observed in horses suffering from naturally occurring tendinopathy following implantation of BMSC [141].

With respect to possible growth factor signaling, in vitro, higher TGF-β bioactivity was found in the BMSC secretome compared to tenocytes [100]. Upon tenogenic differentiation of ASC using BMP-12, VEGF secretion was significantly increased, although no effect on TGF-β was observed [93]. First in vivo evidence regarding the contribution of growth factors in tendon healing following BMSC or ASC implantation was obtained in rat models, in which VEGF, TGF-β, and hepatocyte growth factor expression were increased in the MSC treatment groups [106, 111, 112]. Yet, these studies did not comprehensively reveal whether these factors were released by the MSC or other cells within the tendon lesion.

The brevity of this subsection illustrates that the insight into trophic and protective mechanisms, as well as growth factor release by MSC, in the context of tendon

*Tendons*

**5.1 In vitro evidence**

pathophysiology.

**5.2 In vivo evidence**

In vitro evidence for MSC immunomodulation in tendon disease is scarce. The most comprehensive study investigated whether ASCs influence the effects of differently polarized macrophages on tenocytes in a tri-culture system [98]. In co-cultures of M1 macrophages and tenocytes, release of inflammatory mediators, such as PGE2 and IL-1β, was increased compared to M1 macrophage cultures alone or compared to co-cultures with M0 or M2 macrophages, suggesting inflammatory tenocyte activation. When ASCs were directly co-cultured with the macrophages for 5 days, with the tenocytes added for the last 24 h, tenocyte activation was decreased, with significantly lower release of TNF-α and IL-1β in tri-cultures with M1 macrophages. At the same time, the presence of ASC had increased CD206 expression in M0 and M1 macrophage populations, indicating a switch toward the anti-inflammatory M2 macrophage phenotype and providing insight into the suppressive mechanism. However, ASCs did not effectively counteract inflammatory activation of tenocytes by IL-1β, even when ASCs had been primed with IFN-γ [98]. Interestingly, it has also been shown that tenogenic differentiation of BMSC induced by GDF-5 involves arachidonic acid production and signaling pathways [67], suggesting a link between differentiation and inflammatory processes. In this line, addition of BMP-12 increased IL-6 secretion by ASC and attenuated the suppressive effect of ASC in a mixed lymphocyte reaction [93]. Microvesicles from amniotic membrane mesenchymal cells downregulated TNF-α expression in tenocytes but in contrast to conditioned medium, they had no effect on peripheral blood mononuclear cell proliferation [101, 171]. These studies provide preliminary insight into the modulation of inflammatory tenocyte activation by MSC, while they also suggest that their immunomodulatory potential may be higher when not tenogenically differentiated. Yet, MSC immunomodulation is highly contextspecific and influenced by a variety of factors including three-dimensional culture environments as well as inflammatory priming/licensing [172, 173]. Therefore, it remains crucial to perform further studies specifically mimicking aspects of tendon

The most insightful studies were performed by the same group, shedding light

on ASC-mediated immunomodulation in tendon healing in the canine model [123–126]. Corresponding to the group's in vitro findings, ASC alone, delivered via cell sheets, stimulated the anti-inflammatory M2 macrophage phenotype in healing tendons and reduced total mononuclear cell infiltration. The M2 macrophage markers CD163, MRC1, and CD204 were increased on mRNA and/or protein level, as well as IL-4, prostaglandin reductase-1, and VEGF [123, 126]. Combined administration of ASC and BMP-12 promoted these effects, particularly with respect to IL-4 expression [123]. Furthermore, combined treatment with ASC and CTGF decreased IL-1β, IL-6, and IFN-γ and increased IL-4 expression [125]. These latter findings challenge the hypothesis that tenogenic differentiation decreases the MSC immunomodulatory potential. However, when the inflammatory reaction at the tendon repair site was promoted by a fibrin-based delivery vehicle, ASC and BMP-12 further fostered these unwanted effects [124]. This might indicate that strong inflammation alters the MSC immunomodulatory properties toward a proinflammatory phenotype. In contrast, priming with TNF-α increased the anti-inflammatory effects of BMSC: While nonprimed as well as primed BMSC increased IL-10 and reduced IL-1α, primed BMSC also reduced IL-12 and the numbers of M1 macrophages and increased IL-4 and the numbers of M2 macrophages in

**80**

therapies is still limited. Further research is crucial to improve our ability to exploit these effects and, last not least, to prevent potential negative effects associated with some growth factors, such as hypervascularization in response to VEGF or fibrosis in response to TGF-β.
