**4.1 In vitro evidence**

*Tendons*

capability of homing [139].

cells commit to a tenogenic fate.

**4. Extracellular matrix modulation**

to the tendon lesions in an ovine model of intrasynovial tendon healing [127]. In the equine large animal model, homing of MSC to tendon lesions has been addressed in more detail. Scintigraphic short-term in vivo tracking of technetium-labeled BMSC showed that the cells homed to the tendon lesion after administration by regional limb perfusion, although local administration by direct intralesional injection was more effective, and no homing was observed after intravenous administration. These findings were consistent between artificial tendon lesions [135] and natural tendinopathy [134]. Interestingly, intraarterial limb perfusion showed greater accumulation of BMSC in the lesion on day 10 after surgical lesion induction than on day 3 [135]. This finding illustrates that the stage of tendon disease is of importance to MSC homing mechanisms. However, scintigraphic tracking also revealed that even after local injection, only a relatively small proportion of the injected BMSC remains at the injury site (24% after 24 h) [134]. In accordance with this, we and others demonstrated that ASCs are distributed via the bloodstream within the first few days after their injection into equine tendon lesions, possibly as they are washed away before they can home and attach [136, 139]. We additionally observed that the ASCs were subsequently also found in nontreated tendon lesions, indicating their

Engraftment of MSC within treated tendon lesions was demonstrated in several

studies, albeit results are not conclusive as to the numbers of surviving cells in relation to the cell numbers administered. In rat Achilles tendon defects, BMSC or ASC could be identified histologically at 2, 4, and 8 weeks after cell implantation (injection) [107, 109, 112], as well as 3 weeks after implantation of a BMSC-seeded collagen scaffold [116]. Complementing these small animal studies, MSCs have been traced in large animal studies, including longitudinal in vivo cell tracking. In sheep, green or red fluorescent protein-labeled BMSCs were detected histologically at 1, 2, 3, 4, and 6 weeks following their implantation [128, 129]. In the equine model, we and others could trace superparamagnetic iron oxide-labeled ASC by magnetic resonance imaging during follow-up periods of up to 24 weeks after implantation into artificial tendon lesions [132, 139] and umbilical cord tissuederived MSCs during a follow-up period of 8 weeks in naturally occurring tendinopathy [138]. In the experimental tendon lesions, histological results confirmed the presence of the simultaneously fluorochrome-labeled ASC until week 24 [132, 139]. This provides evidence for a remarkable long-term persistence of part of the locally injected MSC, yet it has neither been proved nor disproved whether these

The restoration of the ECM architecture and functionality is a major goal in regenerative tendon therapies. Based on the early hypothesis of MSC engraftment and tenogenic differentiation, it was assumed that the differentiated cells would subsequently synthesize new tendon ECM. Indeed, MSCs are capable to synthesize a considerable amount of extracellular matrix even in an undifferentiated state [160]. Furthermore, the composition of the ECM synthesized by differentiated MSC reflects the respective tissue lineage, which is well-established for their chondrogenic or osteogenic differentiation. Corresponding in vitro data exist for the differentiation into the tenogenic lineage, although not always consistent between studies. There is also in vivo evidence that MSC transplantation improves tendon ECM structure. However, this is not necessarily due to ECM synthesis by the MSC themselves but might also be a consequence of protective and stimulatory effects on

**76**

As most tenogenic differentiation studies investigated the expression and/ or deposition of tendon-specific extracellular matrix molecules as a marker for successful differentiation, there is quite extensive evidence that the ECM synthesis by MSC is altered during tenogenic differentiation. However, there is some discrepancy between different studies as to whether the ECM molecule expression pattern of tenogenic MSC truly corresponds to that of healthy tendon tissue.

Collagen I, the most abundant protein in healthy tendons, was shown to be upregulated by ectopic mohawk or scleraxis expression [52], in response to treatment with TGF-β superfamily growth factors [60, 67, 88, 93] or scaffold stiffness and alignment [61–63, 74, 81], as well as in three-dimensional dynamic cultures with uniaxial cyclic loading [58, 64, 77, 87]. Furthermore, co-culture with tenocytes in hypoxic conditions or integration of integrin-binding peptides in the scaffold increased collagen I expression on mRNA as well as protein level [69, 72]. However, in other studies, no collagen I upregulation was observed in response to growth factors such as TGF-β [49] or cyclic loading in two-dimensional ASC or BMSC cultures, respectively [66]. Data are particularly conflicting with regard to whether the presence of tendon ECM components promotes or counteracts collagen I expression [46, 47, 58, 64, 65, 83, 84, 88]. Furthermore, even if collagen I is upregulated, which would enable the MSC to contribute to tendon ECM synthesis, this often occurs in conjunction with the upregulation of other extracellular matrix molecules, such as collagen III, decorin, tenascin-C, or cartilage oligomeric matrix protein [60, 61, 69, 70, 72, 74, 77, 83]. While these molecules are important components of native tendon ECM, contributing to collagen organization and fibrillogenesis, their increased presence is also indicative of tendon degeneration or fibrosis [161–163]. Therefore, in order to achieve a beneficial ECM replacement by MSC, their ECM synthesis would have to be highly balanced. It is not yet sufficiently proven that this can be achieved by inducing tenogenic differentiation.

With respect to the hypothesis of active ECM remodeling by MSC, comparatively few data exist so far. Treatment with BMP-12 induced an enhanced secretion of MMP-1 and -8 by ASC [93]. Similarly, ASC culture in collagen scaffolds increased MMP-1, -2, -8, -9, and -13 gene expression and MMP activity compared to two-dimensional culture [46]. For tendon-derived stem cells, it was also found that cyclic mechanical loading did not only upregulate ECM-related genes but also the integrins α1, -α2, and -α11, as well as MMP-9, -13, and -14 [164]. Thus, tenogenic stimuli may increase expression and activation of MMP by MSC. Furthermore, it was found that BMSC inhibits MMP activity in the cell culture medium through secretion of TIMP-1 and TIMP-2, even in an inflammatory environment [165], but that BMSC as well as ASC accumulate active MMP at their cell surface [166]. Although these latter two studies did not focus on tendon therapies, they suggest that MSCs could contribute to matrix remodeling in a highly targeted manner.

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 in future studies.
