**2. Tendon regeneration and defect models**

### **2.1 In vitro and ex vivo models**

*Tendons*

ossifications [15, 16].

review.

and degeneration, culminating in ECM deterioration. Key molecules involved include vascular endothelial growth factor (VEGF), interleukin (IL)-1, tumor necrosis factor (TNF)-α, prostaglandin (PG)E2, glutamate, and substance P [5, 6]. These mediators foster the ingrowth of blood vessels and nerves and the activation of nociceptive pathways. They are also implicated in the upregulation and activation of matrix metalloproteinases (MMP) and downregulation of their endogenous inhibitors (tissue inhibitors of matrix metalloproteinases; TIMP) [7]. This entails ECM degradation which successively alters and weakens the ECM structure [6]. When the accumulated damage and sensitization reach a threshold, clinical manifestation of tendinopathy comprises classical signs of inflammation including pain. Furthermore, provoked by new overload events, massive tissue trauma can occur. The resolution of inflammation is crucial to limit tissue damage, yet this mechanism often fails. Promoting fibrosis, a lack of pro-resolving signals, and persistence of macrophages entails the continuing activation of fibroblasts [8, 9]. Furthermore, macrophages could further contribute to ECM degradation via MMP secretion. Once at a diseased state, the intrinsic regenerative capacity of tendons is poor. Although endogenous mesenchymal stem-like cells with high tenogenic potential reside within tendons [10–12], these are susceptible to damage and suffer age-related changes [13, 14]. In pathological states, they could even contribute to fatty degeneration, fibrosis, and heterotopic

Treatment of tendinopathy still represents an unsolved challenge. Mainly, the use of strict rehabilitation exercise regimens is sufficiently evidence based [17, 18]. Anti-inflammatory drugs are frequently used, but they do not only counteract the active inflammation but also its resolution [19]. Biologicals such as platelet rich plasma have also received much attention, but clinical evidence is not convincing [17, 20, 21]. Research also focuses on the potential of endogenous tendon progenitor cells [22], which may be a promising strategy but will not be addressed in this

Multipotent mesenchymal stromal cells (MSCs) represent a therapeutic tool which might meet the clinical need of an adaptive treatment that simultaneously addresses different aspects of the disease. MSCs reside in virtually any tissue, in close proximity to the vasculature [23, 24]. MSCs derived from bone marrow and adipose tissue (BMSC and ASC, respectively) have been most extensively characterized [25, 26]. The fibroblast-like cells have been defined by a set of inclusion and exclusion antigens, their plastic-adherence, and trilineage differentiation potential in vitro [26]. While their differentiation potential into mesenchymal cell types, including tenocytes [27], has led to their extensive use in tissue engineering, it has become evident that their therapeutic potential by far exceeds cell replacement [24, 28]. While proof of MSC engraftment is often lacking, MSC-based cell therapy has shown beneficial effects in diverse scenarios in animal models, mostly mediated by immunomodulatory and trophic mechanisms [29–33]. Particularly, the immunomodulatory potential is extensively being researched and already exploited

The use of MSC for tendon repair was first suggested in 1998 [37] and, interestingly, has been published as a case report on an equine patient as early as 2003 [38]. Since then, several experimental animal studies—the recent ones being reviewed here—and case series in equine patients [39–41] have raised hope that local implantation of MSC into acute tendon defects improves healing. However, translational progress into human orthopedics is underwhelming, and although equine patients are being treated and few first-in-man clinical trials have been performed or initiated [42–44], convincing evidence from randomized, controlled clinical studies has neither been obtained in equine nor in human patients so far [45]. This

clinically, e.g., for treatment of graft-versus-host disease [34–36].

**70**

In vitro and ex vivo models relevant to MSC mechanisms of action in tendon regeneration comprise two major groups, with some overlap (**Figure 1**). The first includes the wide range of models for tenogenic differentiation [10, 46–94]. Among these, approaches in three-dimensional dynamic cultures appear most representative for MSC mechanisms in vivo [57, 58, 64, 70, 74, 77, 79, 83, 84, 86, 87]. Typically assessed parameters following tenogenic differentiation include the expression of tenogenic transcription factors (scleraxis and, in the more recent studies, mohawk), the transmembrane glycoprotein tenomodulin, as well as the expression and deposition of extracellular matrix components (e.g., collagen I, collagen III, decorin, and tenascin-C) and biomechanical parameters in case of tissue engineered constructs. Upregulation of matrix components such as collagen I or tenascin-C and improved construct strength do not only suggest tenogenic differentiation but also indicate ECM-modulating activities of the MSC. However, it should be acknowledged that no truly specific tendon marker has yet been identified, and that only expression patterns of combined marker sets, e.g., collagen I, scleraxis, and tenascin-C, discriminate healthy tendon from diseased tendon or other musculoskeletal tissues [95].

The second group includes models investigating the interaction of MSC with tenocytes and/or the tendon ECM, using co-cultures of MSC and tenocytes, their respective conditioned media, or tendon explants [48, 69, 74, 75, 88, 91, 92, 94, 96–105].

**Figure 1.** *In vitro models.*

Outcome parameters assessed in these studies are more diverse and include cell viability, proliferation, and metabolic parameters, expression and/ or release of growth factors, cytokines, MMPs and TIMPs, expression of ECM receptors and cytoskeleton formation, ECM protein release or deposition, or modulatory effects on immune cells (e.g., macrophage M1/M2 switch). Consequently, these studies provide insight into MSC trophic effects, immunomodulatory, or matrix-modulatory mechanisms.

The figure gives an overview of the in vitro models included in this review, illustrating the overlap between tenogenic differentiation models and coculture models, and summarizes the most commonly assessed outcome parameters. Note that in this context, the term "coculture" is used to summarize the models investigating the interplay between tenocytes and MSC, thus it does not exclusively refer to cocultures of different cell types but also includes cell culture models using conditioned media or tendon explants.

## **2.2 In vivo models**

In vivo studies on MSC-based tendon therapies need to be discriminated with respect to the animal model used (small vs. large, type of disease or defect model) and the treatment approach (strategy for MSC delivery, possible adjuvant treatments, timing of treatment, MSC source, and cell numbers applied).

Animal species used comprise small (rats [54, 106–118] and rabbits [119–122]) and large animals (dogs [123–126], sheep [127–129], and horses [130–141]). Interestingly, there appears to be a fair balance between small and large animal studies. This suggests preclinical progress, but it is also due to the interest in the equine species within the veterinary community. The tendon defects were created surgically in the majority of studies, with full thickness transections or segmental defects (mostly in the Achilles tendon) used in small animals or dogs and surgically created core lesions in the superficial digital flexor tendons in the equine model. Although there is reason to believe that enzymatical induction of tendon lesions better mimics the ECM degeneration and inflammation in tendon disease, only few among the recent studies used collagenase-based tendinopathy models [106, 108, 110, 129, 137, 139]. Still, neither surgical nor enzyme-based approaches fully reflect the complex tendon pathophysiology. In this light, providing particularly valuable information, some studies in the equine species were performed using horses suffering from naturally occurring tendinopathy [134, 138, 141] (**Figure 2**).

**73**

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

The diagram displays the numbers of studies performed in different animal species which were included in this review and indicates the types of tendon defect

Approaches for MSC implantation include local delivery of MSC suspensions, mostly via (ultrasound-guided) injection [106–112, 119, 120, 127–133, 136–141], coating of suture materials with MSC [113], MSC delivery in fibrin-based vehicles [54, 114, 124] or cell sheets [54, 123, 125, 126], and the use of diverse constructs of MSC and scaffold materials [115–118, 121, 122]. Interestingly, while the delineation between MSC-scaffold constructs for MSC delivery and for tendon replacement is sketchy, it is remarkable that construct-based approaches are almost exclusively used in small animals. This indicates that translational progress using these approaches is poor, possibly due to their incapability to meet the biomechanical

Further aspects of the treatment approach are likely to influence MSC mecha-

Last not least, the MSC source is likely to influence their mechanisms of action, which is an issue with equal relevance for in vitro findings. On the one hand, this applies to the choice of donor in terms of age and health status [143] and in terms of autologous, allogeneic or, in case of many small animal models, even xenogeneic use of MSC. On the other hand, the tissue origin of MSC as well as the donor species impact on the cell characteristics [57, 140, 144] and thus potentially on their mechanisms of action. Therefore, mainly studies focusing on the well-characterized BMSC and ASC were included and their tissue origin discriminated where appropriate. Furthermore, it was attempted to compile only studies which enabled the discrimination of MSC effects from those of possible additional treatments. In this line, in vivo studies using genetically engineered MSC for other purposes than cell

The assumption that MSC engraftment and their tenogenic differentiation after implantation into a tendon lesion lead to the replacement of damaged tenocytes dates back to the earlier days of MSC research and mirrors the general conception of MSC at that time [27, 38]. In the following years, the fact that MSC persistence at the site of tissue damage could not be achieved in models for a wide variety of diseases led to the assumption that differentiation and cell replacement might not even contribute to the regenerative effects observed after MSC transplantation [28]. This hypothesis was fostered by the compelling finding that paracrine factors released by the MSC can lead to similar beneficial effects as the MSC themselves, leading to the concept of cell-free MSC-based therapies [145]. Still, the situation

nisms of action and complicate the coherent interpretation of findings from different studies. Adjuvant treatments, e.g., simultaneous growth factor delivery, or pre-treatment of the MSC, such as pre-differentiation or inflammatory licensing before cell delivery, may support certain mechanisms synergistically but may negatively interfere with other mechanisms. For example, bone morphogenetic protein (BMP)-12 promotes MSC tenogenic differentiation but reduces their immunomodulatory potential [93]. Next, the timing of the treatment is of great importance as different mechanisms of action of MSC are likely to be relevant during different stages of tendon healing. Furthermore, the dosage, i.e., the numbers of MSC applied, may not only play a role with respect to treatment efficacy but also with respect to supporting specific mechanisms of action [120]. For example, interactions between MSC and immune cells depend on the ratio of MSC to leuko-

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

models used in the respective species.

demands in large animals or humans.

tracking were not included in this review.

**3. Engraftment and tenogenic differentiation**

cytes present [142].

**Figure 2.** *In vivo models.*

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

The diagram displays the numbers of studies performed in different animal species which were included in this review and indicates the types of tendon defect models used in the respective species.

Approaches for MSC implantation include local delivery of MSC suspensions, mostly via (ultrasound-guided) injection [106–112, 119, 120, 127–133, 136–141], coating of suture materials with MSC [113], MSC delivery in fibrin-based vehicles [54, 114, 124] or cell sheets [54, 123, 125, 126], and the use of diverse constructs of MSC and scaffold materials [115–118, 121, 122]. Interestingly, while the delineation between MSC-scaffold constructs for MSC delivery and for tendon replacement is sketchy, it is remarkable that construct-based approaches are almost exclusively used in small animals. This indicates that translational progress using these approaches is poor, possibly due to their incapability to meet the biomechanical demands in large animals or humans.

Further aspects of the treatment approach are likely to influence MSC mechanisms of action and complicate the coherent interpretation of findings from different studies. Adjuvant treatments, e.g., simultaneous growth factor delivery, or pre-treatment of the MSC, such as pre-differentiation or inflammatory licensing before cell delivery, may support certain mechanisms synergistically but may negatively interfere with other mechanisms. For example, bone morphogenetic protein (BMP)-12 promotes MSC tenogenic differentiation but reduces their immunomodulatory potential [93]. Next, the timing of the treatment is of great importance as different mechanisms of action of MSC are likely to be relevant during different stages of tendon healing. Furthermore, the dosage, i.e., the numbers of MSC applied, may not only play a role with respect to treatment efficacy but also with respect to supporting specific mechanisms of action [120]. For example, interactions between MSC and immune cells depend on the ratio of MSC to leukocytes present [142].

Last not least, the MSC source is likely to influence their mechanisms of action, which is an issue with equal relevance for in vitro findings. On the one hand, this applies to the choice of donor in terms of age and health status [143] and in terms of autologous, allogeneic or, in case of many small animal models, even xenogeneic use of MSC. On the other hand, the tissue origin of MSC as well as the donor species impact on the cell characteristics [57, 140, 144] and thus potentially on their mechanisms of action. Therefore, mainly studies focusing on the well-characterized BMSC and ASC were included and their tissue origin discriminated where appropriate. Furthermore, it was attempted to compile only studies which enabled the discrimination of MSC effects from those of possible additional treatments. In this line, in vivo studies using genetically engineered MSC for other purposes than cell tracking were not included in this review.

### **3. Engraftment and tenogenic differentiation**

The assumption that MSC engraftment and their tenogenic differentiation after implantation into a tendon lesion lead to the replacement of damaged tenocytes dates back to the earlier days of MSC research and mirrors the general conception of MSC at that time [27, 38]. In the following years, the fact that MSC persistence at the site of tissue damage could not be achieved in models for a wide variety of diseases led to the assumption that differentiation and cell replacement might not even contribute to the regenerative effects observed after MSC transplantation [28]. This hypothesis was fostered by the compelling finding that paracrine factors released by the MSC can lead to similar beneficial effects as the MSC themselves, leading to the concept of cell-free MSC-based therapies [145]. Still, the situation

*Tendons*

media or tendon explants.

**2.2 In vivo models**

Outcome parameters assessed in these studies are more diverse and include cell

viability, proliferation, and metabolic parameters, expression and/ or release of growth factors, cytokines, MMPs and TIMPs, expression of ECM receptors and cytoskeleton formation, ECM protein release or deposition, or modulatory effects on immune cells (e.g., macrophage M1/M2 switch). Consequently, these studies provide insight into MSC trophic effects, immunomodulatory, or matrix-modulatory mechanisms.

The figure gives an overview of the in vitro models included in this review, illustrating the overlap between tenogenic differentiation models and coculture models, and summarizes the most commonly assessed outcome parameters. Note that in this context, the term "coculture" is used to summarize the models investigating the interplay between tenocytes and MSC, thus it does not exclusively refer to cocultures of different cell types but also includes cell culture models using conditioned

In vivo studies on MSC-based tendon therapies need to be discriminated with respect to the animal model used (small vs. large, type of disease or defect model) and the treatment approach (strategy for MSC delivery, possible adjuvant treat-

Animal species used comprise small (rats [54, 106–118] and rabbits [119–122])

ments, timing of treatment, MSC source, and cell numbers applied).

and large animals (dogs [123–126], sheep [127–129], and horses [130–141]). Interestingly, there appears to be a fair balance between small and large animal studies. This suggests preclinical progress, but it is also due to the interest in the equine species within the veterinary community. The tendon defects were created surgically in the majority of studies, with full thickness transections or segmental defects (mostly in the Achilles tendon) used in small animals or dogs and surgically created core lesions in the superficial digital flexor tendons in the equine model. Although there is reason to believe that enzymatical induction of tendon lesions better mimics the ECM degeneration and inflammation in tendon disease, only few among the recent studies used collagenase-based tendinopathy models [106, 108, 110, 129, 137, 139]. Still, neither surgical nor enzyme-based approaches fully reflect the complex tendon pathophysiology. In this light, providing particularly valuable information, some studies in the equine species were performed using horses suf-

fering from naturally occurring tendinopathy [134, 138, 141] (**Figure 2**).

**72**

**Figure 2.** *In vivo models.* might be slightly different in tendon pathologies, and at the moment, it cannot be excluded that tenogenic differentiation of engrafted cells could contribute to regeneration, perhaps as a basis for further trophic and ECM-modulatory mechanisms.

## **3.1 In vitro evidence**

An extensive body of recent literature describes the tenogenic differentiation of MSC in response to a wide range of stimuli, although unfortunately, no generally accepted in vitro model or standard tenogenic differentiation assay exists. Current concepts of tenogenic differentiation are reviewed in detail elsewhere [146, 147]. The most commonly used stimuli to induce tenogenesis in MSC include growth factors, scaffolds with specific topography, and cyclic mechanical loading, with most studies combining two or more of these approaches, based on earlier studies in the field of tissue engineering [37, 148–150].

Growth factors used for induction of tenogenic differentiation mainly include transforming growth factor-β family members (TGF-β [47, 51, 53, 60, 66, 86, 88] and the growth differentiation factors GDF-5/BMP-14 [60, 67, 68, 70, 82, 151], GDF-6/BMP-13 [72], GDF-7/ BMP-12 [56, 60, 80, 93], and GDF-8 [71, 78]) but also fibroblast growth factors (FGF) [49, 89, 90], insulin-like growth factor-1 [53], vascular endothelial growth factor (VEGF) [60], or epidermal growth factor [49]. A promising stepwise differentiation approach has also been reported using TGF-β1 followed by connective tissue growth factor (CTGF) [54]. Growth factors are commonly delivered as culture medium supplements, but, e.g., FGF-2-transduced MSCs have been used as well [89]. Further tenogenic differentiation approaches based on genetic modifications include the forced expression of the tenogenic transcription factors scleraxis [10, 152] or mohawk [52, 116].

Currently used scaffolds comprise decellularized tendon matrices [57, 58, 64, 65, 83, 84, 88] and (synthetic) scaffolds with specifically designed topography and stiffness [59, 61–63, 68, 70, 72–75, 79, 81, 87], both being used based on evidence that physical cues such as scaffold anisotropy and stiffness direct MSC fate. Decellularized tendon matrices provide biochemical cues at the same time. A different approach to exploit the natural tendon biochemical composition is to use tendon ECM or tenocytic extracts as a culture supplement [46, 47, 91].

Mechanical loading of cell cultures, typically MSC-seeded scaffolds, is performed in bioreactors, most commonly by uniaxial cyclic stretching [46, 57, 58, 64, 66, 70, 74, 77, 79, 83, 84, 86, 87]. Different frequencies and strain rates have been used. While results are consistent in that cyclic stretching supports tenogenic differentiation, there is a discrepancy regarding the extent of stretching, with some studies highlighting moderate strain rates of 2 or 3% as beneficial for tenogenic induction [58, 77], while others support the use of higher strain rates (e.g. 10%) [55, 153]. Further approaches to tenogenic differentiation by physical stimulation include the use of extracorporeal shock waves [76], pulsed electromagnetic fields [85], and the activation of mechanosensitive membrane receptors [50].

In addition to using growth factors, scaffolds, and mechanical loading, tenogenic differentiation of MSC has also been reported in co-cultures with tenocytes [48, 69, 74, 75, 92] or in tenocyte-conditioned medium [48].

This overview illustrates that a wide range of stimuli can induce a tenogenic phenotype in MSCs (BMSCs as well as ASCs), although the quality of differentiation cannot be directly compared between studies and certainly varies. With respect to possible MSC tenogenic differentiation in vivo, the studies relying

**75**

integrate.

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

on physiological stimuli, such as mechanical loading, biomimetic scaffolds, or cross-talk with tenocytes, are most insightful. In contrast, the use of growth factors (typically at concentrations exceeding those found in vivo) or genetic modifications is suitable for mechanistic studies and may be helpful for tenogenic pre-differentiation prior to MSC implantation but does not reflect the in vivo situation. To understand if physiological stimuli could promote the same distinct tenogenic phenotype as artificial TGF-β concentrations, it would be helpful to gain further insight into the downstream signaling networks and their possible interfaces. So far, however, tenogenic signaling has mainly been investigated following growth factor stimulation [67, 82, 89, 90]. Only few studies have attempted to elucidate the signaling pathways activated in MSC in response to mechanical load or scaffold topographical cues, focusing on the role of rho/

Yet, although physiological stimuli have repeatedly been shown to induce tenogenic differentiation in MSC, it should not be anticipated that this mechanism is analogously activated when MSCs are implanted into a tendon lesion. Selfevidently, the tendon lesion does not provide a physiological but rather a pathophysiological environment, which may have an entirely different impact on the MSCs. Unfortunately, this issue is still underrepresented in the current literature. Recently, we investigated ASC tenogenic properties in response to physiological tenogenic and simultaneous inflammatory stimulation [84]. This study demonstrated that ASC tenogenic properties are compromised not only in the presence of the pro-inflammatory cytokines IL-1β and TNF-α but also in the presence of leukocytes. Similarly, IL-1β and IL-6 inhibited tenogenic differentiation in tendonderived stem cells [156, 157]. Furthermore, again in tendon-derived stem cells, stiff matrices impeded tenogenic differentiation [158]. Together, these findings suggest that MSC tenogenic differentiation may be impaired in a pathophysiological in vivo environment, which can comprise inflammatory stimuli as well as stiff (fibrotic)

Although extensively investigated in vitro, there is no distinctive evidence of tenogenic differentiation following MSC implantation in vivo. One conceivable explanation is that MSC differentiation is in fact impaired in the pathophysiological lesion environment. Nevertheless, in contrast to studies in other disease models, MSCs have been repeatedly localized in treated tendon lesions, providing a basis for long-term regenerative effects, possibly including differentiation and cell replacement. Furthermore, there is some evidence of homing of MSCs to tendon lesions, although not unambiguous. The mechanism of homing may be of minor importance with respect to cell delivery at the macroscale, as the cells are almost exclusively delivered locally in MSC-based tendon therapies. Yet, the capability of homing is still indicative of MSCs that are capable of identifying regions of tissue damage at the microscale, where they would actively

None of the small animal studies included in this review specifically addressed MSC homing to tendon lesions. However, when bursal tissue was implanted in rotator cuff tendon lesions in a rat model, the green fluorescent protein-labeled mesenchymal stem cells from this tissue infiltrated the healing tendons [159], demonstrating the presence of homing signals. Accordingly, ASC infiltration into the tendons was also evident when cell sheets were used as

delivery vehicle in a canine model [126]. However, when injected into

the tendon sheath, BMSC homed to synovial structures but were not attracted

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

ECM, depending on the stage of disease.

**3.2 In vivo evidence**

ROCK [154, 155].

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

on physiological stimuli, such as mechanical loading, biomimetic scaffolds, or cross-talk with tenocytes, are most insightful. In contrast, the use of growth factors (typically at concentrations exceeding those found in vivo) or genetic modifications is suitable for mechanistic studies and may be helpful for tenogenic pre-differentiation prior to MSC implantation but does not reflect the in vivo situation. To understand if physiological stimuli could promote the same distinct tenogenic phenotype as artificial TGF-β concentrations, it would be helpful to gain further insight into the downstream signaling networks and their possible interfaces. So far, however, tenogenic signaling has mainly been investigated following growth factor stimulation [67, 82, 89, 90]. Only few studies have attempted to elucidate the signaling pathways activated in MSC in response to mechanical load or scaffold topographical cues, focusing on the role of rho/ ROCK [154, 155].

Yet, although physiological stimuli have repeatedly been shown to induce tenogenic differentiation in MSC, it should not be anticipated that this mechanism is analogously activated when MSCs are implanted into a tendon lesion. Selfevidently, the tendon lesion does not provide a physiological but rather a pathophysiological environment, which may have an entirely different impact on the MSCs. Unfortunately, this issue is still underrepresented in the current literature. Recently, we investigated ASC tenogenic properties in response to physiological tenogenic and simultaneous inflammatory stimulation [84]. This study demonstrated that ASC tenogenic properties are compromised not only in the presence of the pro-inflammatory cytokines IL-1β and TNF-α but also in the presence of leukocytes. Similarly, IL-1β and IL-6 inhibited tenogenic differentiation in tendonderived stem cells [156, 157]. Furthermore, again in tendon-derived stem cells, stiff matrices impeded tenogenic differentiation [158]. Together, these findings suggest that MSC tenogenic differentiation may be impaired in a pathophysiological in vivo environment, which can comprise inflammatory stimuli as well as stiff (fibrotic) ECM, depending on the stage of disease.

### **3.2 In vivo evidence**

*Tendons*

mechanisms.

**3.1 In vitro evidence**

field of tissue engineering [37, 148–150].

[10, 152] or mohawk [52, 116].

might be slightly different in tendon pathologies, and at the moment, it cannot be excluded that tenogenic differentiation of engrafted cells could contribute to regeneration, perhaps as a basis for further trophic and ECM-modulatory

An extensive body of recent literature describes the tenogenic differentiation of MSC in response to a wide range of stimuli, although unfortunately, no generally accepted in vitro model or standard tenogenic differentiation assay exists. Current concepts of tenogenic differentiation are reviewed in detail elsewhere [146, 147]. The most commonly used stimuli to induce tenogenesis in MSC include growth factors, scaffolds with specific topography, and cyclic mechanical loading, with most studies combining two or more of these approaches, based on earlier studies in the

Growth factors used for induction of tenogenic differentiation mainly include transforming growth factor-β family members (TGF-β [47, 51, 53, 60, 66, 86, 88] and the growth differentiation factors GDF-5/BMP-14 [60, 67, 68, 70, 82, 151], GDF-6/BMP-13 [72], GDF-7/ BMP-12 [56, 60, 80, 93], and GDF-8 [71, 78]) but also fibroblast growth factors (FGF) [49, 89, 90], insulin-like growth factor-1 [53], vascular endothelial growth factor (VEGF) [60], or epidermal growth factor [49]. A promising stepwise differentiation approach has also been reported using TGF-β1 followed by connective tissue growth factor (CTGF) [54]. Growth factors are commonly delivered as culture medium supplements, but, e.g., FGF-2-transduced MSCs have been used as well [89]. Further tenogenic differentiation approaches based on genetic modifications include the forced expression of the tenogenic transcription factors scleraxis

Currently used scaffolds comprise decellularized tendon matrices [57, 58, 64, 65, 83, 84, 88] and (synthetic) scaffolds with specifically designed topography and stiffness [59, 61–63, 68, 70, 72–75, 79, 81, 87], both being used based on evidence that physical cues such as scaffold anisotropy and stiffness direct MSC fate. Decellularized tendon matrices provide biochemical cues at the same time. A different approach to exploit the natural tendon biochemical composition is to use tendon

Mechanical loading of cell cultures, typically MSC-seeded scaffolds, is performed in bioreactors, most commonly by uniaxial cyclic stretching [46, 57, 58, 64, 66, 70, 74, 77, 79, 83, 84, 86, 87]. Different frequencies and strain rates have been used. While results are consistent in that cyclic stretching supports tenogenic differentiation, there is a discrepancy regarding the extent of stretching, with some studies highlighting moderate strain rates of 2 or 3% as beneficial for tenogenic induction [58, 77], while others support the use of higher strain rates (e.g. 10%) [55, 153]. Further approaches to tenogenic differentiation by physical stimulation include the use of extracorporeal shock waves [76], pulsed electromagnetic fields [85], and the activation of mechanosensitive membrane

In addition to using growth factors, scaffolds, and mechanical loading, tenogenic differentiation of MSC has also been reported in co-cultures with tenocytes

This overview illustrates that a wide range of stimuli can induce a tenogenic phenotype in MSCs (BMSCs as well as ASCs), although the quality of differentiation cannot be directly compared between studies and certainly varies. With respect to possible MSC tenogenic differentiation in vivo, the studies relying

ECM or tenocytic extracts as a culture supplement [46, 47, 91].

[48, 69, 74, 75, 92] or in tenocyte-conditioned medium [48].

**74**

receptors [50].

Although extensively investigated in vitro, there is no distinctive evidence of tenogenic differentiation following MSC implantation in vivo. One conceivable explanation is that MSC differentiation is in fact impaired in the pathophysiological lesion environment. Nevertheless, in contrast to studies in other disease models, MSCs have been repeatedly localized in treated tendon lesions, providing a basis for long-term regenerative effects, possibly including differentiation and cell replacement. Furthermore, there is some evidence of homing of MSCs to tendon lesions, although not unambiguous. The mechanism of homing may be of minor importance with respect to cell delivery at the macroscale, as the cells are almost exclusively delivered locally in MSC-based tendon therapies. Yet, the capability of homing is still indicative of MSCs that are capable of identifying regions of tissue damage at the microscale, where they would actively integrate.

None of the small animal studies included in this review specifically addressed MSC homing to tendon lesions. However, when bursal tissue was implanted in rotator cuff tendon lesions in a rat model, the green fluorescent protein-labeled mesenchymal stem cells from this tissue infiltrated the healing tendons [159], demonstrating the presence of homing signals. Accordingly, ASC infiltration into the tendons was also evident when cell sheets were used as delivery vehicle in a canine model [126]. However, when injected into the tendon sheath, BMSC homed to synovial structures but were not attracted

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 capability of homing [139].

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 cells commit to a tenogenic fate.
