Keywords

1 Introduction

1.1 Tendon Niche

Tendons are dense connective tissues that connect muscles to bone and transmit the mechanical forces generated during contraction to the skeleton, therefore essential for locomotion (Liu et al. 2014; Subramanian and Schilling 2015). Tendons are hypocellular tissues mainly composed of tenocyte, and a stem and progenitor cell population (Bi et al. 2007). However, other cell types may be also present, for instance endothelial cells, mast cells, chondrocytes, synovial and vascular cells. Tenocytes are responsible for extra cellular matrix (ECM) maintenance, which is mainly composed of collagen, in particular fibrillar collagens, namely collagen type I and III, although other types of collagen are also present, such as collagen type III, V, VI, XII, XIV and XV (James et al. 2008; Millar et al. 2015; Rodrigues et al. 2013). Tendon ECM is also composed of proteins and proteoglycans, such as decorin, biglycan, aggrecan and elastin (Aparecida de Aro et al. 2012; Docheva et al. 2015; James et al. 2008; Tan et al. 2015; Subramanian and Schilling 2015).

Morphologically, tendons follow a hierarchical architecture of collagen molecules that gather to form collagen fibrils. These fibrils assemble into fibers to form collagen fiber bundles. Finally, the bundles organize into tendon fascicles. The presence and alignment of collagen fibers is oriented for providing resistance to tendon and increased tensile strength (Docheva et al. 2015; Killian et al. 2012; Lui 2015; Durgam and Stewart 2016), while reducing stress during muscle contraction (Ho et al. 2014).

In tendon milieu, tendon cells and the ECM coordinate actions in promoting damage repair and tissue regeneration. Since vascular supplies in tendon are confined to endotenon and epitenon, it is likely that stem cell recruiting through the vascular system may be restricted to the surrounding areas of these layers. Thus, the resident cell populations, stem cells and tenocytes, have a critical role in physiological homeostasis and regulation of the tendon matrix. When this delicate fine-tuned balance is disturbed the susceptibility to tendon damage increases (Cadby et al. 2014).

Growing evidence supports tendon stem cells, rather than tenocytes, as the main responsible for the healing response in acute injuries. Beyond the self-renewal capacity, proliferation and multi-lineage potential, stem cells are a secretory source of cytokines and growth factors with paracrine and autocrine activities. These soluble factors support the growth and differentiation of stem and progenitor cells and have a angiogenic, chemotatic, anti-apoptotic, anti-scarring or immunomodulatory activity (Meirelles et al. 2009) in local environments. The secretion of a broad range of bioactive molecules with paracrine effects resulting from the dynamic communication between stem cells and niche environment is believed to be the main mechanism by which mesenchymal stem cells achieve their therapeutic effect (Meirelles et al. 2009).

Other intrinsic agents such as age, genetics, nutrition, body habitus and metabolic diseases are also involved in homeostasis as well as extrinsic factors namely pharmacological influences and mechanical stresses, including loading, disuse, compression and exogenous damage (Abate et al. 2009; Riggin et al. 2015). When tendon injuries occur, there is a local failure in physiological conditions, whose attempt to be solved is mediated by tendon healing and regeneration processes.

2 Tendon Repair Mechanisms

The healing process is a prolonged and complex response of the host to injury and is crucial for the mechanisms of tissue regeneration. It is also a window of opportunity envisioning new therapies for improving impaired healing (Stalman et al. 2015) and understanding the molecular entities and mechanisms behind regeneration. Because tendons possess a limited intrinsic regeneration capacity with low cellularity, low vascularization and poor innervation, tendon healing results in healed tissues with impaired mechanical capabilities. The potential of tendon healing also depends on the anatomical location and local environment (Thomopoulos et al. 2015).

Most tendons heal spontaneously upon injury but the load-bearing functions are frequently dominated by fibrotic scaring, which can result in adhesion formation and consequent failure to achieve proper biomechanics (Sharma and Maffulli 2005). Thus, a major challenge in tendon healing is to control the scar tissue formed (over scar remodeling) that deeply compromises the normal function (Rodrigues et al. 2013). Overuse or repetitive stretching during physical activities, which are the major cause of tendon lesions leading to microdisruption of tendon fibers, are known to trigger the release of pro-inflammatory mediators (Rileya et al. 2002; Yang et al. 2005). Increase levels of inflammatory cytokines have been associated to tendon degeneration and disease (Millar et al. 2009).

The lack of understanding on the cell mediated mechanisms disturbing the endogenous repair/regeneration process results in limited knowledge for effective treatment. The inflammation process has a pivotal role in the healing upon injury and failed healing responses contribute to the progression of tendinopathies, which represent a significant medical problem worldwide. The development of tendinopathy compromises tendon structure and function and is characterized by pain, swelling and dysfunction (Magnusson et al. 2010; Zhang et al. 2016), affecting athletes and general population.

The mechanisms supporting tendon healing are still a subject of debate. Two types of tendon healing were proposed: intrinsic and extrinsic. In intrinsic healing, tenocytes from the epitenon and endotenon migrate and proliferate into the site of injury, reorganizing the ECM and giving support to the internal vascular networking (Muller et al. 2015). Conversely, extrinsic healing is achieved by the invasion of cells from the surrounding sheath and synovium. Extrinsic healing has been associated to facilitate scar formation and, consequently inferior biomechanics. Other studies suggest that both intrinsic and extrinsic pathways are fundamental to the early stages of tendon healing (Harrison et al. 2003).

The tendon healing process typically includes three main phases: inflammation, proliferation and remodeling (Docheva et al. 2015) influenced by a temporal and spatially controlled array of mediators and the microenvironment events (Thomopoulos et al. 2015) (Fig. 1).

Fig. 1
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Representation of the main phases of tendon repair. Inflammation, proliferation and remodeling phases and molecular, cellular and matrix changes during these phases

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The first phase is often rapid and of short duration and characterized by the infiltration of inflammatory cells like monocytes, macrophages, neutrophils and platelets. These cells release chemotactic factors that favor the migration and activation of tendon cells from nearby regions of the injury and from tendon sheaths. In the next phase, tendon cells proliferate and produce a collagen rich ECM, re-establishing the alignment of tenocytes between collagen fibrils. Finally, during the remodeling phase, ECM becomes more organized with axial arrangement of collagen fibers. In rat flexor tendons, MMP-9 and MMP-13 mediate tissue degradation, while MMP-2, MMP-3 and MMP-14 were associated to the remodeling phase (Buono et al. 2013). The remodeling of the ECM is a crucial process of tendon healing for gaining biomechanical competence.

During healing, the inflammatory mediators such as Il-6 and TNF-α are secreted by tendon cells (Bauge et al. 2015; Wynn and Vannella 2016) assisting the crosstalk between cells and the ECM synthesis and arrangement contributing for the reparative versus degenerative process that drives tendon remodeling (Dakin et al. 2014; Dean et al. 2017).

Inflammation is the physiological response to injuries and is part of tendon healing process. If the injury is not resolved, the response becomes chronic and pathologic. The magnitude and duration of the inflammatory response is adjusted by regulatory mechanisms at the injury site (Andia et al. 2010; Prisk and Huard 2003).

Persistent inflammation disrupts the balance between MMPs and TIMPs contributing to scared tendon healing and chronic matrix degradation (D’Addona et al. 2017; Tarafder et al. 2017). Scared tissue results in poor rearrangement of collagen fibrils and separation of collagen bundles. The rupture on collagen fibers may be resulted in calcifications (Zabrzyński et al. 2016). Thus, modulation of the inflammatory response is necessary for recovery of tendon function (Shen et al. 2016).

Conservative treatments for tendon healing fight inflammation with anti-inflammatory drugs for tissue recovery in an attempt to diminish an abnormal or prolonged inflammation often associated to pathophysiology conditions. However, interrupting inflammation overruns important beneficial effects that are required for proper healing to occur.

The role of inflammation in tendinopathy is a subject of debate. Although several studies point a relation between inflammation and tendinopathy (D’Addona et al. 2017; Dakin et al. 2014; Dean et al. 2017; Rees et al. 2014), the onset and development of tendinopathy are poorly understood. Growing evidence suggests that inflammation may not be the cause of several tendinopathies but the failure to resolve inflammation will likely contribute to a complex environment of inflammatory mechanisms (stromal, immune-sensing and infiltrating compartments such as immune cells) (Millar et al. 2017) affecting tendon homeostasis and exacerbating symptoms and tissue degeneration.

Tendinopathies are associated to changes in cellularity and in the remodeling activity of tendon ECM resulting in significant structural and biomechanical alterations of the host niche (Lui and Chan 2011; Tempfer and Traweger 2015). Histological examination of tendinopathy tissues showed collagenolytic injuries and an active healing process, focal hypervascularity and metaplasia. Moreover, the collagen fibers show unequal and irregular crimping, loss the transverse bands, separations and rupturing of the fibers with an increase of type III collagen. The type III collagen is deficient in the number of cross-links between and within the tropo-collagen units (Abate et al. 2009; D’Addona et al. 2017; Zabrzyński et al. 2016).

Inflammatory mediators including, IL-1, IL-6 and COX-2 were reported to be increased in Achilles tendinopathy (Legerlotz et al. 2012). In a degenerative tendon model, the expression of IL-6, IL-11, IL-15 and TNF-α was up-regulated and accompanied by increased expression of MMP-13 and IL-1β (Dakin et al. 2014; Legerlotz et al. 2012). MMP-13 levels were also increased in human cuff tendon injuries. MMP-13 together with MMP-1 and MMP-8 participate in the cleavage of type I collagen present in tendons. The excessive collagen degradation during turnover results in chronic injuries (Buono et al. 2013).

2.1 Role of Cytokines in Tendon Healing

Cytokines are small proteins with the ability to influence and regulate biological activities (Dakin et al. 2014; Evans 1999) of cells that contribute to the healing response (Barr and Barbe 2004; Lin et al. 2006; Muller et al. 2015). Cytokines constitute the major mediators of inflammatory response with a relevant role in cell signaling and communication, holding potent immunomodulatory properties. An endogenous expression of inflammatory cytokines, namely TNF-α, IL-1β, IL-6, IL-4 and IL-10 has been demonstrated in human injured and healthy tenocytes (Ackermann et al. 2013; Mobasheri and Shakibaei 2013). Additionally, some of these cytokines may also be involved in the (self)regulation of tenocyte processes as IL-6 stimulation was reported to increase the proliferation capacity of tenocytes and inhibition expression of tendon cell markers (Thomopoulos et al. 2015). Cytokine expression is also affected by external stimuli as mechanical stimuli/exercise, which impacts the cytokine profile (including IL-1β and TNFα) during the tendon healing process (Morita et al. 2017).

The biochemical profile within a tissue niche during healing and other physiological events is of ultimate importance as it can be indicative of homeostatic, inflammatory or pathological conditions. Thus, the biochemical relevance of soluble factors as cytokines in tendon niches anticipates their application as potential diagnosis and therapeutic tools for repair and regeneration strategies (Table 1).

Table 1 Cytokines in homeostasis, inflammation and pathogenesis of tendon tissues
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3 Tendon Derived-Stem Cells (TDSCs) to Modulate Inflammation and Improve Tendon Healing

The interplay of mesenchymal stem/stromal cell (MSCs) with the tendon niche is essential for the modulation of the inflammatory response following injury (Lui and Chan 2011; Zhou et al. 2010) and is strongly dependent on a balance of soluble factors, cell-cell communication and cell-matrix interactions. During inflammation, MSCs interact with resident cells to promote cell migration and proliferation, which could allow a faster re-colonization of the defect, and matrix synthesis (Proffen et al. 2013).

The crosstalk between inflammation cues and stem cells is important to elucidate the mechanisms of how stem cells respond to tissue damage avoiding scar formation and tuning cell-based mechanisms for regenerative approaches.

Previous studies with non-tendon mesenchymal stem/stromal cell suggested that a MSCs treatment could attenuate scar formation and compromised function by improving tissue strength after ligament and tendon injuries. This was due to a paracrine-mediated immunosuppressive effect, through which MSCs modulate macrophage phenotypes (Proffen et al. 2013; Thomopoulos et al. 2015). It is expected that local stem cell populations within different tissues may also exert a similar effect. Thus, tendon stem/progenitor cells hold potential to contribute to the resolution of inflammation and pathophysiology of tendinopathies modulating biological responses at the injury site (Fig. 2).

Fig. 2
figure 2

Schematic representation of the TDSCs in tendon niches as modulators of tissue repair and regeneration

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A local tendon stem cell population could be beneficial over other stem cell sources due to their inherent pro-tenogenic abilities, which are likely more prone to produce tendon components under the influence of tendon environments (Dakin et al. 2014; Snedeker and Foolen 2017).

In 2007, a population of tendon stem/progenitor cells was firstly identified in tendons from mouse and humans, by Bi et al. (Bi et al. 2007). Tendon-derived stem cells (TDSCs) present universal stem cell characteristics such as the ability to self-renewal, clonogenicity and multi-lineage differentiation capacity (Bi et al. 2007). TDSCs were reported to in vitro differentiate into tenocytes, chondrocytes, osteocytes and adipocytes and to originate tendon, cartilage, bone and tendon-to-bone tissues in several animal models such as, nude mouse and rat, rabbit patellar and Achilles tendon (Lui 2013; Zhang and Wang 2010).

TDSCs have also shown evidence as cell source for tendon repair (Lui et al. 2016; Mienaltowski et al. 2014; Tarafder et al. 2017) (Table 2). TDSCs cultured in fibrin glue constructs were shown to promote earlier and improved tissue repair assessed by increased collagen production and fiber alignment in a patellar tendon window defect model (Ni et al. 2013). The TDSCs seeded in knitted silk-collagen sponge scaffolds also demonstrated ability to promoting regeneration of rotator cuff in rabbit model by inducing tenogenic differentiation and secretion of anti-inflammatory cytokines that prevented immunological rejection. (Shen et al. 2012).

Table 2 The role of tendon derived stem cell populations in tendon healing and repair
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The resident stem cell populations present in different regions of the tendon can be subject to different biochemical stimuli and contribute in distinctive ways for the reparative response to injury, and thus play different yet interactive roles in inflammation and healing.

A study by Mienaltowski et al. compared the properties of proper-(TPs) and peritenon-(TPes) derived progenitor cells from embryonic Achilles tendon in an in vitro regenerative tendon construct model. The anatomical origin of TSCs (TPs or TPes) contributed differently for tendon-like tissue formation and the secretome of TPes bolster the expression of tenogenic differentiation markers and matrix assembly genes in TPs and tenocytes. These findings highlight an additional potential role of TPes in tendon repair besides the synthesis of provisional matrix (Mienaltowski et al. 2014).

TDSCs also participate in the regulation of inflammation during healing of acute tendon injuries (Tarafder et al. 2017). Connective tissue growth factor (CTGF) enriched CD146+ TDSCs were shown to reduce pro-inflammatory M1 cells in the early healing phase and express anti-inflammatory IL-10 and TIMP-3 vis JNK/signal transducer and activator of transcription 3 (STAT3) signalling (Tarafder et al. 2017).

The immunomodulatory action and trophic signaling on cytokine modulation are TDSCs parameters to be taken in consideration as they are proposed to impact cellular immunity and immune associated processes, controlling cell responses and holding great promise for a variety of pathologies where inflammation and failed healing could be problematic. Therefore, TDSCs therapy is promising for regenerative medicine approaches aiming to repair tendon injuries to tissues pre-injury functional stage.

4 Conclusions and Future Directions

Despite the insights from recent years on the cellular and molecular cues involved in tendon healing, the knowledge on biological mechanisms to recapitulate tendon regeneration remains at the infancy.

Findings on tendon (stem cell) biology will likely contribute for better understanding of tendon homeostasis and proper healing. Inflammation as a necessary step for healing to occur should not be blocked but modulated and controlled. New studies are required to insight on the role of the mediators involved in unresolved and chronic inflammation to unveil new homeostatic or pathological markers and assist diagnosis tools for the treatment of tendon conditions. Ultimately, the knowledge gathered would enable the control of tendon healing response to injury toward a complete restoration of functional biomechanical cues.