Growth factors (GFs) are biologically active molecules, synthetized by the injured tissue or by other cell types into the inflammatory site, which are released in the extracellular space and modulate the regenerative response. They play a variety of roles in the different stages of muscle regeneration (Table 14.1).

Hepatocyte growth factor (HGF), fibroblast growth factor (FGF), and platelet-derived growth factor (PDGF) are of interest because of their capacity to stimulate satellite cells (Sheehan et al. 2000).

Insulin-like growth factor-1 (IGF-I) appears to be of particular importance for the muscle regeneration process. IGF-I stimulates myoblast proliferation and differentiation (Engert et al. 1996) and is implicated in the regulation of muscle growth (Schiaffino and Mammucari 2011). IGF-I improved muscle healing, and histology of the injected muscle revealed fibrosis within the lacerated site, despite high level of IGF-I production (Lee et al. 2000). However, the efficacy of direct injection of recombinant proteins is limited by the high concentration of the factor typically required to elicit a measurable effect. This is mainly due to the bloodstream rapid clearance of these molecules and their relatively short biological half-lives.

Vascular endothelial growth factor (VEGF) has a potent angiogenic effect and is expressed in high concentration in healing flexor tendons 7–10 days following repair in animal models (Würgler-Hauri et al. 2007). Increased vascularity may improve tendon healing and contribute positively to the repair process. By targeting simultaneously angiogenesis and myogenesis, it was shown that combined delivery of VEGF and IGF-I enhances muscle regenerative process (Borselli et al. 2010).

In this direction, the use of platelet-rich plasma (PRP) is considered as a possible alternative approach based on the ability of autologous growth factors to improve skeletal muscle regeneration (Hamid et al. 2014; Hammond et al. 2009). Approximately 70% of the stored factors are secreted within the first 10 min following activation, and within the first hour almost 100% have been secreted. The degranulation of the α-granules results in the release of PDGF, TGF-β, insulin-like growth factor (IGF), and vascular endothelial growth factor (VEGF), with a host of other growth factors as well. These are native growth factors in their biologically determined ratios. These platelet growth factors enhance DNA synthesis, chemotaxis, and angiogenesis, increase collagen deposition, and stimulate synthesis of extracellular matrix.

Experimental studies on animal models showed that IGF-1, bFGF, and nerve growth factor (NGF) are potent stimulators of myoblast proliferation and fusion. Hammond et al. showed the capability of PRP to promote and accelerate myogenesis in an experimental study investigating the biomechanical and biochemical effects in rat muscle injuries (2009).

In a previous experimental study in a rat model of muscle injury, the effects of a platelet-rich fibrin matrix (PRFM) in the regeneration of damaged muscle tissue were histologically and immunohistochemically evaluated. This morphological study showed that the use of PRFM could improve muscle regeneration and long-term vascularization, suggesting that autologous PRFM may be a suitable and useful tool in the clinical treatment of muscle injuries (Gigante et al. 2012).

A side effect of the use of PRP and/or related products (e.g., PRFM) may be the occurrence of fibrosis. Visser et al. demonstrated that, in vitro, PRFM contains a significantly higher concentration of TGF-β1 compared with whole blood concentrate of similar volume. TGF-β1 has the ability to significantly increase connective cell proliferation over time, thus generating fibrotic tissue. Indeed, in an in vivo study, no increase in fibrotic tissue formation was observed during PRFM treatment in comparison with controls, suggesting that in vivo the amount of TGF-β released by PRFM might be not sufficient for this occurrence (Visser et al. 2010; Gigante et al. 2012).

Furthermore, it has been demonstrated a correlation between the concentration of growth factors and muscle regeneration. In order to closely simulate a clinical approach, Cianforlini et al. injected two different concentrations of PRP intramuscularly 24 h after the surgical trauma and evaluated, by means of histological and immunohistochemical analyses, the dose-dependent effects. Histological results confirmed the effectiveness of PRP in muscle healing and showed that the increase in PRP concentrations (i.e., GFs) in damaged muscle tissue accelerates the tissue regeneration process as well as neovascularization. Immunohistochemical data further strengthened this hypothesis detecting MyoD and myogenin-positive cells, located both inside the basal lamina of the fiber and in the interstitial spaces in the muscle sacrificed at three days and in a dose-dependent manner. It is well known that MyoD and myogenin play a key regulatory role in the processes of plasticity, adaptation, and regeneration in adult muscle. At last no significant side effects related to a higher dose of GFs were detected (Cianforlini et al. 2015).

About the efficacy of GH and IGF-1, Cianforlini et al. wanted to verify the role of GH by means of a single systemic administration in the treatment of acute muscle injury in an experimental model, verifying a possible correlation between the concentration of GH administered and tissue regeneration and fibrosis.

The action of GH is found to be ubiquitous, being increased the amount of muscle tissue and also of the endomisial and perimisiali connective tissue, with a consequent presence of exuberant scar tissue, directly proportional to the concentration of the administered GH. These are only preliminary data, but muscle regeneration and fibrosis could be both dependent from GH concentration, with an effect that would not be positive from a functional point of view, being present abundant scar tissue.

Considered as safe products, autologous PRP injections are increasingly used in patients with sports-related injuries (Engebretsen et al. 2010). Nevertheless, a recent randomized clinical trial shows no significant positive effects of PRP injections, as compared with placebo injections, in patients with muscle injuries, up to one year after injections (Reurink et al. 2014, 2015).

By recent findings, some scientific works combine PRP with Losartan (an angiotensin II type 1 receptor antagonist) (Terada et al. 2013) or PRP with the use of TGF-β1 neutralizing antibodies (Li et al. 2016). These strategies are a promising alternative to promote muscle regeneration while significantly reducing fibrosis.

Transplantation of satellite cell-derived myoblasts has long been explored as a promising approach for treatment of skeletal muscle disorders. After an initial demonstration that normal myoblasts can restore dystrophin expression in mdx mice (Partridge et al. 1989), clinical trials, in which allogeneic normal human myoblasts were injected intramuscularly several times in dystrophic young boys muscles, have not been successful (Law et al. 1990; Mendell et al. 1995). Even recently, despite clear improvement in methodologies that enhance the success of myoblast transplantation in Duchenne patients (Skuk et al. 2007), outcomes of clinical trials are still disappointing. These experiments have raised concerns about the limited migratory and proliferative capacities of human myoblasts, as well as their limited life span in vivo. It led to the investigations of other muscle stem cell sources that could overcome these limitations and outperform the success of muscle cell transplantation. Among all these non-satellite myogenic stem cells, human mesangioblasts, human myogenic-endothelial cells, and human muscle-derived CD133+ have shown myogenic potentials in vitro and in vivo (Sampaolesi et al. 2006; Meng et al. 2014). The use of myogenic progenitor cells for improving muscle healing may become an interesting therapeutic alternative (Tedesco and Cossu 2012).

The functional recovery of muscle in a young rat model of contusion injuries is significantly improved with the combined use of Losartan and muscle-derived stem cells (Kobayashi et al. 2016). New perspectives are provided by the combination of stem cells with anti-fibrotic therapies.

Considering the important role of TGF-β1 in the fibrotic cascade, the neutralization of TGF-β1 expression in injured skeletal muscle should inhibit the formation of scar tissue.

Indeed, the use of anti-fibrotic agents (decorin, relaxin, antibody against TGF-β1, AII antagonist, interferon gamma) that inactivate TGF-β1 signaling pathways reduces muscle fibrosis and, consequently, improves muscle healing, leading to a near complete recovery of lacerated muscle (Fukushima et al. 2001; Li et al. 2007).

The expression of myogenesis factor increased in mice skeletal muscles of the CCl4 + losartan group compared to the corresponding levels in the control group. It could be hypothesize that systemically elevated TGF-β1 as a result of CCl4-induced liver injury causes skeletal muscle injury, while losartan promotes muscle repair from injury via blockade of TGF-β1 signaling (Hwang et al. 2016).

Losartan, an angiotensin II receptor antagonist, neutralizes the effect of TGF-β1 and reduces fibrosis, making it the treatment of choice, since it already has FDA approval to be used clinically (Park et al. 2012; Terada et al. 2013). Suramin, also approved by the FDA, blocks TGF-β1 pathway and reduces muscle fibrosis in experimental model (Chan et al. 2003; Taniguti et al. 2011).

Also studies on the rotator cuff repair suggest the benefit effect of Licofelone (inhibitor of 5-LOX, COX-1, COX-2) on tendon healing, muscle fibrosis, and lipid accumulation (Oak et al. 2014).

In a mouse laceration model, the area of fibrosis decreased when γINF was injected at either 1 or 2 weeks after injury. More importantly, it found to improve muscle function in terms of both fast-twitch and tetanic strength. Demonstrating that γINF is a potent anti-fibrosis agent that can improve muscle healing after laceration injury (Foster et al. 2003).

Appropriately configured materials have the ability to modulate different stages of the healing response by inducing a shift from a process of inflammation and scar tissue formation to one of constructive remodeling and functional tissue restoration. The events that facilitate such a dramatic change during the biomaterial-host interaction are complex and necessarily involve both the immune system and mechanisms of stem cell recruitment, growth, and differentiation. The biological scaffolds derived from animal ECM after a decellularization process that consists in the removal of cells associated antigens, preserving the ultrastructure and composition of the ECM.

When properly manufactured, the scaffold material increases the migration and cell survival of myogenic precursor cells (Boldrin et al. 2007). Controlling the microenvironment of injected myogenic cells using biological scaffolds enhances muscle regeneration (Borselli et al. 2011). Ideally, using an appropriate extracellular matrix (ECM) composition and stiffness, scaffolds should best replicate the in vivo milieu and mechanical microenvironment (Gilbert et al. 2010; Engler et al. 2006).

With enzymatic and chemical decellularization process, we can isolate skeletal muscle ECM; this shows to contain growth factors, glycosaminoglycans, and basement membrane structural proteins. Myogenic cells survive and proliferate on muscle ECM scaffolds in vitro, and when implanted in a rat abdominal wall injury model in vivo shows to induce a constructive remodeling response associate with scaffold degradation and myogenesis in the implant area (Wolf et al. 2012).