Two potential treatments have been investigated to accelerate tissue repair from sites of chronic injury or ischemia, growth factor therapy [32
] and stem cell therapy [33
]. A new approach combining both therapies has been recently studied to induce stem cell differentiation and increase cell differentiation efficiency for tissue repair [34
]. The present study documents a series of experiments aimed at demonstrating potential treatments for patients with SUI. Using a feasible delivery system with synergistic growth factors, we report that implanted autologous USCs were induced to differentiate into a myogenic lineage, and that the growth factor combinations enhanced angiogenesis and innervation, and stimulated resident cells to participate in regeneration of urethra sphincter tissue.
The sphincter muscle unit of the urethra has both internal and external sphincter muscles. The internal sphincter is the extension of the detrusor muscle (the primary muscle for forcing urine out of the bladder), is made of smooth muscle under involuntary or autonomic control. By contrast, the external sphincter is made of skeletal muscle under voluntary control of the somatic nervous system. Other connective tissues around the urethra, including vessels and peripheral nerves, also play important roles in control of micturition. Urinary incontinence occurs in three types of softy tissue injures, i.e. muscle weakness, nerve damage, or vascular (blood supply) changes, all of which are potential targets for stem cell therapies. Unlike using bulking materials to mechanically squeeze the urethra, a long-term strategy to treat SUI is to repair defects of both skeletal and smooth muscle, and to improve the blood supply and innervation in the mid-urethral segment [35
]. Several clinical trials have demonstrated that MSCs isolated from skeletal muscle or fat tissue injected into the middle urethra restored the damaged contractile function of the striated muscles and rhabdosphincter [36
]. The rationale of stem cell therapy is based on the multi-potent differentiation capability and trophic properties of these cells [38
]. Stem cells can give rise to the target cells and secrete paracrine factors, such as angiogenic, neurogenic and cytoprotective factors, to prolong cell survival and facilitate vascularization and innervation. In the present study, we have demonstrated that USCs efficiently gave rise to skeletal myogenic or endothelial lineage cells, and had neuro rescue effect in vivo
via synergistic activity of growth factors released from microbeads vehicles. The growth factors not only improved the environment for the implanted cells by creating angiogenesis and innervation, but also recruited resident cells into the graft site for tissue repair. In addition, the combination of growth factors that facilitated myogenesis, angiogenesis, and innervation was more effective in in vivo
tissue regeneration than the growth factors applied individually. Furthermore, this therapeutic approach would not require a preconditioning for in vitro
stem cell differentiation; thus it shortens the process and increases cell differentiation efficiency.
An adequate blood supply is crucial for survival of cells in cell therapy, particularly in the central core of the implants [39
]. Our previous studies demonstrated that modifying USCs by exposing them to the angiogenic gene VEGF remarkably improved the cell survival rate and myogenic differentiation of USCs by promoting angiogenesis in vivo
]. However, angiogenic gene manipulation causes potential side effects, such as extensive hemorrhaging within the liver [41
] and tumorigenesis [42
] in implanted sites. Except for gene transfection, growth factor injection therapy including cytokines such as VEGF, HGF, IGF, NGF, PDGF, FGF, BMP, and EGF also acted as powerful therapeutic agents in tissue engineering [44
]. However, most growth factors are soluble and disappear quickly due to their short half-life time in vivo
. This growth factor injection approach also requires multiple injections of large doses of proteins that results in several potential side effects, including only transient improvements [42
] or abnormal vascular structure, resulting in insufficient therapeutic effect [44
]. Thus, several growth factor delivery systems, such as chemical conjugation of the growth factor to the matrix, or physical encapsulation of growth factors in the delivery system [45
], have been designed to overcome these disadvantages.
Different types of biomaterials have been used to achieve cytokine or drug delivery, including biologics, polymers, silicon-based materials, carbon-based materials, or metals [46
]. Among those delivery vehicles, alginate hydrogel microbeads are an excellent candidate for cytokine delivery, since they retain the bioactivity of the growth factors as cross-linking occurs under physiological conditions. The alginate microbeads can be easily modified; higher concentrations of alginate yield a tightly cross-linked matrix, resulting in lower porosity and hence slower release of growth factors. Alginate-encapsulated proteins such as FGF-1 [27
], PDGF, and VEGF [47
] have demonstrated a slow, low-level consistent release of growth factors, and the efficacy of the delivery conduit was demonstrated both in vitro
and in vivo
. Unlike gene delivery or protein injection, the effective delivery of proteins, safety, and biocompatibility of microbeads provide promising benefits for angiogenesis [25
Our previous study showed heparin binding to FGF-1 could increase its half-life and retain the normal mitogenic properties of FGF-1. Release time was prolonged when alginate microbeads were combined with the heparin-binding growth factors [48
].The loading efficiency for all growth factors in this study was between 36–40%, which is very comparable to other loading methods [23
]. As alginate beads have a porosity of about 600 kDa, we applied a semi-permeable membrane of PLO coating which reduces the porosity to about 70–80 kDa. This semi-permeable membrane allowed us to control the release of the growth factors from these microbeads. No significant difference in the loading efficiency was observed when the growth factors were loaded into microbeads between 24 to 48 h. As is the case with hydrophilic drug carriers with hydrophilic payload, there is usually an initial burst release that is followed by a sustained release of smaller levels of the encapsulated substance [25
], which explains why about 40–60% of the growth factors were released in one day. Previous studies had shown that this release profile consisting of a high growth factor concentration initially, followed by a decreasing concentration over time was found to result in optimal angiogenic effect [49
]. Thus, it was desirable for such burst release to occur for the enhancement of the bioeffect of the growth factors. In our experiments, we observed a steady and consistent release of smaller levels after the initial burst release during the first day. Although certain variation in release profile was noted when multiple growth factors were combined, the growth factors were still consistently released from the microbeads. The growth factors release efficiency depends on their molecular weights because of their release competition effect. Our data confirmed that biologically-active VEGF was efficiently released from the alginate microbeads, leading endothelial differentiation of USC in vitro
Increasing evidence has shown that cytokine combinations are better than a single cytokine in tissue repair [50
]. In the present study, we investigated the impact of growth factors on angiogenesis with single, dual, or multiple delivery patterns in vivo
. When given together, the growth factors had a synergistic effect that improved implanted cell survival, muscle tissue regeneration, neo-vacuolization, and innervations in vivo
. Although IGF/NGF/FGF acts on innervation, VEGF on angiogenesis, and PDGF/HGF on myogensis, most of them have cross-cutting properties. For example, PDGF not only induces myogenic differentiation of stem cells, but also promote angiogenesis [51
]; furthermore, IGF can enhance innervation as well as promote myogenic differentiation of stem cells [52
]. In addition, FGF-1 also promotes angiogenesis [26
More new nerve fibers appeared in the grafts of USCs implanted with alginate microbeads containing neurogenic growth factors, compared to the other experimental groups. The nerve fibers may have originated from the host tissue, rather than from the implanted USCs, because these regenerated nerve cells were largely not positive for few human nuclei staining. The present study also suggests that revascularization via angiogenic factors released from the microbeads enhanced both muscle regeneration and innervation in vivo.
In summary, we demonstrated that the implanted USCs play an important role in cell survival, myogenesis, angiogenesis, innervation, and recruitment of resident cells. All groups of USCs with added growth factors achieved better outcomes than the groups with the same types of growth factors without USCs. Some USCs could give rise to endothelial cells without the addition of any growth factors, but these effects were strengthened by the presence of angiogenic growth factors in vivo. In addition, as USCs could differentiate into endothelial cells, endothelial differentiated stem cells could replace endothelial cells with no necessity for adding extraneous endothelial cells to implanted grafts. Furthermore, USCs appear the indirect neruogenic and neruoprotective effects during tissue regeneration.