hUCMSCs are advantageous because umbilical cords can be collected at a low cost to provide an inexhaustible stem cell source, without an invasive procedure, which is required for bone marrow MSCs. The present study developed hUCMSC-encapsulating macroporous fibrin construct for the first time, yielding excellent cell viability, proliferation and myogenic differentiation. Comparing with the usual method of cell encapsulation in fibrin without macropores, the novel macroporous fibrin–microbead method greatly increased cell viability. The live cell density of dMB35 at 16 days was increased by 60% compared to that without macropores. High cell density not only indicates good survival but also promotes cell fusion during myogenesis [46
]. The percentage of live cells in dMB35 reached 91% at 16 days. In a previous study, myoblasts were seeded in a macroporous alginate scaffold and growth factors were added to enhance cell viability for muscle tissue engineering [33
]. The percentage of live cells in alginate hydrogel was −60% [33
]. In another study, cold gelatin microbeads were used as a porogen in alginate hydrogel and the live cell percentage was 67–84% [28
]. In a recent study, hUCMSCs were seeded in alginate– fibrin microbeads, and the microbeads were then packed into a RGD-modified alginate matrix and subjected to myogenic induction [8
]. However, the released cells from the degradable microbeads did not survive well inside the alginate matrix, with the percentage of live cells being only 67% or less [8
]. Therefore, the present study developed a novel macroporous fibrin matrix, which greatly enhanced the survival of the encapsulated cells, yielding a percentage of live cells of 91% for dMB35 at 16 days. Furthermore, the hUCMSCs encapsulated in dMB35 were successfully induced to form myotubes in the myogenic medium for 16 days. The formation of myotubes was supported by the following evidence: (1) The hUCMSCs fused into multinucleated cells and the fusion index substantially increased with culture time; and (2) the cells expressed muscle specific proteins, including MYH, ACTN and ACTA1. These two criteria were similarly used to verify the formation of myotubes using bone marrow MSCs in previous studies [39
The good cell viability was likely because the dMBs in the fibrin could start to degrade as early as 4 days [30
]. Macropores were formed in fibrin containing hUCMSCs. Macropores not only enhanced nutrient/waste exchange, but also provided enough space for cell spreading, migration and growth. In addition, it has been demonstrated that cells along the edge of the hydrogel, and not those in the bulk of hydrogel matrix, exhibited superior proliferative activities, which were termed the “edge flourish” (EF) phenomenon [29
]. Previous studies attributed this as a biomechanical response between hydrogel and the encapsulated cell colonies based on real-time microscopy examination, finite-element modeling (FEM) analysis and multiple-particle tracking assay [29
]. These studies showed that the EF phenomenon was induced by the oriented outgrowth of encapsulated cells located at the edge of the hydrogel. The outgrowth of cells subsequently caused significant surface tension at the interface of the hydrogel and medium, which then contributed to the dynamic outgrowth of cells from the hydrogel bulk to the surface [48
]. In the present study, the macropores in the fibrin created a large number of fibrin– culture medium interfaces. Consistent with the EF phenomenon, the encapsulated cells would tend to migrate into the macropores and attach at the fibrin–culture medium interface with enhanced cell viability. Hence, macroporous hydrogels with stem cell encapsulation is expected to have a higher tissue regeneration efficacy than non-macroporous counterparts. The novel macroporous fibrin–dMB–hUCMSC construct is not only promising for muscle tissue engineering, but may be also applicable to other tissue engineering applications for soft and hard tissues.
Several previous studies investigated the effects of fibrinogen and thrombin concentrations on the behavior of encapsulated cells. Fibrinogen solutions at concentrations of 5–50 mg ml−1
were shown to play an important role in cell growth [49
], with dilute fibrinogen solutions (5–10 mg ml−1
) yielding better cell proliferation [17
]. Thrombin might inhibit myogenesis [50
], hence a lower concentration of thrombin is preferred in muscle tissue engineering constructs. Therefore, the present study selected relatively low concentrations of fibrinogen (10 mg ml−1
) and thrombin (5 U ml−1
), following a recent study investigating fibrinogen for muscle tissue engineering [17
]. Fibrin scaffolds made from bovine fibrinogen were used in the present pilot study to prove the concept, because bovine fibrinogen was much less expensive than human fibrinogen in commercial sources. In addition, the use of bovine fibrinogen was consistent with previous studies [51
]. However, further study should apply the novel macroporous fibrin scaffold in muscle defect repair using human fibrinogen instead of bovine fibrinogen.
Another important parameter in myogenic tissue engineering is the cell seeding density. A low cell seeding density may adversely affect myogenesis due to the lack of cell–cell interactions, while a higher cell seeding density can help promote cell fusion to form myotubes [46
]. However, if the cell density is too high, the cell spreading could be suppressed by contact inhibition. Cell spreading is important for myogenic differentiation [53
]. Previous studies using fibrin for muscle tissue engineering investigated a wide range of cell seeding density, from 2.5 × 105
to 1.5 × 107
]. In order to determine a suitable cell seeding density, the present study tested several cell seeding densities in preliminary experiments, ranging from 1 × 106
to 1 × 107
. The preliminary results suggested that 4 × 106
was a suitable cell seeding density, which yielded an appropriate cell density and distribution in the hydrogel constructs, and avoided the cell density being too low, while achieving a healthy cell spreading morphology. Therefore, a seeding density of 4 × 106
was used in the present study.
One drawback of fibrin is that it tends to shrink. If a scaffold shrinks, it cannot maintain adequate room for defect repair. In addition, the growth and spreading of the encapsulated cells are restricted. The prevention of fibrin shrinkage is therefore essential for tissue engineering [44
]. Poly-L-lysine had been used in the culture well to fix the gel [44
]. In the present study, TCPS was used to enhance gel fixation. The addition of dMBs weakened the gel fixation due to the reduced contact area between fibrin and TCPS. To prevent shrinkage, 35% mass fraction of dMBs in fibrin gel was suitable to maximize the macropore formation, without fibrin shrinkage. For in vivo usage of fibrin, attempts have been made to develop long-term stable fibrin gel to prevent this disadvantage [54
]. While the novel macroporous fibrin-microbead construct was advantageous for hUCMSC viability and proliferation without shrinkage at 35% dMBs, further study needs to evaluate the shape stability of the macroporous fibrin construct in vivo.
The degradation products from tissue engineering scaffolds should not adversely affect the cells [55
]. In the present study, dMBs were made of oxidized alginate and fibrin, both of which are biologically safe and have been shown to not produce harmful degradation products [56
]. Previous studies showed that cells survived well when encapsulated in dMBs [30
], indicating that the degradation products had no adverse effect on cell viability. This is consistent with the present study, which showed that the cell viability increased in dMB35 with increasing culture time, while the dMBs degraded and created macropores in the fibrin matrix.
Regarding the degradation of the constructs, it has been reported that the degradation time for fibrin constructs ranged from several days to several weeks in vivo,
and from 3 to 5 weeks in vitro [57
]. The difference in degradation time may result from the different degrees of fibrin crosslinking. Calcium chloride contributes to the crosslinking and subsequent stability of fibrin gel. Furthermore, the addition of protease inhibitors, such as aprotinin, can decrease the degradation rate. In the present study, no obvious fibrin degradation was observed in dMB0 and dMB35 during the 16 days of culture. Cells were observed to grow in a 3-D manner inside the construct. Cells were not observed to grow on a two-dimensional (2-D) surface for both types of constructs during the 16 day period. Further study is needed using longer culture times to determine when fibrin degrades fully, and if the cells will be growing on 2-D or synthesize their own 3-D matrix, both in vitro and in vivo.
Hydrogel–cell constructs without macropores were previously used for muscle tissue engineering. The commonly used hydrogels included fibrin and collagen [17
]. Guo et al. incorporated bone-marrow-derived cardiac stem cells (MCSCs) in fibrin hydrogel for treatment of rat myocardial infarction [17
]. They demonstrated that fibrin provided a good microenvironment for cell survival and migration and promoted the cardiomyogenic differentiation of MCSCs. Other studies investigated hydrogel–cell constructs with macropores due to their superiority for mass transport [33
]. Macroporous alginate fabricated using wire porogens greatly promoted both the viability and migration of seeded myoblasts compared to micro- or nanoporous alginate scaffolds [33
]. In the present study, hUCMSCs encapsulated in the novel macroporous fibrin– dMB construct were successfully differentiated into multinucleated myotubes. The results of myogenic differentiation suggested that the injectable macroporous fibrin fabricated by the addition of dMBs may be promising for muscle tissue engineering. Further studies are required to evaluate its potential in animal models.