Human umbilical cord stem cell encapsulation in novel macroporous and injectable fibrin for muscle tissue engineering

Abstract

There has been little research on the seeding of human umbilical cord mesenchymal stem cells (hUCMSCs) in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were: (i) to seed hUCMSCs in a fibrin hydrogel containing fast-degradable microbeads (dMBs) to create macropores to enhance cell viability; and (ii) to investigate the encapsulated cell proliferation and myogenic differentiation for muscle tissue engineering. Mass fractions of 0-80% of dMBs were tested, and 35% of dMBs in fibrin was shown to avoid fibrin shrinkage while creating macropores and promoting cell viability. This construct was referred to as "dMB35". Fibrin without dMBs was termed "dMB0". Microbead degradation created macropores in fibrin and improved cell viability. The percentage of live cells in dMB35 reached 91% at 16 days, higher than the 81% in dMB0 (p<0.05). Live cell density in dMB35 was 1.6-fold that of dMB0 (p<0.05). The encapsulated hUCMSCs proliferated, increasing the cell density by 2.6 times in dMB35 from 1 to 16 days. MTT activity for dMB35 was substantially higher than that for dMB0 at 16 days (p<0.05). hUCMSCs in dMB35 had high gene expressions of myotube markers of myosin heavy chain 1 (MYH1) and alpha-actinin 3 (ACTN3). Elongated, multinucleated cells were formed with positive staining of myogenic specific proteins including myogenin, MYH, ACTN and actin alpha 1. Moreover, a significant increase in cell fusion was detected with myogenic induction. In conclusion, hUCMSCs were encapsulated in fibrin with degradable microbeads for the first time, achieving greatly enhanced cell viability and successful myogenic differentiation with formation of multinucleated myotubes. The injectable and macroporous fibrin-dMB-hUCMSC construct may be promising for muscle tissue engineering applications.

Published by Elsevier Ltd.

Figures

Fig. 1

(A, B) Schematic of a fibrin construct and a degradable microbead (dMB)-containing fibrin (fibrin–dMB) construct. (C, D) Representative images of dMB35 and dMB80 cultured in growth medium at 16 days. Shrinkage did not happen in dMB0, dMB20 and dMB35 over 16 days, similar to (C). In contrast, shrinkage was observed in some dMB50 and dMB65 constructs, and most dMB80 constructs at 16 days, similar to (D). (E) Relative thiazolyl blue tetrazolium bromide (MTT) absorbance of the encapsulated stem cells. For each experiment, all data were normalized to the MTT absorbance of dMB0 at 1 day (mean ± SD; n = 5).

Fig. 2

Confocal microscope images of live and dead assay of hUCMSCs in dMB0 and dMB35. Live cells were stained green and dead cells were stained red. Representative images were shown at 1 day and 8 days as examples. Cells were primarily alive. Dead cells were relatively few, with examples shown in (C) and (F).

Fig. 3

Quantification of the percentages of live cells (A) and live cell density (B) in dMB0 and dMB35. Five randomly chosen fluorescence images for each sample were analyzed for four constructs, yielding 20 images per group. Each value is mean ± SD; n = 4.

Fig. 4

Representative cryo-SEM images of fibrin-based constructs. dMB0 and dMB35 without cells were cultured in growth medium for up to 16 days. (A, B) Macropores were formed in dMB35 as indicated by “P”. In contrast, dMB0 had no macropores, with an example shown in (C) at 16 days. In (D), the fibrin matrix consisting of a network of fibrin fibers includes thick fibers and thin fibers. The fibrin matrices for both dMB0 and dMB35 had similar microstructures.

Fig. 5

Gene expression assay of myogenic differentiation in dMB35 using real-time RT-PCR. Myosin heavy chain 1 (MYH1) and alpha-actinin 3 (ACTN3) are two key genes associated with the differentiation of muscle fibers. Both (A) MYH1 and (B) ACTN3 expression were significantly up-regulated. hUCMSCs cultured on tissue culture polystyrene (TCPS) in the growth media for 1 day served as calibrator with a value set at 1. Data were reported as mean ± SD (n = 4). The results indicated that hUCMSCs in the dMB35 differentiated into the myogenic lineage.

Fig. 6

(A–I) Immunostaining of dMB35 treated with myogenic medium culture using confocal microscopy. Cells were positive for myogenin (green) staining at 8 days (A–C), myosin heavy chain (MYH; green) staining (D–F), and double staining of sarcomeric alpha-actinin (ACTN; green) and actin alpha 1 (ACTA1; red) (G–I) at 16 days. Nuclei were stained with DAPI (blue). Arrows indicates the multiple nuclei. In (I), an example of a myotube is shown with three nuclei, expressing muscle specific proteins ACTN and ACTA1. (J) The fusion index (mean ± SD; n = 4) increased with time. Values with dissimilar letters are different (p < 0.05).