Fast-degradable microbeads encapsulating human umbilical cord stem cells in alginate for muscle tissue engineering

Abstract

Human umbilical cord mesenchymal stem cells (hUCMSCs) are inexhaustible and can be obtained without an invasive surgery. To date, there has been no report on seeding hUCMSCs in three-dimensional scaffolds for muscle tissue engineering. The objectives of this study were to (1) investigate hUCMSC seeding in a scaffold for muscle engineering and (2) develop a novel construct consisting of hUCMSC-encapsulating and fast-degradable microbeads inside a hydrogel matrix. The rationale was that the hydrogel matrix would maintain the defect volume, while the microbeads would degrade to release the cells and concomitantly create macropores in the matrix. hUCMSCs were encapsulated in alginate-fibrin microbeads, which were packed in an Arg-Gly-Asp (RGD)-modified alginate matrix (AM). This construct is referred to as hUCMSC-microbead-AM. The control consisted of the usual cell encapsulation in AM without microbeads (referred to as hUCMSC-AM). In the hUCMSC-AM construct, the hUCMSCs showed as round dots with no spreading at 1-14 days. In contrast, cells in the hUCMSC-microbead-AM construct had a healthy spreading and elongated morphology. The microbeads successfully degraded and released the cells at 8 days. Myogenic expressions for hUCMSC-microbead-AM were more than threefold those of hUCMSC-AM (p<0.05). Immunofluorescence for myogenic markers was much stronger for hUCMSC-microbead-AM than hUCMSC-AM. Muscle creatine kinase of hUCMSC-microbead-AM at 14 days was twofold that of hUCMSC-AM (p<0.05). In conclusion, hUCMSC encapsulation in novel fast-degradable microbeads inside a hydrogel matrix was investigated for muscle engineering. Compared to the usual method of seeding cells in a hydrogel matrix, hUCMSC-microbead-AM construct had greatly improved cell viability and myogenic differentiation, and hence, is promising to enhance muscle regeneration.

Figures

FIG. 1.

(A) Schematic of Arg-Gly-Asp (RGD)-modified alginate encapsulating human umbilical cord mesenchymal stem cells (hUCMSCs) (referred to as hUCMSC-AM, AM=RGD-modified alginate matrix). (B) hUCMSCs in degradable alginate-fibrin microbeads in AM (referred to as hUCMSC-microbead-AM). Optical photos of hUCMSC-AM (C, E, G) and hUCMSC-microbead-AM (D, F, H). At 1 day, cells were round-shaped in (C) and (D). Arrow in (C) indicates a cell. Arrows in (D) indicate the microbead boundary. Such microbead boundary disappeared due to microbead degradation at 8 days. At 14 days, cells remained as dots in (E), but become elongated in (F). (G) and (H) Higher magnification shows round cells in hUCMSC-AM, and elongated and spreading cells in hUCMSC-microbead-AM at 8 days. Color images available online at www.liebertpub.com/tea

FIG. 2.

Live/dead staining of cells in hUCMSC-AM (A–E), and in hUCMSC-microbead-AM (F–J). In contrast to live cells being green dots in hUCMSC-AM, live cells in hUCMSC-microbead-AM started to have a spreading morphology in (G). Cells spread better in (H) and (I). (E) and (J) Typical higher magnification views showed cells not spreading in hUCMSC-AM, in contrast to healthy spreading and elongated cells in hUCMSC-microbead-AM. Color images available online at www.liebertpub.com/tea

FIG. 3.

Viability of the encapsulated hUCMSCs: (A) percentage of live cells, (B) MTT assay of metabolic activity of cells, and (C) cell spreading size. Each value is mean±sd (n=5). Color images available online at www.liebertpub.com/tea

FIG. 4.

Reverse transcription-polymerase chain reaction (RT-PCR) analysis of myogenic gene expressions: (A)α-actinin 3 (ACTN3), and (B)myosin heavy chain 1 (MYH1). Each value is mean±sd; n=5. The new hUCMSC-microbead-AM construct yielded much higher myogenic gene expressions than hUCMSC-AM. Color images available online at www.liebertpub.com/tea

FIG. 5.

Myogenic differentiation. (A–J) Immunofluorescence images, (K) muscle creatine kinase (MCK) activity. In (A–J), the first row is for hUCMSC-AM, and the second row is for hUCMSC-microbead-AM. MyoD and myogenin, known as early myogenic differentiation markers,42 were stained at 7 days. Sarcomeric α-actinin (ACTN) and myosin heavy chain (MYH), known as late myogenic markers,42 were stained at 14 days. Cells in hUCMSC-AM had a weak staining of MyoD (A, green), myogenin (B, green), ACTN (C, red), and MYH (D, red). In contrast, hUCMSC-microbead-AM had much stronger staining. At 14 days, cells in hUCMSC-microbead-AM had a spreading shape and started to fuse into multinucleated elements (G and H). (I and J) Multinucleated elements of fused cells recovered from hUCMSC-microbead-AM at 14 days. Arrows point to the multiple nulei. In (K), each value is mean±sd; n=5. Color images available online at www.liebertpub.com/tea

FIG. 6.

SEM of hydrogel constructs: (A) hUCMSC-AM at 1 day, and (B, C) hUCMSC-microbead-AM at 7 and 14 days, respectively. No macropores were found in hUCMSC-AM from 1 to 14 days, similar to that shown in (A). In (B), the microbead was degraded and macropore P was formed at 7 days. (C) Macropore formed by the degradation of possibly two microbeads at 14 days. The macropores in alginate matrix had sizes of several hundred microns. Arrows in (B) and (C) demonstrate the macropores. Color images available online at www.liebertpub.com/tea