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. 2007 Jan;28(2):316-25.
doi: 10.1016/j.biomaterials.2006.08.042. Epub 2006 Sep 28.

Continuing Differentiation of Human Mesenchymal Stem Cells and Induced Chondrogenic and Osteogenic Lineages in Electrospun PLGA Nanofiber Scaffold

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Free PMC article

Continuing Differentiation of Human Mesenchymal Stem Cells and Induced Chondrogenic and Osteogenic Lineages in Electrospun PLGA Nanofiber Scaffold

Xuejun Xin et al. Biomaterials. .
Free PMC article

Abstract

Nanofibers have recently gained substantial interest for potential applications in tissue engineering. The objective of this study was to determine whether electrospun nanofibers accommodate the viability, growth, and differentiation of human mesenchymal stem cells (hMSCs) as well as their osteogenic (hMSC-Ob) and chondrogenic (hMSC-Ch) derivatives. Poly(d,l-lactide-co-glycolide) (PLGA) beads with a PLA:PGA ratio of 85:15 were electrospun into non-woven fibers with an average diameter of 760+/-210 nm. The average Young's modulus of electrospun PLGA nanofibers was 42+/-26 kPa, per nanoindentation with atomic force microscopy (AFM). Human MSCs were seeded 1-4 weeks at a density of 2 x 10(6)cells/mL in PLGA nanofiber sheets. After 2 week culture on PLGA nanofiber scaffold, hMSCs remained as precursors upon immunoblotting with hKL12 antibody. SEM taken up to 7 days after cell seeding revealed that hMSCs, hMSC-Ob and hMSC-Ch apparently attached to PLGA nanofibers. The overwhelming majority of hMSCs was viable and proliferating in PLGA nanofiber scaffolds up to the tested 14 days, as assayed live/dead tests, DNA assay and BrdU. In a separate experiment, hMSCs seeded in PLGA nanofiber scaffolds were differentiated into chodrogenic and osteogenic cells. Histological assays revealed that hMSCs continuously differentiated into chondrogenic cells and osteogenic cells after 2 week incubation in PLGA nanofibers. Taken together, these data represent an original investigation of continuous differentiation of hMSCs into chondrogenic and osteogenic cells in PLGA nanofiber scaffold. Consistent with previous work, these findings also suggest that nanofibers may serve as accommodative milieu for not only hMSCs, but also as a 3D carrier vehicle for lineage specific cells.

Figures

Fig. 1
Fig. 1
Electrospun PLGA nanofibers: (A) PLGA nanofibers as non-woven mesh-like structures under SEM. (B) Higher SEM magnification showing irregular pores among PLGA nanofibers. (C) Imaging of single PLGA nanofibers deposited on glass substrate by atomic force microscopy (AFM). (D) AFM imaging of individual PLGA nanofibers on stainless steel substrate. (E) 3D contour of PLGA nanofibers under AFM (scale bars A: 50 μm; B: 10 μm).
Fig. 2
Fig. 2
Human mesenchymal stem cells (hMSCs) and seeding in electrospun PLGA nanofiber scaffold: (A) prior to seeding in nanofiber scaffold, hMSCs in 2D culture were nearly confluent, and were viable by live and dead assay (calcein green indicating live cells). (B) hMSCs seeded in PLGA nanofiber scaffold for 2 weeks showed viability by live and dead assay. (C) DNA contents gradually increased from 1 to 14 days after seeding of hMSCs in PLGA nanofiber scaffold. The DNA contents at each of 3, 7 and 14 days after cell seeding were significantly higher than the DNA content at 1 day following cell seeding. (D) Bromodeoxyuridine (BrdU) immunolocalization showed that a substantial number of seeded hMSCs in nanofiber scaffold were undergoing mitosis (scale bars: 100 μm).
Fig. 3
Fig. 3
Electrospun PLGA nanofibers and the seeding of human mesenchymal stem cells (hMSCs), as well as hMSC-derived osteoblasts and hMSC-derived chondrocytes. (A), (E), (I) Prior to cell seeding, three randomly selected PLGA nanofiber scaffolds showed similar characteristics. Upon seeding of hMSCs (B, C, D), various extracellular matrices were apparently synthesized among PLGA nanofibers over time. By 7 days following hMSC seeding, the seeded cells apparently have attached to nanofiber surface and penetrated into the pores of PLGA nanofiber scaffolds (D), in comparison with 1 and 3 days after cell seeding (B and C, respectively). The morphology of hMSC-derived osteoblasts seeded in PLGA nanofibers also varied between 1, 3 and 7 days following cell seeding (F, G, and H, respectively). By 7 days following cell seeding, hMSC-Ob apparently synthesized a substantial amount of extracellular matrices (H), in comparison with 1 and 3 days after cell seeding (F and G, respectively). Human MSC-derived osteoblasts apparently attached to nanofiber substrates. Human MSC-derived chondrocytes revealed characteristic featured after seeding in PLGA nanofibers (J, K and L). Most PLGA nanofibers were still visible at 7 days following the seeding of hMSC-Ch (L), in comparison with the characteristics of hMSC-Ob seeded in PLGA nanofiber scaffolds after 7 days (H). A number of seeded hMSC-Ch apparently was located in lacunae-like structures, seen arrow (L) (scale bar = 100 μm).
Fig. 4
Fig. 4
Imaging of human mesenchymal stem cells (hMSCs) and nanofiber scaffolds. (A) H&E staining of hMSCs in 2D immediately prior to trypsinization and cell seeding. (B) Day 7 after seeding of hMSCs in PLGA nanofibers. (C) Confocal microscopy showing that hMSCs in 2D assumed somewhat elongated and spindle shape at 14 days following cell seeding. (D) Confocal microscopic image of hMSCs seeded in PLGA nanofibers showing somewhat elongated shape at 14 days following cell seeding. The seeded hMSCs apparently attached to PLGA nanofibers. This verifies that hMSC cultured either in 2D or in 3D of PLGA nanofiber consistently maintain stem cell phenotype after 14 days in vitro (scale bars: A, B: 100 μm; C, D: 50 μm).
Fig. 5
Fig. 5
Differentiation of human mesenchymal stem cells (hMSCs) seeded in PLGA nanofiber scaffold in 3D. (A)–(C) hMSCs cultured in PLGA nanofibers without chondrogenic differentiation showed negative staining to alcian blue, indicating a lack of chondrogenic differentiation. (D)–(F) hMSCs seeded in PLGA nanofibers and treated with chondrogenic medium showed positive staining to alcian blue, suggesting that hMSCs have differentiated into chondrogenic cells with the presence of glycosaminoglycans. (G)–(I) hMSCs cultured in PLGA nanofibers without osteogenic differentiation showed negative staining to alizarin red, indicating a lack of osteogenic differentiation. (J)–(L) hMSCs seeded in PLGA nanofibers and treated with osteogenic medium showed positive staining to alizarin red, suggesting that hMSCs have differentiated into osteogenic cells with the presence of mineral deposition (scale bar: 30 μm).

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