Electrospun gelatin/polycaprolactone nanofibrous membranes combined with a coculture of bone marrow stromal cells and chondrocytes for cartilage engineering

Abstract

Electrospinning has recently received considerable attention, showing notable potential as a novel method of scaffold fabrication for cartilage engineering. The aim of this study was to use a coculture strategy of chondrocytes combined with electrospun gelatin/polycaprolactone (GT/PCL) membranes, instead of pure chondrocytes, to evaluate the formation of cartilaginous tissue. We prepared the GT/PCL membranes, seeded bone marrow stromal cell (BMSC)/chondrocyte cocultures (75% BMSCs and 25% chondrocytes) in a sandwich model in vitro, and then implanted the constructs subcutaneously into nude mice for 12 weeks. Gross observation, histological and immunohistological evaluation, glycosaminoglycan analyses, Young's modulus measurement, and immunofluorescence staining were performed postimplantation. We found that the coculture group formed mature cartilage-like tissue, with no statistically significant difference from the chondrocyte group, and labeled BMSCs could differentiate into chondrocyte-like cells under the chondrogenic niche of chondrocytes. This entire strategy indicates that GT/PCL membranes are also a suitable scaffold for stem cell-based cartilage engineering and may provide a potentially clinically feasible approach for cartilage repairs.

Keywords: cartilage tissue engineering; electrospinning; nanocomposite; nanomaterials; stem cells.

Figure 1

Characterization and biocompatibility of GT/PCL nanofibrous membrane. Notes: (A) Gross view of the sheet of electrospun GT/PCL nanofibrous membrane. (B) SEM image of GT/PCL membrane. (C) Morphology of Passage 1 chondrocytes using a light microscope. Scale bar: 200 μm. (D) Morphology of chondrocytes on GT/PCL membranes after 2 days of cell culture in vitro. (E) Morphology of Passage 2 BMSCs using a light microscope. Scale bar: 200 μm. (F) Morphology of BMSCs on GT/PCL membranes after 2 days of cell culture in vitro. Abbreviations: GT/PCL, gelatin/polycaprolactone; SEM, scanning electron microscopic; BMSC, bone marrow stromal cell.

Figure 2

Engineering cell-scaffold constructs in vitro and the formation of cartilage in vivo. Notes: (A) Gross view of cell-scaffold constructs engineered in the sandwich model in vitro. (B) Cell-scaffold constructs were implanted subcutaneously into nude mice after being cultured for 1 week in vitro. (C) Gross view of cell-scaffold constructs 12 weeks postimplantation. (D–F) Gross view of constructs 12 weeks postimplantation in the chondrocyte group, coculture group, and BMSC group, respectively. Minimum scale: 1 mm. Abbreviation: BMSC, bone marrow stromal cell.

Figure 3

Quantitative analysis of thickness, wet weight, GAG content, and Young’s modulus. Notes: (A) Average thicknesses of cell-scaffold constructs in the chondrocyte group, coculture group, and BMSC group, respectively. (B) Average wet weights of cell-scaffold constructs in the different groups. (C) GAG content normalized to wet weight for cell-scaffold constructs in the different groups. Rabbit auricular cartilages were used as a normal control. (D) Young’s modulus of cell-scaffold constructs in the different groups. The rabbit auricular cartilages were used as a normal control. Mean ± SD (n=4); *P<0.05, **P<0.01. Abbreviations: GAG, glycosaminoglycan; BMSC, bone marrow stromal cell; SD, standard deviation.

Figure 4

Histology of cell-scaffold constructs after 12 weeks in vivo in the different groups. Notes: Tissue sections were stained with H&E. Arrows show nondegraded GT/PCL nanofibrous membranes. Scale bars: 200 μm. Abbreviations: H&E, hematoxylin and eosin; BMSC, bone marrow stromal cell; GT/PCL, gelatin/polycaprolactone.

Figure 5

Special histological staining with Toluidine blue and Safranin O and immunohistochemistry staining with type 11 collagen for cell-scaffold constructs in the different groups. Note: Scale bars: 200 μm. Abbreviation: BMSC, bone marrow stromal cell.

Figure 6

Labeling and tracing chondrogenesis of BMSCs. Notes: (A) Gross view of cell-scaffold constructs formed by GT/PCL membranes seeded with coculture CM-Dil-labeled BMSCs and chondrocytes in vitro. (B) Gross view of cell-scaffold constructs after 12 weeks of implantation in the coculture group. (C–F) Immunofluorescence staining with type II collagen. Arrows show CM-Dil-labeled BMSCs overlapped with the type II collagen-positive cells in the constructs. Scale bars: 200 μm. Abbreviations: DAPI, 4′,6-diamidino-2-phenylindole; BMSC, bone marrow stromal cell; GT/PCL, gelatin/polycaprolactone.