Chondrogenic potential of bone marrow-derived mesenchymal stem cells on a novel, auricular-shaped, nanocomposite scaffold

Abstract

Reconstruction of the human auricle remains a challenge to plastic surgeons, and current approaches are not ideal. Tissue engineering provides a promising alternative. This study aims to evaluate the chondrogenic potential of bone marrow-derived mesenchymal stem cells on a novel, auricular-shaped polymer. The proposed polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea nanocomposite polymer has already been transplanted in patients as the world's first synthetic trachea, tear duct and vascular bypass graft. The nanocomposite scaffold was fabricated via a coagulation/salt-leaching method and shaped into an auricle. Adult bone marrow-derived mesenchymal stem cells were isolated, cultured and seeded onto the scaffold. On day 21, samples were sent for scanning electron microscopy, histology and immunofluorescence to assess for neocartilage formation. Cell viability assay confirmed cytocompatability and normal patterns of cellular growth at 7, 14 and 21 days after culture. This study demonstrates the potential of a novel polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea scaffold for culturing bone marrow-derived mesenchymal stem cells in chondrogenic medium to produce an auricular-shaped construct. This is supported by scanning electron microscopy, histological and immunofluorescence analysis revealing markers of chondrogenesis including collagen type II, SOX-9, glycosaminoglycan and elastin. To the best of our knowledge, this is the first report of stem cell application on an auricular-shaped scaffold for tissue engineering purposes. Although many obstacles remain in producing a functional auricle, this is a promising step forward.

Keywords: Auricle; biomaterial; ear; scaffolds; stem cells; surgery; tissue engineering.

Figure 1.

Images of a novel human-sized, glass mould–designed, porous auricular scaffold made from a nanocomposite POSS-PCU polymer: (a) outer part of the negative glass mould, (b) inner part of the glass mould, (c) a digital photograph of the resultant porous auricular scaffold produced and (d) SEM cross-section of the auricular scaffold fabricated using coagulation/particulate-leaching techniques. POSS-PCU: polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea; SEM: scanning electron microscopy.

Figure 2.

SEM micrographs of (a and b) surface and (c and d) cross-section morphologies of auricular POSS-PCU scaffold indicative of the effect of porogen (NaCl) leaching on construct structure and porosity. SEM: scanning electron microscopy; POSS-PCU: polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea.

Figure 3.

Proliferation of bone marrow–derived mesenchymal stem cells (BMSC) culture on POSS-PCU nanocomposite (blue) and tissue culture polystyrene (TCP) (purple) surfaces after 7, 14 and 21 days. POSS-PCU: polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea; SD: standard deviation. A significant increase in the rate of proliferation was observed in both groups over 21 days. There were proportionally greater increases in cell proliferation at all time-points with POSS-PCU, errors bar = SD. *p < 0.001

Figure 4.

SEM micrographs of auricular scaffold samples seeded with BMSCs on day 21. Images were taken of (a and b) the surface and (c and d) cross-section of the polymer. SEM: scanning electron microscopy; BMSC: bone marrow–derived mesenchymal stem cell; ECM: extracellular matrix. BMSCs (arrows) were found attached to the surface as well as within the polymer pores. ECM production was also evident.

Figure 5.

Histological analysis at day 21 with (a and b) haematoxylin and eosin, (c and d) safranin-O (e and f) and orcein staining. Plane conditions (i.e. tissue culture polystyrene) are presented as a comparison. There is evidence of early chondrogenesis on the POSS-PCU polymer highlighted by the presence of (a) cell nuclei, (c) GAG and (e) elastin. Calibration bar, 20 µm. POSS-PCU: polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea; GAG: glycosaminoglycan.

Figure 6.

Immunofluorescence analysis at day 21 for (a and b) collagen type II and (c and d) SOX-9. Plane conditions (i.e. tissue culture polystyrene) are presented as a comparison. There is evidence of chondrogenesis on the POSS-PCU polymer. Calibration bar, 20 µm. POSS-PCU: polyhedral oligomeric silsesquioxane-modified poly(hexanolactone/carbonate)urethane/urea.