Assessment of Scaffolding Properties for Chondrogenic Differentiation of Adipose-Derived Mesenchymal Stem Cells in Nasal Reconstruction.

Abstract

Nasal reconstruction in patients who are missing a significant amount of structural nasal support remains a difficult challenge. One challenge is the deficiency of cartilage left within the nose as a consequence of rhinectomy or a midline destructive disease. Historically, the standard donor source for large quantities of native cartilage has been costal cartilage. To enable the development of protocols for new mesenchymal stem cell technologies as alternative procedures with reduced donor site morbidity, risk of infection and extrusion. We examined 6 popular scaffold materials in current practice in terms of their biodegradability in tissue culture, effect on adipose-derived mesenchymal stem cell growth, and chondrogenic fate commitment. Various biomaterials of matching size, porosity, and fiber alignment were synthesized by electrospinning and overlaid with rabbit adipose-derived mesenchymal cells in media supplemented or not with chondrogenic factors. Experiments were performed in vitro using as end points biomarkers for cell growth and chondrogenic differentiation. Polydioxanone (PDO), poly-3-hydroxybutyrate-co-3-hydroxyvalerate (PHBV), PHBV-polycaprolactone, poly(L-lactide-co-caprolactone), poly(lactic-co-glycolic acid), and polystyrene scaffolds of 60% to 70% porosity and random fiber alignment were coated with poly(L)-lysine/laminin to promote cell adhesion and incubated for 28 days with 2.5 to 3.5 × 105 rabbit adipose mesenchymal cells. Cell growth was measured by fluorometric DNA quantitation and chondrogenic differentiation of stem cells by spectrophotometric sulfated glycosaminoglycan (sGAG) assay. Microscopic visualization of cell growth and matrix deposition on formalin-fixed, paraffin-embedded tissue sections was performed, respectively, with nuclear fast red and Alcian blue. Of 6 scaffold materials tested using rabbit apidose mesenchymal cells, uncoated scaffolds promoted limited cell adhesion but coating with poly(L)-lysine/laminin enabled efficient cell saturation of scaffold surfaces, albeit with limited involvement of scaffold interiors. Similar growth rates were observed under these conditions, based on DNA content analysis. However, PDO and PHBV/PCL scaffolds supported chondrogenic fate commitment better than other materials, based on soluble sGAG analysis and microscopic observation of chondrogenic matrix deposition. The mean (SD) sGAG scaffold values expressed as fold increase over control were PDO, 2.26 (0.88), PHBV/PCL, 2.09 (0.83), PLCL, 1.36 (0.39), PLGA, 1.34 (0.77), PHBV, 1.07 (0.31), and PS, 0.38 (0.14). These results establish materials, reagents, and protocols for tissue engineering for nasal reconstruction using single-layer, chondrogenically differentiated, adipose-derived mesenchymal stem cells. Stackable, scaffold-supported, multisheet bioengineered tissue may be generated using these protocols. NA.