Comparison of three methods for the derivation of a biologic scaffold composed of adipose tissue extracellular matrix

Abstract

Extracellular matrix (ECM)-based scaffold materials have been used successfully in both preclinical and clinical tissue engineering and regenerative medicine approaches to tissue reconstruction. Results of numerous studies have shown that ECM scaffolds are capable of supporting the growth and differentiation of multiple cell types in vitro and of acting as inductive templates for constructive tissue remodeling after implantation in vivo. Adipose tissue represents a potentially abundant source of ECM and may represent an ideal substrate for the growth and adipogenic differentiation of stem cells harvested from this tissue. Numerous studies have shown that the methods by which ECM scaffold materials are prepared have a dramatic effect upon both the biochemical and structural properties of the resultant ECM scaffold material as well as the ability of the material to support a positive tissue remodeling outcome after implantation. The objective of the present study was to characterize the adipose ECM material resulting from three methods of decellularization to determine the most effective method for the derivation of an adipose tissue ECM scaffold that was largely free of potentially immunogenic cellular content while retaining tissue-specific structural and functional components as well as the ability to support the growth and adipogenic differentiation of adipose-derived stem cells. The results show that each of the decellularization methods produced an adipose ECM scaffold that was distinct from both a structural and biochemical perspective, emphasizing the importance of the decellularization protocol used to produce adipose ECM scaffolds. Further, the results suggest that the adipose ECM scaffolds produced using the methods described herein are capable of supporting the maintenance and adipogenic differentiation of adipose-derived stem cells and may represent effective substrates for use in tissue engineering and regenerative medicine approaches to soft tissue reconstruction.

Figures

FIG. 1.

Gross morphology (A–C) and oil red O staining (D–F, 10 × magnification) of materials resulting from Method A (A, D), Method B (B, E), and Method C (C, F). Red staining is indicative of lipid content. Scale bar = 200 μm. Color images available online at www.liebertonline.com/tec.

FIG. 2.

Hematoxylin and eosin staining (A–C, 20 × magnification), 4′,6-diamidino-2-phenylindole labeling (D–F, 20× magnification), and results of agarose gel electrophoresis of DNA isolated from each scaffold type (G). Scale bar = 100 μm. Lane 1 = DNA ladder, lane 2 = Method A, lane 3 = Method B, lane 4 = Method C. Color images available online at www.liebertonline.com/tec.

FIG. 3.

Scanning electron micrographs of the surfaces of extracellular matrix (ECM) materials decellularized with Method A (A, D), Method B (B, E), and Method C (C, F). Top panel (A–C) = 1000 × magnification, scale bar = 25 μm. Bottom panel (D–F) = 5000 × magnification, scale bar = 5 μm. Asterisks indicate vascular structures. Arrows indicate exposed fibrous ECM structures. Arrowheads represent exposed smooth basement membrane like surfaces. Small lipid droplets can be observed in (A, D). Lipid surfaces indicated by smooth ultrastructure in (B, C, E, F) are contiguous across the surface of the material.

FIG. 4.

Immunolabeling of collagen types I, III, IV, VII, and laminin in samples decellularized with Methods A, B, and C. Immunolabeling of native adipose tissue is shown for comparison. All images 20× magnification. Scale bar = 100 μm. Color images available online at www.liebertonline.com/tec.

FIG. 5.

Growth factor content of adipose ECM materials decellularized with Methods A, B, and C. Basic fibroblast growth factor (bFGF) content and vascular endothelial growth factor (VEGF) content are shown (A and B, respectively). Samples prepared using Methods A and B have higher bFGF and VEGF content than samples prepared with Method C (p < 0.05). Samples prepared using Method A have a higher bFGF content than samples prepared using Method B (p < 0.05). Results are shown as mean ± standard error. **Method A samples have higher content than both Method B and Method C, p < 0.05. *Statistical significance compared to Method C, p < 0.05.

FIG. 6.

Glycosaminoglycan (GAG) content of ECM scaffolds decellularized using Methods A, B, and C. GAG content of scaffolds decellularized with Method A are higher than in samples prepared using Methods B or C (p < 0.05), and levels in samples prepared with Method B are higher than in samples prepared using Method C. Results are shown as mean ± standard error. **Method A samples have higher content than both Method B and Method C, p < 0.05. *Statistical significance compared to Method C, p < 0.05.

FIG. 7.

Quantification of live/dead staining of adipose-derived stem cells seeded onto ECM scaffolds decellularized using Methods A, B, and C at 24 and 72 h postseeding. No significant differences in cell viability were observed for any of the scaffold materials investigated. Percent live cells is represented by black bars. Percent dead cells is represented by white bars. Results are shown as mean ± standard error.

FIG. 8.

Adipo-Red labeling of adipose-derived stem cells seeded onto ECM scaffolds decellularized using Methods A, B, and C, respectively. Scale bar = 50 μm. Color images available online at www.liebertonline.com/tec.