Vitronectin-Based, Biomimetic Encapsulating Hydrogel Scaffolds Support Adipogenesis of Adipose Stem Cells

Abstract

Soft tissue defects are relatively common, yet currently used reconstructive treatments have varying success rates, and serious potential complications such as unpredictable volume loss and reabsorption. Human adipose-derived stem cells (ASCs), isolated from liposuction aspirate have great potential for use in soft tissue regeneration, especially when combined with a supportive scaffold. To design scaffolds that promote differentiation of these cells down an adipogenic lineage, we characterized changes in the surrounding extracellular environment during adipogenic differentiation. We found expression changes in both extracellular matrix proteins, including increases in expression of collagen-IV and vitronectin, as well as changes in the integrin expression profile, with an increase in expression of integrins such as αVβ5 and α1β1. These integrins are known to specifically interact with vitronectin and collagen-IV, respectively, through binding to an Arg-Gly-Asp (RGD) sequence. When three different short RGD-containing peptides were incorporated into three-dimensional (3D) hydrogel cultures, it was found that an RGD-containing peptide derived from vitronectin provided strong initial attachment, maintained the desired morphology, and created optimal conditions for in vitro 3D adipogenic differentiation of ASCs. These results describe a simple, nontoxic encapsulating scaffold, capable of supporting the survival and desired differentiation of ASCs for the treatment of soft tissue defects.

FIG. 1.

Analysis of ECM protein and integrin expression in ASCs before and after adipogenic differentiation. (A) ECM proteins indicated (green) were detected by immunocytochemistry in undifferentiated ASCs (left) and ASCs subjected to adipogenic differentiation for 21 days (right). Nuclei (blue) were detected by Hoechst staining. Scale bar = 50 μm. (B) Integrin subunits indicated (green) were detected by immunocytochemistry in undifferentiated ASCs (left) and ASCs subjected to adipogenic differentiation for 21 days (right). Nuclei (blue) were detected by Hoechst staining. Scale bars = 50 μm. ASCs, adipose-derived stem cells; ECM, extracellular matrix.

FIG. 2.

Effects of full-length ECM proteins on ASCs. (A) Proliferation on different substrates was quantified (*p < 0.05) (B) Secretion of adipogenic factors (adiponectin and leptin) by ASCs cultured on different ECM proteins was quantified by ELISA at day 1 (dark bars) and day 14 (light bars). Secretion of bFGF and SDF1- α (C) and VEGF and HGF (D) by ASCs cultured on different ECM proteins was quantified by ELISA over time (***p < 0.001; **p < 0.01; *p < 0.05). bFGF, basic fibroblast growth factor; Col I, collagen I; Col IV, collagen IV; FN, fibronectin; HGF, hepatocyte growth factor; LM, Laminin; SDF-1α, stromal cell-derived factor 1α; VEGF, vascular endothelial growth factor; VN, vitronectin.

FIG. 3.

ASC Adhesion and spreading on RGD peptides. (A) Sequence of peptides. (B) Average number of cells that attach to peptides in two-dimensional culture, after 3 h, showing varying adhesion, compared to no-peptide control. (C) Average area of cells after attachment on various peptides showing extent of spreading after 3 h of attachment compared to no-peptide control (**p < 0.01, ***p < 0.001).

FIG. 4.

QGel 3D Hydrogel proliferation and adipogenic differentiation. (A) Proliferation of the ASCs in maintenance conditions in 3D culture as determined by a CyQUANT assay. (B) 3D reconstructions of ASCs adipogenically differentiated for 21 days in Qgel containing various attachment peptides. (C) The average number of nuclei visualized per field showing the overall number of cells present after 21 days of adipogenic culture. (D) The percent of total cells present in each field that are associated with a lipid vacuole. (E) The average size of all the lipid vacuoles present for each condition (*p < 0.05, **p < 0.01, ***p < 0.001). 3D, three-dimensional.

FIG. 5.

PEG-DVS 3D hydrogel proliferation and adipogenic differentiation. (A) Proliferation of the ASCs in maintenance conditions in 3D culture over 21 days. (B) 3D reconstructions of ASCs adipogenically differentiated for 21 days in PEG-DVS gels containing various attachment peptides. (C) The average number of nuclei visualized per field showing the overall number of cells present after 21 days of adipogenic culture. (D) The percent of total cells present, in each field that are associated with a lipid vacuole. (E) The average size of all the lipid vacuoles present for each condition (*p < 0.05, **p < 0.01, ***p < 0.001). DVS, divinyl sulfone; PEG, poly(ethylene-glycol).