Effect of Cyclic Dynamic Compressive Loading on Chondrocytes and Adipose-Derived Stem Cells Co-Cultured in Highly Elastic Cryogel Scaffolds

Abstract

In this study, we first used gelatin/chondroitin-6-sulfate/hyaluronan/chitosan highly elastic cryogels, which showed total recovery from large strains during repeated compression cycles, as 3D scaffolds to study the effects of cyclic dynamic compressive loading on chondrocyte gene expression and extracellular matrix (ECM) production. Dynamic culture of porcine chondrocytes was studied at 1 Hz, 10% to 40% strain and 1 to 9 h/day stimulation duration, in a mechanical-driven multi-chamber bioreactor for 14 days. From the experimental results, we could identify the optimum dynamic culture condition (20% and 3 h/day) to enhance the chondrocytic phenotype of chondrocytes from the expression of marker (Col I, Col II, Col X, TNF-α, TGF-β1 and IGF-1) genes by quantitative real-time polymerase chain reactions (qRT-PCR) and production of ECM (GAGs and Col II) by biochemical analysis and immunofluorescence staining. With up-regulated growth factor (TGF-β1 and IGF-1) genes, co-culture of chondrocytes with porcine adipose-derived stem cells (ASCs) was employed to facilitate chondrogenic differentiation of ASCs during dynamic culture in cryogel scaffolds. By replacing half of the chondrocytes with ASCs during co-culture, we could obtain similar production of ECM (GAGs and Col II) and expression of Col II, but reduced expression of Col I, Col X and TNF-α. Subcutaneous implantation of cells/scaffold constructs in nude mice after mono-culture (chondrocytes or ASCs) or co-culture (chondrocytes + ASCs) and subject to static or dynamic culture condition in vitro for 14 days was tested for tissue-engineering applications. The constructs were retrieved 8 weeks post-implantation for histological analysis by Alcian blue, Safranin O and Col II immunohistochemical staining. The most abundant ectopic cartilage tissue was found for the chondrocytes and chondrocytes + ASCs groups using dynamic culture, which showed similar neo-cartilage formation capability with half of the chondrocytes replaced by ASCs for co-culture. This combined co-culture/dynamic culture strategy is expected to cut down the amount of donor chondrocytes needed for cartilage-tissue engineering.

Keywords: adipose-derived stem cells; cartilage-tissue engineering; chondrocytes; compressive loading; cryogel; dynamic culture; mechanical stimulation.

Figure 1

The relative gene expression when chondrocytes were cultured in cryogel scaffolds under different cyclic compressive loading conditions. The expression of Col I (A); Col II (B); Col X (C); TNF-α (D); TGF-β1 (E) and IGF-1 (F) for cells in dynamic culture at 1 Hz was normalized to those in static culture at day 14. The groups under dynamic culture with 10% (40%) strain and 1 h/day (3 h/day) duration are termed D11 (D43) etc. α p < 0.05 with D11; β p < 0.05 compared with D13; χ p < 0.05 compared with D19; δ p < 0.05 compared with D21; ε p < 0.05 compared with D23; Φ p < 0.05 compared with D29; γ p < 0.05 compared with D41; η p < 0.05 compared with D43. Col II: Type II collagen; Col I: Type I collagen; Col X: Type X collagen; TNF-α: Tumor necrosis factor-α; IGF-1: Insulin growth factor-1; TGF-β1: Transforming growth factor-β1.

Figure 2

The proliferation and extracellular matrix (GAGs and Col II) production when chondrocytes were cultured in cryogel scaffolds under different cyclic compressive loading conditions. The DNA (A), GAGs/DNA (B) and Col II/DNA (C) contents of cells dynamically cultured for 14 days at 1 Hz were shown and compared with day 0 (solid lines) and static culture (dotted lines) for 14 days. The groups under dynamic culture with 10% (40%) strain and 1 h/day (3 h/day) duration are termed D11 (D43) etc. α p < 0.05 with D11; β p < 0.05 compared with D13; χ p < 0.05 compared with D19; δ p < 0.05 compared with D21; ε p < 0.05 compared with D23; Φ p < 0.05 compared with D29; γ p < 0.05 compared with D41; η p < 0.05 compared with D43. GAGs: Glycoaminoglycans; Col II: Type II collagen.

Figure 3

Immunofluorescence staining of chondrocytes in the cryogel scaffolds in static or dynamic culture (frequency = 1 Hz) with different cyclic compressive-loading conditions. Blue: cell nucleus (DAPI); red: Col II (Cy 3); green: gelatin (FITC); scale bar = 100 µm. DAPI: 4′,6-diamidino-2-phenylindole; Cy 3: Cyanine 3; FITC: Fluorescein isothiocyanate.

Figure 4

The gene expression of Col I (A); Col II (B); Col X (C) and TNF-α (D) when chondrocytes, adipose-derived stem cells (ASCs) or chondrocytes + ASCs were cultured in cryogel scaffolds for 14 days under static or cyclic compressive loading (frequency = 1 Hz, strain = 20% and duration = 3 h/day) conditions. Cell number: 10⁵ chondrocytes/scaffold for the chondrocytes group; 10⁵ ASCs/scaffold for the ASCs group; 5 × 10⁴ chondrocytes/scaffold and 5 × 10⁴ ASCs/scaffold for the chondrocytes + ASCs group. * p < 0.05 compared with the static group; # p < 0.05 compared with the chondrocytes group; & p < 0.05 compared with the ASCs group.

Figure 5

Proliferation and extracellular matrix (GAGs and Col II) production when chondrocytes, ASCs or chondrocytes + ASCs were cultured in cryogel scaffolds for 14 days under static or cyclic compressive loading (frequency = 1 Hz, strain = 20% and duration = 3 h/day) conditions. Cell number: 10⁵ chondrocytes/scaffold for the chondrocytes group; 10⁵ ASCs/scaffold for the ASCs group; 5 × 10⁴ chondrocytes/scaffold and 5 × 10⁴ ASCs/scaffold for the chondrocytes + ASCs group. (A) DNA; (B) GAGs/DNA; (C) Col II/DNA. * p < 0.05 compared with static; # p < 0.05 compared chondrocytes; & p < 0.05 compared with ASCs.

Figure 6

Immunofluorescence staining of cells/scaffold constructs when chondrocytes, ASCs or chondrocytes + ASCs were cultured in cryogel scaffolds for 14 days under static or cyclic compressive loading (frequency = 1 Hz, strain = 20% and duration = 3 h/day) conditions. Cell number: 10⁵ chondrocytes/scaffold for the chondrocytes group; 10⁵ ASCs/scaffold for the ASCs group; 5 × 10⁴ chondrocytes/scaffold and 5 × 10⁴ ASCs/scaffold for the chondrocytes + ASCs group. Blue: cell nucleus (DAPI); red: Col II (Cy 3); green: gelatin (FITC); Scale bar = 100 µm.

Figure 7

Alcian blue and Safranin O staining of implants eight weeks after subcutaneous implantation in nude mice. The cells/scaffold constructs were cultured in cryogel scaffolds for 14 days under static or cyclic compressive loading (frequency = 1 Hz, strain = 20% and duration = 3 h/day) conditions before implantation. Scale bar = 50 µm.

Figure 8

Immunohistochemical staining of Col II of implants eight weeks after subcutaneous implantation in nude mice. The cells/scaffold constructs were cultured in cryogel scaffolds for 14 days under static or cyclic compressive loading (frequency = 1 Hz, strain = 20% and duration = 3 h/day) conditions before implantation. Scale bar = 50 µm.