Development and Characterization of Acellular Extracellular Matrix Scaffolds from Porcine Menisci for Use in Cartilage Tissue Engineering

Abstract

Given the growing number of arthritis patients and the limitations of current treatments, there is great urgency to explore cartilage substitutes by tissue engineering. In this study, we developed a novel decellularization method for menisci to prepare acellular extracellular matrix (ECM) scaffolds with minimal adverse effects on the ECM. Among all the acid treatments, formic acid treatment removed most of the cellular contents and preserved the highest ECM contents in the decellularized porcine menisci. Compared with fresh porcine menisci, the content of DNA decreased to 4.10%±0.03%, and there was no significant damage to glycosaminoglycan (GAG) or collagen. Histological staining also confirmed the presence of ECM and the absence of cellularity. In addition, a highly hydrophilic scaffold with three-dimensional interconnected porous structure was fabricated from decellularized menisci tissue. Human chondrocytes showed enhanced cell proliferation and synthesis of chondrocyte ECM including type II collagen and GAG when cultured in this acellular scaffold. Moreover, the scaffold effectively supported chondrogenesis of human bone marrow-derived mesenchymal stem cells. Finally, in vivo implantation was conducted in rats to assess the biocompatibility of the scaffolds. No significant inflammatory response was observed. The acellular ECM scaffold provided a native environment for cells with diverse physiological functions to promote cell proliferation and new tissue formation. This study reported a novel way to prepare decellularized meniscus tissue and demonstrated the potential as scaffolds to support cartilage repair.

FIG. 1.

Contents of DNA (a), total collagen (b), and glycosaminoglycan (c) of porcine menisci following various decellularization treatments at different time points are compared to fresh menisci treated with phosphate-buffered saline (PBS) under the same processing steps. Values are presented as the mean±standard deviation (SD) (n=3). Statistical analysis were conducted for treatment at 2 h; *p<0.05 and **p<0.001 compared to fresh menisci treated with PBS. Color images available online at www.liebertpub.com/tec

FIG. 2.

Contents of DNA, glycosaminoglycan, total collagen, and types I and II collagen of porcine menisci treated with formic acid for 2 h are compared to the same weight of fresh menisci treated with PBS under the same processing steps. Values are presented as the mean±SD (n=3). **p<0.001 compared to fresh menisci treated with PBS.

FIG. 3.

Images of fresh porcine menisci (a, c, e, g), and those treated with formic acid for 2 h (b, d, f, h) after staining with hematoxylin and eosin (a, b), Alcian blue (c, d), Masson's trichrome (e, f), and immunohistochemistry for type II collagen (g, h). Scale bar: 100 μm at 100×magnification. Color images available online at www.liebertpub.com/tec

FIG. 4.

Macroscopic view of a fresh porcine meniscus (a, left) and a decellularized one treated with formic acid for 2 h (a, right). Scanning electron microscopy photographs of a decellularized menisci treated with formic acid for 2 h (b) and fresh porcine menisci (c). Scale bar: 100 μm at 300×magnification (b, c).

FIG. 5.

Scanning electron microscopy photographs of acellular extracellular matrix (ECM) scaffolds on which human chondrocytes were seeded for 0 (before cell seeding) (a), 7 (b), 14 (c), 21 (d), and 28 days (e). Scale bar: 50 μm at 800×magnification.

FIG. 6.

Fluorescent-stained photographs of live/dead cells when human chondrocytes were cultured in acellular ECM scaffolds for 7 (a), 14 (b), 21 (c), and 28 days (d). Scale bar: 500 μm. Color images available online at www.liebertpub.com/tec

FIG. 7.

Contents of DNA and cell number (black line) (a), glycosaminoglycan (b), total collagen (c), and type II collagen (d) in acellular ECM scaffolds (day 0) or on which human chondrocytes were cultured at different time points. Values are presented as the mean±SD (n=3). *p<0.05 and **p<0.001 versus DNA content on day 7 (a), and the amount of glycosaminoglycan, total collagen, and type II collagen on day 0 (b, c, d).

FIG. 8.

Hematoxylin and eosin (a1–a4), Alcian blue (b1–b4), Masson's trichrome (c1–c4), and immunohistochemical (d1–d4) staining images of acellular ECM scaffolds onto which human chondrocytes were seeded for 7 (a1, b1, c1, d1), 14 (a2, b2, c2, d2), 21 (a3, b3, c3, d3), and 28 days (a4, b4, c4, d4). Scale bar: 100 μm at 100×magnification. Color images available online at www.liebertpub.com/tec

FIG. 9.

Gene expression levels of aggrecan (a), type II collagen (b), type X collagen (c), and type I collagen (d) in acellular ECM scaffolds seeded with human chondrocytes at different time points. The gene expression levels on days 14, 21, and 28 relative to day 7 were analyzed by comparative C_T method using GAPDH as the internal control. Values are presented as the mean±SD (n=3). *p<0.05; **p<0.001 versus the value on day 7.

FIG. 10.

Relative amounts of glycosaminoglycan/DNA (a), total collagen/DNA (b), and type II collagen/DNA (c) when bone marrow-derived human mesenchymal stem cells (hMSCs) in various of culture conditions. hMSCs were cultured either in a monolayer (tissue culture polystyrene [TCPS]) or on acellular ECM scaffolds (S) and with either growth medium (M) or chondrogenic medium (C) for 14 and 21 days. The values were normalized to that when hMSCs were in a monolayer and with growth medium (TCPS-M) on day 14. Values are presented as the mean±SD (n=3). *p<0.05 and **p<0.001 versus the value of TCPS-M or TCPS-C on day 14.

FIG. 11.

Images of acellular ECM scaffolds seeded with bone marrow-derived hMSCs for 21 days and stained with Alcian blue (a), Masson's trichrome (b), and immunohistochemistry of type II collagen (c). Scale bar: 100 μm at 100×. Color images available online at www.liebertpub.com/tec

FIG. 12.

Gene expression levels of aggrecan (a), type II collagen (b), and type I collagen (c) when bone marrow-derived hMSCs were cultured in a monolayer (TCPS) or on acellular ECM scaffolds (S) and with growth medium (M) or chondrogenic medium (C) for 14 and 21 days. The values were normalized to that when hMSCs were cultured in a monolayer and with growth medium (TCPS-M) on day 14. Values are presented as the mean±SD (n=3). The gene expression levels on various culture conditions relative to TCPS-M on day 14 were analyzed by comparative C_T method using GAPDH as the internal control. *p<0.05 and **p<0.001 versus the value of TCPS-M or TCPS-C on day 14.

FIG. 13.

Hematoxylin and eosin staining images of skin sections from Sprague Dawley rats with sham-operated (a–c) and subcutaneously implanted scaffold (d–f) on days 7, 14, and 28. Areas within the two black dashed lines indicate the implanted scaffolds. Color images available online at www.liebertpub.com/tec