Differential behavior of auricular and articular chondrocytes in hyaluronic acid hydrogels

Abstract

Chondrocytes isolated from a variety of sources, including auricular (AU) and articular (AR) cartilage, can differ in cell behavior, growth, and extracellular matrix (ECM) production, which can impact neocartilage properties in tissue engineering approaches. This behavior is also affected by the surrounding microenvironment, including soluble factors, biomaterials, and mechanical loading. The objective of this study was to investigate differences in juvenile AU and AR chondrocyte behavior when encapsulated in radically polymerized hyaluronic acid hydrogels. When implanted in vivo, differences in macroscopic appearance, mechanical properties, glycosaminoglycan content, and collagen content were observed depending on the chondrocyte type encapsulated. Specifically, AU constructs exhibited construct growth and neocartilage formation with increases in aggregate modulus and ECM accumulation with culture, whereas AR constructs retained their construct size and remained translucent with only a minimal increase in the compressive modulus. When cultured in vitro, both cell types remained viable and differences in gene expression were observed for type I and II collagens. Likewise, differences in gene expression were noted after dynamic mechanical loading, where stimulated AR constructs exhibited 2.3- and 1.5-fold increases in type II collagen and aggrecan over free-swelling controls, while AU samples exhibited smaller fold increases of 1.4- and 1.3-fold, respectively. Thus, these data indicate that the specific cell source, cell/material interactions, and loading environment are important in the final properties of tissue-engineered products.

FIG. 1.

General schematic of chondrocyte isolation, encapsulation, and analysis. Inset: macroscopic image of explanted AU (left) and AR (right) chondrocyte–seeded constructs after 12 weeks of in vivo subcutaneous culture.

FIG. 2.

Mechanical properties of explanted constructs. (A) Modulus of AU (black) and AR (white) chondrocyte–seeded constructs compared to HA hydrogel alone (shaded). Representative stress relaxation curves for AU chondrocyte–seeded (B) and AR chondrocyte–seeded (C) explants after 12 weeks of in vivo culture compared to HA hydrogel alone (D) and to native AU (E) and AR (F) cartilages. Stress relaxation curves are normalized to peak stress. Significant differences (p ≤ 0.05) between AU and AR groups are denoted by asterisks (*).

FIG. 3.

Biochemical content of AU (black) and AR (white) explants reported as millions of chondrocytes per sample (A) and DNA content (B), CS (C), and collagen content (D) per wet weight. Significant differences (p ≤ 0.05) between AU and AR groups are denoted by asterisks (*).

FIG. 4.

Representative sections of AU and AR constructs stained for type I and II collagens and chondroitin sulfate after 12 weeks of subcutaneous in vivo culture compared to native cartilage tissue sections. Scale bars = 100 μm. Color images available online at www.liebertpub.com/ten.

FIG. 5.

(A) Relative mitochondrial activity of AU (black) and AR (white) hydrogels cultured in vitro for 7 and 14 days. Values were normalized to day 0 absorbance readings. (B) Absorbance readings at 570 nm for AU and AR constructs at days 0 (shaded), 7

FIG. 6.

Relative gene expression for AU (A, B) and AR (C, D) constructs after 6 (black) and 14 (white) days of in vitro culture. GAPDH was used as the housekeeping gene, and expression was normalized to cells encapsulated at day 0. Significant differences (p ≤ 0.05) between AU and AR groups are denoted by asterisks (*) using parametric and nonparametric statistics, or hashes (#) using parametric only.

FIG. 7.

Validation of mechanical loading of HA hydrogels at 5% tare, 10% strain, and frequency of 1 Hz (A). Relative gene expression of dynamically loaded AU (B, C) and AR (D, E) hydrogels for 1 (black) and 5 (white) days normalized to free-swelling controls. Significant differences (p ≤ 0.05) between free-swelling and mechanically loaded samples are denoted by asterisks (*) using parametric and nonparametric statistics, and hashes (#) using parametric only.