Dynamic compressive loading enhances cartilage matrix synthesis and distribution and suppresses hypertrophy in hMSC-laden hyaluronic acid hydrogels

Abstract

Mesenchymal stem cells (MSCs) are being recognized as a viable cell source for cartilage repair, and there is growing evidence that mechanical signals play a critical role in the regulation of stem cell chondrogenesis and in cartilage development. In this study we investigated the effect of dynamic compressive loading on chondrogenesis, the production and distribution of cartilage specific matrix, and the hypertrophic differentiation of human MSCs encapsulated in hyaluronic acid (HA) hydrogels during long term culture. After 70 days of culture, dynamic compressive loading increased the mechanical properties, as well as the glycosaminoglycan (GAG) and collagen contents of HA hydrogel constructs in a seeding density dependent manner. The impact of loading on HA hydrogel construct properties was delayed when applied to lower density (20 million MSCs/ml) compared to higher seeding density (60 million MSCs/ml) constructs. Furthermore, loading promoted a more uniform spatial distribution of cartilage matrix in HA hydrogels with both seeding densities, leading to significantly improved mechanical properties as compared to free swelling constructs. Using a previously developed in vitro hypertrophy model, dynamic compressive loading was also shown to significantly reduce the expression of hypertrophic markers by human MSCs and to suppress the degree of calcification in MSC-seeded HA hydrogels. Findings from this study highlight the importance of mechanical loading in stem cell based therapy for cartilage repair in improving neocartilage properties and in potentially maintaining the cartilage phenotype.

FIG. 1.

Viability staining of mesenchymal stem cells (MSCs) seeded at 20 or 60 million cells/ml in hyaluronic acid (HA) hydrogels after 70 days of in vitro culture under free swelling (FS) or dynamic loading (DL) culture conditions. Green: live cells. Red: dead cells. Scale bar=100 μm. Color images available online at www.liebertonline.com/tea

FIG. 2.

Young's modulus (A), dynamic modulus (1Hz) (B), glycosaminoglycan (GAG) (C) and collagen (D) contents normalized to wet weight (w.w.), and GAG (E) and collagen (F) contents normalized to DNA (ng/ng). 20/60FS: constructs seeded with 20 or 60 million cells/ml and cultured under free swelling conditions; 20/60DL: constructs seeded with 20 or 60 million cells/ml and cultured under dynamic loading conditions, *p<0.05 vs. FS group at the same culture time (n=4), ^†p<0.05 vs. 20FS/DL group at the same culture time (n=4).

FIG. 3.

Expression of aggrecan, type II collagen and type I collagen in fold change (relative to day 0) after 14, 42, and 70 days of the culture (n=4) for either 20 or 60 million cells/ml encapsulation density and under FS or DL culture conditions.

FIG. 4.

Alcian blue staining (pH=1.0) (proteoglycans) of all groups after 30 and 70 days of culture; 4× images show the entire construct cross-section, bar=500μm; 20× images show peripheral or central regions of construct, bar=100μm. Color images available online at www.liebertonline.com/tea

FIG. 5.

Picrosirius red staining (collagen) of all the groups after 30 and 70 days of culture; 4× images show entire construct cross-section, bar=500μm; 20× images show peripheral or central regions of the construct, bar=100μm. Color images available online at www.liebertonline.com/tea

FIG. 6.

Expression of selected hypertrophic markers in fold change on day 28 for cells at the 20 million cells/ml seeding density, *p<0.05 vs. FS+T3/−T3 group (n=4).

FIG. 7.

Calcium content (A) and Von Kossa staining (B) of the HA hydrogels on day 30 for cells at the 20 million cells/ml seeding density, *p<0.05 vs. FS+T3 group (n=4); bar=500μm. Color images available online at www.liebertonline.com/tea