A Comparison of Bone Marrow and Cord Blood Mesenchymal Stem Cells for Cartilage Self-Assembly

Abstract

Joint injury is a common cause of premature retirement for the human and equine athlete alike. Implantation of engineered cartilage offers the potential to increase the success rate of surgical intervention and hasten recovery times. Mesenchymal stem cells (MSCs) are a particularly attractive cell source for cartilage engineering. While bone marrow-derived MSCs (BM-MSCs) have been most extensively characterized for musculoskeletal tissue engineering, studies suggest that cord blood MSCs (CB-MSCs) may elicit a more robust chondrogenic phenotype. The objective of this study was to determine a superior equine MSC source for cartilage engineering. MSCs derived from bone marrow or cord blood were stimulated to undergo chondrogenesis through aggregate redifferentiation and used to generate cartilage through the self-assembling process. The resulting neocartilage produced from either BM-MSCs or CB-MSCs was compared by measuring mechanical, biochemical, and histological properties. We found that while BM constructs possessed higher tensile properties and collagen content, CB constructs had superior compressive properties comparable to that of native tissue and higher GAG content. Moreover, CB constructs had alkaline phosphatase activity, collagen type X, and collagen type II on par with native tissue suggesting a more hyaline cartilage-like phenotype. In conclusion, while both BM-MSCs and CB-MSCs were able to form neocartilage, CB-MSCs resulted in tissue more closely resembling native equine articular cartilage as determined by a quantitative functionality index. Therefore, CB-MSCs are deemed a superior source for the purpose of articular cartilage self-assembly.

Keywords: MSCs; cartilage; equine; mesenchymal stem cells; tissue engineering.

FIG. 1.

Construct gross morphology and dimensions presented as mean ± standard deviation in millimeters. Student's t-test was performed using p < 0.05 as statistical significance; starred bars show statistical significance between groups. (A) Representative images of BM-MSC constructs (Bone Marrow) and CB-MSC constructs (Cord Blood). BM-MSCs generated constructs that were generally flatter than CB-MSCs. CB-MSC constructs were significantly wider (B) and thicker (C) compared to BM-MSC constructs. BM-MSC, bone marrow-derived mesenchymal stem cells; CB-MSC, cord blood mesenchymal stem cell.

FIG. 2.

Mechanical properties of BM-MSC and CB-MSC constructs, as well as native equine articular cartilage (Native Tissue). One-way ANOVAs were performed using p < 0.05 for statistical significance; letters above the bars show statistical significance between groups. In terms of compressive properties, both the instantaneous modulus (A) and relaxation modulus (B) were higher in CB-MSC constructs compared to BM-MSC constructs. Native tissue and Cord Blood did not significantly differ for either compressive property as well. BM-MSCs yielded constructs that had both higher tensile stiffness, represented by Young's modulus (C), and ultimate tensile strength (D) compared to CB-MSCs. ANOVAs, analyses of variance.

FIG. 3.

Biochemical properties of both construct groups (Bone Marrow and Cord Blood) and Native Tissue or equine AC-derived tissue (Chondrocytes) in the case of alkaline phosphatase activity. One-way ANOVAs were performed for all assays except Cellularity using p < 0.05 for statistical significance; letters above the bars show statistical significance between groups. Hydration did not differ significantly between Native Tissue and Bone Marrow, while Cord Blood had significantly higher hydration than both groups (A). Collagen/WW basis was significantly higher in Bone Marrow compared to Cord Blood, but both construct groups were significantly lower than Native Tissue (B). GAG/WW was significantly higher in Cord Blood compared to Bone Marrow; however, both of these groups were lower than Native Tissue (C). Cord Blood constructs produced alkaline phosphatase at levels comparable to that of constructs derived from passaged equine ACs. Bone Marrow constructs produced significantly higher levels suggesting a further progression in hypertrophy (D). Cellularity was compared using a Student's t-test with p < 0.05 as statistical significance; the starred bar shows statistical significance between groups. Cord Blood constructs contained more cells than Bone Marrow constructs despite an equal initial seeding density of 2 million cells per construct (E). AC, articular chondrocyte; WW, wet weight.

FIG. 4.

Representative histology images of Bone Marrow constructs, Cord Blood constructs, and equine patellar cartilage (Control). For each section a low magnification image is presented on the left with higher magnification images corresponding to the black boxed regions on the right. Scale bars are presented in the top image of each column and reflect the scale for all images in the column: 200 μm for low magnification images, 50 μm for high magnification and Control images. H&E staining highlights the lacuna structures and hematoxylin intense staining representative of articular cartilage in both the native tissue Control and Cord Blood groups; however, these characteristics are largely absent in the Bone Marrow group. Picrosirius red (PicR) highlights the collagen content, which had higher stain uptake in Bone Marrow compared to Cord Blood, whereas Safranin-O (SafO) with a Fast-Green counterstain highlights GAG content, which had higher stain uptake in Cord Blood. H&E, hematoxylin and eosin.

FIG. 5.

Representative immunohistochemistry images and stain quantification of Bone Marrow constructs, Cord Blood constructs, and positive and negative native tissue controls. For each tissue section, a low magnification image is presented on the left with higher magnification images corresponding to the black boxed regions on the right. Dashed lines define construct edges. Scale bars are presented in the top image of each column and reflect the scale for all images in the column: 200 μm for low magnification images, 50 μm for high magnification and Control images. The percentage of the tissue section that showed DAB staining was quantified and presented as percent area stained for each antibody (right). Student's t-test was performed using p < 0.05 as statistical significance; starred bars show statistical significance between groups. Equine tendon was used as a positive control and equine articular cartilage as a negative control for collagen type I staining (Col I). Bone Marrow constructs had higher levels of staining for this marker of fibrocartilage. Equine articular cartilage was a positive control, and equine tendon was a negative control for collagen type II staining (Col II). Cord Blood constructs possessed higher levels of stain for this hyaline cartilage marker. Osteoarthritic equine articular cartilage served as a positive control, while nonarthritic equine articular cartilage served as a negative control for collagen type X (Col X). Bone Marrow constructs had higher stain uptake for this marker of hypertrophic cartilage.

FIG. 6.

A quantitative functionality index comprised Collagen/WW, GAG/WW, compressive, and tensile properties; a value of 1.0 is equivalent to equine patellar articular cartilage properties. Student's t-test was performed using p < 0.05 as statistical significance; starred bars show statistical significance between groups. Cord Blood had a score closer to 1.0 and was significantly higher than Bone Marrow.