Chondrogenesis and mineralization during in vitro culture of human mesenchymal stem cells on three-dimensional woven scaffolds

Abstract

Human mesenchymal stem cells (hMSCs) and three-dimensional (3D) woven poly(ɛ-caprolactone) (PCL) scaffolds are promising tools for skeletal tissue engineering. We hypothesized that in vitro culture duration and medium additives can individually and interactively influence the structure, composition, mechanical, and molecular properties of engineered tissues based on hMSCs and 3D poly(ɛ-caprolactone). Bone marrow hMSCs were suspended in collagen gel, seeded on scaffolds, and cultured for 1, 21, or 45 days under chondrogenic and/or osteogenic conditions. Structure, composition, biomechanics, and gene expression were analyzed. In chondrogenic medium, cartilaginous tissue formed by day 21, and hypertrophic mineralization was observed in the newly formed extracellular matrix at the interface with underlying scaffold by day 45. Glycosaminoglycan, hydroxyproline, and calcium contents, and alkaline phosphatase activity depended on culture duration and medium additives, with significant interactive effects (all p < 0.0001). The 45-day constructs exhibited mechanical properties on the order of magnitude of native articular cartilage (aggregate, Young's, and shear moduli of 0.15, 0.12, and 0.033 MPa, respectively). Gene expression was characteristic of chondrogenesis and endochondral bone formation, with sequential regulation of Sox-9, collagen type II, aggrecan, core binding factor alpha 1 (Cbfα1)/Runx2, bone sialoprotein, bone morphogenetic protein-2, and osteocalcin. In contrast, osteogenic medium produced limited osteogenesis. Long-term culture of hMSC on 3D scaffolds resulted in chondrogenesis and regional mineralization at the interface between soft, newly formed engineered cartilage, and stiffer underlying scaffold. These findings merit consideration when developing grafts for osteochondral defect repair.

FIG. 1.

Construct appearance at culture days 21 and 45. Representative constructs cultured in (A, B, E, F, I, J, K) chondrogenic and (C, D, G, H, L, M) osteogenic medium shown in (A–D) macroscopic photos and (E–M) histological sections. The arrow in (F) shows how the woven poly(ɛ-caprolactone) scaffold has been forced apart (arrow) by new tissue growth in this histological cross section. Arrows in (I, J, K) show chondrocytic morphology, whereas arrows in (L, M) show fibroblastic morphology. Stain: hematoxylin and eosin. Scale bars: (A–D) 5 mm, (E–H) 1 mm, and (I–M) 50 μm. Color images available online at www.liebertonline.com/ten.

FIG. 2.

Cartilage and bone markers present at culture day 21. Representative constructs cultured in (A, C, E, G) chondrogenic and (B, D, F, H) osteogenic medium showing (A, B) total collagen, (C, D) collagen type II, (E, F) glycosaminoglycan, and (G, H) mineral. Stains were (A, B) Masson's trichrome, (C, D) collagen-type II (green) and DAPI (blue), (E, F) safranin-O/fast green, and (G, H) alizarin red. Scale bars: (A, B, E–H) 50 μm and (C, D) 500 μm. Color images available online at www.liebertonline.com/ten.

FIG. 3.

Cartilage and bone markers present at culture day 45. Representative constructs cultured in (A, C, E, G, H) chondrogenic and (B, D, F, I, J) osteogenic medium showing (A, B) total collagen, (C, D) collagen type II, (E, F) glycosaminoglycan, and (G–J) mineral. Stains were (A, B) Masson's trichrome, (C, D) collagen type II (green) and DAPI (blue), (E, F) safranin-O/fast green, and (G–J) alizarin red. Arrows (H, J) show mineralized cells. Scale bars: (A, B, E–J) 50 μm and (C, D) 500 μm. Color images available online at www.liebertonline.com/ten.

FIG. 4.

Circles indicate regions of interest for comparing Figs. 4C,D,E. Spatial localization of mineralization in the chondrogenic group at culture day 45. (A) Full histological cross section stained with alizarin red. Arrow points to the new tissue that overgrew the confines of the poly(ɛ-caprolactone) scaffold, (B) microcomputerized tomography image looking down onto the construct, (C–E) magnified views of the mineralized region shown as the inset box in (A) in which serial histological sections were stained with (C) alizarin red (D) safranin-O/fast green and (E) Masson's trichrome. Circles indicate regions of interest for comparing C, D, E. Scale bars: (A) 1 mm (B) 2 mm (C–E) 300 μm. Color images available online at www.liebertonline.com/ten.