Gelatin-Based Microribbon Hydrogels Accelerate Cartilage Formation by Mesenchymal Stem Cells in Three Dimensions

Abstract

Hydrogels (HGs) are attractive matrices for cell-based cartilage tissue regeneration given their injectability and ability to fill defects with irregular shapes. However, most HGs developed to date often lack cell scale macroporosity, which restrains the encapsulated cells, leading to delayed new extracellular matrix deposition restricted to pericellular regions. Furthermore, tissue-engineered cartilage using conventional HGs generally suffers from poor mechanical property and fails to restore the load-bearing property of articular cartilage. The goal of this study was to evaluate the potential of macroporous gelatin-based microribbon (μRB) HGs as novel 3D matrices for accelerating chondrogenesis and new cartilage formation by human mesenchymal stem cells (MSCs) in 3D with improved mechanical properties. Unlike conventional HGs, these μRB HGs are inherently macroporous and exhibit cartilage-mimicking shock-absorbing mechanical property. After 21 days of culture, MSC-seeded μRB scaffolds exhibit a 20-fold increase in compressive modulus to 225 kPa, a range that is approaching the level of native cartilage. In contrast, HGs only resulted in a modest increase in compressive modulus of 65 kPa. Compared with conventional HGs, macroporous μRB scaffolds significantly increased the total amount of neocartilage produced by MSCs in 3D, with improved interconnectivity and mechanical strength. Altogether, these results validate gelatin-based μRBs as promising scaffolds for enhancing and accelerating MSC-based cartilage regeneration and may be used to enhance cartilage regeneration using other cell types as well.

Keywords: cartilage; gelatin; hydrogels; macroporous; mesenchymal stem cells; three-dimensional.

FIG. 1.

Gelatin μRB scaffolds displayed improved macroporosity compared with conventional HGs while supporting MSC viability after encapsulation. (A, B) Scanning electron microscopy showed highly interconnected macroporosity in μRB scaffolds, whereas pores were more restricted and smaller in HG scaffolds. (C, D) LIVE/DEAD staining showed comparable high cell viability in both μRB and gelatin HG scaffolds. Only μRB scaffolds supported rapid cell spreading. Green: live cells; red: dead cells; Scale bars: 100 μm (A, B), 200 μm (C, D). μRB, microribbon; HG, hydrogel; MSC, mesenchymal stem cell.

FIG. 2.

μRB scaffolds, but not HGs, led to accelerated increases in compressive moduli of MSC-based neocartilage formation after 21 days of culture in chondrogenic medium in vitro. (A) MSC-seeded μRBs or HG samples showed increased opacity over time, suggesting matrix deposition by MSCs in 3D. Acellular μRB scaffolds degraded and lost integrity over time while no significant change was observed in acellular HG controls. (B) Unconfined compressive testing revealed a 20-fold increase in compressive modulus in MSC-seeded μRBs, while only a three-fold increase was observed in MSC-seeded HGs. The moduli of acellular μRB scaffolds decreased significantly while the compressive modulus of acellular HGs remained the same. Scale bar: 2 mm. ns, nonsignificant (p > 0.05), **p < 0.01, ****p < 0.0001.

FIG. 3.

The μRB scaffolds led to significantly enhanced MSC proliferation and cartilage matrix production compared with HGs in 3D. (A) DNA content per wet weight increased ∼3-fold in μRB scaffolds, and no significant change in DNA was observed in HGs. (B) Quantitative GAG assay showed both μRB and HG scaffolds led to significantly enhanced GAG deposition by MSCs over 21 days, with μRB scaffolds leading to ∼50% more total sGAG deposition than HG scaffolds. ns = nonsignificant (p > 0.05), **p < 0.01, ****p < 0.0001.

FIG. 4.

Macroporous μRB scaffolds, and not HGs, enabled increased amounts of new cartilage matrix deposition in a highly interconnected manner throughout the 3D scaffold at day 21. (A) Safranin-O staining depicted μRB + MSC group with enhanced interconnected GAG deposition, whereas HG + MSCs had limited GAG deposition restricted to pericellular regions. Acellular samples were stained as background controls. (B) Trichrome staining of total collagen showed a similar trend to the GAG staining. Red: GAG; blue: collagen. For both stainings, low-magnification (upper row) and high-magnification (lower row) images are provided. Scale bar in upper row: 1 mm; scale bar in lower row: 200 μm.

FIG. 5.

The μRB scaffolds promoted deposition of collagen that mimics phenotype of articular cartilage. Immunofluorescence staining indicated that collagen produced by MSCs was predominantly type II collagen, a major constituent of native cartilage. Collagen X, a hypertrophy cartilage marker, was observed in HG scaffolds, but not in the μRB group. Minimal collagen I deposition was observed in both μRB and HG samples, suggesting the absence of an undesirable fibrocartilage phenotype. Scale bar: 400 μm.