Mixed Composition Microribbon Hydrogels Induce Rapid and Synergistic Cartilage Regeneration by Mesenchymal Stem Cells in 3D via Paracrine Signaling Exchange

Abstract

Hydrogels are widely used matrices for mesenchymal stem cell (MSC)-based cartilage regeneration but often result in slow cartilage deposition with inferior mechanical strength. We recently reported a gelatin-based microribbon (μRB) scaffold, which contains macroporosity and substantially enhances the speed of cartilage formation by MSCs in 3D. However, our previous method cannot be used to fabricate different polymers into μRBs, and the effects of varying μRB compositions on MSC cartilage regeneration in 3D remain unknown. Here, we report a method that allows fabricating different polymers [gelatin, chondroitin sulfate, hyaluronic acid, and polyethylene glycol (PEG)] into μRB structures, which can be mixed in any ratio and cross-linked into 3D scaffolds in a modular manner. Mixing glycosaminoglycan μRBs with gelatin or PEG μRBs induced great synergy, resulting in fast cartilage deposition. After only 3 weeks of culture, leading mixed μRB composition reached high compressive strength on par with native cartilage. Such synergy can be recapitulated via exchange of soluble factors secreted by MSCs seeded in different μRB compositions in a dose-dependent manner. Tuning the ratio of mixed μRB compositions allowed further optimization of the quantity and speed of cartilage regeneration by MSCs. Together, our results validate mixed μRB compositions as a novel biomaterial tool for inducing synergy and accelerating MSC-based cartilage regeneration with biomimetic mechanical properties through paracrine signal exchange.

Keywords: cartilage; hydrogels; scaffolds; stem cells; tissue engineering.

Figure 1.

Fabrication and characterization of μRB-based hydrogels. (A) Schematic for fabricating μRBs from a variety of natural and synthetic polymers. (B) SEM images of cross-linked μRB-based hydrogels, demonstrating macroporosity. (C) Live/dead staining on day 1 of MSC encapsulation. (D) Cyclic compression testing. (E) Cyclic shear testing. Scale bars: (B) 250 and (C) 100 μm.

Figure 2.

Quantitative analysis of individual composition μRB scaffolds after 21 days in chondrogenic medium. (A) Fold change in proliferation from day 1. (B) sGAG and (C) collagen content in acellular and cellular scaffolds. (D) Compressive stiffness of gels. Data are mean ± SD for n = 3 samples. *p < 0.05; ^**p < 0.001.

Figure 3.

Histologic staining of constructs to visualize distribution of cartilage ECM deposition after 21 days in chondrogenic medium. (A) Safranin-O staining to visualize sGAG and (B) Masson’s trichrome staining to visualize collagen. Immunostaining of (C) collagen II, articular cartilage marker, (D) collagen X, hypertrophic cartilage marker, and (E) collagen I, fibrocartilage marker. Scale bars: (A–B) 200 and (C–E) 100 μm.

Figure 4.

Quantitative analysis of mixed composition μRB scaffolds after 21 days in chondrogenic medium. (A) Fold change in proliferation from day 1. (B) sGAG and (C) collagen content in acellular and cellular scaffolds. (D) Compressive stiffness of gels. Data are mean ± SD for n = 3 samples. *p < 0.05; ^**p < 0.001.

Figure 5.

Interaction indices for (A) proliferation, (B) sGAG, (C) collagen, and (D) compressive moduli quantifying synergy in the mixed μRB constructs. Data are mean ± SD for n = 3 samples. _*p < 0.05; ^**p < 0.001.

Figure 6.

Histologic staining of mixed μRB constructs after 21 days in chondrogenic medium to visualize distribution of (A) sGAG and (B) collagen. Immunostaining of (C) collagen II, articular cartilage marker, (D) collagen X, hypertrophic cartilage marker, and (E) collagen I, fibrocartilage marker. Scale bars: (A,B) 200 and (C–E) 100 μm. n = 2 samples sliced and imaged for histology.

Figure 7.

(A) Schematic of conditioned media experiment. Safranin-O staining after 21 days in chondrogenic medium of MSC-laden (B) CS and (C) PEG scaffolds cultured with conditioned media from MSC-laden PEG and CS scaffolds, respectively. 1× indicates concentrated conditioned media from 1 scaffold; 10× indicates concentrated conditioned media from 10 scaffolds. Scale bars: 1 mm. n = 2 samples sliced and imaged for histology.

Figure 8.

(A) Neocartilage production qualification (A–E) and quantitation (F–H) of MSCs in mixed PEG–CS μRB scaffolds with varying ratios after 21 days in chondrogenic medium. (A) Trichrome staining, (B) Safranin-O staining, (C) collagen II immunohistochemistry, (D) collagen X immunohistochemistry, and (E) collagen I immunohistochemistry. Biochemical analyses for (F) fold change of proliferation, (G) sGAG production, and (H) collagen production. (I) Compressive moduli of constructs. Scale bars: (A,B) 200 μm and (C–E) 100 μm. Data are mean ± SD for n = 3 samples. *p < 0.05; ^**p < 0.001 compared to 100P:0C group. n = 2 samples sliced and imaged for histology.