Highly Organized Porous Gelatin-Based Scaffold by Microfluidic 3D-Foaming Technology and Dynamic Culture for Cartilage Tissue Engineering

Abstract

A gelatin-based hydrogel scaffold with highly uniform pore size and biocompatibility was fabricated for cartilage tissue engineering using microfluidic 3D-foaming technology. Mainly, bubbles with different diameters, such as 100 μm and 160 μm, were produced by introducing an optimized nitrogen gas and gelatin solution at an optimized flow rate, and N₂/gelatin bubbles were formed. Furthermore, a cross-linking agent (1-ethyl-3-(3-dimethyl aminopropyl)-carbodiimide, EDC) was employed for the cross-linking reaction of the gelatin-based hydrogel scaffold with uniform bubbles, and then the interface between the close cells were broken by degassing. The pore uniformity of the gelatin-based hydrogel scaffolds was confirmed by use of a bright field microscope, conjugate focus microscope and scanning electron microscope. The in vitro degradation rate, mechanical properties, and swelling rate of gelatin-based hydrogel scaffolds with highly uniform pore size were studied. Rabbit knee cartilage was cultured, and its extracellular matrix content was analyzed. Histological analysis and immunofluorescence staining were employed to confirm the activity of the rabbit knee chondrocytes. The chondrocytes were seeded into the resulting 3D porous gelatin-based hydrogel scaffolds. The growth conditions of the chondrocyte culture on the resulting 3D porous gelatin-based hydrogel scaffolds were evaluated by MTT analysis, live/dead cell activity analysis, and extracellular matrix content analysis. Additionally, a dynamic culture of cartilage tissue was performed, and the expression of cartilage-specific proteins within the culture time was studied by immunofluorescence staining analysis. The gelatin-based hydrogel scaffold encouraged chondrocyte proliferation, promoting the expression of collagen type II, aggrecan, and sox9 while retaining the structural stability and durability of the cartilage after dynamic compression and promoting cartilage repair.

Keywords: cartilage tissue engineering; dynamic culture; gelatin; microfluidics.

Figure 1

Schematic illustrations for the fabrication process of highly organized EDC-crosslinked porous gelatin-based hydrogel scaffolds, (A) producing N₂/gelatin bubbles by using parallel microfluidic channels, (B) collecting N₂/gelatin bubbles, (C) collected N₂/gelatin bubbles before crosslinking reaction, (D) a crosslinked gelatin-based hydrogel scaffold with uniform close cells, and (E) a designed gelatin-based hydrogel scaffold with uniform open cells.

Figure 2

Microscope results of EDC-crosslinked gelatin-based hydrogel scaffolds having (A) pores with diameter of 160 μm and (B) pores with diameter of 100 μm, scanning electron microscope of (C) pores with diameter of 160 μm and (D) pores with diameter of 100 μm, and conjugate focus fluorescence microscopy of (E) pores with diameter of 160 μm and (F) pores with diameter of 100 μm.

Figure 3

Schematic representing the possible interactions within gelatin molecules, formation of hydrogen bonds during swelling and amide bonds via (A) physical and (B) chemical crosslinking behaviors, in which a crosslinking agent of EDC would be used (C).

Figure 4

(A) Degradation rate, (B) swelling ratio, and (C) tensile strength and elongation at break of EDC-crosslinked gelatin-based hydrogel scaffolds with different pore sizes (160 and 100 μm). Results were expressed as the means ± SEM (n = 3).

Figure 5

O.D. images of (A) culture of rabbit knee articular cartilage fragment (chondrocyte) and (B) primary chondrocytes (P0) (40 x) and immunofluorescence staining results of chondrocytes, green-specific protein, (C) type II collagen, (D) MMP13, (E) aggrecan, and (F) sox9; blue-nucleus and red-cytoskeleton.

Figure 6

Live/dead cytotoxicity viability assay of chondrocytes on the gelatin-based hydrogel scaffold after (A) 1, (B) 4, and (C) 7 days (Scale bar = 300 μm) and (D) MTT assay results of chondrocytes on the gelatin-based hydrogel scaffold. Green: live cells, red: dead cells, orange or yellow: overlap of live and dead cells. Values with different superscripts were significantly different (p < 0.05); *, statistical significance between the groups (p < 0.05); +, nonsignificance within the group (p > 0.05).

Figure 7

Total collagen/DNA of static culturing chondrocytes on EDC-crosslinked gelatin-based hydrogel scaffolds. Values with different superscripts were significantly different (p < 0.05); *, statistical significance between the groups (p < 0.05); +, nonsignificance within the group (p > 0.05).

Figure 8

GAG/DNA of static culturing chondrocytes on gelatin-based hydrogel scaffolds. Values with different superscripts were significantly different (p < 0.05); *, statistical significance between the groups (p < 0.05); +, nonsignificance within the group (p > 0.05); n = 6.

Figure 9

Scanning electron microscope analysis of static culturing chondrocytes on EDC-crosslinked gelatin-based hydrogel scaffolds, (A) after 7 days (100 μm) (surface, scale bar = 120 μm), (B) after 7 days (160 μm) (surface, scale bar = 120 μm), (C) after 14 days (100 μm) (cross section, scale bar = 60 μm), and (D) after 14 days (160 μm) (cross section, scale bar = 60 μm).

Figure 10

SEM morphology of chondrocyte grown on EDC-crosslinked gelatin-based hydrogel scaffold after dynamic culture, wherein (A) surface (scale bar = 200 μm) and (B) cross section (scale bar = 50 μm) after 7 days, (C) surface (scale bar = 2 00 μm) and (D) cross section (scale bar = 50 μm) for 14 days, and (E) surface (scale bar = 200 μm) and (F) cross section (scale bar = 50 μm) for 21 days.

Figure 11

(A) SEM morphology of chondrocyte grown on EDC-crosslinked gelatin-based hydrogel scaffold after dynamic culture for 28 days(cross section) (scale bar = 200 μm), (B) Schematic drawing of chondrocyte growth on EDC-crosslinked gelatin-based hydrogel scaffold after dynamic culture (cross section), and (C) Schematic drawing of chondrocyte growth on EDC-crosslinked gelatin-based hydrogel scaffold after static culture (cross section).

Figure 12

(A) DNA content of dynamic culturing chondrocytes on gelatin-based hydrogel scaffolds, (B) total collagen/DNA of dynamic culturing chondrocytes on gelatin-based hydrogel scaffolds, and (C) GAG/DNA of dynamic culturing chondrocytes on gelatin-based hydrogel scaffolds. Values with different superscripts were significantly different (p < 0.05); *, statistical significance between the groups (p < 0.05); +, nonsignificance within the group (p > 0.05); n = 6.

Figure 13

Immunofluorescence staining of expression from chondrocytes grown on EDC-crosslinked gelatin-based hydrogel scaffold in dynamic culture system for collagen type II (A) after 21 days and (B) after 28 days (blue-Nuclei and green-collagen type II); for MMP13 (C) after 21 days and (D) after 28 days (blue-nucleus and green-MMP13); sox9 (E) after 21 days and (F) after 28 days (blue-nucleus and green-sox9); and aggrecan (G) after 21 days and (H) after 28 days(blue-nucleus and green-aggrecan) (scale bar, 30 μm).

Figure 14

Images of hematoxylin and eosin staining showing growth of chondrocytes on gelatin-based hydrogel scaffolds after (A) 7 days, (B) 14 days, (C) 21 days, and (D) 28 days of dynamic culture. Dark blue-nucleus, light red-cytoplasm, intercellular substance, red-hydrogel scaffold. Scale bar represents 200 μm. .

Figure 15

Images of alcian blue staining showing growth of chondrocytes on gelatin-based hydrogel scaffolds after (A)7 days, (B) 14 days, (C) 21 days, and (D) 28 days of dynamic culture. Blue-glycosaminoglycan, red-nucleus, dark red-hydrogel scaffold. Scale bar represents 200 μm.