Injectable microfluidic hydrogel microspheres based on chitosan and poly(ethylene glycol) diacrylate (PEGDA) as chondrocyte carriers

Abstract

Direct injection of chondrocytes in a minimally invasive way has been regarded as the significantly potential treatment for cartilage repair due to their ability to fill various irregular chondral defects. However, the low cell retention and survival after injection still limited their application in clinical transformation. Herein, we present chondrocyte-laden microspheres as cell carriers based on a double-network hydrogel by the combination of the chitosan and poly(ethylene glycol) diacrylate (PEGDA). The microfluidic technique was applied to prepare size-controllable chitosan/PEGDA hydrogel microspheres (CP-MSs) via the water-in-oil approach after photo-crosslinking and physical-crosslinking. The chondrocytes were laden on CP-MSs, which showed good cell viability and proliferation after long-term cell cultivation. The in vitro investigation further demonstrated that chondrocyte-laden CP-MSs were injectable and the cell viability was still high after injection. In particular, these cell-laden microspheres were self-assembled into a 3D cartilage-like scaffold by a bottom-up strategy based on cell-cell interconnectivity, which suggested that these injectable chondrocyte-laden microspheres showed potential applications as chondrocyte carriers for bottom-to-up cartilage tissue engineering.

Scheme 1. Schematic of uniform microsphere preparation based on CP double-network hydrogel via a core–shell microfluidic chip and injectable chondrocyte-laden CP-MSs scaffold fabrication via a bottom-to-up strategy.

Fig. 1. Synthetic scheme of CP double-network hydrogels. (a) CP double-network hydrogel formation after the photo-crosslinking of PEGDA (step 1) and the physical-crosslinking of chitosan (step 2). (b) Gelation process of CP double-network hydrogels after photo-crosslinking and physical-crosslinking.

Fig. 2. Characterization of the CP double-network hydrogel. (a) FITR spectra of PEGDA hydrogel, chitosan, and CP double-network hydrogel after lyophilization, respectively. (b) Storage modulus (G′) and loss modulus (G′′) of the CP double-network hydrogel in the angular frequency ranging from 0.1 to 10 rad s⁻¹ (1% strain).

Fig. 3. Monodisperse CP-MSs fabrication based on a capillary microfluidic flow-focusing chip. Photograph images of the microfluidic device from the side view (a) and the top view (b). (c) Light microscopic image of the internal composition of the microfluidic device. (d) SEM image showing the uniform spherical CP-MSs after critical point drying. (e) Morphological observation of a single CP-MS. (f) Histograms of diameter distribution of nanofibers on the microsphere surface.

Fig. 4. Monodisperse CP-MSs with different diameter preparation by regulating flow rates and flow rate ratio of oil and water phases. Fluorescence microscope images of CP-MS-1 (a), CP-MS-2 (b), CP-MS-3 (c), CP-MS-4 (d), CP-MS-5 (e), and CP-MS-6 (f) by staining with the FITC dye. Scale bar: 400 μm. (g) Statistical diagram of CP-MSs with different diameters, the X-axis is the water flow rate, the Y-axis is the oil flow rate, and the Z-axis is the diameter of the CP-MSs.

Fig. 5. Cell viability and proliferation of chondrocyte-laden CP-MSs. The top view (a–c) and 3D view (d–f) confocal images of chondrocytes cultured on CP-MS-1 (550 ± 9 μm) after cultivation for 1, 3, and 7 days. The top view (g–i) and 3D view (j–l) confocal images of chondrocytes cultured on CP-MS-4 (220 ± 16 μm) after cultivation for 1, 3, and 7 days. Live cells (green) were stained with calcium AM and dead cells (red) were stained with ethidium homodimer. White dashed lines show approximate borders of microspheres. (m) Quantification of cell viability for TCP, CP-MS-1, and CP-MS-4 groups after 1, 3, and 7 days. (n) Relative cell number of chondrocytes in TCP, CP-MS-1, and CP-MS-4 groups after culturing for 1, 3, and 7 days.

Fig. 6. Cellular morphological properties of chondrocytes cultured on CP-MSs. The top views (a and b) and 3D views (c and d) of the confocal fluorescent images of chondrocytes on CP-MS-1 (a and c) and CP-MS-4 (b and d) stained with F-actin (green) and DAPI (blue) after culturing for 3 days. (e) Statistical analysis shows the nucleus aspect ratios of TCP, CP-MS-1, and CP-MS-4 groups (**P < 0.01).

Fig. 7. Injection and self-assembly of chondrocyte-laden microspheres in vitro. (a) Schematic of the injection process of chondrocyte-laden microspheres. (b) Optical images of injection of chondrocyte-laden CP-MS-4 from a syringe into a Petri dish mold. (c) Cell viability analysis after injection and further culturing for 7 days. NS: not significant. (d–f) Fluorescent images of chondrocyte-laden microspheres after injection and further culturing for 1 (d), 3 (e), and 7 (f) days, where microspheres were stained with nile red dye (red) and chondrocytes were stained with calcium AM (green).