Tissue adhesive, ROS scavenging and injectable PRP-based 'plasticine' for promoting cartilage repair

Abstract

Platelet-rich plasma (PRP) that has various growth factors has been used clinically in cartilage repair. However, the short residence time and release time at the injury site limit its therapeutic effect. The present study fabricated a granular hydrogel that was assembled from gelatin microspheres and tannic acid through their abundant hydrogen bonding. Gelatin microspheres with the gelatin concentration of 10 wt% and the diameter distribution of 1-10 μm were used to assemble by tannic acid to form the granular hydrogel, which exhibited elasticity under low shear strain, but flowability under higher shear strain. The viscosity decreased with the increase in shear rate. Meanwhile, the granular hydrogel exhibited self-healing feature during rheology test. Thus, granular hydrogel carrying PRP not only exhibited well-performed injectability but also performed like a 'plasticine' that possessed good plasticity. The granular hydrogel showed tissue adhesion ability and reactive oxygen species scavenging ability. Granular hydrogel carrying PRP transplanted to full-thickness articular cartilage defects could integrate well with native cartilage, resulting in newly formed cartilage articular fully filled in defects and well-integrated with the native cartilage and subchondral bone. The unique features of the present granular hydrogel, including injectability, plasticity, porous structure, tissue adhesion and reactive oxygen species scavenging provided an ideal PRP carrier toward cartilage tissue engineering.

Keywords: PRP; cartilage regeneration; gelatin; granular hydrogel; tannic acid.

Graphical Abstract

Figure 1.

Gel/TA granular hydrogel carrying PRP for cartilage regeneration.

Figure 2.

Gelatin microspheres preparation. (A) Schematic diagram of gel crosslinking. (B) Maximum failure stress of bulk gelatin hydrogels with different gelatin concentration in compression test. (C) Maximum failure strain of bulk gelatin hydrogels with different gelatin concentration in compression test. (D) The SEM image microsphere (bar scale = 5 μm). (E) The wet microspheres observed by phase-contrast microscope (bar scale = 10 μm). (F) Swelling behavior of the microspheres. (G) The diameter statistics.

Figure 3.

Preparation of gel/TA granular hydrogel. (A) General observation of the granular hydrogel. (B) General observation of the gel/TA granular hydrogel carrying PRP. (C) SEM image of the granular hydrogel (bar scale: 5 μm). (D) SEM image of the granular hydrogel carrying PRP (bar scale: 5 μm). (E) General observation of the plasticine-like behavior of the granular hydrogel carrying PRP. (F) FTIR spectrogram of TA and gel/TA to illustrate their hydrogen bonding. (G) The recording of the storage modulus (G′) and loss modulus (G″) in the scanning range of 0.01–10 Hz (the strain was 0.1%, 20°C).

Figure 4.

Rheology and injectable properties. (A) Oscillatory strain scans of granular hydrogels (0.01–100%, 1 Hz). (B) Viscosity changes under shear. (C–F) Cyclic strain diagrams of gel/TA and gel/TA-PRP granular hydrogels. (G) Extrusion of granular hydrogel with TA concentration of 10 wt%. (H) SEM images of extruded filaments with different TA concentration. (I) Stability of gel/TA-PRP granular hydrogel immersed in PBS at 7 and 14 days, as well as the stability of extruded gel/TA-PRP filament immersed in PBS for 14 days. (J) Accumulative release of TGF-β in vitro.

Figure 5.

Adhesive strength and ROS scavenging ability of granular hydrogels. (A) General observation to show the strong adhesion of the granular hydrogel. (B) Adhesive strength of the granular hydrogels and FibrinGlue (**P < 0.01, *P < 0.05). (C) Fluorescence pictures and the statistical data of HUVECs cells co-cultured with gel/TA granular hydrogels with different TA concentration, with the presence of reactive oxygen stimulant (rosup), (bar scale: 50 μm, *P < 0.01, n = 3). (D) Fibroblasts co-cultured with hydrogels subjected to live/dead staining (bar scale = 50 μm). (E) OD value to show fibroblasts proliferation in vitro (*P < 0.05). (F) OD value to show chondrocytes proliferation in vitro (*P < 0.05). (G) Relative aggrecan gene expression in chondrocytes (*P < 0.05). (H) Relative COL II gene expression in chondrocytes (*P < 0.05).

Figure 6.

General observation of cartilage regeneration in vivo. (A) Articular cartilage defect before and after hydrogel implantation. (B) General observation of cartilage repair in three groups at 8 and 12 weeks post-implantation. (C) Scoring at 8 weeks. (D) Scoring at 12 weeks (****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05).

Figure 7.

Histological evaluation on samples of different groups at 8 weeks in vivo. (A) H&E staining. (B) COL II immunohistochemistry staining. (C) COL I immunohistochemistry staining. (D) Safranin O-Fast Green staining. (The scale of a 20× enlarged image was 600 μm; the scale of a 200× enlarged image was 100 μm.) (E) H&E staining, COL II immunohistochemistry staining, COL I immunohistochemistry staining, Safranin O-Fast Green staining of the normal cartilage (the scale was 100 μm).

Figure 8.

Histological evaluation on samples of different groups at 12 weeks post-implantation. (A) H&E staining. (B) COL II immunohistochemistry staining. (C) COL I immunohistochemistry staining. (D) Safranin O-Fast Green staining. (The scale of a 20× enlarged image was 600 μm; the scale of a 200× enlarged image was 100 μm.)