Repair of full-thickness articular cartilage defects using IEIK13 self-assembling peptide hydrogel in a non-human primate model

Abstract

Articular cartilage is built by chondrocytes which become less active with age. This declining function of the chondrocytes, together with the avascular nature of the cartilage, impedes the spontaneous healing of chondral injuries. These lesions can progress to more serious degenerative articular conditions as in the case of osteoarthritis. As no efficient cure for cartilage lesions exist yet, cartilage tissue engineering has emerged as a promising method aiming at repairing joint defects and restoring articular function. In the present work, we investigated if a new self-assembling peptide (referred as IEIK13), combined with articular chondrocytes treated with a chondrogenic cocktail (BMP-2, insulin and T3, designated BIT) could be efficient to restore full-thickness cartilage defects induced in the femoral condyles of a non-human primate model, the cynomolgus monkey. First, in vitro molecular studies indicated that IEIK13 was efficient to support production of cartilage by monkey articular chondrocytes treated with BIT. In vivo, cartilage implant integration was monitored non-invasively by contrast-enhanced micro-computed tomography, and then by post-mortem histological analysis and immunohistochemical staining of the condyles collected 3 months post-implantation. Our results revealed that the full-thickness cartilage injuries treated with either IEIK13 implants loaded with or devoid of chondrocytes showed similar cartilage-characteristic regeneration. This pilot study demonstrates that IEIK13 can be used as a valuable scaffold to support the in vitro activity of articular chondrocytes and the repair of articular cartilage defects, when implanted alone or with chondrocytes.

Figure 1

Effect of culture conditions on the mRNA steady-state levels of specific markers, as indicated, in macaque and human chondrocytes. After extraction, chondrocytes were amplified on plastic for 14 days (D14) in the presence of FGF-2 and insulin (FI). Then, they were seeded in fibrin or IEIK13 hydrogel and cultivated for 21 days (D21) in control medium (CTRm) or in medium containing BMP-2, insulin and T3 (BIT). Data are presented as box plots (median, quartiles, extreme values). The horizontal dotted lines correspond to mRNA levels of chondrocytes 48 h after their isolation time (values reported as medians). (A,B) Relative mRNA expression of Acan. (C,D) Relative mRNA expression of Col2a1. (E,F) Relative mRNA expression of Col1a1. (G,H) Col2a1:Col1a1 mRNA ratio. The results shown were obtained with chondrocytes isolated from 6 human donors and 9 animals. (¥: significant effects vs. 48 h chondrocytes, as determined using the Mann–Whitney test; $: significant effects vs. D14 chondrocytes amplified with FI, as determined using the Kruskal–Wallis test; £: significant effects vs. D21 chondrocytes cultivated in control medium, as determined using the Wilcoxon test; ¥, $, £, p < 0.05; ¥¥, $$, ££, p < 0.01; ¥¥¥, $$$, £££, p < 0.001).

Figure 2

Representative macroscopic image (A) and histological sections (B–H) of scar tissue and native cartilage observed at day 0, before debridement of the defects. (A) The small arrows indicate the edges of the defects. The square refers to the histological section in (B). (B) Hematoxylin and eosin staining of the border of the defect. The dotted line indicates the interface zone between scar tissue and native cartilage (scale bar: 200 μm). (C–E) and (F–H) depict detailed regions of cartilage and scar tissue, respectively, with staining of GAGs and immunostaining of type I and type II collagen, as indicated (scale bar: 20 μm).

Figure 3

Macroscopic observations of the cartilage defects filled by IEIK13- and fibrin-based hydrogels at the day of implantation (D0) and 82 days after implantation (D82). The hydrogels were loaded or not with chondrocytes, as indicated. The anatomical site of implantation in the knee joint is specified and M1, M2 and M3 correspond to 3 Cynomolgus monkeys. The arrows indicate the original defect margin. The M1 and M2 defects appear globally well resurfaced whereas the central regions of M3 defects appear depressed or filled with rough tissue.

Figure 4

Non-invasive monitoring of IEIK13- and fibrin-based hydrogels implanted in M1 monkey. The hydrogels were loaded or not with cells, as indicated. The site of implantation in knee joint is specified. Examples of segmented CECT images (left and right panels) and 3D surface reconstruction (middle panel) corresponding to different stages of the study: 3 days before first surgery (baseline), 6 days before removal of the scar tissue and implantation (pre-implantation), 41 days after implantation (middle-stage) and 82 days after implantation (end-stage). The green arrows show absence of contrast agent, indicating occupancy of the corresponding regions by the implants.

Figure 5

Non-invasive monitoring of IEIK13- and fibrin-based hydrogels implanted in M2 monkey. The hydrogels were loaded or not with cells, as indicated. The site of implantation in knee joint is specified. Examples of segmented CECT images (left and right panels) and 3D surface reconstruction (middle panel) corresponding to different stages of the study: 3 days before first surgery (baseline), 6 days before removal of the scar tissue and implantation (pre-implantation), 41 days after implantation (middle-stage) and 82 days after implantation (end-stage). The red arrow shows complete filling of the defect by the contrast agent (observed as grey cloud). The yellow arrows show heterogeneous diffusion of the contrast agent. The green arrows show absence of contrast agent, indicating occupancy of the corresponding regions by the implants.

Figure 6

Non-invasive monitoring of IEIK13- and fibrin-based hydrogels implanted in M3 monkey. The hydrogels were loaded or not with cells, as indicated. The site of implantation in knee joint is specified. Examples of segmented CECT images (left and right panels) and 3D surface reconstruction (middle panel) corresponding to different stages of the study: 3 days before first surgery (baseline), 6 days before removal of the scar tissue and implantation (pre-implantation), 41 days after implantation (middle-stage) and 82 days after implantation (end-stage). The red arrows show complete filling of the defects by the contrast agent (observed as grey cloud) and the yellow arrow shows heterogeneous diffusion of the contrast agent. The green arrows show absence of contrast agent, indicating occupancy of the corresponding regions by the implants.

Figure 7

Histological characterization of the reparative tissues in femoral condyles of M1, M2 and M3 monkeys, as indicated. GAG staining and type I and type II collagen immunostaining are shown on coronal sections. The hydrogel type and the anatomical site of implantation are indicated (CL Cell-loaded, NC Native Cartilage, AC Acellular, scale bar: 2 mm). The boundaries between the implants and the surrounding cartilage are represented by dotted lines.

Figure 8

Experimental design of the animal study showing the preparation process of the cartilage constructs made of IEIK13 or fibrin gel combined to chondrocytes. The key times of the study are referred as numbers of days before or after the day of implantation (day 0).