Engineering Inflammation-Resistant Cartilage: Bridging Gene Therapy and Tissue Engineering

Abstract

Articular cartilage defects caused by traumatic injury rarely heal spontaneously and predispose into post-traumatic osteoarthritis. In the current autologous cell-based treatments the regenerative process is often hampered by the poor regenerative capacity of adult cells and the inflammatory state of the injured joint. The lack of ideal treatment options for cartilage injuries motivated the authors to tissue engineer a cartilage tissue which would be more resistant to inflammation. A clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 knockout of TGF-β-activated kinase 1 (TAK1) gene in polydactyly chondrocytes provides multivalent protection against the signals that activate the pro-inflammatory and catabolic NF-κB pathway. The TAK1-KO chondrocytes encapsulate into a hyaluronan hydrogel deposit copious cartilage extracellular matrix proteins and facilitate integration onto native cartilage, even under proinflammatory conditions. Furthermore, when implanted in vivo, compared to WT fewer pro-inflammatory M1 macrophages invade the cartilage, likely due to the lower levels of cytokines secreted by the TAK1-KO polydactyly chondrocytes. The engineered cartilage thus represents a new paradigm-shift for the creation of more potent and functional tissues for use in regenerative medicine.

Keywords: cartilage tissue engineering; gene editing; inflammation.

Figure 1

Overview of the inflammation pathways in cartilage injuries and schematic of the methodology used. A) Illustration of the NF‐κB pathway in the injured joint. Activation of the pathway by IL‐1β, TNFα, and LPS or DAMPS leads to phosphorylation of NF‐κB inhibitory protein (IKKα/β) and NF‐κB translocation to the nucleus, that activates the transcription of proinflammatory genes as IL‐1β and IL‐6 and matrix remodeling enzymes as MMP‐13 and ADAMTS‐5. B) Schematic of our methodology. Chondrocytes are collected by corrective surgeries; TAK1 KO is generated by a single‐step electroporation and can be then encapsulated into a hydrogel for future cartilage defect treatments.

Figure 2

Characterization of the KO by sequencing and protein levels reveals high editing efficiency and low residual levels of TAK1 protein. A) Sanger sequencing of WT and KO samples from 3 different donors, with percentage of off‐frame sequences per total number of alleles (n = 3). B) Sanger sequencing of KO populations for three passages after electroporation from 3 different donors (n = 3). C) Western Blot and D) quantification of TAK1 in WT and KO cells of 3 donors, normalized to GAPDH protein levels. F) Percentages of editing at the 8 most probable off‐target sites by Sanger sequencing (n = 3). Error bars represent SD. OT: off‐target.

Figure 3

NF‐κB levels and activity are reduced in TAK1‐KO cells. A) qPCR‐based gene expression levels of NF‐κB in WT, oxozeaenol‐treated WT (WToxo), and KO cells treated with inflammatory stimuli for 16 h (n = 9 from 3 donors). B) qPCR‐based gene expression levels of TLR4 in adult chondrocytes derived from joint injury reconstruction (jiCh) and infant chondrocytes (iCh) treated with LPS (n = 3). C) Western Blot and D) quantification of Ser‐536 phosphorylated NF‐κB protein in 30 min‐treatment cells compared to GAPDH levels (n = 3 from 3 donors). E) Immunofluorescence of NF‐κB to detect its translocation to the nuclei after 30 min stimulation with inflammatory signals (yellow arrows indicate the positively stained nuclei) and F) percentage of the nuclei that stained strongly (n = 9 from 3 donors). Error bars represent SD. ***p < 0.001, ****p < 0.0001.

Figure 4

Expression of inflammatory genes is lower in TAK1‐KO cells after induction of inflammation. A) qPCR analysis of indicated genes of WT, oxozeaenol‐treated WT (WToxo), and KO cells (n = 9 from 3 donors). B) Diffused IL‐6, TNFα, and MMP13 protein quantification (n = 9 from 3 donors). C) Hierarchical clustering of inflammation‐related genes of WT and KO cells treated with IL‐β; differential gene expression analysis and volcano plot of D) WT and E) KO cells treated with IL‐1β by nSolver (n = 3). Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 5

Cartilage ECM deposition is not impeded in TAK1‐KO cells and is enhanced in highly inflamed environment. A) Safranin O (GAGs), collagen 2 and 1 immunostaining of WT and KO pellets and B) quantification of the staining intensity (paired t test, n = 9 from 3 donors). Scale bar, 500 µm. C) Safranin O (GAGs), collagen 2 and 1 immunostaining of WT, oxozeaenol‐treated WT and KO pellets cultured with inflammatory stimuli in the last week of culture and D) quantification of the intensity (two‐way ANOVA, n = 9 from 3 donors). Scale bar, 200 µm. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 6

TAK1‐KO cells produce high quality ECM when embedded in a hyaluronan‐based hydrogel in inflamed conditions. A) Schematic of the experiment. Rings were formed from bovine cartilage and filled with hydrogel and cells, then cultured for 6 weeks in the presence of indicated inflammatory stimuli before proceeding with the analysis. B) Bern score to assess the quality of the samples maturation (two‐way ANOVA, n = 9 from 3 donors). C,E,G) Histology of the samples by Safranin O, collagen 2 and 1 immunostaining and D,F,H), Quantification of the staining intensity (two‐way ANOVA, n = 9 from 3 donors). Bovine cartilage ring dimensions: outer diameter, 8 mm, inner diameter 4 mm; hydrogel diameter: 4 mm, 1 mm thick. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Figure 7

TAK1‐KO cells‐derived neocartilage strongly integrates into mature cartilage and presents high stiffness in inflamed environment. A) Compressive modulus of the formed neocartilage after 6 weeks in culture with or without indicated inflammatory stimuli, normalized by samples’ diameter (two‐way ANOVA, n = 6 from 2 donors). B) Close‐up on the interface between native bovine cartilage (left) and newly formed cartilage (right) from WT or KO cells. Scale bar, 50 µm. C) Representation of the pushout test for integration assessment and D) pushout test representing the bond strength of the samples to bovine cartilage (two‐way ANOVA, n = 9 from 3 donors). Bovine cartilage ring dimensions: outer diameter, 8 mm, inner diameter 4 mm; hydrogel diameter: 4 mm, 1 mm thick. *p < 0.05, ***p < 0.001, ****p < 0.0001.

Figure 8

TAK1‐KO cells‐derived neocartilage develops into stiff matrix in vivo and recruits less M1 macrophages than WT‐derived one. A) Schematic of the experiment. WT and KO cells were encapsulated, samples were cultured for 3 weeks before subcutaneous implantation into nude rats and explanted or cultured in vitro after further 6 weeks. Sample diameter and thickness were 4 and 1 mm, respectively. C = compression, H = histology. B,C) Compressive modulus of in vivo (B) and in vitro (C) WT and KO samples at 3 and 9 weeks (two‐way ANOVA, 3 donors, n = 6). D) Gene expression analysis of main chemokines by multiplexing of WT and KO cells ± IL1β (n = 3). E) Immunofluorescence of macrophages infiltration in in vivo samples. External part of the construct above the dotted line, construct below. The pro‐inflammatory M1 macrophages were identified with double positivity for CD68 (pan‐macrophage marker) and CD80 (M1 marker). Red: CD68, green: CD80, grey: Hoechst. Orange arrows pointing to double positive cells for CD68 and CD80. Scale bar, 50 µm. F) CD68–CD80 double positive cells per region of interest (ROI) (Unpaired t test, 3 donors, n = 12). ns: non‐significant. Error bars represent SD. *p < 0.05, **p < 0.01, ***p < 0.001.