Figures and data
CRISPR/Cas9 editing of mesenchymal cell lines lead to efficient VEGF knockout in cartilage tissues.
A) Experimental scheme depicting the generation of CRISPR/Cas9-edited MSOD-B lines, and the subsequent in vitro and in vivo tissue formation assessment. B) Overview of the native human VEGF coding sequence composed of eight exons. Designed gRNAs and their targeted binding sites are illustrated, as well as the corresponding expected impact on the coding sequence. gRNAs targeting exon 1 (orange) disrupt translation initiation and inhibit protein expression, gRNA targeting exon 2 (blue) disrupts VEGF receptor binding, and gRNA targeting exon 8 (green) alters the C-Terminal sequence and repress activation of protein. C) ELISA-based quantitative analysis of VEGF protein content in cell culture supernatant from expanded single cell colonies. From all clones, only two had no detectable level of VEGF (1.3_3 and 1.3_6). These clones were subsequently defined as MSOD-BΔV1 and MSOD-BΔV2. D) Histological assessment of living and lyophilized in vitro differentiated constructs using Safranin O staining (Scale bars = 100 µm and 20 µm for magnified areas). Both the MSOD-B and MSOD-BΔV1 displayed glycosaminoglycans (GAG) (orange to reddish in Safranin O), indicating successful cartilage formation (Scale bars = 200 µm). Left bottom inserts show the whole tissue section. E) Quantitative assessment of the total GAG content in MSOD-B and MSOD-BΔV1 in vitro differentiated constructs, post-lyophilization. Unpaired t-test, n = 10 biological replicates, ***p < 0.001. F) ELISA-based quantitative assessment of VEGF protein in in vitro differentiated constructs, post-lyophilization. Unpaired t-test, n = 3-4 biological replicates, ****p < 0.0001. G) Immunofluorescence images of MSOD-B and MSOD-BΔV1 tissues, post-lyophilization. Displayed images consist of 3D-stacks from 80-100 µm thick sections, stained for VEGF (yellow), Collagen Type X (COLX, red) and Collagen Type I (COL1, grey). A clear reduction in the VEGF signal could be observed in the MSOD-BΔV1 tissues, indicating a successful VEGF knockdown (Scale bars = 80 µm). Panel A was created using BioRender.com.
VEGF knockout cartilage tissues retain bone remodeling capacity despite reduced early-stage vascularization
A) Experimental scheme of the Chorioallantoic Membrane (CAM) assay, for ex vivo evaluation of angiogenic potential. B) Macroscopic comparison of in vitro MSOD-B and MSOD-BΔV1 constructs at day 0 (day of implantation) and day 4 (4 days post-implantation), illustrating robust de novo vessel formation perfusing the tissues (Scale bars at 1 mm). C) Quantitative Analysis of Vascular Density in MSOD-B and MSOD-BΔV1. Vascular densities were quantified from macroscopic images obtained on day 4 using ImageJ. Unpaired t-test, n = 3-4 biological replicates, n.s. = not significant. D) overview of the subcutaneous implantation procedure in mice and subsequent in vivo evaluation of vascularization from 2 to 6 weeks post-implantation. E) Immunofluorescence images of MSOD-B and MSOD-BΔV1 tissues two weeks post-in vivo implantation. Displayed images consist of 3D-stacks from 80-100 µm thick sections, vessels stained with mouse CD31 (red) and nuclei with DAPI (cyan) (Scale bars at 500 µm except for magnified white inserts at 80 µm). Box “a” and “c” display the periphery whereas Box “b” and “d” show the central region of MSOD-B and MSOD-BΔV1 constructs respectively. A reduction in tissue vascularization is observed in MSOD-BΔV1 samples. F) Quantitative analysis of the CD31 signal using an isosurface-based strategy (IMARIS software). Unpaired t-test, n = 3-4 biological replicates, *p < 0.05. G) Representative macroscopic and microtomography images of in vivo constructs retrieved at two and six weeks post-implantation. H) Microtomography-based quantification of the sample’s bone/ mineralized volume over their total volume (ratio). No significant differences between MSOD-B and MSOD-BΔV1 could be observed. (BV: Bone Volume, TV: Total Volume). Ordinary One-way ANOVA, n=8 biological replicates, n.s.=not significant I) Histological analysis of in vivo tissues using Safranin O and Masson’s trichrome stains, at two (2W) and six weeks (6W) post-implantation. Both sample types underwent full remodeling into a bone organ after 6 weeks, with presence of bone structures and a bone marrow compartment (Scale bars = 200 µm). CB – cortical bone; BM – bone marrow. TB – trabecular bone.
RUNX2 knockout does not prevent chondrogenic differentiation but impairs hypertrophy
A) Overview of the human RUNX2 coding sequence comprising 8 exons. gRNAs and their corresponding expected protein structure. The gRNA targeting exon 2 (orange) disrupts the DNA binding domain, gRNA targeting exon 5 (violet) disrupts nuclear translocation, gRNAs targeting exon 6 (blue) disrupt the transcriptional activation domain and gRNAs targeting exon 8 (green) disrupt the nuclear matrix targeting signal and repress the protein function. B) Intracellular flow cytometry for RUNX2 detection in MSOD-B and RUNX2-edited clones. A clear protein reduction could be observed in the 6.1_1 and 6.1_23 clones. C) Western blot analysis of RUNX2 in cultured MSOD-B and RUNX2-edited cells. The genetic editing of RUNX2 is confirmed by the detection of the truncated proteins. Actin is used as a control to normalize the protein content. D) Histological analysis of living and lyophilized in vitro differentiated constructs using Safranin O staining (Scale bars = 100 µm and 20 µm for magnified areas), indicating the presence of cartilage matrices. E) Quantitative assessment of the total GAG content in corresponding in vitro generated lyophilized tissues. Unpaired t-test, n=10-11 biological replicates **p < 0.01. F) Immunofluorescence images of MSOD-B and MSOD-BΔR1 lyophilized tissues. Displayed images consist of 3D-stacks from 80-100 µm thick sections, stained for DAPI (blue), Collagen Type II (COL2, yellow) and Collagen Type X (COLX, red). A clear reduction in the COLX signal could be observed in the MSOD-BΔR1 tissues, indicating impaired hypertrophy. (Scale bars = 500 µm) G) Isosurface-based quantification of the COL2 immunofluorescent signal using the IMARIS software. No significant difference between groups confirm the retention of cartilage formation in RUNX2-edited constructs. Unpaired t-test, n=3 biological replicates, n.s.=not significant. H) Isosurface-based quantification of the COLX immunofluorescent signal using the IMARIS software, confirming the disruption of hypertrophy in the MSOD-BΔR1 constructs. Unpaired t-test, n=3, ***p < 0.001. I) Alizarin Red staining evidencing a lack of mineralization in the MSOD-BΔR1 culture compared to the MSOD-B. J) Quantitative PCR analysis displaying the relative expression levels of osteogenesis-related genes: RUNX2, COL1, and ALPL. The expression is normalized to GAPDH as housekeeping gene. n.d= not detected.
RUNX2 knockout in cartilage tissues disrupts effective ectopic bone formation
A) Representative macroscopic and microtomography images of in vivo constructs retrieved at two and six weeks post-implantation. B) Microtomography-based quantification of the sample’s bone/mineralized volume over their total volume (ratio). (BV: Bone Volume, TV: Total Volume). A marked difference is observed as early as two weeks, with a clear lower mineral content in MSOD-BΔR1 samples. Ordinary one way ANOVA, n=3 biological replicates, *p < 0.05, ***p < 0.001. C) Histological analysis of in vivo constructs using Safranin O and Masson’s trichrome stains. After two weeks (2W), a higher bone formation is already evident in the MSOD-B control group. The MSOD-BΔR1 samples explanted after 6 weeks (6W) displayed presence of cortical and trabecular bone, but also large amount of fibrous tissue indicating an incomplete remodeling. (Scale bars = 200 µm).
RUNX2 knockout in cartilage tissues leads to better cartilage regeneration and maintenance in osteochondral defect in rats
A) Experimental scheme for the regenerative potential assessment of MSOD-B & MSOD-BΔR1 cartilage tissues in a rat osteochondral defect. B) Histological analysis of the osteochondral defects for each group using H&E and Masson’s trichrome stains, at three weeks post-implantation. The dash-line marks the defect area. (Scale bars = 500 µm) C) Histological analysis the osteochondral defects using TRAP staining reporting osteoclastic activity, at three weeks post-implantation. The dash-line marks the defect area. (Scale bars = 500 µm and 100 µm for magnified areas) D) Microtomography-based quantification of the sample’s total bone/mineralized volume normalized to the healthy control in percentage. Unpaired t-test, n=3 biological replicates, n.s.=not significant. E) Image J based quantification of Trabecular separation (Tb.Sp). One way ANOVA test, n=3 biological replicates, *p < 0.05 F) Histological analysis of the osteochondral defects using Safranin O staining. After three weeks, a higher regeneration of the surface cartilage is evident in the MSOD-BΔR1 group (a,b,c) (Scale bars = 500 µm and 100 µm for magnified areas). The magnified regions of the subchondral area (d,e,f) show higher cartilage remnants and integration in the MSOD-BΔR1 group. G) Quantitative analysis of cartilage regeneration in the osteochondral surface as compare to the healthy control (100%). Unpaired t-test, n=3 biological replicates, *p < 0.05. Panel A was created using BioRender.com.
Scoring rate for individual categories
Semi-quantitative analysis utilizing a modified histological grading system, including parameters such as cellular morphology, matrix staining, surface regularity, thickness of cartilage, subchondral bone formation and integration of adjacent cartilage, resulting in a comprehensive assessment of tissue regeneration and integration
CRISPR/Cas9 editing of mesenchymal cell lines lead to efficient VEGF knockout in cartilage tissues.
A) Implemented genetic elements of the Mesenchymal Sword Of Damocles BMP-2 (MSOD-B) cells consisting of the human telomerase reverse transcriptase (hTERT), an inducible caspase 9 (iCaspase) death system and the bone morphogenetic protein type-2. All systems are constitutively expressed and further described in Pigeot et al. Advanced Materials 2021. B) Overview of the human VEGF exon structure, nucleotide sequences of targeting gRNAs and their corresponding amino acid sequences.
VEGF knockout cartilage tissues retain bone remodeling capacity despite reduced early-stage vascularization.
A) Sequential processing of a vascular tissue section using Q-VAT, depicting the original image, binary mask creation, and vessel segmentation for quantitative vascular density analysis. B) Microtomography analysis (Bone/mineralized volume) showed significant differences between in vivo constructs at both two weeks and six weeks time points (One way ANOVA, n=3, **p < 0.01, n.s.= not significant). C) Microtomography analysis (total volume) did not show significant difference between in vivo constructs at either two weeks or six weeks time points (One way ANOVA, n=3, n.s.= not significant) (BV: Bone Volume, TV: Total Volume).
RUNX2 knockout does not prevent chondrogenic differentiation but impairs hypertrophy
A) Overview of human RUNX2 exon structure, nucleotide sequences of targeting gRNAs and its corresponding amino acid sequence. B) Intracellular flow cytometry for RUNX2 detection in MSOD-B and RUNX2-edited clones. Clones whose peaks fell left of the dotted line were selected for sequencing. C) Sanger sequencing results of DNA extracted from MSOD-BΔR samples and the modifications compared to MSOD-B DNA sequence. D) Histological analysis of in vitro constructs from using Safranin O and Masson trichrome staining displayed the presence of glycosaminoglycans (GAG) and collagen content respectively, indicating the presence of cartilage formation in MSOD-BΔR2 (Scale bars = 200 µm). E) Quantitative assessment of the total GAG content confirms the presence of cartilage formation in both constructs thus confirming RUNX2 knockout in hMSCs (MSOD-BΔR2) retain cartilage formation. (unpaired t-test, n=3, n.s. = not significant). F) Reduction of RUNX2 expression in cartilage tissues lead to reduced hypertrophy. Representative immunofluorescence staining images of COL2, COLX and DAPI tissue section of in vitro cartilage constructs shows reduction of COLX expression in knockout construct (MSOD-BΔR2) indicating reduction of hypertrophy. (Scale bars = 500 µm). G) Quantitative analysis of the immunofluorescent staining sections using IMARIS software displayed significant difference in the expression of COL2, demonstrating reduction of cartilage formation in knockout constructs (MSOD-BΔR2) (unpaired t-test, n=3, *p < 0.05). H) Quantitative analysis of the immunofluorescent staining sections using IMARIS software displayed a significant reduction in expression of COLX, confirming the disruption of hypertrophy in knockout constructs (unpaired t-test, n=3, **p < 0.01). I) Representative isosurface images of immunofluorescent staining sections constructed using IMARIS software.
RUNX2 knockout in cartilage tissues disrupts effective bone formation.
A) Microtomography analysis (Bone/mineralized volume) showed very high significant difference between in vivo constructs at both two weeks and six weeks time points (One was ANOVA, n=3, ***p < 0.001, ****p < 0.0001). B) Microtomography analysis (total volume) showed significant difference between in vivo constructs at two weeks time point but no significant difference at six weeks time point (One was ANOVA, n=3, n.s. = not significant) (BV: Bone Volume, TV: Total Volume).
Characterization of engineered cartilage tissues post-lyophilization.
A) Quantitative assessment of the total GAG content in MSOD-B in vitro differentiated constructs across three different batch productions. One way ANOVA test, n=6-7 biological replicates, n.s. = not significant. B) Quantitative assessment of the total GAG content in MSOD-BΔV1 in vitro differentiated constructs across three different batch productions. One way ANOVA test, n=3-4 biological replicates, n.s.=not significant. C) Quantitative assessment of the total GAG content in MSOD-BΔR1 in vitro differentiated constructs across three different batch productions. One way ANOVA test, n=3 biological replicates, ***p < 0.001, ****p < 0.0001, n.s =not significant. D) Quantitative assessment of cartilage tissue volume in MSOD-B, MSOD-BΔV1 and MSOD-BΔR1 tissues. One way ANOVA test, n=3 biological replicates, *p < 0.05, n.s.=not significant.
RUNX2 knockout in cartilage tissues exhibit better cartilage regeneration and maintenance in osteochondral defect in rats
A) Quantitative assessment of the total GAG content in MSOD-B and MSOD-BΔR1 in vitro differentiated constructs before (living) and after (Decellularized) the decellularization process. unpaired t-test, n=12 biological replicates, ***p < 0.001, n.s. = not significant B) DNA quantification of in vitro engineered cartilage grafts before (living) and after (Decellularized) the decellularization process. unpaired t-test, n=12 biological replicates, ****p < 0.0001. C) Macroscopic images osteochondral defect model in rats depicting in of empty defect, defect filled with MSOD-B and defect filled with MSOD-BΔR1 respectively. D) Macroscopic images of extracted rat knees depicting healthy control without any defect, defect filled with MSOD-B and defect filled with MSOD-BΔR1 respectively and microtomography images of same respective constructs. E). No significant difference in BV/TV is observed between MSOD-B & MSOD-BΔR1 samples despite significantly reduced BV/TV ratio in both samples compared to healthy control. One way ANOVA test, n=3 biological replicates, **p < 0.01. F) Image J based quantification of Trabecular thickness (tb.Th). One way ANOVA test, n=3 biological replicates, n.s = not significant.
Histological grading system
The histological graded system is modified and adapted from previously established model as mentioned in materials and methods section. This modified scale incorporates six distinct parameters to comprehensively evaluate the quality of tissue repair
