recombinant sema3a  (R&D Systems)

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Elevated expression of neural factors of human and mice knee cartilage during OA progression. a Left , orthotopic view of 3D reconstruction CT of normal and OA knee joints in humans and mice. Right , Kellgren–Lawrence (KL) analysis of the joint space between normal control ( n = 10 patients, n = 8 mice) and OA ( n = 10 patients, n = 8 mice) groups. A two-tailed Mann–Whitney U test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. b Left , H&E staining of cartilage in normal control and OA groups. Scale bar, 200 µm (human), 200 µm (mouse). Middle, Safranin O staining of the cartilage in normal control and OA groups. Scale bar, 200 µm(human), 100 µm(mouse). Right , OARSI scores from normal control ( n = 10 individuals, n = 8 mice) and OA ( n = 10 individuals, n = 8 mice) groups. A two-tailed Mann–Whitney U test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. c Immunofluorescence staining of type X collagen (Col X) in the normal control ( n = 10 individuals, n = 8 mice) and OA ( n = 10 individuals, n = 8 mice) groups. Quantitative analysis of the cells of positive Col X cells per cartilage was performed among groups. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 50 µm. d Immunofluorescence staining of Tuj1 in the normal control ( n = 10 individuals, n = 8 mice) and OA ( n = 10 individuals, n = 8 mice) groups. Scale bar, 50 µm. Quantitative analysis of the area of positive Tuj1 per cartilage area (mm 2 ) was performed among groups. High magnification of Tuj1 staining in knee cartilage was shown in respective groups (arrowhead). A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 25 µm. e Immunofluorescence staining of Tau in the normal control ( n = 8 individuals, n = 8 mice) and OA ( n = 8 individuals, n = 8 mice) groups. Scale bar, 50 µm. Quantitative analysis of the area of positive Tau per cartilage area (mm 2 ) was performed among groups. High magnification of Tau staining in knee cartilage was shown in respective groups (arrowhead). A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. Scale bar, 25 µm. f Immunofluorescence staining of Semaphorin 3A (Sema3A) in the normal control ( n = 10 individuals, n = 8 mice), early OA ( n = 10 individuals, n = 8 4-weeks OA mice), late OA ( n = 10 individuals, n = 8 12-weeks OA mice) groups. Quantitative analysis of the number of positive Sema3a cells in cartilage was performed among groups. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. OA osteoarthritis. OARSI score Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Expressing, Control, Two Tailed Test, MANN-WHITNEY, Staining, Immunofluorescence

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Conditional knockout of Sema3A in chondrocytes before and after ACLT surgery display different knee phenotypes. a Left , orthotopic view of 3D reconstruction CT images of the Sema3A knockout before ACLT surgery (Sema3A KO Pre), Sema3A knockout after ACLT surgery (Sema3A KO Post) and control ACLT groups. Right , Kellgren–Lawrence analysis of the joint space in Sema3A KO Pre ( n = 8), Sema3A KO Post ( n = 8), and ACLT ( n = 8) groups. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative X-ray image from five independent experiments is shown. b Top , H&E staining of the cartilage in Sema3A KO Pre, Sema3A KO Post, and ACLT groups. Scale bar, 100 µm. Bottom , Safranin O staining of cartilage in Sema3A KO Pre, Sema3A KO Post, and ACLT groups. Scale bar, 50 µm. Right , The OARSI score of the Sema3A KO Pre ( n = 8), Sema3A KO Post ( n = 8), and ACLT ( n = 8) groups. Safranin O staining of proteoglycan (red) and bone (blue) in sagittal sections of the distal femur compartment. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative image of each group from five independent experiments is shown. c Immunofluorescence staining of Collagen X in Sema3A KO Pre ( n = 8), Sema3A KO Post ( n = 8), ACLT ( n = 8) groups and the quantitative analysis of the cells of Collagen X positive cells per cartilage. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. d Immunofluorescence staining of Tuj1 in Sema3A KO Pre ( n = 6), Sema3A KO Post ( n = 6), ACLT ( n = 6) groups and the quantitative analysis of the area of Tuj1 positive area per cartilage area (mm 2 ). Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. e Immunofluorescence staining of Sema3A in Sema3A KO Pre ( n = 6), Sema3A KO Post ( n = 6), ACLT ( n = 6) groups and the quantitative analysis of the number of semaphorin-positive cells per cartilage. Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. f Immunofluorescence staining of F-actin in Sema3A KO Pre ( n = 6), Sema3A KO Post ( n = 6), ACLT ( n = 6) groups and the quantitative analysis of the area of F-actin + per cartilage area (mm 2 ). Scale bar, 50 µm. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. A representative result from five independent experiments is shown. g Quantitative analysis of the latency to lick a hind paw following mice from different groups (Sema3A KO Pre ( n = 8), Sema3A KO Post ( n = 8), ACLT ( n = 8)) being placed on a 54 °C plate. A two-tailed unpaired student’s t-test was used for statistical analysis. Mean with SD. ACLT, anterior cruciate ligament transection. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Knock-Out, Control, Two Tailed Test, Staining, Immunofluorescence

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Bidirectional regulation of Sema3A expression in chondrocytes regulates both nerve ingrowth and chondrocyte hypertrophy. a Schematic program of the generation of chondrosphere and dorsal root ganglion (DRG) neurosphere. Sema3A was knocked down in the chondrocytes by siRNA-Sema3A. b Schematic program of the generation of chondrosphere and DRG neurosphere. Sema3A was overexpressed in the chondrosphere by lentivirus CMV-Sema3A-GFP. c The co-culture of chondrospheres and neurospheres, and morphological changes of neuritis growth in the normal control group ( left ) with normal Sema3A expression in the chondrosphere, Sema3A KD group ( middle ) with decreased Sema3A expression in chondrosphere; Sema3A OX group ( right ) with overexpression of sema3A in chondrosphere. Scale bar, 500 µm. d Immunofluorescence staining of Tuji1 in the normal control group with control chondrosphere ( left ), Sema3A KD group with Sema3A knockdown in chondrosphere ( middle ), sema3A OX group with Sema3A overexpressed in chondrosphere ( right ). Scale bar, 100 µm. High magnification of Tuji1 staining in chondrosphere was shown in respective groups (bottom panel, arrowhead). Scale bar, 50 µm. e Quantitative analysis of the intensity of Tuj1 per chondrosphere area (mm 2 ). A two-tailed student’s t-test test was used for statistical analysis. Mean with SD. f Immunofluorescence staining of type X collagen (Col X) in control chondrocyte spheres ( left ), Sema3A knockdown chondrocyte spheres ( middle ) and Sema3A overexpressed chondrocyte spheres ( right ). Scale bar, 50 µm. g Immunofluorescence staining of Runx2 in control chondrosphere ( left ), Sema3a knockdown chondrosphere ( middle ), and Sema3a overexpressed chondrosphere ( right ). Scale bar, 50 µm. h Expression of Col10a1 and Runx2 genes in normal control ( n = 6), sema3A KD ( n = 9), and sema3A OX ( n = 5) groups, A two-tailed student’s t-test was used for statistical analysis. Mean with SD. i Expression of genes involved in the Semaphorin 3A pathway was tested by quantitative RT–PCR ( n = 5 for Ctrl, n = 5 for the knockdown, n = 5 for overexpression). A two-tailed student’s t-test was used for statistical analyses. All data are reported as the mean ± SD

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Expressing, Co-Culture Assay, Control, Over Expression, Immunofluorescence, Staining, Knockdown, Two Tailed Test, Quantitative RT-PCR

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Local administration of Sema3A agents in the articular cartilage arrest degeneration in ACLT mice. a , b 3D reconstruction of micro-CT and Kellgren–Lawrence analysis of the joint space in OA ( n = 10), Sema3A protein ( n = 10), Sema3A overexpressed virus (OS virus) ( n = 10), Anti-Sema3A antibody ( n = 10), Sema3A low-expressed virus (DS virus) ( n = 10) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. c , e , f H&E staining, Safranin O staining, and OARSI scores of the OA ( n = 10), Sema3A protein ( n = 10), OS virus ( n = 10), Anti-Sema3A antibody ( n = 10), DS virus ( n = 10) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 100 µm. A representative result from five independent experiments is shown. d , g Immunofluorescence staining of Collagen X in OA ( n = 8), Anti-Sema3A antibody ( n = 8), Sema3A protein ( n = 8), OS virus ( n = 8), Sema3A DS virus ( n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. h , k Immunofluorescence staining of Tau in OA ( n = 8), Anti-Sema3A antibody ( n = 8), Sema3A protein ( n = 8), OS virus ( n = 8), DS virus ( n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. i , l Immunofluorescence staining of Sema3A in OA ( n = 8), Anti-Sema3A antibody ( n = 8), Sema3A protein ( n = 8), OS virus ( n = 8), DS virus ( n = 8) mice groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. j , m Immunofluorescence staining of F-actin in OA ( n = 8), Anti-Sema3A antibody ( n = 8), Sema3A protein ( n = 8), OS virus ( n = 8), DS virus ( n = 8) mice groups. Statistical significance was determined by multifactorial. ANOVA. All data are reported as the mean ± SD. Scale bar, 50 µm. A representative result from five independent experiments is shown. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Micro-CT, Virus, Staining, Immunofluorescence

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Local administration of Sema3A agents in the articular cartilage arrests the knee joint degeneration in natural OA Rhesus macaques. a , c Representative X-ray images and Kellgren–Lawrence analysis of the joint space in hyaluronate acid (HA) ( n = 8), Sema3A protein ( n = 8), Sema3A overexpressed virus (OS Virus) ( n = 8) Rhesus macaques joint groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. b , d OARSI scores for the HA ( n = 8), Sema3A protein ( n = 8), OS Virus ( n = 8) Rhesus macaques groups. Statistical significance was determined by multifactorial ANOVA. All data are reported as the mean ± SD. A representative result from three independent experiments is shown. Scale bar, 50 µm. e – l Immunofluorescence staining of Col X ( e , i ), Tau ( f , j ), Sema3A ( g , k ), and F-actin ( h , l ) in HA group ( n = 8), Sema3A protein group ( n = 8), OS Virus group ( n = 8) and Rhesus macaques group. All data are reported as the mean ± SD. A representative result from three independent experiments was shown. e , f , h Scale bar, 50 µm. g Scale bar, 25 µm. OARSI score, Osteoarthritis Research Society International scoring system

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Virus, Immunofluorescence, Staining

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Summary of OA patients treated with platelet-rich serum containing sema3A and hyaluronic acid

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Clinical Proteomics

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: The platelet-rich plasma (PRP) containing Sema3A injection rescues knee cartilage during the normal degenerative process in OA patients. a Orthotopic (AP) and lateral (LAT) view of X-ray images of the knee at pre-injection and post-injection (Hidden in the picture is the patient’s private information). b Kellgren–Lawrence analysis of the joint space between control ( n = 10 individuals) and experiment ( n = 10 individuals) groups. Analysis of Variance (ANOVA) with LSD Post Hoc test was used for statistical analysis. The PRP treatment (Pre-treatment: KL, 2.00; 95% confidence interval [CI], 1.50–2.50. Post-treatment: KL, 2.00; 95% CI, 1.50–2.50), HA control group (Pre-treatment: KL, 1.90; 95% CI, 1.50–2.30. Post-treatment: KL, 1.90; 95% CI, 1.50–2.30). c Quantitative T2-weighted MRI scan of the knee cartilage, red parts show soft tissue in the knee joint. d Quantitative analysis of the ROI in T2 value between control ( n = 10 individuals) and experiment ( n = 10 individuals) groups. The PRP treatment group (Pre-treatment: Quantitative MRI ROI, 122.610; 95% CI, 75.509–183.659. Post-treatment: Quantitative MRI ROI, 145.490; 95% CI, 92.289–210.238). The HA control group (Pre-treatment: Quantitative MRI ROI, 63.680; 95% CI, 52.701–76.570. Post-treatment: Quantitative MRI ROI, 80.030; 95% CI, 64.964–98.677). Analysis of Variance (ANOVA) with LSD Post Hoc test was used for statistical analysis. A representative result from nine independent experiments is shown. e – h Gait analysis of the control group ( n = 10 individuals) and experimental group ( n = 10 individuals). e The walking speed of the PRP treatment group (Pre-treatment: Speed, 0.917; 95% CI, 0.850–0.980. Post-treatment: Speed, 1.089; 95% CI, 1.001–1.182) and HA control group (Pre-treatment: Speed, 0.984; 95% CI, 0.846–1.098. Post-treatment: Speed, 1.015; 95% CI, 0.943–1.088). f The Moment max of HA control group (Pre-treatment: Moment max, 0.303; 95% CI, 0.212–0.404. Post-treatment: Moment max, 0.329; 95% CI, 0.235–0.436) a little bit higher than the PRP treatment group (Pre-treatment: Moment max, 0.312; 95% CI, 0.223–0.427. Post-treatment: Moment max, 0.333; 95% CI, 0.272–0.397). g , h The Gait analysis of the PRP treatment group and HA control group. i Pain analysis between control ( n = 10 individuals) and experimental ( n = 10 individuals) groups. PRP treatment group (Pre-treatment: VAS score, 30.60; 95% CI, 21.11–40.40. Post-treatment: VAS score, 12.95; 95% CI, 9.45–17.05) had a higher reduction of pain than the HA control group (Pre-treatment: VAS score, 28.50; 95% CI, 21.20–36.40. Post-treatment: VAS score, 13.90; 95% CI, 11.00–17.40). Analysis of Variance (ANOVA) with LSD Post Hoc test was used for statistical analysis. All data are reported as the mean ± SD

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Clinical Proteomics, Injection, Control

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Neuronal guidance factor Sema3A plays a dual-functional role in the degeneration of knee cartilage. Schematic summarizing the mechanism of Sema3A exerting a dual-functional role in the knee degeneration process of mouse and Rhesus macaque OA models, and in patients with OA. Sema3A expression in chondrocytes regulates both nerve ingrowth and cartilage hypertrophy. (Created in BioRender)

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Functional Assay, Expressing

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Sema3A and Cre primer sequences

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques:

Journal: Bone Research

Article Title: Neuronal guidance factor Sema3A inhibits neurite ingrowth and prevents chondrocyte hypertrophy in the degeneration of knee cartilage in mice, monkeys and humans

doi: 10.1038/s41413-024-00382-0

Figure Lengend Snippet: Primer names (marker genes) and sequences

Article Snippet: These animals received knee injection under anesthesia following approval from the Ethical Review Board of West China Hospital Sichuan University (No. 2018147A) and were divided into three groups: intra-articular injection of Hyaluronate acid (mean Mol. wt: 0.8~1.2 million, ARTZ @ Dispo, JP), Sema3A protein (R&D SYSTEMS, 2150-S3) and lentivirus Rlv-CMV-sema3a-Efla-EGFP (BrainVTA, Wuhan, China) ( n = 12, 4 monkeys in each group, 2 knees tested per monkey).

Techniques: Marker