Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Hyperplastic Human Macromass Cartilage for Joint Regeneration.

doi: 10.1002/advs.202301833

Figure Lengend Snippet: Figure 1. The customized culture enables chondrocytes to proliferate and also express lineage markers. A) Schematic overview of the designed pro- gram for step-wise culture. B) The contribution of different combinations of elements to chondrocytes expansion (n = 3, one-way ANOVA followed by Dunnett’s multiple comparison test). C) The contribution of different additives to the optimization of expansion (n = 3, one-way ANOVA followed by Dunnett’s multiple comparison test). D) Gene expression analysis of chondrocytes markers (COL2A1, ACAN, SOX9, MATN3, FGF2, MMP13, COL1A1 and ADAMTS5) in chondrocytes cultured with/without COL6 (n = 3, unpaired two-tailed Student’s t-tests). E) Representative images of chondrocytes

Article Snippet: Cell Immunofluorescence Staining: For the cell immunofluorescence assay, cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 1% bovine serum albumiin (BSA) for 30 min. After that, cells were incubated with primary antibodies against COL2A1 (1:100, Santa Cruz, sc-52658), SOX9 (1:100, Abcam, ab76997), and COL1 (1:200, Affinity, AF7001) overnight at 4 °C.

Techniques: Comparison, Gene Expression, Cell Culture, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Hyperplastic Human Macromass Cartilage for Joint Regeneration.

doi: 10.1002/advs.202301833

Figure Lengend Snippet: Figure 2. CC-chons can spontaneously aggregate and efficiently proliferate in 3D culture. A) Schematic diagram illustrating the designed program for step-wise scaling up of micro-cartilage. B) Representative bright-field images of CC and TC microtissues over time. Scale bars: 200 μm. C) Diameter of CC and TC microtissues over time (n = 3, unpaired two-tailed Student’s t-tests). D) Frequency distribution of diameter of CC and TC microtissues on day 27 (nCC = 448, nTC = 395). E) Cumulative fold expansion of CC and TC treatments at day 6 and day 27 (n = 3, unpaired two-tailed Student’s t-tests). F) Representative immunostaining images of COL2A1 in CC and TC microtissues. Scale bars: 200 μm. All data were mean ± SEM. n.s p ≥0.05, *p < 0.05, ***p < 0.001.

Article Snippet: Cell Immunofluorescence Staining: For the cell immunofluorescence assay, cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 1% bovine serum albumiin (BSA) for 30 min. After that, cells were incubated with primary antibodies against COL2A1 (1:100, Santa Cruz, sc-52658), SOX9 (1:100, Abcam, ab76997), and COL1 (1:200, Affinity, AF7001) overnight at 4 °C.

Techniques: Two Tailed Test, Immunostaining

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Hyperplastic Human Macromass Cartilage for Joint Regeneration.

doi: 10.1002/advs.202301833

Figure Lengend Snippet: Figure 3. CC-chons can form large-size and ECM-rich macro-cartilage in 3D culture. A) Schematic diagram illustrating the designed program for step- wise pellet culture. B) Representative bright-field images of tissues by CC and TC incubation over time. Scale bar: 1 mm. C) SO/fast green staining of CC and TC tissues. Scale bar: 100 μm. D) Major axis quantification of center section in CC and TC tissues (n = 3, unpaired two-tailed Student’s t-tests). E) Quantification of cell number in the central section (n = 3, unpaired two-tailed Student’s t-tests). F) Representative images of immunofluorescent staining of chondrocyte markers (COL2A1, ACAN, and COL6) in CC and TC tissues. Scale bar: 100 μm. All data were mean ± SEM. ****p < 0.0001.

Article Snippet: Cell Immunofluorescence Staining: For the cell immunofluorescence assay, cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 1% bovine serum albumiin (BSA) for 30 min. After that, cells were incubated with primary antibodies against COL2A1 (1:100, Santa Cruz, sc-52658), SOX9 (1:100, Abcam, ab76997), and COL1 (1:200, Affinity, AF7001) overnight at 4 °C.

Techniques: Incubation, Staining, Two Tailed Test

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

Article Title: Hyperplastic Human Macromass Cartilage for Joint Regeneration.

doi: 10.1002/advs.202301833

Figure Lengend Snippet: Figure 6. CC macro-cartilage maintains lineage phenotype and promotes cartilage repair in vivo. A) Schematic diagram of macro-cartilage implantation in critical-sized cartilage defects. B) Overall repair of cartilage defects 12 weeks after implantation. C) Representative images of repaired cartilage stained by SO/fast green. Scale bar: 200 μm. D) ICRS-II scoring for histological assessment of repaired cartilage (n = 3–6, one-way ANOVA followed by Tukey’s multiple comparison test). E) Immunofluorescence staining of repaired cartilage for detection of COL2A1 and COL1. Scale bar: 200 μm. F) Immunoflu- orescence staining of implanted chondrocyte for detection of human LAMIN in repaired cartilage. Scale bar: 200 μm. G) Quantification of chondrocytes derived from implanted macro-cartilage in repaired cartilage (n = 3, unpaired two-tailed Student’s t-tests). All data were mean ± SEM. n.s p ≥0.05, *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001.

Article Snippet: Cell Immunofluorescence Staining: For the cell immunofluorescence assay, cells were fixed in 4% PFA, permeabilized with 0.3% Triton X-100 for 15 min, and blocked with 1% bovine serum albumiin (BSA) for 30 min. After that, cells were incubated with primary antibodies against COL2A1 (1:100, Santa Cruz, sc-52658), SOX9 (1:100, Abcam, ab76997), and COL1 (1:200, Affinity, AF7001) overnight at 4 °C.

Techniques: In Vivo, Staining, Comparison, Derivative Assay, Two Tailed Test

Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 5. (A) H&E staining. (B) Gene expression levels of ACAN, COL2A1 and SOX9. (C) Western blot detect the protein expression of COL2A1. (D) Semi- quantitation of gray level in (C). (E) Immunofluorescence staining for COL2A1. (F) Semi-quantitation of fluorescence intensity in (E) (scale bars: 170 lm. mean± SD, n ¼ 3; P 0.05, P 0.01, , ###P 0.001. represents the comparison between the experimental group and the control group, and # represents the comparison between the experimental groups).

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Staining, Gene Expression, Western Blot, Expressing, Quantitation Assay, Immunofluorescence, Fluorescence, Comparison, Control

Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 8. Immunohistochemical staining for COL1A1 (A) and COL2A1 (B) in repaired cartilage after 4 and 8 weeks post-surgery (scale bars: 200 and100 lm).

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Immunohistochemical staining, Staining

Journal: Regenerative biomaterials

Article Title: Role of integrin β1 and tenascin C mediate TGF-SMAD2/3 signaling in chondrogenic differentiation of BMSCs induced by type I collagen hydrogel.

doi: 10.1093/rb/rbae017

Figure Lengend Snippet: Figure 9. Collagen hydrogel acts on ITGB1, followed by activating the ITGB1, then induces phosphorylation of SMAD2/3, the p-SMAD2/3 enters into the nucleus and binds to the promoter regions of TNC to initiate its transcription [58]. TNC increases the expression of ITGB1 and further induces activation of ITGB1 and SMAD2/3. p-SMAD2/3 promotes the stability and accumulation of SOX9 protein and forms a transcriptional complex with SOX9, promoting the expression of cartilage-related genes, like COL2A1, etc.

Article Snippet: After blocking with normal goat serum, primary antibodies against TNC (1:100; Proteintech, Wuhan, China), ITGB1 (1:100; Proteintech, Wuhan, China), COL2A1 (1:100; Proteintech, Wuhan, China), COL1A1 (1:100; Proteintech, Wuhan, China), p-SMAD2/3 (1:100; Cell Signaling, USA) and SMAD2/3 (1:100; Cell Signaling, USA) were added and incubated overnight at 4�C.

Techniques: Phospho-proteomics, Expressing, Activation Assay

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 4 | CHIR-99021 restores cartilage injury mitigated by DVL1 silencing. Mice stably administered sh-DVL1 were further treated with the Wnt/β-catenin agonist CHIR-99021 via intra-articular injection. (A) Protein levels of β-catenin in cells determined using WB analysis. (B) Cartilage morphology in the mouse knee joints determined using Safranin O/fast green staining. (C) Protein levels of COL10A1 and COL2A1 in mouse chon- drocytes determined using WB analysis. (D) Positive TRAP staining in the mouse knee joints; in vitro, chondrocytes with stable DVL1 knockdown were treated with 5 μM CHIR-99021 for 24 h. (E) Apoptosis in the extracted chondrocytes determined using TUNEL assay. (F) Protein levels of pro- cleaved-caspase-3 in the extracted chondrocytes determined using WB analysis. (G) Protein levels of expression of MMP13 and SOX9 in the mouse chondrocytes determined using WB analysis. For animal experiments, each group contained five mice. For cell experiments, three biological repli- cates were performed. Differences were compared by the unpaired t-test (A–G).

Article Snippet: The membranes were blocked at room temperature for 1 h in Tris- buffered saline (T5912, Merck) and incubated with antibodies against DVL1 (1:1000, 13- 706, ProSci), COL10A1 (collagen type X alpha 1 chain) (1:1000, A11645, ABclonal Technology Co. Ltd., Wuhan, Hubei, China), MMP13 (matrix metallopeptidase 13) (1:4000, ab39012, Abcam), SOX9 (SRY- box transcription factor 9) (1:1000, orb1258160, Biorbyt LLC, San Francisco, California, United States), cleavedcaspase- 3 (1:1000, PA5- 114687, Thermo Fisher Scientific), βcatenin (1:5000, ab32572, Abcam), COL2A1 (collagen type II alpha 1 chain) (1:1000, orb1259434, Biorbyt), SP2 (1:1000, PA5- 103254, Thermo Fisher Scientific), and GAPDH (1:2500, ab9485, Abcam) overnight at 4°C.

Techniques: Stable Transfection, Injection, Staining, In Vitro, Knockdown, TUNEL Assay, Expressing

Journal: The journal of gene medicine

Article Title: Sp2 Transcription Factor Alleviates Chondrocyte Loss in Osteoarthritis by Repressing the DVL1-Dependent Wnt/β-Catenin Signaling Pathway.

doi: 10.1002/jgm.70021

Figure Lengend Snippet: FIGURE 7 | DVL1 overexpression increases chondrocyte loss suppressed by SP2. Chondrocytes were administered lentiviral vectors encapsu- lating OE-NC/OE-SP2 or the additional OE-NC/OE-DVL1. (A) mRNA expression of DVL1 in cells determined using RT-qPCR. (B) Apoptosis in cells determined using TUNEL assay. (C) Protein levels of β-catenin in chondrocytes determined using WB analysis. (D) Transcriptional activity of β-catenin in chondrocytes analyzed by TOP/FOPFlash assays. (E) Protein levels of SOX9 and COL2A1 in the chondrocytes determined using WB analysis. Three biological replicates were performed. Differences were compared by unpaired t-test (A) or ANOVA (B–E).

Article Snippet: The membranes were blocked at room temperature for 1 h in Tris- buffered saline (T5912, Merck) and incubated with antibodies against DVL1 (1:1000, 13- 706, ProSci), COL10A1 (collagen type X alpha 1 chain) (1:1000, A11645, ABclonal Technology Co. Ltd., Wuhan, Hubei, China), MMP13 (matrix metallopeptidase 13) (1:4000, ab39012, Abcam), SOX9 (SRY- box transcription factor 9) (1:1000, orb1258160, Biorbyt LLC, San Francisco, California, United States), cleavedcaspase- 3 (1:1000, PA5- 114687, Thermo Fisher Scientific), βcatenin (1:5000, ab32572, Abcam), COL2A1 (collagen type II alpha 1 chain) (1:1000, orb1259434, Biorbyt), SP2 (1:1000, PA5- 103254, Thermo Fisher Scientific), and GAPDH (1:2500, ab9485, Abcam) overnight at 4°C.

Techniques: Over Expression, Expressing, Quantitative RT-PCR, TUNEL Assay, Activity Assay

Journal: PLOS ONE

Article Title: Low-frequency whole-body vibration can enhance cartilage degradation with slight changes in subchondral bone in mice with knee osteoarthritis and does not have any morphologic effect on normal joints

doi: 10.1371/journal.pone.0270074

Figure Lengend Snippet: Oligonucleotide primers for RT-PCR.

Article Snippet: Immunohistochemistry staining for Collagen II (Col2a1) (1:100, bs-10589R; Bioss, Beijing, China), Aggrecan (Acan) (1:200, bs-11655R; Bioss), MMP3(1:50, Ab52915; Abcam, Cambridge, UK), MMP13 (1:200, Ab39012; Abcam), IL6 (1:100, ab290735; Abcam) and TNFα(1:1000, ab307164; Abcam) were conducted on paraffin sections following appropriate antigen retrieval methods.

Techniques:

Journal: PLOS ONE

Article Title: Low-frequency whole-body vibration can enhance cartilage degradation with slight changes in subchondral bone in mice with knee osteoarthritis and does not have any morphologic effect on normal joints

doi: 10.1371/journal.pone.0270074

Figure Lengend Snippet: Compared to the baseline group with sham DMM, the group that underwent DMM surgery exhibited an obvious increase in Acan, Col2a1, MMP3 and MMP13. The mouse model of OA eight weeks after surgery showed a further increase in MMP3 and MMP13, while Acan and Col2a1 showed a decrease, compared with those of the baseline OA group. Obvious increases in TNFα and IL6 were also presented in the mouse model of eight weeks after surgery compared to those in the matched group four weeks after DMM surgery. All the genes detected had a significant decrease in the group with OA and WBV compared with the non-vibrated group. (n = 6). Data are expressed as the mean ± SD.** P <0.01.

Article Snippet: Immunohistochemistry staining for Collagen II (Col2a1) (1:100, bs-10589R; Bioss, Beijing, China), Aggrecan (Acan) (1:200, bs-11655R; Bioss), MMP3(1:50, Ab52915; Abcam, Cambridge, UK), MMP13 (1:200, Ab39012; Abcam), IL6 (1:100, ab290735; Abcam) and TNFα(1:1000, ab307164; Abcam) were conducted on paraffin sections following appropriate antigen retrieval methods.

Techniques:

Journal: PLOS ONE

Article Title: Low-frequency whole-body vibration can enhance cartilage degradation with slight changes in subchondral bone in mice with knee osteoarthritis and does not have any morphologic effect on normal joints

doi: 10.1371/journal.pone.0270074

Figure Lengend Snippet: Compared to the baseline group with sham DMM, the group that underwent DMM surgery exhibited an obvious decrease in Acan and Col2a1, and increase in MMP3, MMP13, IL6 and TNFα. The mouse model of OA eight weeks after surgery showed a further increase in MMP3, MMP13, and an obivious decrease in Acan, Col2a1, compared with those of the baseline OA group. All the proteins detected had a significant decrease in the group DMM+WBV compared with the DMM+NON-WBV group. (n = 6).

Article Snippet: Immunohistochemistry staining for Collagen II (Col2a1) (1:100, bs-10589R; Bioss, Beijing, China), Aggrecan (Acan) (1:200, bs-11655R; Bioss), MMP3(1:50, Ab52915; Abcam, Cambridge, UK), MMP13 (1:200, Ab39012; Abcam), IL6 (1:100, ab290735; Abcam) and TNFα(1:1000, ab307164; Abcam) were conducted on paraffin sections following appropriate antigen retrieval methods.

Techniques:

Journal: Cardiovascular Research

Article Title: Regulation of Ptbp1-controlled alternative splicing of pyruvate kinase muscle by liver kinase B1 governs vascular smooth muscle cell plasticity in vivo

doi: 10.1093/cvr/cvae187

Figure Lengend Snippet: Histological analysis of the aortas from WT and Lkb1 SMiKO mice. ( A ) H&E, elastin, and Masson trichrome stains of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.0–1.5 months post-TAM induction. Scale bars: 200 µm. ( B ) H&E staining and immunofluorescence staining (α-actin) of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.0–1.5 months post-TAM induction. Scale bars: 20 and 50 µm, respectively. ( C ) Quantification of the medial wall thickness of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.5 months post-TAM induction. Data represent mean ± SEM ( n = 6 for WT mice and n = 8 for Lkb1 SMiKO mice). ( D ) Quantification of the internal and external elastic laminae (IEL and EEL, respectively) perimeters of the thoracic aortas from WT and Lkb1 SMiKO mice at 1.5 and 4.5 months post-TAM induction. Data represent mean ± SEM ( n = 6 for WT mice and n = 8 for Lkb1 SMiKO mice). ( E ) H&E, elastin, and Masson stains of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. ( F ) Alcian Blue, Safranin O, and Von Kossa staining of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. ( G and H ) Immunofluorescence staining for α-actin ( G ), SM22α ( G ), and Col2a1 ( H ) of the thoracic aortas from WT and Lkb1 SMiKO mice at 4.5 months post-TAM induction ( n = 6 mice per genotype). Where indicated, nuclei were counterstained with DAPI. Dashed lines denote the IEL and EEL of the aortas. Scale bars: 200 µm. Boxes indicate fields shown at higher magnification in images to the immediate right. P -values were determined using Student’s t -test ( C and D ). For all panels, * P < 0.05; ** P < 0.01.

Article Snippet: Briefly, OCT-embedded sections were washed with phosphate-buffered saline (PBS), fixed with acetone at 4°C for 15 min, and permeabilized with 0.2% Triton X-100 for 10 min. After blocking with goat serum (protein block) for 30 min, the sections were incubated with primary antibodies against α-actin (Abcam, Cat #ab7817, 1:200), SM22α (Abcam, Cat #ab14106, 1:200), Col2A1 (Boster, Cat #PA2141-1, 1:200), and Sox9 (Millipore, Cat #ABE2868, 1:500) overnight.

Techniques: Staining, Immunofluorescence