Journal: NPJ Regenerative Medicine

Article Title: Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis

doi: 10.1038/s41536-022-00272-1

Figure Lengend Snippet: a PD sheets were handled using a circular PVDF support membrane. b Representative images of cross-section of PD sheet. Histological staining with hematoxylin and eosin (HE), immunohistochemical staining for type I collagen (COL1), type II collagen (COL2), aggrecan (ACAN), and fibronectin (FN). scale bar; 50 μm. c – e Distribution of transplanted PD sheet characteristics of nine lots: The box represents the interquartile range of the values. The top and bottom of the whiskers represent the maximum and minimum values. The line inside the box represents the median values. Cell number (c) and cell viability (d) were determined after enzymatic digestion of a cell sheet. e Thickness of PD sheet was measured using microscopic images of cross sections. f Flow cytometric analysis of surface markers for the cells in the PD sheets. The PD sheets were digested enzymatically and analyzed by single-color staining. Data are expressed as the mean ± SD of the percentage of surface marker-expressing cells from nine lot of sheets (Supplementary Fig. 2). Cells were positive for CD81, CD90, CD44, and CD105 and negative for CD31 and CD45. Staining for CD146, CD49a, and GD2 differed between the lots. g Concentrations of humoral factors secreted by PD sheets. Each mark indicates a lot of sheets. Some marks overlap. h The gene expression profile of transplanted sheets was analyzed using qPCR for cartilage-related genes, and the results are reported relative to the expression of GAPDH . A total of nine lots of PD sheets were fabricated using cryopreserved cells from three polydactyly patient donors. Three lots were from donor 1 and were used to create sheets 1-1, 1-2, and 1–3 in h . Four lots were from donor 2 and were used to create sheets 2-1, 2-2, 2-3, and 2–4. Two lots were from donor 3 and were used to create sheets 3-1 and 3-2. Each lot was used for one patient, except for sheet 3-2, which was transplanted into two patients (patients 9 and 10). Data with the same color were from the same lot of PD sheets in g , h .

Article Snippet: Sections 20 μm thick were immunostained with anti-human COL1 (1:200; 1310-01, Southern Biotech, Birmingham, AL, USA), COL2 (1:200; F-57, Kyowa Pharma Chemical, Toyama, Japan), FN (1:1000; MA5-11981, Thermo Fisher Scientific, Waltham, MA, USA), and ACAN (1:20; 967800, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight.

Techniques: Membrane, Staining, Immunohistochemical staining, Marker, Expressing, Gene Expression

Journal: NPJ Regenerative Medicine

Article Title: Polydactyly-derived allogeneic chondrocyte cell-sheet transplantation with high tibial osteotomy as regenerative therapy for knee osteoarthritis

doi: 10.1038/s41536-022-00272-1

Figure Lengend Snippet: a KOOS was used to assess the effects on knee-related parameters. The patient averages for the KOOS subscales, symptom, pain, ADL, and QOL improved significantly from the baseline. * p < 0.05, ** p < 0.01. ADL activities of daily living, QOL quality of life. b Patient average for LKS improved significantly from the baseline. The box represents the interquartile range of the scores. The top and bottom of the whiskers represent the maximum and minimum scores, except for the data at 12 months that contains the outlier. The line inside the box represents the median score. * p < 0.05, ** p < 0.01. c – f Correlational analysis was used to select a gene marker set that was predictive of each of the outcome measures. The scatter plot shows the correlations between the gene scores and KOOS pain at 12 months ( c ), LKS at 12 months ( d ), and OARSI ( e ) and ICRS II ( f ). The gene score was calculated from the gene expression of the predictive gene marker set for KOOS pain ( PTGS2 , TGFB1 , MIA , and G0S2 ), the LKS ( COL1A2 , MATN2 , SOX9 , and TGFB1 ), and the OARSI histological score ( CCN2 , COMP , ACKR4 , ESM1 , and GREM1 ), and ICRS II overall assessment ( COL2A1 , COL27A1 , ACKR4 , and ESM1 ), as described in the Methods section (Supplementary Tables – ). The number assigned to each symbol indicates the corresponding patient. g Histological results of patient biopsies and results from clinical evaluations. C = articular cartilage, B = subchondral bone, Dashed line = boundary between articular cartilage region and subchondral bone region. Biopsies of femoral medial condyle were taken from areas of regenerated cartilage 12 months postoperatively. scale bar = 1 mm. Histological sections from all patients stained for Safranin O. Immunostaining showed the expression of type II collagen (COL2) in all patients.

Article Snippet: Sections 20 μm thick were immunostained with anti-human COL1 (1:200; 1310-01, Southern Biotech, Birmingham, AL, USA), COL2 (1:200; F-57, Kyowa Pharma Chemical, Toyama, Japan), FN (1:1000; MA5-11981, Thermo Fisher Scientific, Waltham, MA, USA), and ACAN (1:20; 967800, R&D Systems, Minneapolis, MN, USA) at 4 °C overnight.

Techniques: Marker, Gene Expression, Staining, Immunostaining, Expressing

Journal: PLoS ONE

Article Title: Endogenous PTH Deficiency Impairs Fracture Healing and Impedes the Fracture-Healing Efficacy of Exogenous PTH(1-34)

doi: 10.1371/journal.pone.0023060

Figure Lengend Snippet: Real-time RT–PCR was performed on callus extracts from V-WT and V-KO mice and PTH-WT and PTH-KO mice at 1 week and 2 weeks post-fracture for gene expression of (A, B) ALP and (C, D) type I collagen (Col I). Messenger RNA expression was calculated as a ratio relative to the GAPDH mRNA level and expressed relative to levels of V-WT mice. (E) Western blots for protein expression of Cbfa1, and insulin-like growth factor-1 (IGF-1), of callus extracts from V-WT and V-KO mice and PTH-WT and PTH-KO mice at 1 week and 2 weeks post-fracture. β-tubulin was used as loading control for Western blots. (F, G) Cbfa1 and (H, I) IGF-1 protein levels relative to β-tubulin protein level were assessed by densitometric analysis and expressed relative to levels of V-WT mice. Each value is the mean ± SEM of determinations in 6 animals from each group. *, P<0.05; **, P<0.01; ***, P<0.001 compared with WT mice of the same group. #, P<0.05; ##, P<0.01; ###, P<0.001 compared with genotype-matched V-treated mice.

Article Snippet: Immunohistochemical staining for type I collagen was performed on paraffin sections using the avidin-biotin-peroxidase complex technique with affinity-purified goat anti-human type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL, USA).

Techniques: Quantitative RT-PCR, Expressing, RNA Expression, Western Blot

Journal: PLoS ONE

Article Title: Endogenous PTH Deficiency Impairs Fracture Healing and Impedes the Fracture-Healing Efficacy of Exogenous PTH(1-34)

doi: 10.1371/journal.pone.0023060

Figure Lengend Snippet: Representative micrographs of paraffin sections of calluses from V-WT and V-KO mice and PTH-WT and PTH-KO mice at 4 weeks post-fracture stained with (A) Sirius Red for total collagen, (B) hematoxylin and eosin (HE), (C) histochemically for ALP and (D) immunohistochemically for type I collagen (Col I). (E) Total collagen positive bony callus areas, (F) osteoblast number relative to bone perimeter (N.Ob/B.Pm, #/mm), (G) ALP positive areas and (H) Col I immunopositive areas were measured by computer-assisted image analysis. Each value is the mean ± SEM of determinations in 6 animals from each group. **, P<0.01; ***, P<0.001 compared with WT mice of the same group. #, P<0.05; ##, P<0.01; ###, P<0.001 compared with genotype-matched V-treated mice.

Article Snippet: Immunohistochemical staining for type I collagen was performed on paraffin sections using the avidin-biotin-peroxidase complex technique with affinity-purified goat anti-human type I collagen antibody (Southern Biotechnology Associates, Birmingham, AL, USA).

Techniques: Staining