vitamin c 2-phospho-l-ascorbic acid trisodium salt  (Millipore)

Journal: bioRxiv

Article Title: Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

doi: 10.1101/2024.12.04.626830

Figure Lengend Snippet: (A and B) Second harmonic generation microscopy images of non-regenerating (NR) and regenerating (R) digits 14 days post-amputation (DPA), highlighting collagen I fibers. The dashed yellow line indicates the border of the second (P2) or third (P3) phalanx bone. Scale bars = 250 µm. The bottom panels show magnified views of the green boxed regions of fibrosing (A’) and blastema (A”) tissues. Scale bars = 100 µm. Fiber number was quantified in (B). n = 5 mice per condition. (C and D) Atomic force microscopy stiffness maps of the 14DPA NR wound (green arrowhead) and blastema (magenta arrowhead). The dashed blue line indicates the border of the P2 or P3 bone. Scale bars = 500 µm. Average stiffnesses were quantified in (D), which were determined by applying the Hertz model. n = 4 mice per condition. (I) (E) Schematic of the single-cell RNA sequencing experiment of 14DPA NR and R digit tissues. Two 14DPA non-regenerative datasets were original datasets generated from this paper. n = 2 replicates of 4 mice per replicate. The remaining datasets were acquired from the public domain. n = 1, 14DPA non-regenerative dataset (GEO: GSE135985); n = 3, 14DPA blastema datasets (GEO: GSE135985 and GSE143888). (F-H) Uniform Manifold Approximation and Projection (UMAP) of 20,536 Pdgfrα -expressing cells from 14DPA NR and blastema datasets. In (F), cells are color-coded according to clusters, which include four fibroblast (Fibroblast 1, 2, 3, and 4), osteochondral-lineage, osteo-lineage, cycling, and bone marrow stromal cell (BMSC) clusters. In (G), cells are color-coded by NR and blastema datasets. In (H), each cell was assigned a score based upon its expression of fibrillar collagen genes, where cells with a low score appear as yellow, and a high score dark purple. (I) Violin plots of Col1a1 and Col1a2 expression among Fibroblast 1 (Fibro 1), Fibroblast 2 (Fibro 2), and Fibroblast 3 (Fibro 3) cells. (J) Dot plot showing top differentially expressed genes for each fibroblast sub-type. The size of the circle represents the fraction of cells expressing the gene, and average gene expression is shown according to the key, in which dark blue and white represent high and low expression, respectively. Pie charts represent the relative proportion of NR (blue) and blastema (gold) cells within a specific fibroblast sub-type. (K and L) Immunofluorescence images of 14DPA NR and R digits stained for TNX (white) in (K) and THBS4 (white) in (L). The yellow dashed line indicates the border of the P2 or P3 bone. All images were counterstained with Hoechst (blue). Scale bars = 250 µm. Insets show the orange boxed regions of fibrosing (K’ and L’) and blastema (K” and L”) tissues at higher magnification. Green arrowheads indicate dim TNX and THBS4 expression in the blastema. Scale bars = 50 µm. (M) Schematic of the experimental design to test the effects of substrate stiffness on fibrotic ECM synthesis. Ascorbic acid was supplemented to induce collagen fibrillogenesis. (N and O) Immunofluorescence images of fibroblasts cultured on stiff (50 kPa; N) or soft (0.7 kPa; O) hydrogels, treated with ascorbic acid, and stained for COLI (magenta), F-actin (yellow), THBS4 (cyan), and Hoechst (blue). In (N), green arrowheads point at collagen fibers co-localizing with THBS4. In (O), orange arrowheads point at intracellular collagen. Scale bars = 50 µm. (P) Working model of the cellular and ECM composition of the wounded P2 digit after non-regenerative amputations. Bidirectional arrows indicate cell-ECM feedback. Data are shown as mean ± SEM. Statistical significance was determined by two-tailed unpaired student’s t -test. *p < 0.05, **p<0.01, ***p<0.001, ns = not significant.

Article Snippet: To induce collagen synthesis and fibrillogenesis, 25 µg/ml 2-phospho-L-ascorbic acid trisodium salt (Merck, 49752-10G) was supplemented into the medium and replenished every other day until the experimental endpoint.

Techniques: Microscopy, RNA Sequencing Assay, Generated, Expressing, Immunofluorescence, Staining, Cell Culture, Two Tailed Test

Journal: bioRxiv

Article Title: Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

doi: 10.1101/2024.12.04.626830

Figure Lengend Snippet: (A) Schematic of the experimental design testing the impact of hyaluronidase (HAse) degradation of hyaluronic acid on collagen fibrillogenesis. SHG = second harmonic generation microscopy. (B-D) Second harmonic generation (SHG) microscopy images of 14 days post-amputation (DPA) control and HAse digits highlighting collagen I fibers. The yellow dashed line indicates the border of the third phalanx (P3) bone. Scale bars = 100 µm. Fiber number (C) and orientation (D) were quantified using CT-FIRE. n = 3 mice per condition. (E) Schematic of the experimental design testing the impact of 4-MU depletion of hyaluronic acid on collagen and tissue mechanics. AFM = atomic force microscopy, SHG = second harmonic generation microscopy. (F-H) Second harmonic generation microscopy images of 14DPA control and 4-MU digits highlighting collagen I fibers. Scale bars = 100 µm. Fiber number (G) and orientation (H) were quantified using CT-FIRE. n = 3 mice per condition. (I and J) Atomic force microscopy stiffness maps of the 14DPA blastema (magenta arrowhead) and 4-MU wound (green arrowhead). The blue dashed line indicates the border of the P3 bone. Scale bars = 500 µm. Average stiffnesses were quantified in (J), which were determined by applying the Hertz model. n = 4 mice per condition. (K) Schematic of single-cell RNA sequencing and immunostaining experiments to determine the impact of 4-MU depletion of hyaluronic acid on cellular behavior. (L-M) UMAP of 8,995 Pdgfrα -expressing cells from the 4-MU and control datasets. Given the reduced biological replicates in the 4-MU experiment, prior 14DPA blastema datasets (gray) were integrated only to strengthen dimensionality reduction plotting; all downstream analyses were performed comparing 4-MU versus control samples only. In (M), three distinct cell sub-types were identified. They included an osteo-lineage (OL, purple), Fibroblast 3 (green), and cycling cell (brown) clusters. n = 2 replicates per condition, 6 mice pooled per replicate. (N) Volcano plot of differentially expressed genes in 4-MU versus control Pdgfrα -expressing cells, determined by the two-part generalized linear model MAST. Genes of interest have been annotated. (O-Q) Immunofluorescence images of 14DPA (O) and 28DPA (P) control (top panels) and 4-MU (bottom panels) digits stained for pSMAD1/5/8 (white) and Hoechst (blue). Scale bars = 100 µm. The right-hand panels show the yellow boxed regions of the blastema (O’ and P’) and 4-MU wound (O” and P”) tissues at higher magnification. Scale bars = 10 µm. Quantification of the average pSMAD1/5/8 fluorescence intensities in arbitrary units (A.U.) is shown in (Q). n = 3 mice per condition. Data are shown as mean ± SEM. Statistical significance was determined by two-tailed unpaired student’s t -test or two-way ANOVA with Tukey’s multiple comparisons test. *p < 0.05, **p<0.01, ***p<0.001, ns = not significant.

Article Snippet: To induce collagen synthesis and fibrillogenesis, 25 µg/ml 2-phospho-L-ascorbic acid trisodium salt (Merck, 49752-10G) was supplemented into the medium and replenished every other day until the experimental endpoint.

Techniques: Microscopy, Control, RNA Sequencing Assay, Immunostaining, Expressing, Immunofluorescence, Staining, Fluorescence, Two Tailed Test

Journal: bioRxiv

Article Title: Hyaluronic Acid and Emergent Tissue Mechanics Orchestrate Digit Tip Regeneration

doi: 10.1101/2024.12.04.626830

Figure Lengend Snippet: (A) Immunofluorescence images of uninjured digits stained for HABP (magenta), HAPLN1 (green), and Hoechst (blue). The yellow dashed line indicates the border of the third phalanx (P3) bone, and the orange dashed line the border of the second phalanx (P2) bone. Scale bar = 300 µm. The right-hand panels (A’ and A”) show the white boxed regions of the P3 and P2 periosteum (blue arrowheads) at higher magnification. Scale bars = 50 µm. (B) Schematic of the experimental design testing the expression of Hapln1 in uninjured P2 and P3 cells. (C) qPCR analysis of Hapln1 expression in uninjured P2 and P3 cells. n = 3 independent experiments. (D) Schematic of the experimental design testing the impact of Hapln1 overexpression (Hapln1 OE ) on pericellular hyaluronic acid in a stiff, fibrosis-like mechanical environment, with or without high-molecular-weight hyaluronic acid (HA). (E) Immunofluorescence images of mCherry Control and Hapln1 OE fibroblasts cultured on stiff (50 kPa) hydrogels, with or without high-molecular-weight (HMW) hyaluronic acid (HA) and stained for mCherry (yellow), HAPLN1 (white), HABP (magenta), and Hoechst (blue). The green arrowheads point towards bright regions of extracellular HAPLN1, co-localizing with aggregates of HA. Scale bars = 50 µm. (F) Immunoblotting for HAPLN1 with GAPDH as the loading control in mCherry Control and Hapln1 OE fibroblasts cultured on stiff (50 kPa) hydrogels, with or without HMW HA. Corresponding HA coverage per field of view (FOV) was quantified, related to . Quantification of HAPLN1 integrated band intensity normalized to that of GAPDH. n = 3 independent experiments. (G) Schematic of the experimental design testing the impact of Hapln1 overexpression (Hapln1 OE ) on collagen fibrillogenesis, induced by ascorbic acid. (H and I) Immunofluorescence images of mCherry Control and Hapln1 OE fibroblasts cultured on stiff (50 kPa) hydrogels with ascorbic acid and stained for HABP (magenta), mCherry (yellow), COLI (white), and Hoechst (blue). The green arrowheads highlight dim regions of extracellular HA, co-localizing with bright collagen fibers. The orange arrowheads highlight bright regions of HA, co-localizing with dim collagen fibers. Scale bars = 50 µm. The number of collagen fibers per field of view was quantified in (I). n = 3 independent experiments. Data are shown as mean ± SEM. Statistical significance was determined by two-tailed unpaired student’s t -test, three-way ANOVA with Tukey’s multiple comparisons test, or one-way ANOVA with Tukey’s multiple comparisons test. qPCR data were normalized to P2 cells and shown as log2FC, with statistical analyses performed on -11CT values. *p < 0.05, **p<0.01, ***p<0.001, ns = not significant.

Article Snippet: To induce collagen synthesis and fibrillogenesis, 25 µg/ml 2-phospho-L-ascorbic acid trisodium salt (Merck, 49752-10G) was supplemented into the medium and replenished every other day until the experimental endpoint.

Techniques: Immunofluorescence, Staining, Expressing, Over Expression, Molecular Weight, Control, Cell Culture, Western Blot, Two Tailed Test