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Injectable biomimetic liquid crystalline scaffolds enhance muscle stem cell transplantation

  1. Samuel I. Stuppa,d,e,i,j4,5
  1. aSimpson Querrey Institute for BioNanotechnology, Northwestern University, Chicago, IL 60611-2875;
  2. bBaxter Laboratory for Stem Cell Biology, Stanford University School of Medicine, Stanford, CA 94305-5175;
  3. cMeinig School of Biomedical Engineering, Cornell University, Ithaca, NY 14853;
  4. dDepartment of Chemistry, Northwestern University, Evanston, IL 60208;
  5. eDepartment of Biomedical Engineering, Northwestern University, Evanston, IL 60208;
  6. fDepartment of Molecular Biosciences, Northwestern University, Evanston, IL 60208;
  7. gDepartment of Neurobiology, Northwestern University, Evanston, IL 60208;
  8. hDepartment of Radiology, Northwestern University, Evanston, IL 60208;
  9. iDepartment of Materials Science and Engineering, Northwestern University, Evanston, IL 60208;
  10. jDepartment of Medicine, Northwestern University, Chicago, IL 60611
  1. Contributed by Helen M. Blau, August 4, 2017 (sent for review November 6, 2016; reviewed by Kristi S. Anseth and Jason A. Burdick)

  1. Fig. 2.

    Encapsulation and survival of myogenic precursor cells in aligned aPA scaffolds. (A) Schematic representation of the cell encapsulation and aPA/cell construct formation process. A 13-mM PA solution (containing self-assembled nanofibers) is annealed and then used to resuspend cells to form a homogeneous dispersion of cells in the aPA solution. Finally, this aPA/cell suspension is pipetted out into a glass slide containing a gelling solution, which immediately turns the aPA solution into a gel, thus encapsulating the cells within the construct. The confocal fluorescent micrograph shows encapsulated C2C12-GFP cells (green) inside the aPA scaffold (propidium iodide, red) at day 4 after formation at a density of 20,000 cells per microliter. (Scale bar, 100 μm.) (B) Stiffness (G′) values (mean ± SEM) from frequency sweeps of aPA gels (n = 3–4 measurements per sample). (C) Young’s modulus values (E) measured on oriented aPA gels by atomic force microscopy (AFM) represented as a box and whiskers plot of the 5–95 percentile with n = 163–526 measurements per sample. (D) Representative confocal micrographs of C2C12-GFP cells in different aPA scaffolds and at different times in culture, stained with calcein AM (green, alive) and propidium iodide (red, dead). White arrowheads point to dead cells (red dots), which is not to be confused with the nonspecific staining of the borders of aPA scaffolds by propidium iodide. (Scale bar, 250 μm.) (E) Viability analysis from the images in C (mean + SEM); *P < 0.05; **P < 0.01; ns, nonsignificant; two-way ANOVA with Bonferroni post hoc test.

  2. Fig. 3.

    aPA scaffold nanofiber alignment and stiffness modulate the alignment, maturation, and proliferation of C2C12-GFP cells. C2C12-GFPs were encapsulated at a density of 20,000 cells per microliter in nonaligned mid G′ aPA scaffolds or in aligned, aPA scaffolds with different stiffness magnitudes (low, mid, or high G′) and cultured for several days. (A) Representative confocal fluorescent micrographs of calcein AM-stained C2C12 cells in aPA scaffolds at different time points showing differential elongation of cells or cell clusters. (Scale bar, 200 μm.) (B) Directionality analysis from the images in A. The histograms show the angular distribution of the directions in which cells or cell clusters elongate (black bars) and the Gaussian equation that fits the data (red line). (C) As a measure of directionality, we divided the maximum by the minimum value of the Gaussian fit to calculate an order parameter (mean + SEM). When this order parameter is 1 (red dotted line), there is no preferred direction in the sample; greater values indicate greater directionality. *P < 0.5; **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant; one-way ANOVA with Bonferroni post hoc test. (D) Representative confocal fluorescent micrographs of C2C12-GFP cells after 10 d in culture in aPA scaffolds displaying GFP (green) and stained for total nuclei (propidium iodide, red) and myosin heavy chain (MF20 antibody) or alpha-actinin,(ACTN) (blue). White arrows point to differentiated cells or cluster of cells that have likely fused with each other. (Scale bar, 200 μm.) (E, Left) Representative confocal micrographs of C2C12-GFP cells at day 8 in culture in aPA scaffolds displaying GFP (green) and stained for EdU incorporation (red) and total nuclei (DAPI, blue). (Scale bar, 250 μm.) (Right) Quantification of the percentage of cells that have incorporated EdU (24-h incubation), from the images above; ***P < 0.001; ns, nonsignificant; one-way ANOVA with Bonferroni post hoc test.

  3. Fig. 4.

    Injector apparatus and its use with Gd(III)-labeled aPA to visualize and track aligned biomimetic scaffolds over time in vivo. (A) The injection device used for all injections is represented by this schematic showing a Hamilton syringe mounted onto a retracting leadscrew linear actuator platform. (B) The smaller zoomed Inset shows the aPA solution (blue) being injected into muscle tissue (Movie S2). (C) Macroscopic image of harvested TA muscles after injection with an Evans blue-dyed mid G′ aPA solution. (D) Two coronal MRI scans of the same mouse leg spaced 200 μm apart. The aPA scaffold can be seen throughout the length of the TA muscle (white arrows). (Scale bar, 1 cm.) (E) TEM showing TA muscle sarcomere banding next to injected aPA scaffold. The direction of the long axis of the muscle and of the aPA scaffold nanofibers are indicated by the black arrow. (F and G) Higher magnification Insets showing the muscle and the scaffold nanofibers, respectively; both the myofibers and nanofibers are oriented along the vertical direction parallel to the long axis of the muscle. (Scale bar, 1 μm.) (H) Molecular structure of the gadolinium chelate (green sphere) and PA molecule used for tracking the scaffold in vivo and molecular graphics of the supramolecular PA nanofiber composed of 5% Gd(III)-labeled PA and 95% C16V3A3E3 PA. (I) Axial MRI scan showing the Gd-aPA scaffold (arrows) in the same leg for 8 d postinjection; the Gd-aPA is no longer distinguishable by day 15. (Scale bar, 500 μm.) (J) MRI signal quantified by T1 relaxation time of the Gd-aPA area compared with the background muscle tissue (shown are the individual values as dots and the mean as a horizontal line). We were only able to discern the Gd-aPA signal from the background in fewer slices so the T1 average at day 8 was most likely overestimated. (K) ICP-MS quantification of Gd(III) content in mice legs at different times postinjection (mean ± SEM). The one phase decay fit yields a half-life of 13.5 d for the scaffold.

  4. Fig. 5.

    Proliferation and differentiation of primary myoblasts in aPA scaffolds and GF encapsulation and release by aPA scaffolds in vitro. Primary myoblasts were encapsulated at a density of 10,000 cells per microliter. (A and C) Representative confocal micrographs of primary myoblasts in the mid G′ aPA scaffold at different time point culture conditions. Proliferating cells were stained for EdU incorporation (green) and total nuclei were stained with 7-AAD (red). Myosin heavy chain was stained using the MF20 antibody (A, blue). (Scale bar, 100 μm.) (B and D) Graph showing the quantification of EdU incorporation (24-h incubation), from the images in A and C (mean + SEM). (C and D) After the first day in culture, all media were changed to DM alone. **P < 0.01; ***P < 0.001; ****P < 0.0001; ns, nonsignificant; one (D) or two-way (B) ANOVA with Bonferroni post hoc test. (E) Schematic representation of the protein encapsulation and release experiment. (F) Cumulative release (Left), and amount retained (Right), of VEGF and bFGF from mid G′ aPA gels in two different media, expressed as percentage of total (released + retained) (mean ± SEM).

  5. Fig. 6.

    Biomimetic scaffolds enhance MuSC transplantation therapy. (A) Primary mouse muscle stem cells were isolated from Gfp/Luciferase double-transgenic mice by GFP+/CD34+/integrin-α7+ FACS sorting and mixed with 13 mM mid G′ aPA solution containing bFGF and FBS (“+GF”) or no serum/growth factors (“?GFs”; in G and H only) at 200 cells μL?1. Biomimetic scaffold/MuSC mixtures (1 μL per muscle) were extruded into the TA muscles of preirradiated NOD/Scid by intramuscular injection to form biomimetic scaffolds in situ. In contralateral hindlimbs, control MuSC injections were performed in resuspension buffer ±GFs. Injections were performed with or without DMSO (1.8% final) to evaluate the effect of carrier in drug resuspension studies. No statistically significant effects between control (DMSO-free) and DMSO condition were observed for any comparison so n = 10 samples were grouped per method. Some hindlimbs were injured by intramuscular injection of notexin 3 d pretransplant in G. (BF) BLI and immunohistochemical detection transplanted MuSC engraftment and myofiber repair in uninjured muscles. Engraftment threshold (dashed line) corresponding to histological detection of one or more donor-derived (GFP+) myofibers (as in refs. 21 and 22). (B) BLI normalized to injected cell number at 0–5 wk posttransplant of n = 10 total (five control, five DMSO) transplants grouped by injection method (p, photons). ***P < 0.0001 by two-way ANOVA with Bonferroni post hoc test for comparison of time courses. (C) Normalized BLI values at 5 wk posttransplant. Scatterplot overlain on box (50%) and whisker (full range) with median line. **P < 0.01 by Mann–Whitney U test on confidence intervals of endpoints. (D) Engraftment analysis using threshold BLI value. **P < 0.01 by Fisher’s test on endpoint values. (E and F) Detection of transplant-derived (GFP+) myofibers by anti-GFP and anti-laminin immunohistochemistry from DMSO-free transplants. (E) Representative immunohistological images. (Scale bar, 500 μm.) (F) GFP+ myofibers per recipient TA muscle (median line). n = 4 transplants per method. (G and H) BLI detection (5 wk posttransplant) of MuSC engraftment into either uninjured (H) or notexin preinjured hindlimb muscles (G) via biomimetic scaffold encapsulation, with and without bFGF/FBS-loading but not DMSO. Scatterplot shows n = 4 transplants per condition with median line. In FH, *P < 0.05 by Mann–Whitney U test. ns, not significant.

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