Electrosensitive Polyacrylic Acid/Fibrin Hydrogel Facilitates Cell Seeding and Alignment

Three-dimensional cell culture and conditioning is an effective means to guide cell distribution and patterning for tissue engineered constructs such as vascular grafts. Polyacrylic acid is known as an electroresponsive polymer, capable of transforming environmental stimuli like electrical energy to mechanical forces. In this study, we developed an electrosensitive and biocompatible hydrogel-based smart device composed of acrylic acid and fibrin as a tissue engineered construct to mechanically stimulate cells. Structural properties of the hydrogel were assessed by FTIR-ATR, scanning electron microscopy, prosimetry, and swelling measurement. Distribution and alignment of porcine smooth muscle cells (pSMCs) seeded on the surface of lyophilized hydrogels were evaluated and quantified by two-photon laser scanning microscopy. Smooth muscle cell tissue constructs exposed to 2 h of pulsatile electrical stimulation showed significantly enhanced cell penetration and alignment due to dynamic changes produced by alternative swelling and deswelling, in comparison with static samples. On the basis of the results, this hydrogel under electrical stimulation works as a mechanical pump, which can direct SMC alignment and facilitate infiltration and distribution of cells throughout the structure

Visualization of eGFP-macrophages in muscle tissue (n=4).

<p>A, Overview of muscle tissue after ligation and macrophage injection. B, eGFP-macrophages located in the injection site at post-operative day 2 (scale bar = 100 µm). C, Negative control eGFP staining. Injected macrophages could be detected in the perivascular space of collateral vessels at day 2 (D), day 4 (E) and day 6 (F) (scale bar = 20µm).</p

Wound Administration of M2-Polarized Macrophages Does Not Improve Murine Cutaneous Healing Responses

<div><p>Macrophages play a crucial role in all stages of cutaneous wound healing responses and dysregulation of macrophage function can result in derailed wound repair. The phenotype of macrophages is influenced by the wound microenvironment and evolves during healing from a more pro-inflammatory (M1) profile in early stages, to a less inflammatory pro-healing (M2) phenotype in later stages of repair. The aim of the current study was to investigate the potential of exogenous administration of M2 macrophages to promote wound healing in an experimental mouse model of cutaneous injury. Bone marrow derived macrophages were stimulated in-vitro with IL-4 or IL-10 to obtain two different subsets of M2-polarized cells, M2a or M2c respectively. Polarized macrophages were injected into full-thickness excisional skin wounds of either C57BL/6 or diabetic db/db mice. Control groups were injected with non-polarized (M0) macrophages or saline. Our data indicate that despite M2 macrophages exhibit an anti-inflammatory phenotype in-vitro, they do not improve wound closure in wild type mice while they delay healing in diabetic mice. Examination of wounds on day 15 post-injury indicated delayed re-epithelialization and persistence of neutrophils in M2 macrophage treated diabetic wounds. Therefore, topical application of ex-vivo generated M2 macrophages is not beneficial and contraindicated for cell therapy of skin wounds.</p></div

Histological analysis of db/db wounds isolated at day 15 post-wounding.

<p>Wounds from saline or macrophage injected db/db mice were isolated 15 days after wounding and processed for generation of paraffin sections. (A-D) H&E stained sections indicating migrating epithelial tongues (dotted line) in M2a and M2c wounds while complete re-epithelialization is present in saline and M0 wounds. (E-H) MSB trichrome staining indicating increased presence of fibrin (marked by arrowheads) and erythrocytes (marked by stars) in M2 macrophage injected wounds. Magnification 40x with 100x insets. Graphs are quantifications of (A) re-epithelialization and (B) fibrin/erythrocyte staining respectively. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test. n = 6 mice (wounds)/group.</p

Gene expression analysis of db/db wounds isolated at day 15 post-wounding.

<p>Wounds were isolated from db/db mice 15 days after wounding and analyzed by real-time PCR for expression levels of (A) macrophage M2 markers Arginase-1, YM-1, Fizz-1 and HMOX-1; (B) inflammatory markers TNF, IL-6, IL-12, IL-1β and IL-10; (C) chemokines CXCL1 and CXCL2 and (D) matrix metalloproteinases MMP2 and MMP9. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test. *p<0.05. n = 6 mice (wounds)/group.</p

Immunohistochemical analysis of db/db wounds isolated at day 15 post-wounding.

<p>Immunohistochemical stainings were performed in cryosections. (A-D) NIMP staining for wound neutrophils indicated higher numbers of neutrophils in M2a and M2c treated wounds compared to saline and M0 groups. Mice analyzed per group: salines = 3; M0 = 5; M2a = 6; M2c = 5. (E-H) F4/80 staining for macrophages indicated not significant differences between the groups. Mice analyzed per group: salines = 4; M0 = 4; M2a = 6; M2c = 6. (I-L) CD31 staining indicated similar staining for endothelial cells in the different groups. Mice analyzed per group: salines = 4; M0 = 5; M2a = 5; M2c = 4. Magnification 40x with 100x insets. Graphs are quantifications of (A) NIMP, (B) F4/80 and (C) angiogenesis staining respectively. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test.</p

Wound healing in M2 macrophage injected wounds generated in C57BL/6 mice.

<p>(A) Macroscopic appearance of representative saline, M0, M2a, and M2c injected wounds. Day 0 pictures were taken immediately after wounding. (B) Quantification of wound closure rate. At the indicated days, wound areas were determined using image analysis and expressed as percentage of wound area immediately post-injury as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0102994#s2" target="_blank">methods</a> (n = 6 mice/group). Statistical significance was evaluated with a two-way ANOVA with Bonferroni post-test.</p

Effect of chlodronate liposomes on blood monocyte and granulocyte population (n=6).

<p>Clodronate liposome treatment reduces the endogenous monocyte population significantly compared to PBS liposome treatment (***: p<0.001) and compared to single clodronate injection (#: p<0.05). A, Single injection with clodronate reduces the monocyte population with 53%, whereas a second injection results in 68% reduction. B, Granulocyte population increases upon clodronate treatment after double injections with clodronate compared to PBS-liposome treatment (***: p<0.001) and single clodronate injection (#: p<0.001), most probably due the depletion of the monocyte population.</p

Histological analysis of db/db wounds isolated at day 15 post-wounding.

<p>Wounds from saline or macrophage injected db/db mice were isolated 15 days after wounding and processed for generation of paraffin sections. (A-D) H&E stained sections indicating migrating epithelial tongues (dotted line) in M2a and M2c wounds while complete re-epithelialization is present in saline and M0 wounds. (E-H) MSB trichrome staining indicating increased presence of fibrin (marked by arrowheads) and erythrocytes (marked by stars) in M2 macrophage injected wounds. Magnification 40x with 100x insets. Graphs are quantifications of (A) re-epithelialization and (B) fibrin/erythrocyte staining respectively. Statistical significance was evaluated analyzing M0, M2a and M2c expression levels compared to salines by t-test. n = 6 mice (wounds)/group.</p

Induction of M2 macrophages.

<p>Phenotypic characterization of polarized macrophages. (A) Gene expression analysis for M2 markers in M0 control, M2a and M2c bone marrow derived macrophages (BMDM) after 24 h of IL-4 (for M2a) or IL-10 (for M2c) polarization. (B-D) Gene expression analysis for inflammatory markers in M0 control, M2a and M2c polarized cells after 24 h of polarization and an additional 24 h of LPS stimulation. Cells were analyzed in triplicates and the mean expression of 2–3 independent experiments is indicated. M0 control levels were arbitrarily set to 1 in each experiment and for each gene and statistical significance was evaluated analyzing M2a and M2c expression levels compared to M0, using a one-sample t-test analysis against the hypothetical value 1 (M0). *p≤0.05.</p