PCG-EPC-treated wound healing mimics normal wound healing process.

<p>Histopathological analyses of wound healing process from d 2 to 10. Non-diabetic controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g007" target="_blank">Figure 7</a>; images ‘a’ to ‘e’), untreated diabetic controls (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g007" target="_blank">Figure 7</a>; images ‘f’ to ‘j’), PLLA-EPC-treated diabetic wounds (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g007" target="_blank">Figure 7</a>; images ‘k’ to ‘o’) and PCG-EPC-treated diabetic wound (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g007" target="_blank">Figure 7</a>; images ‘p’ to ‘t’) (10× magnification) show that PCG-EPC-treated wounds mimic normal wound healing process. All data are representative of at least 3 independent experiments with at least 3 mice per time point.</p

% viability of migrated cells at 24, 48 and 72h.

<p>PCG-EPCs exhibited higher viability post-migration as compared to VN and PLLA-EPCs. Although a progressive decrease in % cellular viability was observed over 24, 48 and 72 h time points in all groups, PCG-EPCs show significantly higher maintenance of cellular viability as compared to its PLLA and VN-EPC counterparts at all time points (*<i>p</i><0.001). Results are represented as mean ± SD of at least 3 independent experiments (n = 4/5).</p

PCG-EPCs migrate deep into the wound beds.

<p>VN-EPCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g008" target="_blank">Figure 8</a>; images ‘a’ and ‘d’), PLLA-EPCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g008" target="_blank">Figure 8</a>; images ‘b’ and ‘e’) and PCG-EPCs (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0069960#pone-0069960-g008" target="_blank">Figure 8</a>; images ‘c’ and ‘f’), pre-stained with cell tracker-Orange, were delivered at d 0 onto diabetic wounds for 48 h. Cryosections were counterstained with DAPI and analyzed for incorporation of pre-stained EPCs into wound beds of diabetic mice. Scale bars for images = 100 µm; Scale bars for insets = 20 µm. The data are representative of 4 mice in each group.</p

Chemotactic migration of EPCs grown on various matrices.

<p>Comparative EPC migration towards VEGF (50 ng/ml) in vitro at 24, 48 and 72 h from VN, PLLA and PCG matrix is graphically illustrated. Data are represented as mean of at least 3 independent experiments (n = 3) ± SD. Each experiment had at least 4 wells per group. EPCs from both electro-spun matrices consistently exhibited a slower cellular migration compared to those from VN at all time points (*p<0.001).</p

Comparative growth of EPCs on various matrices.

<p>(A). A representative phase contrast image of EPCs growing on VN, PLLA and PCG matrix is depicted (200× magnification). (B). Characterization of VN, PLLA and PCG-EPCs. EPCs were co-positive for Alexaflour 488-Ac-LDL uptake and UEA1-TRITC staining. The nuclei are stained with DAPI. Presence or absence of nano-fibre matrix is visible in the phase contrast merged image. (scale bar = 50 µm).</p

(A) PCG-EPCs accelerate wound healing process.

<p>Photographic evidence of wound healing in non-diabetic animals in comparison to untreated diabetic, VN-EPC-treated, PLLA-EPC-treated and, PCG-EPC-treated diabetic animals, from d 0 to d 10. Photographs are from one representative experimental set from among 3. At least 3 mice were analyzed for each time point. (B). Graphical representation of percent wound closure in various groups. Percent wound closure in PCG-EPCs is significantly higher compared to VN-EPC-treated, PLLA-EPC-treated and control groups on days 2 to 8 (*p<0.010) as well as on day 10 (**p<0.050). (C) Photographic evidence of scar tissue formation in non-diabetic (ND), untreated diabetic (Diab), VN-EPC-treated (VN-EPC), PLLA-EPC-treated and PCG-EPC-treated diabetic mice (PCG-EPCs) on day 22. Fibrotic tissue formed in untreated diabetic mouse is encircled in black. The PCG-EPC-treated wounds healed without the formation of any scar tissue or deposition of collagen. The data are representative of at least 4 mice per group.</p

Enhanced Growth of Endothelial Precursor Cells on PCG-Matrix Facilitates Accelerated, Fibrosis-Free, Wound Healing: A Diabetic Mouse Model

<div><p>Diabetes mellitus (DM)-induced endothelial progenitor cell (EPC) dysfunction causes impaired wound healing, which can be rescued by delivery of large numbers of ‘normal’ EPCs onto such wounds. The principal challenges herein are (a) the high number of EPCs required and (b) their sustained delivery onto the wounds. Most of the currently available scaffolds either serve as passive devices for cellular delivery or allow adherence and proliferation, but not both. This clearly indicates that matrices possessing both attributes are ‘the need of the day’ for efficient healing of diabetic wounds. Therefore, we developed a system that not only allows selective enrichment and expansion of EPCs, but also efficiently delivers them onto the wounds. Murine bone marrow-derived mononuclear cells (MNCs) were seeded onto a PolyCaprolactone-Gelatin (PCG) nano-fiber matrix that offers a combined advantage of strength, biocompatibility wettability; and cultured them in EGM2 to allow EPC growth. The efficacy of the PCG matrix in supporting the EPC growth and delivery was assessed by various in vitro parameters. Its efficacy in diabetic wound healing was assessed by a topical application of the PCG-EPCs onto diabetic wounds. The PCG matrix promoted a high-level attachment of EPCs and enhanced their growth, colony formation, and proliferation without compromising their viability as compared to Poly L-lactic acid (PLLA) and Vitronectin (VN), the matrix and non-matrix controls respectively. The PCG-matrix also allowed a sustained chemotactic migration of EPCs in vitro. The matrix-effected sustained delivery of EPCs onto the diabetic wounds resulted in an enhanced fibrosis-free wound healing as compared to the controls. Our data, thus, highlight the novel therapeutic potential of PCG-EPCs as a combined ‘growth and delivery system’ to achieve an accelerated fibrosis-free healing of dermal lesions, including diabetic wounds.</p></div

Characterization of PCG-matrix by electron microscopy.

<p>(A) SEM images depict morphology of electrospun PCG nanofibers showing random orientation and smooth morphology. Image on the left: 1000× magnification (scale bar = 50 µm) and image on the right: 2000× magnification (scale bar = 20 µm). (B). TEM images of electrospun nanofiber of PCG matrix showing smooth surface (scale bar on left = 500 nm; scale bar on right = 5000 nm).</p

Biocompatibility assessment of PCG- matrix Biocompatibility of the PCG matrix was assessed by estimation of cell adhesion (A), colony forming cells/units (B), % viability (C) and proliferation potential (D) on d 14, in comparison with the parallel VN- and PLLA-grown EPCs used as controls.

<p>Data are represented as mean of triplicate wells from 5 independent experiments (n = 5) ± SD. *<i>p</i><0.001.</p