Developing Hybrid Polymer Scaffolds Using Peptide Modified Biopolymers for Cell Implantation

Polymeric scaffolds containing biomimics offer exciting therapies with broad potential impact for cellular therapies and thereby potentially improve success rates. Here we report the designing and fabrication of a hybrid scaffold that can prevent a foreign body reaction and maintain cell viability. A biodegradable acrylic based cross-linkable polycaprolactone based polymer was developed and using a multihead electrospinning station to fabricate hybrid scaffolds. This consists of cell growth factor mimics and factors to prevent a foreign body reaction. Transplantation studies were performed subcutaneously and in epididymal fat pad of immuno-competent Balb/c mice and immuno-suppressed B6 Rag1 mice and we demonstrated extensive neo-vascularization and maintenance of islet cell viability in subcutaneously implanted neonatal porcine islet cells for up to 20 weeks of post-transplant. This novel approach for cell transplantation can improve the revascularization and allow the integration of bioactive molecules such as cell adhesion molecules, growth factors, etc

Expression of pro-angiogenic/pro-islet cytokines in subcutaneous implants in diabetic mice.

<p>The expression of GM-CSF (<b>A</b>), SCF (<b>B</b>), SDF-1α (<b>C</b>), VCAM-1 (<b>D</b>) and VEGF (<b>E</b>) protein in hydrogels explanted at 1 (black bars), 2 (grey bars) and 6 weeks (white bars) was normalized to the levels in the collagen hydrogel at their respective time point (<i>n</i>=3 each). <i>P</i>-values in (A): *<i>p</i>=0.007 and <i>p</i>=0.04 vs. 1 week collagen-chitosan and collagen-chitosan+CAC implants, respectively; ^<b>†</b><i>p</i>=0.02; ^{<b>††</b>}<i>p</i>=0.005; and ^{<b>†††</b>}<i>p</i><0.05). In (B): *<i>p</i>≤0.004 vs. collagen+CAC and collagen-chitosan+CAC implants at 1 week; ^<b>†</b><i>p</i>=0.002; and ^{<b>††</b>}<i>p</i>=0.01. In (C): *<i>p</i>=0.01 vs. collagen+CAC at 1 week. In (D): *<i>p</i>=0.01 vs. collagen at 2 weeks; **<i>p</i>=0.004 vs. collagen+CAC at 6 weeks; ***<i>p</i>=0.0002 vs. collagen+CAC at 6 weeks. In (E): *<i>p</i>≤0.007 vs. collagen+CAC and collagen-chitosan+CAC at 1 week; and ^<b>†</b><i>p</i>=0.03.</p

Expression of pro-angiogenic/anti-islet cytokines in subcutaneous implants in diabetic mice.

<p>The expression of IL-1β (<b>A</b>), lymphotactin (<b>B</b>), MCP-1 (<b>C</b>), MCP-5 (<b>D</b>), M-CSF (<b>E</b>), RANTES (<b>F</b>), TARC (<b>G</b>) and TNF-α (<b>H</b>) protein in hydrogels explanted at 1 (black bars), 2 (grey bars) and 6 weeks (white bars) was normalized to the levels in the collagen hydrogel at their respective time point (<i>n</i>=3 each). <i>P</i>-values in (A): *<i>p</i>=0.05 vs. collagen-chitosan+CAC at 1 week; and ^<b>†</b><i>p</i>≤0.003. In (B): *<i>p</i>=0.03 vs. collagen+CAC at 1 week; **<i>p</i>=0.04 vs. collagen at 2 weeks; and ^<b>†</b><i>p</i>≤0.02. In (C): *<i>p</i>=0.04 vs. collagen at 1 week; **<i>p</i>=0.003 vs. collagen at 1 week; ***<i>p</i>≤0.01 vs. collagen and collagen+CAC hydrogels at 2 weeks; ****<i>p</i>=0.01 vs. collagen and collagen+CAC at 6 weeks; and ^<b>†</b><i>p</i>≤0.006 vs. collagen+CAC at 2 and 6 weeks. In (D): ^<b>†</b><i>p</i>=0.04. In (E): *<i>p</i>≤0.04 vs. all other hydrogels at 1 week; **<i>p</i>≤0.003 vs. all other hydrogels at 2 weeks; ***<i>p</i>=0.02 vs. collagen at 6 weeks; and ^<b>†</b><i>p</i>≤0.02. In (F): *<i>p</i>=0.009 vs. collagen+CAC at 1 week; **<i>p</i>≤0.01 vs. collagen and collagen+CAC at 2 weeks; ^<b>†</b><i>p</i>=0.04; and ^{<b>††</b>}<i>p</i>≤0.02. In (G): *<i>p</i>≤0.02 vs. all other implants at 1 week; ^<b>†</b><i>p</i>≤0.007; ^{<b>††</b>}<i>p</i>=0.03; and ^{<b>†††</b>}<i>p</i>≤0.008.</p

Degradation and elastic modulus of hydrogels.

<p>(<b>A</b>) Collagen (black bars) and collagen-chitosan (gray bars) hydrogels were incubated in 100U collagenase and the residual mass was determined over time (*<i>p</i>≤0.03 vs. collagen at the same time-point; **<i>p</i><0.0001 vs. collagen at 2 hours; ***<i>p</i><0.0001 vs. collagen-chitosan at 3h; <i>n</i>=4 each). (<b>B</b>) Stress/strain curve for collagen and collagen-chitosan hydrogels. (<b>C</b>) The elastic modulus for the collagen and collagen-chitosan hydrogel samples (*<i>p</i><0.0001; <i>n</i>=8).</p

Characterization of hydrogel fiber cross-linking.

<p>Representative SEM images of collagen (<b>A</b>) or collagen (coll)-chitosan (<b>B</b>) hydrogels. Total cross-links per image (<b>C</b>) as well as distance (µm) between cross-links (<b>D</b>) were quantified (*<i>p</i>=0.046; **<i>p</i>=0.03; <i>n</i>=4 each). </p

Evaluation of a Collagen-Chitosan Hydrogel for Potential Use as a Pro-Angiogenic Site for Islet Transplantation

<div><p>Islet transplantation to treat type 1 diabetes (T1D) has shown varied long-term success, due in part to insufficient blood supply to maintain the islets. In the current study, collagen and collagen:chitosan (10:1) hydrogels, +/- circulating angiogenic cells (CACs), were compared for their ability to produce a pro-angiogenic environment in a streptozotocin-induced mouse model of T1D. Initial characterization showed that collagen-chitosan gels were mechanically stronger than the collagen gels (0.7kPa vs. 0.4kPa elastic modulus, respectively), had more cross-links (9.2 vs. 7.4/µm²), and were degraded more slowly by collagenase. After gelation with CACs, live/dead staining showed greater CAC viability in the collagen-chitosan gels after 18h compared to collagen (79% vs. 69%). <i>In vivo</i>, collagen-chitosan gels, subcutaneously implanted for up to 6 weeks in a T1D mouse, showed increased levels of pro-angiogenic cytokines over time. By 6 weeks, anti-islet cytokine levels were decreased in all matrix formulations ± CACs. The 6-week implants demonstrated increased expression of VCAM-1 in collagen-chitosan implants. Despite this, infiltrating vWF⁺ and CXCR4⁺ angiogenic cell numbers were not different between the implant types, which may be due to a delayed and reduced cytokine response in a T1D versus non-diabetic setting. The mechanical, degradation and cytokine data all suggest that the collagen-chitosan gel may be a suitable candidate for use as a pro-angiogenic ectopic islet transplant site. </p> </div

Expression of anti-angiogenic/anti-islet cytokines in subcutaneous implants in diabetic mice.

<p>The expression of BLC (<b>A</b>), IFN-λ (<b>B</b>), IL12p70 (<b>C</b>), MIG (<b>D</b>), and PF-4 (<b>E</b>) protein in hydrogels explanted at 1 (black bars), 2 (grey bars) and 6 weeks (white bars) was normalized to the levels in the collagen hydrogel at their respective time point (<i>n</i>=3 each). <i>P</i>-values in (A): ^<b>†</b><i>p</i>=0.02. In (B): *<i>p</i>=0.02 vs. collagen at 6 weeks; ^<b>†</b><i>p</i>=0.01; and ^{<b>††</b>}<i>p</i>=0.04. In (C): *<i>p</i>≤0.02 vs. collagen and collagen+CAC implants at 1 week; ^<b>†</b><i>p</i>=0.02; and ^{<b>††</b>}<i>p</i>=0.02). In (D): *<i>p</i>=0.02 vs. collagen-chitosan at 1 week; **<i>p</i>≤0.02 vs. collagen and collagen-chitosan at 1 week; ***<i>p</i>≤0.01 vs. all other implants at 2 weeks; ****<i>p</i>≤0.001 vs. all other implants at 6 weeks; *****<i>p</i>=0.03 vs. collagen+CAC at 6 weeks; ^<b>†</b><i>p</i>=0.02; ^{<b>††</b>}<i>p</i><0.0001; and ^{<b>†††</b>}<i>p</i>=0.009. In (E): *<i>p</i>≤0.03 vs. collagen-chitosan+CAC and collagen+CAC implants at 1 week; **<i>p</i>≤0.006 vs. all other implants at 2 weeks; ***<i>p</i>≤0.01 vs. collagen-chitosan and collagen-chitosan+CAC at 6 weeks; ****<i>p</i>=0.007 vs. collagen-chitosan+CAC at 6 weeks; ^<b>†</b><i>p</i><0.0001; and ^{<b>††</b>}<i>p</i><0.0001.</p

Representative images of HPS-stained collagen and collagen-chitosan hydrogels (±CACs) explanted at 2 and 6 weeks.

<p>Representative images of HPS-stained collagen and collagen-chitosan hydrogels (±CACs) explanted at 2 and 6 weeks.</p

Effect of hepatocyte growth factor (HGF) on human islet total cellular insulin content and insulin secretory capacity after exposure to pro-inflammatory cytokines.

<p>Results are reported as % recovery of total cellular insulin relative to untreated controls (islets alone). Islet function is assessed by a static glucose stimulated insulin release assay. The stimulation index (SI) is calculated as a ratio of insulin release at high glucose versus low glucose. Insulin release (% insulin content) is reported as insulin secreted at 2.8 mM glucose or 20.0 mM glucose divided by insulin content for corresponding islets. Values are expressed as mean ± SEM.</p>*<p>p<0.05 for islet vs. islet + cytokine.</p>†<p>p<0.05 for islet vs. islet + HGF (10 ng/mL) + cytokine.</p>‡<p>p<0.05 for islet + cytokines vs. islet + HGF (10 ng/mL) + cytokine.</p

Protection of human islets from cytokine induced apoptosis by bone marrow derived MSCs.

<p>A-C) 500 Islets, D-F) 500 Islets + cytokines, G-I) 500 Islets +1.0×10⁶ bMSCs, + cytokines. Tissues were stained for insulin (A,D,G) in red and TUNEL (B,E,H) in green. The merge of the red and green images are presented in panels C, F, and I. Islets cultured without cytokines demonstrated minimal TUNEL positive cells. After cytokine exposure, the number of TUNEL positive cells increased; TUNEL and insulin co-expression was also increased with cytokine treatment. Alteration of native islet organization was observed. After cytokine exposure, co-expression of insulin and TUNEL did not increase in the islets + bMSCs group; cytokines – cocktail of IFNγ, TNFα and IL-1β described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0038189#s2" target="_blank">materials and methods</a>. Scale bar represents 100 µm.</p