279733 Microfabrication of Proangiogenic Cell-Laden Alginate-g-Pyrrole Hydrogels
Over the past several decades, cells have been increasingly studied as a new generation of medicine because of their capacity to produce multiple therapeutic proteins in a sustainable manner. A promising application for these therapies is to promote neovascularization by transplanting cells that can endogenously secrete proangiogenic growth factors or that have been genetically engineered to produce them; however, it still remains a challenge to sustainably trigger cells to produce therapeutic proteins at a transplant site over a desired treatment period, due to a lack of necessary tools.
Recently, several studies have demonstrated that certain soluble factors found in inflammatory tissues stimulate cellular angiogenic pathways through receptor recognition of certain molecular motifs. One good example is (carboxyalkyl)pyrrole proteins (CAPs) overexpressed in wounds. These CAPs have been shown to promote cell proliferation and angiogenic factor expression, such as vascular endothelial growth factor (VEGF), via the recognition of pyrrole motifs; however, it is still a challenging task to sustainably present CAPs at an implantation site.
We therefore hypothesize that pyrrole units conjugated to water soluble polymers, such as alginate, can chemically cross-link to form a cell adherent hydrogel that retains its structural integrity at an implantation site and further biochemically stimulates proliferation and VEGF expression of cells adhered to the gel. In addition, the introduction of micro-sized posts with varying densities on the hydrogel surface would further tune the cellular proangiogenic activities, because of an increased number of pyrrole units on the gel surface. The capacity of this hydrogel for stimulating neovessel formation was examined both in vitro and in vivo.
Pyrrole was chemically linked to sodium alginate through an amide linkage using N-(2-aminopropyl)pyrrole, synthesized from 1-(2-cyanoethyl)pyrrole. Additionally, methacrylic groups were linked to alginate through amide linkage using 2-aminoethyl methacrylate. Alginate-g-pyrrole hydrogels were formed through the oxidative cross-linking between pyrrole and/or methacrylic molecules conjugated to sodium alginate. The elastic modulus and the degree of swelling of the hydrogels were examined to determine the gels cross-linking densities. Mouse fibroblasts were cultured on the hydrogels to examine the role of the pyrrole cross-linking units on cellular angiogenic activities, including proliferation and VEGF expression. The micro-sized posts were incorporated on the surface of alginate-g-pyrrole hydrogels by preparing the gels on micropatterned molds, via an in situ cross-linking reaction. Finally, the micropatterned alginate-g-pyrrole gels laden with cells were implanted on chick chorioallantoic membranes (CAMs) to examine their capacity to enhance neovascularization.
RESULTS AND DISCUSSION
The alginate molecule was substituted with varied amounts of pyrrole units and methacrylic groups, while keeping the total degree of substitution of pyrrole and methacrylic groups constant. The addition of an oxidative cross-linking agent to pre-gel solutions therefore activated the formation of gels with statistically similar mechanical and water adsorption properties, independent of the gels’ overall pyrrole content. Fibroblasts cultured on the hydrogels with varied pyrrole content exhibited an increased proliferation and VEGF expression modulated by the pyrrole content of the gels throughout 4 days of cell culture. Through immuno-blocking experiments, it was determined that the cells were interacting with the pyrrole units of the hydrogels through the β3 integrin, which has been demonstrated to modulate angiogenic activities.
Alginate-g-pyrrole hydrogels were further prepared with varied densities of micro-posts through an in situ polymerization. The proliferation and VEGF expression of fibroblasts cultured on these gels was enhanced proportionally to the density of microposts on the gel’s surfaces due to the increase surface area of the gels.
Finally, alginate-g-pyrrole micropatterned hydrogels seeded with therapeutic protein releasing fibroblasts were implanted on CAMs, to examine their neovascularization capacity. The pyrrole cross-linked alginate gels demonstrated an enhancement in the formation of vasculature compared to control samples, determined by increases in blood vessel size and densities.
We have demonstrated that the use of pyrrole units has the capacity to form hydrogels with tunable mechanical and biochemical properties, both of which are beneficial to support proangiogenic activities of transplanted cells. Our approach to recapitulate the function of natural CAPs over an extended time period in a target tissue serves to advance the efficacy of a wide array of cell-based hydrogel therapies.