Paracrine cross-talk between human adipose tissue-derived endothelial cells and perivascular cells accelerates the endothelialization of an electrospun ionomeric polyurethane scaffold.

The ex vivo endothelialization of small diameter vascular prostheses can prolong their patency. Here, we demonstrate that heterotypic interactions between human adipose tissue-derived endothelial cells and perivascular cells can be exploited to accelerate the endothelialization of an electrospun ionomeric polyurethane scaffold. The scaffold was used to physically separate endothelial cells from perivascular cells to prevent their diffuse neo-intimal hyperplasia and spontaneous tubulogenesis, yet enable their paracrine cross-talk to accelerate the integration of the endothelial cells into a temporally stable endothelial lining of a continuous, elongated, and aligned morphology. Perivascular cells stimulated endothelial basement membrane protein production and suppressed their angiogenic and inflammatory activation to accelerate this biomimetic morphogenesis of the endothelium. These findings demonstrate the feasibility and underscore the value of exploiting heterotypic interactions between endothelial cells and perivascular cells for the fabrication of an endothelial lining intended for small diameter arterial reconstruction. STATEMENT OF SIGNIFICANCE: Adipose tissue is an abundant, accessible, and uniquely dispensable source of endothelial cells and perivascular cells for vascular tissue engineering. While their spontaneous self-assembly into microvascular networks is routinely exploited for the vascularization of engineered tissues, it threatens the temporal stability of an endothelial lining intended for small diameter arterial reconstruction. Here, we demonstrate that an electrospun polyurethane scaffold can be used to physically separate endothelial cells from perivascular cells to prevent their spontaneous capillary morphogenesis, yet enable their cross-talk to promote the formation of a stable endothelium. Our findings demonstrate the feasibility of engineering an endothelial lining from human adipose tissue, poising it for the rapid ex vivo endothelialization of small diameter vascular prostheses in an autologous, patient-specific manner.

Self-Assembled Oligo-Urethane Nanoparticles: Their Characterization and Use for the Delivery of Active Biomolecules into Mammalian Cells.

Developing safe and effective strategies to deliver biomolecules such as oligonucleotides and proteins into cells has grown in importance over recent years, with an increasing demand for non-viral methods that enable clinical translation. Here, we investigate uniquely configured oligo-urethane nanoparticles based on synthetic chemistries that minimize the release of pro-inflammatory biomarkers from immune cells, show low cytotoxicity in a broad range of cells, and efficiently deliver oligonucleotides and proteins into mammalian cells. The mechanism of cell uptake for the self-assembled oligo-urethane nanoparticles was shown to be directed by caveolae-dependent endocytosis in murine myoblasts (C2C12) cells. Inhibiting caveolae functions with genistein and methyl-ß-cyclodextrin limited nanoparticle internalization. The nanoparticles showed a very high delivery efficiency for the genetic material (a 47-base oligonucleotide) (∼80% incorporation into cells) as well as the purified protein (full length firefly luciferase, 67 kDa) into human embryonic kidney (HEK293T) cells. Luciferase enzyme activity in HEK293T cells demonstrated that intact and functional proteins could be delivered and showed a significant extension of activity retention up to 24 h, well beyond the 2 h half-life of the free enzyme. This study introduces a novel self-assembled oligo-urethane nanoparticle delivery platform with very low associated production costs, enabled by their scalable chemistry (the benchwork cost is $ 0.152/mg vs $ 974.6/mg for typical lipid carriers) that has potential to deliver both oligonucleotides and proteins for biomedical purposes.

Proteome analysis of secretions from human monocyte-derived macrophages post-exposure to biomaterials and the effect of secretions on cardiac fibroblast fibrotic character.

The use of exogenous biomolecules (BM) for the purpose of repairing and regenerating damaged cardiac tissue can yield serious side effects if used for prolonged periods. As well, such strategies can be cost prohibitive depending on the regiment and period of time applied. Alternatively, autologous monocytes/monocyte-derived macrophages (MDM) can provide a viable path towards generating an endogenous source of stimulatory BM. Biomaterials are often considered as delivery vehicles to generate unique profiles of such BM in tissues or to deliver autologous cells, that can influence the nature of BM produced by the cells. MDM cultured on a degradable polar hydrophobic ionic (D-PHI) polyurethane has previously demonstrated a propensity to increase select anti-inflammatory cytokines, and therefore there is good rationale to further investigate a broader spectrum of the cells' BM in order to provide a more complete proteomic analysis of human MDM secretions induced by D-PHI. Further, it is of interest to assess the potential of such BM to influence cells involved in the reparative state of vital tissues such as those that affect cardiac cell function. Hence, this current study examines the proteomic profile of MDM secretions using mass spectrometry for the first time, along with ELISA, following their culture on D-PHI, and compares them to two important reference materials, poly(lactic-co-glycolic acid) (PLGA) and tissue culture polystyrene (TCPS). Secretions collected from D-PHI cultured MDM led to higher levels of regenerative BM, AGRN, TGFBI and ANXA5, but lower levels of pro-fibrotic BM, MMP7, IL-1ß, IL-6 and TNFα,  when compared to MDM secretions collected from PLGA and TCPS. In the application to cardiac cell function, the secretion collected from D-PHI cultured MDM led to more human cardiac fibroblast (HCFs) migration. A lower collagen gel contraction induced by MDM secretions collected from D-PHI was supported by gene array analysis for human fibrosis-related genes. The implication of these findings is that more tailored biomaterials such as D-PHI, may lead to a lower pro-inflammatory phenotype of macrophages when used in cardiac tissue constructs, thereby enabling the development of vehicles for the delivery of interventional therapies, or be applied as coatings for sensor implants in cardiac tissue that minimize fibrosis. The general approach of using synthetic biomaterials in order to induce MDM secretions in a manner that will guide favorable regeneration will be critical in making the choice of biomaterials for tissue regeneration work in the future. STATEMENT OF SIGNIFICANCE: Immune modulation strategies currently applied in cardiac tissue repair are mainly based on the delivery of defined exogenous biomolecules. However, the use of such biomolecules may pose wide ranging systemic effects, thereby rendering them clinically less practical. The chemistry of biomaterials (used as a potential targeted delivery modality to circumvent the broad systemic effects of biomolecules) can not only affect acute and chronic toxicity but also alters the timeframe of the wound healing cascade. In this context, monocytes/monocyte-derive macrophages (MDM) can be harnessed as an immune modulating strategy to promote wound healing by an appropriate choice of the biomaterial. However, there are limited reports on the complete proteome analysis of MDM and their reaction of biomaterial related interventions on cardiac tissues and cells. No studies to date have demonstrated the complete proteome of MDM secretions when these cells were cultured on a non-traditional immune modulatory ionomeric polyurethane D-PHI film. This study demonstrated that MDM cultured on D-PHI expressed significantly higher levels of AGRN, TGFBI and ANXA5 but lower levels of MMP7, IL-1ß, IL-6 and TNFα when compared to MDM cultured on a well-established degradable biomaterials in the medical field, e.g. PLGA and TCPS, which are often used as the relative standards for cell culture work in the biomaterials field. The implications of these findings have relevance to the repair of cardiac tissues. In another aspect of the work, human cardiac fibroblasts showed significantly lower contractility (low collagen gel contraction and low levels of ACTA2) when cultured in the presence of MDM secretions collected after culturing them on D-PHI compared to PLGA and TCPS. The findings place emphasis on the importance of making the choice of biomaterials for tissue engineering and regenerative medicine applied to their use in cardiac tissue repair.