A limiting factor of traditional electrospinning is that the electrospun scaffolds consist entirely of tightly packed nanofiber layers that only provide a superficial porous structure due to the sheetlike assembly process. This unavoidable characteristic hinders cell infiltration and growth throughout the nanofibrous scaffolds. Numerous strategies have been tried to overcome this challenge, including the incorporation of nanoparticles, using larger microfibers, or removing embedded salt or water-soluble fibers to increase porosity. However, these methods still produce sheet-like nanofibrous scaffolds, failing to create a porous three-dimensional scaffold with good structural integrity. Thus, we have developed a three-dimensional cotton ball-like electrospun scaffold that consists of an accumulation of nanofibers in a low density and uncompressed manner. Instead of a traditional flat-plate collector, a grounded spherical dish and an array of needle-like probes were used to create a Focused, Low density, Uncompressed nanoFiber (FLUF) mesh scaffold. Scanning electron microscopy showed that the cotton ball-like scaffold consisted of electrospun nanofibers with a similar diameter but larger pores and less dense structure compared to the traditional electrospun scaffolds. In addition, laser confocal microscopy demonstrated an open porosity and loosely packed structure throughout the depth of the cotton ball-like scaffold, contrasting the superficially porous and tightly packed structure of the traditional electrospun scaffold. Cells seeded on the cotton ball-like scaffold infiltrated into the scaffold after 7 days of growth, compared to no penetrating growth for the traditional electrospun scaffold. Quantitative analysis showed approximately a 40% higher growth rate for cells on the cotton ball-like scaffold over a 7 day period, possibly due to the increased space for in-growth within the three-dimensional scaffolds. Overall, this method assembles a nanofibrous scaffold that is more advantageous for highly porous interconnectivity and demonstrates great potential for tackling current challenges of electrospun scaffolds.

Cardiovascular disease is the number one cause of death in the United States. Deployment of stents and vascular grafts has been a major therapeutic method for treatment. However, restenosis, incomplete endothelialization, and thrombosis hamper the long term clinical success. As a solution to meet these current challenges, we have developed a native endothelial ECM mimicking selfassembled nanofibrous matrix to serve as a new treatment model. The nanofibrous matrix is formed by self-assembly of peptide amphiphiles (PAs), which contain nitric oxide (NO) donating residues, endothelial cell adhesive ligands composed of YIGSR peptide sequence, and enzyme-mediated degradable sites. NO was successfully released from the nanofibrous matrix rapidly within 48 hours, followed by sustained release over period of 30 days. The NO releasing nanofibrous matrix demonstrated a significantly enhanced proliferation of endothelial cells (51 ± 3 % to 67 ± 2 %) but reduced proliferation of smooth muscle cells (35 ± 2 % to 16 ± 3 %) after 48 hrs of incubation. There was also a 150-fold decrease in platelet attachment on the NO releasing nanofibrous matrix (470 ± 220 platelets/cm 2 ) compared to the collagen-I (73 ± 22 × 10 3 platelets/cm 2 ) coated surface. The nanofibrous matrix has the potential to be applied to various cardiovascular implants as a selfassembled coating, thereby providing a native endothelial extracellular matrix (ECM) mimicking environment.

Peptide amphiphiles (PAs) are self-assembling molecules that form interwoven nanofiber gel networks. They have gained lots of attention because of their excellent biocompatibility, adaptable peptide structure that allows for specific biochemical functionality, and nanofibrous assembly that mimics natural tissue formation. However, variations in molecule length, charge, and intermolecular bonding between different bioactive PAs cause contrasting mechanical properties. This potentially limits cell-delivery therapies because scaffold durability is needed to withstand the rigors of clinician handling and transport to wound implant sites. Additionally, the mechanical properties have critical influence on cellular behavior, as the elasticity and stiffness of biomaterials have been shown to affect cell spreading, migration, contraction, and differentiation. Several different PAs have been synthesized, each endowed with specific cellular adhesive ligands for directed biological response. We have investigated mechanical means for modulating and stabilizing the gelation properties of PA hydrogels in a controlled manner. A more stable, biologically-inert PA (PA-S) was synthesized and combined with each of the bioactive PAs. Molar ratio (Mr = PA/PA-S) combinations of 3:1, 1:1, and 1:3 were tested. All PA composites were characterized by observed nanostructure and rheological analysis measuring viscoelasticity. It was found that the PAs could be combined to successfully control and stabilize the gelation properties, allowing for a mechanically-tunable scaffold with increased durability. Thus, the biological functionality and natural degradability of PAs can be provided in a more physiologically-relevant microenvironment using our composite approach to modulate the mechanical properties, thereby improving the vast potential for cell encapsulation and other tissue engineering applications. KeywordsPeptide amphiphile; hydrogel; viscoelasticity; biomimetic material; ECM (extracellular matrix)With ever expanding research into synthetic biomaterials, the importance of recapitulating the innate characteristics of biological tissue microenvironments, including the biochemical and mechanical properties, has gained greater importance. In particular, synthetic microenvironments need to provide both the spatial and temporal complexities necessary to guide cell-specific tissue development and function. 1 Peptide-based hydrogels offer one such promising biomaterial, crafted to simplistically mimic the native extracellular matrix (ECM). *Corresponding author: Dr. Ho-Wook Jun; hwjun@uab.edu; University of Alabama at Birmingham, Shelby 806, 1825 University Blvd., Birmingham, AL 35294-2182, United States; office: 205-996-6938; fax: 205-974-6938. Supporting Information Available: (1) Macroscopic gelation properties of peptide amphiphile composite hydrogels modulated at a 3:1 molar ratio. (2) Viscoelastic characterization using dynamic oscillatory rheometry to measure storage modulus in relation to frequency for peptide amphiphile compo...

BACKGROUND Human pluripotent stem cell (hPSC)-derived endothelial cells (ECs) have limited clinical utility due to undefined components in the differentiation system and poor cell survival in vivo. Here, we aimed to develop a fully defined and clinically compatible system to differentiate hPSCs into ECs. Further, we aimed to enhance cell survival, vessel-formation, and therapeutic potential by encapsulating hPSC-ECs with a peptide amphiphile (PA) nanomatrix gel. METHODS We induced differentiation of hPSCs into the mesodermal lineage by culturing on collagen-coated plates with a GSK3β inhibitor. Next, VEGF, EGF, and bFGF were added for endothelial lineage differentiation followed by sorting for CDH5 (VE-Cadherin). We constructed an extracellular matrix-mimicking PA nanomatrix gel (PA-RGDS) by incorporating the cell adhesive ligand Arg-Gly-Asp-Ser (RGDS) and a matrix metalloproteinase-2 degradable sequence. We then evaluated whether the encapsulation of hPSC-CDH5+ cells in PA-RGDS could enhance long-term cell survival and vascular regenerative effects in a hindlimb ischemia model using Laser Doppler perfusion imaging, bioluminescence imaging, real-time RT-PCR, and histological analysis. RESULTS The resultant hPSC-derived CDH5+ cells (hPSC-ECs) showed highly enriched and genuine EC characteristics and pro-angiogenic activities. When injected into ischemic hindlimbs, hPSC-ECs showed better perfusion recovery and higher vessel-forming capacity compared to media-, PA-RGDS-, or HUVEC-injected groups. However, the group receiving the PA-RGDS-encapsulated hPSC-ECs showed better perfusion recovery, more robust and longer cell survival (> 10 months), and higher and prolonged angiogenic and vascular incorporation capabilities than the bare hPSC-EC-injected group. Surprisingly, the engrafted hPSC-ECs demonstrated previously unknown sustained and dynamic vessel-forming behavior: initial perivascular concentration, a guiding role for new vessel formation, and progressive incorporation into the vessels over 10 months. CONCLUSION We generated highly enriched hPSC-ECs via a clinically compatible system. Further, this study demonstrated that a biocompatible PA-RGDS nanomatrix gel substantially improved long-term survival of hPSC-ECs in an ischemic environment and improved neovascularization effects of hPSC-ECs via prolonged and unique angiogenic and vessel-forming properties. This PA-RGDS-mediated transplantation of hPSC-ECs can serve as a novel platform for cell-based therapy and investigation of long-term behavior of hPSC-ECs.

Current cardiovascular therapies are limited by loss of endothelium, restenosis, and thrombosis. The goal of this study is to develop a biomimetic hybrid nanomatrix that combines unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but they are hampered by lack of surface bioactivity to control cellular behavior. It is hypothesized that PAs can self-assemble onto the surface of ePCL nanofibers and endow them with characteristic properties of native endothelium. PAs, which comprise hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell adhesive ligands (YIGSR) or ploylysine (KKKKK) nitric oxide (NO) donors. Two different PAs (PA-YIGSR and PA-KKKKK) were successfully synthesized and mixed in a 90:10 (YK) ratio to obtain PA-YK. PA-YK was reacted with pure NO to develop PA-YK-NO, which was then self-assembled onto ePCL nanofibers to generate a hybrid nanomatrix, ePCL-PA-YK-NO. Uniform coating of self-assembled PA nanofibers on ePCL was confirmed by TEM. Successful NO release from ePCL-PA-YK-NO was observed. ePCL-YK and ePCL-PA-YK-NO showed significantly increased adhesion of human umbilical vein endothelial cells (HUVECs). Also, ePCL-PA-YK-NO showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL-PA-YK-NO also displayed significantly reduced platelet adhesion when compared to ePCL, ePCL-PA-YK, and collagen control. These results indicate that this hybrid nanomatrix has great potential applications in cardiovascular implants.Corresponding author: Dr. Ho-Wook Jun, Assistant Professor, 1825 University Boulevard, Shelby 806, Birmingham, AL, 35211. Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final citable form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. Electrospinning has been garnering a lot of attention recently [7][8][9][10][11], due to its ability to fabricate highly interconnected, non-woven fibers with diameters in the nanoscale ranges, which are structurally similar to nanofibrillar extracellular matrix (ECM) proteins [12]. Due to their ability to physically resemble natural ECM protein structure, several studies have been conducted into using electrospun materials as cardiovascular devices such as vascular grafts [13][14][15][16][17]. An important feature of electrospinning is its ability to deposit these nanofibers on a rotating mandrel to form a tubular structure, which is essential for vascular grafts [18,19], and it is also possible to generate scaff...