Ho-Wook Jun

Cell infiltration and growth in a low density, uncompressed three-dimensional electrospun nanofibrous scaffold.

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 sheet-like 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.

A Nitric Oxide Releasing, Self Assembled Peptide Amphiphile Matrix that Mimics Native Endothelium for Coating Implantable Cardiovascular Devices

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 self-assembled 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 h, 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 h 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 x 10(3)platelets/cm(2)) coated surface. The nanofibrous matrix has the potential to be applied to various cardiovascular implants as a self-assembled coating, thereby providing a native endothelial extracellular matrix (ECM) mimicking environment.

Self-Assembling Peptide Amphiphile-Based Nanofiber Gel for Bioresponsive Cisplatin Delivery

The aim of this study is to develop a bioresponsive cisplatin (CDDP) delivery system with a self-assembling peptide amphiphile (PA) comprising a cell-adhesive matrix metalloproteinase-2 (MMP-2)-sensitive GTAGLIGQRGDS and a fatty acid. A biomimetic CDDP-PA gel was spontaneously formed upon incubating a mixture of CDDP and the PA for 5 h at 37 degrees C. CDDP-PA gel formation was confirmed by rheological analysis. The structure of self-assembled CDDP-PA nanofibers inside the gel was determined by transmission electron microscopy (TEM). Bioresponsive drug release from the biomimetic gel was demonstrated by in vitro MMP-2-triggered CDDP release. The MMP-2-sensitive CDDP release was dependent on the enzyme concentration in the medium. Enzymatic degradation of the CDDP-PA gel was confirmed by TEM images of the gel degraded in an MMP-2 containing medium. The MMP-2-triggered CDDP release as well as the presentation of RGDS in the gel would potentially provide a spatially and temporally controlled delivery system for targeted anticancer drug delivery.

Osteogenic differentiation of human mesenchymal stem cells directed by extracellular matrix-mimicking ligands in a biomimetic self-assembled peptide amphiphile nanomatrix

This study investigated the ability of nanoscale, biomimetic peptide amphiphile (PA) scaffolds inscribed with specific cellular adhesive ligands to direct the osteogenic differentiation of human mesenchymal stem cells (hMSCs) without osteogenic supplements. PA sequences were synthesized to mimic the native bone extracellular matrix (ECM), expressing different isolated ligands (i.e., RGDS, DGEA, KRSR). All PAs were presented as self-assembled two-dimensional coatings for the seeded hMSCs. Initial attachment results demonstrated that the different PAs could be individually recognized based on the incorporated adhesive ligands. Long-term studies assessed osteogenic differentiation up to 35 days. The RGDS-containing PA nanomatrix expressed significantly greater alkaline phosphatase activity, indicating the early promotion of osteogenic differentiation. A progressive shift toward osteogenic morphology and positive staining for mineral deposition provided further confirmation of the RGDS-containing PA nanomatrix. Overall, the PA nanomatrix clearly has great promise for directing the osteogenic differentiation of hMSCs without the aid of supplements by mimicking the native ECM, providing an adaptable environment that allows for different adhesive ligands to control cellular behaviors. This research model establishes the beginnings of a new versatile approach to regenerate bone tissues by closely following the principles of natural tissue formation.

Modulating the Gelation Properties of Self-Assembling Peptide Amphiphiles

Peptide amphiphiles (PAs) are self-assembling molecules that form interwoven nanofiber gel networks. They have gained a lot 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 (M(r) = 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.

Development of a YIGSR-peptide-modified polyurethaneurea to enhance endothelialization

Polyurethanes have been investigated for use as vascular grafts due to their excellent mechanical properties and relatively good biocompatibility. However, poor retention of endothelial cells and thrombogenicity in vivo remain problematic for vascular graft applications. The peptide YIGSR has been shown to increase endothelial cell adhesion but not attachment of platelets, suggesting its possible utility for vascular graft applications. In this study, a bioactive polyurethaneurea has been synthesized by incorporating GGGYIGSRGGGK peptide sequences into the polymer backbone. Successful incorporation of the peptides was confirmed by NMR, contact angle measurement and ESCA. Uniform distribution of peptides on the surface was observed using a fluorescent probe capable of reacting with tyrosine residues on the peptides. Hard segment domains were visualized using tapping mode AFM. Endothelial cell adhesion, spreading, proliferation, migration and extra-cellular matrix production were improved on bioactive polyurethaneurea compared to control polyurethaneurea. Competitive inhibition of endothelial cell attachment and spreading by soluble YIGSR peptides indicated that cell adhesion and spreading were specifically mediated by YIGSR-sensitive cell adhesion receptor, not just by changed surface properties. There was no significant difference in the number of adherent platelets. Therefore, this bioactive polyurethanurea may improve vascular graft endothelialization without increasing thrombogenicity.

A hybrid biomimetic nanomatrix composed of electrospun polycaprolactone and bioactive peptide amphiphiles for cardiovascular implants

Current cardiovascular therapies are limited by the loss of endothelium, restenosis and thrombosis. The goal of this study was to develop a biomimetic hybrid nanomatrix that combined the unique properties of electrospun polycaprolactone (ePCL) nanofibers with self-assembled peptide amphiphiles (PAs). ePCL nanofibers have interconnected nanoporous structures, but are hampered by a lack of surface bioactivity to control cellular behavior. It has been hypothesized that PAs could self-assemble onto the surface of ePCL nanofibers and endow them with the characteristic properties of native endothelium. The PAs, which comprised hydrophobic alkyl tails attached to functional hydrophilic peptide sequences, contained enzyme-mediated degradable sites coupled to either endothelial cell-adhesive ligands (YIGSR) or polylysine (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 transmission electron microscopy. 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). ePCL-PA-YK-NO also showed significantly increased proliferation of HUVECs and reduced smooth muscle cell proliferation. ePCL-PA-YK-NO also displayed significantly reduced platelet adhesion compared with ePCL, ePCL-PA-YK and a collagen control. These results indicate that this hybrid nanomatrix has great potential application in cardiovascular implants.

Biomimetic self-assembled nanofibers

Peptide-amphiphiles, peptides to which a non-peptidic hydrophobic moiety has been added to the N or C terminal end, have been demonstrated to be a versatile method for simultaneously controlling nanostructure and chemical functionality. These amphiphiles are able to self-assemble, in a controlled fashion, into nanofibers with diameter between 6-10 nm and with length in excess of 1000 nm. At proper concentration these nanofibers form a viscoelastic gel capable of entrapping living cells and eliciting specific responses from them. Because of the flexibility of the display of chemical functionality on a controlled nanofibrous scaffold, applications for peptide-amphiphiles have been proposed including heterogeneous catalysis, nanoelectronics, drug delivery, and tissue engineering.

Osteogenic differentiation of human mesenchymal stem cells synergistically enhanced by biomimetic peptide amphiphiles combined with conditioned medium.

An attractive strategy for bone tissue engineering is the use of extracellular matrix (ECM) analogous biomaterials capable of governing biological response based on synthetic cell-ECM interactions. In this study, peptide amphiphiles (PAs) were investigated as an ECM-mimicking biomaterial to provide an instructive microenvironment for human mesenchymal stem cells (hMSCs) in an effort to guide osteogenic differentiation. PAs were biologically functionalized with ECM isolated ligand sequences (i.e. RGDS, DGEA), and the osteoinductive potential was studied with or without conditioned medium, containing the supplemental factors of dexamethasone, β-glycerol phosphate and ascorbic acid. It was hypothesized that the ligand-functionalized PAs would synergistically enhance osteogenic differentiation in combination with conditioned medium. Concurrently, comparative evaluations independent of osteogenic supplements investigated the differentiating potential of the functionalized PA scaffolds as promoted exclusively by the inscribed ligand signals, thus offering the potential for therapeutic effectiveness under physiological conditions. Osteoinductivity was assessed by histochemical staining for alkaline phosphatase (ALP) and quantitative real-time polymerase chain reaction analysis of key osteogenic markers. Both of the ligand-functionalized PAs were found to synergistically enhance the level of visualized ALP activity and osteogenic gene expression compared to the control surfaces lacking biofunctionality. Guided osteoinduction was also observed without supplemental aid on the PA scaffolds, but at a delayed response and not to the same phenotypic levels. Thus, the biomimetic PAs foster a symbiotic enhancement of osteogenic differentiation, demonstrating the potential of ligand-functionalized biomaterials for future bone tissue repair.

Effect of endothelium mimicking self-assembled nanomatrices on cell adhesion and spreading of human endothelial cells and smooth muscle cells

UNLABELLED The goal of this study is to develop unique native endothelium mimicking nanomatrices and evaluate their effects on adhesion and spreading of human umbilical vein endothelial cells (HUVECs) and aortic smooth muscle cells (AoSMCs). These nanomatrices were developed by self-assembly of peptide amphiphiles (PAs) through a solvent evaporation technique. Three PAs, one containing the Tyr-Ile-Gly-Ser-Arg (YIGSR) ligand, the second containing the Val-Ala-Pro-Gly (VAPG) ligand, and a third without cell adhesive ligands, were developed. Cell adhesion and spreading were evaluated by a PicoGreen-DNA assay and live/dead assay, respectively. Our results show that PA-YIGSR significantly enhances HUVEC adhesion (26,704 +/- 2708), spreading (84 +/- 8%), and proliferation (50 +/- 2%) compared with that of other PAs. PA-VAPG and PA-YIGSR showed significantly greater AoSMC adhesion compared with that of PA-S. PA-VAPG also showed significantly greater spreading of AoSMCs (63 +/- 11%) compared with that of other PAs. Also, all the PAs showed significantly reduced platelet adhesion compared with that of collagen I (control). These findings would facilitate the development of novel vascular grafts, heart valves, and cell-based therapies for cardiovascular diseases. FROM THE CLINICAL EDITOR The goal of this study was to develop unique native endothelium mimicking nanomatrices and evaluate their effects on adhesion and spreading of human umbilical vein endothelial cells (HUVECs) and aortic smooth muscle cells (AoSMCs). These nanomatrices were developed by self-assembly of peptide amphiphiles through a solvent evaporation technique. The findings are expected to facilitate the development of novel vascular grafts, heart valves, and cell based therapies for cardiovascular diseases.