Lesley W. Chow

Nitric oxide and nanotechnology: A novel approach to inhibit neointimal hyperplasia

OBJECTIVE Nitric oxide (NO) has been shown to inhibit neointimal hyperplasia after arterial interventions in several animal models. To date, however, NO-based therapies have not been used in the clinical arena. Our objective was to combine nanofiber delivery vehicles with NO chemistry to create a novel, more potent NO-releasing therapy that can be used clinically. Thus, the aim of this study was to evaluate the perivascular application of spontaneously self-assembling NO-releasing nanofiber gels. Our hypothesis was that this application would prevent neointimal hyperplasia. METHODS Gels consisted of a peptide amphiphile, heparin, and a diazeniumdiolate NO donor (1-[N-(3-Aminopropyl)-N-(3-ammoniopropyl)]diazen-1-ium-1,2-diolate [DPTA/NO] or disodium 1-[(2-Carboxylato)pyrrolidin-1-yl]diazen-1-ium-1,2-diolate [PROLI/NO]). Nitric oxide release from the gels was evaluated by the Griess reaction, and scanning electron microscopy confirmed nanofiber formation. Vascular smooth muscle cell (VSMC) proliferation and cell death were assessed in vitro by (3)H-thymidine incorporation and Personal Cell Analysis (PCA) system (Guava Technologies, Hayward, Calif). For the in vivo work, gels were modified by reducing the free-water content. Neointimal hyperplasia after periadventitial gel application was evaluated using the rat carotid artery injury model at 14 days (n = 6 per group). Inflammation and proliferation were examined in vivo with immunofluorescent staining against CD45, ED1, and Ki67 at 3 days (n = 2 per group), and graded by blinded observers. Endothelialization was assessed by Evans blue injection at 7 days (n = 3 per group). RESULTS Both DPTA/NO and PROLI/NO, combined with the peptide amphiphile and heparin, formed nanofiber gels and released NO for 4 days. In vitro, DPTA/NO inhibited VSMC proliferation and induced cell death to a greater extent than PROLI/NO. However, the DPTA/NO nanofiber gel only reduced neointimal hyperplasia by 45% (intima/media [I/M] area ratio, 0.45 +/- 0.07), whereas the PROLI/NO nanofiber gel reduced neointimal hyperplasia by 77% (I/M area ratio, 0.19 +/- 0.03, P < .05) vs control (injury alone I/M area ratio, 0.83 +/- 0.07; P < .05). Both DPTA/NO and PROLI/NO nanofiber gels significantly inhibited proliferation in vivo (1.06 +/- 0.30 and 0.19 +/- 0.11 vs injury alone, 2.02 +/- 0.20, P < .05), yet had minimal effect on apoptosis. Only the PROLI/NO nanofiber gel inhibited inflammation (monocytes and leukocytes). Both NO-releasing nanofiber gels stimulated re-endothelialization. CONCLUSIONS Perivascular application of NO-releasing self-assembling nanofiber gels is an effective and simple therapy to prevent neointimal hyperplasia after arterial injury. Our study demonstrates that the PROLI/NO nanofiber gel most effectively prevented neointimal hyperplasia and resulted in less inflammation than the DPTA/NO nanofiber gel. This therapy has great clinical potential to prevent neointimal hyperplasia after open vascular interventions in patients.

A bioactive self-assembled membrane to promote angiogenesis.

We report here on a bioactive hierarchically structured membrane formed by self-assembly. The membrane is formed with hyaluronic acid and peptide amphiphiles with binding affinity for heparin, and its hierarchical structure contains both an amorphous zone and a layer of fibrils oriented perpendicular to the membrane plane. The design of bioactivity is based on the potential ability to bind and slowly release heparin-binding growth factors. Human mesenchymal stem cells (hMSCs) seeded on these membranes attached and remained viable. Basic fibroblast growth factor (FGF2) and vascular endothelial growth factor (VEGF) were incorporated within the membrane structure prior to self-assembly and released into media over a prolonged period of time (14 days). Using the chicken chorioallantoic membrane (CAM) assay, we also found that these membranes induced a significant and rapid enhancement of angiogenesis relative to controls.

Growth Factor Delivery From Self-Assembling Nanofibers to Facilitate Islet Transplantation

Recent advances in nanotechnology and molecular self-assembly may provide novel solutions to current cell transplantation deficiencies. Heparin-binding peptide amphiphiles (HBPAs) self-assemble from aqueous media into nanofibers that bind growth factors through interactions with the bioactive polymer heparin. In this report, we demonstrate that delivery of vascular endothelial growth factor and fibroblast growth factor-2 from HBPA scaffolds significantly increases blood vessel density in the mouse omentum over control scaffolds without growth factors (P<0.0005) and significantly enhances islet engraftment. Diabetic recipients transplanted with 250 isologous islets and HBPA scaffolds containing vascular endothelial growth factor/fibroblast growth factor-2 achieved normoglycemia at a higher rate (78%) than control animals receiving identical scaffolds without growth factors (30%; P<0.05) or growth factors alone (20%). These data indicate that the enhanced engraftment can be attributed to specific growth factor effects that were made possible by the delivery mechanism of HBPA nanostructures.

Collagen-mimetic peptide-modifiable hydrogels for articular cartilage regeneration.

Regenerative medicine strategies for restoring articular cartilage face significant challenges to recreate the complex and dynamic biochemical and biomechanical functions of native tissues. As an approach to recapitulate the complexity of the extracellular matrix, collagen-mimetic proteins offer a modular template to incorporate bioactive and biodegradable moieties into a single construct. We modified a Streptococcal collagen-like 2 protein with hyaluronic acid (HA) or chondroitin sulfate (CS)-binding peptides and then cross-linked with a matrix metalloproteinase 7 (MMP7)-sensitive peptide to form biodegradable hydrogels. Human mesenchymal stem cells (hMSCs) encapsulated in these hydrogels exhibited improved viability and significantly enhanced chondrogenic differentiation compared to controls that were not functionalized with glycosaminoglycan-binding peptides. Hydrogels functionalized with CS-binding peptides also led to significantly higher MMP7 gene expression and activity while the HA-binding peptides significantly increased chondrogenic differentiation of the hMSCs. Our results highlight the potential of this novel biomaterial to modulate cell-mediated processes and create functional tissue engineered constructs for regenerative medicine applications.

Peptide-directed spatial organization of biomolecules in dynamic gradient scaffolds.

Specific binding peptides are used to spatially organize biomolecule gradients within an electrospun fiber scaffold. Different biomolecule-binding peptide-polymer conjugates are sequentially co-electrospun with a fiber-forming host polymer to generate opposing gradients of peptide functionalization. The binding peptides specifically and non-covalently guide the spatial arrangement of biomolecules into dynamic gradients within the scaffold, mimicking biological gradients found in native tissues.

Mapping Local Cytosolic Enzymatic Activity in Human Esophageal Mucosa with Porous Silicon Nanoneedles

Porous silicon nanoneedles can map Cathepsin B activity across normal and tumor human esophageal mucosa. Assembling a peptide‐based Cathepsin B cleavable sensor over a large array of nanoneedles allows the discrimination of cancer cells from healthy ones in mixed culture. The same sensor applied to tissue can map Cathepsin B activity with high resolution across the tumor margin area of esophageal adenocarcinoma.

The promotion of functional urinary bladder regeneration using anti-inflammatory nanofibers

Current attempts at tissue regeneration utilizing synthetic and decellularized biologic-based materials have typically been met in part by innate immune responses in the form of a robust inflammatory reaction at the site of implantation or grafting. This can ultimately lead to tissue fibrosis with direct negative impact on tissue growth, development, and function. In order to temper the innate inflammatory response, anti-inflammatory signals were incorporated through display on self-assembling peptide nanofibers to promote tissue healing and subsequent graft compliance throughout the regenerative process. Utilizing an established urinary bladder augmentation model, the highly pro-inflammatory biologic scaffold (decellularized small intestinal submucosa) was treated with anti-inflammatory peptide amphiphiles (AIF-PAs) or control peptide amphiphiles and used for augmentation. Significant regenerative advantages of the AIF-PAs were observed including potent angiogenic responses, limited tissue collagen accumulation, and the modulation of macrophage and neutrophil responses in regenerated bladder tissue. Upon further characterization, a reduction in the levels of M2 macrophages was observed, but not in M1 macrophages in control groups, while treatment groups exhibited decreased levels of M1 macrophages and stabilized levels of M2 macrophages. Pro-inflammatory cytokine production was decreased while anti-inflammatory cytokines were up-regulated in treatment groups. This resulted in far fewer incidences of tissue granuloma and bladder stone formation. Finally, functional urinary bladder testing revealed greater bladder compliance and similar capacities in groups treated with AIF-PAs. Data demonstrate that AIF-PAs can alleviate galvanic innate immune responses and provide a highly conducive regenerative milieu that may be applicable in a variety of clinical settings.

Electrostatic Control of Structure in Self‐Assembled Membranes

Self-assembling peptide amphiphiles (PAs) can form hierarchically ordered membranes when brought in contact with aqueous polyelectrolytes of the opposite charge by rapidly creating a diffusion barrier composed of filamentous nanostructures parallel to the plane of the incipient membrane. Following this event, osmotic forces and charge complexation template nanofiber growth perpendicular to the plane of the membrane in a dynamic self-assembly process. In this work, we show that this hierarchical structure requires massive interfacial aggregation of PA molecules, suggesting the importance of rapid diffusion barrier formation. Strong PA aggregation is induced here through the use of heparin-binding PAs with heparin and also with polyelectrolytes of varying charge density. Small angle X-ray scattering shows that in the case of weak PA-polyelectrolyte interaction, membranes formed display a cubic phase ordering on the nanoscale that likely results from clusters of PA nanostructures surrounded by polyelectrolyte chains.

Temporally degradable collagen–mimetic hydrogels tuned to chondrogenesis of human mesenchymal stem cells

Tissue engineering strategies for repairing and regenerating articular cartilage face critical challenges to recapitulate the dynamic and complex biochemical microenvironment of native tissues. One approach to mimic the biochemical complexity of articular cartilage is through the use of recombinant bacterial collagens as they provide a well–defined biological ‘blank template’ that can be modified to incorporate bioactive and biodegradable peptide sequences within a precisely defined three–dimensional system. We customized the backbone of a Streptococcal collagen–like 2 (Scl2) protein with heparin–binding, integrin–binding, and hyaluronic acid–binding peptide sequences previously shown to modulate chondrogenesis and then cross–linked the recombinant Scl2 protein with a combination of matrix metalloproteinase 7 (MMP7)– and aggrecanase (ADAMTS4)–cleavable peptides at varying ratios to form biodegradable hydrogels with degradation characteristics matching the temporal expression pattern of these enzymes in human mesenchymal stem cells (hMSCs) during chondrogenesis. hMSCs encapsulated within the hydrogels cross–linked with both degradable peptides exhibited enhanced chondrogenic characteristics as demonstrated by gene expression and extracellular matrix deposition compared to the hydrogels cross–linked with a single peptide. Additionally, these combined peptide hydrogels displayed increased MMP7 and ADAMTS4 activities and yet increased compression moduli after 6 weeks, suggesting a positive correlation between the degradation of the hydrogels and the accumulation of matrix by hMSCs undergoing chondrogenesis. Our results suggest that including dual degradation motifs designed to respond to enzymatic activity of hMSCs going through chondrogenic differentiation led to improvements in chondrogenesis. Our hydrogel system demonstrates a bimodal enzymatically degradable biological platform that can mimic native cellular processes in a temporal manner. As such, this novel collagen–mimetic protein, cross–linked via multiple enzymatically degradable peptides, provides a highly adaptable and well defined platform to recapitulate a high degree of biological complexity, which could be applicable to numerous tissue engineering and regenerative medicine applications.

Modular and Versatile Spatial Functionalization of Tissue Engineering Scaffolds through Fiber-Initiated Controlled Radical Polymerization

Native tissues are typically heterogeneous and hierarchically organized, and generating scaffolds that can mimic these properties is critical for tissue engineering applications. By uniquely combining controlled radical polymerization (CRP), end‐functionalization of polymers, and advanced electrospinning techniques, a modular and versatile approach is introduced to generate scaffolds with spatially organized functionality. Poly‐ε‐caprolactone is end functionalized with either a polymerization‐initiating group or a cell‐binding peptide motif cyclic Arg‐Gly‐Asp‐Ser (cRGDS), and are each sequentially electrospun to produce zonally discrete bilayers within a continuous fiber scaffold. The polymerization‐initiating group is then used to graft an antifouling polymer bottlebrush based on poly(ethylene glycol) from the fiber surface using CRP exclusively within one bilayer of the scaffold. The ability to include additional multifunctionality during CRP is showcased by integrating a biotinylated monomer unit into the polymerization step allowing postmodification of the scaffold with streptavidin‐coupled moieties. These combined processing techniques result in an effective bilayered and dual‐functionality scaffold with a cell‐adhesive surface and an opposing antifouling non‐cell‐adhesive surface in zonally specific regions across the thickness of the scaffold, demonstrated through fluorescent labelling and cell adhesion studies. This modular and versatile approach combines strategies to produce scaffolds with tailorable properties for many applications in tissue engineering and regenerative medicine.