Vivek A. Kumar

Cell surface engineering with polyelectrolyte multilayer thin films.

Layer-by-layer assembly of polyelectrolyte multilayer (PEM) films represents a bottom-up approach for re-engineering the molecular landscape of cell surfaces with spatially continuous and molecularly uniform ultrathin films. However, fabricating PEMs on viable cells has proven challenging owing to the high cytotoxicity of polycations. Here, we report the rational engineering of a new class of PEMs with modular biological functionality and tunable physicochemical properties which have been engineered to abrogate cytotoxicity. Specifically, we have discovered a subset of cationic copolymers that undergoes a conformational change, which mitigates membrane disruption and facilitates the deposition of PEMs on cell surfaces that are tailorable in composition, reactivity, thickness, and mechanical properties. Furthermore, we demonstrate the first successful in vivo application of PEM-engineered cells, which maintained viability and function upon transplantation and were used as carriers for in vivo delivery of PEMs containing biomolecular payloads. This new class of polymeric film and the design strategies developed herein establish an enabling technology for cell transplantation and other therapies based on engineered cells.

The use of microfiber composites of elastin-like protein matrix reinforced with synthetic collagen in the design of vascular grafts

Collagen and elastin networks contribute to highly specialized biomechanical responses in numerous tissues and species. Biomechanical properties such as modulus, elasticity, and strength ultimately affect tissue function and durability, as well as local cellular behavior. In the case of vascular bypass grafts, compliance at physiologic pressures is correlated with increased patency due to a reduction in anastomotic intimal hyperplasia. In this report, we combine extracellular matrix (ECM) protein analogues to yield multilamellar vascular grafts comprised of a recombinant elastin-like protein matrix reinforced with synthetic collagen microfibers. Structural analysis revealed that the fabrication scheme permits a range of fiber orientations and volume fractions, leading to tunable mechanical properties. Burst strengths of 239-2760 mm Hg, compliances of 2.8-8.4%/100 mm Hg, and suture retention strengths of 35-192 gf were observed. The design most closely approximating all target criteria displayed a burst strength of 1483 +/- 143 mm Hg, a compliance of 5.1 +/- 0.8%/100 mm Hg, and a suture retention strength of 173 +/- 4 gf. These results indicate that through incorporation of reinforcing collagen microfibers, recombinant elastomeric protein-based biomaterials can play a significant role in load bearing tissue substitutes. We believe that similar composites can be incorporated into tissue engineering schemes that seek to integrate cells within the structure, prior to or after implantation in vivo.

Highly Angiogenic Peptide Nanofibers

Major limitations of current tissue regeneration approaches using artificial scaffolds are fibrous encapsulation, lack of host cellular infiltration, unwanted immune responses, surface degradation preceding biointegration, and artificial degradation byproducts. Specifically, for scaffolds larger than 200-500 μm, implants must be accompanied by host angiogenesis in order to provide adequate nutrient/waste exchange in the newly forming tissue. In the current work, we design a peptide-based self-assembling nanofibrous hydrogel containing cell-mediated degradation and proangiogenic moieties that specifically address these challenges. This hydrogel can be easily delivered by syringe, is rapidly infiltrated by cells of hematopoietic and mesenchymal origin, and rapidly forms an extremely robust mature vascular network. Scaffolds show no signs of fibrous encapsulation and after 3 weeks are resorbed into the native tissue. These supramolecular assemblies may prove a vital paradigm for tissue regeneration and specifically for ischemic tissue disease.

Self-assembling multidomain peptides tailor biological responses through biphasic release

Delivery of small molecules and drugs to tissues is a mainstay of several tissue engineering strategies. Next generation treatments focused on localized drug delivery offer a more effective means in dealing with refractory healing when compared to systemic approaches. Here we describe a novel multidomain peptide hydrogel that capitalizes on synthetic peptide chemistry, supramolecular self-assembly and cytokine delivery to tailor biological responses. This material is biomimetic, shows shear stress recovery and offers a nanofibrous matrix that sequesters cytokines. The biphasic pattern of cytokine release results in the spatio-temporal activation of THP-1 monocytes and macrophages. Furthermore, macrophage-material interactions are promoted without generation of a proinflammatory environment. Subcutaneous implantation of injectable scaffolds showed a marked increase in macrophage infiltration and polarization dictated by cytokine loading as early as 3 days, with complete scaffold resorption by day 14. Macrophage interaction and response to the peptide composite facilitated the (i) recruitment of monocytes/macrophages, (ii) sustained residence of immune cells until degradation, and (iii) promotion of a pro-resolution M2 environment. Our results suggest the potential use of this injectable cytokine loaded hydrogel scaffold in a variety of tissue engineering applications.

A Nanostructured Synthetic Collagen Mimic for Hemostasis

Collagen is a major component of the extracellular matrix and plays a wide variety of important roles in blood clotting, healing, and tissue remodeling. Natural, animal derived, collagen is used in many clinical applications but concerns exist with respect to its role in inflammation, batch-to-batch variability, and possible disease transfection. Therefore, development of synthetic nanomaterials that can mimic the nanostructure and properties of natural collagen has been a heavily pursued goal in biomaterials. Previously, we reported on the design and multihierarchial self-assembly of a 36 amino acid collagen mimetic peptide (KOD) that forms nanofibrous triple helices that entangle to form a hydrogel. In this report, we utilize this nanofiber forming collagen mimetic peptide as a synthetic biomimetic matrix useful in thrombosis. We demonstrate that nanofibrous KOD synthetic collagen matrices adhere platelets, activate them (indicated by soluble P-selectin secretion), and clot plasma and blood similar to animal derived collagen and control surfaces. In addition to the thrombotic potential, THP-1 monocytes incubated with our KOD collagen mimetic showed minimal proinflammatory cytokine (TNF-α or IL-1β) production. Together, the data presented demonstrates the potential of a novel synthetic collagen mimetic as a hemostat.

Fibrillogenesis in Continuously Spun Synthetic Collagen Fiber

The universal structural role of collagen fiber networks has motivated the development of collagen gels, films, coatings, injectables, and other formulations. However, reported synthetic collagen fiber fabrication schemes have either culminated in short, discontinuous fiber segments at unsuitably low production rates, or have incompletely replicated the internal fibrillar structure that dictates fiber mechanical and biological properties. We report a continuous extrusion system with an off-line phosphate buffer incubation step for the manufacture of synthetic collagen fiber. Fiber with a cross-section of 53+ or - 14 by 21 + or - 3 microm and an ultimate tensile strength of 94 + or - 19 MPa was continuously produced at 60 m/hr from an ultrafiltered monomeric collagen solution. The effect of collagen solution concentration, flow rate, and spinneret size on fiber size was investigated. The fiber was further characterized by microdifferential scanning calorimetry, transmission electron microscopy (TEM), second harmonic generation (SHG) analysis, and in a subcutaneous murine implant model. Calorimetry demonstrated stabilization of the collagen triple helical structure, while TEM and SHG revealed a dense, axially aligned D-periodic fibril structure throughout the fiber cross-section. Implantation of glutaraldehyde crosslinked and noncrosslinked fiber in the subcutaneous tissue of mice demonstrated limited inflammatory response and biodegradation after a 6-week implant period.

Two-Step Self-Assembly of Liposome-Multidomain Peptide Nanofiber Hydrogel for Time-Controlled Release

Progress in self-assembly and supramolecular chemistry has been directed toward obtaining macromolecular assemblies with higher degrees of complexity, simulating the highly structured environment in natural systems. One approach to this type of complexity are multistep, multicomponent, self-assembling systems that allow approaches comparable to traditional multistep synthetic organic chemistry; however, only a few examples of this approach have appeared in the literature. Our previous work demonstrated nanofibrous mimics of the extracellular matrix. Here we demonstrate the ability to create a unique hydrogel, developed by stepwise self-assembly of multidomain peptide fibers and liposomes. The two-component system allows for controlled release of bioactive factors at multiple time points. The individual components of the self-assembled gel and the composite hydrogel were characterized by TEM, SEM, and rheometry, demonstrating that peptide nanofibers and lipid vesicles both retain their structural integrity in the composite gel. The rheological robustness of the hydrogel is shown to be largely unaffected by the presence of liposomes. Release studies from the composite gels loaded with different growth factors EGF, MCP-1, and PlGF-1 showed delay and prolongation of release by liposomes entrapped in the hydrogel compared to more rapid release from the hydrogel alone. This bimodal release system may have utility in systems where timed cascades of biological signals may be valuable, such as in tissue regeneration.

Biodegradable poly(diol citrate) nanocomposite elastomers for soft tissue engineering

At present, synthetic biodegradable polymers commonly used for scaffolds in tissue engineering have a limited range of mechanical properties. This limitation is a challenge to in vivo tissue engineering, as the cell-scaffold construct is expected to maintain or restore normal tissue biomechanics during new tissue formation. Herein we report the synthesis and characterization of biodegradable elastomeric nanocomposite materials whose mechanical properties can be tailored to meet the requirements of soft tissue engineering applications. The nanocomposite consists of a nanofibrous poly(L-lactic acid) (PLLA) nanophase and an elastomeric poly(diol citrate) macrophase. Incorporation of a PLLA nanophase provides reinforcement to the poly(diol citrate) as demonstrated by an increase in tensile strength, modulus, and elongation at break with minimal permanent deformation. The mechanical properties of the nanocomposite were altered with the concentration of PLLA, choice of poly(diol citrate), and polymerization conditions. More importantly, the tensile mechanical properties compare favorably to those of human cartilage, ligament, and blood vessel. Furthermore, the compressive modulus is very similar to those of human and bovine articular cartilage. These results suggest that poly(diol citrate) nanocomposite elastomers are promising candidate biomaterials for soft tissue engineering.

Microcrimped Collagen Fiber-Elastin Composites

Emerging biomaterials based upon analogues of native extracellular matrix proteins provide an opportunity to create protein scaffolds that mimic tissue mechanical behavior and guide cellular responses. However, in order to reproduce macroscale tissue properties, protein analogues must be endowed with appropriate microstructural features. In particular, the crimped or wavy microstructure of native collagen fibers, with a periodicity of 10 – 200 μm, contributes in a significant manner to the compliance, strength, and durability of soft tissues. In this report, we describe a templating strategy based upon the application of micropatterned elastomeric substrates, which yields dense, aligned arrays of synthetic collagen microfibers that display a well-defined microcrimped pattern. Following crosslinking with glutaraldehyde vapor, fiber arrays were embedded in a recombinant elastin protein polymer,[1] which contributes to the resilience of the composite structure by bearing tensile loads at low strains, analogous to a native elastin fiber network.[2, 3] We demonstrate the preservation of fiber crimp after repetitive cyclic loading, as well as the assembly of hierarchical microcrimped multilamellar composites with mechanical responses similar to native tissues. The periodic waviness of fibrous collagen is observed in nearly all human tissues, including blood vessels, valve leaflets, intestine, tendon, and intervertebral discs.[4-6] The morphological features of crimp structure has been characterized as planar zig-zag,[7] sinusoid,[8] or helical[9, 10] with wavelengths between 10 to 200 μm. Crimp ensures that at low levels of tensile strain, loads are sustained both by the surrounding matrix and the fiber network. Typically, fibers straighten as a load is imposed with an observed transition from low to high tissue stiffness.[4, 5, 11, 12] These mechanisms serve to enhance compliance at low strain while generating greater strength as load increases. Since physiologic strains are imposed at levels of stress where fibers are often not fully extended, the propensity for fatigue-related fiber damage is minimized. All told, fiber crimp has evolved as an important bioengineering principle that affords a favorable combination of compliance, strength, and durability. A set of techniques using soft, contracting substrates to shape thin coatings of high modulus materials into crimped, wrinkled, and wavy structures has recently emerged.[13-19] For example, Bowden and colleagues deposited metal films on heated PDMS and noted that, upon cooling, the contraction of the PDMS buckled the metal layer into sophisticated patterns of wrinkles.[13] However, in these cases the extent of waviness is limited by the extent of inducible thermal shrinkage. Alternatively, an elastomeric substrate may be mechanically stretched prior to the application of a thin film,[15] array of nanoribbons,[14, 16] integrated circuit,[17] or carbon nanotubes,[18] with relaxation of stretch producing defined wavy structures. Collectively, these studies have lead to the fabrication of controlled micro- and nano-scale waveforms. However, microcrimping techniques have not been developed that are suitable for biological materials, such as collagen fibers.

Treatment of hind limb ischemia using angiogenic peptide nanofibers

For a proangiogenic therapy to be successful, it must promote the development of mature vasculature for rapid reperfusion of ischemic tissue. Whole growth factor, stem cell, and gene therapies have yet to achieve the clinical success needed to become FDA-approved revascularization therapies. Herein, we characterize a biodegradable peptide-based scaffold engineered to mimic VEGF and self-assemble into a nanofibrous, thixotropic hydrogel, SLanc. We found that this injectable hydrogel was rapidly infiltrated by host cells and could be degraded while promoting the generation of neovessels. In mice with induced hind limb ischemia, this synthetic peptide scaffold promoted angiogenesis and ischemic tissue recovery, as shown by Doppler-quantified limb perfusion and a treadmill endurance test. Thirteen-month-old mice showed significant recovery within 7 days of treatment. Biodistribution studies in healthy mice showed that the hydrogel is safe when administered intramuscularly, subcutaneously, or intravenously. These preclinical studies help establish the efficacy of this treatment for peripheral artery disease due to diminished microvascular perfusion, a necessary step before clinical translation. This peptide-based approach eliminates the need for cell transplantation or viral gene transfection (therapies currently being assessed in clinical trials) and could be a more effective regenerative medicine approach to microvascular tissue engineering.