In natural situations, angiogenic growth factors such as vascular endothelial growth factor (VEGF) are present in a matrix-bound form, yet therapeutic use of such growth factors has focused on application in soluble form. To explore matrix immobilization of angiogenic growth factors, we have explored three approaches: enzymatic conjugation of variant forms of the growth factors, complexation with recombinant variants of fibronectin, and engineering growth factors fused to a domain that binds strongly to extracellular matrix proteins. In the approach of enzymatic conjugation, which has been taken to clinical studies, growth factors are engineered so as to contain a substrate domain for the coagulation transglutaminase factor XIIIa, modeled after the N terminus of alpha2 plasmin inhibitor. To provide a release mechanism, an enzymatic substrate was included in the growth factor variant between the transglutaminase substrate and the growth factor domain, taken as either a plasmin substrate domain or a matrix metalloproteinase domain. Thus, we have explored the activity of tripartite fusion proteins for inducing angiogenesis. In a second approach, to explore noncovalent immobilization upon matrices, we have engineered a fibrin-binding domain of fibronectin, containing the 12th-14th type III repeat (which was known to bind VEGF-A). In studies of the 12th-14th type III repeat, we determined that the growth factor binding activity of this domain was rather promiscuous, binding to VEGF-A, VEGF-C, PDGF-AA, PDGF-BB and PDGF-AB, for example, in addition to a wide number of other growth factors. Incorporation of this domain into fibrin, also through transglutaminase activity, provides a powerful and generalizable method to retain such growth factors into surgical matrices. In a third approach, broad extracellular matrix binding capacity is engineered into the growth factors themselves, by fusion with a domain from placenta growth factor-2 (PlGF-2), which we determined to have such favorable binding characteristics. PlGF-2, and fusions containing a domain of PlGF-2, were shown to bind with nM affinity to fibronectin, fibrinogen, tenascin C and other matrix proteins. In this way, either a fibrin matrix or a tissue itself may be turned into a reservoir for growth factor sequestration and presentation. Examples in bone and chronic wound repair have demonstrated the power of this approach.

The current challenge in the treatment of autoimmune diseases is the development of therapies that induce antigen-specific immunological tolerance, which would in turn circumvent the present need for generalized immunosuppression that results in increased susceptibilities to potentially life-threatening infections and the development of neoplasia. We had previously shown that antigen (Ag) coupled to apoptotic splenocytes was capable of inducing Ag-specific immunological tolerance that ultimately resulted in the prevention and treatment of relapsing-remitting experimental autoimmune encephalomyelitis (R-EAE). This approach is the focus of a Phase I/IIa clinical trial examining the effects of immunological tolerance using multiple myelin peptides coupled to autologous peripheral blood leukocytes in early relapsing-remitting multiple sclerosis (MS) patients. In order to facilitate translation into the clinic and to more elegantly and precisely probe the mechanisms of tolerance induction, we have engineered a robust multi-functional particle-based platform for the delivery of Ag in a fashion that induces Ag-specific immunological tolerance in the R-EAE SJL/J mouse model of MS. Particles were prepared and characterized by photon correlation spectroscopy and scanning electron microscopy. Antigen (PLP139-151 peptide or OVA323-339 peptide) was attached to the particles by chemical cross-linking. R-EAE was induced in SJL/J mice by immunization with PLP139-151 peptide in complete Freund&’s adjuvant (CFA). The establishment of immunological tolerance in R-EAE was determined by the prevention of paralysis development in SJL/J mice post-immunization, relative to the control. We have shown that particles chemically cross-linked with the myelin epitope PLP139-151 were capable of completely preventing and treating R-EAE. Therapy was initiated either before the induction of disease or at peak disease severity during the acute onset of disease. In particular, PLP139-151-coupled particles inhibit the development and prevent the relapse of R-EAE more effectively than PLP139-151-coupled apoptotic splenocytes. Finally, we have determined that fluorescent particles cross-linked with Ag localize to the marginal zones within the spleen, an organ critical for the induction of immunological tolerance. Progress on understanding the mechanisms leading to the induction of immunological tolerance will also be discussed. We envision that our multi-functional particles will provide a robust off-the-shelf Ag delivery platform for the induction of Ag-specific immunological tolerance, thus enabling the facile treatment of autoimmune diseases, with minimal side-effects. In addition, our particle-based platform can be easily functionalized with various immunogenic and tolerogenic signals to allow for the intricate interrogation of the mechanisms underlying the induction of Ag-specific immunologic tolerance.

Recent research into the immune system has revealed that foreign pathogens are detected through a series of receptors on antigen presenting cells. These receptors are synergistically activated by multiple pathogen associated molecular patterns. Materials scientists have a large role to play in the coordinated design of vaccines and synthetic activators of the immune system. We report on our development of chemical and polymeric tools for interacting with the immune system. Our methods involve the bioconjugation of multiple different PAMPs to polymeric scaffolds. These synergistic PAMP scaffolds have been tested against dendritic cells and provide enhanced immune activation. We will report on the results. Additionally, we will report on our work coupling these synergistic combinations to cell-surface and other antigen rich environments.

The design of chemically defined biomaterials for use as vaccines or adjuvants requires a careful balancing of multiple desired functionalities: (i) strong immunogenicity, to raise a robust antibody or T cell response, (ii) a non-inflammatory response profile, and (iii) ready incorporation of one or more antigens, particularly epitopes for B and T cells, ideally without the use of carrier proteins, which complicate the design and manufacture of peptide- and carbohydrate-based vaccines.

We recently developed a self-adjuvanting vaccine platform based on self-assembled beta-sheet nanofibers of the peptide Q11 (QQKFQFQFEQQ), which can present conjugated antigens at high density and raise long-lived antibody responses against them in mice. Now, we show for a model peptide antigen, ovalbumin(323-339), that Q11 fibers with this antigen (OVA-Q11) raised a protective and high-affinity antibody response without inducing detectable inflammation at the injection site, in contrast to aluminum and magnesium hydroxide particles, a common but relatively inflammatory clinical adjuvant. OVA-Q11 fibers induced negligible swelling after injection into the mouse footpad, and failed to induce recruitment of inflammatory cells or secretion of any of a panel of six inflammatory cytokines after injection into the peritoneal cavity. Nevertheless, serum from OVA-Q11 immunized mice was protective in an in vitro assay of haemagglutinin inhibition against an OVA-expressing influenza virus.
Furthermore, we utilized the modular self-assembly of these fibers to combine a peptide antigen that contained only a B cell (antibody) epitope from Staphylococcus aureus with a separate T cell epitope peptide. Co-assembly of B and T epitopes on Q11 fibers facilitated testing of different B : T mol ratios to optimize antibody titer, affinity, and protection. This straightforward co-assembly approach eliminated the need for conjugation of peptide antigens to carrier proteins, which must be produced anew for each mol ratio and antigen of interest. In summary, the Q11 nanofiber system described here may be useful as a non-inflammatory vaccine platform for modular combination of B and T epitopes, so that protective antibody responses can be raised by a fully chemically defined material.

Engineered nano- and microparticles that co-deliver antigen and immunostimulatory molecules are of interest as next-generation subunit vaccines and so-called ‘smart&’ adjuvants, given their ability to mimic pathogens while avoiding toxicity and anti-vector immune responses. To demonstrate that mesoporous oxide nanoparticles warrant development as particulate vaccines and adjuvants, we co-loaded mesoporous silica nanoparticles (MSNPs) with a model protein antigen, ovalbumin (OVA), and an immunostimulatory RNA (isRNA) known to activate Toll-like receptor (TLR) 7 and TLR8 and then encapsulated cargo-loaded MSNPs in a supported lipid bilayer (SLB) that we further modified with various targeting and endosomolytic moieties. We found that high-surface-area MSNPs are able to encapsulate 50-60 wt% of OVA or isRNA individually and simultaneously encapsulate ~30 wt% of both OVA and isRNA, capacities that exceed those of state-of the-art liposomes and polymeric nanoparticles by 2 to 100-fold. We, furthermore, found that a SLB composition of DOPC modified with 5 wt% of the TLR4 agonist, monophosphoryl lipid A (MPLA) triggers efficient uptake of MSNPs by human dendritic cells (DCs) derived from peripheral blood monocytes. We have previously shown that endolysosome acidification destabilizes the SLB, thereby exposing the MSNP core and stimulating its dissolution (see Nature Materials (2011) 10: 389-397); to confirm this result, we showed that MSNPs stably encapsulate OVA and isRNA for two months when stored in a pH 7.4 buffer at 4°C but release 90% of their encapsulated cargo within 12 hours when exposed to a pH 5 buffer at 37°C. We also employed fluorescently-labeled OVA and isRNA to demonstrate that incorporating endosomolytic peptides (e.g. ‘H5WYG&’) and/or lipids (e.g. DOPE) in the SLB enables endosomal release of isRNA and cytosolic dispersion of OVA, which, in turn, trigger DC maturation (determined by staining for CD80 and CD86) and cross-presentation of OVA-derived peptides (determined by staining for MHC class I H-2K^b molecules complexed with SIINFEKL). Furthermore, DCs pulsed with MPLA-targeted MSNPs loaded with OVA and isRNA induce vigorous proliferation of OVA-specific CD8⁺ T cells, whereas DCs pulsed with free OVA or OVA complexed with Imject Alum trigger weak T cell responses. Upon immunization of C57Bl/6 mice, MPLA-targeted MSNPs loaded with OVA and isRNA, additionally, induce high-titer (>10⁵), OVA-specific IgG responses that are 100- and 10,000-fold higher than the titers achieved using OVA complexed to Imject Alum and free OVA, respectively. Our results indicate that mesoporous oxide nanoparticles are an important class of antigen delivery vehicles and warrant further development. We are currently working to enhance the efficacy of antigen presenting cell-targeted, antigen-loaded oxide nanoparticles by utilizing mesoporous alum for antigen adsorption, presentation, and delivery.

Molecular conjugates and nanoparticles that rapidly transport antigen and immunostimulatory adjuvant molecules from injection sites through lymphatics to lymph nodes are of great interest as synthetic vaccine vectors. To design molecular vaccines that combine precise physical characteristics with the manufacturing and scalability advantages of bottom-up systems, we explored well-defined polymeric amphiphiles with the general structure (lipid mimic)-(polar block)-(cargo), where the lipid block controls interactions with serum proteins and the polar moiety controls self-assembly and solubility properties of the constructs. Incorporation of a polar block comprised of an oligoguanine repeat gave amphiphiles that self-assemble into nanoparticles with tunable serum stability determined by G-quartet-formation between adjacent G repeats. By varying the composition of the lipophilic tail and polar blocks of these amphiphiles, we defined characteristics for optimized lymph node targeting of peptide antigens and oligonucleotide adjuvants, which led to >10-fold enhancements in the immune response stimulated by these molecular vaccines. Application of this approach to vaccines for infectious disease and strategies for tumor immunotherapy will be discussed.

Current treatments for Th2-associated allergic diseases, including allergic asthma and food allergies, are inefficient, with immunotherapies carrying significant risks of inducing anaphylaxis. As such, there exists an urgent need to develop new treatments that safely and efficiently induce antigen-specific immunological tolerance in Th2-associated allergic diseases. We previously showed that antigen (Ag) coupled to apoptotic splenocytes was capable of inducing Ag-specific immunological tolerance that ultimately resulted in the prevention and treatment of both allergic asthma and peanut-induced food allergy. This approach is the focus of a Phase I/IIa clinical trial examining the effects of immunological tolerance using multiple myelin peptides in early relapsing-remitting multiple sclerosis (MS) patients. We have engineered robust multi-functional particle-based platforms for the surface delivery of cross-linked Ag or the sustained release of soluble encapsulated Ag, to induce Ag-specific immunological tolerance in an ovalbumin-alum induced mouse model of allergic asthma. Particles were prepared and characterized by photon correlation spectroscopy and scanning electron microscopy. Antigen (ovalbumin or myelin basic protein) was attached to the surface of the particles by chemical cross-linking. We have shown that particles with ovalbumin cross-linked on their surfaces were capable of inducing protective tolerance when administered prior to disease induction. Production of ovalbumin-specific IgE antibodies and lung inflammation in response to aerosolized ovalbumin were inhibited. In addition, lymphocyte proliferation and Th2 cytokine production in response to ovalbumin were prevented. Progress on investigating the therapeutic potential of particles with surface-cross-linked-Ag by administering them post-disease induction and the prophylactic and therapeutic potential of particles containing soluble encapsulated Ag will also be discussed. In conclusion, particle-based antigen-delivery platforms are promising systems for the treatment of allergic diseases.

Transplantation of pancreatic islets (cell clusters) has emerged as a promising treatment for Type 1 diabetes. However its clinical application remains limited due to adverse effects of immunosuppression which puts increase demand for efficient islet coating that can preserve islet viability and function. We developed an ultrathin polymer coating with immunomodulatory and anti-inflammatory responses to protect living pancreatic islets. The coating was comprised of cytocompatible polymers of hydrogen-bonded natural polyphenol (tannic acid) and poly(N-vinylpyrrolidone) deposited on the islet surface via non-ionic layer-by-layer assembly. The coating was conformal over the surfaces of rat, non-human primate, and human islets. In contrast to unmodified controls, the coated islets maintained their viability and β-cell functionality for at least 96 hours in vitro. We also determined that the coating demonstrated immunomodulatory cytoprotective properties suppressing pro-inflammatory cytokine synthesis in stimulated bone marrow-derived macrophages and diabetogenic CD4+ T cells. A decrease in Th1 cytokine responses was observed when antigen-stimulated BDC-2.5 T cells and LPS-stimulated bone marrow-derived macrophages were co-treated with the coating material, suggesting the efficacy of this encapsulation strategy to provide physical islet protection and prevent efficient autoreactive T cell responses. The developed material combines high chemical stability under physiologically relevant conditions with capability of scavenging free-radicals, two crucial parameters for prolonged islet integrity, viability, and function in vitro/in vivo. Our study offers new opportunities in the area of advanced transplant materials to be used in Type 1 diabetes treatment as well as in various areas of cell-based therapy.

Bone grafting surgeries are widely needed to repair or replace defects caused by trauma, tumor resection, pathological degeneration and congenital malformations. In the U.S., estimately more than 500,000 bone grafting surgeries are operated every year. As an alternative to autologous bone grafting, an absorbable collagen carrier combined with rhBMP-2 is developed and approved by FDA for the treatment of certain bone diseases and fractures. However, the low affinity of BMP2 to the collagen matrix greatly increases the dosing and therefore the cost. In addition, the inflammatory reaction associated with the BMP2 is also limiting its successful applications. In our research, we are presenting a polymeric core-shell BMP2 nanocapsule with each BMP2 enwrapped in an individual degradable polymer shell. The polymeric shell protects the BMP2 from the recognition of immune system. After implanting the BMP2 nanocapsule-loaded nanocapsules, the polymer shell gradually degrades in the local basic environment, releasing its core protein to function. With this strategy, satisfactory level of spine fusion was achieved with inflammatory reaction greatly reduced compared with the conventional collagen/BMP2 system.

Injury and diseases that affect articular cartilage present a daunting challenge in orthopedic medicine. During the onset of injury or disease, low oxygen environments decrease healthy cartilage cell growth prohibiting the efficacy of tissue scaffolds in the defective joint. In this study, preparation, characterization, and fabrication of a therapeutic injectable composite hydrogel system containing poly(N-vinylcaprolactam)[PVCL], hyaluronic acid (HA), and a synthetic antioxidant has been investigated. Hydrogels were characterized using nuclear magnetic resonance (NMR) spectroscopy, photometry, and temperature-controlled rheology. We hypothesize that: i) the PVCL-based hydrogels will have tunable mechanics upon temperature change; and ii) the incorporation of antioxidants may increase extracellular matrix (ECM) production of articular cartilage cells (chondrocytes) under low oxygen conditions by quenching reactive oxygen species. Chondrocytes were harvested and seeded on various formulations of PVCL-graft-HA [PVCL-g-HA] and therapeutic hydrogels under normal (21%) oxygen and low (hypoxic) (1%) oxygen conditions. Extracellular matrix (ECM) synthesis and biochemical assays were evaluated after 1, 2 and 3 weeks of incubation. Upon comparing cloud point measurements and rheology, the correlations between LCST and viscoelastic properties were investigated. PVCL polymers showed a dramatic decrease in elastic modulus (G') and viscous modulus (G'') at 38°C indicating insolubility. PVCL-g-HA gels had lower G' and G'' values near LCST, but increased above physiological temperatures. Cell culture experiments of chondrocytes and stem cells remained viable for up to 10 days on all hydrogels under normal and hypoxic conditions. PVCL-g-HA had higher chondrocyte viability than HA and therapeutic samples under hypoxia. This work suggests that injectable PVCL-based therapeutic systems have tunable mechanics and show promise in protecting cells against hypoxic environments via the antioxidant component.

Neurons interact with their mechanical environment. While most neuron-biomechanics studies reported today focus on central nervous system neurons, we report on the response of peripheral nerve neurons and Schwann cells to substrate rigidity and topography in vitro. These efforts proceed in the context of defining robust strategies to promote functional regeneration after nerve injury in vivo.
Silicone-based substrates with controlled stiffness and topography are prepared by distributing on the surface arrays of elastomeric pillars with a sub-cellular size of a few microns. The flexible nature of the micropillar geometry allows for an artificially reduced stiffness to be sensed by the cell, compared to that of the bulk material. By modulating the geometrical characteristics of the micro-pillars, i.e. their diameter, height and spacing, we produce surfaces with effective elastic modulus ranging from sim;200Pa (micropillar spring constant of 0.28nN/um) to sim;50kPa (micropillar spring constant of 70nN/um) and topographically rich. We review the design and microfabrication of these elastomeric surfaces with dense arrays of high aspect ratio silicone micro-pillars. Before seeding with dorsal root ganglions (either from rat neonates or adult animals), the micropillar substrates are bio-functionalized by coating their top surfaces with neuron-promoting molecules (e.g. Poly-D-Lysine and laminin) while micropillar shafts are coated with repulsive molecules (e.g. Pluronics or PEG).
We report on our initial results in terms of adhesion, spreading, and motility of peripheral nerve neurons and Schwann cells with increasing substrate stiffness.

Introduction: The rigidity and density of adhesion sites of cell culture substrates strongly influences cell phenotype and gene expression. Interactions between integrin receptors and the substrate mediate these phenomena at the cell membrane, but previous studies have focused on a limited number of intracellular signals that relay these physical cues to the nucleus. To investigate the signaling pathways that determine the intrinsic response of cell to its surroundings, we have applied a novel systems biology approach for large scale, dynamic quantification of transcription factor (TF) activity, which represents the output of complex intracellular signaling networks in response to various extrinsic factors including substrate mechanics and ligand presentation. These studies provide a more holistic view of the signaling pathways that determine a cell's response to mechanical and adhesive stimuli and can provide a mechanistic connection between biomaterial properties and cell phenotype.
Materials and Methods: We have developed polyethylene glycol (PEG)-based hydrogels in which the adhesive ligand density (e.g., RGD) and mechanical properties can be independently tuned across three orders of magnitude. Briefly, 4-arm PEG-acryloyl was dissolved at 5, 10 or 20% w/v in HEPES-buffered saline (pH 7.4) and conjugated to peptides containing RGD via Michael addition between acrylates and cysteines and then photocrosslinked using Irgacure 2959 as a photoinitiator. Elastic and viscous moduli were determined by a frequency sweep using a parallel plate rheometer set up (Anton Paar Physica). Human foreskin fibroblasts (HFFs) were cultured on hydrogels where modulus and RGD concentration were independently varied. Prior to seeding on PEG hydrogels, HFFs were transduced by lentiviral reporter constructs in which luciferase expression is driven by consensus sequences known to bind certain TFs. Luciferase levels were then measured non-invasively by bioluminescence imaging of HFFs cultured in a 384-well plate format, providing a platform with rapid data acquisition to quantify TF activity. Measurements were taken at 3, 6, 9, 12 and 27 h. after HFF seeding.
Results and Discussion: In these large-scale arrays, we employed over 50 reporter constructs that encompass the major intracellular signaling pathways and have identified at least 10 binding elements that display differential dynamics in HFFs cultured on substrates with varying elastic moduli. These elements include those known to bind TFs involved in mechanotransduction (e.g., AP-1, NFAT and Elk-1). Similar patterns of signaling pathway activity were observed when HFFs were cultured on substrates with varying ligand density. Overall, this work has identified the dynamic activation of multiple intracellular signaling networks that occur simultaneously during fibroblast attachment.

In this contribution, we report on the behavior of human mesenchymal stem cells (MSCs) and osteoblasts (OB) on top of various nano-structured SiO2 substrates. The aim of this interdisciplinary project between Physics and Life Science is to quantitatively investigate the cellular adhesion, proliferation and differentiation of MSCs and OB as influenced by systematically nano-structured surfaces in order to develop optimized interfacial interactions between cells and substrate. We present a novel nanofabrication technique based on the production of highly ordered metal nanoparticles in two-dimensional arrays, which, subsequently, are used as nanomasks for anisotropic reactive ion etching (RIE). For this purpose, diblock-copolymer micelle nanolithography (BCML) is applied in combination with photochemical growth to obtain Au nanoparticles. By this approach, particle diameters as well as interparticle distances can be controlled and exhibit narrow distributions. Cyclic combination of photochemical growth and RIE results in well-defined hexagonally ordered SiO2 nanopillar arrays with heights up to 320 nm (maximum aspect ratio ~9:1).
The human MSC and OB cells were taken in accordance with the local ethics committee from patients as part of routine operations. For the morphological characterization of cell-substrate interaction, high resolution scanning electron microscope was used. Osteogenic differentiation was analyzed by immunofluorescence. Ten SiO2 nanopillar arrays combining heights of 20 and 50 nm with interpillar distances of 50, 100, 120 nm and diameters of 10, 30 nm were investigated. For all surfaces including a reference plain SiO2 substrate, cell adhesion, proliferation and viability were analyzed for day 1 and 7. For MSC, the optimum interpillar distance for the highest proliferation rate was observed at 100 nm. MSC response to 10 nm width nanopillars was significant. Highest proliferation rate for MSC was observed on the nanopillar arrays with dimensions of 10-100-50 nm (width, distance, height). For all topographies except the reference, the cell numbers were increased in a week. For OB, higher proliferation rate for wider pillars was observed (30 nm). It was demonstrated that for both pillar heights (20, 50 nm), the substrates shows good MSC proliferation. Osteogenic differentiation, however, is significantly induced by shorter pillar heights. For the reference substrate, cell death was observed after 1 week. SEM examination showed that regardless of the 3D structure of the cells, they mainly adhered to the very tops of the pillars.
In summary, systematically varying geometric parameters of nanopillar arrays on SiO2 surfaces offers new opportunities to study their effect on cellular processes with implications for orthopedic device design and application.

Adhesion and activation of integrins have been shown to be important for the differentiation and proliferation of keratinocytes, the predominant cell type in skin [1]. Integrins are a group of transmembrane receptors mediating the attachment to the extracellular environment. Attaching to a ligand can change the conformation of the intracellular domain of the protein, and vice-versa, thus facilitating signal transmission across the membrane. In vivo the keratinocytes are known to adhere to the basal membrane through various integrins until committing to terminal differentiation and ceasing proliferation, at which point the expression of specific integrins is down regulated leading to cell detachment from the basal membrane to become part of the upper layers of the skin [2].
Here nanopatterns of integrin ligand islands of varying size are used to restrict the size of focal adhesions formed by adherent human primary keratinocytes in vitro. These materials enable studies of the role of focal adhesion development in cellular adhesion and differentiation. These surfaces present patches of absorbed protein on a protein repellant background. Adhesion should then be restricted to these patches. In order to examine the adhesion and morphology of the cells afterwards, fluorescence microscopy can be employed.
Disperse colloidal lithography allows for the fabrication of large area nanopatterns exhibiting short range order, by exploiting the absorption of charged polystyrene nanoparticles to a polymer coated gold surface possessing the opposite charge. The particles will absorb to the surface in a single layer while keeping a characteristic distance from each other due to mutual repulsion. These patterns can then be functionalized to result in adhesive protein islands of tunable sizes ranging from 100 nm to 1000 nm on an otherwise protein repellant background covered with poly(ethylene glycol) [3]. In this case the method was used to create 100 nm, 300 nm and 800 nm islands of collagen IV, a constituent of the basal membrane and a known ligand for the α2β1 integrin, one of the integrins involved in regulating differentiation in keratinocytes[1].
The method used for creating these patterns will be presented, as well as preliminary data regarding the adhesion of primary human keratinocytes to systematically varied collagen IV nanopatterns.
[1]: Fiona M. Watt: Role of Integrins in Regulating Epidermal Adhesion, Growth and Differentiation, The EMBO Journal, 2002, Vol. 21, No. 15
[2]: P. Kaur and A. Li: Adhesive Properties of Human Basal Epidermal Cells: An Analysis of Keratinocyte Stem Cells, Transit Amplifying Cells, and Postmitotic Differentiating Cells, Journal of Investigative Dermatology, 2000, Vol. 114
[3]: J. Malmström, B. Christensen, H.P. Jakobsen, J. Lovmand, R. Foldbjerg, E.S. Soslash;rensen and D.S. Sutherland: Large Area Protein Patterning Reveals Nanoscale Control of Focal Adhesion Development, Nano Letters, 2010, 10

Nipah virus (NiV), a highly pathogenic member of the Paramyxoviridae family depicted in the 2011 film,Contagion, was first isolated after a 1998-1999 outbreak of fatal encephalitis among pig farmers and abattoir workers in Southeast Asia. NiV and its close relative, Hendra virus (HeV), have been classified as BSL-4 agents due to their broad host range, their numerous routes of transmission, and the high rates of mortality associated with infection. Despite recent advances in understanding the tropism of NiV and HeV, however, there are currently no available vaccines or therapeutics. To reduce the time and cost associated with developing, for example, subunit vaccines or therapeutic antibodies for emerging pathogens, we have constructed complex (~10¹⁰ members), random peptide libraries displayed on MS2 phage virus-like particles (VLPs), which self-assemble from 180 copies of a single coat protein into a 28-nm icosahedron that can display up to 90 peptides/particle in an accessible surface loop. We have shown that affinity-selection against neutralizing antibodies or convalescent anti-sera yields MS2 VLPs that display peptide mimics of natural antigen epitopes, regardless of whether the epitope is linear, conformational, or carbohydrate-based. Furthermore, since MS2 VLPs display peptide mimotopes in dense, repetitive arrays, they provoke efficient oligomerization of B cell receptors and induce potent anti-peptide responses. In order to identify NiV and HeV vaccine candidates, we performed affinity selections against several neutralizing monoclonal antibodies that recognize linear epitopes from the NiV attachment glycoprotein (G). After two rounds of selection at high valency (90 peptides/VLP) and two rounds at low valency (~3 peptides/VLP), we obtained peptides for each antibody that map directly onto the NiV-G and HeV-G sequences. VLPs that display these peptides in high valency bind to their respective antibodies with high affinity (K_d = 1-20 nM) and induce peptide-specific, high titer (10⁴-10⁵), adjuvant-independent IgG responses when injected into C57Bl/6 mice. Furthermore, anti-sera collected from immunized mice completely neutralize NiV and HeV pseudoviruses at dilutions as high as 1:10,000 and provide complete protection of ex ovo avian embryos challenged with a lethal dose of pseudovirus. Our results demonstrate that MS2 VLPs are, to date, the only nanoparticles that can be used both for epitope identification and for vaccination. This technology holds great promise for rapidly (< 1 month) identifying vaccine candidates, since it requires only convalescent anti-sera against an emerging pathogen and, thus, negates the time-consuming steps of pathogen isolation and characterization. As importantly, MS2 VLP-based vaccines can be produced in large quantities using either bacterial expression systems or cell-free protein synthesis at a fraction the cost of subunit vaccines or therapeutic antibodies.

Immunotherapy has emerged as a new approach toward eliminating immune-evading diseases and producing stronger vaccines. Recently, immune-stimulating molecules have been used to combat disease, where vaccines are the classic example. Vaccines can use attenuated viruses to activate the immune system and provide immunity to targeted diseases. Current dendritic cell vaccines function by stimulating dendritic cells through pathogen-response receptor (PRR) ligands ex vivo. Subsequently, the stimulated cells are injected back into the body to stimulate the immune system. However, the stimulated dendritic cells are not always potent enough or may lose activation once in vivo. In general, immunotherapies still lack a modular system that can easily and specifically target multiple receptors at once. Our strategy improves upon past methods by directly attaching agonists to target cells, which results in effective immune system stimulation. Using general chemistry to chemically modify target cells allows us to create an immune-stimulating faccedil;ade using single or different combinations of agonists. We report the bioconjugation of an agonist faccedil;ade onto Lewis Lung Carcinoma (LLC), which contains CpG-DNA, a Toll-like receptor (TLR) 9 agonist. The CpG-DNA modified LLCs were recognized as bacteria by the JAWS II dendritic cell line and resulted in dendritic cell stimulation. The effects from using single or multiple agonists will teach us more about how dendritic and T-cells interact and function within the immune system. In general, we intend to address current challenges in immunotherapy by redirecting the immune system toward combating elusive diseases, such as cancer and HIV, using chemical methods.

The creation of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels presents a major challenge to the field of microvascular research and tissue engineering. Current methods for formation of microvascular channels are limited with non-circular channel cross-sections (rectangular, square or trapezoidal shapes), complicated fabrication, and less flexibility in microchannel network designs. Such channels impose widely varying fluid shear stresses and non-physiological geometries on cells in different channel positions that can induce significant variations in cell physiology. It is well accepted that the degree of shear stress imposed on luminal endothelial cells, which depends strongly on the geometry of the flow channel, affects their differentiation state, alignment and elongation, tight-junction formation, gene expression, and response to inflammatory stimuli. For these reasons, the geometry of the channel should replicate as closely as possible to the geometry of in vivo microvessels because of its essential role in the formation and maturation of vessels.
To address current limitations in creating of engineered microvascular channels with complex three-dimensional (3-D) geometries in the shape of microvessels, we developed and presented a convenient, cost-effective, and reproducible microfabrication approach to create a circular cross-section of multi-depth microchannel network in polydimethylsiloxane (PDMS) and human interstitial collagen, by the combination of photolithographic photoresist reflow techniques, soft lithography, and sacrificial molding. The design of the microchannel network was based on Murray&’s Law to mimic the geometry of microvascular microvessels in vivo, which adopt roughly circular cross-sections with radii between 30 and 300 mu;m. Further examination of the channel dimensions and surface profiles at different branching levels showed that the shape of the microchannel was well approximated by a circular surface, and a multi-level and multi-depth channel network were created. In addition, our results showed that primary human umbilical vein endothelial cells (HUVECs) were successfully growing and lining along the cylindrical channels under shear flow conditions. The multi-depth microfluidic channel network would allow the flow patterns are more physiological relevant to in vivo conditions and expected to benefit microvascular research, such as the microfabricated cylindrical microchannels and networks can either work as a scaffold for investigating microvascular cells seeding and growing under shear flows and luminal pressures or as a mold for further fabrication of tissue scaffolds for morphogenesis and tubulogenesis.

Type 1 Diabetes is a chronic disease that occurs when the body can no longer produce insulin and requires lifelong treatment. Current standard treatment of care requires multiple daily insulin injections with regular blood glucose testing. Lack of patient compliance can lead to severe short and long-term health complications. While islet transplantation has had moderate clinical success, only one third of clinical islet transplants achieve insulin independent after two years. Little is known about the proper microenvironment necessary for islet survival and health, particularly with regard to cell organization and biophysical cues.
To elucidate the micro-environmental conditions necessary for improved islet function and survival, we have microfabricated a series of 3D polyacrylamide cell scaffolds to regulate the spatial and mechanical control of bio-signals. Specifically, we have shown a significant size-dependent increase in the glucose stimulated insulin response per cell for 2D and 3D beta cell clusters 100um in diameter. Our data suggests that the physical interactions with the microenvironment, including stiffness of the matrix, may regulate cell function. Additionally, to account for the host immune response we have created a thin film macro-encapsulation device that: (1) allows cellular signal exchange between the transplanted islets and the host while protecting the islets from immune destruction and (2) supports islet microenvironment to maintain healthy islets response. In order to ensure successful transplantation both cells immediate exchange and interactions with microenvironment and host nutrients need to be achieved. By combining the optimal microenvironmental cues with macroencapsulation stratagies, we hope to improve long-term islet viability while providing the recipient with a dynamic glucose-responsive source of insulin.

Introduction. Biomaterials can serve as 3D scaffolds for engineering stem cell niche with tunable properties. However, few biomaterials systems developed to-date allow independent tuning of niche properties such as biochemical signals and mechanical stiffness. To allow independent tuning of niche properties in 3D, interpenetrating network (IPN) hydrogels have emerged as an artificial stem cell niche comprising two or more polymer networks, which can be used to individually. Previous research has resulted in the formation of semi-IPNs, which composed of physical entanglement of a covalently crosslinked network and dynamic chains. However, the distribution and stability of the biochemical network within such semi-IPN is hard to control, and batch-to-batch variance could be high. To overcome these limitations, here we report the development of a novel method for constructing stable, homogeneous interpenetrating network (IPN) hydrogel as stem cell niche with independently tunable niche properties (biochemical, mechanical and degradation) using cell-friendly processes. The effects of interactive niche signaling on osteogenic differentiation of human adipose-derived stem cells in 3D were evaluated using combinatorial IPN hydrogels.
Materials and Methods. To create a 3D stem cell niche with independently tunable biochemical and mechanical property, we chose two mechanisms that can crosslink independently and simultaneously, namely amine-NHS coupling for biochemical network and thiol-ene radical addition for mechanical network . Both approaches can be carried out at physiological conditions and are cell-friendly. We chose poly(ethylene-glycol) (PEG) as the backbone structure due to its “blank slate” structure and amenability to chemical modification. To incorporate biochemical ligand, PEG-diol blocks were coupled by trans-5-norbornene 2,3-diacrybonyl chloride to introduce norbornene residual to the backbone, followed by incorporation of cysteine containing peptide by radical addition. To make the IPN hydrogel degradable, enzymatically cleavable peptide CGPQGIWGQC with two thiol groups at the terminal cysteine residuals were utilized replacing linear PEG-dithiol in mechanical network to introduce the enzymatic degradation.
Results and Discussion. Using a modular design approach, we have developed a novel stable interpenetrating network hydrogel as stem cell niche with independently tunable niche properties. Uniform presentation of biochemical ligands was achieved by using specifically designed and synthesized PEG derivative with bio-active peptide incorporation. A combinatorial IPN microarray with independently tunable RGD concentration and mechanical stiffness was fabricated to examine osteogenic differentiation of human adipose stem cells (hADSCs). The IPN developed here may provide a powerful tool for elucidating how interactive niche signals regulate cell fate in 3D in a high-throughput manner.

Introduction. Polyethylene glycol (PEG) hydrogels are attractive candidates as drug delivery platforms due to their biocompatibility and wide use for tissue engineering applications. Most PEG platforms reported so far utilized radial polymerization such as PEG-diarcylate (PEG-DA), which often led to burst release of encapsulated proteins. However, long-term sustained protein release is desirable for guiding cellular processes and tissue regeneration in situ. We hypothesize that enhancing PEG hydrogel network homogeneity will decrease the burst release, and protein release from PEG hydrogels can be tailored by the ratio of the size of the protein vs. the mesh size of hydrogel network. The overall goal of this project is to develop a facile strategy for sustained protein release from PEG hydrogels by tuning the PEG hydrogel network structure, homogeneity and degradation.
Materials and Methods. To enhance the homogeneity of PEG hydrogel network, we utilized thiolene addition to crosslink multi-arm, norbornene-terminated PEG with thiol-terminated PEG (multi-arm or linear). To tailor the mesh size of hydrogel, two different number-average molecular weights between cross-links (Mc), 4K and 2.5K, were obtained by crosslinking 8-arm PEG 10K with linear PEG 1.5K and 8-arm PEG 10K with 8-arm PEG 10K correspondingly. To tailor the degradation profile, three different degradable structures, non-degradable (ND), mercapto-acetic ester (MAE), mercapto-propionic ester (MPE) were introduced to the network. Bovine serum albumin (BSA) was used as a model protein for testing the release profile from the hydrogel. Basic fibroblast growth factor (bFGF) was also used as a model protein for testing the retention of the bio-functionality after release from the hydrogel by checking the cell proliferation response to the collected medium.
Result and Discussion. Increasing hydrogel concentration to 20 % (w/v) avoided burst release of proteins from all hydrogel compositions at the initial stage. Hydrogels with higher Mc (4K) showed a faster release than the 2.5K groups. Protein release increased as the PEG degradation increased, with the degradable MAE-PEG groups leading to a sustained release. Protein release from the non-degradable PEG groups and MPE-PEG groups reached a plateau early on due to the extremely slow degradation. Using the optimal hydrogel formulation (2.5K-MAE), linear and sustained protein release was achieved over 50 days. bFGF was encapsulated and released from the optimal hydrogel formulations over time. Released bFGF stimulated cell proliferation of human adipose-derived stromal cells, which confirmed the retention of biological activity of released growth factors. Given PEG is a bio-inert polymer, the platform reported herein is solely diffusion driven and can be used for controlled release of a broad range of proteins.

The individual role of pico, nano and submicron titanium topography on stem cell dynamics is not only critical to understanding the fundamentals of stem cell dynamics, but can also reveal instructive information to guide the smart design of effective surface structures for accelerating strong osseointegration around endosseous implants. This study provides a comprehensive evaluation of mesenchymal stem cell responses on pico, nano and nano-submicron hybrid titanium surface structures by using two different cell lineages, primary mouse bone marrow mesenchymal stem cell (mBMSC) and human bone marrow mesenchymal stem cell (hMSC) for future applications in the surface treatment of bone-interfacing permanent metallic implants. Although threshold surface height for directing favorable early cellular responses in mBMSC and hMSC was around 2-3 nm, the threshold height of the surface features for influencing significant osteoblast phenotype differentiation was greater than 20 nm with submicron lateral dimensions (larger than 100 nm).

Pathogen associated molecular patterns (PAMPs) were covalently linked as hetero-dimers using PAMP reactive α,omega;-heterotelechelic polymers. The resulting multi-PAMP conjugates were used to stimulate a range of immune cells. Dendritic cells communicate with T cells and B cells to shape responses within the mammalian adaptive immune system. Dendritic cells contain pattern recognition receptors (PRRs) that vary by dendritic cell type, and may be present on the cell surface or in compartments within the cell. PRRs are highly sensitive to a wide array of PAMPs, and mixtures of PAMPs can cause immunostimulatory synergy in dendritic cells. Although the presentation of multiple PAMPs by a pathogen occurs over discrete proximities and spatial orientations, the effect of inter-PAMP spatial relationships on this synergy is not understood. Inter-PAMP spatial orientation, whereby each PAMP is held at a well-defined proximity, has yet to be systematically investigated. Proximity effects are important in understanding how the adaptive immune system recognizes PAMP presentation on pathogens. Additionally, controlling inter-PAMP spatial relationships allows for PAMP heterodimer constructs that can mimic the spacing that occurs on native pathogens. We produced well-defined inter-PAMP proximities by linking synergistic PAMP combinations with α,omega;-heterotelechelic PAMP-reactive polymers (HPRPs) to produce PAMP heterodimers. A range of inter-PAMP proximities were examined by varying degree of polymerization across different HPRPs. The resulting PAMP heterodimers were used to stimulate a range of cell lines including RAW-Blue, JAWS II, and primary murine myeloid dendritic cells. Synergistic effects have been observed between PAMPs and this has allowed for a more refined understanding of the mechanisms of dendritic cell stimulation.

Myocardial infarction (MI) and congestive heart failure (CHF) are major cardiovascular diseases with high mortality rates. Cardiac patch tissue engineering has become an advanced strategy for surgical intervention of MI and CHF therapy. The criteria of scaffold for cardiac patch tissue engineering include porous, elastic, biocompatible, biodegradable, and mimic biomechanical properties of the cardiac muscle. Electrospinning is a promising technique to fabricate nanofibrous scaffold which is structurally similar to the native extracellular matrix (ECM) and provides high surface-to-volume ratios with interconnected pores. In this presentation, we will demonstrate the successfully developed scaffold by electrospinning technique. It features significant biodegradability, biocompatibility, and mechanical properties (high tensile strength, modulate Young&’s modulus, high elongation and flexibility).
Elastic biodegradable polyurethane (PU) was used in scaffold fabrication. In order to increase the biocompatibility, we blended the PU with ethyl cellulose (EC) for the first time in different weight ratios. By optimizing the processing parameters of electrospinning (flow rate, voltage, deposition distance, solution concentration, etc.), we are able to obtain fibers with controlled diameters ranged from <100 nm to 1 mu;m. The mechanical properties tests including tensile strength, Young&’s modulus and elongation were performed with respective to the fibrous scaffolds of different PU/EC ratios and diameters. The pristine PU reveals low tensile strength and low Young&’s modulus. However, blending proper amounts of EC into PU enhances these properties but still remains high elongation and flexibility. It indicates the inclusion of EC can improve the biocompatibility without sacrifice the mechanical properties. Additionally, the mechanical properties of fibrous scaffolds are significantly size-dependent (from 100 nm to 1 mu;m) and systematically studied in the present work. In vitro test of biocompatibility and cytotoxicity was performed by culturing the H9C2 cells (rat cardiomyoblast cell line) and seeding on scaffolds. The morphology of H9C2 cells on fibrous scaffolds were observed by scanning electron microscope. The cell adhesion and spreading ensures the subsequent proliferation, migration and extracellular matrix production. Moreover, the biodegradation of the fabricated fibrous scaffolds was also tested by examining the reduction of mass and molecular weight in phosphate buffered saline (PBS). Finally, we also fabricated aligned fibrous scaffolds, which can instruct the growth of H9C2 cells more anisotropically organized and improve the differentiated phenotype of H9C2 cells as indicated by rod-shaped cell morphology. To sum up, the successfully developed PU/EC fibrous scaffold by electrospinning considerably advance the current technology in cardiac patch tissue engineering which awaits for further in vivo test and practical applications.

Cells sense various stimuli in the extracellular matrix and integrate these numerous signals to respond appropriately. Therefore, the precise bio-functionalization of surfaces with multiple signaling molecules, adhesion peptides and receptor ligands is essential to control cell-material interactions and influences the cell response to synthetic interfaces. Here, we present the formation of patterned gold nanoparticles on glass surfaces and the precise functionalization of these surfaces with two different bioactive molecules such as peptides, proteins and small molecules and their applications in cell systems. First, quasi-hexagonal gold nanoparticle arrays on glass are prepared by micellar nanolithography, a technique established in our lab, where the spacing and the size of the gold nanoparticles can be precisely controlled. Then, the glass surface between the gold nano-particles is functionalized with PEG self-assembled monolayers, which contain an alkyne functionality at the end (click-PEG). This combination allows for specific functionalization of the surfaces with a first signaling molecule on the click-PEG via the click-reaction and then with a second signaling molecule on the gold via thiol chemistry. Thus, it is possible to study cell responses to surfaces functionalized with two different signaling molecules with precise density and arrangements. Consequently, we will be able to characterize the interplay between the two immobilized signals and clustering effects in ligand-receptor interactions.

Introduction: Hydrogels are widely used as tissue engineering scaffolds due to their injectability, tissue like water content and tunable properties. Macroporosity within hydrogels is important to facilitate nutrient transfer, cell proliferation, migration and matrix deposition. Most techniques for producing macroporous hydrogels are not cell-friendly and often result in poor cell penetration and distribution. Furthermore, methods to introduce internal channels as macroporosity within hydrogels remain lacking. To overcome these limitations, here we aim to develop a novel method for engineering macroporous hydrogels with internal channels using stimuli-responsive microfibers as porogens. In addition to their roles in internal channel formation, we also explore the potential of utilizing stimuli-degradable porogens as delivery vehicles for dispatching cells or biological cues to promote desired cellular processes and tissue formation.
Materials and Methods: We have chosen to utilize calcium alginate (Ca-Alg) to make microfibers, which serve as a “porogen” that dissolves in the presence of ethylenediaminetetraacetic acid (EDTA), leaving interconnected microchannels. Microfibers with sizes ranging 80-200 µm were fabricated using coaxial flow of alginate solution (2% w/v) and calcium chloride solution (1% w/v). Microfibers containing human embryonic kidney (HEK) cells were encapsulated into a 3D gelatin hydrogel, then dissolved using EDTA solution (8mM or 16mM; 30min, 1hr, 2hr). Scaffold internal morphology was examined using scanning electron microscopy (SEM). Cell viability and morphology were examined using live-dead staining and fluorescence microscopy.
Results and Discussion: SEM confirmed successful removal of microfibers after EDTA treatment, leaving smooth internal channel structures within the 3D gelatin hydrogels. Under optimal EDTA treatment condition, the encapsulated cells were released from microfibers, attached and spread on the internal surface of microchannels within the 3D gelatin hydrogel. Live/dead assay indicated that the process was cell-friendly for HEK cells, and showed enhanced spreading of HEKs compared to non-EDTA treated control samples. In summary, here we report a novel, cell-friendly process of fabricating 3D macroporous hydrogels with internal channel structure. The stimuli-responsive microfibers allow dynamic control of internal architecture within 3D hdyrogels, and provide a powerful tool to deliver cells in 3D for patterning desirable tissue structures or engineering tubular tissues such as blood vessel network.

The pathogenic amoeba Acanthamoeba castellanii can cause severe diseases like Acanthamoeba keratitis and encephalitis. Most patients suffering from Acanthamoeba keratitis are contact lens users, who are infected with Acanthamoeba due to wrong contact lens care. Through small lesions of the epithelial cells the amoebae get into the eye and start to destroy target cells by an extracellular killing mechanism. A crucial first step during this cytotoxic reaction is the formation of a close contact between the Acanthamoeba and the target cells. This contact appears to be mediated by carbohydrate molecules.
In particular, mannose has proven to be strongly involved in the specific recognition of target cells by amoebae. In order to investigate this recognition and adhesion process in detail, we are using carbohydrate-functionalized surfaces to mimick the target cell coat. Employing hexagonal patterns of gold nanodots fabricated with diblock-copolymer micelle nanolithography, we can precisely define the density and distance of anchorage sites for cellular carbohydrate-binding molecules. We have found that there is a significant influence of carbohydrate density on the number of adhering amoebae and on their adhesion area. Our experiments show that a precise functionalization of surfaces at the micro- and nanometer scale allows studying carbohydrate-mediated target cell interactions of amoebae in great detail.

Assembly of microparticles into a scaffold provides a promising modular material for tissue engineering and drug delivery. Microgels have previously been formed via the precipitation of poly(ethylene glyol)-diacrylate (PEGDA); these microgels were then assembled into scaffolds to investigate nerve outgrowth. In addition to using the microgels as a scaffold support, we are interested in encapsulating neurotrophic factors to enhance the properties of the scaffold. In this study, poly(ethylene glycol) (PEG) was modified with lactic acid (LA), mixed with PEG-dithiol (PEGDT), and microgels were formed via precipitation reaction that utilized mixed-mode polymerization. Nerve growth factor (NGF) was included in the PEGLA:PEGDT solution and encapsulated within the microgel during the precipitation. The diameter of microgels was measured through dynamic light scattering and mesh size was calculated from its swelling ratio. In addition, FTIR was used to measure the thiol conversion and confocal and electron microscopy were used to examine the structure. The microgels were then assembled into a scaffold by mixing the pre-formed microgels containing NGF and 8-arm PEG vinylsufone. Free thiol groups near the surface of microgels react with the vinylsulfone, which construct the modular hydrogels. Atomic force microscopy, rheology, and confocal microscopy were used to characterize the scaffold. Drug release profiles were recorded for up to one month and the activity of released NGF was examined through PC12 cell differentiation. The diameter of the microgels after mixed-mode polymerization was 1.42 ± 0.25 mu;m, which compared favorably to our previously published work with PEGDA. Unlike scaffolds formed from PEGDA microgels, the scaffolds formed from these mixed-mode polymerization microgels and thiol:vinylsufone crosslinking reaction were optically clear and had a 3-fold increase in shear modulus over scaffolds formed using an amine:succinimide reaction. Preliminary data indicates that inclusion of NGF in the microgels does not alter diameter and that NGF released completely from microgels by day 21, with minimal burst release. However, the bioactivity of NGF was significantly reduced. Current work is investigating methods to maintain the bioactivity of the NGF for longer periods of time. Overall, expanding the library of microgels will provide us with building blocks that can be used to design modular scaffolds. Overall, the hydrogels designed in this research will allow for the development of scaffolds with specific delivery profiles that can be used for neural tissue engineering applications.

Limited vascularization, caused by an inadequate migration of microvascular endothelial cells into the wound bed, plays a crucial role in the etiology of diabetic ulcers. Thus, promoting neovascularization is a critical step to improving material transport and ensuring rapid wound resolution. Sonic hedgehog (Shh) is an important growth and differentiation factor that up-regulates multiple angiogenic signaling pathways, and exogenous delivery of Shh can significantly improve the rate and quality of diabetic would closure. However, poor control over growth factor localization and bioactivity during drug delivery has limited its use as a wound healing clinical treatment. We have previously developed multivalent protein-polymer bioconjugates to enhance the potency and stability of growth factors following targeted in vivo administration. This method, based on carbodiimide and maleimide chemistry, allows for stoichiometric control over the conjugation of proteins to a soluble biopolymer, such as hyaluronic acid (HyA). In this project, we hypothesized that multivalent conjugation would increase the bioactivity of Shh and promote neovascularization relative to unconjugated Shh at identical molar ratios. We synthesized soluble clusters of Shh conjugated to single-chains of HyA at the following defined ratios of Shh to HyA: 1:1, 3.5:1, 7:1, 14:1, and 22:1. The polyvalency of the HyA conjugates was determined by SEC-MALS. We then evaluated the effect of Shh valency and concentration on neovascularization using an excisional wound-healing model in diabetic mice. We have verified that high-valency Shh conjugates (i.e. 14:1 / Shh:HyA) promote more rapid migration of CD31+ endothelial cells into the wound bed by a factor of 3 compared to negative controls and a factor of 2 compared to unconjugated Shh, drastically increasing the density of vascular structures during early phases of wound healing. As a result, diabetic wounds treated with the multivalent Shh conjugates demonstrated an improvement in healing based on the overall wound appearance and closure rate. Taken together, our findings suggest that our method of multivalent conjugation Shh to soluble HyA biopolymers may be an enabling approach for developing a clinical therapy for diabetic wound healing.

A key goal in designing biomaterials is to harness the functionality found in nature to create materials that mimic critical components of the natural extracellular matrix (ECM). We design such materials using the tools of recombinant protein engineering to synthesize block-copeptide scaffolds that combine elements derived from elastin and fibronectin. Dictating the specific amino acid sequences of these designed proteins affords us molecular-level control to independently tailor the biochemical and biomechanical properties of the resulting scaffolds. Given their cytocompatibility and bioactivity, these materials are ideal candidates for use as cell and drug delivery vehicles, implant coatings, and biomaterials for reconstructive surgeries. Through a series of systematic, three-dimensional (3D) culture studies, we are beginning to elucidate the optimal biochemical and biomechanical scaffold properties for two different tissue systems: (1) embryonic stem cell-derived cardiomyocytes and (2) multi-cellular neural tissue. These experiments demonstrate that both scaffold stiffness and integrin ligand density are critical material properties that can instruct cellular behavior in 3D cultures.

About 85% of the US population requires repair or replacement of one or more teeth as the dental pulp tissue is vulnerable to infection. Micro/nano patterning technology the positioning of cells in-vitro in desired locations allowing to recreate the geometrical organization of natural tissues in laboratory settings. Objective: To generate an in-vitro cell culture platform that is able to create the highly organized and densely packed odontoblast at the dentin-pulp interface in-vitro. Method: Silicon molds are processed with two step lithography/Deep Reactive Ion Etching to create a master mold for spin casting of a porous polycaprolactone film . Dentin tubules are mimicked by 5~10um thick PCL membrane which is designed to have one through-hole pore. Each pore is surrounded by eight pillars(3-5um width and 10um height) to confine single cell and prevent from spreading on membrane. About one million pockets were fabricated with single batch process. Membranes were used in transwell setting with a gradient of serum between compartments to induce protrusion of cell processes through pores. Results: NIH-3T3 cells readily adhered and spread across the membrane without indication of protrusion through pores in the absence of pillars. With pillar structures, cell spreading was restricted on the surface. Confocal microscopy revealed that cells form protrusions when pore size was 1-2 µm while 3µm pores allowed cell migration through the pores. At 3 days of incubation, cells exhibited a polarization perpendicular to the micropatterned membrane mimicking odontoblast morphology. Conclusion: A porous membranes engineered with micropatterning technology can be used to assign one cell to single pocket while inducing cell protrusion successfully, providing us the possibility of recreating the odontoblast layer in the dental pulp in-vitro. Such in-vitro mimicries will serve as the precursors for exploring interactions of odontoblasts with matrix molecules or epithelial cells and may set primary guidelines for dental tissue regeneration.

The development of three dimensional micro-channel networks having circular cross sections can provide structure and morphology similar to that of vascular networks in-vivo. The size and dimensions of these networks are of interest in the biomedical field for studies related to nutrition and molecular flow, blood clotting, and drug delivery applications. Micro-channels made of PDMS having circular cross sections were developed using a new, cost-effective, facile method. Specifically, soft lithography was employed to cast multiple strands of thin filament wire in PDMS to produce a network of micro-channels. The structure of the PDMS was unchanged during the wire removal process and resulted in circular channels. An embryonic mouse cell line (B2) that had been differentiated partially towards a mesodermal fate was cultured in the tubular network. The results showed that cells were capable of adhering to the walls of the channels after additional treatment with standard matrix coating. The B2 cells have two intrinsic fluorescent markers, Green Fluorescent Protein under the Tie-2 promoter showing endothelial fate, and Red Fluorescent Protein under the alpha smooth muscle promoter showing smooth muscle fate. After 10 days in culture, results show increased RFP in tubular structures of 160mu;m in diameter over standard 2D culturing methods.

Polymeric substrates for cell culturing were developed through a series of surface treatments in order to ultimately control topographic dimensions, structure, and patterning for the understanding of cellular behavior. Shrinky Dinks®, which are low-cost pre-stressed polystyrene sheets, were treated with organic solvents and/or heat in order to generate multi-scale topographic dimensionality. Specifically, PSP sheets were treated either with acetone, or gold coated and clamped in place while heat was applied, or both, in order to produce wrinkle morphologies of varying length scales. Wrinkle size could be controlled based upon the timing and duration of acetone treatment, as well as by varying gold film thickness and heat treatment conditions. Scanning Electron Microscopy (SEM) showed alignment of wrinkle morphology based on previously applied strains to the sheet. Acetone treatment results in crazing along the surface and the development of wrinkles ranging in width from 90 mu;m to 290 mu;m, with an average width of 166 mu;m. Gold-coating and heat treatment results in wrinkles ranging in width from 1 mu;m to 20 mu;m, with an average width of 6 mu;m. The multi-scale patterned PSP sheets can then be used to mold PDMS in order to create bio-chips for cell culture experiments. Optical microscopy shows cells were successfully cultured on these platforms and that cellular behavior could be controlled by varying solvent and heat treatments to produce winkles of different length scales. This extremely simple, low-cost, and time-effective method of fabrication allows for the understanding of cell-surface interactions including cellular alignment, surface adhesion, and cell orientation, which are crucial areas of research for tissue engineering in vitro and biomedical technologies.

Introduction: Stem cells reside in a multi-factorial environment containing biochemical and mechanical signals. Changing biochemical signals in most scaffolds often lead to simultaneous changes in mechanical properties, which make it difficult to elucidate the complex interplay between niche cues. Combinatorial studies on cell-material interactions provide novel tools to facilitate analyses of stem cell responses to various niche cues, but most studies to date have been performed on 2D culture. The goal of this study is to develop 3D combinatorial hydrogels containing photocrosslinkable extracelluar matrix proteins, with independent control of biochemical and mechanical properties to promote osteogenesis of human adipose-derived stem cells (hADSCs) in 3D.
Materials and Methods: To vary the biochemical cues within the combinatorial hydrogels, 4 types of extracellular matrix proteins were incorporated into the hydrogel network at varying concentrations. All ECM proteins were modified with methacrylate end groups for photocrosslinking to facilitate homogenous presentation in 3D hydrogels. To vary the mechanical properties of the hydrogels, poly-(ethylene glycol) diacrylate (PEGDA) (5kDa) with varying concentration was used (5, 10, and 15% w/v). Gelatin, a digested form of collagen was included at a constant concentration to facilitate cell adhesion. A total of 27 combinatorial hydrogel compositions with independently tunable biochemical and mechanical cues were examined. Human adipose-derived stem cells (passage 5) were encapsulated in combinatorial hydrogels at 15 million cells/ml. All samples were cultured in osteogenic medium for 21 days before harvested for analyses. Outcome analyses performed at day 21 including compression mechanical testing and quantitative gene expression of early and late osteogenic markers.
Results and Discussion: Mechanical testing showed that hydrogel stiffness was controlled by PEG concentration, with a range of 4-80kPa when increasing PEG concentration from 5% to 15%. Within the examined range of ECM concentration, varying ECM type or concentration had less dominant effects on osteogenesis. Increasing hydrogel stiffness from 5% PEG to 10% led to 5-20 fold increase in CBFA1 gene expression, whereas further increase of hydrogel stiffness (from 10 to 15%) only led to slight changes in CBFA1 or osteocalcin expression. This may be due to the relative narrow range of ECM concentration examined. Further work will expand the concentration of ECM proteins to a broader range. In sum, we have developed combinatorial hydrogels containing photocrosslinkable extracellular matrix proteins as biomimetic niche for stem cell culture in 3D. The combinatorial hydrogels with independently tunable biochemical and mechanical properties can also provide useful platforms to aid in elucidating how interactive niche signals regulate stem cells fate in 3D.

Introduction: Recent studies have shown that the stiffness of extracellular matrix (ECM) plays an important role in regulating cell fate and tissue development. To study the interplay between the matrix stiffness and cell behavior, poly(ethylene glycol) (PEG) hydrogels have been widely used due to their easily tunable physical properties by varying molecular weight (MW) or concentration of PEG. However, varying crosslinking density also leads to simultaneous changes in the mesh size of hydrogel network, which may affect protein diffusion and cell behavior within the hydrogels. Furthermore, different hydrogel formation mechanisms lead to network with different homogeneity, which may also change protein diffusion in 3D hydrogels. In this study, we aim to examine the effects of hydrogel crosslinking density and network homogeneity on protein diffusion in PEG hydrogels using combined experimental and computational approach.
Materials and Methods: Two different hydrogel structures were formed via chain growth polymerization (CG) and step growth (SG) polymerization. To form CG hydrogels with varying crosslinking density, PEGDA powder with different MW (2k,3k,4k,5k,10k) and concentration (10,15,20% (w/v)) was dissolved in phosphate buffered saline (PBS). For SG hydrogels, norbornene-terminated PEG and thiol-terminated PEG with different MW (2.5k,4k,12.5k) and concentration (10,15,20% (w/v)) were used. SG gel MW was defined as MW between two adjacent crosslinks. Hydrogels were immersed in BSA solution (4g/l) at room temperature for 24h for protein loading. For release test, hydrogels were immersed in fresh PBS, and moved to fresh PBS every 5-30 min to maximize driving force for diffusion. To determine BSA diffusivity within different hydrogels, the measured release profiles were fitted by three-dimensional Fickian diffusion model.
Results and Discussion: Increasing PEG MW or decreasing PEG concentration led to an increase in accumulated protein release. We also found that higher gel homogeneity leads to less protein accumulation. CG gels (more heterogeneous) accumulated more protein than SG gels (more homogeneous). For CG gels, increasing PEG MW from 2k to 5K did not change the diffusion profiles significantly, while further increase from 5k to 10k led to 30% increase in accumulated protein mass. For SG gels, increasing MW from 4k to 12.5k led to a 10-fold increase in accumulated protein mass. The change of PEG concentration has a more mild effect on protein diffusion, with the largest drop observed from 10% to 15%. In sum, we have shown that protein diffusion is affected by varying gel crosslinking density and gel homogeneity using 3D hydrogels and a 3D Fickian diffusion model. The experimental and computational platforms reported here can provide useful tools for characterizing scaffold diffusion property to appropriately interpret cellular responses in scaffolds with different stiffness.

Here we describe a technique that uses nano/micro molding process to fabricate polymeric micro/nanopillars for manipulating the fate of stem cells. The micro/nanopillars are fabricated by a combination of standard photo lithography and micro/nanomolding processes. Polymeric micro/nanopillars were made of polydimethylsiloxane (PDMS), UV curable adhesive (NOA 62) and biodegradable polymers. These micro/nanopillars were further modified with peptides derived from ECM molecules and then used as the substrates for culturing the human iPS and embryonic stem cells. Our results indicated that it is possible to maintain iPS and embryonic stem cells on the pillar surface with defined media. It was also found that the differentiation of the iPS cells depended on the aspect ratios of the micro/nano-pillar as well as the surface modification. The morphology of the iPS was also influenced by the aspect ratios of the pillars.

We identified the novel peptide sequences that can specifically interact with collagenous collagen matrices. Cancer is a major public health problem in the world. Early diagnosis and appropriate therapy are essential for the reduction in cancer death rates. For that reason, new biomarkers and therapeutics have been developed for the cancer diagnosis. Collagen, the most abundant protein of the extracellular matrix (ECM) in human body, is emerging as a new biomarker for cancer diagnosis and the prediction of cancer prognosis. ECM in tumor site is modified in close association with tumor stromal cells and tumor cells. Tumor stromal cells (i.e., cancer associated fibroblasts, tumor endothelial cells, and tumor-associated macrophages) express growth factors sustaining tumor growth, and proteolytic enzymes catalyzing the degradation of the ECM facilitating tumor cell growth and invasion. Structural change of collagen for tumor progress is well investigated in the breast cancer, named a tumor-associated collagen signature (TACS). Collagen type I is over-expressed in the breast cancer, the importance of studying stromal interactions in breast tissue is further reinforced by the fact that patients with collagen-dense breast tissue possess the increased risk of breast carcinoma, and a tumor-associated collagen signature-3 (TACS-3) is correlated with the long-term survival rate of human patients in reverse. In addition to breast cancer, various types of collagen are over-expressed in many kinds of cancer such as type IV collagen in the tumor stroma of pancreas, collagen type IV and type VII in colorectal cancer, collagen type I and III in epithelial ovarian cancer, and collagen type I in medulloblastoma. Here, we identified novel peptides that can specifically bind collagenous collagen matrices using a phage display. We first constructed collagen-like peptide library that carrying (Gly-Pro-Xaa) and (Gly-Xaa-Yaa) deca-repeats on the pIII minor coat protein of M13 bacteriophages (phages; Xaa and Yaa are random twenty amino acids). We utilized engineered M13 phages as a novel bio-nanomaterial for clinical applications for the cancer targeting, due to their manageability and safety to mammalian cells. Our phage-display discovered peptides exhibited selective and specific binding capability for the cancerous collagen matrices. In addition, our engineered phages that display collagen binding peptides will provide a sensitive and selective binding for collagen in cancer cells, a cancer biomarker, and might be useful for cancer imaging and therapy during cancer progression in the future.

Biodegradable scaffolds with deterministic mechanical properties, nano- and microscale topographical features, and precisely defined surface chemistry are desirable for tissue engineering applications. Among these, chitin is a naturally abundant polysaccharide, which is mechanically stable, biodegradable, nontoxic, and physiologically inert. In this study, we have developed a “chitin nanofiber ink” that self-assembles into ultrafine (3nm) nanofibers upon drying. We merge this ink with replica molding and microcontact printing technique to fabricate scaffolds with precisely defined topographical features and mechanical properties. We utilized these scaffolds to guide and align the assembly of 3T3 cells and create self-supporting cell sheets that can be transimplanted for tissue engineering applications. Our preliminary results confirmed pronounced 3T3 cell alignment along the chitin patterns, while random distribution of the cell was only observed on plain chitin film substrates (control substrate).

In the field of tissue engineering, electrospun fibrous scaffolds can deliver inducible factors which promote tissue regeneration, and also provide physical support for cell migration and proliferation. However, conventional electrospun scaffolds possess 2-dimensional sheet-like structures with small pore sizes and densely packed layers where cells can reside on the surface. In order to give higher degree of mimicking the native extracellular matrix structure and the efficient 3-dimensional environmental signaling such as cell-cell interaction and mechanical strength, cellular penetration ability needs to be improved. In this study, scaffolds composed of rigid polymer with hydrophobicity and hydrogel with swelling property were fabricated by side-by-side electrospinning approach. Simultaneous electrospinning of two polymer solutions from the end of the two needles allowed fabrication of bicomponent fibrous scaffold. We hypothesized that the swelling property of hydrogel can contribute to make micro-scale inter-fiber space in the scaffold, followed by the improvement of cellular infiltration ability, while enough mechanical strength was given by the rigid polymer. The alternate structure and bent fibers of the scaffold were confirmed by confocal laser scanning microscope after the scaffold had swollen. We expect that this phenomenon leads to enhanced cellular infiltration ability of the scaffold. This scaffold fabrication technique may promote functional construction of specific tissue by creating 3-d microenvironment, thereby contribute to various tissue engineering processes.

Geometrically patterning substrates has been demonstrated to control the microenvironment of stem cells to direct cell fate via multiple regulator mechanisms, including cell-cell contact, cell focal adhesion, and cytoskeletal tension. In our work, we developed a PEG-PEG diacrylate photo-crosslinked polymer network, covalently bonded to an oxygen plasma treated polystyrene surface. This non-fouling hydrogel was patterned via a soft lithography technique using a PDMS stencil protection during a 6 minute, 154W oxygen plasma etch, which creates hydrophilic regions that favor protein adsorption. This transparent, 40 mu;m thick PEG-based non-fouling surface was used to pattern human induced pluripotent stem cells (hiPSCs) with different sizes and shapes to study the effect of geometric factors on hiPSC self-renewal and differentiation. We demonstrated that hiPSCs were able to maintain their pluripotency on different patterned domains with adsorbed fibronectin, vitronectin, or a commercial peptide-based surface (Sythemax). We treated circularly- and triangularly-patterned hiPSCs cultures with 50 ng/mL Activin A for one day to induce mesoderm differentiation, and found that hiPSCs still expressed the pluripotent marker Oct3/4 only along the perimeter of the culture, where cells were condensed with higher local cell density (31 ± 3.3 cells/2500µm2) compared to the center (20 ± 2.9 cells/2500µm2). Smaller size (200 µm circle, 64 ± 5.9% Oct3/4 positive) and higher curvature (triangle, 56 ± 2.4%) patterns maintained more pluripotent hiPSCs than the other patterns (400 µm circle, 47 ± 2.5%) during the mesoderm induction. Due to the high cell-cell contact at the edge of patterns, higher E-cadherin expression was co-localized with these Oct3/4-positive cells. We have developed a practical and robust patterning technique to study the effects of culture geometry on hiPSC differentiation.

The extracellular matrix is often a composite network of molecularly and structurally distinct fibers, and cells engage the matrix through a variety of adhesion molecules. A number of recent studies demonstrate that cellular mechanosensing can depend strongly on the type of ECM protein presented to cells, and hence on the integrin(s) that is engaged for cell adhesion. Thus, mechanobiology studies on surfaces with a single ECM ligand may not be predictive of cell behavior on composite matrices. In order to study how cells probe and respond to more complex environments with multiple ECM structures, we developed a micropatterning technique that permits the replication of arrays of multiple, spatially separated adhesive islands of cell adhesion molecules with features as small as 1 um in diameter on soft hydrogels. In addition, the patterning tool allows for independent control of cell shape, substrate rigidity, and the topographical pattern of adhesion ligands. Furthermore, we will describe how this multicomponent system can be used to simultaneously do three things: (1) measures the cellular traction forces (CTF); (2) measures the spatial distribution of shear modulus in the cell; (3) measures the distribution of cell contractile prestress. Through patterning of fibronectin and laminin into discrete 1 um islands, we will describe how CTFs are segregated between each of the ligands during cell spreading, migration, in single versus multi-cell clusters, and during the course of cellular remodeling of the extracellular matrix after up to one week in culture. The development of a comprehensive system for measuring CTFs and the mechanical properties of cells will have a transformative impact on a number of fields within the cell mechanics, tissue engineering, and regenerative medicine fields.

Diffuse infiltration of single cells into brain parenchyma is a hallmark of malignant glioma. This extreme invasiveness makes complete tumor resection difficult and contributes to the high mortality rate associated with this disease. Brain parenchyma has a distinct physical structure characterized by densely packed neural cell processes and sub-micron extracellular space, and is largely devoid of the fibrillar collagen scaffolding typically found in stromal tissue. Consequently, glioma cells migrating in brain slices exhibit a distinct type of motility, with branched protrusions and hourglass-shaped cell-body deformations that help squeeze cells through tight spaces (Beadle et al., Mol. Biol. Cell 2008; 19(8):3357-68). However, the mechanistic details of this unique mode of motility remain incompletely understood. To address this question, we synthesized brain-mimetic nanoporous, non-fibrillar extracellular matrices (ECMs) based on cross-linked hyaluronic acid (HA), the major component of brain ECM, and functionalized them with Arg-Gly-Asp (RGD)-containing peptides to facilitate integrin-mediated cell adhesion. In a three-dimensional (3D) spheroid invasion paradigm, glioma cell motility was highly reminiscent of that seen in brain slices, validating the use of these ECMs a model system (Ananthanarayanan et al. Biomaterials 2011; 32(31):7913-23). Eliminating RGD peptides from the ECM abolished invasion, suggesting a requirement for integrin-mediated adhesions for this mode of motility. Increasing matrix density, or inhibiting myosin-based cellular contractility by blebbistatin or shRNA-induced knockdown of Myosin IIA also severely impaired motility. We also report preliminary results from two sets of studies: First, we investigate the balance of protrusive and contractile forces in glioma cell motility by pharmacologically and genetically manipulating the Rho GTPases RhoA and Rac1. Second, we explore the involvement of hyaluronidase-mediated enzymatic remodeling of the HA matrix in this mode of motility. Our studies collectively help define the mechanotransductive signaling mechanisms that underlie the distinct mode of glioma cell motility observed in non-fibrillar matrices.

The ability of living cells to generate and detect mechanical forces plays a central role in diverse biological processes, including wound healing, growth and development, and cancer metastasis. At present the vast majority of work characterizing cellular migration and force generation has examined cells on 2D substrates. As a result, cellular mechanisms for migration and extracellular matrix (ECM) remodeling in more realistic, 3D environments remain poorly understood. Here we present a quantitative analysis of how epithelial cells and fibroblasts, two canonical cell types with central roles in wound healing, couple ECM degradation and cell motility in fibrin hydrogels. The fibrin matrix serves as a simple model for the ECM present at a wound site, and moreover is elastic up to 50% deformations, allowing us to equate matrix displacements with spatiotemporal changes in strain energies. We used live-cell confocal imaging to simultaneously track migration, matrix distortion, and fibrin degradation by Madin-Darby Canine Kidney (MDCK) epithelial cells and human foreskin fibroblasts (HFFs) on timescales ranging from seconds to hours. Consistent with previous studies of fibroblastic cell types, HFFs generate spindle-like protrusions and contract the fibrin matrix. In marked contrast, MDCK epithelial cells adopt a rounded phenotype, display dramatic cytoskeletal activity on the seconds timescale, and actively degrade the fibrin matrix. Despite markedly differing in their mode of interaction with the fibrin network, both cells generate large fluctuations in strain energies within the surrounding matrix. We propose that the dramatically different cellular phenotypes we observe recapitulate the distinct functions of fibroblasts and epithelial cells during wound healing, in which fibroblasts reinforce the transient ECM at the wound site while epithelial cells dissolve and replace it with a functional epithelium. Ongoing work examines the intracellular signaling events that mediate these dramatic differences between cell types, a topic about which little is presently known.

Biophysical cues from the cellular micro-environment, including extracellular matrix (ECM) elasticity, have been shown to dramatically influence stem cell fate in vitro, but the influence of those cues on stem cell-mediated tissue regeneration remains unclear. A key challenge hampering in vivo studies linking matrix elasticity to stem cell behavior is a lack of macroporous materials that allow precise control over the molecular cell-material interface under conditions that are amenable to cell encapsulation. To allow analysis of the effects of matrix elasticity on stem cell mediated tissue regeneration, we fabricated cell-encapsulating, “void-forming” hydrogel materials that form pores in situ, after injection, to facilitate cell deployment to injured tissues without affecting the adhesion ligand presentation or mechanical properties of the hydrogel surrounding pores. We applied these materials towards bone regeneration using mesenchymal stem cells (MSC).
Void-forming hydrogels were fabricated by co-encapsulating cells along with rapidly degrading, gel-based porogens (typically 150mu;m diameter) into a more slowly degrading “bulk” hydrogel network. Sacrificial porogens were formed using oxidized alginate that exhibits rapid hydrolytic degradation, which was subsequently extruded under co-axial airflow into a calcium chloride bath. The bulk phase was formed from a high molecular weight alginate polymer modified with integrin-binding RGD peptides. Void density and formation were verified by scanning electron microscopy and mechanical testing. By modulating the extent of crosslinking and succeptibility to hydrolytic degradation of porogens, it was possible to vary the initiation of MSC release from 5-50 days in vitro or 5-20 days in vivo. Further, in cranial defect studies, human MSC (hMSC) deployed from void-forming hydrogels led to more extensive bone regeneration than hMSC deployed either in standard, non-degrading hydrogel materials or saline.
To study the effects of matrix elasticity on hMSC-mediated bone regeneration, we first performed in vitro studies to confirm that modulating the rigidity of the “bulk” component of gels surrounding voids could regulate MSC behaviors. These studies revealed a strong role for matrix elasticity, with MSC proliferation and osteogenic commitment being optimal at an intermediate range (20-60 kPa). Strikingly, in cranial defect studies, bone regeneration by hMSC delivered in void-forming hydrogels depended strongly on matrix elasticity, with regeneration occurring optimally when cells were deployed from matrices of intermediate elastic modulus (60 kPa versus 5 or 110 kPa). This verifies that matrix elasticity can directly control cell behaviors in vivo. More broadly, this strategy for fabricating void-forming materials may be useful for studying how specific cues from biomaterials, in combination with cues from host tissues, regulate transplanted stem cell fate.

Cell adhesion typically relies on the specific binding of proteins to their ligands.
We recently found that in the mammalian RGD-integrin adhesion system the spacing between integrin binding sites controls cell adhesion forces, cell adhesion reinforcement and cell elasticity. Importantly, the spacing of the binding sites and not their density is controlling adhesion.
Here we use micro-patterned nanostructured surfaces to further study the effect of ligand spacing on cell adhesion. Micro-structured domains on surfaces can be easily obtained with photolithography followed by an etching step. Subsequently, we create hexagonal patterns of nanometer- sized gold dots within the micro-domains using diblock-copolymer micelle nanolithography. The spacing between the gold dots can be varied from about 20 to 200 nm with nanometer precision. With our approach we are able to generate micro-patterns with different gold dot spacings on one single substrate. By biofunctionalization of the gold dots with ligands for cell adhesion, e.g. peptides, we can anchor cellular adhesion receptors to the gold dots. Thus, our micro-patterned nanostructured surfaces now provide a versatile platform for studying many different cellular adhesion processes that are influenced by micro-nanostructured ligand spacing and ligand density effects.
References:
(1) Selhuber-Unkel, C., Erdmann, T., Loacute;pez-García, M., Kessler, H., Schwarz, U. S. and Spatz, J. P. Biophysical Journal (2010), 98, 543-551.
(2) Selhuber-Unkel, C., Loacute;pez-García, M., Kessler, H., Spatz, J. P. Biophysical Journal (2008), 95, 5424-5431.

There are four primary technical challenges for the successful development and fabrication
of an engineered tissue, 1) cell source, 2) structural matrix, 3) host immune response and 4) tissue vascularization. The vasculature is necessary to provide for the tissue&’s metabolic needs and without it, only very thin organs that can survive by diffusion can be generated. Therefore to make a clinically relevant sized tissue will require a functional and mature vascular system integrated into the tissue. For many years the vasculature was viewed as a passive plumbing system, delivering oxygen and nutrients then carrying away metabolic waste. Now we clearly understand that proper development and maturation of parenchymal cells occurs in parallel to and by cross-talk with the developing vasculature. Of critical importance to this discussion, how do we progress from immature, leaky vessels (which are easy to make) to a fully mature, perfused and stable vasculature (not so easy to make)? In addition to the basic architectural difficulties of making vessels, as with the elemental tissue
parenchyma, where will the cells be sourced and how do we deal with the host immune system? In this presentation we will discuss these issues and present potential strategies
for addressing this critical obstacle.

Damage of coronary arteries limits the supply of oxygen rich-blood to heart leading to the death of functional cardiomyocytes. Revascularization can aid in recovery and improve cardiac function by restoring the blood flow to the heart. In this work, we have investigated the influence of hyaluronic acid (HyA) based hydrogels on vascular tube formation via the differentiation of endogenous cardiac progenitor Sca-1+CD45- cells (CPCs) into the endothelial cell lineage. We explored a modular HyA based hydrogel system that contains bspRGD (15) peptide for cell adhesion, heparin for sustained presentation of growth factors, and matrix metalloproteinase (MMP-13) sensitive peptide crosslinks for matrix modulation. Viscoelastic storage modulus of these hydrogels were tuned from 10Pa to 850Pa. Covalent conjugation of heparin in the HyA network retained over 70% of transforming growth factor β1 (TGF β1) for three weeks. Encapsulated Sca-1+CD45- CPCs within the hydrogel network were viable, proliferated, and formed vessel-like networks inside the hydrogel. Average diameter and density of the vessel-like tubes were tuned by altering the peptide density and modulus of the hydrogel. This HyA hydrogel/CPC system is a promising candidate for tissue regeneration where revascularization is a prime objective.

The wound healing process is an intricate integration of overlapping events involving a multitude of cells, cytokines, and extracellular matrix components. In healthy individuals, this process is generally carried out to completion, resulting in timely wound resolution. However, elderly individuals and those suffering from diseases such as obesity and diabetes display an increased risk of disruption in this process, resulting in non-healing chronic wounds. While growth factor therapy has demonstrated a limited ability to promote healing, the current therapeutic modalities do not accurately recreate the natural temporal dynamics of wound healing. I will discuss the development new therapeutic dressings based on layer-by-layer technology that more accurately recreate the dynamic signaling observed in vivo. Results show that these new dressings retain the biological activity of multiple incorporated cytokines both in vitro and in vivo and impact multiple wound healing processes in a chronic wound mouse model of type-2 diabetes.

Endothelial cells (ECs) exposed to laminar shear stress align in the direction of blood flow and express low levels of adhesion molecules. However, it is unknown whether these biological responses result from shear stress per se, or if they can be recapitulated using topography cues from the underlying extracellular matrix (ECM). We hypothesized that collagen topographical patterning can regulate EC assembly and function in the absence of shear stress. We created spatially patterned substrates composed of parallel-oriented polydimethylsiloxane (PDMS) microchannels coated with collagen, as well as parallel-aligned nanofibrillar collagen generated from a novel extrusion approach. When grown on oriented collagen substrates, the ECs underwent striking re-organization of their actin cytoskeleton (aligned within 10 degrees of the microchannel direction) and their focal adhesions. Mimicking the reported EC responses to laminar shear stress, aligned ECs were less adhesive for monocytes and platelets (with 50% reductions in the expression of intercellular adhesion molecule 1, and in monocyte adhesion in a functional binding assay). DNA microarrays revealed a transcriptional signature of 905 genes that were significantly up-regulated in the patterned cells and 3,303 genes were significantly down-regulated, including genes previously not associated with EC function. These results demonstrate that topographical cues from the underlying ECM are potent regulators of EC morphology and function and mimic the effects of laminar shear-stress. This work highlights the importance of cell-ECM interactions in maintenance of EC phenotype and has implications in the design of vascular conduits to minimize atherogenesis.

Work into synthesizing microvascular structures for tissue engineering has undergone significant advancement in the last few years. Many researchers have been developing methods to print, place and grow vascular and tissue constructs. We report on our new synthetic technique, VESSEL, (Vascularization of Endothelial Scaffolds through Sacrificial ELements) that enables the formation of 3D micro-structures for the placement of tissue and vasculature in precise 3-dimensional orientations. We have shown that VESSEL can be used to grow vascular structures of complex topologies. We will also report on our recent advances in using VESSEL to create tissue constructs that mimic the liver and lung. We detail the VESSEL process as well as methods for creating more complex 3D microstructures for tissue engineering applications.

Central nervous systems diseases, such as stroke, spinal cord injury and blindness, require innovative solutions such as stem cell transplantation to promote tissue and functional repair; however, stem cells often die soon after transplantation, necessitating the design of cell delivery vehicles for enhanced survival. We have investigated a series of hyaluronan-methyl cellulose hydrogels for the delivery of neural stem cells to the brain, spinal cord and retina. The hydrogel itself has shown a pro-survival benefit on cells both in vitro and in vivo, and the mechanism for this pro-survival effect has been elucidated. Combination with additional factors that promote host tissue integration further enhance survival. Chemical modification with peptides and growth factors promote greater cell survival and differentiation both in vitro and in vivo. These well-defined strategies will be described for neural (or retinal) stem cells tested in vitro and in animal models.

Recombinant proteins have been explored for numerous biotechnological applications. In particular, artificial proteins based on structural repeats of elastin and silk have been investigated for use in tissue engineering and drug delivery applications. Compared to synthetic or natural materials, recombinant proteins have a number of advantages that include: exquisite control over sequence and composition; precise molecular weight; and a modular nature, which allows for the functional domains to be easily altered.
We have developed a family of modular protein biomaterials composed of bioactive domains and resilin structural repeats. Resilin is an elastic protein from insect joints and tendons that contributes to flight and jumping. Resilin has a high resilience, low stiffness, and a high fatigue lifetime, which makes it ideal for use in repetitive environments. We observed rapid crosslinking of resilin-based proteins with tris(hydroxymethyl)phosphine. Crosslinked hydrogels had a complex modulus of 22 kPa, and an unconfined compressive modulus of 2.4 MPa. Furthermore, mesenchymal stem cells (MSCs) had a high viability one day after being encapsulated within the hydrogel.
We incorporated two different bioactive domains within our resilin-based biomaterials. One family of proteins contained the RGD cell-binding domain derived from fibronectin. Our studies showed that MSCs recognized the cell adhesion sequence in a sequence-specific manner and had a cell area 1.4 times higher than when cells were seeded on a sequence-scrambled negative control protein. Another family of resilin-based proteins incorporated a peptide derived from bone morphogenetic protein-2 (BMP-2). MSCs seeded on proteins containing the BMP-2 peptide had higher levels of mineralization after 11 and 13 days of bone differentiation compared to cells seeded on sequence-scrambled negative control proteins or tissue culture polystyrene. Thus, our results demonstrate that incorporation of the BMP-2 peptide within our resilin-based biomaterial accelerated osteogenesis.

The natural extracellular matrix (ECM) contains a heterogeneous composition of proteins that are presented to residing cells within discrete locations (not a homogeneous concentration or distribution) and precise times (determined by the developmental stage of the tissue), however engineered ECMs (eECMs) that attempt to recapitulate the natural ECM have not yet been able to successfully mimic this heterogeneity. We believe that the natural heterogeneity of ECMs is important to recover and in its absence the construction of complex tissue architectures is difficult to achieve. To capture both spatial and temporal material heterogeneity we have produced a photoactive-enzymatic hybrid platform that uses cell compatible light to activate specific volumes within eECMs, followed by enzyme catalyzed immobilization of active soluble factors (e.g. adhesive peptides, growth factors, etc.). This platform provides an advanced tool for the study of in vivo and in vitro tissue growth, regeneration, and development.

In order to understand the complex interactions between the stem cells and micro/nano-scale ECM which guide the cell phenotype and tissue formation, reproducible in vitro models that can provide biochemical, mechanical and topographic cues are needed. Additionally, the whole proteins are required to best mimic the complex native ECM microenvironment. To address this need, the whole proteins 3D micro/nanostructure are fabricated by multiphoton excited photochemistry provides both the biological and morphological properties necessary to better examine the stem cell-ECM interaction. A series of “modules”, each compromised of four adjacent arches forming a tunnel-like structure, are synthesized from laminin (LN/BSA) mixtures and from pure BSA alone. This afforded studying the migration dynamics of mesenchymal stem cells (MSCs) and separating the roles of topographic cues (BSA only) and ECM binding cues (LN). The strong interaction between the MSCs and modules are shown by time-lapse migration measurements, static SEM and as well as 3D fluorescence lifetime microscopy (FLIM) imaging. In addition, we found that MSCs have significant longer interaction times with LN/BSA than the pure BSA modules, presumably pointing to the role of LN binding cues in conjunction with 3D morphology. Some of material properties including the volumetric swelling ratio, pore size distribution, and fractal dimension, were characterized and indicated that the LN/BSA and pure BSA modules had similar microstructures, underscoring the importance of the LN binding cues on the migration. Critical to this work was the development of an online monitoring system with active feedback based on fluorescence imaging to provide quality control when synthesizing multiple identical constructs under the automatic control. The optimized fabrication process can be utilized to create nearly any 3D structure to model the ECM.

Reconstructing organs in vitro requires understanding how multiple cell types self-organize into a specific three-dimensional structure. Although intrinsic signals such as differential cell adhesion are known to contribute to the self-organization of dissociated tissues, the effect of extrinsic signals such as the physicochemical properties of the reconstituted ECM are poorly characterized. To better define the role of the ECM in directing the self-organization process, we are attempting to reconstruct the human mammary gland in vitro. Using mammary epithelial cells derived from human breast reduction mammoplasties, we sort cells into their luminal and myoepithelial lineages and reassemble them in precisely defined 3D co-cultures using a combination of photolithographic and chemical strategies. By tuning the adhesivity and elasticity of the biomimetic hydrogels used to accommodate these heterotypic cell-populations, we can control the 3D positioning of the different cell-types and promote the formation of bilayered acini that resemble their normal in vivo architecture. Remarkably, changes to the material properties of the ECM can trigger complete inversion of the glandular architecture - reversing the relative positioning of the two cell types. We use these assays to identify cell-cell/cell-ECM adhesion molecules as well as downstream effectors responsible for the self-organization of epithelial cells in different material contexts. Current efforts aim to use the findings towards the reconstruction of an entire human mammary gland in vitro.

Cells sense changes in their environment and respond by altering their behavior. We investigate how cells manipulate their membrane surface chemistry, which has profound effects on disease progression. Cells orchestrate the density and organization of proteins and lipids to govern adhesion, migration, and proliferation. From this knowledge, we can engineer drug delivery vehicles that complement the molecular patterns observed on cells to achieve strong, cooperative binding. We observe relationships between multiple cell membrane proteins that impact vehicle binding. We have employed these strategies in a model system of inflammation and in breast cancer metastasis. Our synergistic approach to targeted drug delivery may transform how we design therapeutics.

Fibrosis is implicated in a variety of disease states including scar contractures in skin, cardiac complications after a heart attack, and cancer metastasis. This pathology is mediated by connective tissue cells known as fibroblasts, which have been found to behave differently based on their substrate. These cells prefer to grow on fiber-like substrates such as collagen and we hypothesized that introducing high aspect ratio particles would decrease the amount of pathological contraction and scarring which result in fibrosis. We tested this hypothesis by investigating the cell response to incubation with multi-wall carbon nanotubes (MWNT), single-wall carbon nanotubes (SWNT), or spherical carbon black.
We modified an established model of skin contraction by adding concentrations of 100 µg/mL, 10 µg/mL, and 1 µg/mL of each nanoparticle type in phosphate buffered saline (PBS) and pluronic F-127 to type I collagen gels with or without fibroblasts. After 7 days of incubation, the gels containing MWNT and SWNT significantly inhibited contraction at every concentration tested. The carbon black gels did not significantly alter contraction at any concentration tested.
There have been conflicting reports of carbon nanoparticle toxicity, and the viability of the cells is especially critical in this case because contraction is a cell-mediated process; decreased contraction could also be achieved merely by killing the cells. However, in both a live/dead assay at day 3 and counts of live cells using trypan blue exclusion at day 7, the nanoparticle gels consistently had more viable cells. In the MWNT and SWNT gels, a significant change occurred only with the highest concentration of particles, which is suggestive of a percolation threshold. These gels had a roughly 2.5-fold increase in viability. In the carbon black gels, the intermediate concentration of 10 µg/mL yielded a significantly higher viability than the control, though it was not significantly different than either of the other carbon black concentrations which where not significantly different than the control.
We have found that the presence of rod-shaped carbon nanotubes, whether single-wall or multi-wall, inhibits pathological contraction while the spherical carbon nanoparticle carbon black does not. Our data also show that this decrease in contraction occurs without harming the cells. MWNTs and SWNTs are a promising area of research for treatment of fibrosis, for which there are currently no therapies that treat the underlying cause.

Hydrogels based on poly (ethylene glycol) (PEG) have been widely used as tissue engineering scaffolds due to their biocompatibility, injectability as well as tunable chemical and physical properties. However, several key hurdles remain before PEG hydrogels can be applied broadly for engineering load bearing tissues. First, PEG based hydrogels are often associated with poor flexibility when subject to cyclic loading, which is present in load bearing tissues such as articular joints. Second, most PEG hydrogels are nanoporous, whereas macroporosity is desirable for efficient nutrient diffusion, cell proliferation and matrix deposition. Third, few hydrogels developed-to-date allow independent tuning of niche cues such as macroporosity, biochemical ligand density and mechanical stiffness. In this study, we aim to develop a novel method for fabricating PEG-based microribbons with decoupled biochemical and mechanical properties, which can crosslink into 3D macroporous scaffolds with high flexibility in a cell-friendly manner.
The microribbons were produced by wet-spinning 8-arm-poly(ethylene glycol)-n-Hydroxysuccinimide ester (PEG-8NHS) in isopropanol containing tris(2-aminoethyl)amine as crosslinker. To tune mechanical property, the ratio of NHS-groups to PEG-arms was adjusted by ethylamine prior to wet-spinning, which led to various crosslinking rates and rigidities. Maleimide groups were introduced to incorporate biochemical ligands, and methacrylate groups were introduced to allow photocrosslinking of microribbons into 3D scaffolds. The macroporosity, biochemical and mechanical properties of resulting scaffold that cells are sensing were independently tuned by varying microribbon density, choice of biochemical ligands (cysteine or peptide CRGDS), and microribbon rigidity.
Our fabrication process led to successful formation of PEG-based, microribbon-like elastomers that can photocrosslink into macroporous scaffolds for direct cell encapsulation with high viability. Cellular responses to the microribbon-based scaffold can be tuned by independently changing microribbon density, choice of biochemical ligands, and microribbon rigidity. By tuning the biochemical ligand density of CRGDS, enhanced spreading and alignment of human adipose derived stromal cells (hADSCs) can be observed by day 5. When encapsulated in 3D microribbon-based scaffold, hADSCs proliferated up to 20-fold within 2 weeks. Increasing the ratio of NHS-groups to PEG-arms from 0.625 to 0.8 increased the microribbon rigidity non-linearly from about 10 kPa to 100 kPa and enhanced cell spreading. Microribbon-based scaffold can sustain up to 70% cyclic deformation without failing. Such synthetic, microribbon-based hydrogels can serve as novel scaffolds for engineering load bearing tissues. Furthermore, the decoupled niche properties of such scaffolds would provide novel platforms to facilitate studying how interactive effects of niches signals regulate cell fate and tissue formation in 3D.

The ability to cue cellular response based on scaffold properties has been well established in the field of tissue engineering. Particularly, scaffold morphology has been shown to promote differentiation in adult stem cells. However, the mechanisms by which scaffold morphology directs cellular processes such as differentiation is not fully understood. The present work examines the influence of scaffold morphology on osteogenesis and gene expression of primary human bone marrow stromal cells, adult stem cells isolated from bone marrow with the capacity to undergo differentiation into osteogenic, condrogenic, or adipogenic lineages. Cells were cultured on both 3D nanofibers and 2D film scaffolds and mRNA expression was analyzed by cDNA microarray. Gene expression patterns were correlated with gene ontology analysis to elucidate biological processes influenced by scaffold morphology. Our results indicate that the 3D nanofiber scaffolds mimic the effect of soluble osteogenic supplements in promoting osteogenesis via the TGF-β signaling pathway, whereas 2D films do not.

Coronary heart disease is a critical challenge in the US with over one million myocardial infarctions (MI) each year. Despite optimal care in the acute setting of MI, subsequent development of scar tissue and a lack of treatments for this maladaptive response often lead to a poor prognosis. Therapies targeting scar tissue prevention and promoting contractile tissue regeneration need to be identified to treat this growing patient population in the wake of increasing rates of obesity and heart disease. Previously, the Desai lab has designed a system of polymeric microrods that have been shown to decrease fibroblast proliferation and promote cardiomyocyte hypertrophy in 2D and 3D culture. Furthermore, preliminary experiments from the lab examining microrod injections into infarcts in a rat model of MI have shown promising reductions in scar tissue and notable improvements in cardiac function. Developing a stronger understanding of how cardiac fibroblasts interact with microtopographical cues in a mechanically altered extracellular matrix containing microrods will provide valuable insight into this therapeutic strategy. Using sensitive imaging and gene expression characterization, we have studied the mechanisms by which the presence of microrods directly affects the behavior of cardiac fibroblasts through interaction with specific pathways of mechanotransduction and cell communication, in vitro. We have also demonstrated the therapeutic viability and efficacy of this approach in a rat model of MI, showing improvements in cardiac function and changes in scar tissue density and quality. The results of this work will enable us to harness capabilities in engineering of the extracellular environment using materials to influence cell behavior and signal transduction in order to design informed translational strategies for cardiac repair after hypoxic injury.

Active cellular division is a hallmark of tissue repair as well as multiple other pathologies, and therefore methods to modulate gene expression profiles in proliferating cells would have enormous therapeutic advantages. However, while many types of cells will efficiently internalize nucleic acid assemblies, poorly controlled subcellular trafficking and disassembly represent key hurdles preventing clinical realization. We hypothesized that utilizing sequences from nature&’s DNA packaging scaffolds - histone proteins - might enable recapitulation, on recombinant plasmids, of native nuclear localization and controlled release mechanisms. Accordingly, we engineered gene delivery polyplexes by packaging plasmid DNAs (pDNAs) with histone tail sequences containing nuclear localization sequences (NLSs) and key modifications known to activate transcription on chromatin. We have demonstrated that these histone-mimetic polyplexes exhibit robust gene transfer activity in cultured fibroblasts relevant to tissue repair. Furthermore, we have used a combination of cell synchronization, cellular microinjection, and confocal microscopy to identify key steps in polyplex trafficking to the nucleus, including retrograde transport through the Golgi apparatus and endoplasmic reticulum, as well as NLS-dependent nuclear retention during cellular mitosis. Finally, we have shown that these polyplexes interact with key nuclear proteins to activate rapid unpackaging and transcription in the nucleus. Thus, our studies add new understanding of cellular gene transfer mechanisms, with direct relevance to wound repair and potential applicability to a wide range of other therapeutic applications involving gene modulation in dividing cells.

Introduction. Gene delivery is a promising tool for the induction of therapeutic factors in regenerative medicine; however, clearance of gene therapy vectors from the intended site has been problematic historically. Micro-porous PLG scaffolds are utilized in our laboratory to provide a platform for the controlled delivery of vectors to enhance their transduction efficiency and expression in vivo. To expand upon our previous work, these studies modify the surface properties of PLG scaffolds to extend vector retention in vitro and enhance their expression in vivo.
Methods. Microporous PLG scaffolds were fabricated using a gas foaming/particle leaching based technique. Naturally-derived polysaccharides were conjugated onto surfaces of these scaffolds using EDC/NHS chemistry. Chitosan, a positively-charged amine-rich polymer, was conjugated directly to the carboxylic acids of PLG using this chemistry. Heparin and hyaluronan, negatively-charged carboxylic acid-rich polymers, were conjugated onto PLG using 2 EDC/NHS reactions—the first to attach an amine-containing spacer to the PLG and the second to attach the polysaccharides onto the spacer. Lentivirus was adsorbed onto the surface-modified scaffolds and then assessed in vitro for their ability to retain the virus and enhance the virus&’ transduction efficiency. In vivo gene delivery was assessed in the spinal cord which required the development of a porous, multiple-channel PLG bridge for a mouse injury model. Neurotrophin 3- and sonic hedgehog-encoding lentiviruses were delivered using a heparin-modified bridge.
Results. All polysaccharides enhanced the incorporation of lentivirus onto the biomaterial compared to unmodified PLG scaffolds; however, only chitosan- and heparin- modified scaffolds enhanced the transduction and gene expression of human endothelial kidney cells (HEK 293T). Studies revealed the enhanced transduction in vitro was in part due to increased retention and extended half-life after surface modification. Assessment in vivo revealed surface modification did not grossly affect the extent of cell infiltration and behavior at acute time-points, in particular axon extension and myelination. Yet, surface modification enhanced the number of cells transduced inside and near the bridge. Lentivirus delivery utilizing our bridges produced transgene expression that persisted for weeks, regardless of surface modification. However, heparin- and chitosan-modified bridges enhanced gene expression levels in vivo compared to unmodified bridges. Use of heparin-modified bridges for delivery of vectors encoding neurotrophic factors enhanced the cellular response after injury.
Conclusion. We present a surface modification strategy to enhance the incorporation and retention of lentivirus onto PLG scaffolds of multiple architectures and to increase their transduction efficiency and expression for in vitro and in vivo applications.

One of the primary challenges that hinder successful implantation of tissue engineering scaffolds is achieving proper formation of blood vessels, or angiogenesis, within the scaffold for appropriate tissue integration during the wound healing cascade. We aim to create a hydrogel system that will promote angiogenesis by incorporating bioactive signals (DNA polyplexes) with a highly porous hydrogel structure. We believe the pores within the hydrogel will (i) promote cellular infiltration and transgene expression, (ii) allow for vascular in-growth, and (iii) provide a means for oxygen and nutrient diffusion as well as removal of metabolic waste. We first investigated the effect of pore size (53-63 µm and 90-106 µm) in hyaluronic acid (HA) hydrogels in vitro. Although we saw differences in mechanical properties due to pore size, this appeared to have a minimal effect on cell viability, spreading, and proliferation in vitro. Our lab has previously developed a caged nanoparticle encapsulation (CnE) technique to load our HA hydrogels with a high concentration of non-viral pDNA/L-PEI polyplexes. Using this technique for all investigated pore sizes, we incorporated 1 µg/µl pDNA encoding for Gaussia luciferase and observed sustained DNA release and transfection for up to 10 days but with no significant differences between pore sizes. Currently, we are investigating gene delivery of a GFP-luciferase reporter plasmid from porous HA hydrogels in two different murine models for subcutaneous implantation and wound healing. We have incorporated 2.5 µg/µl pDNA and have observed gene transfer at 3 and 6 weeks, with higher expression at 3 weeks in the subcutaneous implantation model. In our wound healing model, preliminary results show high cellular infiltration in our porous hydrogels as compared to our nanogels (containing no macropores), which had infiltration limited to the periphery of the implant. Moreover, we observed transfection and blood vessel formation within the granulation tissue of our porous hydrogels at 14 days, while the nanogels had noticeably less granulation tissue, and therefore decreased transfection and angiogenesis. Investigation of wound closure rates indicated that the incorporation of pores into the hydrogel significantly decreased the wound area over two weeks. Thus, from our in vivo investigations of cellular infiltration, angiogenesis, transfection, and wound closure rates, the importance of the incorporation of pores into a scaffold is highlighted. We are currently aiming to better promote angiogenesis through the incorporation of therapeutic plasmids encoding for VEGF and PDGF.

Adult bone marrow derived mesenchymal stem cells (BMSC) represent an important source of cells for tissue regeneration, and biomaterials that bypass the need for ex vivo BMSC manipulation and transplantation by instead recruiting native BMSCs to aid in regeneration are very promising. The specific aims of this study were to (i) identify a chemokine to recruit BMSCs, and (ii) design a biomaterial system to deliver this chemokine in a bone defect in order to recruit BMSCs and provide recruited cells an environment to proliferate and differentiate
Identification of a chemokine to recruit BMSC&’s: Using a transwell assay to screen a number of candidate chemokines, it was observed that primary BMSC&’s showed best migratory response to Thymus Chemokine 1 (TCK 1)(upto 2.5 fold increase, p<0.05). BMSC&’s were also shown to respond in a dosage dependent manner when exposed to TCK 1 (10^-11,-10,-9,-8,-7 M; p<0.05). Furthermore, in a 3D migration assay using a micro-fabrication setup, BMSC&’s were shown to migrate to a TCK 1 gradient in a directional manner (chemotaxis, p<0.05). Further, no inhibitory effects of TCK 1 were observed on MSC proliferation and differentiation in vitro.
Design of a biomaterial system to deliver TCK 1: Two compartment, macroporous scaffolds were fabricated from alginate to allow for TCK 1 delivery and cell recruitment. One compartment was comprised of a slowly degrading alginate “bulk” polymer, and was intended for sustained slow release of TCK 1. The second compartment was fabricated from a rapidly degrading oxidized alginate “porogen”s in order to release TCK 1 rapidly. TCK 1 was encapsulated in each of the two compartments, and its subsequent release quantified. Encapsulation of TCK 1 in the nanoporous (control), bulk, and porogen phase&’s resulted in different release profiles; nanoprorous with burst release (30-35ng/day), bulk (15-20ng/day), porogen (10-15ng/day). Using the two component system it was possible to tune the release rate of TCK 1 at 15-20 ng/ day as desired for our application. The bulk alginate in the two component system was RGD modified such that when the rapidly degrading porogens result in voids, the recruited BMSC&’s would infiltrate the bulk gels, attach, and proliferate inside the biomaterial.
Five in vivo groups were used to test our hypothesis in a rat femur defect model: (i) control- empty defect, (ii) chemokine (TCK 1) encapsulated in nanoporous, (iii) empty nanoporous gels, (iv) chemokine encapsulated in macroporous gels, and (v) empty macroporous gels. 1.2 mm defects were made in rat femurs and the gels were placed in the defects and monitored postoperatively at 3 and 10 Days. Qualitative immune-histology for MSC markers, and microCT results indicate gels with chemokine to perform best in MSC recruitment and bone formation. The preliminary results from this study demonstrate the use of biomaterial based delivery of a chemokine (TCK-1) to recruit bone marrow mesenchymal stem cells.

Injectable, shear-thinning, hydrogels can be used for localized delivery of therapeutic factors to promote target cell behaviors. However, the sustained delivery of therapeutic factors from hydrogels remains a significant challenge. Our approach uses a novel hetero-assembling protein hydrogel that allows for direct loading and prolonged release of therapeutic factors including genes, small molecules, and protein drugs. In our Mixing-Induced Two-Component Hydrogel (MITCH), bulk assembly is driven by molecular recognition and stoichiometric binding between cross-linking domains within two polypeptide chains. By integrating recombinant protein design with polymer physics models, we manipulate the polypeptide chain interactions and demonstrate the direct ability to tune the cross-linking density, gelation behavior, and mechanical properties. These MITCH materials hetero-assemble under physiological conditions, an advantage over other commonly used natural and synthetic hydrogels, which may improve the activity of therapeutic factors following encapsulation. Furthermore, as gelation is initiated upon mixing, therapeutic factors may be directly loaded during assembly, minimizing the need for multiple loading and wash steps. Through a series of in vitro studies, we establish the versatility of this material to encapsulate several therapeutic factors, including small peptides, model growth factors, and non-viral gene vectors. We further demonstrate the potential of MITCH materials to allow for prolonged and tunable release up to at least 10 days. Current experiments are quantifying the stability and activity of the released therapeutic factors by analyzing their effects on cell behavior. Initial selection of therapeutic factors is targeted at promoting angiogenesis for applications in stroke and peripheral arterial disease.

Slanted columnar thin film (SCTF) substrates possess intricate sculptured features on the nanoscale with applications in photovoltaics, sensing (chemical, biological, optical, and pressure), micro- and nano-fluidics, biomaterials, and nanoelectronics, including field emitters, supercapacitors, and transistors. When fabricated by glancing angle deposition, these nanoscale thin films consisting of uniform nanocolumns, which possess intricate and complex architectures ranging in size from the sub-nano to micro-scales with precise intercolumnar spacing. In this work, SCTFs were investigated for their potential to serve as cell-instructive interfaces as well as loading and release depots for various organic materials such as proteins, polymers, and DNA complexes loaded within the intercolumnar spaces of the nancolumns. We employed a novel, combinatorial analytical approach - spectroscopic ellipsometry and quartz crystal microbalance with dissipation (SE/QCM-D) - to dynamically characterize the adsorption processes and effective porosities of biomolecule adsorption within three-dimensional thin films. SE/QCM-D was used to analyze the adsorption of fibronectin (FN) and bovine serum albumin (BSA) within silicon and titanium SCTFs, which demonstrated that increased surface area of SCTFs significantly increases the loading capacity of proteins. Furthermore, both SE and QCM-D reported greater adsorbed mass and porosity for FN adsorption relative to BSA adsorption within SCTFs. We have also demonstrated the ability to functionalize individual silicon SCTF nanocolumns with stimuli-responsive PAA polymer brush coatings. SE data was collected and modeled before and after brush grafting. The ellipsometric model revealed polymer brush fractions ranging between 12-33% of the SCTF layer were present between the columns of the SCTFs. SEM was able to validate that polymer material penetrates within the intercolumnar void space of the SCTF film layer. Combinatorial SE/QCM-D measurements demonstrated the reversible, pH-dependent swelling characteristics of the polymer brushes, which could be used for biomolecule loading and release. We are also investigating the ability to load DNA and DNA complexes within the intercolumnar void space of the native and polymer-brush functionalized SCTFs, as an innovative substrate-mediated delivery approach, which takes advantage of the increased surface area afforded by the nanotopopgraphy, as well as providing a mechanism to alter surface properties that can enhance cellular responsiveness to gene delivery. These studies show that SCTFs are capable of loading and releasing biomolecules, as well as supporting cell culture and stimuli-responsive polymer brush functionalization. SCTFs demonstrate immense potential as biomaterial coatings for use in tissue engineering, therapeutic, and biosensing applications.

In order for gene therapy to be used in clinical settings, safe and efficacious delivery systems must be developed that simultaneously address numerous extra- and intracellular challenges. Non-viral delivery systems, which typically employ cationic lipids or polymers to complex and condense DNA through attractive electrostatic forces, are easier to construct and have superior safety profiles when compared to viral vectors. Despite numerous efforts to resolve their limitations, however, most lipo- and polyplexes suffer from low DNA packaging efficiency, modest targeting specificity, and a high degree of undesired cytotoxicity. To this end, we have engineered mesoporous silica nanoparticle-supported lipid bilayers (‘protocells&’ - see Nature Materials (2011), 10: 389-397) to selectively deliver a plasmid (pCB1) that encodes the reporter protein, ZsGreen, and a cyclin B1-specific small hairpin RNA (shRNA) to dividing and non-dividing hepatocellular carcinoma (HCC) cells with nearly 100% efficiency. We use histones to package pCB1 into highly compact nanoparticles that are subsequently modified with a nuclear localization sequence (NLS) and incorporated within the pores of mesoporous silica nanoparticles. Zwitterionic liposomes are then fused to pCB1-loaded cores to form a supported lipid bilayer (SLB) that we modify with PEG to enhance colloidal stability, an endosomolytic peptide to promote endosomal escape, and a targeting peptide (MC40) that binds to hepatocyte growth factor receptor, a protein known to be over-expressed by many types of HCC. We have found that protocells have a 100-fold higher capacity for DNA than similarly-sized lipoplexes and retain histone-packaged pCB1 for > 4 weeks when exposed to a simulated body fluid at 37°C. MC40-targeted protocells have a 10³-fold higher affinity for HCC (Hep3B) than for untransformed hepatocytes and are selectively internalized by Hep3B via receptor-mediated endocytosis. Endosome acidification destabilizes the SLB and protonates the endosomolytic peptide, both of which promote efficient cytosolic dispersion of histone-packaged pCB1, while the NLS induces nuclear accumulation and enables transfection of nearly 100% of dividing and non-dividing Hep3B cells, which express ZsGreen for up to 4 weeks. When exposed to Hep3B in vitro, MC40-targeted, pCB1-loaded protocells, furthermore, induce dose and time-dependent decreases in the expression of cyclin B1 mRNA and protein, which results in rapid G₂/M arrest and apoptosis. We are currently assessing the ability of pCB1-loaded protocells to selectively transfect human cancer cells injected into ex ovo avian embryos; our preliminary findings suggest that target cells express 50-fold more ZsGreen when embryos are injected with protocells versus corresponding lipoplexes. In conclusion, protocells possess the capacity, specificity, and stability necessary to overcome many of the barriers to effective gene therapy.

Transgenic DNA must penetrate three biological barriers, including cellular membrane, nuclear membrane, and chromosomal integrity, to achieve successful gene therapy. To overcome these barriers, three functional components were integrated into liposome-based nanoparticles to enable efficient gene transfection in rat mesenchymal stem cells (rMSCs): a novel rMSC-targeting peptide, a reported nuclear localization signal (NLS) peptide and a constructed Sleeping Beauty (SB) transposon system. The rMSC-targeting peptide was selected using phage display technique and allowed the nanoparticles to recognize and bind to rMSCs. The NLS peptide promoted the translocation of gene to nuclei. The SB transposon system used is a plasmid system that allows efficient insertion of transgenes into the host genome and is capable of expressing GFP-VEGF protein. Gene transfer study showed that the rMSC-targeting peptide could direct the nanoparticles to target the MSCs whereas NLS peptide could direct transposon to accumulate into nuclei once the nanoparticles are internalized inside the cells. After entering the nuclei, SB transposon increased the insertion rates into cellular chromosomes, leading to increased gene expression. The targeting nanoparticles did not show obvious cell toxicity, nor did they influence the osteogenic differentiation potential of rMSCs. Therefore, the nanoparticles bearing the rMSC-targeting peptide, NLS peptide and SB transposon system are promising non-viral gene delivery vectors in stem cell based gene therapy.

Drug delivery via biomaterials-based approaches has the potential impact on therapeutic angiogenesis. The nature of the multifactorial process underlying the neovessel growth inspires researchers to design and engineer advanced synthetic devices to better understand the synchronized effect of various parameters on neovascularization. This talk will present our recent work to assemble a composite hydrogel enabling sequential dual drug delivery. This gel comprises of two distinct domains; a stable hydrogel of poly(ethyleneglycol) dimethacrylate (PEGDMA) and a rapidly degrading gel of polyethyleneimine (PEI) & polyethylenediacrylate (PEGDA). The composite hydrogel was fabricated by initially generating PEGDMA microwells via soft lithography and then filling the microwells with the PEI-PEGDA hydrogel. To demonstrate the capability of this gel to releasing dual drugs in a sequential manner, two different types of angiogenic growth factors, VEGF 165 and VEGF 121, were incorporated into these two hydrogel domains. VEGF 165 was encapsulated in the poly(lactic acid-co-glycolide) (PLGA) particles and then loaded into the PEGDMA hydrogel, while VEGF 121 was directly loaded in the degradable PEI-PEGDA hydrogel assembled in the PEGDMA microwells. VEGF 165 was released from the composite material over two weeks, while VEGF 121 was being released from the material within one day. This hydrogel successfully increased the number of new blood vessels at an implantation site, as compared to a condition to release single type of simultaneously released VEGF 165 and VEGF 121. The biomaterial system assembled in this study will be broadly useful for controlling dual drug release rates in a more sophisticated manner and further improving therapeutic efficacy of drug molecules.

Transcription is a fundamental gene regulatory mechanism mediated by transcription factor (TF) proteins, and regulates all aspects of cellular function including proliferation, apoptosis, and differentiation. The structure of TFs is comprised of two fundamental domains that function synergistically to initiate transcription: a DNA binding domain (DBD) and an activation domain (AD). Synthetic analogues of each domain have been developed and constructed into small molecules called synthetic transcription factors (STFs).
STFs have been explored by several groups, however, none of these first generation STFs were able to induce potent gene expression in live cells, as most experiments were conducted using nuclear extracts. In order to overcome the limitations associated with STFs and to introduce a nanoparticle-based platform to mimic the function of TFs, we integrated gold nanoparticles with the STFs. Gold nanoparticles have a small size, are bio-compatible, and have surface properties that allow attachment of multiple biomolecules.
Herein, we demonstrate that a gold nanoparticle functionalized with STFs and other components, is capable of penetrating the plasma membrane, localizing within the nucleus, activating transcription of a reporter gene, and upregulating expression of targeted endogenous genes within live HeLa cell cultures. In order to mimic the function TFs, this multi-component gold nanoparticle (termed NanoScript) is comprised of the three functional ligands: (i) a nuclear localization signal (NLS) peptide for nuclear targeting, (ii) a hairpin polyamide (the DBD) that recognizes and binds to pre-determined DNA sequences, and (iii) a non-natural trans-activation peptide (the AD) that recruits proteins and enzymes to initiate transcription.
Advantages of the gold nanoparticle backbone of NanoScript include capability of adding nuclear targeting moieties, tunable DBD:AD ratios, and protection against protease degradation, while maintaining high cell viability. First, using a combination of fluorescence microscopy and transmission electron microscopy, we confirmed the presence of NanoScript within the nucleus while remaining intact. Secondly, NanoScript was demonstrated to initiate strong transcriptional activity of a reporter plasmid in a dose-dependent manner in live HeLa cell cultures. And lastly, we show that NanoScript can bind to select sequences on native DNA in HeLa cells overexpress several targeted endogenous genes.
In conclusion, we have successfully developed NanoScript to mimic the function and fundamental structure of transcription factors. The versatile and tunable properties of NanoScript establish this as an effective tool for applications that require endogenous gene regulation such as cellular reprogramming or stem cell differentiation.

Endothelial cells play significant roles in conditioning the environment in local tissues after insult and injury by the production and secretion of angiocrine factors. At least two distinct subset of macrophages, CD45+/CD11b+/Ly6C+/CX3CR1- inflammatory macrophages and CD45+/CD11b+/Ly6C-/CX3CR1+ anti-inflammatory macrophages, respond to these angiocrine factors differentially and promote pathogen/debris clearance and arteriogenesis and tissue regeneration, respectively. We demonstrate here that S1P₃ activation primes endothelial cells to produce angiocrine factors that recruit anti-inflammatory macrophages to remodeling vessels in ischemic and inflamed tissues. Poly (lactic-co-glycolic acid) thin films were used to deliver FTY720, an S1P_1/3 agonist to tissues. FTY720 enhanced the hydrophilicy of these films and did not activate monocytes. When implanted in vivo, FTY720-loaded films lead to a reduction in pro-inflammatory cytokine secretion, an increase in SDF-1 production and a shift in the recruitment of anti-inflammatory macrophages relative to inflammatory macrophages. These highly expressing CX3CR1 cells were localized peri-vascularly and promoted arteriolar diameter expansion, length density and vessel tortuosity: all classic signs of arteriogenesis. This work establishes a role for S1P receptor signaling in the local conditioning of tissues by angiocrine factors that preferentially recruit regenerative macrophages that can enhance healing outcomes, tissue regeneration and biomaterial implant functionality.

Statement of Purpose: Osteoarthritis (OA) is the predominant form of arthritis in our aging population. With individuals living increasingly longer lives, we face new problems relating to the degradation of tissues within the body. Regenerative tissue engineering utilizing bio-polymer scaffolds allows treatment to begin at the first sign of OA, alleviating pain as well as returning them to a pre-osteoarthritic condition. As the osteochondral tissues display a gradient of properties and structure, an integrated multi-phasic scaffold made of extracellular matrix (ECM) components was designed to mimic natural tissue. Our goal is to fabricate an integrated multi-phasic scaffold in the presence of drug-encapsulated microspheres to direct osteochondral tissue regeneration. Methods: Methacrylation of hyaluronan (HA) was performed through a novel method in dimethyl sulfoxide by ion exchange with an ammonium salt. The solution was reacted with methacrylic anhydride for 24 hours, hydrolyzed, lyophilized, and characterized by 1H-NMR. Cytocompatibility of the HA-MA was previously verified with primary human mesenchymal stem cells (hMSCs). HA-MA (2-4% w/v) blends containing varying concentrations of collagen IV, laminin, and heparan sulfate were photocrosslinked in the presence of a photoinitiator. Integrated multi-phasic scaffolds were formed by successive layering and polymerization of varied polymer blends in a custom mold. In addition, scaffolds were formed in the presence of PEG-modified alginate (AA-PEG) microspheres for the purpose of controlled drug release. Briefly, AA was modified with methoxy-terminated PEG amine using EDC/NHS chemistry an characterized by 1H-NMR. FITC-encapsulated AA-PEG microspheres were fabricated using a water-oil emulsion reaction and calcium crosslinking. Microsphere morphology and diameter were characterized by scanning electron microscopy (SEM). Results and Discussion: A layer-by-layer polymerization of HA-MA based blends was successful in fabricating an integrated multi-phasic scaffold. The individual layers of the scaffold were in-separable yet distinguishable in the presence UV-safe dyes. AA-PEG microspheres with nominal diameters of 500nm-1mu;m were homogenously dispersed through-out the polymerized scaffold. AA-PEG microspheres were uniform in size (per batch) and spherical in shape. Conclusions: The HA-MA-based scaffolds demonstrate efficacy of incorporating ECM components and AA-PEG microspheres into the hydrogel network. Future work will include the incorporation of 100-200mu;m AA-PEG microspheres into the hydrogel solution prior to photocrosslinking and subsequent dissolution of the microspheres to create a macroporous scaffold. Mechanical testing will be performed to optimize properties of each scaffold layer. Differentiation and proliferation of hMSCs within our scaffold will be investigated to determine the optimal concentration of ECM components and drug release profiles within each layer of the scaffold.

Contact guidance reactions are highly conserved, active cellular responses to structural components in the extracellular matrix. Cell-topography responses are likely to govern decision-making processes regarding interactions with micro- and nano-particles such as opsonizaiton and phagocytosis versus frustrated phagocytosis. Contact guidance has also been implicated in fundamental processes in development, cell migration, and neural regeneration. Here I will present recent advancements in from our laboratory in the rationale design of programmable substrate topography to elucidate the mechanism of contact guidance in mammalian cells. A facile processing strategy for the fabrication of these surfaces will be presented. This new class of materials will be used as a critical tool in combination with high-resolution live-cell imaging to measure filopodia dynamics of IC-21 macrophages. Filopodia dynamics will be measured in response to the instantaneous presentation and removal of topographic grating cues with feature sizes of approximately 1 micron in wavelength and 300 nm in amplitude. Empirical observations from these experiments are used to inform a new hypothesis for contact guidance that is based on passive filopodia diffusion. Specifically, these data will be used to support a working model that is based on biased filopodia migration dynamics in a potential field via transition state theory. Possible implications for this model of contact guidance will be discussed in the context of designing micro/nanoparticles and biomaterials interfaces.

Cell-cell interactions play an important role in stem cell differentiation and tissue development. In conventional co-culture systems such as transwell co-culture and mixed cell population in 2D monolayer or pellet co-culture have been widely used to study cell-cell interactions. However, these co-culture models provide limited control over cell density and distribution. In addition, with an increasing volume of studies implicating the differences in cell behavior in two- versus three-dimensions, there is a need to develop a co-culture model to study cell-cell interactions in 3 dimensional (3D). Here we report the effects of interactions between adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (ACs) on the fate of each cell type and cartilage matrix production in 3D. We hypothesized that the local concentration of soluble signaling (i.e. proximity between the two cell types) plays a crucial role in cell-cell interactions. We designed various co-culture models were designed using 3D biomimetic hydrogel, including: 1) mixed co-culture at different cell ratios; 2) bi-layered co-culture that confined the two cell types in separate layers; and 3) cells cultured with supplementation of conditioned medium from the other cell type. Unlike traditional pellet culture, the utilization of a 3D hydrogel enables us to create a truly 3D cellular microenvironment with control of cell density. In addition, we can engineer the effective distance between the two cell types through varying the ratio (mixed co-culture) and distribution of the two cell types (bi-layer) while keeping the cell density constant. We demonstrated that in mixed co-culture, ADSC chondrogenesis was enhanced while ACs maintained their chondrogenic phenotype. Mixed co-culture led to a significant increase in cell proliferation and cartilage matrix production that surpassed culture containing chondrocytes alone. The extent of cell proliferation, and neo-tissue production as well as organization depended on the ratio of the two cell type. These results demonstrated that enhanced cartilage formation observed in mixed co-culture resulted from ADSC chondrogenesis and increased cell proliferation. The dependence of these results on cell ratios in mixed co-culture, along with the relatively weak response in conditioned-medium and bi-layered co-culture, suggested that local concentration and distribution of paracrine factors play a crucial role in mediating cell-cell crosstalk and the subsequent neo-tissue formation. Taken together, this study suggests that manipulating cell-cell interactions and signal transduction through engineered 3D hydrogels may be a promising strategy for cartilage tissue engineering.

Supermacroporous, continuous monolithic cryogel could be produced by gelation of polymer precursors in moderate frozen conditions. In the reaction system, polymeric precursors and crosslinkers are mixed in aqueous media and incubated at sub-zero temperatures. The solvent freezes and forms ice crystals whereas solute molecules concentrate in the nonfrozen microzones, within which the crosslinking reaction proceeds and leads to the formation of a polymeric network with continuous macro-scale channels. The ice crystals formed from the solvent act as a porogen, which can be easily removed from the polymeric network by thawing and creates voids, which lead to a macroporous structure. The macroporosity allows the unhindered influx of high molecular weight solutes and nutrients, as well as the transport of cellular waste. This also enhances the cellular proliferation and provides increased matrix diffusion in the gel when used as scaffolds in tissue engineering. We prepare three-dimensional macroporous gelatin-hyaluronic acid (GE-HA) cryogels and use them in adipose tissue engineering. The cryogel scaffolds are with high porosity (>90%), inter-connected pores of 100~200 mu;m pore size, and good mechanical properties. Porcine adipose-derived stem cells (pASCs) isolated and purified from pig inguinal fat tissue were seeded in GE-HA cryogel scaffolds to confirm the proliferation and differentiation of pASCs in vitro and in vivo. The in vitro study showed good proliferation and adipogenic differentiation of pASCs in the cryogel as evidence from the morphological change in cell shape from fibroblastic-like to spherical appearance, the decreased cytoskeleton expression, the secretion of adipogenesis marker protein leptin, and the expression of early (LPL, PPARγ) and late (aP2 and leptin) adipose-specific genes. The in vivo studies in nude mice and pigs demonstrated the adipose tissue formation by presentation of PPARγ and leptin from immunohistochemistry stains. The specimens in the experimental groups also show high adipose-specific genes expression than control groups at 2, 4 and 8 weeks in both animal models. In conclusion, the GE-HA cryogel with its three-dimensional, macroporous structure and inter-pore connection can provide an extracellular matrix-like environment for proliferation and adipogenesis of pASCs.

Cell state and fate is regulated by physical signals in the microenvironment that are converted into biochemical signals in the cell, a process called mechanotransduction. Moreover, cells receive signals from neighbouring cells via membrane-bound ligands and receptors. It has been proposed that receptor-ligand signaling depends on the formation of higher order complexes at the membrane that are subject to high spatial control. However, direct regulation of receptor activation by the distribution of ligands at the nanoscale has not been shown. Here we demonstrate that DNA origami nanostructures can be conjugated with ligands at specific positions. We have used these nanostructures to present ligands with different proximities and local stoichiometries to human breast cancer cells, and we show that the nanoscale distribution of ligands regulates the levels of receptor activation. We hereby demonstrate a tool to control spatial receptor signalling directed by spatial distribution of ligands at the nanoscale. These results hold promise to provide knowledge necessary for the development of pharmacological interventions that target the physical context of the ligand/receptor interaction.

Injuries to the central nervous system often combine a loss of crucial motor or sensory function with a troubling lack of potential for spontaneous regeneration. In particular, acute spinal cord injuries result in a notoriously degenerative microenvironment bathed in growth-inhibiting proteoglycans. This degenerative environment can produce a sizeable injury gap, across which neurites must elongate. We propose that an implantable hydrogel capable of delivering neurotrophic factors across the injury gap may improve the regenerative potential of the spinal cord. To this end, we have developed an in vitro platform to quantify the neurotrophin and hydrogel properties that best support the outgrowth of injured neurites.
Previously, we reported the design of elastin-like proteins (ELPs) that enable control of mechanical stiffness and the presentation of cell-adhesive RGD ligands. Here a two-step fabrication process is developed to create ELP constructs comprising of a thin film, drug-delivery region and a hydrogel, cell-outgrowth region. The material properties of the two regions are independently defined, resulting in composite scaffolds that produce defined soluble gradients within three-dimensional matrices to promote neurite elongation. Scanning electron microscopy (SEM) and Quartz Crystal Microbalance with Dissipation (QCM-D) measurements reveal our processed films to be reproducible and radially uniform with a thickness ranging between 0.1 and 1 µm. Depending on the molecular weight of the encapsulated drug and the film properties, we can achieve release kinetics ranging from a burst-like profile over several hours to a burst-and-sustained release profile over the course of weeks.
The biomaterials-enabled release of three neurotrophins (Nerve Growth Factor (NGF), Brain-Derived Growth Factor (BDNF), and Neurotrophin-3 (NT-3)) are studied in isolation and combination using embryonic chick Dorsal Root Ganglia (DRGs). This mixed cell population is evaluated by immunostaining and confocal microscopy for beta-III-tubulin and S100, enabling us to distinguish between neurites and Schwann cells, respectively. Three-dimensional neurite outgrowth distance, Schwann cell migration, and extent of fasciculation (i.e., neurite bundling) are evaluated over three days. These experiments are being used to explore the contextual relationship between biomaterial properties (e.g., pore size, stiffness, and integrin ligand density) and neurotrophic gradient properties (e.g., neutrophin identity, release kinetics, and concentration profile) in instructing neurite outgrowth.

Electrospinning has been shown as an efficient and cost-effective way to fabricate the nanofibrous structures. It has many advantages in tissue engineering applications, such as high surface/weight ratio, versatile and controllable substructures and morphology, and especially biomimicity due to their extracellular matrix (ECM)-like structures. Nevertheless, to be used in actual medicinal practice, electrospun fibres have a critical disadvantage: it can only be fabricated into two-dimensional constructs instead of three-dimensional structures.
Recently, Xu et al. [1] presented a novel fabrication method of fluffy scaffolds using polystyrene (PS) without manipulation of the apparatus or the collector. PS, however, is not a biodegradable material which limits the utilization of PS fluffy scaffolds in tissue engineering applications. In this study, a novel methodology of fabricating three-dimensional scaffolds by employing clay-like electrospun matrices that can be shaped freely with a mold is presented. We focused on developing the novel methodology of producing electrospun clay and molding to generate a variety of representative shapes. This could be used in many regenerative medicinal applications via the specific leaching PS out of PS/poly(ε-caprolactone) (PCL) composite fluffy electrospun fibres. Furthermore, the combinatorial effect of using electrospun clay and viral vectors [2] was also investigated. The viral vectors (AAV), widely known as safe, non-pathogenic and efficient vectors for human gene therapies, were incorporated with the scaffolds in two ways as follows: i) encapsulated in the fibres and ii) adsorbed on the fibres. In the present study, mechanical, chemical and biological properties of electrospun clay-based scaffolds such as biocompatibility, cell viability and cellular penetration were characterized and, finally, sustained viral transduction was observed in the overall scaffold.
Reference: [1] B Sun et al. Nanoscale, 2012, 4, 2134, [2] S Lee et al. Acta Biomaterialia, 2011, 7(11), 3868

To understand and redirect cellular signaling and response in a controlled in vitro environment, it is valuable if not essential to localize cells spatiotemporally in desired relationships to each other, and to genetically reprogram cells. We report here advances on both fronts, based on controlled biofabrication through chemical/electrochemical means in biomicrosystems that simultaneously achieves both goals.
Our research team has previously developed techniques for localizing polysaccharide gels in microfluidic environments through controllable electrical and chemical signals which enable biomolecular and cellular assembly/immobilization and detailed real-time in-situ visualization [1-7]. We have discovered that this process can be combined with nucleic acids to achieve nonviral nucleic acid delivery to the cells as they become entrapped in the hydrogels. Specifically, cells are mixed with a nucleic acid, alginate, and calcium carbonate particles. A 30 V/cm DC field is applied to the solution, producing an abundance of protons at the anode surface. These protons migrate from the anode, dissolving calcium carbonate that they encounter. This does two things: it releases calcium ions to gel the alginate and it releases carbonate anion, which buffers the surrounding area from drastic pH changes. As the alginate hydrogel forms, the cells are reprogrammed by uptake of the nucleic acid from the immediate environment. Experimental and simulation results indicate that the nucleic acid delivery is accomplished by means of a synergistic interaction of the DC electrical field, Joule heating, and ionic/osmotic effects.
This technique is site-specific, as only cells in the vicinity of both the active electrode and the nucleic acids are reprogrammed. Furthermore, additional populations of cells can be assembled at the same electrode, allowing researchers greater flexibility in designing experiments to study interactions between populations of cells or between cells and their environment. The nucleic acid delivery and immobilization is reagentless and mild, entrapping cells in a mechanical and chemical environment conducive to growth. Additionally, the immobilization is reversible, allowing the electroaddressed population of cells to be released and recovered when desired for further investigation and experimentation. This technique represents a new, gentler means to such nonviral nucleic acid delivery than conventional approaches using high voltages, and it presents a qualitatively new strategy for understanding and redirecting cellular behavior in controlled in vitro environments.
1. Shi, et al., Adv. Funct. Mater., 2009, 19, 2074-2080.
2. Cheng, et al., Soft Matter, 2010, 6, 3177-3183.
3. Cheng, et al., Soft Matter, 2011, 7, 5677-5684.
4. Cheng, et al., Lab Chip, 2011, 11, 2316-2318.
5. Terrell, et al., Biotechnol. J., 2011, 7, 428-439.
6. Luo, et al., Biomaterials, 2012, 33, 5136-5143.
7. Cheng, et al., Adv. Funct. Mater., 2012, 22, 519-528.