Branching Analysis of Multivalent Conjugates Using Size Exclusion Chromatography-Multiangle Light Scattering.

Multivalent conjugates (MVCs) (conjugation of multiple proteins to a linear polymer chain) are powerful for improving the bioactivity and pharmacokinetics of a bioactive molecule. Since this effect is highly dependent upon the valency of the conjugated proteins, it is imperative to have a technique for analysis of the conjugation ratio. Studies of MVCs have used size exclusion chromatography-multiangle light scattering (SEC-MALS), which allows for the separate and individual analysis of the protein and biopolymer components based on their specific refractive index increment and UV extinction coefficient constants to determine the number of proteins bound per biopolymer molecule. In this work, we have applied traditional branching analysis to the SEC-MALS data, with the primary assumption that the polymer backbone can be used as the linear counterpart. We demonstrated good agreement between the branching values and the valency determined by traditional analysis, demonstrating that branching analysis can be used as an alternative technique to approximate the valency of MVCs. The branching analysis method also provides a more complete picture of the distribution of the measured values, provides important branching information about the molecules, and lowers the cost and complexity of the characterization. However, since MVC molecules are both conjugate molecules and branched molecules, the most powerful approach to their characterization would be to use both traditional multivalent conjugate analysis and branching analysis in conjunction.

Low Fouling Electrospun Scaffolds with Clicked Bioactive Peptides for Specific Cell Attachment.

While electrospun fibers are of interest as scaffolds for tissue engineering applications, nonspecific surface interactions such as protein adsorption often prevent researchers from controlling the exact interactions between cells and the underlying material. In this study we prepared electrospun fibers from a polystyrene-based macroinitiator, which were then grafted with polymer brushes using surface-initiated atom transfer radical polymerization (SI-ATRP). These brush coatings incorporated a trimethylsilyl-protected PEG-alkyne monomer, allowing azide functional molecules to be covalently attached, while simultaneously reducing nonspecific protein adsorption on the fibers. Cells were able to attach and spread on fibrous substrates functionalized with a pendant RGD-containing peptide, while spreading was significantly reduced on nonfunctionalized fibers and those with the equivalent RGE control peptide. This effect was observed both in the presence and absence of serum in the culture media, indicating that protein adsorption on the fibers was minimal and cell adhesion within the fibrous scaffold was mediated almost entirely through the cell-adhesive RGD-containing peptide.

Chemical patterning of ultrathin polymer films by direct-write multiphoton lithography.

We applied 2-photon laser ablation to write subdiffraction nanoscale chemical patterns into ultrathin polymer films under ambient conditions. Poly(ethylene glycol) methacrylate brush layers were prepared on quartz substrates via surface-initiated atom-transfer radical polymerization and ablated to expose the underlying substrate using the nonlinear 2-photon absorbance of a frequency-doubled Ti:sapphire femtosecond laser. Single-shot ablation thresholds of polymer films were ~1.5 times smaller than that of a quartz substrate, which allowed patterning of nanoscale features without damage to the underlying substrate. At a 1/e(2) laser spot diameter of 0.86 µm, the features of exposed substrate approached ~80 nm, well below the diffraction limit for 400 nm light. Ablated features were chemically distinct and amenable to chemical modification.

Surface creasing instability of soft polyacrylamide cell culture substrates.

Efforts to understand and engineer cell behavior in mechanically soft environments frequently employ two-dimensional cell culture substrates consisting of thin hydrogel layers with low elastic modulus supported on rigid substrates to facilitate culturing, imaging, and analysis. Here we characterize how an elastic creasing instability of the gel surface may occur for the most widely used soft cell culture substrate, polyacrylamide hydrogels, and show that stem cells respond to and change their behavior due to these surface features. The regions of stability and corresponding achievable ranges of modulus are elucidated in terms of the monomer and cross-linker concentrations, providing guidance for the synthesis of both smooth and creased soft cell substrates for basic and applied cell engineering efforts.

Myocardial injection of a thermoresponsive hydrogel with reactive oxygen species scavenger properties improves border zone contractility.

The decrease in contractility in myocardium adjacent (border zone; BZ) to a myocardial infarction (MI) is correlated with an increase in reactive oxygen species (ROS). We hypothesized that injection of a thermoresponsive hydrogel, with ROS scavenging properties, into the MI would decrease ROS and improve BZ function. Fourteen sheep underwent antero-apical MI. Seven sheep had a comb-like copolymer synthesized from N-isopropyl acrylamide (NIPAAm) and 1500 MW methoxy poly(ethylene glycol) methacrylate, (NIPAAm-PEG1500), injected (20 × 0.5 mL) into the MI zone 40 min after MI (MI + NIPAAm-PEG1500) and seven sheep were MI controls. Cardiac MRI was performed 2 weeks before and 6 weeks after MI + NIPAAm-PEG1500. BZ wall thickness at end systole was significantly higher for MI + NIPAAm-PEG1500 (12.32 ± 0.51 mm/m2 MI + NIPAAm-PEG1500 vs. 9.88 ± 0.30 MI; p = .023). Demembranated muscle force development for BZ myocardium 6 weeks after MI was significantly higher for MI + NIPAAm-PEG1500 (67.67 ± 2.61 mN/m2 MI + NIPAAm-PEG1500 vs. 40.53 ± 1.04 MI; p < .0001) but not significantly different from remote myocardium or BZ or non-operated controls. Levels of ROS in BZ tissue were significantly lower in the MI + NIPAAm-PEG1500 treatment group (hydroxyl p = .0031; superoxide p = .0182). We conclude that infarct injection of the NIPAAm-PEG1500 hydrogel with ROS scavenging properties decreased ROS and improved contractile protein function in the border zone 6 weeks after MI.

A combined hiPSC-derived endothelial cell and in vitro microfluidic platform for assessing biomaterial-based angiogenesis.

Human induced pluripotent stem cell (hiPSC) derived angiogenesis models present a unique opportunity for patient-specific platforms to study the complex process of angiogenesis and the endothelial cell response to biomaterial and biophysical changes in a defined microenvironment. We present a refined method for differentiating hiPSCs into a CD31 + endothelial cell population (hiPSC-ECs) using a single basal medium from pluripotency to the final stage of differentiation. This protocol produces endothelial cells that are functionally competent in assays following purification. Subsequently, an in vitro angiogenesis model was developed by encapsulating the hiPSC-ECs into a tunable, growth factor sequestering hyaluronic acid (HyA) matrix where they formed stable, capillary-like networks that responded to environmental stimuli. Perfusion of the networks was demonstrated using fluorescent beads in a microfluidic device designed to study angiogenesis. The combination of hiPSC-ECs, bioinspired hydrogel, and the microfluidic platform creates a unique testbed for rapidly assessing the performance of angiogenic biomaterials.

Substrate modulus directs neural stem cell behavior.

Although biochemical signals that modulate stem cell self-renewal and differentiation were extensively studied, only recently were the mechanical properties of a stem cell's microenvironment shown to regulate its behavior. It would be desirable to have independent control over biochemical and mechanical cues, to analyze their relative and combined effects on stem-cell function. We developed a synthetic, interfacial hydrogel culture system, termed variable moduli interpenetrating polymer networks (vmIPNs), to assess the effects of soluble signals, adhesion ligand presentation, and material moduli from 10-10,000 Pa on adult neural stem-cell (aNSC) behavior. The aNSCs proliferated when cultured in serum-free growth media on peptide-modified vmIPNs with moduli of >/=100 Pa. In serum-free neuronal differentiation media, a peak level of the neuronal marker, beta-tubulin III, was observed on vmIPNs of 500 Pa, near the physiological stiffness of brain tissue. Furthermore, under mixed differentiation conditions with serum, softer gels ( approximately 100-500 Pa) greatly favored neurons, whereas harder gels ( approximately 1,000-10,000 Pa) promoted glial cultures. In contrast, cell spreading, self-renewal, and differentiation were inhibited on substrata with moduli of approximately 10 Pa. This work demonstrates that the mechanical and biochemical properties of an aNSC microenvironment can be tuned to regulate the self-renewal and differentiation of aNSCs.

Multivalency of Sonic hedgehog conjugated to linear polymer chains modulates protein potency.

A potently active multivalent form of the protein Sonic hedgehog (Shh) was produced by bioconjugation of a modified recombinant form of Shh to the linear polymers poly(acrylic acid) (pAAc) and hyaluronic acid (HyA) via a two-step reaction exploiting carboimiide and maleimide chemistry. Efficiency of the conjugation was approximately 75% even at stoichiometric ratios of 30 Shh molecules per linear HyA chain (i.e., 30:1 Shh/HyA). Bioactivity of the conjugates was tested via a cellular assay across a range of stoichiometric ratios of Shh molecules to HyA linear chains, which was varied from 0.6:1 Shh/HyA to 22:1 Shh/HyA. Results indicate that low conjugation ratios decrease Shh bioactivity and high ratios increase this activity beyond the potency of monomeric Shh, with approximately equal activity between monomeric soluble Shh and conjugated Shh at 7:1 Shh/HyA. In addition, high-ratio constructs increased angiogenesis determined by the in vivo chick chorioallantoic membrane (CAM) assay. These results are captured by a kinetic model of multiple interactions between the Shh/HyA conjugates and cell surface receptors resulting in higher cell signaling at lower bulk Shh concentrations.

Gellan Gum Hydrogels with Enzyme-Sensitive Biodegradation and Endothelial Cell Biorecognition Sites.

The survival of a biomaterial or tissue engineered construct is mainly hampered by the deficient microcirculation in its core, and limited nutrients and oxygen availability to the implanted or colonizing host cells. Aiming to address these issues, we herein propose bioresponsive gellan gum (GG) hydrogels that are biodegradable by metalloproteinase 1 (MMP-1) and enable endothelial cells adhesion and proliferation. GG is chemically functionalized with divinyl sulfone (DVS) and then biofunctionalized with thiol cell-adhesive peptides (T1 or C16) to confer GG endothelial cell biorecognition cues. Biodegradable hydrogels are then formed by Michael type addition of GGDVS or/and peptide-functionalized GGDVS with a dithiol peptide crosslinker sensitive to MMP-1. The mechanical properties (6 to 5580 Pa), swelling (17 to 11), MMP-1-driven degradation (up to 70%), and molecules diffusion coefficients of hydrogels are tuned by increasing the polymer amount and crosslinking density. Human umbilical cord vein endothelial cells depict a polarized elongated morphology when encapsulated within T1-containing hydrogels, in contrast to the round morphology observed in C16-containing hydrogels. Cell organization is favored as early as 1 d of cell culture within the T1-modified hydrogels with higher concentration of peptide, while cell proliferation is higher in T1-modified hydrogels with higher modulus. In conclusion, biodegradable and bioresponsive GGDVS hydrogels are promising endothelial cell responsive materials that can be used for vascularization strategies.

Theoretical impact of the injection of material into the myocardium: a finite element model simulation.

BACKGROUND: To treat cardiac injuries created by myocardial infarcts, current approaches seek to add cells and/or synthetic extracellular matrices to the damaged ventricle to restore function. Because definitive myocardial regeneration remains undemonstrated, we propose that cardiac changes observed from implanted materials may result from altered mechanisms of the ventricle. METHODS AND RESULTS: We exploited a validated finite element model of an ovine left ventricle with an anteroapical infarct to examine the short-term effect of injecting material to the left ventricular wall. The model's mesh and regional material properties were modified to simulate expected changes. Three sets of simulations were run: (1) single injection to the anterior border zone; (2) therapeutic multiple border zone injections; and (3) injection of material to the infarct region. Results indicate that additions to the border zone decrease end-systolic fiber stress proportionally to the fractional volume added, with stiffer materials improving this attenuation. As a potential therapy, small changes in wall volume (approximately 4.5%) reduce elevated border zone fiber stresses from mean end-systole levels of 28.2 kPa (control) to 23.3 kPa (treatment), similar to levels of 22.5 kPA computed in remote regions. In the infarct, injection improves ejection fraction and the stroke volume/end-diastolic volume relationship but has no effect on the stroke volume/end-diastolic pressure relationship. CONCLUSIONS: Simulations indicate that the addition of noncontractile material to a damaged left ventricular wall has important effects on cardiac mechanics, with potentially beneficial reduction of elevated myofiber stresses, as well as confounding changes to clinical left ventricular metrics.

Regulation of endothelial cell function by GRGDSP peptide grafted on interpenetrating polymers.

Vascular endothelium plays an important role in preventing thrombogenesis. Bioactive molecules such as fibronectin-derived peptide Gly-Arg-Gly-Asp-Ser-Pro (GRGDSP) can be used to modify the surface of cardiovascular implants such as vascular grafts to promote endothelialization. Here we conjugated GRGDSP peptide to the nonfouling surface of an interpenetrating polymer network (IPN), and investigated the effects of the immobilized GRGDSP molecules on EC functions under static and flow conditions at well-defined GRGDSP surface densities (approximately 0 to 3 pmol/cm2). EC adhesion and spreading increased with GRGDSP surface density, reached a plateau at 1.5 pmol/cm2, and increased further beyond 2.8 pmol/cm2. Cell adhesion and spreading on GRGDSP induced two waves of extracellular signal-regulated kinase (ERK) activation, and 0.2 pmol/cm2 density of GRGDSP was sufficient to activate ERK. EC proliferation rate was not sensitive to GRGDSP surface density, suggesting that cell spreading at low-density of GRGDSP is sufficient to maintain EC proliferation. EC migration on lower-density GRGDSP-IPN surfaces was faster under static condition. With the increase of GRGDSP density, the speed and persistence of EC migration dropped quickly (0.2-0.8 pmol/cm2) and reached a plateau, followed by a slower and gradual decrease (1.5-3.0 pmol/cm2). These data suggest that the changes of EC functions were more sensitive to the increase of GRGDSP density at lower range. Under flow condition with shear stress at 12 dyn/cm2, EC migration was inhibited on GRGDSP-IPN surfaces, which may be attributed to the assembly of large focal adhesions induced by shear stress, suggesting a catch-bond characteristic for RGD-integrin binding. This study provides a rational base for surface engineering of cardiovascular implants.

Peri-implant bone formation and implant integration strength of peptide-modified p(AAM-co-EG/AAC) interpenetrating polymer network-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly (acrylamide-co-ethylene glycol/acrylic acid) functionalized with an -Arg-Gly-Asp- (RGD) containing 15 amino acid peptides, derived from rat bone sialoprotein (bsp-RGD(15), were grafted to titanium implants in an effort to modulate bone formation in the peri-implant region in the rat femoral ablation model. Bone-implant contact (BIC) and bone formation within the medullary canal were determined using microcomputed tomography at 2 and 4 weeks postimplantation. BIC for bsp-RGD(15)-IPN implants was enhanced relative to hydroxyapatite tricalcium phosphate (HA-TCP) coated implants, but was similar to all other groups. Aggregate bone formation neither indicated a dose-dependent effect of bsp-RGD(15) nor a meaningful trend. Mechanical testing of implant fixation revealed that only the HA-TCP coated implants supported significant (>1 MPa) interfacial shear strength, despite exhibiting lower overall BIC, an indication that bone ingrowth into the rougher coating was the primary mode of implant fixation. While no evidence was found to support the hypothesis that bsp-RGD(15)-modified IPN coated implants significantly impacted bone-implant bonding, these results point to the lack of correlation between in vitro studies employing primary osteoblasts and in vivo wound healing in the peri-implant region.

Biomimetic interfacial interpenetrating polymer networks control neural stem cell behavior.

Highly-regulated signals surrounding stem cells, such as growth factors at specific concentrations and matrix mechanical stiffness, have been implicated in modulating stem cell proliferation and maturation. However, tight control of proliferation and lineage commitment signals is rarely achieved during growth outside the body, since the spectrum of biochemical and mechanical signals that govern stem cell renewal and maturation are not fully understood. Therefore, stem cell control can potentially be enhanced through the development of material platforms that more precisely orchestrate signal presentation to stem cells. Using a biomimetic interfacial interpenetrating polymer network (IPN), we define a robust synthetic and highly-defined platform for the culture of adult neural stem cells. IPNs modified with two cell-binding ligands, CGGNGEPRGDTYRAY from bone sialoprotein [bsp-RGD(15)] and CSRARKQAASIKVAVSADR from laminin [lam-IKVAV(19)], were assayed for their ability to regulate self-renewal and differentiation in a dose-dependent manner. IPNs with >5.3 pmol/cm(2) bsp-RGD(15) supported both self-renewal and differentiation, whereas IPNs with lam-IKVAV(19) failed to support stem cell adhesion and did not influence differentiation. The IPN platform is highly tunable to probe stem cell signal transduction mechanisms and to control stem cell behavior in vitro.

The effect of enzymatically degradable IPN coatings on peri-implant bone formation and implant fixation.

Short-term osseointegration of orthopedic implants is critical for the long-term stability of the implant-bone interface. To improve initial implant stability, one strategy under consideration involves the presentation of adhesion ligands on the implant surface to stimulate bone regeneration in the peri-implant region. To assess the relative effects of implant surface chemistry and topography on osseointegration within the rat femoral ablation implant model, a nonfouling, enzymatically degradable interpenetrating polymer network (edIPN) of poly(AAm-co-EG/AAc) amenable to presenting the cell signaling domain Arg-Gly-Asp (RGD), was developed. Moderate enhancement of peri-implant bone formation was found after 28 days using the edIPN without peptide modification (p = 0.032). However, no data supported a benefit of peptide modification, as bone-implant contact, normalized bone volume and normalized fixation strength was equivalent or poorer than dual acid-etched (DAE) treated implants after 28 days. Surface topography was determined to be the dominant factor in modulating osseointegration, as DAE implants produced equivalent roughness-normalized fixation strength versus previously reported data on plasma-sprayed hydroxyapatite/tricalcium phosphate-coated implants (Barber et al., J Biomed Mater Res A, forthcoming). An ideal osseointegrated implant will require optimization of all three aforementioned parameters, and may take the form of biomolecule delivery from thin degradable polymer networks.

A Bioengineering Approach to Myopia Control Tested in a Guinea Pig Model.

Purpose: To investigate the biocompatibility of an injectable hydrogel and its ability to control myopia progression in guinea pigs. Methods: The study used a hydrogel synthesized from acrylated hyaluronic acid with a conjugated cell-binding peptide and enzymatically degradable crosslinker. Seven-day-old guinea pigs were first form deprived (FD) with diffusers for 1 week. One group was kept as an FD-only control; two groups received a sub-Tenon's capsule injection of either hydrogel or buffer (sham surgery) at the posterior pole of the eye. Form deprivation treatments were then continued for 3 additional weeks. Treatment effects were evaluated in terms of ocular axial length and refractive error. Safety was evaluated via intraocular pressure (IOP), visual acuity, flash electroretinograms (ERG), and histology. Results: Both hydrogel and sham surgery groups showed significantly reduced axial elongation and myopia progression compared to the FD-only group. For axial lengths, net changes in interocular difference (treated minus control) were 0.04 ± 0.06, 0.02 ± 0.09, and 0.24 ± 0.08 mm for hydrogel, sham, and FD-only groups, respectively (P = 0.0006). Intraocular pressures, visual acuities, and ERGs of treated eyes were not significantly different from contralateral controls. Extensive cell migration into the implants was evident. Both surgery groups showed noticeable Tenon's capsule thickening. Conclusions: Sub-Tenon's capsule injections of both hydrogel and buffer inhibited myopia progression, with no adverse effects on ocular health. The latter unexpected effect warrants further investigation as a potential novel myopia control therapy. That the hydrogel implant supported significant cell infiltration offers further proof of its biocompatibility, with potential application as a tool for drug and cell delivery.

In vitro characterization of peptide-modified p(AAm-co-EG/AAc) IPN-coated titanium implants.

Interpenetrating polymer networks (IPNs) of poly(acrylamide-co-ethylene glycol/acrylic acid) [p(AAm-co-EG/AAc)] functionalized with an -Arg-Gly-Asp- containing peptide derived from rat bone sialoprotein [bsp-RGD(15)] were grafted to titanium implants in an effort to modulate osteoblast behavior in vitro. Surface characterization data were consistent with the presence of an IPN, and ligand density measurements established that the range of peptide density on the modified implants spanned three orders of magnitude (0.01-20 pmol/cm2). In vitro biological characterization of the modified implants employing the primary rat calvarial osteoblast (RCO) model resulted in the identification of a critical ligand density (0.01

Hydrogels as artificial matrices for human embryonic stem cell self-renewal.

Human embryonic stem cells (hESCs) have the potential to differentiate into all cell types in the body and hold great promise for regenerative medicine; however, large-scale expansion of undifferentiated hESCs remains a major challenge. Self-renewal of hESCs requires culturing these cells on either mouse or human fibroblast cells (i.e., a feeder layer of cells), or on artificial extracellular matrices (ECMs) while supplementing the media with soluble growth factors. Here we report a completely synthetic ECM system composed of a semi-interpenetrating polymer network (sIPN), a polymer hydrogel, which was designed to allow the independent manipulation of cell adhesion ligand presentation and matrix stiffness. In the short term, hESCs that were cultured on the sIPN adhered to the surface, remained viable, maintained the morphology, and expressed the markers of undifferentiated hESCs. This was the first demonstration that a completely synthetic ECM can support short-term self-renewal of hESCs.

In vitro cardiac tissue models: Current status and future prospects.

Cardiovascular disease is the leading cause of death worldwide. Achieving the next phase of potential treatment strategies and better prognostic tools will require a concerted effort from interdisciplinary fields. Biomaterials-based cardiac tissue models are revolutionizing the area of preclinical research and translational applications. The goal of in vitro cardiac tissue modeling is to create physiological functional models of the human myocardium, which is a difficult task due to the complex structure and function of the human heart. This review describes the advances made in area of in vitro cardiac models using biomaterials and bioinspired platforms. The field has progressed extensively in the past decade, and we envision its applications in the areas of drug screening, disease modeling, and precision medicine.

Matrix metalloproteinase-13 mediated degradation of hyaluronic acid-based matrices orchestrates stem cell engraftment through vascular integration.

A critical design parameter for the function of synthetic extracellular matrices is to synchronize the gradual cell-mediated degradation of the matrix with the endogenous secretion of natural extracellular matrix (ECM) (e.g., creeping substitution). In hyaluronic acid (HyA)-based hydrogel matrices, we have investigated the effects of peptide crosslinkers with different matrix metalloproteinases (MMP) sensitivities on network degradation and neovascularization in vivo. The HyA hydrogel matrices consisted of cell adhesive peptides, heparin for both the presentation of exogenous and sequestration of endogenously synthesized growth factors, and MMP cleavable peptide linkages (i.e., QPQGLAK, GPLGMHGK, and GPLGLSLGK). Sca1(+)/CD45(-)/CD34(+)/CD44(+) cardiac progenitor cells (CPCs) cultured in the matrices with the slowly degradable QPQGLAK hydrogels supported the highest production of MMP-2, MMP-9, MMP-13, VEGF165, and a range of angiogenesis related proteins. Hydrogels with QPQGLAK crosslinks supported prolonged retention of these proteins via heparin within the matrix, stimulating rapid vascular development, and anastomosis with the host vasculature when implanted in the murine hindlimb.

Multivalent hyaluronic acid bioconjugates improve sFlt-1 activity in vitro.

Anti-VEGF drugs that are used in conjunction with laser ablation to treat patients with diabetic retinopathy suffer from short half-lives in the vitreous of the eye resulting in the need for frequent intravitreal injections. To improve the intravitreal half-life of anti-VEGF drugs, such as the VEGF decoy receptor sFlt-1, we developed multivalent bioconjugates of sFlt-1 grafted to linear hyaluronic acid (HyA) chains termed mvsFlt. Using size exclusion chromatography with multiangle light scattering (SEC-MALS), SDS-PAGE, and dynamic light scattering (DLS), we characterized the mvsFlt with a focus on the molecular weight contribution of protein and HyA components to the overall bioconjugate size. We found that mvsFlt activity was independent of HyA conjugation using a sandwich ELISA and in vitro angiogenesis assays including cell survival, migration and tube formation. Using an in vitro model of the vitreous with crosslinked HyA gels, we demonstrated that larger mvsFlt bioconjugates showed slowed release and mobility in these hydrogels compared to low molecular weight mvsFlt and unconjugated sFlt-1. Finally, we used an enzyme specific to sFlt-1 to show that conjugation to HyA shields sFlt-1 from protein degradation. Taken together, our findings suggest that mvsFlt bioconjugates retain VEGF binding affinity, shield sFlt-1 from enzymatic degradation, and their movement in hydrogel networks (in vitro model of the vitreous) is controlled by both bioconjugate size and hydrogel network mesh size. These results suggest that a strategy of multivalent conjugation could substantially improve drug residence time in the eye and potentially improve therapeutics for the treatment of diabetic retinopathy.