Benjamin F. Pierce

An entropy–elastic gelatin-based hydrogel system

Gelatin is a non-immunogenic and degradable biopolymer, which is widely applied in the biomedical field e.g. for drug capsules or as absorbable hemostats. However, gelatin materials present limited and hardly reproducible mechanical properties especially in aqueous systems, particularly caused by the uncontrollable partial renaturation of collagen-like triple helices. Therefore, mechanically demanding applications for gelatin-based materials, such as vascular patches, i.e. hydrogel films that seal large incisions in vessel walls, and for induced autoregeneration, are basically excluded if this challenge is not addressed. Through the synthesis of a defined chemical network of gelatin with hexamethylene diisocyanate (HDI) in DMSO, the self-organization of gelatin chains could be hindered and amorphous gelatin films were successfully prepared having Youngs moduli of 60–530 kPa. Transferring the crosslinking reaction with HDI and, alternatively, ethyl lysine diisocyanate (LDI), to water as reaction medium allowed the tailoring of swelling behaviour and mechanical properties by variation of crosslinker content while suppressing the formation of helices. The hydrogels had Youngs moduli of 70–740 kPa, compressive moduli of 16–48 kPa, and degrees of swelling of 300–800 vol%. Test reactions investigated by ESI mass spectrometry allowed the identification and quantification of reaction products of the crosslinking reaction. The HDI crosslinked networks were stabilized by direct covalent crosslinks (ca. 10 mol%), supported by grafting (50 mol%) and blending of hydrophobic oligomeric chains. For the LDI-based networks, less crosslinked (3 mol%) and grafted species (5 mol%) and much higher amounts of oligomers were observed. The adjustable hydrogel system enables the application of gelatin-based materials in physiological environments.

One Step Creation of Multifunctional 3D Architectured Hydrogels Inducing Bone Regeneration

Structured hydrogels showing form stability and elastic properties individually tailorable on different length scales are accessible in a one-step process. They support cell adhesion and differentiation and display growing pore size during degradation. In vivo experiments demonstrate their efficacy in biomaterial-induced bone regeneration, not requiring addition of cells or growth factors.

Influence of Tyrosine-Derived Moieties and Drying Conditions on the Formation of Helices in Gelatin

The single and triple helical organization of protein chains strongly influences the mechanical properties of gelatin-based materials. A chemical method for obtaining different degrees of helical organization in gelatin is covalent functionalization, while a physical method for achieving the same goal is the variation of the drying conditions of gelatin solutions. Here we explored how the introduction of desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT) linked to lysine residues of gelatin influenced the kinetics and thermodynamic equilibrium of the helicalization process of single and triple helices following different drying conditions. Drying at a temperature above the helix-to-coil transition temperature of gelatin (T > T(c), called v(short)) generally resulted in gelatins with relatively lower triple helical content (X(c,t) = 1-2%) than lower temperature drying (T < T(c), called v(long)) (X(c,t) = 8-10%), where the DAT(T) functional groups generally disrupted helix formation. While different helical contents affected the thermal transition temperatures only slightly, the mechanical properties were strongly affected for swollen hydrogels (E = 4-13 kPa for samples treated by v(long) and E = 120-700 kPa for samples treated by v(short)). This study shows that side group functionalization and different drying conditions are viable options to control the helicalization and macroscopic properties of gelatin-based materials.

Knowledge-Based Tailoring of Gelatin-Based Materials by Functionalization with Tyrosine-Derived Groups

Molecular models of gelatin-based materials formed the basis for the knowledge-based design of a physically cross-linked polymer system. The computational models with 25 wt.-% water content were validated by comparison of the calculated structural properties with experimental data and were then used as predictive tools to study chain organization, cross-link formation, and estimation of mechanical properties. The introduced tyrosine-derived side groups, desaminotyrosine (DAT) and desaminotyrosyl tyrosine (DATT), led to the reduction of the residual helical conformation and to the formation of physical net-points by π-π interactions and hydrogen bonds. At 25 wt.-% water content, the simulated and experimentally determined mechanical properties were in the same order of magnitude. The degree of swelling in water decreased with increasing the number of inserted aromatic functions, while Youngs modulus, elongation at break, and maximum tensile strength increased.

Viability of human mesenchymal stem cells seeded on crosslinked entropy-elastic gelatin-based hydrogels.

Biomimetic polymer network systems with tailorable properties based on biopolymers represent a class of degradable hydrogels that provides sequences for protein adsorption and cell adhesion. Such materials show potential for in vitro MSC proliferation as well as in vivo applications and were obtained by crosslinking different concentrations of gelatin using varying amounts of ethyl lysine diisocyanate in the presence of a surfactant in pH 7.4 PBS solution. Material extracts, which were tested for cytotoxic effects using L929 mouse fibroblasts, were non-toxic. The hydrogels were seeded with human bone marrow-derived MSCs and supported viable MSCs for the incubation time of 9 d. Preadsorption of fibronectin on materials improved this biofunctionality.

Demonstrating the influence of water on shape-memory polymer networks based on poly[(rac-lactide)-co-glycolide] segments in vitro.

Thermally-responsive shape-memory polymers (SMP) are highly promising implant materials for applications in minimally-invasive surgery since the shape-memory effect (SME) enables the implantation of a bulky device in a compressed temporary state through a small incision. When heated to a temperature exceeding the material switching temperature (Tsw), the device recovers its original bulky shape. Therefore, SMP implants with Tsw ~ 37°C are required for such applications because the body cannot withstand excessive applications of heat. Here, Tsw of networks based on poly[(rac-lactide)-co-glycolide] star-shaped macrotriol or macrotetrols with 19–22 wt% glycolide content, varying oligomer molecular weight (Mn=3000–10000 g·mol−1), and netpoint functionality (f=3 or 4) were lowered from 55–66°C to below body temperature via the uptake of water, which also induced SME at body temperature. Programmed samples kept their temporary shape at room temperature in water as well as at 37°C under dry conditions but recovered in 37°C water. Water uptake/swelling studies and FTIR analysis indicated that the mechanism of solvent-induced SME involved the plasticization of water in switching domains as opposed to changes in swelling or hydrogen bonding. This indirect actuation of SME by using a combination of solvent and heat could be exploited in easy-to-handle shape-memory implant with slower degradation kinetics.

Why are so few degradable polymeric biomaterials currently established in clinical applications

Most of today’s established polymeric implant materials were not originally designed for exclusive use in medical applications; rather, they were selected from a group of available engineering plastics. It was a revolutionary concept to introduce polymers intended to degrade within the human body after temporarily serving as structural supports, as matrices for controlling drug release, or as substitutes for the extracellular matrix capable of inducing the regeneration of tissue (1-3). The classic approach to designing degradable polymers is to incorporate chemical bonds that are cleavable under physiological conditions (see Fig. 1). This cleavage can occur through hydrolysis (e.g., in polyesters), enzymatic cleavage, or via other mechanisms such as redox reactions (e.g., the reduction of disulfide bonds). Such chain scission can occur along the main chain or the side chains, depending on their repeating units and on the architecture of the macromolecules. The cleavage of bonds results in water-soluble polymer fragments, which can be excreted from the body via renal clearance or through metabolization, thereby obviating the need for a second surgical procedure to explant the device. Although the development of medical implants based on degradable biomaterials has become a promising strategy for the medical device industry since many decades, in reality the full potential of the market for applicable degradable polymers has not yet been realized (4, 5). Currently, degradable materials comprise only a small fraction of biomaterials in use. Given the market and clinical potential of degradable materials (5), the question arises as to why only a few degradable polymeric systems have been successfully established in clinical applications thus far. Our definition of “degradable” focuses on materials deWhy are so few degradable polymeric biomaterials currently established in clinical applications?

Endothelial cell response to (co)polymer nanoparticles depending on the inflammatory environment and comonomer ratio.

Endothelial cells lining the lumen of blood vessels serve as a physiological barrier controlling nanoparticle movement from the vasculature into the tissue. For exploring the effect of polymer hydrophilicity on nanoparticle interactions with human umbilical vein endothelial cells (HUVECs) in vitro, a series of monomodal poly[acrylonitrile-co-(N-vinylpyrrolidone)] model nanoparticles with increasing hydrophilicity as related to their increasing content (0-30 mol.%) of N-vinylpyrrolidone (NVP) were synthesized by miniemulsion polymerization. Nanoparticles with a low NVP content were rapidly endocytized into all cells independent from the particle dose with toxic effects only observed at high particle concentrations, while only 10-30% of the cells incorporated particles with ≥20 mol.% NVP. Since pathologies are often related to inflammation, an inflammatory HUVEC culture condition with IL-1β stimulation has been introduced and suggested to be widely applied for studying nanocarriers, since cellular uptake in this assay was clearly increased for NVP contents ≥20 mol.%. Importantly, the secretion of functional biological mediators by HUVECs was not relevantly influenced by the nanoparticles for both homeostatic and inflammatory conditions. These findings may motivate concepts for nanocarriers specifically targeted to pathologic regions. Additionally, rapidly endocytized RhodaminB loaded particles with low NVP content may be explored for cell labeling and tracking.

A molecular dynamic analysis of gelatin as an amorphous material: Prediction of mechanical properties of gelatin systems

Biomaterials are used in regenerative medicine for induced autoregeneration and tissue engineering. This is often challenging, however, due to difficulties in tailoring and controlling the respective material properties. Since functionalization is expected to offer better control, in this study gelatin chains were modified with physically interacting groups based on tyrosine with the aim of causing the formation of physical crosslinks. This method permits application-specific properties like swelling and better tailoring of mechanical properties. The design of the crosslink strategy was supported by molecular dynamic (MD) simulations of amorphous bulk models for gelatin and functionalized gelatins at different water contents (0.8 and 25 wt.-%). The results permitted predictions to be formulated about the expected crosslink density and its influence on equilibrium swelling behavior and on elastic material properties. The models of pure gelatin were used to validate the strategy by comparison between simulated and experimental data such as density, backbone conformation angle distribution, and X-ray scattering spectra. A key result of the simulations was the prediction that increasing the number of aromatic functions attached to the gelatin chain leads to an increase in the number of physical netpoints observed in the simulated bulk packing models. By comparison with the Flory-Rehner model, this suggested reduced equilibrium swelling of the functionalized materials in water, a prediction that was subsequently confirmed by our experimental work. The reduction and control of the equilibrium degree of swelling in water is a key criterion for the applicability of functionalized gelatins when used, for example, as matrices for induced autoregeneration of tissues.

Design of Decorin‐Based Peptides That Bind to Collagen I and their Potential as Adhesion Moieties in Biomaterials

Mimicking the binding epitopes of protein-protein interactions by using small peptides is important for generating modular biomimetic systems. A strategy is described for the design of such bioactive peptides without accessible structural data for the targeted interaction, and the effect of incorporating such adhesion peptides in complex biomaterial systems is demonstrated. The highly repetitive structure of decorin was analyzed to identify peptides that are representative of the inner and outer surface, and it was shown that only peptides based on the inner surface of decorin bind to collagen. The peptide with the highest binding affinity for collagen I, LHERHLNNN, served to slow down the diffusion of a conjugated dye in a collagen gel, while its dimer could physically crosslink collagen, thereby enhancing the elastic modulus of the gel by one order of magnitude. These results show the potential of the identified peptides for the design of biomaterials for applications in regenerative medicine.