A polyelectrolyte multilayer platform for investigating growth factor delivery modes in human liver cultures.

Polyelectrolyte multilayers (PEMs) of chitosan and heparin are useful for mimicking growth factor (GF) binding to extracellular matrix (ECM) as in vivo. Here, we developed a PEM platform for delivering bound/adsorbed GFs to monocultures of primary human hepatocytes (PHHs) and PHH/non-parenchymal cell (NPC) co-cultures, which are useful for drug development and regenerative medicine. The effects of ECM protein coating (collagen I, fibronectin, and Matrigel®) and terminal PEM layer on PHH attachment/functions were determined. Then, heparin-terminated/fibronectin-coated PEMs were used to deliver varying concentrations of an adsorbed model GF, transforming growth factor ß (TGFß), to PHH monocultures while using soluble TGFß delivery via culture medium as the conventional control. Soluble TGFß delivery caused a severe, monotonic, and sustained downregulation of all PHH functions measured (albumin and urea secretions, cytochrome-P450 2A6 and 3A4 enzyme activities), whereas adsorbed TGFß delivery caused transient upregulation of 3 out of 4 functions. Finally, functionally stable co-cultures of PHHs and 3T3-J2 murine embryonic fibroblasts were created on the heparin-terminated/fibronectin-coated PEMs modified with adsorbed TGFß to elucidate similarities and differences in functional response relative to the monocultures. In conclusion, chitosan-heparin PEMs constitute a robust platform for investigating the effects of GF delivery modes on PHH monocultures and PHH/NPC co-cultures. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 971-984, 2018.

Mechanical Properties and Cell Compatibility of Agarose Hydrogels Containing Proteoglycan Mimetic Graft Copolymers.

Proteoglycans have vital biochemical and biomechanical functions. Their proteolytic degradation results in loss of these functions. We have previously reported nonprotein proteoglycan-mimetic graft copolymers that stabilize and deliver growth factors and are not subject to proteases. Here we expand our investigation of these proteoglycan mimics by also investigating their effects on hydrogel mechanical properties. Four polysaccharide side chains, chondroitin sulfate, heparin, dextran, and dextran sulfate, are each grafted to a hyaluronan backbone. The polysaccharides and graft copolymers are added to agarose hydrogels. Cyclic compression and stress relaxation tests reveal how the addition of the polysaccharides and graft copolymers influence hydrogel modulus. Cells encapsulated in agarose hydrogels containing chondroitin sulfate and the chondroitin sulfate graft copolymer have decreased cell viability and metabolic activity compared to cells in unmodified agarose hydrogels. These multifunctional additives can be used to improve both the biochemistry and biomechanics of materials, warranting further optimization to overcome the potentially negative effects these may have on cell viability and activity.

Two-Phase Electrospinning to Incorporate Polyelectrolyte Complexes and Growth Factors into Electrospun Chitosan Nanofibers.

Growth factors are potent signaling proteins for tissue engineering, but they are susceptible to loss of activity when exposed to solvents used for polymer processing. This work explores preservation of fibroblast growth factor-2 (FGF-2) activity in chitosan nanofibers using two-phase electrospinning via a compound coaxial needle and from a water-in-oil emulsion FGF-2 in aqueous poly(vinyl alcohol) is added on either the inside (A/O) or the outside (O/A) of an organic chitosan phase, using the compound needle. FGF-2 is further stabilized by complexation to heparin-based nanoparticles. The emulsion method does not result in detectable incorporation of FGF-2. The A/O fibers incorporate the highest amount of FGF-2. Nanoparticle-stabilized FGF-2 in A/O nanofibers is most active toward bone-marrow stromal cells.

Preparation of Proteoglycan Mimetic Graft Copolymers.

Proteoglycans are proteins with pendant glycosaminoglycan polysaccharide side chains. The method described here enables the preparation of graft copolymers with glycosaminoglycan side chains, which mimic the structure and composition of proteoglycans. By controlling the stoichiometry, graft copolymers can be obtained with a wide range of glycosaminoglycan side-chain densities. The method presented here uses a three-step reaction mechanism to first functionalize a hyaluronic acid backbone, followed by reductive amination to couple the glycosaminoglycan side chain to the backbone, by the reducing end. Proteoglycan mimics like the ones proposed here could be used to study the structure-property relationships of proteoglycans and to introduce the biochemical and biomechanical properties of proteoglycans into biomaterials and therapeutic formulations.

Synthesis and characterization of proteoglycan-mimetic graft copolymers with tunable glycosaminoglycan density.

Proteoglycans (PGs) are important glycosylated proteins found on the cell surface and in the extracellular matrix. They are made up of a core protein with glycosaminoglycan (GAG) side chains. Variations in composition and number of GAG side chains lead to a vast array of PG sizes and functions. Here we present a method for the synthesis of proteoglycan-mimetic graft copolymers (or neoproteoglycans) with tunable GAG side-chain composition. This is done using three different GAGs: hyaluronan, chondroitin sulfate, and heparin. Hyaluronan is functionalized with a hydrazide-presenting linker. Either chondroitin sulfate or heparin is grafted by the reducing end on to the hyaluronan backbone through reductive amination. PG mimics with heparin or chondroitin sulfate side chains and four different ratios of GAG side chain result in graft copolymers with a wide range of sizes. The chemistry is confirmed through attentuated total reflectance Fourier transform infrared spectroscopy (ATR-FTIR) and (1)H NMR. Effective hydrodynamic diameter and zeta potential are determined using dynamic light scattering and electrophoretic mobility measurements. Graft copolymers were tested for their ability to bind and deliver basic fibroblast growth factor (FGF-2) to mesenchymal stem cells (MSCs). The chondroitin sulfate-containing graft copolymers successfully deliver FGF-2 to cells from surfaces. The lowest graft density of heparin-containing PG mimic also performs well with respect to growth factor delivery from a surface. This new method for preparation of GAG-based graft copolymers enables a wide range of graft density, and can be used to explore applications of PG mimics as new biomaterials with tunable biochemical and biomechanical functions.

Nanostructured biomaterials from electrospun demineralized bone matrix: a survey of processing and crosslinking strategies.

In the design of scaffolds for tissue engineering biochemical function and nanoscale features are of particular interest. Natural polymers provide a wealth of biochemical function, but do not have the processability of synthetic polymers, limiting their ability to mimic the hierarchy of structures in the natural extracellular matrix. Thus, they are often combined with synthetic carrier polymers to enable processing. Demineralized bone matrix (DBM), a natural polymer, is allograft bone with inorganic material removed. DBM contains the protein components of bone, which includes adhesion ligands and osteoinductive signals, such as important growth factors. Herein we describe a novel method for tuning the nanostructure of DBM through electrospinning without the use of a carrier polymer. This work surveys solvents and solvent blends for electrospinning DBM. Blends of hexafluoroisopropanol and trifluoroacetic acid are studied in detail. The effects of DBM concentration and dissolution time on solution viscosity are also reported and correlated to observed differences in electrospun fiber morphology. We also present a survey of techniques to stabilize the resultant fibers with respect to aqueous environments. Glutaraldehyde vapor treatment is successful at maintaining both macroscopic and microscopic structure of the electrospun DBM fibers. Finally, we report results from tensile testing of stabilized DBM nanofiber mats, and preliminary evaluation of their cytocompatibility. The DBM nanofiber mats exhibit good cytocompatibility toward human dermal fibroblasts (HDF) in a 4-day culture; neither the electrospun solvents nor the cross-linking results in any measurable residual cytotoxicity toward HDF.

Aggrecan-mimetic, glycosaminoglycan-containing nanoparticles for growth factor stabilization and delivery.

The direct delivery of growth factors to sites of tissue healing is complicated by their relative instability. In many tissues, the glycosaminoglycan (GAG) side chains of proteoglycans like aggrecan stabilize growth factors in the pericellular and extracellular space, creating a local reservoir that can be accessed during a wound healing response. GAGs also regulate growth factor-receptor interactions at the cell surface. Here we report the development of nanoparticles for growth factor delivery that mimic the size, GAG composition, and growth factor binding and stabilization of aggrecan. The aggrecan-mimetic nanoparticles are easy to assemble, and their structure and composition can be readily tuned to alter their physical and biological properties. We use basic fibroblast growth factor (FGF-2) as a model heparin-binding growth factor, demonstrating that aggrecan-mimetic nanoparticles can preserve its activity for more than three weeks. We evaluate FGF-2 activity by measuring both the proliferation and metabolic activity of bone marrow stromal cells to demonstrate that chondroitin sulfate-based aggrecan mimics are as effective as aggrecan, and heparin-based aggrecan mimics are superior to aggrecan as delivery vehicles for FGF-2.

Layer-by-layer assembly of polysaccharide-based polyelectrolyte multilayers: a spectroscopic study of hydrophilicity, composition, and ion pairing.

Polyelectrolyte multilayers using the polycations chitosan and N,N,N-trimethyl chitosan and the polyanions hyaluronan, chondroitin sulfate, and heparin are studied. Chitosan and hyaluronan behave as a weak polycation and weak polyanion, respectively, whereas N,N,N-trimethyl chitosan, chondroitin sulfate, and heparin behave as strong polyelectrolytes. Hydrophilicity is determined by water contact angle measurements and by comparing wet and dry film thickness measurements. Wet thickness is obtained using Fourier transform surface plasmon resonance, whereas dry thickness is obtained through ellipsometry. For the very thin PEMs studied here, the surface hydrophilicity and swelling in water are highly correlated. The multilayer chemistry is assessed by FT-IR and X-ray photoelectron spectroscopy (XPS). FT-IR and XPS provide information about the composition, degree of ionization, and by inference, the ion pairing. We find that hydrophilicity and swelling are reduced when one polyelectrolyte is strong and the other is weak, whereas ion pairing is increased. By this combination of techniques, we are able to compose a unified description of how the PEM swelling is dictated by the ion pairing in thin polysaccharide-based PEMs.

Enhanced osteoblastic differentiation of mesenchymal stem cells seeded in RGD-functionalized PLLA scaffolds and cultured in a flow perfusion bioreactor.

The present study combines chemical and mechanical stimuli to modulate the osteogenic differentiation of mesenchymal stem cells (MSCs). Arg-Gly-Asp (RGD) peptides incorporated into biomaterials have been shown to upregulate MSC osteoblastic differentiation. However, these effects have been assessed under static culture conditions, while it has been reported that flow perfusion also has an enhancing effect on MSC osteoblastic differentiation. It is clear that there is a need to combine RGD modification of biomaterials with mechanical stimulation of MSCs via flow perfusion and evaluate its effects on MSC differentiation down the osteogenic lineage. In this study, the effect of different levels of RGD modification of poly(L-lactic acid) scaffolds on MSC osteogenesis was evaluated under conditions of flow perfusion. It was found that there is a synergistic enhancement of different osteogenic markers, due to the combination of flow perfusion and RGD surface modification when compared to their individual effects. Furthermore, under conditions of flow perfusion, there is an RGD surface concentration optimal for differentiation, and it is flow rate-dependent. This report underlines the significance of incorporating combined biomimesis via biochemical and mechanical microenvironments that modulate in vivo cell behaviour and tissue function for more efficient tissue-engineering strategies.