Surface modification of copolymerized films from three-armed biodegradable macromers - An analytical platform for modified tissue engineering scaffolds.

The concept of macromers allows for a broad adjustment of biomaterial properties by macromer chemistry or copolymerization. Copolymerization strategies can also be used to introduce reactive sites for subsequent surface modification. Control over surface features enables adjustment of cellular reactions with regard to site and object of implantation. We designed macromer-derived polymer films which function as non-implantable analytical substrates for the investigation of surface properties of equally composed scaffolds for bone tissue engineering. To this end, a toolbox of nine different biodegradable, three-armed macromers was thermally cross-copolymerized with poly(ethylene glycol)-methacrylate (PEG-MA) to films. Subsequent activation of PEG-hydroxyl groups with succinic anhydride and N-hydroxysuccinimid allowed for covalent surface modification. We quantified the capacity to immobilize analytes of low (amino-functionalized fluorescent dye, Fcad, and RGD-peptides) and high (alkaline phosphatase, ALP) molecular weight. Fcad grafting level was controlled by macromer chemistry, content and molecular weight of PEG-MA, but also the solvent used for film synthesis. Fcad molar amount per surface area was twentyfive times higher on high-swelling compared to low-swelling films, but differences became smaller when large ALP (appr. 2:1) were employed. Similarly, small differences were observed on RGD peptide functionalized films that were investigated by cell adhesion studies. Presentation of PEG-derivatives on surfaces was visualized by atomic force microscopy (AFM) which unraveled composition-dependent domain formation influencing fluorescent dye immobilization. Surface wetting characteristics were investigated via static water contact angle. We conclude that macromer ethoxylation and lactic acid content determined film swelling, PEG domain formation and eventually efficiency of surface decoration. STATEMENT OF SIGNIFICANCE: Surfaces of implantable biomaterials are the site of interaction with a host tissue. Accordingly, modifications in the composition of the surface will determine cellular response towards the material which is crucial for the success of innovations and control of tissue regeneration. We employed a macromer approach which is most flexible for the design of biomaterials with a broad spectrum of physicochemical characteristics. For ideal analytical accessibility of the material platform, we cross-copolymerized films on solid supports. Films allowed for the covalent immobilization of fluorescent labels, peptides and enzymes and thorough analytical characterization revealed that macromer hydrophilicity is the most relevant design parameter for surface analyte presentation in these materials. All analytical results were combined in a model describing PEG linker domain formation and ligand presentation.

Artificial extracellular matrices support cell growth and matrix synthesis of human dermal fibroblasts in macroporous 3D scaffolds.

Surface modification of materials designed for regenerative medicine may improve biocompatibility and functionality. The application of glycosaminoglycans (GAGs) and chemically sulphated GAG derivatives is a promising approach for designing functional biomaterials, since GAGs interact with cell-derived growth factors and have been shown to support fibroblast growth in two-dimensional (2D) cultures. Here, coatings with artificial extracellular matrix (aECM), consisting of the structural protein collagen I and the GAG hyaluronan (HA) or sulphated HA derivatives, were investigated for their applicability in a three-dimensional (3D) system. As a model, macroporous poly(lactic-co-glycolic acid) (PLGA) scaffolds were homogeneously coated with aECM. The resulting scaffolds were characterized by compressive moduli of 0.9-1.2 MPa and pore sizes of 40-420 µm. Human dermal fibroblasts (dFbs) colonized these aECM-coated PLGA scaffolds to a depth of 400 µm within 14 days. In aECM-coated scaffolds, collagen I(α1) and collagen III(α1) mRNA expression was reduced, while matrix metalloproteinase-1 (MMP-1) mRNA expression was increased within 7 days, suggesting matrix-degradation processes. Stimulation with TGFß1 generally increased cell density and collagen synthesis, demonstrating the efficiency of bioactive molecules in this 3D model. Thus, aECM with sulphated HA may modulate the effectivity of TGFß1-induced collagen I(α1) expression, as demonstrated previously in 2D systems. Overall, the tested aECM with modified HA is also a suitable material for fibroblast growth under 3D conditions. Copyright © 2015 John Wiley & Sons, Ltd.

Matrix metalloproteinase 9 (MMP-9) mediated release of MMP-9 resistant stromal cell-derived factor 1α (SDF-1α) from surface modified polymer films.

Preparation of smart materials by coatings of established surfaces with biomolecules will lead to the next generation of functionalized biomaterials. Rejection of implants is still a major problem in medical applications but masking the implant material with protein coatings is a promising approach. These layers not only disguise the material but also equip it with a certain biological function. The anti-inflammatory chemokine stromal cell-derived factor 1α (SDF-1α) is well suited to take over this function, because it efficiently attracts stem cells and promotes their differentiation and proliferation. At least the initial stem cell homing requires the formation of a concentration gradient. Thus, a reliable and robust release mechanism of SDF-1α from the material is essential. Several proteases, most notably matrix metalloproteinases, are upregulated during inflammation, which, in principle, can be exploited for a tightly controlled release of SDF-1α. Herein, we present the covalent immobilization of M-[S4V]-SDF-1α on novel biodegradable polymer films, which consist of heterobifunctional poly(ethylene glycol) and oligolactide-based functionalized macromers. A peptidic linker with a trimeric matrix metalloproteinase 9 (MMP-9) cleavage site (MCS) was used as connection and the linkage between the three components was achieved by combination of expressed protein ligation and Cu(I) catalyzed azide/alkyne cycloaddition. The MCS was used for MMP-9 mediated release of M-[S4V]-SDF-1α from the biomaterial and the released SDF-1α derivative was biologically active and induced strong cell migration, which demonstrates the great potential of this system.

On-resin synthesis of an acylated and fluorescence-labeled cyclic integrin ligand for modification of poly(lactic-co-glycolic acid).

Cyclic Arg-Gly-Asp (RGD) peptides show remarkable affinity and specificity to integrin receptors and mediate important physiological effects in tumor angiogenesis. Additionally, they are one of the keyplayers in improving the biocompatibility of biomaterials. The fully biodegradable polymer poly(lactic-co-glycolic acid) (PLGA) is frequently used for biomedical implants and can be applied as nanoparticles for drug delivery. The aim of this work was the generation of a lipidated c[RGDfK] peptide including a second functionality for coating of hydrophobic PLGA. Therefore, we established a general and straightforward strategy for the introduction of two different modifications into the same c[RGDfK] peptide. This allowed the generation of a palmitoylated integrin-binding lipopeptide that shows high affinity to PLGA. Additionally, we coupled 5(6)-carboxyfluorescein to the second site for modification to enable sensitive quantification of the immobilized lipopeptide on PLGA. In conclusion, we present a synthesis protocol that enables the preparation of c[RGDfK] lipopeptides with a strong affinity to PLGA and an additional site for modifications. This will provide the opportunity to introduce a variety of effector molecules site-specifically to the c[RGDfK] lipopeptide, which will enable the introduction of multifunctionality into c[RGDfK]-coated PLGA devices or nanoparticles.

Traumatic brain injury elicits similar alterations in α7 nicotinic receptor density in two different experimental models.

Traumatic brain injury (TBI) is a major cause of death and disability worldwide, especially in children and young adults. Previous studies have shown alterations in the central cholinergic neurotransmission after TBI. We therefore determined α7 nicotinic acetylcholine receptor (nAChR) densities in newborn piglets and adult rats after experimental TBI. Thirteen newborn piglets (post-TBI survival time: 6 h) underwent fluid percussion (FP) injury (n = 7) or sham operation (n = 6). Furthermore, adult rats randomized into three groups of post-TBI survival times (2, 24, 72 h) received controlled cortical impact injury (CCI, n = 8) or sham operation (n = 8). Brains were frozen, sagittally cut and incubated with the α7-specific radioligand [(125)I]α-bungarotoxin for autoradiography. In injured newborn piglets, decreased α7 receptor densities were observed in the hippocampus (-38%), the hippocampus CA1 (-40%), thalamus (-30%) and colliculus superior (-30%). In adult rats, CCI decreased the receptor densities (between -16 and -47%) in almost any brain region within 2 and 24 h. In conclusion, widespread and significantly lowered α7 nAChR densities were demonstrated in both TBI models. Our results suggest that a nearly similar TBI-induced decrease in the α7 density in the brain of immature and adult animals is found, even with the differences in species, age and experimental procedures. The alterations make the α7 nAChR a suitable target for drug development and neuroimaging after TBI.