Faculty Bibliography

A poly(ethylene glycol) (PEG)-based hydrogel was used as a scaffold for chondrocyte culture. Branched PEG-vinylsulfone macromers were end-linked with thiol-bearing matrix metalloproteinase (MMP)-sensitive peptides (GCRDGPQGIWGQDRCG) to form a three-dimensional network in situ under physiologic conditions. Both four- and eight-armed PEG macromer building blocks were examined. Increasing the number of PEG arms increased the elastic modulus of the hydrogels from 4.5 to 13.5 kPa. PEG-dithiol was used to prepare hydrogels that were not sensitive to degradation by cell-derived MMPs. Primary bovine calf chondrocytes were cultured in both MMP-sensitive and MMP-insensitive hydrogels, formed from either four- or eight-armed PEG. Most (>90%) of the cells inside the gels were viable after 1 month of culture and formed cell clusters. Gel matrices with lower elastic modulus and sensitivity to MMP-based matrix remodeling demonstrated larger clusters and more diffuse, less cell surface-constrained cell-derived matrix in the chondron, as determined by light and electron microscopy. Gene expression experiments by real-time RT-PCR showed that the expression of type II collagen and aggrecan was increased in the MMP-sensitive hydrogels, whereas the expression level of MMP-13 was increased in the MMP-insensitive hydrogels. These results indicate that cellular activity can be modulated by the composition of the hydrogel. This study represents one of the first examples of chondrocyte culture in a bioactive synthetic material that can be remodeled by cellular protease activity.

A method is presented for high-throughput cell manipulation that is capable of trapping and transporting biological cells in liquid using acoustic forces in an ultrasound field. The authors applied this technique for concentrating several cell types such as HeLa cells and human mesencymal stem cells. More than 90% of the cells were successfully concentrated into desired patterns. They also investigated cell viability in ultrasound fields and found little adverse effect. This work demonstrates that ultrasonic cell manipulation is suitable for being integrated into lab-on-a-chip systems for trapping and transporting large numbers of cells rapidly and is promising in cell fractioning.

Cell adhesion molecule L1 was implicated in angiogenic processes, tumor formation and metastasis. Here, we provide evidence that the sixth Ig-like domain of L1 (L1Ig6) interacts with alpha(v)beta3 to induce process extension of human umbilical vein endothelial cells (HUVECs) in vitro and angiogenesis in vivo. HUVECs formed network-like structures on full-length L1 or L1Ig6 substrates comparable to structures found on matrigel. In the presence of mab alpha(v)beta3 or cyclic RGD, apoptosis was induced. In fibrin matrices where L1Ig6 was covalently incorporated, HUVECs formed multicellular and hollow processes through interactions between cell-surface alpha(v)beta3 and RGD-sites of matrix-immobilized L1Ig6. No such processes were induced by L1Ig6 having non-functional RDG-sites, or in the presence of mab alpha(v)beta3 or cyclic RGD. In those matrices, increased apoptosis was found. Co-immunoprecipitation of L1 or L1Ig6 with alpha(v)beta3 suggests close interactions. Furthermore, L1Ig6 stimulated HUVECs showed increased tyrosine phosphorylation of alpha(v)beta3 and phosphorylation of MAP kinases (ERK1 and ERK2) but not AKT indicating specific activation of alpha(v) and alpha(v)beta3 followed by activation of downstream kinases. Application of L1Ig6-modified fibrin matrices on CAMs induced 50-60% increased alpha(v) and alpha(v)beta3 protein expression and in vivo angiogenesis indicated by approximately 50% increased mean vascular length density. The results demonstrate angiogenic potential of L1Ig6 involving ligation and activation of alpha(v)beta3.

A hydrogel system that is designed to gel due to oxidation by molecular oxygen is described. This is achieved by the use of a branched poly(ethylene glycol) modified with thiol end groups. A stable precursor molecule, starPEG thioacetate, was synthesized by radical addition of thioacetic acid to an intermediate starPEG allyl ether. Rapid deprotection of the thiol can be achieved using a base, e.g. sodium hydroxide, quantitatively liberating a thiol group and a non-toxic acetate ion. This step can be carried out under anaerobic conditions, yielding a solution with known thiol content that can be stored. The reaction with oxygen is accelerated by the use of a catalyst based on Fenton chemistry, which makes the material useful for biomedical applications where in situ polymerization of an injectable material is beneficial. This gelation takes place under near physiological conditions without the need for a cross-linking agent.

Local, controlled induction of angiogenesis remains a challenge that limits tissue engineering approaches to replace or restore diseased tissues. We present a new class of bioactive synthetic hydrogel matrices based on poly(ethylene glycol) (PEG) and synthetic peptides that exploits the activity of vascular endothelial growth factor (VEGF) alongside the base matrix functionality for cellular ingrowth, that is, induction of cell adhesion by pendant RGD-containing peptides and provision of cell-mediated remodeling by cross-linking matrix metalloproteinase substrate peptides. By using a Michael-type addition reaction, we incorporated variants of VEGF121 and VEGF165 covalently within the matrix, available for cells as they invade and locally remodel the material. The functionality of the matrix-conjugated VEGF was preserved and was critical for in vitro endothelial cell survival and migration within the matrix environment. Consistent with a scheme of locally restricted availability of VEGF, grafting of these VEGF-modified hydrogel matrices atop the chick chorioallontoic membrane evoked strong new blood vessel formation precisely at the area of graft-membrane contact. When implanted subcutaneously in rats, these VEGF-containing matrices were completely remodeled into native, vascularized tissue. This type of synthetic, biointeractive matrix with integrated angiogenic growth factor activity, presented and released only upon local cellular demand, could become highly useful in a number of clinical healing applications of local therapeutic angiogenesis.

The graft copolymer poly(L-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) and its RGD- and RDG-functionalized derivatives (PLL-g-PEG/PEG-peptide) were assembled from aqueous solutions on titanium (oxide) surfaces. The polymers were characterized by NMR in order to determine quantitatively the grafting ratio, g (Lys monomer units/PEG side chains), and the fraction of the PEG side chains carrying the terminal peptide group. The titanium surfaces modified with the polymeric monomolecular adlayers were exposed to full heparinized blood plasma. The adsorbed masses were measured by in situ ellipsometry. The different PLL-g-PEG-coated surfaces showed, within the detection limit of the ellipsometric technique, no statistically significant protein adsorption during exposure to plasma for 30 min at 22 degrees C or 37 degrees C, whereas clean, uncoated titanium surfaces adsorbed approximately 350 ng/cm2 of plasma proteins. The high degree of resistance of the PEGylated surface to non-specific adsorption makes peptide-modified PLL-g-PEG a useful candidate for the surface modification of biomedical devices such as implants that are capable of eliciting specific interactions with integrin-type cell receptors even in the presence of full blood plasma. The results refer to short-term blood plasma exposure that cannot be extrapolated a priori to long-term clinical performance.

alpha-dystroglycan is a cell surface receptor that is expressed in many tissues including the nervous system. The study shows that a recombinant, non-glycosylated N-terminal fragment of alpha-dystroglycan comprising residues 30 to 315 [alphaDG (30-315)] bound to laminin-2/-4 and laminin-1, fibronectin and fibrinogen, all molecules highly upregulated in the regenerating peripheral nerve. The interaction was concentration dependent and saturable and could not be inhibited by heparin suggesting only minor involvement of sulfated carbohydrate moieties. In contrast to published data, addition of bivalent cations increased the binding affinity by only ten fold.alphaDG (30-315) promotes neurite extension of PC12 cells in a similar amount as described for laminin isoforms and could be inhibited in a concentration dependent manner by alphaDG (30-315) itself, soluble laminin-1, partially by heparin, EDTA, and an RGD-peptide. Furthermore, co-immunoprecipitations between alpha-dystroglycan and beta1-integrin from PC12 cell surfaces suggested complex interactions between neuronal dystroglycan, integrins, and the ECM that induce neurite extension in vitro.

Within native tissues cells are held within the extracellular matrix (ECM), which has a role in maintaining homeostasis, guiding development and directing regeneration. Efforts in tissue engineering have aimed to mimick the ECM to help guide morphogenesis and tissue repair. Studies have not only looked at ways to mimick the structure and characteristics of the ECM, but have also considered ways to reproduce its molecular properties including its bioadhesive character, proteolytic susceptibility and ability to bind growth factors.

Anionic pH-sensitive membrane-disruptive polymers have evolved as a new class of bioactive excipients for the cytosolic delivery of therapeutic macromolecules. A large variety of anionic copolymers and analogues of poly(acrylic acid) (PA) was investigated and compared to a cationic PA copolymer. The pH-responsive membrane-disruptive properties were characterized by employing three in vitro models, such as pH dependent shift of pyrene fluorescence, liposome leakage and lysis of red blood cells. The pH-dependent increase of polarity and membrane disruption in the different model systems was in good agreement for all tested PA polymers. The efficacy of polymer-induced membrane disruption was concentration-dependent and significantly affected by the composition of the membrane. The sensitivity of relatively complex membranes of mammalian cells can be ranked between plain diphosphatidylcholine (DPPC) liposomal membranes and the more rigid cholesterol-containing DPPC membranes. Among the various studied PA polymers, medium and low molecular poly(ethacrylic acid) (PEA) and poly(propacrylic acid) (PPA) were identified as displaying significant pH-dependent disruptive activity. Relative to the disruptive cationic PA polymer (PDMAEM) the ranking is PEA < PPA < PDMAEM. The fine tuning of the pH-responsive hydrophilic-hydrophobic balance is likely to be responsible for the superior effect of PEA and PPA compared to other anionic PA polymers. This thorough investigation of a large variety of different anionic PA polymers and the comparison with an efficient, although rather toxic cationic PA polymer provides a good assessment for further therapeutic applications.