Osteonectin-derived peptide increases the modulus of a bone-mimetic nanocomposite

Many factors contribute to the toughness of bone including the presence of nano-size apatite crystals, a dense network of collagen fibers, and acidic proteins with the ability to link the mineral phase to the gelatinous collagen phase. We investigated the effect of a glutamic acid (negatively charged) peptide (Glu6), which mimics the terminal region of the osteonectin glycoprotein of bone, on the shear modulus of a synthetic hydrogel/apatite nanocomposite. One end of the synthesized peptide was functionalized with an acrylate group (Ac-Glu6) to covalently attach the peptide to the hydrogel phase of the composite matrix. When microapatite crystals (5 μm diameter) were used, addition of Ac-Glu6 peptide did not affect the modulus of the microcomposite. However, when nanoapatite crystals (100 nm diameter) were used, addition of Ac-Glu6 resulted in significant reinforcement of the shear modulus of the nanocomposite (∼100% in elastic shear modulus). Furthermore, addition of Ac-Gly6 (a neutral glycine sequence) or Ac-Lys6 (a positively charged sequence) did not reinforce the nanocomposite. These results demonstrate that the reinforcement effect of the Glu6 peptide, a sequence in the terminal region of osteonectin, is modulated by the size of the apatite crystals. The findings of this work can be used to develop advanced biomimetic composites for skeletal tissue regeneration.     

Modeling and experimental investigation of rheological properties of injectable poly(lactide ethylene oxide fumarate)/hydroxyapatite nanocomposites

Injectable multiphasic polymer/ceramic composites are attractive as bioresorbable scaffolds for bone regeneration because they can be cross-linked in situ and are osteoconductive. The injectability of the composite depends on the nanoparticle content and the energetic interactions at the polymer/particle interface. The objective of this research was to determine experimentally the rheological properties of the PLEOF/apatite composite as an injectable biomaterial and to compare the viscoelastic response with the predictions of a linear elastic dumbbell model. A degradable in situ cross-linkable terpolymer based on low molecular weight poly(L-lactide) and poly(ethylene oxide) linked by unsaturated fumarate groups is synthesized. The poly(L-lactide-co-ethylene oxide-co-fumarate) (PLEOF) terpolymer interacts with the surface of the apatite nanoparticles by polar interactions and hydrogen bonding. A kinetic model is developed that takes into account the adsorption/desorption of polymer chains to/from the nanoparticle surface. Rheological properties of the aqueous dispersion of PLEOF terpolymer reinforced with nanosized hydroxyapatite (HA) particles are investigated using mechanical rheometry. To this end, we performed a series of rheological experiments on un-cross-linked PLEOF reinforced with different volume fractions of HA nanoparticles. The results demonstrate that the observed nonlinear viscoelasticity at higher shear rates is controlled by the energetic interactions between the polymer chains and dispersed particle aggregates and by the rate of the adsorption/desorption of the chains to/from the surface of the nanoparticles.     

Material properties and cytocompatibility of injectable MMP degradable poly(lactide ethylene oxide fumarate) hydrogel as a carrier for marrow stromal cells

Injectable in situ crosslinkable biomaterials seeded with multipotent progenitor cells and coupled with minimally invasive arthroscopic techniques are an attractive alternative for treating irregularly shaped osteochondral defects. An in situ crosslinkable poly(lactide-co-ethylene oxide-co-fumarate) (PLEOF) macromer has been developed with ultralow molecular weight poly(L-lactide) and poly(ethylene glycol) (PEG) units linked by fumaryl unit. The PLEOF macromer was crosslinked with the MMP-13 degradable peptide sequence QPQGLAK with acrylate end-groups or the methylene bisacrylamide (BISAM) crosslinker to form enzymatically or hydrolytically degradable hydrogels, respectively. Cell viability of the peptide crosslinker was significantly higher than that of BISAM. The relatively higher molecular weight peptide crosslinker significantly affected the water content and the rate of crosslinking (e.g., sol vs gel fraction). The addition of a small fraction of a highly reactive BISAM crosslinker to the PLEOF/peptide mixture reduced the gelation time and increased the elastic modulus while retaining enzymatic degradability of the hydrogel. Bone marrow stromal (BMS) cells were encapsulated in the peptide crosslinked PLEOF hydrogel; 84% of the encapsulated cells was viable after 1 week of incubation in osteogenic media. The encapsulated BMS cells differentiated to osteoblasts and produced a mineralized matrix, as measured by ALPase activity and calcium content. The degradation rate of the hydrogel depended on the ratio of the peptide to the BISAM crosslinker, MMP-13 concentration, and incubation time. The results demonstrate that the peptide crosslinked PLEOF hydrogel with tunable degradation characteristics is potentially useful as an injectable in situ crosslinkable carrier for bone marrow stromal cells.     

Heparin interacting protein mediated assembly of nano-fibrous hydrogel scaffolds for guided stem cell differentiation

A new methodology is developed to conjugate hyaluronic acid (HA) hydrogel with novel nano-fibrous architectures via non-covalent assembly that specifically allows for targeted adipose-derived stem cells (ASCs) differentiation and soft tissue engineering. The assembly of non-covalently associated hydrogel network produced via the interaction of a low molecular weight heparin (LMWH) modified HA derivative and heparin interacting protein (HIP). The multifunctional star poly(ethylene glycol) (PEG) and HIP copolymer has the capability to mediate the non-covalent assembly of nano-fibrous HA hydrogel networks via affinity interactions with LMWH. The effect of the HIP mediation on in vitro gelation, rheological characteristics, degradation, equilibrium swelling, adipose-derived stem cells (ASCs) proliferation and differentiation of nano-fibrous hydrogel is examined. The results suggest the potential utility of this unique design of the bioactive nano-fibrous HA hydrogel in directing the differentiation of ASCs and adipogenesis in ECM-mimetic scaffolds in vitro. These studies demonstrate that this nano-fibrous HA hydrogel can render the formulation of a therapeutically effective platform for in vitro adipogenesis applications.     

Bone‐Inspired Tube Filling Decellularized Matrix of Toad Cartilage Provided an Osteoinductive Microenvironment for Mesenchymal Stem Cells to Facilitate the Radius Defect Repair of Rabbit

Toad bone not only contains the rich cartilage‐like matrix but also presents low immunogenicity. It is inferred that decellularized toad bone matrix (dBECM) may provide the more profitable osteoinductive microenvironment for mesenchymal stem cells (MSCs) to promote the repair of bone defects. Herein, a hollow bone‐inspired tube is first made from hydroxyapatite (HA) and poly (γ‐glutamic acid) (PGA), and then MSCs/dBECM hydrogel is uniformly filled to its central cavity, constructing a biomimetic bone (dBECM + MSCs ? PGA + HA). In vitro scratch and transwell experiments show that dBECM hydrogel not only effectively promotes migration and proliferation of MSCs but also induces their osteogenic differentiation. Moreover, the less inflammatory macrophages infiltrate at rat skin after subcutaneously injecting dBECM hydrogel, indicating its low potential for inflammatory attack. After implanting dBECM + MSCs ? PGA + HA to critical radius defect of rabbit, X‐ray and CT imaging shows that the cortex is effectively regenerated and the medullary cavity recanalization is completed at 20 weeks. Moreover, the expression of Collagen‐II and OCN are obviously increased in the defect after implanting dBECM + MSCs ? PGA + HA. The therapeutic mechanism of dBECM + MSCs ? PGA + HA scaffold are highly associated with the enhanced angiogenesis. Collectively, the biomimetic dBECM + MSCs ? PGA + HA scaffold may be a promising strategy to improve radius defect healing efficiency.     

Solid-phase synthesis of reactive peptide crosslinker by selective deprotection

An effective and simple strategy for preparing peptide crosslinkers is described. An MMP-13 degradable peptide QPQGLAK-NH(2) was prepared on the solid-phase using Fmoc chemistry. The peptide crosslinker was synthesized on-bead by the coupling reaction between acrylic acid and the amine groups of glutamine and lysine residues. The synthetic procedure employed the acid-labile Fmoc-Lys (Mtt)-OH and base-labile Fmoc-AA-OH derivatives. Selective deprotection, of -Mtt and -Fmoc groups on-bead, freed the amine end-groups on glutamine and lysine residues for coupling reaction with acrylic acid while maintaining the peptide attached to the resin. Subsequent cleavage from the resin yielded a peptide crosslinker with two unsaturated acrylate end-groups with high yield and purity. This method can be generally employed for the synthesis of a wide range of peptides with one or more reactive groups for grafting in the fabrication of biomimetic scaffolds in tissue engineering applications.     

Gelation characteristics and osteogenic differentiation of stromal cells in inert hydrolytically degradable micellar polyethylene glycol hydrogels

The use of poly(ethylene glycol) (PEG) hydrogels in tissue engineering is limited by their persistence in the site of regeneration. In an attempt to produce inert hydrolytically degradable PEG-based hydrogels, star (SPELA) poly(ethylene glycol-co-lactide) acrylate macromonomers with short lactide segments (<15 lactides per macromonomer) were synthesized. The SPELA hydrogel was characterized with respect to gelation time, modulus, water content, sol fraction, degradation, and osteogenic differentiation of encapsulated marrow stromal cells (MSCs). The properties of SPELA hydrogel were compared with those of the linear poly(ethylene glycol-co-lactide) acrylate (LPELA). The SPELA hydrogel had higher modulus, lower water content, and lower sol fraction than the LPELA. The shear modulus of SPELA hydrogel was 2.2 times higher than LPELA, whereas the sol fraction of SPELA hydrogel was 5 times lower than LPELA. The degradation of SPELA hydrogel depended strongly on the number of lactide monomers per macromonomer (nL) and showed a biphasic behavior. For example, as nL increased from 0 to 3.4, 6.4, 11.6, and 14.8, mass loss increased from 7 to 37, 80, 100% and then deceased to 87%, respectively, after 6 weeks of incubation. The addition of 3.4 lactides per macromonomer (<10 wt % dry macromonomer or <2 wt % swollen hydrogel) increased mass loss to 50% after 6 weeks. Molecular dynamic simulations demonstrated that the biphasic degradation behavior was related to aggregation and micelle formation of lactide monomers in the macromonomer in aqueous solution. MSCs encapsulated in SPELA hydrogel expressed osteogenic markers Dlx5, Runx2, osteopontin, and osteocalcin and formed a mineralized matrix. The expression of osteogenic markers and extent of mineralization was significantly higher when MSCs were encapsulated in SPELA hydrogel with the addition of bone morphogenetic protein-2 (BMP2). Results demonstrate that hydrolytically degradable PEG-based hydrogels are potentially useful as a delivery matrix for stem cells in regenerative medicine.     

Helix packing motif common to the crystal structures of two undecapeptides containing dehydrophenylalanine residues: implications for the de novo design of helical bundle super secondary structural modules

De novo designed peptide based super secondary structures are expected to provide scaffolds for the incorporation of functional sites as in proteins. Self-association of peptide helices of similar screw sense, mediated by weak interactions, has been probed by the crystal structure determination of two closely related peptides: Ac-Gly1-Ala2-Delta Phe3-Leu4-Val5-DeltaPhe6-Leu7-Val8-DeltaPhe9-Ala10-Gly11-NH2 (I) and Ac-Gly1-Ala2-DeltaPhe3-Leu4-Ala5-DeltaPhe6-Leu7-Ala8-DeltaPhe9-Ala10-Gly11-NH2 (II). The crystal structures determined to atomic resolution and refined to R factors 8.12 and 4.01%, respectively, reveal right-handed 3(10)-helical conformations for both peptides. CD has also revealed the preferential formation of right-handed 3(10)-helical conformations for both molecules. Our aim was to critically analyze the packing of the helices in the solid state with a view to elicit clues for the design of super secondary structural motifs such as two, three, and four helical bundles based on helix-helix interactions. An important finding is that a packing motif could be identified common to both the structures, in which a given peptide helix is surrounded by six other helices reminiscent of transmembrane seven helical bundles. The outer helices are oriented either parallel or antiparallel to the central helix. The helices interact laterally through a combination of N--H...O, C--H...O, and C--H...pi hydrogen bonds. Layers of interacting leucine residues are seen in both peptide crystal structures. The packing of the peptide helices in the solid state appears to provide valuable leads for the design of super secondary structural modules such as two, three, or four helix bundles by connecting adjacent antiparallel helices through suitable linkers such as tetraglycine segments.     

Mesenchymal stem cell responses to bone-mimetic electrospun matrices composed of polycaprolactone, collagen I and nanoparticulate hydroxyapatite

The performance of biomaterials designed for bone repair depends, in part, on the ability of the material to support the adhesion and survival of mesenchymal stem cells (MSCs). In this study, a nanofibrous bone-mimicking scaffold was electrospun from a mixture of polycaprolactone (PCL), collagen I, and hydroxyapatite (HA) nanoparticles with a dry weight ratio of 50/30/20 respectively (PCL/col/HA). The cytocompatibility of this tri-component scaffold was compared with three other scaffold formulations: 100% PCL (PCL), 100% collagen I (col), and a bi-component scaffold containing 80% PCL/20% HA (PCL/HA). Scanning electron microscopy, fluorescent live cell imaging, and MTS assays showed that MSCs adhered to the PCL, PCL/HA and PCL/col/HA scaffolds, however more rapid cell spreading and significantly greater cell proliferation was observed for MSCs on the tri-component bone-mimetic scaffolds. In contrast, the col scaffolds did not support cell spreading or survival, possibly due to the low tensile modulus of this material. PCL/col/HA scaffolds adsorbed a substantially greater quantity of the adhesive proteins, fibronectin and vitronectin, than PCL or PCL/HA following in vitro exposure to serum, or placement into rat tibiae, which may have contributed to the favorable cell responses to the tri-component substrates. In addition, cells seeded onto PCL/col/HA scaffolds showed markedly increased levels of phosphorylated FAK, a marker of integrin activation and a signaling molecule known to be important for directing cell survival and osteoblastic differentiation. Collectively these results suggest that electrospun bone-mimetic matrices serve as promising degradable substrates for bone regenerative applications.     

Effect of chemistry and morphology on the biofunctionality of self-assembling diblock copolypeptide hydrogels

Amphiphilic, diblock copolypeptides of hydrophilic lysine or glutamic acid and hydrophobic leucine or valine have been observed to self-assemble into rigid hydrogels in aqueous solution at neutral pH and very low volume fraction of polymer, > or =0.5 wt % polypeptide. Laser scanning confocal microscopy and ultra small angle neutron scattering revealed a heterogeneous microstructure with distinct domains of hydrogel matrix and pure water pores. In situ nanoscale characterization, using cryogenic transmission electron microscopy, revealed a porous, interconnected membranous network of assembled polypeptides. At concentrations of polypeptide below gelation, diblocks containing lysine were cytotoxic to cells, whereas those containing glutamic acid were noncytotoxic. At higher polypeptide concentrations, within rigid gel scaffolds, both lysine and glutamic acid based diblocks were noncytotoxic but did not support cell attachment/proliferation. The cationic chemistry observed as cytotoxic in the fluid state was essentially inert in the intact, rigid hydrogel state.     

Hydrolytically degradable hyaluronic acid hydrogels with controlled temporal structures

Polysaccharides are being processed into biomaterials for numerous biological applications due to their native source in numerous tissues and biological functions. For instance, hyaluronic acid (HA) is found abundantly in the body, interacts with cells through surface receptors, and can regulate cellular behavior (e.g., proliferation, migration). HA was previously modified with reactive groups to form hydrogels that are degraded by hyaluronidases, either added exogenously or produced by cells. However, these hydrogels may be inhibitory and their applications are limited if the appropriate enzymes are not present. Here, for the first time, we synthesized HA macromers and hydrogels that are both hydrolytically (via ester group hydrolysis) and enzymatically degradable. The hydrogel degradation and growth factor release was tailored through the hydrogel cross-linking density (i.e., macromer concentration) and copolymerization with purely enzymatically degradable macromers. When mesenchymal stem cells (MSCs) were encapsulated in the hydrogels, cellular organization and tissue distribution was influenced by the copolymer concentration. Importantly, the distribution of released extracellular matrix molecules (e.g., chondroitin sulfate) was improved with increasing amounts of the hydrolytically degradable component. Overall, this new macromer allows for enhanced control over the structural evolution of the HA hydrogels toward applications as biomaterials.     

Kinetics of in vivo bone deposition by bone marrow stromal cells within a resorbable porous calcium phosphate scaffold: an X-ray computed microtomography study

Resorbable ceramic scaffolds based on Silicon stabilized tricalcium phosphate (Si-TCP) were seeded with bone marrow stromal cells (BMSC) and ectopically implanted for 2, 4, and 6 months in immunodeficient mice. Qualitative and quantitative evaluation of the scaffold material was performed by X-ray synchrotron radiation computed microtomography (microCT) with a spatial resolution lower than 5 microm. Unique to these experiments was that microCT data were first collected on the scaffolds before implantation and then on the same scaffolds after they were seeded with BMSC, implanted in the mice and rescued after different times. Volume fraction, mean thickness and thickness distribution were evaluated for both new bone and scaffold phases as a function of the implantation time. New bone thickness increased from week 8 to week 16. Data for the implanted scaffolds were compared with those derived from the analysis of the same scaffolds prior to implantation and with data derived from 100% hydroxyapatite (HA) scaffold treated and analyzed in the same way. At variance with findings with the 100% HA scaffolds a significant variation in the density of the different Si-TCP scaffold regions in the pre- and post-implantation samples was observed. In particular a post-implantation decrease in the density of the scaffolds, together with major changes in the scaffold phase composition, was noticeable in areas adjacent to newly formed bone. Histology confirmed a better integration between new bone and scaffold in the Si-TCP composites in comparison to 100% HA composites where new bone and scaffold phases remained well distinct.     

Synthesis of [1, 6-cyclo (acetyl-1-L-glutamic acid, 2-D-phenylalanine, 3-D-tryptophan, 6-D-lysine)] luteinizing hormone-releasing hormone on poly-N-acrylylpyrrolidine resin

The protected peptide, Ac-Glu(OBut)-D-Phe-D-Trp-Ser(But)-Tyr(But)-D-Lys (Z)-Leu-Arg(Tos)-Pro-Gly-NH2 was synthesized in a stepwise manner on a resin of poly-N-acrylylpyrrolidine using both acid cleavable N alpha-tert.-butyloxycarbonyl and base cleavable N alpha-fluorenylmethyloxycarbonyl protecting groups. After cleavage by ammonolysis in methanol, the tert.-butyl and benzyloxy-carbonyl side-chain protecting groups were cleaved with CF3-CO2H-thioanisole and the 1-6 amide ring formed by cyclization with diphenylphosphorylazide, after which the remaining tosyl protecting group was cleaved in HF-anisole. [1,6-Cyclo(Ac-Glu1, D-Phe2, D-Trp3, D-Lys6]LH-RH exhibited less than 10% of the antiovulatory potency of [D less than Glu1, D-Phe2, D-Trp3,6] LH-RH, a potent linear antagonist.     

Osteotropic Peptide that differentiates functional domains of the skeleton

HPMA copolymer-d-aspartic acid octapeptide (D-Asp8) conjugates have been found to target the entire skeleton after systemic administration. In a recent study using the ovariectomized rat model of osteoporosis, we surprisingly discovered that D-Asp8 would favorably recognize resorption sites in skeletal tissues, while another bone-targeting moiety, alendronate (ALN), directs the delivery system to both formation and resorption sites. Atomic force microscopy (AFM) analyses reveal that ALN has a stronger binding force to hydroxyapatite (HA) than D-Asp8. In vitro HA binding studies indicate that D-Asp8 is more sensitive to change of HA crystallinity than ALN. Because the bone apatite in the newly formed bone (formation sites) usually has lower crystallinity than the resorption sites (mainly mature bone), we believe that the favorable recognition of D-Asp8 to the bone resorption sites could be attributed to its relatively weak binding to apatite, when compared to bisphosphonates, and the different levels of crystallinity of bone apatite at different functional domains of the skeleton.     

A single amino acid can switch the oligomerization state of the alpha-helical coiled-coil domain of cartilage matrix protein.

We have studied the oligomerization of an alpha-helical coiled-coil using as an example a peptide corresponding to the C-terminal domain of cartilage matrix protein. By replacing one arginine residue, which forms an interchain ionic interaction with a glutamic acid residue, with glutamine, we found that this peptide assembles into a homotetramer at neutral pH in contrast to the native molecule which forms homotrimers. At acidic and basic pH, however, we again observed the trimer conformation. Another arginine, which is probably involved in an intrachain salt bridge, has no effect on the assembly. Our data demonstrate that besides the specific distribution of hydrophobic residues, interchain ionic interactions can be crucial in modulating the association behavior of alpha-helical coiled-coil domains.     

Membrane binding of pH-sensitive influenza fusion peptides. positioning, configuration, and induced leakage in a lipid vesicle model

pH-sensitive HA2 fusion peptides from influenza virus hemagglutinin have potential as endosomal escape-inducing components in peptide-based drug delivery. Polarized light spectroscopy and tryptophan fluorescence were used to assess the conformation, orientation, effect on lipid order, and binding kinetics of wild-type peptide HA2(1-23) and a glutamic acid-enriched analogue (INF7) in large unilamellar POPC or POPC/POPG (4:1) lipid vesicles (LUVs). pH-sensitive membrane leakage was established for INF7 but not HA2(1-23) using an entrapped-dye assay. A correlation is indicated between leakage and a low degree of lipid chain order (assessed by linear dichroism, LD, of the membrane orientation probe retinoic acid). Both peptides display poor alignment in zwitterionic POPC LUVs compared to POPC/POPG (4:1) LUVs, and it was found that peptide-lipid interactions display slow kinetics (hours), resulting in reduced lipid order and increased tryptophan shielding. At pH 7.4, INF7 displays tryptophan emission and LD features indicative of a surface-orientated peptide, suggesting that its N-terminal glutamic acid residues prevent deep penetration into the hydrocarbon core. At pH 5.0, INF7 displays weaker LD signals, indicating poor orientation, possibly due to aggregation. By contrast, the orientation of the HA2(1-23) peptide backbone supports previously reported oblique insertion ( approximately 60-65 degrees relative to the membrane normal), and aromatic side-chain orientations are consistent with an interfacial (pH-independent) location of the C-terminus. We propose that a conformational change upon reduction of pH is limited to minor rearrangements of the peptide "hinge region" around Trp14 and repositioning of this residue.     

Osteogenic Properties of PBLG-g-HA/PLLA Nanocomposites

New development of biomaterial scaffolds remains a prominent issue for the regeneration of lost or fractured bone. Of these scaffolds, a number of bioactive polymers have been synthesized and fabricated for diverse biological roles. Although recent evidence has demonstrated that composite scaffolds such as HA/PLLA have improved properties when compared to either HA or PLLA alone, recent investigations have demonstrated that the phase compatibility between HA and PLLA layers is weak preventing optimal enhancement of the mechanical properties and making the composites prone to breakdown. In the present study, poly (γ-benzyl-L-glutamate) modified hydroxyapatite/(poly (L-lactic acid)) (PBLG-g-HA/PLLA) composite scaffolds were fabricated with improved phase compatibility and tested for their osteogenic properties in 18 Wistar female rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. At time points, 2, 4 and 8 weeks post surgery, bone formation was evaluated by µ-CT and histological analysis by comparing 4 treatment groups; 1) blank defect, 2) PLLA, 3) HA/PLLA and 4) PBLG-g-HA/PLLA scaffolds. The in vivo analysis demonstrated that new bone formation was much more prominent in HA/PLLA and PBLG-g-HA/PLLA groups as depicted by µ-CT, H&E staining and immunohistochemistry for collagen I. TRAP staining was also utilized to determine the influence of osteoclast cell number and staining intensity to the various scaffolds. No significant differences in either staining intensity or osteoclast numbers between all treatment modalities was observed, however blank defects did contain a higher number of osteoclast-like cells. The results from the present study illustrate the potential of PBLG-g-HA/PLLA scaffolds for bone tissue engineering applications by demonstrating favorable osteogenic properties.     

Monosaccharide-Responsive Phenylboronate-Polyol Cell Scaffolds for Cell Sheet and Tissue Engineering Applications

Analyte-responsive smart polymeric materials are of great interest and have been actively investigated in the field of regenerative medicine. Phenylboronate containing copolymers form gels with polyols under alkaline conditions. Monosaccharides, by virtue of their higher affinity towards boronate, can displace polyols and solubilize such gels. In the present study, we investigate the possibility of utilizing phenylboronate-polyol interactions at physiological pH in order to develop monosaccharide-responsive degradable scaffold materials for systems dealing with cells and tissues. Amine assisted phenylboronate-polyol interactions were employed to develop novel hydrogel and cryogel scaffolds at neutral pH. The scaffolds displayed monosaccharide inducible gel-sol phase transformability. In vitro cell culture studies demonstrated the ability of scaffolds to support cell adhesion, viability and proliferation. Fructose induced gel degradation is used to recover cells cultured on the hydrogels. The cryogels displayed open macroporous structure and superior mechanical properties. These novel phase transformable phenylboronate-polyol based scaffolds displayed a great potential for various cell sheet and tissue engineering applications. Their monosaccharide responsiveness at physiological pH is very useful and can be utilized in the fields of cell immobilization, spheroid culture, saccharide recognition and analyte-responsive drug delivery.     

Studies on the mechanism of membrane fusion: site-specific mutagenesis of the hemagglutinin of influenza virus

Oligonucleotide-directed mutagenesis of a cDNA encoding the hemagglutinin of influenza virus has been used to introduce single base changes into the sequence that codes for the conserved apolar "fusion peptide" at the amino-terminus of the HA2 subunit. The mutant sequences replaced the wild-type gene in SV40-HA recombinant virus vectors, and the altered HA proteins were expressed in simian cells. Three mutants have been constructed that introduce single, nonconservative amino acid changes in the fusion peptide, and three fusion phenotypes were observed: substitution of glutamic acid for the glycine residue at the amino-terminus of HA2 abolished all fusion activity; substitution of glutamic acid for the glycine residue at position 4 in HA2 raised the threshold pH and decreased the efficiency of fusion; and, finally, extension of the hydrophobic stretch by replacement of the glutamic acid at position 11 with glycine yielded a mutant protein that induced fusion of erythrocytes with cells with the same efficiency and pH profile as the wild-type protein. However, the ability of this mutant to induce polykaryon formation was greatly impaired. Nevertheless, all the mutant proteins underwent a pH-dependent conformational change and bound to liposomes. These results are discussed in terms of the mechanism of HA-induced membrane fusion.     

Facile synthesis and characterization of disulfide-cross-linked hyaluronic acid hydrogels for protein delivery and cell encapsulation

Injectable hyaluronic acid (HA) hydrogels cross-linked via disulfide bond are synthesized using a thiol-disulfide exchange reaction. The production of small-molecule reaction product, pyridine-2-thione, allows the hydrogel formation process to be monitored quantitatively in real-time by UV spectroscopy. Rheological tests show that the hydrogels formed within minutes at 37 °C. Mechanical properties and equilibrium swelling degree of the hydrogels can be controlled by varying the ratio of HA pyridyl disulfide and macro-cross-linker PEG-dithiol. Degradation of the hydrogels was achieved both enzymatically and chemically by disulfide reduction with distinctly different kinetics and profiles. In the presence of hyaluronidase, hydrogel mass loss over time was linear and the degradation was faster at higher enzyme concentrations, suggesting surface-limited degradation. The kinetics of hydrogel erosion by glutathione was not linear, nor did the erosion rate correlate linearly with glutathione concentration, suggesting a bulk erosion mechanism. A cysteine-containing chemokine, stromal cell-derived factor 1α, was successfully encapsulated in the hydrogel and released in vitro without chemical alteration. Several different cell types, including fibroblasts, endothelial cells, and mesenchymal stem cells, were successfully encapsulated in the hydrogels with high cell viability during and after the encapsulation process. Substantial cell viability in the hydrogels was maintained up to 7 days in culture despite the lack of adhesion between the HA matrix and the cells. The facile synthesis of disulfide-cross-linked, dual-responsive degradable HA hydrogels may enable further development of bioactive matrices potentially suitable for tissue engineering and drug delivery applications.