Fast-Curing Injectable Microporous Hydrogel for In Situ Cell Encapsulation.

Injectable hydrogels have previously demonstrated potential as a temporary scaffold for tissue regeneration or as a delivery vehicle for cells, growth factors, or drugs. However, most injectable hydrogel systems lack a microporous structure, preventing host cell migration into the hydrogel interior and limiting spreading and proliferation of encapsulated cells. Herein, an injectable microporous hydrogel assembled from gelatin/gelatin methacryloyl (GelMA) composite microgels is described. Microgels are produced by a water-in-oil emulsion using a gelatin/GelMA aqueous mixture. These microgels show improved thermal stability compared to GelMA-only microgels and benefit from combined photopolymerization using UV irradiation (365 nm) in the presence of a photoinitiator (PI) and enzymatic reaction by microbial transglutaminase (mTG), which together enable fast curing and tissue adhesion of the hydrogel. The dual-crosslinking approach also allows for the reduction of PI concentration and minimizes cytotoxicity during photopolymerization. When applied for in situ cell encapsulation, encapsulated human dermal fibroblasts and human mesenchymal stem cells (hMSCs) are able to rapidly spread and proliferate in the pore space of the hydrogel. This hydrogel has the potential to enhance hMSC anti-inflammatory behavior through the demonstrated secretion of prostaglandin E2 (PGE2) and interleukin-6 (IL-6) by encapsulated cells. Altogether, this injectable formulation has the potential to be used as a cell delivery vehicle for various applications in regenerative medicine.

Nanocomposite gold-silk nanofibers.

Cell-biomaterial interactions can be controlled by modifying the surface chemistry or nanotopography of the material, to induce cell proliferation and differentiation if desired. Here we combine both approaches in forming silk nanofibers (SNFs) containing gold nanoparticles (AuNPs) and subsequently chemically modifying the fibers. Silk fibroin mixed with gold seed nanoparticles was electrospun to form SNFs doped with gold seed nanoparticles (SNF(seed)). Following gold reduction, there was a 2-fold increase in particle diameter confirmed by the appearance of a strong absorption peak at 525 nm. AuNPs were dispersed throughout the AuNP-doped silk nanofibers (SNFs(Au)). The Young's modulus of the SNFs(Au) was almost 70% higher than that of SNFs. SNFs(Au) were modified with the arginine-glycine-aspartic acid (RGD) peptide. Human mesenchymal stem cells that were cultured on RGD-modified SNF(Au) had a more than 2-fold larger cell area compared to the cells cultured on bare SNFs; SNF(Au) also increased cell size. This approach may be used to alter the cell-material interface in tissue engineering and other applications.

Raman spectroscopic investigation of peptide-glycosaminoglycan interactions.

Protein-glycosaminoglycan (GAG) interactions play a central role in tissue engineering and drug delivery. A rapid and efficacious method for screening these interactions is essential. Raman spectroscopy was used to identify chemical interactions and conformational changes occurring upon binding between a synthetic peptide (QRRFMQYSARRF) and two glycosaminoglycans (GAGs), heparin and chondroitin 6-sulfate (C6S). The results identify three main chemical groups that are involved in the binding of the synthetic peptide with heparin and C6S. Tyrosine formed hydrogen bonds with the GAGs via its hydroxyl group. The amide I band demonstrated substantial shifts in Raman wavenumbers when bound to heparin and C6S (Deltaomega=-10.2+/-0.7 cm(-1) and Deltaomega=-11.9+/-0.3 cm(-1), respectively), suggesting that the peptide underwent planar conformational changes after binding occurred. Upon binding to the peptide, the sulfate peak of heparin displayed a substantially greater shift in the Raman wavenumber (-7.5+/-0.5 cm(-1)) than that of C6S (-2.6+/-0.5 cm(-1)). The greater amide I and sulfate band shifts seen during peptide-heparin interactions are indicative of a stronger association compared to that between the peptide and C6S. This observation was confirmed by capillary electrophoresis, which demonstrated a lower dissociation constant (KD) between the peptide and heparin (KD of 19.2+/-3.3 microM) than between the peptide and C6S (26.7+/-2.5 microM). We conclude that the shift in the Raman wavenumbers of amide I and sulfate groups can be used for high-throughput screening of interaction affinities between libraries of peptides and GAGs.

Interplay between covalent and physical interactions within environment sensitive hydrogels.

A systematic study is carried out to understand how physical and covalent crosslinks affect the mechanical properties of an eight-arm poly(ethyleneglycol)-based hydrogel. Heparin and heparin-binding peptide are used as a physical crosslinker, and an enzymatically cleavable peptide with a cysteine on each end serves as a covalent crosslinker. While physical crosslinks alone do not induce gelation due to the low binding affinity between heparin and heparin-binding peptide, the addition of covalent crosslinks leads to gel formation. Strikingly, the addition of the covalent crosslinks not only leads to gel formation, but also enhances the contribution from the physical crosslinks to the overall shear moduli, which are negligible in the absence of covalent crosslinks. The gels, which contain both covalent and physical crosslinks, are able to reversibly respond to external stimuli such as temperature and oscillatory shear unlike the purely covalent gel in which the moduli remain largely insensitive to such stimuli. Two explanations are provided for this striking phenomenon. First, the addition of covalent crosslinks increased the stress relaxation time of the gel enabling the physical interactions to contribute to the moduli. This is contrasted to the case of physically crosslinked material, which relaxes the stress too quickly, preventing the physical interactions from contributing to the low frequency moduli. Second, it is believed that the physical interactions within the covalent network were further enhanced by "macromolecular confinement", which favors the formation of compact conformational structures in the confined space. Quartz crystal microbalance (QCM) was used to measure the dissociation constant (K(d)) within the hydrogel and to demonstrate that the binding between heparin and heparin-binding peptide is stronger within the gel compared to that within the solution phase. Because extracellular matrix (ECM) contains both covalent and physical interactions between its constituents, and the mechanical properties of the ECM are important factors to control cell functions, the findings of this research may have important implications in various fields of tissue engineering and cell biology.

A novel assay to probe heparin-peptide interactions using pentapeptide-stabilized gold nanoparticles.

In this article, we present a novel assay to probe the interactions between heparin and heparin-binding peptides based on CALNN pentapeptide-stabilized gold nanoparticles. This assay relies on rapid aggregation of gold nanoparticles and dramatic retardation in the presence of a large excess of heparin due to the binding of peptides to heparin. Using this method, the dissociation constant ( K d) and melting temperature ( T m) of three different peptides against heparin were determined. The results from capillary electrophoresis demonstrated that K d values measured by this method were comparatively accurate. It was found that the peptide with the lowest K d did not have the highest T m. Structural analysis by circular dichroism was performed to explain this phenomenon. A comparison with the results from affinity chromatography indicates that electrostatic interactions only are not the major determinant of the affinity between heparin and peptide, but other interactions such as hydrogen-bonding and hydrophobic interactions may play important roles in the overall interactions. This novel assay is inexpensive, label-free, and easy to implement in the laboratories, does not suffer precipitation of the heparin-peptide complex or their conformational changes caused by surface immobilization, and is expected to be a useful complement to other existing methods.