Enhanced osteogenesis of mesenchymal stem cells encapsulated in injectable microporous hydrogel.

Delivery of therapeutic stem cells to treat bone tissue damage is a promising strategy that faces many hurdles to clinical translation. Among them is the design of a delivery vehicle which promotes desired cell behavior for new bone formation. In this work, we describe the use of an injectable microporous hydrogel, made of crosslinked gelatin microgels, for the encapsulation and delivery of human mesenchymal stem cells (MSCs) and compared it to a traditional nonporous injectable hydrogel. MSCs encapsulated in the microporous hydrogel showed rapid cell spreading with direct cell-cell connections whereas the MSCs in the nonporous hydrogel were entrapped by the surrounding polymer mesh and isolated from each other. On a per-cell basis, encapsulation in microporous hydrogel induced a 4 × increase in alkaline phosphatase (ALP) activity and calcium mineral deposition in comparison to nonporous hydrogel, as measured by ALP and calcium assays, which indicates more robust osteogenic differentiation. RNA-seq confirmed the upregulation of the genes and pathways that are associated with cell spreading and cell-cell connections, as well as the osteogenesis in the microporous hydrogel. These results demonstrate that microgel-based injectable hydrogels can be useful tools for therapeutic cell delivery for bone tissue repair.

Enhanced Osteogenesis of Mesenchymal Stem Cells Encapsulated in Injectable Microporous Hydrogel.

Delivery of therapeutic stem cells to treat bone tissue damage is a promising strategy that faces many hurdles to clinical translation. Among them is the design of a delivery vehicle which promotes desired cell behavior for new bone formation. In this work, we describe the use of an injectable microporous hydrogel, made of crosslinked gelatin microgels, for the encapsulation and delivery of human mesenchymal stem cells (MSCs) and compared it to a traditional nonporous injectable hydrogel. MSCs encapsulated in the microporous hydrogel showed rapid cell spreading with direct cell-cell connections whereas the MSCs in the nonporous hydrogel were entrapped by the surrounding polymer mesh and isolated from each other. Microporous hydrogel induced more robust osteogenic differentiation of MSCs and calcium mineral deposition than the nonporous hydrogel confirmed by alkaline phosphatase (ALP) assay and calcium assay. RNA-seq confirmed the upregulation of the genes and pathways that are associated with cell spreading and cell-cell connections, as well as the osteogenesis in the microporous hydrogel. These results demonstrate that the microgel-based injectable hydrogels can be useful tools for therapeutic cell delivery for bone tissue repair.

Synthesis of biologically derived poly(pyrogallol) nanofibers for antibacterial applications.

Herein, we present the facile synthesis of poly(pyrogallol) biopolymers and their application as antibacterial agents. Pyrogallol is a class of phenolic compounds that can be found in various plants. Polymerization was performed by the auto-oxidation of pyrogallol under a hydrated condition. The microscopic image of poly(pyrogallol) shows a highly homogenous nanofibrous structure with a diameter of 100.3 ± 16.3 nm. Spectroscopic analysis by FT-IR spectroscopy, Raman spectroscopy, and XPS corroborated the formation of ether (C-O-C) bonds between the hydroxyl group and adjacent carbons of pyrogallol during polymerization. The FT-IR and XPS spectra also revealed redox-active gallol functional groups on poly(pyrogallol) nanofibers, which can be used to release free electrons and protons during oxidation followed by the generation of reactive oxygen species (ROS). The generated ROS from poly(pyrogallol) was used to inhibit the growth of bacteria, Escherichia coli, at a inhibition rates of 56.3 ± 9.7% and 95.5 ± 2.0% within 0.5 and 2 h, respectively. This finding suggests that poly(pyrogallol) can be used as a naturally occurring antibacterial agent for various biomedical and environmental applications.

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.

Matrix Metalloproteinase-Deactivating Contact Lens for Corneal Melting.

Corneal melting is an uncontrolled, excessive degradation of cellular and extracellular components of the cornea. This potential cause of corneal blindness is caused by excessive expression of zinc-dependent matrix metalloproteinases (MMPs) and has no satisfying cure as of now. Herein, we introduce a novel therapeutic hydrogel which can be made into a contact lens to slow down the progression of corneal melting by deactivating MMPs. The hydrogel backbone is comprised of poly(2-hydroxyetyl methacrylate) (pHEMA), a main material for commercial contact lenses, and dipicolylamine (DPA) which has high affinity and selectivity towards zinc ion. Due to the high affinity towards zinc ions, the DPA-conjugated pHEMA (pDPA-HEMA) hydrogel selectively removes zinc ions from a physiological buffer and deactivates MMP-1, MMP-2 and MMP-9 within 2 hours. pDPA-HEMA hydrogel also effectively prevents degradation of porcine corneas by collagenase A, a zinc-dependent protease, whereas the corneas completely degrades within 15 hours when incubated with pHEMA hydrogel. The presence of pDPA-HEMA hydrogel does not affect the viability of keratocytes and corneal epithelial cells. Unlike the conventional MMP inhibitors (MMPi), the pDPA-HEMA hydrogel minimizes the risk of serious non-specific side effects, and provides a method to slow down the progression of corneal melting and other related ocular diseases.

A multi-interpenetrating network (IPN) hydrogel with gelatin and silk fibroin.

A mechanically strong composite hydrogel was produced based on an interpenetrating network (IPN) between gelatin and silk fibroin. When two layers of the IPN were created, the resulting hydrogel exhibited much improved mechanical properties. This hydrogel is biodegradable and non-cytotoxic and allows for cell adhesion and proliferation on the surface.

Injectable Macroporous Hydrogel Formed by Enzymatic Cross-Linking of Gelatin Microgels.

Injectable hydrogels can be useful tools for facilitating wound healing since they conform to the irregular shapes of wounds, serving as a temporary matrix during the healing process. However, the lack of inherent pore structures of most injectable hydrogels prohibits desired interactions with the cells of the surrounding tissues limiting their clinical efficacy. Here, we introduce a simple, cost-effective and highly biofunctional injectable macroporous hydrogel made of gelatin microgels crosslinked by microbial transglutaminase (mTG). Pores are created by the interstitial space among the microgels. A water-in-oil emulsion technique was used to create gelatin microgels of an average size of 250µm in diameter. When crosslinked with mTG, the microgels adhered to each other to form a bulk hydrogel with inherent pores large enough for cell migration. The viscoelastic properties of the porous hydrogel were similar to those of nonporous gelatin hydrogel made by adding mTG to a homogeneous gelatin solution. The porous hydrogel supported higher cellular proliferation of human dermal fibroblasts (hDFs) than the nonporous hydrogel over two weeks, and allowed the migration of hDFs into the pores. Conversely, the hDFs were unable to permeate the surface of the nonporous hydrogel. To demonstrate its potential use in wound healing, the gelatin microgels were injected with mTG into a cut out section of an excised porcine cornea. Due to the action of mTG, the porous hydrogel stably adhered to the cornea tissue for two weeks. Confocal images showed that a large number of cells from the cornea tissue migrated into the interstitial space of the porous hydrogel. The porous hydrogel was also used for the controlled release of platelet-derived growth factor (PDGF), increasing the proliferation of hDFs compared to the nonporous hydrogel. This gelatin microgel-based porous hydrogel will be a useful tool for wound healing and tissue engineering.

Gelatin Hydrogel Combined with Polydopamine Coating to Enhance Tissue Integration of Medical Implants.

Soft tissue integration of medical implants is important to prevent bacterial infection and implant failure. A bioadhesive that forms firm binding between the implant and the surrounding tissue and facilitates the wound-healing process will be a great tool to establish the desired tissue-implant integration. In this project, we introduce a novel method that can be used to enhance integration between any implant material and any tissue using an enzyme-crosslinked gelatin hydrogel combined with polydopamine (PDA) coating. PDA coating was shown to enhance the binding between the gelatin hydrogel and three model implant materials - aluminum, poly(methyl methacrylate) (PMMA) and titanium. When combined with the gelatin hydrogel, pig cornea tissue adhered more strongly to the PDA coated surfaces than to the uncoated surfaces. The enzyme-crosslinked gelatin hydrogel was non-cytotoxic to human dermal fibroblasts and it also allowed the cells to adhere and proliferate. Altogether, the results indicate that the combination of PDA coating with gelatin hydrogel can be used to enhance the integration of various medical implants.

Titanium Coating of the Boston Keratoprosthesis.

PURPOSE: We tested the feasibility of using titanium to enhance adhesion of the Boston Keratoprosthesis (B-KPro), ultimately to decrease the risk of implant-associated complications. METHODS: Cylindrical rods were made of poly(methyl methacrylate) (PMMA), PMMA coated with titanium dioxide (TiO2) over a layer of polydopamine (PMMATiO2), smooth (Ti) and sandblasted (TiSB) titanium, and titanium treated with oxygen plasma (Tiox and TiSBox). Topography and surface chemistry were analyzed by scanning electron microscopy (SEM), atomic force microscopy (AFM), and X-ray photoelectron spectroscopy (XPS). Adhesion force between rods and porcine corneas was measured ex vivo. Titanium sleeves, smooth and sandblasted, were inserted around the stem of the B-KPro and implanted in rabbits. Tissue adhesion to the stem was assessed and compared to an unmodified B-Kpro after 1 month. RESULTS: X-ray photoelectron spectroscopy demonstrated successful deposition of TiO2 on polydopamine-coated PMMA. Oxygen plasma treatment did not change the XPS spectra of titanium rods (Ti and TiSB), although it increased their hydrophilicity. The materials did not show cell toxicity. After 14 days of incubation, PMMATiO2, smooth titanium treated with oxygen plasma (Tiox), and sandblasted titanium rods (TiSB, TiSBox) showed significantly higher adhesion forces than PMMA ex vivo. In vivo, the use of a TiSB sleeve around the stem of the B-KPro induced a significant increase in tissue adhesion compared to a Ti sleeve or bare PMMA. CONCLUSIONS: Sandblasted titanium sleeves greatly enhanced adherence of the B-KPro to the rabbit cornea. This approach may improve adhesion with the donor cornea in humans as well. TRANSLATIONAL RELEVANCE: This approach may improve adhesion with donor corneas in humans.

Synthetic ligand-coated magnetic nanoparticles for microfluidic bacterial separation from blood.

Bacterial sepsis is a serious clinical condition that can lead to multiple organ dysfunction and death despite timely treatment with antibiotics and fluid resuscitation. We have developed an approach to clearing bacteria and endotoxin from the bloodstream, using magnetic nanoparticles (MNPs) modified with bis-Zn-DPA, a synthetic ligand that binds to both Gram-positive and Gram-negative bacteria. Magnetic microfluidic devices were used to remove MNPs bound to Escherichia coli , a Gram-negative bacterium commonly implicated in bacterial sepsis, from bovine whole blood at flows as high as 60 mL/h, resulting in almost 100% clearance. Such devices could be adapted to clear bacteria from septicemic patients.

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.

Hydroxyapatite for keratoprosthesis biointegration.

PURPOSE: Integration of keratoprosthesis with the surrounding cornea is very important in preventing bacterial invasion, which may cause ocular injury. Here the authors investigated whether hydroxyapatite (HAp) coating can improve keratoprosthesis (KPro) biointegration, using polymethyl methacrylate (PMMA)--the principal component of the Boston KPro--as a model polymer. METHODS: HAp coatings were induced on PMMA discs after treatment with concentrated NaOH and coating with poly-dopamine (PDA) or polydopamine and then with 11-mercaptoundecanoic acid (11-MUA). Coatings were characterized chemically (Fourier transform infrared spectroscopy [FTIR], energy dispersive X-ray spectroscopy [EDX]) and morphologically (SEM) and were used as substrates for keratocyte growth in vitro. Cylinders of coated PMMA were implanted in porcine corneas ex vivo for 2 weeks, and the force required to pull them out was measured. The inflammatory reaction to coated discs was assessed in the rabbit cornea in vivo. RESULTS: FTIR of the coatings showed absorption bands characteristic of phosphate groups, and EDX showed that the Ca/P ratios were close to those of HAp. By SEM, each method resulted in morphologically distinct HAp films; the 11-MUA group had the most uniform coating. The hydroxyapatite coatings caused comparable enhancement of keratocyte proliferation compared with unmodified PMMA surfaces. HAp coating significantly increased the force and work required to pull PMMA cylinders out of porcine corneas ex vivo. HAp coating of implants reduced the inflammatory response around the PMMA implants in vivo. CONCLUSIONS: These results are encouraging for the potential of HAp-coated surfaces for use in keratoprostheses.

Surface modification and drug delivery for biointegration.

Biointegration refers to the interconnection between a biomedical device and the recipient tissue. In many implant devices, the lack of proper biointegration can cause device failure and potentially serious medical problems. This review summarizes the recent progress in surface chemistry, drug delivery and antifouling methods to improve the biointegration of implants. Much progress has been made as our understanding of biological systems and material properties expands and as new technologies become available. This article addresses methods of enhancing biointegration by means of modifying implant surface chemistry and by drug-delivery approaches.

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.