Self-Assembling Peptide-Carbon Nanotube Dispersions and Hydrogels for Tissue Engineering and Biosensor Applications

Carbon nanotubes (CNTs) are attractive functional materials for use in a broad range of fields due to their unique mechanical and electrical properties. However, their hydrophobic nature is a major problem for some of these applications. Several approaches such as dispersing them in organic solutions and covalently or non-covalently modifying them have been developed to make CNTs usable for desired applications. Since organic solutions can be problematic for bio-applications and covalent modification can introduce defects into the CNT structure (responsible for its unique properties), the approach of making non-covalent modification is more promising. Different types of polymers and surfactants have been used so far in this way. In the past two decades, self-assembling peptides have emerged as promising functional nanomaterials capable of use for different bio-applications. Employing biocompatible self-assembling peptides for CNT dispersion not only removes the first obstacle to use CNTs in solution, but also can result in a new class of hybrid nanomaterials benefitting from the synergistic effects of peptides and CNTs. To the best of our knowledge, this is the first work reporting the dispersion of CNTs using β-sheet-forming self-assembling ionic-complementary peptides. Also this is the first study on the application of peptide-CNT hybrid dispersions and hydrogels for biosensor and tissue engineering applications. This thesis focuses on the modification of CNTs with self-assembling peptides, characterization of the resulting hybrid dispersions and their application for biosensor development and scaffolding for tissue engineering and cancer spheroid studies. In particular, the study includes the following topics: (i) characterization of the dispersions of multi-walled carbon nanotubes (MWNTs) modified with EFK16-II peptide, (ii) AFM characterization of dispersions of single-walled carbon nanotubes (SWNTs) modified with EFK8 peptide, (iii) formation of hybrid EFK8-SWNT hydrogels, (iv) application of the hybrid EFK8-SWNT dispersion in electrode modification and design of a hemoglobin biosensor, and (v) application of the EFK8 and EFK8-SWNT hydrogels as scaffolds for tissue engineering and 3D cancer cell spheroid formation. First, we have shown that by non-covalently modifying MWNTs with the ionic-complementary peptide EFK16-II, very stable dispersions of MWNTs can be formed due to the electrostatic repulsion between self-assembled peptides on the MWNTs. Zeta potential and DLS measurements indicated that as the pH diverges from the isoelectric point of ~ 6.7 for EFK16-II, the repulsion between the particles increases and their resulting sizes decrease. AFM, SEM and TEM studies revealed a uniform distribution of individual modified MWNTs. Finally, tissue culture plates treated with these hybrid dispersions were found to have enough biocompatibility for cell attachment and growth. In the next step, the peptide EFK8, a shorter version of EFK16-II, was used to disperse SWNTs in water. Scanning probe microscopy (SPM) techniques based on nano-mechanical measurements and electric force microscopy (EFM) were used to more closely examine the structure formed between nanotubes and peptides. The SPM images reveal a structure consistent with EFK8 fibers wrapping around SWNTs and rendering their outer surfaces hydrophilic which enables their dispersion in water. Also it was shown that the hybrid dispersions can form uniform composite EFK8-SWNT hydrogels upon adding solutions containing ≥1mM monovalent salts. In the third part of the study, EFK8 and EFK8-SWNT hybrid hydrogels were prepared and used to culture NIH-3T3 fibroblast and A549 lung cancer cells. The effect of the presence of SWNTs in the peptide hydrogel on NIH-3T3 cells behavior cultured on hydrogels was first investigated. Inverted light and confocal microscopy images showed that although cells grow they tend to maintain spherical morphology and form colonies on the EFK8 hydrogel. The presence of SWNTs significantly improved cell behavior so that they exhibited a stretched morphology, spread individually and homogeneously over the surface and proliferated faster. In addition, the cells were observed to migrate into the hydrogel after being seeded on top of the hydrogel. Micro-indentation tests showed that increasing EFK8 solution concentration led to an increase in the hydrogel compressive modulus, whereas the presence of SWNTs did not have any effect in this case. So the beneficial effect of SWNTs on cell behavior cannot be attributed to mechanical property modification and is probably due to their providing locations for cell anchorage that facilitate attachment, spreading and migration. The cells encapsulated in both hydrogels showed the same behavior as in 2D environments (i.e., forming colonies on EFK8 and spreading individually on the hybrid hydrogel). In the second part of this study, the potential of EFK8 hydrogels for spheroid formation of cancer cells was explored. These cancer cell spheroids can be used as models for real tumors, to carry out drug screening in 3D cell cultures and to investigate the effect of the microenvironment on tumor progression and metastasis. It was observed that cells formed spheroids on EFK8 hydrogels at normal peptide concentrations, but exhibited a more stretched morphology and migratory phenotype when seeded on the stiffer hydrogel. The cells also adopted a stretched morphology with higher migration when seeded on the EFK8-SWNT hydrogels. Again this behavior can be attributed to the effect of SWNTs to facilitate cell adhesion and migration. This effect can be used to study another effect of the microenvironment, namely cell-binding motifs, on tumor progression and metastasis. In the last step of the study, the application of the hybrid EFK8-SWNT dispersion was investigated for immobilization and direct electrochemistry of hemoglobin (Hb) on a glassy carbon electrode (GCE) as well as the efficacy of this platform for making a hydrogen peroxide (H2O2) biosensor. Cyclic voltammetry (CV) and electrochemical impedance spectroscopy (EIS) experiments showed that the presence of SWNTs in the modifying peptide layer on GCE significantly enhances the electrochemical response of the electrode. Furthermore, this response was further increased as more layers of the EFK8-SWNT dispersion were applied. The next step was to immobilize hemoglobin on the electrode by a casting method. The effectiveness of this approach was confirmed by CV and EIS experiments which showed that immobilized Hb retained its bio-catalytic activity for Fe ions in Hb chains and could form the basis of a mediatorless H2O2 biosensor. Overall, by expanding the functionalities of both CNTs and self-assembling peptides, this work has introduced a new hybrid nanomaterial for bio-applications, especially biosensors, 3D cell cultures and tissue engineering.4 month

Peptide and peptide-carbon nanotube hydrogels as scaffolds for tissue & 3D tumor engineering

The final publication is available at Elsevier via http://dx.doi.org/10.1016/j.actbio.2017.12.012 © 2018. This manuscript version is made available under the CC-BY-NC-ND 4.0 license https://creativecommons.org/licenses/by-nc-nd/4.0/The use of hybrid self-assembling peptide (EFK8)-carbon nanotube (SWNT) hydrogels for tissue engineering and in vitro 3D cancer spheroid formation is reported. These hybrid hydrogels are shown to enhance the attachment, spreading, proliferation and movement of NIH-3T3 cells relative to that observed using EFK8-only hydrogels. After five days, ∼30% more cells are counted when the hydrogel contains SWNTs. Also, 3D encapsulation of these cells when injected in hydrogels does not adversely affect their behavior. Compressive modulus measurements and microscopic examination suggest that SWNTs have this beneficial effect by providing sites for cell anchorage, spreading and movement rather than by increasing hydrogel stiffness. This shows that the cells have a particular interaction with SWNTs not shared with EFK8 nanofibers despite a similar morphology. The effect of EFK8 and EFK8-SWNT hydrogels on A549 lung cancer cell behavior is also investigated. Increasing stiffness of EFK8-only hydrogels from about 44 Pa to 104 Pa promotes a change in A549 morphology from spheroidal to a stretched one similar to migratory phenotype. EFK8-SWNT hydrogels also promote a stretched morphology, but at lower stiffness. These results are discussed in terms of the roles of both microenvironment stiffness and cell-scaffold adhesion in cancer cell invasion. Overall, this study demonstrates that applications of peptide hydrogels in vitro can be expanded by incorporating SWNTs into their structure which further provides insight into cell-biomaterial interactions. Statement of significance For the first time we used hybrid self-assembling peptide-carbon nanotube hybrid hydrogels (that we have recently introduced briefly in the “Carbon” journal in 2014) for tissue engineering and 3D tumor engineering. We showed the potential of these hybrid hydrogels to enhance the efficiency of the peptide hydrogels for tissue engineering application in terms of cell behavior (cell attachment, spreading and migration). This opens up new rooms for the peptide hydrogels and can expand their applications. Also our system (peptide and peptide-CNT hydrogels) was used for cancer cell spheroid formation showing the effect of both tumor microenvironment stiffness and cell-scaffold adhesion on cancer cell invasion. This was only possible based on the presence of CNTs in the hydrogel while the stiffness kept constant. Finally it should be noted that these hybrid hydrogels expand applications of peptide hydrogels through enhancing their capabilities and/or adding new properties to them.Natural Sciences and Engineering Research Council of Canada (NSERC)Canada Foundation for Innovation (CFI)Canada Research Chairs (CRC) progra

Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing

Abstract Background Compromised wound healing has become a global public health challenge which presents a significant psychological, financial, and emotional burden on patients and physicians. We recently reported that acellular gelatinous Wharton’s jelly of the human umbilical cord enhances skin wound healing in vitro and in vivo in a murine model; however, the key player in the jelly which enhances wound healing is still unknown. Methods We performed mass spectrometry on acellular gelatinous Wharton’s jelly to elucidate the chemical structures of the molecules. Using an ultracentrifugation protocol, we isolated exosomes and treated fibroblasts with these exosomes to assess their proliferation and migration. Mice were subjected to a full-thickness skin biopsy experiment and treated with either control vehicle or vehicle containing exosomes. Isolated exosomes were subjected to further mass spectrometry analysis to determine their cargo. Results Subjecting the acellular gelatinous Wharton’s jelly to proteomics approaches, we detected a large amount of proteins that are characteristic of exosomes. Here, we show that the exosomes isolated from the acellular gelatinous Wharton’s jelly enhance cell viability and cell migration in vitro and enhance skin wound healing in the punch biopsy wound model in mice. Mass spectrometry analysis revealed that exosomes of Wharton’s jelly umbilical cord contain a large amount of alpha-2-macroglobulin, a protein which mimics the effect of acellular gelatinous Wharton’s jelly exosomes on wound healing. Conclusions Exosomes are being enriched in the native niche of the umbilical cord and can enhance wound healing in vivo through their cargo. Exosomes from the acellular gelatinous Wharton’s jelly and the cargo protein alpha-2-macroglobulin have tremendous potential as a noncellular, off-the-shelf therapeutic modality for wound healing

Additional file 1: of Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing

Figure S1. Scanning electron microscopy (SEM) images of cord cross-section. SEM images show two zones. Zone 1 displays a section in between the umbilical vein and two arteries, and zone 2 displays a region closer to the umbilical vein. (A–E) Magnification of zone 1. (F–J) Magnification of zone 2. (TIF 10880 kb

Additional file 4: of Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing

Figure S3. Cell viability quantification for keratinocytes. Control medium-treated keratinocytes compared with exosome-treated and α2M (100 ng/ml)-treated keratinocytes. *p < 0.05, ***p < 0.001. N = 6 for control and exosomes; N = 12 for α2M. (TIF 4635 kb

Additional file 2: of Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing

Figure S2. Schematic representation of the exosome isolation protocol. (TIF 3668 kb

Additional file 3: of Exosomes from acellular Wharton’s jelly of the human umbilical cord promotes skin wound healing

Video S1. Time-lapse imaging showing that exosomes concentrate to the cell periphery and, by 3 h, disappear as depicted by a decrease in red fluorescence. (WMV 32791 kb

In vitro corrosion and biocompatibility behavior of CoCrMo alloy manufactured by laser powder bed fusion parallel and perpendicular to the build direction

Biomedical cobalt-chromium-molybdenum alloys (CoCrMo) are frequently used for orthopedic implant and dental materials exposed to mechanical stressors, such as wear and cyclic load. Due to the high demand for customizable implant shapes, these alloys are increasingly manufactured by additive manufacturing methods such as laser powder bed fusion (LPBF). LPBF results in different microstructures and surface roughness as a function of the building direction. This study investigated the corrosion resistance, bioactivity, biocompatibility, and microstructure of LPBF CoCrMo (low carbon content, heat-treated) in the XY (perpendicular) and XZ (parallel) plane of the building direction for as-printed (as-received) and abraded surfaces. A distinct microstructure and different surface roughness were found for the XY and XZ planes. The as-received XY surface showed the lowest corrosion resistance but was still passive in phosphate-buffered saline (PBS, pH 7.4). As-received surfaces were less corrosion-resistant than abraded surfaces. All specimens exhibited lower corrosion resistance in PBS containing citric acid at pH 7.4 than in PBS and citric acid alone. As-received surfaces showed better hydroxyapatite precipitation and cell viability; however, all surfaces had satisfactory biocompatibility and bioactivity. This study showed that the building direction had a minor effect on the corrosion of LPBF CoCrMo