Characteristics of Serum Exosomes after Burn Injury and Dermal Fibroblast Regulation by Exosomes In Vitro.

(1) Background: Exosomes (EXOs) have been considered a new target thought to be involved in and treat wound healing. More research is needed to fully understand EXO characteristics and the mechanisms of EXO-mediated wound healing, especially wound healing after burn injury. (2) Methods: All EXOs were isolated from 85 serum samples of 29 burn patients and 13 healthy individuals. We characterized the EXOs for morphology and density, serum concentration, protein level, marker expression, size distribution, and cytokine content. After a confirmation of EXO uptake by dermal fibroblasts, we also explored the functional regulation of primary human normal skin and hypertrophic scar fibroblast cell lines by the EXOs in vitro, including cell proliferation and apoptosis. (3) Results: EXOs dynamically changed their morphology, density, size, and cytokine level during wound healing in burn patients, which were correlated with burn severity and the stages of wound healing. EXOs both from burn patients and healthy individuals stimulated dermal fibroblast proliferation and apoptosis. (4) Conclusions: EXO features may be important signals that influence wound healing after burn injury; however, to understand the mechanisms by which EXOs regulates the fibroblasts in healing wounds, further studies will be required.

Substance P analogs devoid of key residues fail to activate human mast cells via MRGPRX2.

Mast cells play an important role in disease pathogenesis by secreting immunomodulatory molecules. Mast cells are primarily activated by the crosslinking of their high affinity IgE receptors (FcεRI) by antigen bound immunoglobulin (Ig)E antibody complexes. However, mast cells can also be activated by the mas related G protein-coupled receptor X2 (MRGPRX2), in response to a range of cationic secretagogues, such as substance P (SP), which is associated with pseudo-allergic reactions. We have previously reported that the in vitro activation of mouse mast cells by basic secretagogues is mediated by the mouse orthologue of the human MRGPRX2, MRGPRB2. To further elucidate the mechanism of MRGPRX2 activation, we studied the time-dependent internalization of MRGPRX2 by human mast cells (LAD2) upon stimulation with the neuropeptide SP. In addition, we performed computational studies to identify the intermolecular forces that facilitate ligand-MRGPRX2 interaction using SP. The computational predictions were tested experimentally by activating LAD2 with SP analogs, which were missing key amino acid residues. Our data suggest that mast cell activation by SP causes internalization of MRGPRX2 within 1 min of stimulation. Hydrogen bonds (h-bonds) and salt bridges govern the biding of SP to MRGPRX2. Arg1 and Lys3 in SP are key residues that are involved in both h-bonding and salt bridge formations with Glu164 and Asp184 of MRGPRX2, respectively. In accordance, SP analogs devoid of key residues (SP1 and SP2) failed to activate MRGPRX2 degranulation. However, both SP1 and SP2 caused a comparable release of chemokine CCL2. Further, SP analogs SP1, SP2 and SP4 did not activate tumor necrosis factor (TNF) production. We further show that SP1 and SP2 limit the activity of SP on mast cells. The results provide important mechanistic insight into the events that result in mast cell activation through MRGPRX2 and highlight the important physiochemical characteristics of a peptide ligand that facilitates ligand-MRGPRX2 interactions. The results are important in understanding activation through MRGPRX2, and the intermolecular forces that govern ligand-MRGPRX2 interaction. The elucidation of important physiochemical properties within a ligand that are needed for receptor interaction will aid in designing novel therapeutics and antagonists for MRGPRX2.

Targeting active sites of inflammation using inherent properties of tissue-resident mast cells.

Mast cells play a pivotal role in initiating and directing host's immune response. They reside in tissues that primarily interface with the external environment. Activated mast cells respond to environmental cues throughout acute and chronic inflammation through releasing immune mediators via rapid degranulation, or long-term de novo expression. Mast cell activation results in the rapid release of a variety of unique enzymes and reactive oxygen species. Furthermore, the increased density of mast cell unique receptors like mas related G protein-coupled receptor X2 also characterizes the inflamed tissues. The presence of these molecules (either released mediators or surface receptors) are particular to the sites of active inflammation, and are a result of mast cell activation. Herein, the molecular design principles for capitalizing on these novel mast cell properties is discussed with the goal of manipulating localized inflammation. STATEMENT OF SIGNIFICANCE: Mast cells are immune regulating cells that play a crucial role in both innate and adaptive immune responses. The activation of mast cells causes the release of multiple unique profiles of biomolecules, which are specific to both tissue and disease. These unique characteristics are tightly regulated and afford a localized stimulus for targeting inflammatory diseases. Herein, these important mast cell attributes are discussed in the frame of highlighting strategies for the design of bioresponsive functional materials to target regions of inflammations.

Screening Peptides that Activate MRGPRX2 using Engineered HEK Cells.

Identifying ligands specific to therapeutically significant cell receptors is crucial for many applications, including the design and development of new therapeutics. Mas related G-protein receptor-X2 (MRGPRX2) is an important receptor that regulates mast cell activation and, thus, directs the general immune response. Numerous ligands for MRGPRX2 have been identified and include endogenous peptides like PAMPs, defensins, LL-37 and other protein fragments (i.e., degraded albumin). Further identification of MRGPRX2 specific ligands requires the screening of a large number of peptides (i.e., peptide library); however, mast cells are difficult and expensive to maintain in vitro and, therefore, not economical to use for screening large numbers of molecules. The present paper demonstrates a method to design, develop, and screen a library of small peptide molecules using MRGPRX2 expressing HEK cells. This cell line is relatively easy and inexpensive to maintain and can be used for in vitro high-throughput analysis. A calcium sensitive Fura-2 fluorescent dye to mark intracellular calcium flux upon activation was used to monitor the activation. The ratio of fluorescence intensity of Fura-2 at 510 nm against excitation wavelengths of 340 and 380 nm was used to calculate calcium concentration. The peptide library used to verify this system was based on the endogenous proadrenomedullin N-terminal 12 (PAMP-12) secretagogue, which is known to bind MRGPRX2 with high specificity and affinity. Subsequent peptides were generated through amino acid truncation and alanine scanning techniques applied to PAMP-12. The method described here is simple and inexpensive yet robust for screening a large library of compounds to identify binding domains and other important parameters that play an important role in receptor activation.

Identification of short peptide sequences that activate human mast cells via Mas-related G-protein coupled receptor member X2.

Peptide based therapeutics are desirable owing to their high biological specificity. However, a number of these fail in clinical testing due to an adverse inflammatory response. Mast cells play a key role in directing the host response to drugs and related products. Although the role of FcεRI receptor is well known, Mas-related G-protein coupled receptor X2 (MRGPRX2) binding of endogenous peptides, and drugs will activate mast cells independent of FcεRI. Identifying peptides that activate mast cells through MRGPRX2, and their respective activation potency, can be used to reduce the failure rate of peptide therapeutics at clinical trial. Moreover, it will allow for peptide design where mast cell activation is actually desired. It was found that FRKKW and WNKWAL are two motifs that activate human LAD2 cells similar to PAMP-12 controls. Peptide activators of MRGPRX2 could be reduced to Xa-(Y)(n ≥ 3)-Xb where: Xa is an aromatic residue; Xb is a hydrophobic residue; and Y is a minimum 3 residue long sequence, containing a minimum of one positively charged residue with the remainder being uncharged residues. Artificial peptides WKKKW and FKKKF were constructed to test this structural functionality and were similar to PAMP-12 controls. Peptides with different activation potentials were found where FRKKW = WKKKW = FKKKF > PAMP-12 = WNKWAL > YKKKY > FRKKANKWALSR = FRKKWNKAALSR > KWKWK > FRKK = WNKWA > KYKYK > NKWALSR = YKKY = WNK. These sequences should be considered when designing peptide-based therapeutics. STATEMENT OF SIGNIFICANCE: Mast cells release immune regulating molecules upon activation that direct host's immune response. MRGPRX2 receptor provides an alternate pathway for mast cell activation that is independent of FcεRI receptor. It is thought that mast cell activation through MRGPRX2 plays a critical role in high failure rates of drugs in clinical trials. Identifying peptide sequences that activate mast cells through MRGPRX2 can serve two important purposes, namely, sequences to avoid when designing peptide therapeutics, and artificial peptides with different activation potentials for mast cells. Herein, we have identified a general amino acid sequence that induces mast cell activation through MRGPRX2. Furthermore, by modulating the identified sequence, artificial peptides have been designed which activate mast cells by varying degrees for therapeutic applications.

Developing Hybrid Polymer Scaffolds Using Peptide Modified Biopolymers for Cell Implantation.

Polymeric scaffolds containing biomimics offer exciting therapies with broad potential impact for cellular therapies and thereby potentially improve success rates. Here we report the designing and fabrication of a hybrid scaffold that can prevent a foreign body reaction and maintain cell viability. A biodegradable acrylic based cross-linkable polycaprolactone based polymer was developed and using a multihead electrospinning station to fabricate hybrid scaffolds. This consists of cell growth factor mimics and factors to prevent a foreign body reaction. Transplantation studies were performed subcutaneously and in epididymal fat pad of immuno-competent Balb/c mice and immuno-suppressed B6 Rag1 mice and we demonstrated extensive neo-vascularization and maintenance of islet cell viability in subcutaneously implanted neonatal porcine islet cells for up to 20 weeks of post-transplant. This novel approach for cell transplantation can improve the revascularization and allow the integration of bioactive molecules such as cell adhesion molecules, growth factors, etc.

Engineering a multi-biofunctional composite using poly(ethylenimine) decorated graphene oxide for bone tissue regeneration.

Toward preparing strong multi-biofunctional materials, poly(ethylenimine) (PEI) conjugated graphene oxide (GO_PEI) was synthesized using poly(acrylic acid) (PAA) as a spacer and incorporated in poly(ε-caprolactone) (PCL) at different fractions. GO_PEI significantly promoted the proliferation and formation of focal adhesions in human mesenchymal stem cells (hMSCs) on PCL. GO_PEI was highly potent in inducing stem cell osteogenesis leading to near doubling of alkaline phosphatase expression and mineralization over neat PCL with 5% filler content and was ≈50% better than GO. Remarkably, 5% GO_PEI was as potent as soluble osteoinductive factors. Increased adsorption of osteogenic factors due to the amine and oxygen containing functional groups on GO_PEI augment stem cell differentiation. GO_PEI was also highly efficient in imparting bactericidal activity with 85% reduction in counts of E. coli colonies compared to neat PCL at 5% filler content and was more than twice as efficient as GO. This may be attributed to the synergistic effect of the sharp edges of the particles along with the presence of the different chemical moieties. Thus, GO_PEI based polymer composites can be utilized to prepare bioactive resorbable biomaterials as an alternative to using labile biomolecules for fabricating orthopedic devices for fracture fixation and tissue engineering.

3D scaffold alters cellular response to graphene in a polymer composite for orthopedic applications.

Graphene-based polymer nanocomposites are being studied for biomedical applications. Polymer nanocomposites can be processed differently to generate planar two-dimensional (2D) substrates and porous three-dimensional (3D) scaffolds. The objective of this work was to investigate potential differences in biological response to graphene in polymer composites in the form of 2D substrates and 3D scaffolds. Polycaprolactone (PCL) nanocomposites were prepared by incorporating 1% of graphene oxide (GO) and reduced graphene oxide (RGO). GO increased modulus and strength of PCL by 44 and 22% respectively, whereas RGO increased modulus and strength by 22 and 16%, respectively. RGO increased the water contact angle of PCL from 81° to 87° whereas GO decreased it to 77°. In 2D, osteoblast proliferated 15% more on GO composites than on PCL whereas RGO composite showed 17% decrease in cell proliferation, which may be attributed to differences in water wettability. In 3D, initial cell proliferation was markedly retarded in both GO (36% lower) and RGO (55% lower) composites owing to increased roughness due to the presence of the protruding nanoparticles. Cells organized into aggregates in 3D in contrast to spread and randomly distributed cells on 2D discs due to the macro-porous architecture of the scaffolds. Increased cell-cell contact and altered cellular morphology led to significantly higher mineralization in 3D. This study demonstrates that the cellular response to nanoparticles in composites can change markedly by varying the processing route and has implications for designing orthopedic implants such as resorbable fracture fixation devices and tissue scaffolds using such nanocomposites.

Chemical functionalization of graphene to augment stem cell osteogenesis and inhibit biofilm formation on polymer composites for orthopedic applications.

Toward designing the next generation of resorbable biomaterials for orthopedic applications, we studied poly(ε-caprolactone) (PCL) composites containing graphene. The role, if any, of the functionalization of graphene on mechanical properties, stem cell response, and biofilm formation was systematically evaluated. PCL composites of graphene oxide (GO), reduced GO (RGO), and amine-functionalized GO (AGO) were prepared at different filler contents (1%, 3%, and 5%). Although the addition of the nanoparticles to PCL markedly increased the storage modulus, this increase was largest for GO followed by AGO and RGO. In vitro cell studies revealed that the AGO and GO particles significantly increased human mesenchymal stem cell proliferation. AGO was most effective in augmenting stem cell osteogenesis leading to mineralization. Bacterial studies revealed that interaction with functionalized GO induced bacterial cell death because of membrane damage, which was further accentuated by amine groups in AGO. As a result, AGO composites were best at inhibiting biofilm formation. The synergistic effect of oxygen containing functional groups and amine groups on AGO imparts the optimal combination of improved modulus, favorable stem cell response, and biofilm inhibition in AGO-reinforced composites desired for orthopedic applications. This work elucidates the importance of chemical functionalization of graphene in polymer composites for biomedical applications.

Engineering a nanostructured "super surface" with superhydrophobic and superkilling properties.

We present a nanostructured "super surface" fabricated using a simple recipe based on deep reactive ion etching of a silicon wafer. The topography of the surface is inspired by the surface topographical features of dragonfly wings. The super surface is comprised of nanopillars 4 µm in height and 220 nm in diameter with random inter-pillar spacing. The surface exhibited superhydrophobicity with a static water contact angle of 154.0° and contact angle hysteresis of 8.3°. Bacterial studies revealed the bactericidal property of the surface against both gram negative (Escherichia coli) and gram positive (Staphylococcus aureus) strains through mechanical rupture of the cells by the sharp nanopillars. The cell viability on these nanostructured surfaces was nearly six-fold lower than on the unmodified silicon wafer. The nanostructured surface also killed mammalian cells (mouse osteoblasts) through mechanical rupture of the cell membrane. Thus, such nanostructured super surfaces could find applications for designing self-cleaning and anti-bacterial surfaces in diverse applications such as microfluidics, surgical instruments, pipelines and food packaging.