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In order to achieve effective bone regeneration, the architectural/structural and biological environment must be considered. In this study, we investigated the influence of structure of extracellular matrix-mimicking electrospun poly ( l -lactic acid) (PLLA) fibrous scaffolds and incorporated growth factors (bone morphogenetic protein2 (BMP2) and platelet-derived growth factor (PDGF)) (GFs) on in vitro differentiation of human mesenchymal stem cells (hMSCs) cultured onto the scaffolds. Furthermore, Cargo GFs were first encapsulated into coacervate (Coa). Coa-coated nano-sized PLLA (Coa-nPLLA) exhibited relatively higher coating efficacy, than Coa-coated micro-sized PLLA (Coa-mPLLA) due to large mesh hole area of mPLLA. Consequently, a faster GF release pattern was observed in nPLLA groups. As a result of osteogenic differentiation of hMSC which cultured onto GFs loaded Coa-PLLA, the architectural cue of fiber diameters effectively modulated early osteogenic differentiation of hMSCs. Facilitated long-term differentiation of hMSCs could be achieved by synergistic effect of dual BMP2 and PDGF in nanofbrous environments.

Salmon fish farmers face remarkable problems in fish rearing and handling due to the spread of disease by infectious pancreatic necrosis virus (IPNV). Therefore, we developed a straightforward and sensitive technique to detect IPNV-based on recombinant human apoferritin heavy chain (hAFN-H) protein nanoparticles. In this study, the 24 subunits of the hAFN-H were genetically modified to express 6×His-tag and protein-G at their C-terminal site using Escherichia coli. We thus achieved a two-step signal amplifying strategy that utilizes a recombinant hAFN-H nanoprobe having a protein-G-binding site that targets the Fc region of monoclonal antibodies and a 6×His-tag that actively interacts with the functionalized Ni-NTA derivatives. In this study, we report a considerable advancement in magnetic bead-based detection systems that use Ni-NTA-Atto 550, reliably exhibiting detection limits of 1.02 TCID50/mL (50% tissue culture infective dose). Additionally, we propose a lateral flow chip-based detection method that uses the hAFN-H surface functionalized with 5 nm of the Ni-NTA-nanogold complex as a nanoprobe; the limit of detection towards IPNV was 0.88 TCID50/mL. The detection of IPNV by this recombinant hAFN-H nanoprobe was linear to virus titers in the range of 101-103 TCID50/mL.

Here we report optimization and scale-up of a biodegradable polycation, poly(ethylene argininylaspartate diglyceride) (PEAD) for the purpose of clinical translation. This polycation complexes heparin to form a coacervate. The resultant complex coacervate acts as a vehicle for controlled delivery of therapeutic proteins and through the bioactivity of heparin, greatly increases the half lives of many therapeutic proteins. In an effort to translate this drug delivery platform commercially and clinically, it is necessary to consider the scale of the synthesis, the environmental impact and the cost of the production. Therefore, we substituted the more expensive and hazardous solvents in the reactions, optimized the reaction parameters for a higher yield, scaled up the synthesis capacity up to hundreds of grams per batch and reduced the polymer cost by nearly 90%. The resultant PEAD bears more positive charges than the counterpart made using our previous method, and thus less polymer is needed to complex heparin for coacervation. The material demonstrated good cytocompatibility with human umbilical vein endothelial cells at concentrations up to 5 mg/ml, nearly 500-times more cytocompatible than the commercially resourced polyethyleneimine. Basic fibroblast growth factor was loaded in the coacervate with loading efficiency above 99%. The cargo steadily released approximately 1% over 37 days in vitro without an initial burst, indicating a highly stable coacervate for sustained release. This study paves the way for the translation of this delivery platform and may inform the scale-up of similar polymers.

Although there are numerous medical applications to recover damaged skin tissue, scarless wound healing is being extensively investigated to provide a better therapeutic outcome. The exogenous delivery of therapeutic growth factors (GFs) is one of the engineering strategies for skin regeneration. This study presents an exogenous GF delivery platform developed using coacervates (Coa), a tertiary complex of poly(ethylene argininyl aspartate diglyceride) (PEAD) polycation, heparin, and cargo GFs (i.e., transforming growth factor beta 3 (TGF-β3) and interleukin 10 (IL-10)). Coa encompasses the advantage of high biocompatibility, facile preparation, protection of cargo GFs, and sustained GF release. We therefore speculated that coacervate-mediated dual delivery of TGF-β3/IL-10 would exhibit synergistic effects for the reduction of scar formation during physiological wound healing. Our results indicate that the exogenous administration of dual GF via Coa enhances the proliferation and migration of skin-related cells. Gene expression profiles using RT-PCR revealed up-regulation of ECM formation at early stage of wound healing and down-regulation of scar-related genes at later stages. Furthermore, direct injection of the dual GF Coa into the edges of damaged skin in a rat skin wound defect model demonstrated accelerated wound closure and skin regeneration after 3 weeks. Histological evaluation and immunohistochemical staining also revealed enhanced formation of the epidermal layer along with facilitated angiogenesis following dual GF Coa delivery. Based on these results, we conclude that polycation-mediated Coa fabrication and exogenous dual GF delivery via the Coa platform effectively augments both the quantity and quality of regenerated skin tissues without scar formation. Statement of Significance This study was conducted to develop a simple administration platform for scarless skin regeneration using polycation-based coacervates with dual GFs. Both in vitro and in vivo studies were performed to confirm the therapeutic efficacy of this platform toward scarless wound healing. Our results demonstrate that the platform developed by us enhances the proliferation and migration of skin-related cells. Sequential modulation in various gene expression profiles suggests a balanced collagen-remodeling process by dual GFs. Furthermore, in vivo histological evaluation demonstrates that our technique enhances clear epidermis formation with less scab and thicker woven structure of collagen bundle, similar to that of a normal tissue. We propose that simple administration of dual GFs with Coa has the potential to be applied as a clinical approach for fundamental scarless skin regeneration.

Nonunion following bone fracture and segmental bone defects are challenging clinical conditions. To combat this clinical dilemma, development of new bone tissue engineering therapies using biocompatible materials to deliver bone growth factors is desirable. This aim of this study is to use a heparin/polycation coacervate sustained-release platform to compare 5 bone morphogenetic proteins (BMPs) for promoting bone defect healing in a critical sized calvarial defect model. The in vitro 3D osteogenic pellet cultures assays demonstrated that BMPs 2, 4, 6, 7 and 9 all enhanced mineralization in vitro compared to the control group. BMP2 resulted in higher mineralized volume than BMP4 and BMP6. All BMPs and the control group activated the pSMAD5 signaling pathway and expressed osterix (OSX). The binding of BMP2 with coacervate significantly increased the coacervate average particle size. BMP2, 4, 6, & 7 bound to coacervate significantly increased the Zeta potential of the coacervate while BMP9 binding showed insignificant increase. Furthermore, using a monolayer culture osteogenic assay, it was found that hMDSCs cultured in the coacervate BMP2 osteogenic medium expressed higher levels of RUNX2, OSX, ALP and COX-2 compared to the control and BMPs 4, 6, 7 & 9. Additionally, the coacervate complex can be loaded with up to 2 μg of BMP proteins for sustained release. In vivo, when BMPs were delivered using the coacervate sustained release system, BMP2 was identified to be the most potent BMP promoting bone regeneration and regenerated 10 times of new bone than BMPs 4, 6 & 9. BMP7 also stimulated robust bone regeneration when compared to BMPs 4, 6 & 9. The quality of the newly regenerated bone by all BMPs delivered by coacervate is equivalent to the host bone consisting of bone matrix and bone marrow with normal bone architecture. Although the defect was not completely healed at 6 weeks, coacervate sustain release BMPs, particularly BMP2 and BMP7, could represent a new strategy for treatment of bone defects and non-unions.

Direct administration of bone morphogenetic protein-2 (BMP-2) for bone regeneration could cause various clinical side effects such as osteoclast activation, inflammation, adipogenesis, and bone cyst formation. In this study, thiolated gelatin/poly(ethylene glycol) diacrylate (PEGDA) interpenetrating (IPN) composite hydrogels were developed for guided skull bone regeneration. To promote bone regeneration, either polycation-based coacervates (Coa) or gelatin microparticles (GMPs) were incorporated within IPN gels as BMP-2 carriers. Both BMP-2 loaded Coa and BMP-2 loaded GMPs showed significantly enhanced in vitro alkaline phosphate (ALP) activity of human mesenchymal stem cells (hMSCs) than non-BMP-2 treated control. Moreover, BMP-2 loaded GMPs group exhibited statistically increased ALP activity compared to both bolus BMP-2 administration and BMP-2 loaded Coa group, indicating that our carriers could protect and maintain biological activity of cargo BMP-2. Sustained release kinetics of BMP-2 from IPN composite hydrogels could be controlled by different formulations. For in vivo bone regeneration, various IPN gel formulations (i.e., (1) control, (2) only hydrogel, (3) hydrogel with bolus BMP-2, (4) hydrogel with BMP-2-loaded Coa, and (5) hydrogel with BMP-2-loaded GMPs) were bilaterally implanted into 5 mm-sized rat calvarial defects. After 4 weeks, micro-CT and histological analysis were performed to evaluate new bone formation. Significantly higher scores for bony bridging and union were observed in BMP-2-loaded Coa and BMP-2-loaded GMP groups as compared to other formulations. In addition, rats treated with BMP-2-loaded GMPs showed a significantly higher ratio of bone volume/total volume and lower trabecular separation scores than others. Finally, rats treated with either Coa or GMP groups exhibited a significant increase in bone formation area, as assessed via histomorphometric analysis. Taken together, it could be concluded that Coa and GMPs were effective carriers to maintain the bioactivity of cargo BMP-2 during its sustained release. Consequently, our IPN composite hydrogel system that combines such BMP-2 carriers could effectively promote skull bone regeneration.

Melanoma is the deadliest type of skin cancer with one of the fastest increasing incidence rates among solid tumors. The use of checkpoint inhibitors (e.g. αPD-1 antibody) has recently emerged as a viable alternative to conventional modes of therapy. However, increasing evidence points towards the need for a tumor priming step to improve intratumoral immune cell infiltration. IL-12 is an immune-activating cytokine with such potential and was explored in earlier clinical trials as a highly concentrated systemic infusion. This unfortunately led to severe adverse effects. From this perspective, the localization and gradual release of such a potent immunotherapeutic agent in the tumor microenvironment is desired. This manuscript reports the use of a heparin-based complex coacervate to deliver IL-12, in which heparin-binding motifs on IL-12 allow for its effective encapsulation. IL-12-encapsulated complex coacervates significantly improved the bioactivity of IL-12 and provided protection from proteolytic cleavage in-vitro. Indeed, a single injection of IL-12 coacervate significantly inhibits the in-vivo growth of treated and untreated, contralateral tumor growth in a syngeneic B16F10 mouse melanoma model. Furthermore, tumors in mice receiving IL-12 complex coacervate treatment displayed increased infiltration by natural killer (NK) cells and CD8α(+) T cells, and a decreased presence of CD4(+)Foxp3(+) regulatory T cells. This study provides proof-of-concept data supporting the use of complex coacervates for sustained delivery of immunostimulatory proteins as an effective therapeutic strategy against disseminated tumors.

Current cell-based therapies administered after myocardial infarction (MI) show limited efficacy due to subpar cell retention in a dynamically beating heart. In particular, cardiac patches generally provide a cursory level of cell attachment due to the lack of an adequate microenvironment. From this perspective, decellularized cell-derived ECM (CDM) is attractive in its recapitulation of a natural biophysical environment for cells. Unfortunately, its weak physical property renders it difficult to retain in its original form, limiting its full potential. Here, a novel strategy to peel CDM off from its underlying substrate is proposed. By physically stamping it onto a polyvinyl alcohol hydrogel, the resulting stretchable extracellular matrix (ECM) membrane preserves the natural microenvironment of CDM, thereby conferring a biological interface to a viscoelastic membrane. Its various mechanical and biological properties are characterized and its capacity to improve cardiomyocyte functionality is demonstrated. Finally, evidence of enhanced stem cell delivery using the stretchable ECM membrane is presented, which leads to improved cardiac remodeling in a rat MI model. A new class of material based on natural CDM is envisioned for the enhanced delivery of cells and growth factors that have a known affinity with ECM.

The extracellular matrix (ECM) provides both structural support and biochemical and biophysical cues through a complex and highly-organized conglomerate of macromolecules. Such roles have led to interest in harnessing ECM for various regenerative applications. To capture the full potential of ECM, the various biophysical and biochemical cues of ECM must be fully defined and decoded. Frequently used platforms employ coatings of a single ECM protein on synthetic substrates. While such platforms have undeniably led to advancements in the field it is important to recognize that they are based on a reductionist approach in that the natural microenvironment is resolved into a single variable (e.g. stiffness, roughness, topography, etc.). In this chapter, organ, tissue, and cell-derived ECM – collectively called decellularized ECM (dECM) –are taken comprehensively, in which an entire spectrum of biophysical variables are integrated into one platform. Given that stiffness plays a large role in determining biological responses, we assess different chemical, biological, and physical methods to modulate the stiffness, and subsequently the global biophysical profile, of dECM. We also examine the use of such stiffness-tuned dECM platforms for various regenerative applications. Ultimately, the use of stiffness-tuned dECM should provide valuable information parallel to that obtained through traditional means such as synthetic substrates.

Osteoblast and osteoclast communication (i.e. osteocoupling) is an intricate process, in which the biophysical profile of bone ECM is an aggregate product of their activities. While the effect of microenvironmental cues on osteoblast and osteoclast maturation has been resolved into individual variables (e.g. stiffness or topography), a single cue can be limited with regards to reflecting the full biophysical scope of natural bone ECM. Additionally, the natural modulation of bone ECM, which involves collagenous fibril and elastin crosslinking via lysyl oxidase, has yet to be reflected in current synthetic platforms. Here, we move beyond traditional substrates and use cell-derived ECM to examine individual and coupled osteoblast and osteoclast behavior on a physiological platform. Specifically, preosteoblast-derived ECM is crosslinked with genipin, a biocompatible crosslinker, to emulate physiological lysyl oxidase-mediated ECM crosslinking. We demonstrate that different concentrations of genipin yield changes to ECM density, stiffness, and roughness while retaining biocompatibility. By approximating various bone ECM profiles, we examine how individual and coupled osteoblast and osteoclast behavior are affected. Ultimately, we demonstrate an increase in osteoblast and osteoclast differentiation on compact and loose ECM, respectively, and identify ECM crosslinking density as an underlying force in osteocoupling behavior.

Extracellular matrix (ECM), comprised of multiple cues (chemical, physiomechanical), provides a niche for cell attachment, migration, and differentiation. Given that different cells give rise to distinct physiological milieus, the role of such microenvironmental cues on various cells has been well-studied. Particularly, the effect of various physiomechanical factors on stem cell lineage has been resolved into individual variables via ECM protein-coated polymeric systems. Such platforms, while providing a reductionist approach as a means to remove any confounding factors, unfortunately fall short of capturing the full biophysical scope of the natural microenvironment. Herein, the use of a cell-derived ECM platform is reported in which its crosslinking density is tunable; varying concentrations (0, 0.5, 1, 2% w/v) of genipin (GN), a naturally derived crosslinker with low toxicity, are used to form inter- and intrafibril crosslinks. ECM crosslinking produces GN concentration-dependent changes in ECM stiffness (

The aqueous nature of complex coacervates provides a biologically-relevant context for various therapeutic applications. In this sense, biological applications demand a corresponding level of biocompatibility from the polyelectrolytes that participate in complex coacervation. Continued development with naturally-occurring polyelectrolytes such as heparin and chitosan underscore such aims. Herein, we design a synthetic polycation, in which betaine is conjugated to a biodegradable polyester backbone. Betaine is a naturally-occurring methylated amino acid that is ubiquitously present in human plasma. Inspired by its vast range of benefits - including but not limited to anti-inflammation, anti-cancer, anti-bacterial, anti-oxidant, protein stabilization, and cardiovascular health - we aim to impart additional functionality to a polycation for eventual use in a complex coacervate with heparin. We report on its in vitro and in vivo biocompatibility, in vitro and in vivo effect on angiogenesis, in vitro effect on microbial growth, and ability to form complex coacervates with heparin.

Growth factors (GFs) are major biochemical cues for tissue regeneration. Herein, a novel dual GF delivery system is designed composed of poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) and alginate microcapsules (MCs) via an electrodropping method. While bone morphogenetic protein (BMP)-2 is encapsulated in the PLGA NPs, vascular endothelial growth factor (VEGF) is included in the alginate MCs, where BMP-2-loaded PLGA NPs are entrapped together in the fabrication process. The initial loading efficiencies of BMP-2 and VEGF are 78% ± 3.6% and 43% ± 1.7%, respectively. When our dual GF-loaded MCs are assessed for in vitro osteogenesis of umbilical cord blood-derived mesenchymal stem cells (UCB-MSCs) on 2D and 3D environment, MCs contribute to much better UCB-MSCs osteogenesis as confirmed by von Kossa staining, immunofluorescence (osteocalcin, collagen 1), calcium content measurement, and osteogenic markers expression. In addition, when dual GF-encapsulated MCs are combined with collagen and then applied to 8 mm diameter rat calvarial defect model, the positive effects on vascularized bone regeneration are much more pronounced; micro computed tomography (CT) and histology analyses exhibit 82.3% bone healing coupled with 12.6% vessel occupied area. Put together, current study indicates a synergistic effect of BMP-2/VEGF and highlights the great potential of dual GF delivery modality (PLGA NPs-in-MC) for regeneration of vascularized bone.

Growth factors (GFs) are very critical in stem cell differentiation and tissue regeneration. Therefore GF delivery carriers have been a major subject in tissue engineering research. In this study, we prepare and optimize core-shell microcapsules (C-S MCs) for dual GF delivery. The C-S MCs, composed of an alginate shell and poly(lactic-co-glycolic) acid (PLGA) core, are fabricated using an electrodropping method via custom-made coaxial needles. They are 198±38 µm in diameter with an average core size of 90±13 µm, and they are fabricated using an alginate concentration of 1% (w/v), an electrical voltage of 11 kV, and an inner syringe flow rate of 50 µL/min. Using this platform, dual GFs, bone morphogenetic protein (BMP-2) and vascular endothelial growth factor (VEGF) are encapsulated in the alginate shell and PLGA core, respectively. In vitro release tests of dual GF-loaded C-S MCs reveal early release of BMP-2, followed by VEGF on a temporal release profile of 28 days. In vitro study of the dual GF-loaded MCs demonstrates their osteogenic activity with preosteoblasts; osteogenic markers (osteocalcin and type I collagen) are upregulated and both calcium content and alkaline phosphatase (ALP) activity also increased. In addition, C-S MCs combined with collagen and preosteoblasts were subcutaneously transplanted to the dorsal region of nude mice for 3 weeks. Analysis of the retrieved constructs exhibits that both osteogenesis and angiogenesis were more active in the group containing dual GF-loaded MCs, along with deep penetration of blood vessels inside the construct, compared to blank MCs or single GF (BMP-2)-loaded MCs. This study proposes a dual GF delivery carrier using C-S MCs and demonstrates the feasibility of C-S MCs in the induction of osteogenesis and angiogenesis.

Vascular endothelial growth factor (VEGF) is one of the most important signaling cues during angiogenesis. Since many delivery systems of VEGF have been reported, the presentation of VEGF using a more physiologically relevant extracellular matrix (ECM), however, has yet to be thoroughly examined. In this study, we propose that fibroblast-derived extracellular matrix (FDM) is a novel platform for angiogenic growth factor delivery and that FDM-mediated VEGF delivery can result in an advanced angiogenic response. The FDMs, activated by EDC/NHS chemistry, were loaded with varying amounts of heparin. Different doses of VEGF were subsequently immobilized onto the heparin-grafted FDM (hep-FDM); 19.6 ± 0.6, 39.2 ± 3.2, and 54.8 ± 8.9 ng of VEGF were tethered using 100, 300, and 500 ng of initial VEGF, respectively. VEGF-tethered FDM was found chemoattractive and VEGF dose-dependent in triggering human umbilical vein endothelial cells (ECs) migration in vitro. When hep-FDM-bound VEGF (H-F/V) was encapsulated into alginate capsules (A/H-F/V) and subjected to release test for 28 days, it exhibited a significantly reduced burst release at early time point compared to that of A/V. The cell proliferation results indicated a substantially extended temporal effect of A/H-F/V on EC proliferation compared to those treated with soluble VEGF. For a further study, A/H-F/V was transplanted subcutaneously into ICR mice for up to 4 weeks to assess its in vivo effect on angiogenesis; VEGF delivered by hep-FDM was more competitive in promoting blood vessel ingrowth and maturation compared to other groups. Taken together, this study successfully engineered an FDM-mediated VEGF delivery system, documented its capacity to convey VEGF in a sustained manner, and demonstrated the positive effects of angiogenic activity in vivo as well as in vitro.

We obtained fibroblast- (FDM) and preosteoblast- (PDM) derived matrices in vitro from their respective cells. Our hypothesis was that these naturally occurring cell-derived matrices (CDMs) would provide a better microenvironment for the multi-lineage differentiation of human mesenchymal stromal cells (hMSCs) than those based on traditional single-protein-based platforms. Cells cultured for 5-6 days were decellularized with detergents and enzymes. The resulting matrices showed a fibrillar surface texture. Under osteogenic conditions, human bone-marrow-derived stromal cells (HS-5) exhibited higher amounts of both mineralized nodule formation and alkaline phosphatase (ALP) expression than those cultured on plastic or gelatin. Osteogenic markers (Col I, osteopontin, and cbfa1) and ALP activity from cells cultured on PDM were notably upregulated at 4 weeks. The use of FDM significantly improved the cellular expression of chondrogenic markers (Sox 9 and Col II), while downregulating that of Col I at 4 weeks. Both CDMs were more effective in inducing cellular synthesis of glycosaminoglycan content than control substrates. We also investigated the effect of matrix surface texture on hMSC (PT-2501) differentiation; soluble matrix (S-matrix)-coated substrates exhibited a localized fibronectin (FN) alignment, whereas natural matrix (N-matrix)-coated substrates preserved the naturally formed FN fibrillar alignment. hMSCs cultured for 4 weeks on N-matrices under osteogenic or chondrogenic conditions deposited a greater amount of calcium and proteoglycan than those cultured on S-matrices as assessed by von Kossa and Safranin O staining. In contrast to the expression levels of lineage-specific markers for cells cultured on gelatin, FN, or S-matrices, those cultured on N-matrices yielded highly upregulated levels. This study demonstrates not only the capacity of CDM for being an effective inductive template for the multi-lineage differentiation of hMSCs, but also the critical biophysical role that the matrix fibrillar texture itself plays on the induction of stem cell differentiation.

Herein, we report on the preparation of red fluorescent Si nanoparticles stabilized with styrene. Nano-sized Si particles emit fluorescence under UV excitation, which could be used to open up new applications in the fields of optics and semi-conductor research. Unfortunately, conventional methods for the preparation of red fluorescent Si nanoparticles suffer from the lack of a fully-established standard synthesis protocol. A common initial approach during the preparation of semi-conductors is the etching of crystalline Si wafers in a HF/ ethanol/H2O bath, which provides a uniformly-etched surface of nanopores amenable for further nano-sized modifications via tuning of various parameters. Subsequent sonication of the etched surface crumbles the pores on the wafer, resulting in the dispersion of particles into the solution. In this study, we use styrene to occupy these platforms to stabilize the surface. We determine that the liberated silicon particles in ethanol solution interact with styrene, resulting in the substitution of Si-H bonds with those of Si-C as determined via UV photocatalysis. The synthesized styrene-coated Si nanoparticles exhibit a stable, bright, red fluorescence under excitation with a 365 nm UV light, and yield approximately 100 mg per wafer with a synthesis time of 2 h. We believe this protocol could be further expanded as a cost-effective and high-throughput standard method in the preparation of red fluorescent Si nanoparticles.

The emergence of biomarkers as key players in the paradigm shift towards preventative medicine underscores the need for their detection and quantification. Advances made in the field of nanotechnology have played a crucial role in achieving these needs, and have contributed to recent advances in the field of medicine. Nanoparticle-based immunomagnetic assays, in particular, offer numerous advantages that utilize the unique physical properties of magnetic nanoparticles. In this review, we focus on recent developments and trends with regards to immunomagnetic assays used for detection of biomarkers. The various immunomagnetic assays are categorized into the following: particle-based multiplexing, signal control, microfluidics, microarray, and automation. Herein, we analyze each category and discuss their advantages and disadvantages.

Bacteriophages are traditionally used for the development of phage display technology. Recently, their nanosized dimensions and ease with which genetic modifications can be made to their structure and function have put them in the spotlight towards their use in a variety of biosensors. In particular, the expression of any protein or peptide on the extraluminal surface of bacteriophages is possible by genetically engineering the genome. In addition, the relatively short replication time of bacteriophages offers researchers the ability to generate mass quantities of any given bacteriophage-based biosensor. Coupled with the emergence of various biomarkers in the clinic as a means to determine pathophysiological states, the development of current and novel technologies for their detection and quantification is imperative. In this review, we categorize bacteriophages by their morphology into M13-based filamentous bacteriophages and T4- or T7-based icosahedral bacteriophages, and examine how such advantages are utilized across a variety of biosensors. In essence, we take a comprehensive approach towards recent trends in bacteriophage-based biosensor applications and discuss their outlook with regards to the field of biotechnology.

The generation of nanosized probes often requires time-intensive and application-specific optimization processes that involve conjugating a nanoconstruct to a targeting moiety. Herein, we genetically modify apoferritin and generate a universal interface system composed of protein G and 6×His-tag. The resulting construct, conferred with modularity and high targeting efficiency, is applied toward two distinct applications in the detection of a pancreatic cancer biomarker and used to demonstrate its potential in the facile exchange of nanoprobe components.

After myocardial infarction (MI), the heart undergoes fibrotic pathological remodeling instead of repair and regeneration. With multiple pathologies developing after MI, treatment using several proteins is expected to address this range of pathologies more effectively than a single-agent therapy. A factorial design of experiments study guided us to combine three complementary factors in one injection: tissue inhibitor of metalloproteinases-3 (TIMP-3) was embedded in a fibrin gel for signaling in the initial phase of the treatment, while basic fibroblast growth factor (FGF-2) and stromal cell-derived factor 1-alpha (SDF-1α) were embedded in heparin-based coacervates for sustained release and distributed within the same fibrin gel to exert their effects over a longer period. The gel was then tested in a rat model of myocardial infarction. Contractility of rat hearts treated with the protein coacervate-gel composite stabilized and slightly improved after the first week while contractility continued to decrease in rats treated with free proteins or saline over the 8 week study period. Hearts receiving the protein coacervate-gel composite treatment also exhibited reduced ventricular dilation, inflammation, fibrosis, and extracellular matrix (ECM) degradation. Revascularization, cardiomyocyte preservation, stem cell homing, and increased myocardial strain likely all contributed to the repair. This study demonstrates the potential of a multifactorial therapeutic approach in MI, using three complementary proteins delivered sequentially for comprehensive healing. The study also shows the necessity of controlled delivery for growth factors and cytokines to be an effective treatment.

Heavy metal pollution has been a problem since the advent of modern transportation, which despite efforts to curb emissions, continues to play a critical role in environmental pollution. Copper ions (Cu2+), in particular, are one of the more prevalent metals that have widespread detrimental ramifications. From this perspective, a simple and inexpensive method of detecting Cu2+ at the micromolar level would be highly desirable. In this study, we use porous silicon nanoparticles (NPs), obtained via anodic etching of Si wafers, as a basis for undecylenic acid (UDA)- or acrylic acid (AA)-mediated hydrosilylation. The resulting alkyl-terminated porous silicon nanoparticles (APS NPs) have enhanced fluorescence stability and intensity, and importantly, exhibit [Cu2+]-dependent quenching of fluorescence. After determining various aqueous sensing conditions for Cu2+, we demonstrate the use of APS NPs in two separate applications – a standard well-based paper kit and a portable layer-by-layer stick kit. Collectively, we demonstrate the potential of APS NPs in sensors for the effective detection of Cu2+.

Ischemic heart disease is a leading cause of death worldwide. After the onset of myocardial infarction, many pathological changes take place and progress the disease towards heart failure. Pathologies such as ischemia, inflammation, cardiomyocyte death, ventricular remodeling and dilation, and interstitial fibrosis, develop and involve the signaling of many proteins. Proteins can play important roles in limiting or countering pathological changes after infarction. However, they typically have short half-lives in vivo in their free form and can benefit from the advantages offered by controlled release systems to overcome their challenges. The controlled delivery of an optimal combination of proteins per their physiologic spatiotemporal cues to the infarcted myocardium holds great potential to repair and regenerate the heart. The effectiveness of therapeutic interventions depends on the elucidation of the molecular mechanisms of the cargo proteins and the spatiotemporal control of their release. It is likely that multiple proteins will provide a more comprehensive and functional recovery of the heart in a controlled release strategy.

Although the use of reactive oxygen species (ROS) has been extensively studied, current systems employ external stimuli such as light or electrical energy to produce ROS, which limits their practical usage. In this report, biocompatible metals were used to construct a novel electrochemical system that can spontaneously generate H2O2 without any external light or voltage. The corrosion of Mg transfers electrons to Au-decorated oxidized Ti in an energetically favorable process, and the spontaneous generation of H2O2 in an oxygen reduction reaction was revealed to occur at titanium by combined spectroscopic and electrochemical analyses. The controlled release of H2O2 noticeably enhanced in vitro angiogenesis even in the absence of growth factors. Finally, a new titanium implant prototype was developed by Mg incorporation, and its potential for promoting angiogenesis was demonstrated.

The aberrant glycosylation profile on the surface of cancer cells has been recognized for its potential diagnostic value towards assessing tumor progression. In this study, we initially investigate N-glycan profiles on the surface of normal (HPDE) and cancerous (Capan-1, Panc-1, and MIA PaCa-2) pancreatic cell lines, which are from different sites of pancreatic tumor. The enzymatically deglycosylated total N-glycans are permethylated via a quantitative solid-phase method and then analyzed by using MALDI-TOF MS and MALDI-QIT-TOF MS. We demonstrate that the level of high-mannose type glycans is higher among Capan-1 cells—pancreatic cancer cells that have metastasized to the liver—than that observed among Panc-1 and MIA PaCa-2 cells—pancreatic cancer cells from the pancreas duct head and tail regions, respectively. Furthermore, the relative abundance of highly-branched sialyted N-glycans is significantly up-regulated on Panc-1 and MIA PaCa-2 pancreatic cancer cells compared to that of normal HPDE pancreas cells. Taken together, these results indicate that specific N-glycosylation profile changes in pancreatic cancer cells can be used to not only distinguish between normal and cancerous cells but also provide more information on their location and metastatic potential.

Francisella tularensis is a human zoonotic pathogen and the causative agent of tularemia, a severe infectious disease. Given the extreme infectivity of F. tularensis and its potential to be used as a biological warfare agent, a fast and sensitive detection method is highly desirable. Herein, we construct a novel detection platform composed of two units: (1) Magnetic beads conjugated with multiple capturing antibodies against F. tularensis for its simple and rapid separation and (2) Genetically-engineered apoferritin protein constructs conjugated with multiple quantum dots and a detection antibody against F. tularensis for the amplification of signal. We demonstrate a 10-fold increase in the sensitivity relative to traditional lateral flow devices that utilize enzyme-based detection methods. We ultimately envision the use of our novel nanoprobe detection platform in future applications that require the highly-sensitive on-site detection of high-risk pathogens.

Successful bone tissue engineering generally requires an osteoconductive scaffold that consists of extracellular matrix (ECM) to mimic the natural environment. In this study, we developed a PLGA/PLA-based mesh scaffold coated with cell-derived extracellular matrix (CDM) for the delivery of bone morphogenic protein (BMP-2), and assessed the capacity of this system to provide an osteogenic microenvironment. Decellularized ECM from human lung fibroblasts (hFDM) was coated onto the surface of the polymer mesh scaffolds, upon which heparin was then conjugated onto hFDM via EDC chemistry. BMP-2 was subsequently immobilized onto the mesh scaffolds via heparin, and released at a controlled rate. Human placenta-derived mesenchymal stem cells (hPMSCs) were cultured in such scaffolds and subjected to osteogenic differentiation for 28 days in vitro. The results showed that alkaline phosphatase (ALP) activity, mineralization, and osteogenic marker expression were significantly improved with hPMSCs cultured in the hFDM-coated mesh scaffolds compared to the control and fibronectin-coated ones. In addition, a mouse ectopic and rat calvarial bone defect model was used to examine the feasibility of current platform to induce osteogenesis as well as bone regeneration. All hFDM-coated mesh groups exhibited a significant increase of newly formed bone and in particular, hFDM-coated mesh scaffold loaded with a high dose of BMP-2 exhibited a nearly complete bone defect healing as confirmed via micro-CT and histological observation. This work proposes a great potency of using hFDM (biophysical) coupled with BMP-2 (biochemical) as a promising osteogenic microenvironment for bone tissue engineering applications.

Jonghoon Choi,1,2 Mintai P Hwang,3 Jong-Wook Lee,3 Kwan Hyi Lee3,41Institute of Research Strategy and Development (IRSD), Seoul, Republic of Korea; 2Department of Bionano Engineering, Hanyang University, Ansan, Republic of Korea; 3Center for Biomaterials, Biomedical Research Institute, Korea Institute of Science and Technology, Seoul, Republic of Korea; 4Department of Biomedical Engineering, University of Science and Technology, Seoul, Republic of KoreaAbstract: Mesenchymal stem cells (MSCs) have been thought to hold potential as a mode of therapy for immuno-related pathologies, particularly for autoimmune diseases. Despite their potential, the interaction between MSCs and T cells, key players in the pathophysiology of autoimmune diseases, is not yet well understood, thereby preventing further clinical progress. A major obstacle is the highly heterogeneous nature of MSCs in vitro. Unfortunately, bulk assays do not provide information with regard to cell–cell contributions that may play a critical role in the overall cellular response. To address these issues, we investigated the interaction between smaller subsets of MSCs and CD4 T cells in a microwell array. We demonstrate that MSCs appear capable of modulating the T cell proliferation rate in response to persistent cell–cell interactions, and we anticipate the use of our microwell array in the classification of subpopulations within MSCs, ultimately leading to specific therapeutic interventions.Keywords: mesenchymal stem cell, T cell, microwell array

Cardiovascular disease (CVD) is a potent cause of mortality in both advanced and developing countries. While soluble CD40L (sCD40L) has been implicated as a correlative factor among CVD patients, methods to quantify sCD40L are not yet well-established. In this paper, we present an ability to separate and quantify sCD40L via a simple immunomagnetic assay. Composed of functionalized magnetic beads conferred with directionality and on-demand detachable quantum dots for subsequent optical analysis, our system utilizes the competitive nature of imidazole and nickel ions for histidine. In essence, we demonstrate the capacity to effectively separate and detect sCD40L within a clinically relevant range that contains the cut-off value for acute coronary disease. While sCD40L was used to conduct this study, we envision the use of our system for the separation and quantification of other biomarkers.

Lanthanide ion-doped nanophosphors are an emerging group of nanomaterials with excellent optical properties, and have been suggested as alternatives to quantum dots. In this letter, we determine the in-vitro and in-vivo toxicity of β-NaYF4:Ce,Tb nanophosphors using Capan-1 cells and embryonic zebrafish, respectively. In particular, we are the first to report on the in-vivo toxicity of β-phase nanophosphors and examine phenotypic developmental abnormalities (growth retardation, heart deformity, and bent tail), apoptotic cell death, and changes in heart function due to the nanophosphors. This study suggests the use of β-NaYF4:Ce,Tb nanophosphors as alternatives for QDs in a wide variety of biomedical imaging applications.

The field of biomarker quantification has experienced a growth parallel to the discovery of new materials. In this paper, we propose an innovative system for the separation and quantification of biomarkers using a simple magnetic bead (MB)–quantum dot (QD) sandwich assay. The basis of the system lies in the interaction between histidine residues on protein G and Ni ions on QDs, and the use of imidazole to selectively detach QDs bound to target biomarkers, in effect enumerating the absolute number of biomarker units. We used C-reactive protein (CRP) as a proof-of-concept and demonstrated a detection sensitivity of 82.5 fmoles in 50 μl of sample volume, a commonly used analytical volume (e.g. ELISA). Although CRP was used as a model to conduct this study, the sensitivity and simplicity of this detachable system make it a viable approach in the quantification of other target analytes.

We report a simple and green procedure for the preparation of magnetic iron oxide nanocrystals via solvothermal synthesis. The nanocrystal synthesis was carried out under mild conditions in the water–ethanol–oleic acid solvent system with the use of the oleate anion as a surface modifier of nanocrystals and glucose as a reducing agent. Specific conditions for homogenous precipitation achieved in such a reaction system lead to the formation of uniform high-quality nanocrystals down to 5 nm in diameter. The obtained hydrophobic nanocrystals can easily be converted to hydrophilic magnetic nanoparticles by being immobilized in a poly(l-lactide)–polyethyleneimine polymeric matrix. These hybrid nano-constructs may find various biomedical applications, such as magnetic separation, gene transfection and/or magnetic resonance imaging. This is the peer-reviewed version of the paper:Stojanović, Zoran, Mojca Otoničar, Jongwook Lee, Magdalena M. Stevanović, Mintai P. Hwang, Kwan Hyi Lee, Jonghoon Choi, and Dragan Uskoković. 2013. “The Solvothermal Synthesis of Magnetic Iron Oxide Nanocrystals and the Preparation of Hybrid Poly(l-lactide)–polyethyleneimine Magnetic Particles.” Colloids and Surfaces B: Biointerfaces 109 (September 1): 236–243. doi:[https://dx.doi.org/10.1016/j.colsurfb.2013.03.053] Published version: [https://vinar.vin.bg.ac.rs/handle/123456789/7339]

Mesenchymal stem cells (MSCs) have been thought to be a potential mode of therapy for immuno-related pathologies, particularly autoimmune diseases. Specifically, the ability to impart immunomodulatory effects on target inflammatory sites makes the use of MSCs particularly attractive for patients affected by taking immunosuppressive medicine, which works to downregulate the immune system on a systemic level. Despite its promises, the interaction between MSCs and T-cells, a key player in the pathophysiology of autoimmune diseases, is not yet well understood, thereby preventing further progress in the clinic. A major obstacle is the highly heterogeneous nature of MSCs in-vitro. Bulk assays commonly carried out discount the inherent heterogeneity, and thus do not provide information with regards to single-cell contributions that may play a critical role in the overall bulk response. To address these issues, we propose to study the single-cell interaction between MSCs and CD4 T cells in a nanowell array. Using the nanowell assay, we demonstrate that individual MSCs appear capable of dynamically modulating the T cell proliferation rate in response to persistent cell-cell interactions in a microenviroment. Based on these results, we anticipate the use of our nanowell assay in the classification of subpopulations within MSCs, ultimately leading to specific therapeutic interventions.

An artificial matrix (Fn-Tigra), consisting of graphene oxide (GO) and fibronectin (Fn), is developed on pure titanium (Ti) substrates via an electrodropping technique assisted with a custom-made coaxial needle. The morphology and topography of the resulting artificial matrix is orderly aligned and composed of porous microcavities. In addition, Fn is homogenously distributed and firmly bound onto GO as determined via immunofluorescence and elemental mapping, respectively. The artificial matrix is moderately hydrophobic (63.7°), and exhibits an average roughness of 546 nm and a Young's modulus (E) of approximately 4.8 GPa. The biocompatibility, cellular behavior, and osteogenic potential of preosteoblasts on Fn-Tigra are compared to those of cells cultured on Ti and Ti-GO (Tigra). Cell proliferation and viability are significantly higher on Fn-Tigra and Tigra than that of cells grown on Ti. Focal adhesion molecule (vinculin) expression is highly activated at the central and peripheral area of preosteoblasts when cultured on Fn-Tigra. Furthermore, we demonstrate enhanced in vitro osteogenic differentiation of preosteoblasts cultured on Fn-Tigra over those cultured on bare Ti, as determined via Alizarin red and von Kossa staining, and the analysis of osteocalcin, type I collagen, alkaline phosphatase activity, and calcium contents. Finally, we investigate the biophysical and biomechanical properties of the cells using AFM. While the height and roughness of preosteoblasts increased with time, cell surface area decreased during in vitro osteogenesis over 2 weeks. In addition, the E of cells cultured on Tigra and Fn-Tigra increase in a statistically significant and time-dependent manner by 30%, while those cultured on bare Ti retain a relatively consistent E. In summary, we engineer a biocompatible artificial matrix (Fn-Tigra) capable of osteogenic induction and consequently demonstrate its potential in bone tissue engineering applications.

Background Topography, stiffness, and composition of biomaterials play a crucial role in cell behaviors. In this study, we have investigated biochemical (gene markers), biophysical (roughness), and biomechanical (stiffness) changes during the osteogenic differentiation of preosteoblasts on gelatin matrices. Results Our results demonstrate that gelatin matrices offer a favorable microenvironment for preosteoblasts as determined by focal adhesion and filopodia formation. The osteogenic differentiation potential of preosteoblasts on gelatin matrices is confirmed by qualitative (Alizarin red, von kossa staining, immunofluorescence, and gene expression) and quantitative analyses (alkaline phosphatase activity and calcium content). The biomechanical and biophysical properties of differentiating preosteoblasts are analyzed using atomic force microscopy (AFM) and micro indentation. The results show sequential and significant increases in preosteoblasts roughness and stiffness during osteogenic differentiation, both of which are directly proportional to the progress of osteogenesis. Cell proliferation, height, and spreading area seem to have no direct correlation with differentiation; however, they may be indirectly related to osteogenesis. Conclusions The increased stiffness and roughness is attributed to the mineralized bone matrix and enhanced osteogenic extracellular matrix protein. This report indicates that biophysical and biomechanical aspects during in vitro cellular/extracellular changes can be used as biomarkers for the analysis of cell differentiation.