The use of heparin/polycation coacervate sustain release system to compare the bone regenerative potentials of 5 BMPs using a critical sized calvarial bone defect model.

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.

Single injection of IL-12 coacervate as an effective therapy against B16-F10 melanoma in mice.

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.

Enhanced Detection of Infectious Pancreatic Necrosis Virus via Lateral Flow Chip and Fluorometric Biosensors Based on Self-Assembled Protein Nanoprobes.

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.

Stretchable ECM Patch Enhances Stem Cell Delivery for Post-MI Cardiovascular Repair.

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.

Coacervate-mediated exogenous growth factor delivery for scarless skin regeneration.

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-ß3) 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-ß3/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.

Enhanced Skull Bone Regeneration by Sustained Release of BMP-2 in Interpenetrating Composite Hydrogels.

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.

A biocompatible betaine-functionalized polycation for coacervation.

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.

Sensitive detection of copper ions via ion-responsive fluorescence quenching of engineered porous silicon nanoparticles.

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+.

Tunable Crosslinked Cell-Derived Extracellular Matrix Guides Cell Fate.

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 (<0.1-9.4 kPa), roughness (96-280 nm), and chemical composition (100-60% amine content). The effect of the various crosslinked ECM profiles on human mesenchymal stem cell differentiation, vascular morphogenesis, and cardiomyogenesis are then evaluated. Taken together, this study demonstrates that tunable crosslinked cell-derived ECM platform is capable of providing a comprehensive physiological platform, and envisions its use in future tissue engineering applications.

Approximating bone ECM: Crosslinking directs individual and coupled osteoblast/osteoclast behavior.

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.

Towards comprehensive cardiac repair and regeneration after myocardial infarction: Aspects to consider and proteins to deliver.

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.

A novel nanoprobe for the sensitive detection of Francisella tularensis.

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.

Bioactive cell-derived matrices combined with polymer mesh scaffold for osteogenesis and bone healing.

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.

Fibronectin-tethered graphene oxide as an artificial matrix for osteogenesis.

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.

Antibacterial activity and cytotoxicity of multi-walled carbon nanotubes decorated with silver nanoparticles.

Recently, various nanoscale materials, including silver (Ag) nanoparticles, have been actively studied for their capacity to effectively prevent bacterial growth. A critical challenge is to enhance the antibacterial properties of nanomaterials while maintaining their biocompatibility. The conjugation of multiple nanomaterials with different dimensions, such as spherical nanoparticles and high-aspect-ratio nanotubes, may increase the target-specific antibacterial capacity of the consequent nanostructure while retaining an optimal biocompatibility. In this study, multi-walled carbon nanotubes (MWCNTs) were treated with a mixture of acids and decorated with Ag nanoparticles via a chemical reduction of Ag cations by ethanol solution. The synthesized Ag-MWCNT complexes were characterized by transmission electron microscopy, X-ray diffractometry, and energy-dispersive X-ray spectroscopy. The antibacterial function of Ag-MWCNTs was evaluated against Methylobacterium spp. and Sphingomonas spp. In addition, the biocompatibility of Ag-MWCNTs was evaluated using both mouse liver hepatocytes (AML 12) and human peripheral blood mononuclear cells. Finally, we determined the minimum amount of Ag-MWCNTs required for a biocompatible yet effective antibacterial treatment modality. We report that 30 µg/mL of Ag-MWCNTs confers antibacterial functionality while maintaining minimal cytotoxicity toward both human and animal cells. The results reported herein would be beneficial for researchers interested in the efficient preparation of hybrid nanostructures and in determining the minimum amount of Ag-MWCNTs necessary to effectively hinder the growth of bacteria.

Fibroblast-derived matrix (FDM) as a novel vascular endothelial growth factor delivery platform.

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.

Multi-lineage differentiation of human mesenchymal stromal cells on the biophysical microenvironment of cell-derived matrix.

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.

A glimpse into the interactions of cells in a microenvironment: the modulation of T cells by mesenchymal stem cells.

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.

A systematic in-vivo toxicity evaluation of nanophosphor particles via zebrafish models.

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 ß-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 ß-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 ß-NaYF4:Ce,Tb nanophosphors as alternatives for QDs in a wide variety of biomedical imaging applications.

Immunomagnetic nanoparticle-based assays for detection of biomarkers.

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.