Indicators of HSV1 Infection, ECM-Receptor Interaction, and Chromatin Modulation in a Nuclear Family with Schizophrenia.

Schizophrenia (SCZ) is a complex psychiatric disorder with high heritability; identifying risk genes is essential for deciphering the disorder's pathogenesis and developing novel treatments. Using whole-exome sequencing, we screened for mutations within protein-coding sequences in a single family of patients with SCZ. In a pathway enrichment analysis, we found multiple transmitted variant genes associated with two KEGG pathways: herpes simplex virus 1 (HSV1) infection and the extracellular matrix (ECM)-receptor interaction. When searching for rare variants, six variants, SLC6A19p.L541R, CYP2E1p.T376S, NAT10p.E811D, N4BP1p.L7V, CBX2p.S520C, and ZNF460p.K190E, segregated with SCZ. A bioinformatic analysis showed that three of these mutated genes were associated with chromatin modulation. We found that HSV1 infection, ECM-receptor interaction pathways, and epigenetic mechanisms may contribute to the pathogenesis of SCZ in certain families. The identified polygenetic risk factors from the sample family provide distinctive underlying biological mechanisms of the pathophysiology of SCZ and may be useful in clinical practice and patient care.

Tuning the Crystallinity and Coverage of SiO2-ZnIn2S4 Core-Shell Nanoparticles for Efficient Hydrogen Generation.

The coverage, thickness, and crystallinity of ZnIn2S4 (ZIS) shells on SiO2 core nanoparticles (SiO2@ZIS) were systematically investigated using microwave-assisted solvothermal methods aided by the addition of acid in ethanolic medium. The surface modification of the SiO2 cores with (3-mercaptopropyl)trimethoxysilane was found to be critical to generate a homogeneous coverage of ZnIn2S4. The SiO2@ZIS core-shell nanoparticles exhibited the best coverage but poor crystallinity when synthesized in pure ethanol, whereas best crystallinity but poor coverage was observed when synthesized in an aqueous solution. The addition of selected amounts of acid (HCl) led to improved crystallinity in the ethanolic medium. The thickness of the ZIS shell could be controlled in an ethanolic solution by judiciously varying the amounts of acid and the concentration of the ZIS precursor. Increasing the concentration of the ZIS precursor to twice the standard concentration in ethanolic solution with the addition of 100 µL of HCl afforded better crystallinity, homogeneous coverage, and optimal photocatalytic hydrogen production.

RNU (Foxn1 RNU-Nude) Rats Demonstrate an Improved Ability to Regenerate Muscle in a Volumetric Muscle Injury Compared to Sprague Dawley Rats.

Products developed for skeletal muscle regeneration frequently incorporate allogeneic and xenogeneic materials to elicit a regenerative response to heal skeletal muscle wounds. To avoid graft rejection in preclinical studies, immunodeficient rodents are used. Whether the immunodeficiency alters the host response to the material in skeletal muscle has not been studied. In this study, we hypothesized that an allogeneic acellular skeletal muscle grafts implanted in an immunodeficient rat (RNU, Foxn1-deficient) would exhibit better new muscle fiber formation compared to grafts implanted in immunocompetent Sprague Dawley (SD) rats. Decellularized SD skeletal muscle matrix (DMM) was implanted in the gastrocnemius (N = 8 rats/group). 56 days after surgery, animal gait was examined and animals were euthanized. Muscle force was assessed and fiber number as well as immune cell infiltrate was measured by histomorphometry and immunohistochemistry. Animal gait and percent recovery of muscle force were unchanged in both groups, but newly regenerated muscle fibers increased in RNU rats. Macrophage staining for CD68 was higher in RNU rats than in SD rats. These data show differences in muscle regeneration between animal models using the same biomaterial treatment, but these differences could not be ascribed to the immune response. Overall, our data provide awareness that more studies are needed to understand how host responses to biomaterials differ based on the animal model used.

Injectable Allograft Adipose Matrix Supports Adipogenic Tissue Remodeling in the Nude Mouse and Human.

BACKGROUND: Adipose tissue reaches cellular stasis after puberty, leaving adipocytes unable to significantly expand or renew under normal physiologic conditions. This is problematic in progressive lipodystrophies, in instances of scarring, and in soft-tissue damage resulting from lumpectomy and traumatic deformities, because adipose tissue will not self-renew once damaged. This yields significant clinical necessity for an off-the-shelf de novo soft-tissue replacement mechanism. METHODS: A process comprising separate steps of removing lipid and cellular materials from adipose tissue has been developed, creating an ambient temperature-stable allograft adipose matrix. Growth factors and matrix proteins relevant to angiogenesis and adipogenesis were identified by enzyme-linked immunosorbent assay and immunohistochemistry, and subcutaneous soft-tissue integration of the allograft adipose matrix was investigated in vivo in both the athymic mouse and the dorsum of the human wrist. RESULTS: Allograft adipose matrix maintained structural components and endogenous growth factors. In vitro, adipose-derived stem cells cultured on allograft adipose matrix underwent adipogenesis in the absence of media-based cues. In vivo, animal modeling showed vasculature formation followed by perilipin A-positive tissue segments. Allograft adipose matrix maintained soft-tissue volume in the dorsal wrist in a 4-month investigation with no severe adverse events, becoming palpably consistent with subcutaneous adipose. CONCLUSIONS: Subcutaneous implantation of allograft adipose matrix laden with retained angiogenic and adipogenic factors served as an inductive scaffold for sustaining adipogenesis. Tissue incorporation assessed histologically from both the subcutaneous injection site of the athymic nude mouse over 6 months and human dorsal wrist presented adipocyte morphology residing within the injected scaffold.

Decellularized Muscle Supports New Muscle Fibers and Improves Function Following Volumetric Injury.

Current strategies to treat volumetric muscle loss use primarily pedicle or free muscle transfers, but these grafts fail to adequately regenerate functional tissue. Decellularized soft tissue grafts possess physical and chemical cues to promote muscle regeneration, suggesting their potential for use in large muscle defects. In this study, we developed a decellularized muscle matrix (DMM) graft using rat gastrocnemius. Anisotropy and chemical components of the extracellular matrix were retained, including laminin, fibronectin, and collagen. We compared the ability of DMM, autologous muscle grafts (clinical standard), and type I collagen plugs (negative control) to support muscle regeneration. DMM supported regeneration over a 56-day period in 1 × 1 cm and 1.5 × 1 cm gastrocnemius defects in rats. Muscle function tests demonstrated improved muscle recovery in rats with DMM grafts when compared to collagen. Histological sections were assessed using morphometrics and immunostaining. DMM supported muscle regeneration with less fibrosis and more de novo neuromuscular receptors than either autograft or collagen. Overall, our results indicate that DMM may be used as a muscle replacement graft based on its ability to improve muscle function recovery, promote muscle regeneration, and support new neuromuscular junctions.

A Novel Reticular Dermal Graft Leverages Architectural and Biological Properties to Support Wound Repair.

BACKGROUND: Acellular dermal matrices (ADMs) are frequently used in reconstructive surgery and as scaffolds to treat chronic wounds. The 3-dimensional architecture and extracellular matrix provide structural and signaling cues for repair and remodeling. However, most ADMs are not uniformly porous, which can lead to heterogeneous host engraftment. In this study, we hypothesized that a novel human reticular ADM (HR-ADM; AlloPatch Pliable, Musculoskeletal Transplant Foundation, Edison, N.J.) when aseptically processed would have a more open uniform structure with retention of biological components known to facilitate wound healing. METHODS: The reticular and papillary layers were compared through histology and scanning electron microscopy. Biomechanical properties were assessed through tensile testing. The impact of aseptic processing was evaluated by comparing unprocessed with processed reticular grafts. In vitro cell culture on fibroblasts and endothelial cells were performed to showcase functional cell activities on HR-ADMs. RESULTS: Aseptically processed HR-ADMs have an open, interconnected uniform scaffold with preserved collagens, elastin, glycosaminoglycans, and hyaluronic acid. HR-ADMs had significantly lower ultimate tensile strength and Young's modulus versus the papillary layer, with a higher percentage elongation at break, providing graft flexibility. These preserved biological components facilitated fibroblast and endothelial cell attachment, cell infiltration, and new matrix synthesis (collagen IV, fibronectin, von Willebrand factor), which support granulation and angiogenic activities. CONCLUSIONS: The novel HR-ADMs provide an open, interconnected scaffold with native dermal mechanical and biological properties. Furthermore, aseptic processing retains key extracellular matrix elements in an organized framework and supports functional activities of fibroblasts and endothelial cells.

Do Processing Methods Make a Difference in Acellular Dermal Matrix Properties?

BACKGROUND: The use of acellular dermal matrices (ADMs) has become the standard of practice in many reconstructive and aesthetic surgical applications. Different methods used to prepare the allograft tissue for surgical use can alter the ADMs natural properties. Aseptic processing has been shown to retain the natural properties of ADMs more favorably than terminally sterilized ADMs. Terminal sterilization has been historically linked to alteration of biological materials. In vitro work was conducted to compare ADM processing methods. OBJECTIVES: Characterize aseptically processed ADMs and compare cell-matrix interaction characteristics to terminally sterilized ADMs. METHODS: Two aseptically processed ADMs, FlexHD Pliable and BellaDerm, were characterized via histological evaluation, biomechanical integrity, enzymatic degradation, and in vitro cell studies. FlexHD Pliable was compared to Alloderm Ready-to-Use (RTU). RESULTS: Histological evaluation revealed that FlexHD Pliable had a uniform, open structure compared to BellaDerm. Mechanical characterization demonstrated that BellaDerm had higher strength and stiffness compared to FlexHD Pliable, which maintained higher elasticity. Immunohistochemical analysis verified that key matrix proteins remained intact after aseptic processing. Cell studies found that fibroblasts attached more readily, and proliferated faster on FlexHD Pliable compared to BellaDerm. Additionally, fibroblasts infiltrated into FlexHD Pliable from both sides and on the dermal side in BellaDerm and produced an abundance of multi-layered matrix proteins (collagen, fibronectin) when compared to AlloDerm RTU which was sparse. CONCLUSIONS: Aseptically processed FlexHD Pliable and BellaDerm provide a suitable, biocompatible option for tissue repair and regeneration in aesthetic and reconstructive surgical applications.

The designs and applications of a scanning interface with electrical signal detection on the scalp for the severely disabled.

This study discussed a computer-aided program development that meets the requirements of people with physical disabilities. A number of control modes, such as electrode signal recorded on the scalp and blink control, were combined with the scanning human-machine interface to improve the external input/output device. Moreover, a novel and precise algorithm, which filters noise and reduces misrecognition of the system, was proposed. A convenient assistive device can assist people with physical disabilities to meet their requirements for independent living and communication with the outside. The traditional scanning keyboard is changed, and only the phonetic notations are typed instead of characters, thus the time of tone and function selection could be saved, and the typing time could be also reduced. Barrier-free computer assistive devices and interface for people with physical disabilities in typing or speech could allow them to use a scanning keyboard to select phonetic symbols instead of Chinese characters to express their thoughts. The human-machine interface controls can obtain more reliable results as 99.8% connection success rate and 95% typing success rate.

Endogenous regeneration of critical-size chondral defects in immunocompromised rat xiphoid cartilage using decellularized human bone matrix scaffolds.

Clinical efforts to repair cartilage defects delivering cells or engineered cartilage implants into the lesions have met with limited success. This study used a critical-size chondral defect model in immunocompromised rat xiphoid cartilage to test whether endogenous chondrogenesis could be achieved using human bone matrix scaffolds to deliver human cartilage particles and/or a variant isoform of fibroblast growth factor-2 (FGF2-variant). Seventy-two male athymic RNU rats were enrolled in this study with eight rats per experimental group. Decellularized and demineralized human bone matrix scaffolds loaded with human articular cartilage particles or heat-inactivated cartilage particles were combined with different doses of the FGF2-variant. Scaffolds were implanted into 3-mm-diameter critical-size defects prepared using a biopsy punch through the center of the xiphoid. The samples were evaluated 28 days postsurgery using X-ray, equilibrium partitioning of ionic contrast microcomputed tomography, and safranin O-stained histological sagittal sections. Scaffolds containing cartilage particles plus the FGF2-variant induced dose-dependent increases in the formation of neocartilage (p<0.05), which was distributed homogeneously throughout the defects in comparison to scaffolds containing only the FGF2-variant. These effects were less pronounced when scaffolds with heat-inactivated cartilage particles were used. These results demonstrate that endogenous repair of chondral defects can be achieved in the absence of exogenous cells or bone marrow, suggesting that a similar approach may be successful for treating chondral lesions clinically.

Quantifying the relation between bond number and myoblast proliferation.

Many functions of the extracellular matrix can be mimicked by small peptide fragments (e.g., arginine-glycine-aspartic acid (RGD) sequence) of the entire molecule, but the presentation of the peptides is critical to their effects on cells. It is likely that some effects of peptide presentation from biomaterials simply relate to the number of bonds formed between cell receptors and the adhesion ligands, but a lack of tools to quantify bond number limits direct investigation of this assumption. The impact of different ligand presentations (density, affinity, and nanoscale distribution) on the proliferation of C2C12 and human primary myoblasts was first examined in this study. Increasing the ligand density or binding affinity led to a similar enhancement in proliferation of C2C12 cells and human primary myoblasts. The nanoscale distribution of clustered RGD ligands also influenced C2C12 cells and human primary myoblast proliferation, but in an opposing manner. A theological technique and a FRET technique were then utilized to quantify the number of receptor-ligand interactions as a function of peptide presentation. Higher numbers of bonds were formed when the RGD density and affinity were increased, as measured with both techniques, and bond number correlated with cell growth rates. However, the influence of the nanoscale peptide distribution did not appear to be solely a function of bond number. Altogether, these findings provide significant insight to the role of peptide presentation in the regulation of cell proliferation, and the approaches developed in this work may have significant utility in probing how adhesion regulates a variety of other cellular functions and aid in developing design criterion for cell-interactive materials.

Design of biodegradable hydrogel for the local and sustained delivery of angiogenic plasmid DNA.

PURPOSE: To attain the effective local and sustained delivery of plasmid DNA (pDNA) encoding for a growth factor. METHODS: We hypothesized that controlling the degradation rate of biomaterials encapsulating pDNA via concurrent physical dissociation of the cross-linked structure and hydrolytic chain breakage of polymers would allow one to significantly broaden the range of pDNA release rate. This hypothesis was examined using ionically cross-linked polysaccharide hydrogels which were previously designed to rapidly degrade via engineering of ionic cross-linking junction and partial oxidation of polysaccharide chains. RESULTS: The hydrogel degradation rates were varied over the broad range, and pDNA release correlated with the gel degradation rate. Degradable hydrogels were used for the local and sustained delivery of a pDNA encoding for vascular endothelial growth factor (VEGF) in the ischemic hindlimbs of mice, and local pDNA release significantly improved the recovery of blood perfusion as compared with a bolus injection of VEGFencoding pDNA. CONCLUSION: This strategy to control the hydrogel degradation rate may be useful in regulating the delivery of a broad array of macromolecular drugs, and subsequently improve their therapeutic efficacy.

Analysis of a spatially dispersive displacement sensor utilizing an AlGaInP chip.

We present a demonstration and analysis of an industrialized design of a spatially dispersive displacement sensor, which is composed of an AlGaInP gain chip in visible range, optical assembly, and a spectrum analyzer. The sensor utilizes the spatial dispersion of focus from the optical assembly and wavelength spectrum's deviation induced by the displacement of the target. As a result, the sensor delivers a quick and simple way of measuring displacement. By adapting the magnification and resolution of the optical assembly, a displacement sensor with a middle measurement range, ~10 microm, was obtained. However, we should note that 25 nm resolution is limited by the bandwidth and temperature fluctuation of the gain chip.

Homeostatic lymphoid chemokines synergize with adhesion ligands to trigger T and B lymphocyte chemokinesis.

Homeostatic chemokines such as CCL19, CCL21, and CXCL13 are known to elicit chemotaxis from naive T and B cells and play a critical role in lymphocyte homing to appropriate zones within secondary lymphoid organs (SLO). Here we tested whether CCL21 and CXCL13 modulate murine lymphocyte motility in the absence of concentration gradients, using videomicroscopy to directly observe the migration of single cells. CCL21 treatment of T cells induced rapid polarization and sustained random migration with average speeds of 5.16 +/- 2.08 microm/min; B cell migration (average velocity 4.10 +/- 1.58 microm/min) was similarly induced by CXCL13. Migration required the presence of both chemokine and adhesion ligands and was sustained for >24 h. Furthermore, in in vitro assays modeling the relative infrequency of Ag-specific T cell-dendritic cell (DC) encounters during primary immune responses, we found that CCL21 addition to T-DC cocultures accelerated the kinetics of CD69 up-regulation and enhanced by 2-fold the proliferation of Ag-specific T cells in a manner dependent on G-protein-coupled receptor signaling in T cells. These results suggest that homeostatic chemokines could substantially impact the dynamics and priming of lymphocytes within SLO even in the absence of significant concentration gradients.

Characteristics of a paired surface plasma waves biosensor.

A novel paired surface plasma wave biosensor (PSPWB) is described and setup. By integrating the features of a common-path optical heterodyne interferometer and the amplitude-ratio detection mode, the PSPWB not only produces a high detection sensitivity but also provides a large dynamic measurement range for effective refractive index (Deltan(eff)) based on amplitude-sensitive detection method. Thus, the performance of PSPWB becomes equivalent to shot-noise limited of a conventional SPR biosensor. To our knowledge, this novel PSPWB shows the highest detection sensitivity on (Deltan(eff)) when compared with conventional SPR biosensors using either a non-interferometric or interferometric technique. The experimental results correctly verify the properties of a PSPWB that the detection sensitivity is an order of 10(-7) refractive index unit (RIU) when measuring a 0.001% sucrose-water solution. This result confirms the detection sensitivity up to 10(-9) RIU of the IgG/anti-IgG interaction in real time successfully. Furthermore, a dynamic range of 10(5) using PSPWB was also obtained.

Long-term in vivo gene expression via delivery of PEI-DNA condensates from porous polymer scaffolds.

Nonviral delivery vectors are attractive for gene therapy approaches in tissue engineering, but suffer from low transfection efficiency and short-term gene expression. We hypothesized that the sustained delivery of poly(ethylenimine) (PEI)-condensed DNA from three-dimensional biodegradable scaffolds that encourage cell infiltration could greatly enhance gene expression. To test this hypothesis, a PEI-condensed plasmid encoding beta-galactosidase was incorporated into porous poly(lactide-co-glycolide) (PLG) scaffolds, using a gas foaming process. Four conditions were examined: condensed DNA and uncondensed DNA encapsulated into PLG scaffolds, blank scaffolds, and bolus delivery of condensed DNA in combination with implantation of PLG scaffolds. Implantation of scaffolds incorporating condensed beta-galactosidase plasmid into the subcutaneous tissue of rats resulted in a high level of gene expression for the entire 15-week duration of the experiment, as exemplified by extensive positive staining for beta-galactosidase gene expression observed on the exterior surface and throughout the cross-sections of the explanted scaffolds. No positive staining could be observed for the control conditions either on the exterior surface or in the cross-section at 8- and 15-week time points. In addition, a high percentage (55-60%) of cells within scaffolds incorporating condensed DNA at 15 weeks demonstrated expression of the DNA, confirming the sustained uptake and expression of the encapsulated plasmid DNA. Quantitative analysis of beta-galactosidase gene expression revealed that expression levels in scaffolds incorporating condensed DNA were one order of magnitude higher than those of other conditions at the 2- week time point and nearly two orders of magnitude higher than those of the control conditions at the 8- and 15-week time points. This study demonstrated that the sustained delivery of PEI-condensed plasmid DNA from PLG scaffolds led to an in vivo long-term and high level of gene expression, and this system may find application in areas such as bone tissue engineering.

Combined angiogenic and osteogenic factor delivery enhances bone marrow stromal cell-driven bone regeneration.

UNLABELLED: Bone formation is a coordinated process involving various biological factors. We have developed a scaffold system capable of sustained and localized presentation of osteogenic (BMP-4) and angiogenic (VEGF) growth factors and human bone marrow stromal cells to promote bone formation at an ectopic site. Combined delivery of these factors significantly enhanced bone formation compared with other conditions. INTRODUCTION: Tissue regeneration entails complex interactions between multiple signals and materials platforms. Orchestrating the presentation of these signals may greatly enhance the regeneration of lost tissue mass. Bone formation, for example, is dependent on the signaling of BMPs, molecules initiating vascularization (e.g., vascular endothelial growth factor [VEGF]), and osteogenic precursor cells capable of responding to these cues and forming bone tissue. It was hypothesized that combined and concerted delivery of these factors from biodegradable scaffolds would lead to enhanced bone formation. MATERIALS AND METHODS: Poly(lactic-co-glycolic acid) scaffolds containing combinations of condensed plasmid DNA encoding for BMP-4, VEGF, and human bone marrow stromal cells (hBMSCs) were implanted into the subcutaneous tissue of SCID mice. Implants (n = 6) were retrieved at 3, 8, and 15 weeks after implantation. Bone and blood vessel formation was determined qualitatively and quantitatively by methods including histology, immmunostaining, and muCT. RESULTS: Scaffolds delivering VEGF resulted in a prominent increase in blood vessel formation relative to the conditions without VEGF. BMP-4 expression in scaffolds encapsulating condensed DNA was also confirmed at the 15-week time-point, showing the characteristic of long-term delivery in this system. Combined delivery of all three types of factors resulted in a significant increase in the quantity of regenerated bone compared with any factor alone or any two factors combined, as measured with DXA, X-ray, and histomorphometric analysis. Furthermore, bone formed with all three factors had elastic moduli significantly higher than any other condition. CONCLUSIONS: Concerted delivery of BMP-4, VEGF, and hBMSCs promoted greater bone formation relative to any single factor or combination of two factors. Materials systems that allows multifactorial presentation more closely mimic natural developmental processes, and these results may have important implications for bone regeneration therapeutics.

Endothelial cell modulation of bone marrow stromal cell osteogenic potential.

In the context of bone development and regeneration, the intimate association of the vascular endothelium with osteogenic cells suggests that endothelial cells (ECs) may directly regulate the differentiation of osteoprogenitor cells. To investigate this question, bone marrow stromal cells (BMSCs) were cultured: in the presence of EC-conditioned medium, on EC extracellular matrix, and in EC cocultures with and without cell contact. RNA and protein were isolated from ECs and analyzed by reverse transcriptase-polymerase chain reaction and Western blotting, respectively, for expression of bone morphogenetic protein 2 (BMP-2). In animal studies, BMSCs and ECs were cotransplanted into severe combined immunodeficient mice on biodegradable polymer matrices, and histomorphometric analysis was performed to determine the extent of new bone and blood vessel formation. ECs significantly increased BMSC osteogenic differentiation in vitro only when cultured in direct contact. ECs expressed BMP-2, and experiments employing interfering RNA inhibition confirmed its production as contributing to the increased BMSC osteogenic differentiation. In vivo, cotransplantation of ECs with BMSCs resulted in greater bone formation than did transplantation of BMSCs alone. These data suggest that ECs function not only to form the microvasculature that delivers nutrients to developing bone but also to modulate the differentiation of osteoprogenitor cells in vitro and in vivo.

Fabrication and in vitro testing of polymeric delivery system for condensed DNA.

Polyethylenimine (PEI) was combined with plasmid DNA and freeze dried following the addition of sucrose as a lyoprotectant and pore-forming agent. Freeze-dried PEI DNA condensates were dry mixed with granular polylactideglycolic acid (PLGA) then compression molded and sponged to encapsulated PEI DNA. A measurement of the elastic modulus indicated that 91 wt% sucrose substituted for 95 wt% sodium chloride as a porogen, resulting in PLGA sponges with a mechanical modulus of 100 kPa. The PEI DNA was retained (80%) within PLGA sponges prepared with sucrose during the leaching and subsequent 2-week release studies, whereas sodium chloride PLGA sponges caused the premature release (100%) of PEI DNA within 2 days. In vitro gene transfer studies with PEI DNA PLGA sponges established that adherent and infiltrating fibroblasts expressed reporter gene for 15 days compared with the short, 3-day expression mediated by direct gene of PEI DNA on cells in culture. The results demonstrate an approach to encapsulate condensed DNA in a PLGA sponge for the purpose of retaining DNA within the matrices and creating efficient gene transfer during tissue engineering.