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Williford J.-M.,Johns Hopkins University | Wu J.,Johns Hopkins University | Ren Y.,Johns Hopkins University | Archang M.M.,Johns Hopkins University | And 3 more authors.
Annual Review of Biomedical Engineering | Year: 2014

Inhibiting specific gene expression by short interfering RNA (siRNA) offers a new therapeutic strategy to tackle many diseases, including cancer, metabolic disorders, and viral infections, at the molecular level. The macromolecular and polar nature of siRNA hinders its cellular access to exert its effect. Nanoparticulate delivery systems can promote efficient intracellular delivery. Despite showing promise in many preclinical studies and potential in some clinical trials, siRNA has poor delivery efficiency, which continues to demand innovations, from carrier design to formulation, in order to overcome transport barriers. Previous findings for optimal plasmid DNA delivery cannot be generalized to siRNA delivery owing to significant discrepancy in size and subtle differences in chain flexibility between the two types of nucleic acids. In this review, we highlight the recent advances in improving the stability of siRNA nanoparticles, understanding their intracellular trafficking and release mechanisms, and applying judiciously the promising formulations to disease models. Copyright © 2014 by Annual Reviews. All rights reserved.


Jiang X.,Johns Hopkins University | Mao H.-Q.,Johns Hopkins University | Mao H.-Q.,Translational Tissue Engineering Center | Wang T.-H.,Johns Hopkins University
Nano Letters | Year: 2014

Nonviral gene delivery holds great promise not just as a safer alternative to viral vectors in traditional gene therapy applications, but also for regenerative medicine, induction of pluripotency in somatic cells, and RNA interference for gene silencing. Although it continues to be an active area of research, there remain many challenges to the rational design of vectors. Among these, the inability to characterize the composition of nanoparticles and its distribution has made it difficult to probe the mechanism of gene transfection process, since differences in the nanoparticle-mediated transfection exist even when the same vector is used. There is a lack of sensitive methods that allow for full characterization of DNA content in single nanoparticles and its distribution among particles in the same preparation. Here we report a novel spectroscopic approach that is capable of interrogating nanoparticles on a particle-by-particle basis. Using PEI/DNA and PEI-g-PEG/DNA nanoparticles as examples, we have shown that the distribution of DNA content among these nanoparticles was relatively narrow, with the average numbers of DNA of 4.8 and 6.7 per particle, respectively, in PEI/DNA and PEI-g-PEG/DNA nanoparticles. This analysis enables a more accurate description of DNA content in polycation/DNA nanoparticles. It paves the way toward comparative assessments of various types of gene carriers and provides insights into bridging the efficiency gap between viral and nonviral vehicles. © 2014 American Chemical Society.


News Article | October 25, 2016
Site: www.cemag.us

In a proof-of-concept study with mice, scientists at The Johns Hopkins University show that a novel coating they made with antibiotic-releasing nanofibers has the potential to better prevent at least some serious bacterial infections related to total joint replacement surgery. A report on the study, published in Proceedings of the National Academy of Sciences, was conducted on the rodents’ knee joints, but, the researchers say, the technology would have “broad applicability” in the use of orthopedic prostheses, such as hip and knee total joint replacements, as well pacemakers, stents and other implantable medical devices. In contrast to other coatings in development, the researchers report the new material can release multiple antibiotics in a strategically timed way for an optimal effect. “We can potentially coat any metallic implant that we put into patients, from prosthetic joints, rods, screws and plates to pacemakers, implantable defibrillators and dental hardware,” says co-senior study author Lloyd S. Miller, M.D., Ph.D., an associate professor of dermatology and orthopedic surgery at the Johns Hopkins University School of Medicine. Surgeons and biomedical engineers have for years looked for better ways —including antibiotic coatings — to reduce the risk of infections that are a known complication of implanting artificial hip, knee, and shoulder joints. Every year in the U.S., an estimated 1 to 2 percent of the more than 1 million hip and knee replacement surgeries are followed by infections linked to the formation of biofilms — layers of bacteria that adhere to a surface, forming a dense, impenetrable matrix of proteins, sugars and DNA. Immediately after surgery, an acute infection causes swelling and redness that can often be treated with intravenous antibiotics. But in some people, low-grade chronic infections can last for months, causing bone loss that leads to implant loosening and ultimately failure of the new prosthesis. These infections are very difficult to treat and, in many cases of chronic infection, prostheses must be removed and patients placed on long courses of antibiotics before a new prosthesis can be implanted. The cost per patient often exceeds $100,000 to treat a biofilm-associated prosthesis infection, Miller says. Major downsides to existing options for local antibiotic delivery, such as antibiotic-loaded cement, beads, spacers or powder, during the implantation of medical devices are that they can typically only deliver one antibiotic at a time and the release rate is not well-controlled. To develop a better approach that addresses those problems, Miller teamed up with Hai-Quan Mao, Ph.D., a professor of materials science and engineering at the Johns Hopkins University Whiting School of Engineering, and a member of the Institute for NanoBioTechnology, Whitaker Biomedical Engineering Institute and Translational Tissue Engineering Center. Over three years, the team focused on designing a thin, biodegradable plastic coating that could release multiple antibiotics at desired rates. This coating is composed of a nanofiber mesh embedded in a thin film; both components are made of polymers used for degradable sutures. To test the technology’s ability to prevent infection, the researchers loaded the nanofiber coating with the antibiotic rifampin in combination with one of three other antibiotics: vancomycin, daptomycin, or linezolid. “Rifampin has excellent anti-biofilm activity but cannot be used alone because bacteria would rapidly develop resistance,” says Miller. The coatings released vancomycin, daptomycin or linezolid for seven to 14 days and rifampin over three to five days. “We were able to deploy two antibiotics against potential infection while ensuring rifampin was never present as a single agent,” Miller says. The team then used each combination to coat titanium Kirschner wires — a type of pin used in orthopedic surgery to fix bone in place after wrist fractures — inserted them into the knee joints of anesthetized mice and introduced a strain of Staphylococcus aureus, a bacterium that commonly causes biofilm-associated infections in orthopedic surgeries. The bacteria were engineered to give off light, allowing the researchers to noninvasively track infection over time. Miller says that after 14 days of infection in mice that received an antibiotic-free coating on the pins, all of the mice had abundant bacteria in the infected tissue around the knee joint, and 80 percent had bacteria on the surface of the implant. In contrast, after the same time period in mice that received pins with either linezolid-rifampin or daptomycin-rifampin coating, none of the mice had detectable bacteria either on the implants or in the surrounding tissue. “We were able to completely eradicate infection with this coating,” says Miller. “Most other approaches only decrease the number of bacteria but don’t generally or reliably prevent infections.” After the two-week test, each of the rodents’ joints and adjacent bones were removed for further study. Miller and Mao found that not only had infection been prevented, but the bone loss often seen near infected joints — which creates the prosthetic loosening in patients — had also been completely avoided in animals that received pins with the antibiotic-loaded coating. Miller emphasized that further research is needed to test the efficacy and safety of the coating in humans, and in sorting out which patients would best benefit from the coating — people with a previous prosthesis joint infection receiving a new replacement joint, for example. The polymers they used to generate the nanofiber coating have already been used in many approved devices by the U.S. Food and Drug Administration, such as degradable sutures, bone plates, and drug delivery systems. In addition to Miller and Mao, the study’s authors are Alyssa Ashbaugh, Xuesong Jiang, Jesse Zheng, Andrew Tsai, Woo-Shin Kim, John Thompson, Robert Miller, Jonathan Shahbazian, Yu Wang, Carly Dillen, Alvaro Ordonez, Yong Chang, Sanjay Jain, Lynne Jones, and Robert Sterling of The Johns Hopkins University. Funding for this work was provided by a Nexus Award from the Johns Hopkins Institute for Clinical and Translational Research, which has been funded by the National Center for Advancing Translational Sciences and the National Institutes of Health Roadmap for Medical Research.


News Article | October 25, 2016
Site: www.rdmag.com

In a proof-of-concept study with mice, scientists at The Johns Hopkins University show that a novel coating they made with antibiotic-releasing nanofibers has the potential to better prevent at least some serious bacterial infections related to total joint replacement surgery. A report on the study, published online the week of Oct. 24 in Proceedings of the National Academy of Sciences, was conducted on the rodents' knee joints, but, the researchers say, the technology would have "broad applicability" in the use of orthopaedic prostheses, such as hip and knee total joint replacements, as well pacemakers, stents and other implantable medical devices. In contrast to other coatings in development, the researchers report the new material can release multiple antibiotics in a strategically timed way for an optimal effect. "We can potentially coat any metallic implant that we put into patients, from prosthetic joints, rods, screws and plates to pacemakers, implantable defibrillators and dental hardware," says co-senior study author Lloyd S. Miller, M.D., Ph.D., an associate professor of dermatology and orthopaedic surgery at the Johns Hopkins University School of Medicine. Surgeons and biomedical engineers have for years looked for better ways --including antibiotic coatings -- to reduce the risk of infections that are a known complication of implanting artificial hip, knee and shoulder joints. Every year in the U.S., an estimated 1 to 2 percent of the more than 1 million hip and knee replacement surgeries are followed by infections linked to the formation of biofilms -- layers of bacteria that adhere to a surface, forming a dense, impenetrable matrix of proteins, sugars and DNA. Immediately after surgery, an acute infection causes swelling and redness that can often be treated with intravenous antibiotics. But in some people, low-grade chronic infections can last for months, causing bone loss that leads to implant loosening and ultimately failure of the new prosthesis. These infections are very difficult to treat and, in many cases of chronic infection, prostheses must be removed and patients placed on long courses of antibiotics before a new prosthesis can be implanted. The cost per patient often exceeds $100,000 to treat a biofilm-associated prosthesis infection, Miller says. Major downsides to existing options for local antibiotic delivery, such as antibiotic-loaded cement, beads, spacers or powder, during the implantation of medical devices are that they can typically only deliver one antibiotic at a time and the release rate is not well-controlled. To develop a better approach that addresses those problems, Miller teamed up with Hai-Quan Mao, Ph.D., a professor of materials science and engineering at the Johns Hopkins University Whiting School of Engineering, and a member of the Institute for NanoBioTechnology, Whitaker Biomedical Engineering Institute and Translational Tissue Engineering Center. Over three years, the team focused on designing a thin, biodegradable plastic coating that could release multiple antibiotics at desired rates. This coating is composed of a nanofiber mesh embedded in a thin film; both components are made of polymers used for degradable sutures. To test the technology's ability to prevent infection, the researchers loaded the nanofiber coating with the antibiotic rifampin in combination with one of three other antibiotics: vancomycin, daptomycin or linezolid. "Rifampin has excellent anti-biofilm activity but cannot be used alone because bacteria would rapidly develop resistance," says Miller. The coatings released vancomycin, daptomycin or linezolid for seven to 14 days and rifampin over three to five days. "We were able to deploy two antibiotics against potential infection while ensuring rifampin was never present as a single agent," Miller says. The team then used each combination to coat titanium Kirschner wires -- a type of pin used in orthopaedic surgery to fix bone in place after wrist fractures -- inserted them into the knee joints of anesthetized mice and introduced a strain of Staphylococcus aureus, a bacterium that commonly causes biofilm-associated infections in orthopaedic surgeries. The bacteria were engineered to give off light, allowing the researchers to noninvasively track infection over time. Miller says that after 14 days of infection in mice that received an antibiotic-free coating on the pins, all of the mice had abundant bacteria in the infected tissue around the knee joint, and 80 percent had bacteria on the surface of the implant. In contrast, after the same time period in mice that received pins with either linezolid-rifampin or daptomycin-rifampin coating, none of the mice had detectable bacteria either on the implants or in the surrounding tissue. "We were able to completely eradicate infection with this coating," says Miller. "Most other approaches only decrease the number of bacteria but don't generally or reliably prevent infections." After the two-week test, each of the rodents' joints and adjacent bones were removed for further study. Miller and Mao found that not only had infection been prevented, but the bone loss often seen near infected joints -- which creates the prosthetic loosening in patients -- had also been completely avoided in animals that received pins with the antibiotic-loaded coating. Miller emphasized that further research is needed to test the efficacy and safety of the coating in humans, and in sorting out which patients would best benefit from the coating -- people with a previous prosthesis joint infection receiving a new replacement joint, for example. The polymers they used to generate the nanofiber coating have already been used in many approved devices by the U.S. Food and Drug Administration, such as degradable sutures, bone plates and drug delivery systems.


Barreto-Ortiz S.F.,Johns Hopkins University | Zhang S.,Johns Hopkins University | Zhang S.,Translational Tissue Engineering Center | Davenport M.,Johns Hopkins University | And 6 more authors.
PLoS ONE | Year: 2013

In microvascular vessels, endothelial cells are aligned longitudinally whereas several components of the extracellular matrix (ECM) are organized circumferentially. While current three-dimensional (3D) in vitro models for microvasculature have allowed the study of ECM-regulated tubulogenesis, they have limited control over topographical cues presented by the ECM and impart a barrier for the high-resolution and dynamic study of multicellular and extracellular organization. Here we exploit a 3D fibrin microfiber scaffold to develop a novel in vitro model of the microvasculature that recapitulates endothelial alignment and ECM deposition in a setting that also allows the sequential co-culture of mural cells. We show that the microfibers' nanotopography induces longitudinal adhesion and alignment of endothelial colony-forming cells (ECFCs), and that these deposit circumferentially organized ECM. We found that ECM wrapping on the microfibers is independent of ECFCs' actin and microtubule organization, but it is dependent on the curvature of the microfiber. Microfibers with smaller diameters (100-400 μm) guided circumferential ECM deposition, whereas microfibers with larger diameters (450 μm) failed to support wrapping ECM. Finally, we demonstrate that vascular smooth muscle cells attached on ECFC-seeded microfibers, depositing collagen I and elastin. Collectively, we establish a novel in vitro model for the sequential control and study of microvasculature development and reveal the unprecedented role of the endothelium in organized ECM deposition regulated by the microfiber curvature. © 2013 Barreto-Ortiz et al.


Jiang X.,Johns Hopkins University | Jiang X.,Translational Tissue Engineering Center | Christopherson G.T.,Johns Hopkins University | Mao H.-Q.,Johns Hopkins University | And 2 more authors.
Interface Focus | Year: 2011

Previous studies have shown that substrate surface chemistry and topography exhibit significant impact on haematopoietic progenitor cell adhesion, proliferation and differentiation. In the present study, the effect of surface amine density and structure of grafted polymer chains on the adhesion and expansion of haematopoietic progenitor cells was investigated. Cryopreserved human umbilical cord blood CD133 + cells were expanded in cytokine-supplemented medium on ethylenediamine (EDA)- or 2-aminoethyl methacrylate hydrochloride (AEMA)-grafted polyethersulphone (PES) nanofibre scaffolds for 10 days. Although the percentage of CD34 + cells among the expanded cells increased with the surface amine density, the maximum fold expansion of CD34 + cells was obtained at a moderate amine density of 20-80 nmol cm -2.When comparing nanofibre matrices with similar amine densities, but prepared with two different methods, cells cultured on the AEMA-grafted PES nanofibre matrix showed lower fold expansion in terms of total cell number (300+84 fold) and CD34{thorn} cell number (68+19-fold) in comparison with those cultured on EDA-grafted nanofibres (787+84-fold and 185+84-fold, respectively). These results indicate that the surface amine density and the conjugate structure are important determinants for the preservation of CD34 surface marker and expansion efficiency of CD34 + cells. © 2011 The Royal Society.


Ren Y.-J.,Johns Hopkins University | Ren Y.-J.,Translational Tissue Engineering Center | Zhang S.,Johns Hopkins University | Zhang S.,Translational Tissue Engineering Center | And 7 more authors.
Acta Biomaterialia | Year: 2013

Human pluripotent stem cell-derived neural crest stem cells (NCSCs) provide a promising cell source for generating Schwann cells in the treatment of neurodegenerative diseases and traumatic injuries in the peripheral nervous system. Influencing cell behavior through a synthetic matrix topography has been shown to be an effective approach to directing stem cell proliferation and differentiation. Here we have investigated the effect of nanofiber topography on the differentiation of human embryonic stem cellderived NCSCs towards the Schwann cell lineage. Using electrospun fibers of different diameters and alignments we demonstrated that aligned fiber matrices effectively induced cell alignment, and that fiber matrices with average diameters of 600 nm and 1.6 lm most effectively promoted NCSC differentiation towards the Schwann cell lineage compared with random fibers and two-dimensional tissue culture plates. More importantly, human NCSCs that were predifferentiated in Schwann cell medium for 2 weeks exhibited higher sensitivity to the aligned fiber topography than undifferentiated NCSCs. This study provides an efficient protocol for Schwann cell derivation by combining an aligned nanofiber matrix and an optimized differentiation medium, and highlights the importance of matching extrinsic matrix signaling with cell intrinsic programming in a temporally specific manner. © 2013 Acta Materialia Inc. Published by Elsevier Ltd.


PubMed | Johns Hopkins University and Translational Tissue Engineering Center
Type: Journal Article | Journal: Biomaterials science | Year: 2016

Central nervous system (CNS) diseases and injuries are accompanied by reactive gliosis and scarring involving the activation and proliferation of astrocytes to form hypertrophic and dense structures, which present a significant barrier to neural regeneration. Engineering astrocytes to functional neurons or oligodendrocytes may constitute a novel therapeutic strategy for CNS diseases and injuries. Such direct cellular programming has been successfully demonstrated using viral vectors via the transduction of transcriptional factors, such as Sox2, which could program resident astrocytes into neurons in the adult brain and spinal cord, albeit the efficiency was low. Here we report a non-viral nanoparticle-based transfection method to deliver Sox2 or Olig2 into primary human astrocytes and demonstrate the effective conversion of the astrocytes into neurons and oligodendrocyte progenitors following the transgene expression of Sox2 and Olig2, respectively. This approach is highly translatable for engineering astrocytes to repair injured CNS tissues.


PubMed | Virginia Commonwealth University, Johns Hopkins University and Translational Tissue Engineering Center
Type: | Journal: Biomaterials | Year: 2016

Strategies to enhance survival and direct the differentiation of stem cells invivo following transplantation in tissue repair site are critical to realizing the potential of stem cell-based therapies. Here we demonstrated an effective approach to promote neuronal differentiation and maturation of human fetal tissue-derived neural stem cells (hNSCs) in a brain lesion site of a rat traumatic brain injury model using biodegradable nanoparticle-mediated transfection method to deliver key transcriptional factor neurogenin-2 to hNSCs when transplanted with a tailored hyaluronic acid (HA) hydrogel, generating larger number of more mature neurons engrafted to the host brain tissue than non-transfected cells. The nanoparticle-mediated transcription activation method together with an HA hydrogel delivery matrix provides a translatable approach for stem cell-based regenerative therapy.


News Article | November 18, 2016
Site: www.rdmag.com

Bone regeneration is a critical need in the United States. Each year, 209,000 procedures require craniomaxillofacial grafts for patients undergoing facial reconstructive surgery. Currently the sole option surgeons have is to take another bone, break it and fit it into a patient's facial structure, which is not the ideal solution. In comes Prof. Warren Grayson, Ph.D., and his team who have created a ready-to-implant plastic bone that can turn into living tissue and dramatically improve life for patients. “Can we regenerate bone with appropriate anatomical shape and vascular supply?” Grayson asked during his presentation at the second annual R&D 100 Conference that took place earlier this month at the Gaylord National Resort & Convention Center in Washington, D.C. The answer is yes. In his presentation titled “3D Printing and the Search for Better Bone Replacement,” Grayson spoke about breakthrough technologies that allow for precise control of the cellular microenvironment and addressed fundamental questions regarding the application of biophysical cues to regulate stem cell differentiation. The Associate Professor in the Department of Biomedical Engineering & Translational Tissue Engineering Center at Johns Hopkins University School of Medicine and his team at the Grayson Lab have developed a successful combination for 3D bone printing by creating an effective framework for filling in missing bone. They have achieved this goal by mixing at least 30 percent of pulverized natural bone with special man-made plastic, which then they molded into the necessary shape using a 3D printer. While the world of 3D bioprinting is still very new and ambiguous, a few scientists have started utilizing this cutting-edge technology for various areas of regenerative medicine to help fill the tissue-and-organ shortage void. In their experiments on mice, Grayson and his team decided to make a composite material that would combine the strength and printability of plastic with the biological “information” contained in natural bone. The team held several experiments before finding a successful combination. The scaffolds were tested on rodents with relatively large holes in their skulls where bones were input experimentally. Without intervention, the bone wounds were too large to heal. Mice that got scaffold implants laden with stem cells saw new bone growth within the hole over the 12 weeks of the experiment. CT scans showed that at least 50 percent more bone grew in scaffolds containing 30 or 70 percent bone powder, compared to those with pure Polycaprolactone (PCL- a biodegradable polyester). Grayson’s work represents a significant step forward in the 3D printing of human bones. The Grayson Lab also conducts research on stem cell biology, bioreactor technology and scaffold development.

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