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Biomedical subjects

Robert Langer

Publications and source records attributed to Robert Langer.

At least 91 records · Page 5Linked to original sources

Direct patterning of mammalian cells onto porous tissue engineering substrates using agarose stamps.

This paper describes simple, inexpensive, and potentially generic methodology for generating patterns of mammalian cells on porous scaffolds for tissue engineering using replica printing. Circular patterns (diameter: 200, 700, and 1000 microm) of human osteoblasts were transferred directly from topographically patterned agarose stamps onto porous hydroxyapatite scaffolds or onto fibronectin-coated glass slides. The use of hydrogel stamps provided a "wet", biocompatible surface and maintained the viability of cells adsorbed on stamps during the patterning process. Stamps inked once with suspensions of cells allowed the repeated patterning of substrates. Direct stamping of human osteoblasts (and, potentially other mammalian cells) can be used to control the size, spacing, and geometry of patterns of cells printed on porous tissue engineering substrates. This approach may find use in controlling the spatial invasion of scaffolds, promoting the hierarchical organization of cells, and in controlling cell-cell interactions as a step in preservation of phenotypes of cells.

Actins↗

Structure/property studies of polymeric gene delivery using a library of poly(beta-amino esters).

Here we describe the synthesis and characterization of a library of 486 second-generation poly(beta-amino esters). To understand better the structure/property relationships governing polymeric gene delivery, we synthesized polymers with 70 different primary structures, at 6 to 12 different molecular weights, using monomers previously identified as common to effective gene delivery polymers. This library was characterized by (1) molecular weight, (2) particle size upon complexation with DNA, (3) surface charge upon complexation with DNA, (4) optimal polymer/DNA ratio, and (5) transfection efficiency. In this library, polymers with 20 of the 70 primary structures possess transfection efficiencies as good as or better than one of the best commercially available lipid reagents, Lipofectamine 2000. In general, the most effective polymers condense DNA into sub-150-nm complexes with positive surface charge. Among this group, the 2 most effective polymers condensed DNA to the smallest particle sizes (71 and 79 nm). Interestingly, the top 9 polymers were all formed from amino alcohols, and the structure of the 3 top performing polymers differs by only one carbon. This convergence in structure of the top performing polymers suggests a common mode of action and provides a framework with which future polymers can be designed.

Animals↗

Beta-amino ester polymers facilitate in vivo DNA transfection and adjuvant plasmid DNA immunization.

Increased in vivo expression of intramuscularly delivered plasmid DNA will be essential for clinical success in gene therapy and plasmid DNA vaccination. We screened polymers from a library of beta-amino esters for their ability to augment transgene expression as measured by beta-galactosidase activity and cellular immune responses. Among the candidates identified in this screen, poly[(1,6-di(acryloxyethoxy)hexane)-co-(4-aminobutanol)] enhanced plasmid DNA transgene expression by sevenfold (P=0.0001) and its immunogenicity by 70% (P=0.03). We found that polymers with moderately hydrophobic backbones and terminal alcohol groups facilitated transfection most effectively in vivo. We also observed a log-linear correlation (R2=0.93) between peak cellular immune responses and transgene activity in all evaluated polymer-plasmid DNA formulations, clarifying the relationship between immunogenicity and the quantity of expressed antigen.

Animals↗

Midmembranous vocal fold lamina propria proteoglycans across selected species.

OBJECTIVES: We examined the proteoglycan (PG) and associated sulfated glycosaminoglycan (GAG) content of the midmembranous vocal fold lamina propria (LP) of humans, dogs, pigs, and ferrets. METHODS: The LP PG levels were assessed indirectly by quantifying the associated sulfated GAGs, and immunohistochemical analyses of specific PGs and/or GAGs (PGs/GAGs) were conducted. RESULTS: Sulfated GAGs constituted approximately (average +/- SEM) 14.7 +/- 2.1 microg per milligram of tissue total protein in the human LP--similar to levels in canine, porcine, and ferret LPs (p > .05). Immunohistochemical analysis identified versican, chondroitin 4- and 6-sulfate, and heparan sulfate in the LP extracellular matrix--PGs/GAGs previously believed to be localized only intracellularly and in the basement membrane. Observations of PG/GAG staining patterns resulted in identification of microstructurally based subdivisions of canine, porcine, and ferret LPs. CONCLUSIONS: The sulfated GAG concentration in human LP was similar to that of dermis. In contrast to the interspecies similarity in LP sulfated GAG levels, immunohistochemical analysis indicated notable interspecies differences in specific PG/GAG distributions. Moreover, spatial variations in the presence of several PGs/GAGs were observed--variations that may be integral in maintaining normal LP physiology. Finally, the noted canine, porcine, and ferret LP subdivisions may yield insight into the adaptation of LP microstructure to the phonatory needs of each species.

Adult↗

Functional assembly of engineered myocardium by electrical stimulation of cardiac myocytes cultured on scaffolds.

The major challenge of tissue engineering is directing the cells to establish the physiological structure and function of the tissue being replaced across different hierarchical scales. To engineer myocardium, biophysical regulation of the cells needs to recapitulate multiple signals present in the native heart. We hypothesized that excitation-contraction coupling, critical for the development and function of a normal heart, determines the development and function of engineered myocardium. To induce synchronous contractions of cultured cardiac constructs, we applied electrical signals designed to mimic those in the native heart. Over only 8 days in vitro, electrical field stimulation induced cell alignment and coupling, increased the amplitude of synchronous construct contractions by a factor of 7, and resulted in a remarkable level of ultrastructural organization. Development of conductive and contractile properties of cardiac constructs was concurrent, with strong dependence on the initiation and duration of electrical stimulation.

Animals↗

Repeated in vivo electrochemical activation and the biological effects of microelectromechanical systems drug delivery device.

The repeated activation of a microelectromechanical systems (MEMS) drug delivery device was studied in vivo in rats to examine the effect of implantation on the device operation and the effect of electrochemical activation on the inflammatory and wound-healing response. The MEMS devices were fabricated from a silicon wafer into which reservoirs were etched and covered with gold membranes. The membranes were electrochemically removed when an anodic voltage was applied. Devices were implanted subcutaneously both with and without stainless steel mesh cages for 4, 7, 14, 21, or 28 days before activation. Devices were activated every other day for five activations. Leukocyte concentrations indicated that both the application of voltage and the gold corrosion products elevated the inflammatory response which was resolved within 48 h after each activation. The efficiency of gold membrane removal was not impaired throughout the implantation, although a bimodal distribution of background current densities was observed after long implantation times. The thickness of the fibrous capsule surrounding the MEMS devices was similar between activated and control devices explanted at each time point. It was concluded that the repeated activation of MEMS drug delivery devices was successful and the activation produced an acceptable biological response that resolved promptly.

Animals↗

Adhesion-mediated signal transduction in human articular chondrocytes: the influence of biomaterial chemistry and tenascin-C.

Chondrocyte 'dedifferentiation' involves the switching of the cell phenotype to one that no longer secretes extracellular matrix found in normal cartilage and occurs frequently during chondrocyte expansion in culture. It is also characterized by the differential expression of receptors and intracellular proteins that are involved in signal transduction pathways, including those associated with cell shape and actin microfilament organization. The objective of this study was to examine the modulation of chondrocyte phenotype by cultivation on polymer substrates containing poly(ethylene glycol) (PEG). We observed differential arrangement of actin organization in articular chondrocytes, depending on PEG length. When cultivated on 300 g/mol PEG substrates at day 19, chondrocytes had lost intracellular markers characteristic of the differentiated phenotype, including type II collagen and protein kinase C (PKC). On these surfaces, chondrocytes also expressed focal adhesion and signaling proteins indicative of cell attachment, spreading, and FA turnover, including RhoA, focal adhesion kinase, and vinculin. The switch to a dedifferentiated chondrocyte phenotype correlated with integrin expression. Conversely, the expression of CD44 receptors coincided with chondrogenic characteristics, suggesting that binding via these receptors could play a role in maintaining the differentiated phenotype on such substrates. These effects can be similar to those of compounds that interfere in intracellular signaling pathways and can be utilized to engineer cellular response.

Actins↗

In vivo release from a drug delivery MEMS device.

A drug delivery microelectromechanical systems (MEMS) device was designed to release complex profiles of multiple substances in order to maximize the effectiveness of drug therapies. The device is based on micro-reservoirs etched into a silicon substrate that contain individual doses of drug. Each dose is released by the electrochemical dissolution of the gold membrane that covers the reservoir. The first in vivo operation of this device was reported in this study. Subcutaneous release was demonstrated in rats using two tracer molecules, fluorescein dye and radiolabeled mannitol, and one radiolabeled chemotherapeutic agent, carmustine (BCNU). BCNU was chosen because of the need to improve the direct delivery of chemotherapy to malignant tumors. The spatial profile of fluorescein dye release from the drug delivery device was evaluated by fluorimetry, the temporal profile of 14C labeled mannitol release was evaluated by liquid scintillation counting, and the temporal profile of 14C labeled BCNU release was evaluated by accelerator mass spectrometry (AMS). Release profiles obtained from injected controls were compared with those from activated devices. The in vivo dye release results showed high concentration of fluorescein in the flank tissue surrounding the devices 1 h after activation. The 14C labeled mannitol released from the drug delivery devices was rapidly cleared (1 day) from the rat urine. In vivo release of 14C labeled BCNU from activated devices showed slightly slower kinetics than the injected and in vitro controls, and the time to reach the steady-state plasma 14C concentration was on the order of 1 h. All these results demonstrated the capability of this drug delivery device to achieve localized delivery of various compounds with well-defined temporal profiles.

Animals↗

Mathematical model of oxygen distribution in engineered cardiac tissue with parallel channel array perfused with culture medium containing oxygen carriers.

A steady-state model of oxygen distribution in a cardiac tissue construct with a parallel channel array was developed and solved for a set of parameters using the finite element method and commercial software (FEMLAB). The effects of an oxygen carrier [Oxygent; 32% volume perfluorocarbon (PFC) emulsion] were evaluated. The parallel channel array mimics the in vivo capillary tissue bed, and the PFC emulsion has a similar role as the natural oxygen carrier hemoglobin in increasing total oxygen content. The construct was divided into an array of cylindrical domains with a channel in the center and tissue space surrounding the channel. In the channel, the main modes of mass transfer were axial convection and radial diffusion. In the tissue region, mass transfer was by axial and radial diffusion, and the consumption of oxygen was by Michaelis-Menten kinetics. Neumann boundary conditions were imposed at the channel centerline and the half distance between the domains. Supplementation of culture medium by PFC emulsion improved mass transport by increasing convective term and effective diffusivity of culture medium. The model was first implemented for the following set of experimentally obtained parameters: construct thickness of 0.2 cm, channel diameter of 330 mum, channel center-to-center spacingof 700 microm, and average linear velocity per channel of 0.049 cm/s, in conjunction with PFC supplemented and unsupplemented culture medium. Subsequently, the model was used to define favorable scaffold geometry and flow conditions necessary to cultivate cardiac constructs of high cell density (10(8) cells/ml) and clinically relevant thickness (0.5 cm). In future work, the model can be utilized as a tool for optimization of scaffold geometry and flow conditions.

Animals↗

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds.

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.

Absorbable Implants↗

A polymer library approach to suicide gene therapy for cancer.

Optimal gene therapy for cancer must (i) deliver DNA to tumor cells with high efficiency, (ii) induce minimal toxicity, and (iii) avoid gene expression in healthy tissues. To this end, we generated a library of >500 degradable, poly(beta-amino esters) for potential use as nonviral DNA vectors. Using high-throughput methods, we screened this library in vitro for transfection efficiency and cytotoxicity. We tested the best performing polymer, C32, in mice for toxicity and DNA delivery after intratumor and i.m. injection. C32 delivered DNA intratumorally approximately 4-fold better than one of the best commercially available reagents, jetPEI (polyethyleneimine), and 26-fold better than naked DNA. Conversely, the highest transfection levels after i.m. administration were achieved with naked DNA, followed by polyethyleneimine; transfection was rarely observed with C32. Additionally, polyethyleneimine induced significant local toxicity after i.m. injection, whereas C32 demonstrated no toxicity. Finally, we used C32 to deliver a DNA construct encoding the A chain of diphtheria toxin (DT-A) to xenografts derived from LNCaP human prostate cancer cells. This construct regulates toxin expression both at the transcriptional level by the use of a chimeric-modified enhancer/promoter sequence of the human prostate-specific antigen gene and by DNA recombination mediated by Flp recombinase. C32 delivery of the A chain of diphtheria toxin DNA to LNCaP xenografts suppressed tumor growth and even caused 40% of tumors to regress in size. Because C32 transfects tumors locally at high levels, transfects healthy muscle poorly, and displays no toxicity, it may provide a vehicle for the local treatment of cancer.

Animals↗

Nanoparticle-aptamer bioconjugates: a new approach for targeting prostate cancer cells.

Nucleic acid ligands (aptamers) are potentially well suited for the therapeutic targeting of drug encapsulated controlled release polymer particles in a cell- or tissue-specific manner. We synthesized a bioconjugate composed of controlled release polymer nanoparticles and aptamers and examined its efficacy for targeted delivery to prostate cancer cells. Specifically, we synthesized poly(lactic acid)-block-polyethylene glycol (PEG) copolymer with a terminal carboxylic acid functional group (PLA-PEG-COOH), and encapsulated rhodamine-labeled dextran (as a model drug) within PLA-PEG-COOH nanoparticles. These nanoparticles have the following desirable characteristics: (a) negative surface charge (-50 +/- 3 mV, mean +/- SD, n = 3), which may minimize nonspecific interaction with the negatively charged nucleic acid aptamers; (b) carboxylic acid groups on the particle surface for potential modification and covalent conjugation to amine-modified aptamers; and (c) presence of PEG on particle surface, which enhances circulating half-life while contributing to decreased uptake in nontargeted cells. Next, we generated nanoparticle-aptamer bioconjugates with RNA aptamers that bind to the prostate-specific membrane antigen, a well-known prostate cancer tumor marker that is overexpressed on prostate acinar epithelial cells. We demonstrated that these bioconjugates can efficiently target and get taken up by the prostate LNCaP epithelial cells, which express the prostate-specific membrane antigen protein (77-fold increase in binding versus control, n = 150 cells per group). In contrast to LNCaP cells, the uptake of these particles is not enhanced in cells that do not express the prostate-specific membrane antigen protein. To our knowledge, this represents the first report of targeted drug delivery with nanoparticle-aptamer bioconjugates.

Antigens, Surface↗

Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds.

Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation.

Absorbable Implants↗

Injectable microparticle-gel system for prolonged and localized lidocaine release. I. In vitro characterization.

Current treatment protocol for postoperative pain is to infuse anesthetic solution around nerves or into the epidural space. This clinical practice is beset by the short duration of the anesthetic effect unless the infusion is continuous. Continuous infusion, however, requires hospitalization of the patients, thereby increasing medical costs. In addition, it also causes systemic accumulation of the drug. We reported herein a novel treatment for the postoperative pain by applying to the surgical site a biodegradable microsphere-gel system for prolonged and localized release of encapsulated anesthetic drugs. This lidocaine-containing biodegradable poly(D,L-lactic acid) (PLA) microsphere system, although being established previously by other investigators, was hindered by a burst release and a followed rapid release of the drug within several hours in vitro. In this article, we demonstrated that by a step-by-step modification of the formulation, prolonged release of lidocaine, up to several days in vitro, could be achieved. Differential scanning calorimetry revealed a lower glass transition temperature for these lidocaine-loaded microspheres comparing to that of lidocaine-free microspheres. This decreased Tg explained for the tendency of the lidocaine-loaded microspheres to physically fuse at higher temperatures. In vitro studies showed that microspheres, when loaded with 35% lidocaine, yielded a threefold increase in the degradation rate. The molecular weight of PLA of the drug-loaded microspheres was reduced by 50% within a period of 1 month. Based on the results (of prolonged lidocaine release and rapid PLA microsphere degradation), this lidocaine-loaded PLA microsphere system could offer a simple solution to the treatment of postoperative pain.

Anesthetics, Local↗

Injectable microparticle-gel system for prolonged and localized lidocaine release. II. In vivo anesthetic effects.

Current treatment protocols for postoperative pain are beset by either the short duration of the anesthetic effect or requirement of hospitalization of the patients. We reported herein a novel treatment by applying to the surgical site a biodegradable microparticle-gel system for prolonged and localized release of encapsulated anesthetic drugs. In a previous publication, lidocaine-loaded poly(D,L-lactic acid) microspheres were fabricated and their formulations were optimized. In vitro characterization of these lidocaine-loaded microspheres, however, revealed a shortcoming of this system; that is, microspheres tend to fuse physically. Fusion of the microspheres could hinder their clinical applications, as it would clog the needle. In this article, we demonstrated that fabricating microspheres with high molecular weight (approximately 60 KDa) poly(lactic-co-glycolic acid) would increase the glass transition temperature of the microspheres after lidocaine loading, thereby increasing their mechanical stability and eliminating their fusion during storage. Such microspheres containing 31% (w/w) lidocaine in the presence or absence of 25% (w/v) poloxamer 407 gel were then evaluated in vivo by monitoring the sensory and motor functions of the rats after sciatic nerve block, using the previously established hot-plate and weight-bearing testing methods. Results showed that microspheres formulated with poloxamer 407 gel yielded the longest duration of sensory and motor block for a period of approximately 8.5 h, compared to 5 h by microspheres in saline, 5 h by lidocaine in poloxamer 407 gel, and 2 h by lidocaine in saline. This study suggests that the microsphere-gel system containing lidocaine could potentially be applied clinically to the treatment of postoperative pain.

Anesthetics, Local↗

pH-triggered microparticles for peptide vaccination.

Improving vaccine delivery to human APCs is a way to increase the CTL response to vaccines. We report the use of a novel pH-triggered microparticle that exploits the ability of APCs to cross-present MHC I-restricted Ags that have been engulfed in the low pH environment of the phagosome. A model MHC class I-restricted peptide Ag from the influenza A matrix protein was encapsulated in spray-dried microparticles composed of dipalmitoylphosphatidylcholine and the pH-sensitive polymethacrylate Eudragit E100. Release of the peptide from the particle was triggered by a drop in pH to the acidity normally found in the phagosome. The particles were efficiently phagocytosed by human monocytes and dendritic cells with minimal cellular toxicity and no functional impairment. Encapsulation of the peptide in the microparticles resulted in efficient presentation of the peptide to CD8(+) T cells by human dendritic cells in vitro, and was superior to unencapsulated peptide or peptide encapsulated in an analogous pH-insensitive particle. Vaccination of human HLA-A*0201 transgenic mice with peptide encapsulated in pH-triggering microparticles resulted in priming of CTL responses. These microparticles can be modified to coencapsulate a range of adjuvants along with the Ag of interest. Encapsulation of MHC I epitopes in pH-triggered microparticles increases Ag presentation and may improve CD8(+) T cell priming to peptide vaccines against viruses and cancer.

1,2-Dipalmitoylphosphatidylcholine↗

Molded polyethylene glycol microstructures for capturing cells within microfluidic channels.

The ability to control the deposition and location of adherent and non-adherent cells within microfluidic devices is beneficial for the development of micro-scale bioanalytical tools and high-throughput screening systems. Here, we introduce a simple technique to fabricate poly(ethylene glycol)(PEG) microstructures within microfluidic channels that can be used to dock cells within pre-defined locations. Microstructures of various shapes were used to capture and shear-protect cells despite medium flow in the channel. Using this approach, PEG microwells were fabricated either with exposed or non-exposed substrates. Proteins and cells adhered within microwells with exposed substrates, while non-exposed substrates prevented protein and cell adhesion (although the cells were captured inside the features). Furthermore, immobilized cells remained viable and were stained for cell surface receptors by sequential flow of antibodies and secondary fluorescent probes. With its unique strengths in utility and control, this approach is potentially beneficial for the development of cell-based analytical devices and microreactors that enable the capture and real-time analysis of cells within microchannels, irrespective of cell anchorage properties.

Animals↗