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Biomedical subjects

R Langer

Publications and source records attributed to R Langer.

At least 127 records · Page 7Linked to original sources

A novel biotinylated degradable polymer for cell-interactive applications.

We describe the development of a novel biodegradable polymer designed to present bioactive motifs at the surfaces of materials of any architecture. The polymer is a block copolymer of biotinylated poly(ethylene glycol) (PEG) with poly(lactic acid) (PLA); it utilizes the high-affinity coupling of the biotin-avidin system to undergo postfabrication surface engineering. We show, using surface plasmon resonance analysis (SPR) and confocal microscopy that surface engineering can be achieved under aqueous conditions in short time periods. These surfaces interact with cell surface molecules and generate beneficial responses as demonstrated by the model study of integrin-mediated spreading of endothelial cells on polymer surfaces presenting RGD peptide adhesion sequences.

Animals↗

Drug delivery and targeting.

When a pharmaceutical agent is encapsulated within, or attached to, a polymer or lipid, drug safety and efficacy can be greatly improved and new therapies are possible. This has provided the impetus for active study of the design of degradable materials, intelligent delivery systems and approaches for delivery through different portals in the body.

Drug Administration Routes↗

Chondrogenesis in a cell-polymer-bioreactor system.

Chondrogenesis was studied under controlled in vitro conditions using a cell-polymer-bioreactor system. Bovine calf articular chondrocytes were seeded onto biodegradable polymer scaffolds and cultured in rotating bioreactor vessels. Concomitant increases in the amounts of glycosaminoglycan (GAG) and type II collagen resulted in cell-polymer constructs with continuous cartilaginous matrix over their entire cross sections (6.7 mm diameter x 5 mm thick) after 40 days of cultivation. As compared to natural calf cartilage, constructs had comparable cellularities, 68% as much GAG and 33% as much type II collagen per gram wet weight. The progression of chondrogenesis in chondrocyte-polymer constructs was similar to that suggested previously for precursor cells in vitro and developing limbs in vivo. In particular, the polymer scaffold provided a three-dimensional structure that could be seeded with chondrocytes at high cell densities in order to establish cell-to-cell contacts and initiate cartilage tissue development, whereas the bioreactor vessel provided a permissive microenvironment for chondrogenesis. This work demonstrates the promise of using tissue engineered constructs for in vitro studies of cell interactions and differentiation.

Animals↗

Relating the phagocytosis of microparticles by alveolar macrophages to surface chemistry: the effect of 1,2-dipalmitoylphosphatidylcholine.

This study examines the potential of 1,2-dipalmitoylphosphatidylcholine (DPPC), a major component of lung surfactant, to reduce the phagocytosis of microspheres by altering the cellular interactions occurring in the alveoli. These microspheres could be designed to act as a controlled delivery system for small molecules, peptides or proteins for pulmonary administration. Microspheres were prepared using poly (lactic-co-glycolic acid) (PLGA, 50/50 and encapsulated peroxidase as a model protein. DPPC was included in some formulations. The interaction of PLGA and DPPC-PLGA microspheres with phagocytic cells was evaluated using lung macrophages in culture. X-ray Photoelectron Spectra (XPS) results indicate that the inclusion of DPPC in the microspheres alters the microsphere surface chemistry, with the DPPC covering a large portion of the microsphere surface. The dominance of DPPC on the microsphere surface is highly beneficial in moderating the interaction occurring between the microspheres and phagocytic cells in the lung. Fluorescent confocal microscopy indicates that only 25% of cells internalized DPPC-coated particles, whereas 70% of those cells exposed to particles without the DPPC coating internalized particles after one hour of incubation.

1,2-Dipalmitoylphosphatidylcholine↗

A tissue-engineered conduit for peripheral nerve repair.

BACKGROUND: Peripheral nerve repair using autograft material has several shortcomings, including donor site morbidity, inadequate return of function, and aberrant regeneration. Recently, peripheral nerve research has focused on the generation of synthetic nerve guidance conduits that might overcome these phenomena to improve regeneration. In our laboratory, we use the unique chemical and physical properties of synthetic polymers in conjunction with the biological properties of Schwann cells to create a superior prosthesis for the repair of multiply branched peripheral nerves, such as the facial nerve. OBJECTIVES: To create a polymeric facial nerve analog approximating the fascicular architecture of the extratemporal facial nerve, to introduce a population of Schwann cells into the analog, and to implant the prosthesis into an animal model for assessment of regeneration. RESULTS: Tubes of poly-L-lactic acid (molecular weight, 100000) or polylactic-co-glycolic acid copolymer were formed using a dip-molding technique. They were created containing 1, 2, 4, or 5 sublumina, or "fascicular analogs." Populations of Schwann cells were isolated, expanded in culture, and plated onto these polymer films, where they demonstrated excellent adherence to the polymer surfaces. Regeneration was demonstrated through several constructs. CONCLUSIONS: A tubular nerve guidance conduit possessing the macroarchitecture of a polyfascicular peripheral nerve was created. The establishment of resident Schwann cells onto poly-L-lactic acid and polylactic-co-glycolic acid surfaces was demonstrated, and the feasibility of in vivo regeneration through the conduit was shown. It is hypothesized that these tissue-engineered devices, composed of widely used biocompatible, biodegradable polymer materials and adherent Schwann cells, will be useful in promoting both more robust and more precisely directed peripheral nerve regeneration.

Animals↗

Preliminary in vivo report on the osteocompatibility of poly(anhydride-co-imides) evaluated in a tibial model.

A novel class of polymers with mechanical properties similar to cancellous bone are being investigated for their ability to be used in weight-bearing areas for orthopedic applications. The poly(anhydride-co-imide) polymers based on poly[trimellitylimidoglycine-co-1,6-bis(carboxyphenoxy)hexan e] (TMA-Gly:CPH) and poly[pyromellitylimidoalanine-co-1,6-bis(carboxyphenoxy)hexa ne] (PMA-Ala:CPH) in molar ratios of 30:70 were investigated for osteocompatibility, with effects on the healing of unicortical 3-mm defects in rat tibias examined over a 30-day period. Defects were made with surgical drill bits (3-mm diameter) and sites were filled with poly(anhydride-co-imide) matrices and compared to the control poly(lactic acid-glycolic acid) (PLAGA) (50:50), a well-characterized matrix frequently used in bone regeneration studies, and defects without polymeric implants. At predetermined time intervals (3, 6, 9, 12, 20, and 30 days), animals were sacrificed and tissue histology was examined for bone formation, polymer-tissue interaction, and local tissue response by light microscopy. The studies revealed that matrices of TMA-Gly:CPH and PMA-Ala:CPH produced responses similar to the control PLAGA with tissue compatibility characterized by a mild response involving neutrophils, macrophages, and giant cells throughout the experiment for all matrices studied. Matrices of PLAGA were nearly completely degraded by 21 days in contrast to matrices of TMA-Gly:CPH and PMA-Ala:CPH that displayed slow erosion characteristics and maintenance of shape. Defects in control rats without polymer healed by day 12, defects containing PLAGA healed after 20 days, and defects containing poly(anhydride-co-imide) matrices produced endosteal bone growth as early as day 3 and formed bridges of cortical bone around matrices by 30 days. In addition, there was marrow reconstitution at the defect site for all matrices studied along with matured bone-forming cells. This study suggests that novel poly(anhydride-co-imides) are promising polymers that may be suitable for use as implants in bone surgery, especially in weight-bearing areas.

Animals↗

Altered membrane skeleton of hydroxyethylstarch-cryopreserved human erythrocytes.

Attempts have been made to use hydroxyethylstarch (HES) as an alternative to glycerol for cryopreservation of erythrocytes. However, HES cryopreservation causes significant transient rheological alterations in erythrocytes. Membrane proteins play a critical role for erythrocyte rheology. This study was undertaken to analyze erythrocyte membrane proteins during HES cryopreservation. Erythrocyte membranes with submembrane skeleton (ghosts) and the submembrane skeleton alone (unstripped skeletons) were prepared before freezing (native), after thawing and following 3 h reconditioning in glucose-enriched Ringer's solution (Ringer plus glucose), or in autologous fresh frozen plasma (AFFP). After electrophoresis protein concentrations (percentage of total protein) were determined by densitometry. In ghosts, no significant changes were found, whereas in unstripped skeletons the following results could be seen: beta-Spectrin: 31.8 +/- 2.2% (native), 22.1 +/- 0.8% (postthawing, P < 0.05 vs native), 22.4 +/- 1.6% (Ringer plus glucose, P < 0.05 vs native), 31.0 +/- 2.8% (AFFP). Other proteins remained unchanged. Since a significant decrease in beta-spectrin concentration after HES cryopreservation and after subsequent reconditioning in Ringer's solution with glucose was only detected in unstripped skeletons, this cannot be interpreted as in vivo protein loss. More likely, HES cryopreservation may have created changes in protein-protein associations. The course of beta-spectrin concentration parallels certain rheological and biochemical changes and might explain the transient rheological changes seen after HES cryopreservation.

Cryopreservation↗

Creation of viable pulmonary artery autografts through tissue engineering.

BACKGROUND: "Repair" of many congenital cardiac defects requires the use of conduits to establish right ventricle to pulmonary artery continuity. At present, available homografts or prosthetic conduits lack growth potential and can become obstructed by tissue ingrowth or calcification leading to the need for multiple conduit replacements. Tissue engineering is an approach by which cells are grown in vitro onto biodegradable polymers to construct "tissues" for implantation. A tissue engineering approach has recently been used to construct living cardiac valve leaflets from autologous cells in our laboratory. This study assesses the feasibility of a tissue engineering approach to constructing tissue-engineered "living" pulmonary artery conduits. MATERIALS AND METHODS: Ovine artery (group A, n = 4) or vein (group V, n = 3) segments were harvested, separated into individual cells, expanded in tissue culture, and seeded onto synthetic biodegradable (polyglactin/polyglycolic acid) tubular scaffolds (20 mm long x 15 mm diameter). After 7 days of in vitro culture, the autologous cell/polymer vascular constructs were used to replace a 2 cm segment of pulmonary artery in lambs (age 68.4 +/- 15.5 days, weight 18.7 +/- 2.0 kg). One other control animal received an acellular polymer tube sealed with fibrin glue without autologous cells. Animals were sacrificed at intervals of 11 to 24 weeks (mean follow-up 130.3 +/- 30.8 days, mean weight 38.9 +/- 13.0 kg) after echocardiographic and angiographic studies. Explanted tissue-engineered conduits were assayed for collagen (4-hydroxyproline) and calcium content, and a tissue deoxyribonucleic acid assay (bis-benzimide dye) was used to estimate number of cell nuclei as an index of tissue maturity. RESULTS: The acellular control graft developed progressive obstruction and thrombosis. All seven tissue-engineered grafts were patent and demonstrated a nonaneurysmal increase in diameter (group A = 18.3 +/- 1.3 mm = 95.3% of native pulmonary artery; group V = 17.1 +/- 1.2 mm = 86.8% of native pulmonary artery). Histologically, none of the biodegradable polymer scaffold remained in any tissue-engineered graft by 11 weeks. Collagen content in tissue-engineered grafts was 73.9% +/- 8.0% of adjacent native pulmonary artery. Histologically, elastic fibers were present in the media layer of tissue-engineered vessel wall and endothelial specific factor VIII was identified on the luminal surface. Deoxyribonucleic acid assay showed a progressive decrease in numbers of cell nuclei over 11 and 24 weeks, suggesting an ongoing tissue remodeling. Calcium content of tissue-engineered grafts was elevated (group A = 7.95 +/- 5.09; group V = 13.2 +/- 5.48; native pulmonary artery = 1.2 +/- 0.8 mg/gm dry weight), but no macroscopic calcification was found. CONCLUSIONS: Living vascular grafts engineered from autologous cells and biodegradable polymers functioned well in the pulmonary circulation as a pulmonary artery replacement. They demonstrated an increase in diameter suggesting growth and development of endothelial lining and extracellular matrix, including collagen and elastic fibers. This tissue-engineering approach may ultimately allow the development of viable autologous vascular grafts for clinical use.

Animals↗

Chemical changes during in vivo degradation of poly(anhydride-imide) matrices.

The in vivo degradation characteristics of a novel class of biodegradable polymers, poly(anhydride-imides), were investigated. The poly(anhydride-imides) examined were poly[trimellitylimidoglycine-co-1,6-bis(p-carboxyphenoxy)hex ane] (TMA-gly:CPH) in 10:90, 30:70 and 50:50 molar ratios and poly[pyromellitylimidoalanine-co-1,6-bis(p-carboxyphenoxy)he xane] (PMA-ala:CPH) in 10:90 and 30:70 molar ratios. The polymer matrices were compression-molded into circular discs, then implanted in rat subcutaneous tissues for nearly two months. At defined time intervals, the animals were sacrificed and explants analyzed. Proton NMR spectroscopic analysis revealed a complete absence of imide monomer units in PMA-ala: CPH compositions after 28 d and complete removal of imide units at 56 d from TMA-gly matrices. Gross observation of the implants closely correlated to the imide content: with decreasing imide content, the explants darkened and fragmented at a faster rate. The chemical compositions of the poly(anhydride imide) explants were also monitored using IR spectroscopy. The residual amount of anhydride bonds in the polymer backbone following implantation were calculated from peaks specific to the anhydride bonds relative to the total amount of carbonyl bonds present. Initially, the imide (TMA-gly or PMA-ala) anhydride bonds were rapidly hydrolyzed then solubilized, followed by the slower hydrolysis of the CPH monomer anhydride bonds.

Animals↗

Skin electroporation: rapid measurements of the transdermal voltage and flux of four fluorescent molecules show a transition to large fluxes near 50 V.

Large molecular fluxes and tight control are highly desired for transdermal drug delivery, which motivated this study of molecular transport due to high voltage pulsing. We used a flow-through sampling system (time resolution of approximately 14 s) to measure the response of human skin in vitro to a series of exponential pulses (time constant of 1 ms; peak transdermal voltages [Uskin,0] of 0 V to approximately 300 V, one pulse every 5.6 s). Four negatively charged, hydrophilic fluorescent tracer molecules were employed: sulforhodamine, lucifer yellow, cascade blue, and calcein (molecular weights of 450 to 625 Da). Although differences in their molecular transport profiles were observed, all four molecules exhibited a transition from small to large fluxes at Uskin,0 approximately 50 V. This behavior may reflect a transition from electroporation of the skin's appendages to electroporation of the multilamellar bilayer membranes within the stratum corneum.

Electroporation↗

Stealth PLA-PEG nanoparticles as protein carriers for nasal administration.

PURPOSE: The aim of the study was to encapsulate a model protein antigen, tetanus toxoid (TT), within hydrophobic (PLA) and surface hydrophilic (PLA-PEG) nanoparticles and to evaluate the potential of these colloidal carriers for the transport of proteins through the nasal mucosa. METHODS: TT-loaded nanoparticles, prepared by a modified water-in-oil-in-water solvent evaporation technique, were characterized in their size, zeta potential and hydrophobicity. Nanoparticles were also assayed in vitro for their ability to deliver active antigen for extended periods of time. Finally, 125I-TT-loaded nanoparticles were administered intranasally to rats and the amount of radioactivity recovered in the blood compartment, lymph nodes and other relevant tissues was monitored for up to 48 h. RESULTS: PLA and PLA-PEG nanoparticles had a similar particle size (137-156 nm) and negative surface charge, but differed in their surface hydrophobicity: PLA were more hydrophobic than PLA-PEG nanoparticles. PLA-PEG nanoparticles, especially those containing gelatine as an stabilizer, provided extended delivery of the active protein. The transport of the radiolabeled protein through the rat nasal mucosa was highly affected by the surface properties of the nanoparticles: PLA-PEG nanoparticles led to a much greater penetration of TT into the blood circulation and the lymph nodes than PLA nanoparticles. Furthermore, after administration of 125I-TT-loaded PLA-PEG nanoparticles, it was found that a high amount of radioactivity persisted in the blood compartment for at least 48 h. CONCLUSIONS: A novel nanoparticulate system has been developed with excellent characteristics for the transport of proteins through the nasal mucosa.

Administration, Intranasal↗

Skin electroporation causes molecular transport across the stratum corneum through localized transport regions.

High voltage pulsing of human skin (approximately 100 V across the skin, 1 ms pulses) has been hypothesized to cause electroporation of the stratum corneum, and to cause large fluxes of drugs and other molecules across the skin, through newly created aqueous pathways. In contrast, iontophoresis (<0.5 mA per cm2, <1 V across the skin) has long been used in transdermal drug delivery, and is believed to involve pre-existing pathways associated with hair follicles and sweat ducts. Either high voltage pulsing or iontophoresis was applied to human, hairless rat, or black rat snake skin. Hairless rat skin contains more hair follicles than human skin, and snake skin does not contain any hair follicles. All three types of skin had comparable electrical resistances at low voltages; however, the iontophoretic transport of charged fluorescent molecules was significant for human and hairless rat skin, but no transport occured across snake skin, indicating that hair follicles and sweat ducts play a major role in iontophoresis. Electroporation caused large molecular transport for all three types of skin, and involved spontaneously forming localized transport regions, not associated with appendages. These experiments thus provide further support for the hypothesis that high voltage pulsing causes electroporation in the stratum corneum, and that this transport mechanism is fundamentally different from iontophoresis.

Animals↗

[Osteitis diagnosis in a group of angiology patients: a comparison of X-ray and nuclear medicine studies].

PURPOSE: Of the study was to examine the ranking of radiographs and bone scans in the diagnosis of osteitis. PATIENTS AND METHODS: 115 patients with arterial occlusive disease in stage Fontaine i.v. were referred for examination of the peripheral skeletal parts of the lower extremity. 126 radiographs and bone scans were taken. The time between the two examinations was 10 days maximum to enable direct comparison. Leukocyte scintigraphy was employed as gold standard. RESULTS: The sensitivity of radiographs was 47% with a specificity of 91%. Sensitivity and specificity of bone scans was 78% for both values. The positive predictive values for radiographs and bone scans were 88% and 85%, the negative predictive values were between 80% and 97%. CONCLUSION: In the majority of cases osteitis can be diagnosed with relatively inexpensive methods such as conventional x-rays and bone scans. Only in unclear cases further examinations such as leukocyte scintigraphy or magnetic resonance imaging should supplement the diagnosis.

Adult↗

[MRI with supermagnetic iron particles versus double-spiral CT in identification of malignant liver lesions].

PURPOSE: The purpose of this study was to evaluate the efficacy of MRI with superparamagnetic iron oxide (SPIO) and double-spiral CT in the detection of liver metastases and hepatocellular carcinoma. METHODS: 38 patients with a total of 144 malignant hepatic lesions underwent CT and MRI. A panel of experts defined the gold standard. Five experienced judges performed independently blinded evaluation of the number of detectable lesions. Multifactorial variance analysis was used to determine the statistical significance. RESULTS: SPIO-MRI shows the highest rate of detection and is significantly superior to native MRI and native CT. The highest rate of detection by CT is shown in the portal-venous phase of contrast; nevertheless, the rate is significantly inferior to SPIO-MRI. In general, the native phases of CT and MRI are significantly inferior to the contrast phases of both. SPIO-MRI shows a higher rate of false positive findings. CONCLUSION: The time-consuming and cost-intensive SPIO-MRI significantly increases the rate of detectability for malignant liver lesions compared with double-spiral CT but it also increases the rate of false positive findings.

Adult↗

Formation of spheroidal aggregates of hepatocytes on biodegradable polymers under continuous-flow bioreactor conditions.

Our laboratory has investigated heterotopic hepatocyte transplantation on biodegradable polymer matrices as an experimental treatment for end-stage liver disease. One of the limitations has been survival of sufficient cell mass after transplantation. We hypothesize that in vitro conditioning of cells within polymer matrices prior to implantation may increase hepatocyte survival and function. In this preliminary study we investigated the effect of continuous flow on hepatocytes and sinusoidal endothelial cells on poly-L-lactic acid (PLLA) discs in vitro. Highly porous PLLA discs were manufactured measuring 18 mm diameter by 1 mm thickness using previously described techniques. Hepatocytes were isolated from adult, male Lewis rats (200-300 g) using a two-step collagenase digestion. Sinusoidal endothelial cells were isolated using a two-step collagenase digestion, differential sedimentation, Percoll gradient centrifugation, and selective adherence. PLLA discs were seeded with hepatocytes alone or with co-cultures of hepatocytes and sinusoidal endothelial cells. Seeded discs were then secured within a flow bioreactor chamber and exposed to continuous flow of culture media at a rate of 20 ml/minute through the chamber. Seeded discs placed in static culture conditions served as controls. Specimens seeded with only hepatocytes were harvested at 24 hours, 48 hours, and 168 hours after seeding. Co-culture specimens were harvested after 168 hours. Specimens were viewed under phase-contrast microscopy and then formalin-fixed and prepared for histologic sectioning. Sections were stained with Hematoxylin and Eosin and then analyzed with light microscopy. Hepatocytes under flow conditions formed spheroidal aggregates of cells of 50 to 200 microns in diameter by 24 hours in culture. Hepatocytes in static conditions showed decreased aggregation of cells and spheroid formation was absent. Co-cultured specimens under flow also showed spheroid formation with endothelial cells lining the outside of hepatocyte spheroids. Co-cultured specimens in static culture showed no spheroid formation and no organization between sinusoidal endothelial cells and hepatocytes. These results suggest that continuous flow increases organization of hepatocytes cultured within biodegradable polymer matrices.

Animals↗

Pelvic splenosis mimicking endometriosis, causing low abdominal mass and pain.

Splenosis is the heterotopic autotransplantation of splenic tissue that usually follows traumatic splenectomy. The clinical significance of these splenic implants and the need for surgical removal is debatable. A case of a 35 year old woman, with post-traumatic splenectomy presenting with low abdominal pain and pelvic mass is reported. Laparoscopic removal of the pelvic splenic implants dispelled all complaints.

Abdominal Pain↗

Spatially controlled cell engineering on biodegradable polymer surfaces.

Controlling receptor-mediated interactions between cells and template surfaces is a central principle in many tissue engineering procedures (1-3). Biomaterial surfaces engineered to present cell adhesion ligands undergo integrin-mediated molecular interactions with cells (1, 4, 5), stimulating cell spreading, and differentiation (6-8). This provides a mechanism for mimicking natural cell-to-matrix interactions. Further sophistication in the control of cell interactions can be achieved by fabricating surfaces on which the spatial distribution of ligands is restricted to micron-scale pattern features (9-14). Patterning technology promises to facilitate spatially controlled tissue engineering with applications in the regeneration of highly organized tissues. These new applications require the formation of ligand patterns on biocompatible and biodegradable templates, which control tissue regeneration processes, before removal by metabolism. We have developed a method of generating micron-scale patterns of any biotinylated ligand on the surface of a biodegradable block copolymer, polylactide-poly(ethylene glycol). The technique achieves control of biomolecule deposition with nanometer precision. Spatial control over cell development has been observed when using these templates to culture bovine aortic endothelial cells and PC12 nerve cells. Furthermore, neurite extension on the biodegradable polymer surface is directed by pattern features composed of peptides containing the IKVAV sequence (15, 16), suggesting that directional control over nerve regeneration on biodegradable biomaterials can be achieved.

Animals↗