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Biomedical subjects

R Langer

Publications and source records attributed to R Langer.

At least 271 records · Page 15Linked to original sources
PubMed1994

Solid-phase aggregation of proteins under pharmaceutically relevant conditions.

In order to successfully employ proteins as pharmaceuticals, it is essential to understand mechanistically the stability issues relevant to their formulation and delivery. Various deleterious processes may occur in protein formulations, thereby diminishing their therapeutic value. This review focuses upon one aspect of this problem, namely aggregation of solid proteins under pharmaceutically relevant conditions (elevated temperature and water activity). Strategies to pursue such studies are presented with an emphasis on a mechanistic analysis of aggregate formation. Both covalent and noncovalent aggregation pathways have been elucidated. Proteins that contain disulfide bonds as well as free thiol residues may aggregate via thiol-disulfide interchange. For proteins which contain disulfides but not free thiol residues, intermolecular disulfide bonding may still occur when intact disulfides undergo beta-elimination, yielding free thiols which can catalyze disulfide scrambling. Finally, proteins containing no cysteine/cystine residues may aggregate by other covalent pathways or by noncovalent routes. On the basis of these pathways, some rational stabilization strategies have been proposed and verified. Ultimately, application of this knowledge should lead to more stable and effective pharmaceutical protein formulations.

Chemical Phenomena↗
PubMed1994

Enhanced protein blotting from PhastGel media to membranes by irradiation of low-intensity ultrasound.

A novel approach to protein blotting based on application of ultrasound is proposed. Three minutes of ultrasound exposure (1 MHz, 2.5 W/cm2) was sufficient for a very clear transfer of proteins from a polyacrylamide gel (PhastGel) to nitrocellulose or nylon 66 Biotrans membranes. The proteins evaluated were prestained sodium dodecyl sulfate-polyacrylamide gel electrophoresis standards (18,500-106,000 Da) and 14C-labeled Rainbow protein molecular weight markers (14,300-200,000 Da). In control experiments, which were performed following similar procedures without turning the ultrasonic generator on, no protein blotting could be seen. For comparable blotting results, 30 min for electroelution or 240 min for elution by convection blotting was required.

Electrophoresis, Polyacrylamide Gel↗
PubMed1994

Design of nasoseptal cartilage replacements synthesized from biodegradable polymers and chondrocytes.

Reconstructive and aesthetic surgery of the nose is a challenging problem in facial plastic surgery. In this study, biodegradable polymers composed of polyglycolic acid (PGA) and poly-L-lactic acid (PLLA) and their co-polymers were used to produce templates to transplant cells and promote regeneration of structural cartilage. A highly porous anatomically shaped three-dimensional non-woven PGA fibre network was sprayed with a coating polymer solution. Reinforcement of the outer circumference of the 12 nasoseptal constructs using high molecular weight PLLA further stabilized the constructs during the process of neomorphogenesis of cartilage, both during in vitro incubation and in vivo implantation. These cell transplantation devices also proved to be adhesive substrates for dissociated bovine chondrocytes. When implanted subcutaneously into nude mice, the polymer templates guided the reorganization after 8 wk of the bovine chondrocytes into neocartilage in the precisely designed size and shape of the original size and shape of the polymer delivery device. All implants loaded with chondrocytes showed evidence of formation of histologically organized hyaline cartilage. The implantation of nasal scaffolds without cells did not show cartilage formation. The technique of tissue engineered growth of cartilage has potential applications in orthopaedic, plastic and reconstructive, and craniomaxillofacial surgery.

Animals↗
PubMed1994

Wetting of poly(L-lactic acid) and poly(DL-lactic-co-glycolic acid) foams for tissue culture.

Biodegradable foams of hydrophobic polymers can be efficiently wet by two-step immersion in ethanol and water, which overcomes the hindered entry of water into air-filled pores. Ethanol readily enters into the porous polymer, after which it is diluted and replaced by water. This method was evaluated for porous disks of poly(L-lactic acid) (PLLA) and poly(DL-lactic-co-glycolic acid) (PLGA) foams of copolymer ratios 85:15 and 50:50. For PLLA disks of 0.88 porosity and 1730 microns thickness, prewetting with ethanol for 1 h increased the percentage of void volume filled with water after 48 h from 23 to 79%. The same enhanced entry of water was also observed for prewet PLGA 85:15 disks of 0.86 porosity and 1300 microns thickness, which exhibited an increase from 59 to 97% void volume occupied by water. Furthermore, the water entry even after 1 h was very close to its plateau value for all prewet polymers tested. In recent studies, this method has been useful in uniformly seeding three-dimensional biodegradable polymer substrates for cell and tissue culture.

Culture Techniques↗
PubMed1994

Biodegradable microspheres as controlled-release tetanus toxoid delivery systems.

Purified tetanus toxoid, a high-molecular-weight protein, was entrapped within poly(L-lactic acid) (PLA) and poly(D,L-lactic/glycolic acid) (PLGA) microspheres prepared by either a solvent extraction or a solvent evaporation method carried out in a multiple emulsion system (water-in-oil-in-water). The physical integrity and antigenicity of the protein treated under different processing conditions were investigated. A reduction of antigenicity that was related to the percentage of aggregated protein was noticed under some experimental conditions. This partial loss of antigenicity was associated with the lyophilization process and affected by the nature of the organic solvent. All types of microspheres prepared with different molecular weight PLA and PLGA displayed a high protein-loading efficiency (> 80%) but their size was strongly influenced by polymer molecular weight (3000 versus 100,000). Protein release pattern was influenced by both polymer molecular weight and composition (PLA versus PLGA). A constant release pattern after an induction period of 10 days was observed for microspheres composed of high-molecular-weight polymers (PLA and PLGA). The release rate was lower from PLA microspheres than from PLGA microspheres. In contrast, a continuously increasing release rate preceded by a burst was observed for low-molecular-weight (3000) PLGA microspheres. Microencapsulated tetanus toxoid was significantly more immunogenic in mice than fluid toxoid as determined by IgG anti-tetanus antibody levels and neutralizing antibodies. However, the magnitude and duration of the antibody response did not differ significantly from a similar dose of aluminium phosphate-adsorbed toxoid. We conclude that microencapsulated tetanus toxoid shows significant adjuvant activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1994

Quantitative study of molecular transport due to electroporation: uptake of bovine serum albumin by erythrocyte ghosts.

Electroporation is believed to involve the creation of aqueous pathways in lipid bilayer membranes by transient elevation of the transmembrane voltage to approximately 1 V. Here, results are presented for a quantitative study of the number of bovine serum albumin (BSA) molecules transported into erythrocyte ghosts caused by electroportion. 1) Uptake of BSA was found to plateau at high field strength. However, this was not necessarily an absolute maximum in transport. Instead, it represented the maximum effect of increasing field strength for a particular pulse protocol. 2) Maximum uptake under any conditions used in this study corresponded to approximately one-fourth of apparent equilibrium with the external solution. 3) Multiple and longer pulses each increased uptake of BSA, where the total time integral of field strength correlated with uptake, independent of inter-pulse spacing. 4) Pre-pulse adsorption of BSA to ghost membranes appears to have increased transport. 5) Most transport of BSA probably occurred by electrically driven transport during pulses; post-pulse uptake occurred, but to a much lesser extent. Finally, approaches to increasing transport are discussed.

Adsorption↗
PubMed1994

Novel approaches to controlled-release antigen delivery.

Two strategies for vaccine-delivery systems, both relying on concepts of controlled-release technology, are described in this review. The first strategy involves using biodegradable polymer microspheres for parenteral and oral delivery of antigens. The other strategy combines two technologies, the encapsulation of antigen within liposomes and liposome encapsulation in hydrogels, to protect them from a rapid degradation in vivo. Both strategies have shown promise in terms of increasing the immunogenicity of poorly immunogenic peptides and protein vaccines. The microencapsulation process, antigen stability, mechanism of antigen release, and optimal release kinetics for vaccine delivery are reviewed, and the strengths and weaknesses of each approach are discussed.

Antigens↗
PubMed1994

Development and characterization of microencapsulated microspheres.

A process for the coating of polymer microspheres with the same or different polymers and the characterization of these particles is described. Coated microspheres were manufactured from degradable and non-degradable polymers. Several physicochemical methods were used to establish that the particles were fully coated. Polarized light microscopy revealed strong birefringence of coated microspheres resulting in the appearance of Maltese Crosses on coated microspheres. After staining the core and the coating of particles using different fluorescent dyes, the uneven distribution of the dyes in the core and on the surface allows one to verify the coating success. After cutting microspheres using a cryomicrotome we were able to assess the microstructure of the coated microspheres. Electron Spectroscopy for Chemical Analysis (ESCA) was used to determine the surface composition of coated microspheres. Determining the carbon and oxygen content of samples we were able to verify the completeness of the coating procedure. To examine the benefit of coating microspheres, the effect of coating on the release of tetanus toxoid from polylactide microspheres was studied as a possible pharmaceutical application.

Delayed-Action Preparations↗
PubMed1994

Moisture-induced aggregation of lyophilized insulin.

A critical problem in the storage and delivery of pharmaceutical proteins is aggregation in the solid state induced by elevated temperature and moisture. These conditions are particularly relevant for studies of protein stability during accelerated storage or for proteins loaded in polymeric delivery devices in vivo. In the present investigation, we have found that, when exposed to an environment simulating these conditions, lyophilized insulin undergoes both covalent and noncovalent aggregation. The covalent process has been elucidated to be intermolecular thiol-catalyzed disulfide interchange following beta-elimination of an intact disulfide bridge in the insulin molecule. This process is accelerated by increasing the temperature and water content of the insulin powder or by performing lyophilization and/or dissolution of insulin in alkaline media. The aggregation can be ameliorated by the presence of Cu2+, which presumably catalyzes the oxidization of free thiols. The water sorption isotherm for insulin reveals that the extent of aggregation directly correlates with the water uptake by the lyophilized insulin powder, thus pointing to the critical role of protein conformational mobility in the aggregation process.

Animals↗
PubMed1994

Biodegradable polymer scaffolds for tissue engineering.

Synthetic polymer scaffolds designed for cell transplantation were reproducibly made on a large scale and studied with respect to biocompatibility, structure and biodegradation rate. Polyglycolic acid (PGA) was extruded and oriented to form 13 microns diameter fibers with desired tenacity. Textile processing techniques were used to produce fibrous scaffolds with a porosity of 97% and sufficient structural integrity to maintain their dimensions when seeded with isolated cartilage cells (chondrocytes) and cultured in vitro at 37 degrees C for 8 weeks. Cartilaginous tissue consisting of glycosaminoglycan and collagen was regenerated in the shape of the original PGA scaffold. The resulting cell-polymer constructs were the largest grown in vitro to date (1 cm diameter x 0.35 cm thick). Construct mass was accurately predicted by accounting for accumulation of tissue components and scaffold degradation. The scaffold induced chondrocyte differentiation with respect to morphology and phenotype and represents a model cell culture substrate that may be useful for a variety of tissue engineering applications.

Biocompatible Materials↗
PubMed1994

[Cerebral aneurysms: their 3-dimensional imaging with spiral CT].

In this study, the possibility of non-invasive, three-dimensional demonstration of aneurysms of the basal cerebral arteries by means of spiral CT was investigated. The first step was to obtain exact definition of optimal examination parameters. Angio CTs at appropriate levels were performed on 10 subjects and time/density curves of the arterial and venous phases obtained in order to optimise the beginning of the arterial spiral CT series. The second step in this investigation was to examine 7 patients; in 6 of these basal aneurysms had been demonstrated by DSA. By means of multiplanar three-dimensional reconstruction from the data of the spiral CT it was possible to demonstrate 7 aneurysms with a diameter between 5 and 18 mm. Their position and relationship to the bony skull was also shown.

Adult↗
PubMed1994

[Indications for the application of a high resolution coil in MR tomography].

This is the first study with high spatial resolution Magnetic Resonance Imaging (MRI), performed with a high resolution coil in a clinical whole-body system. Measurements were recorded with a slice thickness of 2 mm. A 256 x 256 matrix and a 2.5 cm field of view were used resulting in a pixel size of 0.01 mm2. MR images of the skin, hand, wrist, knee and ankle of 14 healthy volunteers, of three anatomic pathology tissue specimens and of three formalin-fixed specimens were obtained. Normal anatomy was identified and compared with the three gross anatomic pathology sections. The skin, hands, wrists and ankles of 30 patients were examined and a variety of pathological lesions were detected, including cutaneous neoplasms and ulceration, acute and chronic tendon lesions and pathologic features of the bone and wrist. Based on the excellent imaging of anatomic detail and superior contrast resolution, high resolution MRI proved an important complement for preoperative diagnosis.

Adult↗
PubMed1994

[Color-coded duplex sonography in the differential diagnosis of cervical lymph node enlargements].

55 patients with cervical lymph node enlargement were studied prospectively by colour coded duplex sonography. The aim was to demonstrate the perfusion of individual lymph nodes and to determine whether the resistance and pulsatile index are able to show the cause of the enlargement. The lymph nodes were subsequently examined histologically. Useful perfusion measurements were obtained in 177 lymph nodes out of 216. Perfusion index < 1.6 and resistance index < 0.8 distinguishes between reactive lymph node enlargement and lymph node metastases with an accuracy of 91%. Reliable differentiation between lymphoma and metastases was not possible. Tuberculous lymph nodes and cysts showed significantly reduced resistance and pulsatile index when compared with metastases. Further information on the type of disease was obtained from the perfusion patterns. Reactive lymph nodes showed increased central perfusion of the hilum, whereas metastases tended to show increased peripheral perfusion.

Diagnosis, Differential↗
PubMed1994

[Cerebral arteriovenous angiomas: 3-dimensional demonstration by spiral CT].

In 20 patients with known or suspected supratentorial arteriovenous malformations, an attempt was made to see how far CT angiography with 3-dimensional reconstructions is able to make a diagnosis and to differentiate the various components of the angioma. Spiral CT was performed following an intravenous bolus injection of 60-80 ml of iodine containing contrast medium. In all patients the diagnosis was confirmed by intra-arterial DSA of the vertebral vessels. In 13 patients, av malformations could be diagnosed following multiplanar 3-D reconstructions which agreed with the findings on DSA. The large supplying vessels, the nidus and the large draining veins could be defined with certainty. In 6 patients follow-up examination after embolisation was performed. The results could be demonstrated in three dimensions and the success of treatment could be documented unequivocally. CT angiography with 3-D reconstruction is able to supply important information in the majority of intracranial av malformations, both during initial investigation and following treatment.

Adolescent↗
PubMed1994

Extracellular matrix controls tubulin monomer levels in hepatocytes by regulating protein turnover.

Cells have evolved an autoregulatory mechanism to dampen variations in the concentration of tubulin monomer that is available to polymerize into microtubules (MTs), a process that is known as tubulin autoregulation. However, thermodynamic analysis of MT polymerization predicts that the concentration of free tubulin monomer must vary if MTs are to remain stable under different mechanical loads that result from changes in cell adhesion to the extracellular matrix (ECM). To determine how these seemingly contradictory regulatory mechanisms coexist in cells, we measured changes in the masses of tubulin monomer and polymer that resulted from altering cell-ECM contacts. Primary rat hepatocytes were cultured in chemically defined medium on bacteriological petri dishes that were precoated with different densities of laminin (LM). Increasing the LM density from low to high (1-1000 ng/cm2), promoted cell spreading (average projected cell area increased from 1200 to 6000 microns2) and resulted in formation of a greatly extended MT network. Nevertheless, the steady-state mass of tubulin polymer was similar at 48 h, regardless of cell shape or ECM density. In contrast, round hepatocytes on low LM contained a threefold higher mass of tubulin monomer when compared with spread cells on high LM. Furthermore, similar results were obtained whether LM, fibronectin, or type I collagen were used for cell attachment. Tubulin autoregulation appeared to function normally in these cells because tubulin mRNA levels and protein synthetic rates were greatly depressed in round cells that contained the highest level of free tubulin monomer. However, the rate of tubulin protein degradation slowed, causing the tubulin half-life to increase from approximately 24 to 55 h as the LM density was lowered from high to low and cell rounding was promoted. These results indicate that the set-point for the tubulin monomer mass in hepatocytes can be regulated by altering the density of ECM contacts and changing cell shape. This finding is consistent with a mechanism of MT regulation in which the ECM stabilizes MTs by both accepting transfer of mechanical loads and altering tubulin degradation in cells that continue to autoregulate tubulin synthesis.

Animals↗
PubMed1994

A new colpo-needle suspension for the surgical treatment of stress incontinence: a 2-year follow-up.

Sixty-three patients with urinary stress incontinence underwent a new colpo-needle suspension procedure. It differs from the other transvaginal bladder neck suspension procedures by using the fixed and stable Cooper ligament as the fixation point for the suspension sutures rather than the unstable and compressible anterior abdominal wall. At 2-year follow-up we obtained a 90.5% cure rate of urinary stress incontinence. We consider the procedure suitable for all patients with genuine stress incontinence. Long-term follow-up is required for further evaluation of this new technique.

Adult↗
PubMed1994

[Characterization of 24-hour survival rate and duration of survival of hydroxyethyl starch cryopreserved erythrocytes after autologous transfusion in the dog].

BACKGROUND: Cryopreservation of erythrocytes using hydroxyethyl starch (HES) as cryoprotecting additive could result in a nearly unlimited storage stability of preserved red cells. In addition, it would allow its immediate use for transfusion. In order to assess the therapeutic efficacy of erythrocytes cryopreserved with HES, their 24-hour post-transfusion survival and long-term survival was evaluated. MATERIALS AND METHODS: The experiments were carried out with dog erythrocytes as an animal model for human erythrocytes. To each of 6 German shepherd dogs a 15-ml sample of erythrocyte suspension, labeled with 51Cr (25 microCi) after thawing, was autologously injected. Caused by hemolysis 29% of the formerly cryopreserved erythrocytes have not been labeled. To each of 6 control animals 15 ml of a suspension of freshly drawn and 51Cr-labeled erythrocytes was injected. The 51Cr radioactivity in later taken blood samples was a measure for the number of injected erythrocytes having remained in the circulation until the moment of blood withdrawal. The effect of cryopreservation was assessed by comparison of the test group with the control group. RESULTS: In both groups 30% of the applied cells left the circulation within 30 min. This was effected by pharmacological enlargement of the dogs' spleen and not by hemolysis of the erythrocytes. After the first 24 h all of the cryopreserved labeled erythrocytes had survived to the same amount (> 95%) as the labeled fresh red cells. Between 12 h and 20 days after injection, in both groups the 51Cr activity decreased exponentially by 4.8 and 4.5%/d. This difference was not significant. The area under the curve amounted to 1253 and 1257% d, respectively. CONCLUSIONS: There exists a subpopulation of red cells that is destroyed by freezing stress. As a result the freed stroma would be a serious transfusion risk. All erythrocytes having survived the cryopreservation procedure resemble the fresh erythrocytes with regard to the in-vivo survival; their therapeutic efficacy is not impaired. In the context of in-vitro results with human erythrocytes it can be expected that at the present developmental state of the cryopreservation procedure at least 93% of the human erythrocytes cryopreserved with HES have a normal 24-hour and long-term post-transfusion survival.

Animals↗