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Biomedical subjects

W M Saltzman

Publications and source records attributed to W M Saltzman.

13 recordsLinked to original sources

Three-dimensional cell cultures mimic tissues.

Three-dimensional cell culture using gels of type I collagen is a flexible method for studying cell behavior in a tissuelike environment. With only small changes in the basic protocol, we were able to encapsulate neutrophils, hepatocytes, and PC12 cells. As demonstrated by cell-specific assays for migration, protein secretion, and growth factor induction, the encapsulated cells were viable and functional. In future studies, we will focus on using these cell cultures to study cell movement, cell growth, and cell function in carefully controlled tissuelike environments.

Animals

Quantification of human neutrophil motility in three-dimensional collagen gels. Effect of collagen concentration.

Leukocytes must migrate through tissues to fulfill their role in the immune response, but direct methods for observing and quantifying cell motility have mostly been limited to migration on two-dimensional surfaces. We have now developed methods for examining neutrophil movement in a three-dimensional gel containing 0.1 to 0.7 mg/ml rat tail tendon collagen. Neutrophil-populated collagen gels were formed within flat glass capillary tubes, permitting direct observation with light microscopy. By following the tracks of individual cells over a 13.5-min observation period and comparing them to a stochastic model of cell movement, we quantified cell speed within a given gel by estimating a random motility coefficient (mu) and persistence time (P). The random motility coefficient changed significantly with collagen concentration in the gel, varying from 1.6 to 13.3 x 10(-9) cm2/s, with the maximum occurring at a collagen gel concentration of 0.3 mg/ml. The methods described may be useful for studying tissue dynamics and for evaluating the mechanism of cell movement in three-dimensional gels of extracellular matrix (ECM) molecules.

Biophysical Phenomena

Controlled antibody delivery systems.

We have developed methods for controlling the release of antibodies (Ab) from biocompatible polymers. Human Ab, human Ab fragments, and mouse monoclonal antibody (mAb) directed against human chorionic gonadotropin (anti-hCG) were incorporated into matrices of poly(ethylene-co-vinyl acetate), which is stable in biological environments. Human Ab and bovine gamma-globulin were also incorporated in biodegradable matrices of a poly-anhydride copolymer composed of a stearic acid dimer and sebacic acid. Abs were slowly released from all the polymeric carriers during 30 days of continuous immersion in buffered saline. The ability of anti-hCG to bind antigen was retained following release from EVAc matrices. Only minor Ab aggregation was observed following release from either polymer. Polymeric delivery systems, similar to those described here, may become an important element in the delivery of mAbs to humans for immunoprotection against infectious diseases or the delivery of mAb-conjugates for immunotherapy against cancer.

Anhydrides

Controlled vaginal delivery of antibodies in the mouse.

Controlled delivery of monoclonal antibodies to the mucus secretions of the vagina might provide women with passive immunoprotection against both sexually transmitted diseases and unwanted pregnancy. We have developed intravaginal devices composed of poly(ethylene-co-vinyl acetate) (EVAc) that continuously release IgG antibodies for over 30 days into buffered saline, and we have tested these devices in the vagina of mice. Polymeric devices containing either BSA (as a test reagent for proteins) or anti-hCG antibody, when inserted into the vaginas of mice, provided a continuous supply of either BSA or hCG-binding antibodies to the vaginal mucus for 30 days. Antibodies released by the devices achieved high concentration in the mucus within the lumen of the vagina, but did not significantly ascend into the uterine horns, as determined by epifluorescence microscopy of fluorescently labeled mouse IgG and by immunohistochemical localization of rabbit IgG. Our results suggest that long-term intravaginal delivery of functionally intact antibodies can be achieved with devices composed of EVAc.

Administration, Intravaginal

Fibroblast and hepatocyte behavior on synthetic polymer surfaces.

Biodegradable poly(phosphoesters) with varying side group chemistry and copolymers of styrene and methyl vinyl ketone (MVK) with varying degrees of hydrophobicity were used to study the growth and behavior of surface-attached fibroblasts and hepatocytes. Mouse 3T3 fibroblasts and chicken embryo fibroblasts attached and proliferated on all of the polymers tested. Fewer cells attached to copolymers of styrene and MVK than to glass or tissue culture polystyrene controls; cell attachment to several poly(phosphoester) surfaces was indistinguishable from controls. The mean speed of fibroblast migration was faster on surfaces where fewer cells attached (59 to 84 microns/h on low attachment surfaces compared with 40 to 46 microns/h on high attachment surfaces). When surface-attached cells were stained with fluorescently labeled phalloidin, only a fraction of the cells on low attachment surfaces were shown to have prominent arrays of actin filament bundles. Chicken hepatocytes also attached to the polymer surfaces. When a suspension containing a large number of cells was placed over the polymer surfaces, approximately 50% of the hepatocytes attached during the first 9 h. Surprisingly, hepatocyte attachment and viability in culture were relatively insensitive to the chemistry of the synthetic polymer substrates. Cell number increased by about a factor of 2 over the first 48 h of culture, then decreased back to approximately 50% of initial cell number over the next several days. Cell morphology did depend on the chemical structure of the substrates.

Actins

Spermicide permeation through biocompatible polymers.

Although spermicides are safe and effective contraceptive/prophylactic agents, they are inconvenient to use. Formulations that provide a controlled release of spermicide may improve user acceptance, and therefore effectiveness. Using a two-chamber diffusion cell, we measured the rates of permeation of nonoxynol-9 (N9), benzalkonium chloride (BC), and chlorhexidine (CH) through films of ethylene-vinyl acetate copolymer (EVAc) and silicone elastomer (SILASTIC). In addition, we encapsulated N9, BC, and CH into solid polymer matrices and measured the rate of spermicide release following immersion in water. We also developed equations for predicting the release rate of spermicide from a vaginal ring containing encapsulated spermicide, and tested these equations using hollow SILASTIC rings containing pure N9 or BC. N9 diffuses through a thin film of SILASTIC several orders of magnitude slower than through water. The rates of permeation of N9 through EVAc, BC through SILASTIC, and CH through SILASTIC were too low to detect over a one-week experiment. Polymer matrices of EVAc or SILASTIC released N9 at a controlled rate for several days. Based on these measurements, we predict that a vaginal ring containing an inner core of EVAc/N9 surrounded by a thin, permeable layer of SILASTIC will provide a controlled, constant release of N9 for over 30 days. Because of its low permeability in SILASTIC, BC is probably not a good spermicide for a long-acting vaginal ring. Because of its low solubility in water, CH is also not a good candidate for controlled release into the vaginal mucus.

Benzalkonium Compounds

Controlled release of nerve growth factor from a polymeric implant.

Recent studies suggest that neurotrophic factors applied directly to brain tissue may enhance regeneration in the central nervous system. Biocompatible polymeric implants providing a controlled release of nerve growth factor (NGF) for over one month were developed. The released nerve growth factor stimulated neurite sprouting in cultured PC12 cells. While a model polymer with demonstrated biocompatibility was used for the present study, the methods can be extended to other polymer systems. Controlled release implants may be useful in the treatment of Alzheimer's disease.

Animals

Macromolecules released from polymers: diffusion into unstirred fluids.

Polymers that release macromolecules may be useful for preventing and treating human disease. In certain applications of polymeric controlled release, like drug therapy of brain disease and immunoprotection of mucus epithelia, effectiveness may be limited by diffusion through an unstirred fluid near the polymer. Using computer-assisted epifluorescence microscopy, we have examined the local distribution of fluorescently labelled macromolecules released from an ethylene-vinyl acetate copolymer matrix into unstirred layers of phosphate-buffered water and mid-cycle human cervical mucus. Diffusion coefficients in the fluid were determined by observing the concentration profiles as a function of time. Diffusion coefficients determined for fluorescein, bovine serum albumin, and three classes of human immunoglobulins (IgG, sIgA and IgM) in phosphate-buffered water were in good agreement with literature values. For fluorescein, albumin and IgG, diffusion in mucus was comparable with diffusion in water: the largest molecule tested was slowed by only a factor of 3.

Biocompatible Materials

Controlled release of dopamine from a polymeric brain implant: in vivo characterization.

Intracerebral microdialysis was used to evaluate the long-term in vivo release of dopamine from ethylene-vinyl acetate (EVAc)-dopamine copolymer matrix discs for up to 65 days following striatal implantation. Dopamine release occurred through a single cavity present on one side of the disc, which was otherwise fully coated with an additional, impermeable layer of EVAc. At 20 days following implantation of the device, extracellular concentrations of dopamine within the striatum reached micromolar levels, over 200-fold greater than control values. Release of dopamine was shown to be stable and maintained for the 2-month duration of the experiment. Histological examination confirmed the biocompatible nature of the implant. There are potential applications of this technology to the treatment of Parkinson's disease and other neurological and psychiatric disorders.

Animals

Controlled release of dopamine from a polymeric brain implant: in vitro characterization.

A biocompatible polymeric matrix system for the long-term controlled release of dopamine has been developed. Solid particles of this bioactive agent were encapsulated in ethylene-vinyl acetate copolymer (EVAc). Following immersion in an aqueous buffer solution, the release rate of dopamine from the polymer matrix was found to depend on the initial concentration of dopamine in the polymer. After coating the matrix devices with an additional impermeable layer of EVAc, constant rates of release were obtained by creating a cavity in this impermeable layer. The observed experiments are consistent with a diffusion-limited model of dopamine release; all the in vitro experimental results were therefore correlated by the effective diffusion coefficient of dopamine through the porous polymer network. These results are discussed in terms of potential design modifications to achieve desired release characteristics for a variety of neuroactive substances, including neurotransmitters or their precursors.

Brain

Transport rates of proteins in porous materials with known microgeometry.

Many biological and biotechnological systems involve the diffusion of macromolecules through complicated macroporous (pore size on the order of 10-100 microns) environments. In this report, we present and evaluate an experimental system for measuring the rate of protein transport in an inert, macroporous membrane. For this particular membrane system, the microgeometry was characterized in terms of distribution of pore size, position, and orientation. Although the rate of protein desorption was much less than expected based on continuum diffusion models, we demonstrate that the measured transport rates are consistent with diffusion of protein in a complex, interconnected network of water-filled pores. The porous systems exhibit transitional behavior in quantitative agreement with the behavior of percolation lattices (mean square error 7%, n = 29). Predictive mathematical models of the diffusion process were developed: these models used percolation concepts to describe pore topology, continuum models of diffusion/dissolution to describe protein movement at each single pore, and measured pore size distributions. Effective diffusion coefficients for protein transport in aqueous, constricted macropores were predicted by this technique. Predicted diffusion coefficients, based on measured and derived microstructural parameters, agree with experimentally measured diffusion coefficients within a factor of 2. This approach may be useful in the design of porous polymer systems for biological applications and for evaluating other biological systems where conduction of mass, heat, momentum, or charge occurs in a heterogeneous environment.

Kinetics

Selective cell transplantation using bioabsorbable artificial polymers as matrices.

To date, selective cell transplantation has involved injecting cell suspensions into tissues or the vascular system. This study describes attaching cell preparations to bioerodable artificial polymers in cell culture and then implanting this polymer-cell scaffold into animals. Using standard techniques of cell harvest, single cells and clusters of fetal and adult rat and mouse hepatocytes, pancreatic islet cells, and small intestinal cells have been seeded onto biodegradable polymers of polyglactin 910, polyanhydrides, and polyorthoester. Sixty-five fetuses and 14 adult animals served as donors. One hundred fifteen polymer scaffolds were implanted into 70 recipient animals: 66 seeded with hepatocytes; 23 with intestinal cells and clusters; and 26 with pancreatic islet preparations. The cells remained viable in culture, and in the case of fetal intestine and fetal hepatocytes, appeared to proliferate while on the polymer. After four days in culture, the cell-polymer scaffolds were implanted into host animals, either in the omentum, the interscapular fat pad, or the mesentery. In three cases of fetal intestinal implantation coupled with partial hepatectomy, successful engraftment occurred in the omentum, one forming a visible 6.0 mm cyst. Three cases of hepatocyte implantation, one using adult cells and two using fetal cells, have also engrafted, showing viability of hepatocytes, mitotic figures, and vascularization of the cell mass. To date, no pancreatic islets have survived implantation. This method of cell transplantation, which we have termed "chimeric neomorphogenesis," is an alternative to current methods and requires further study.

Animals

Dextran retention in the rat brain following release from a polymer implant.

Intracranial controlled release polymers may improve drug administration to the brain, where therapy is frequently limited due to the low permeability of brain capillaries to therapeutic agents. On the basis of drug transport and elimination rates, we proposed that high molecular weight, water-soluble molecules would be retained in the brain space following release from an intracranial implant. To test this hypothesis, solid particles of different molecular weight fractions of fluorescein isothiocyanate labeled dextran (FITC-dextran; 4 x 10(3) Da (4 kDa) < weight-averaged molecular weight (Mw) < 150 kDa) or fluorescein were uniformly dispersed in matrices of a polyanhydride copolymer synthesized from a fatty acid dimer and sebacic acid in a 50:50 ratio, P(FAD:SA). When incubated in buffered saline, FITC-dextran fractions of 70 kDa Mw were released from the polymer within 48 h; 4 kDa Mw FITC-dextran and fluorescein were released more slowly. Following implantation of P(FAD:SA) matrices containing either 70 kDa Mw FITC-dextran, 4 kDa Mw FITC-dextran, or fluorescein into the brains of normal rats, fluorescent tracers were continuously released into the brain tissue for 30 days. Tracer concentrations within the brain were significantly higher for large molecular weight tracers (70 kDa Mw FITC-dextran >> 4 kDa Mw FITC-dextran > fluorescein). The rate of elimination, kapp, of each tracer from the brain was determined by comparing experimental data with a model describing tracer diffusion/elimination in the brain extracellular space; kapp decreased with increasing molecular weight (fluorescein > 4 kDa Mw FITC-dextran > 70 kDa Mw FITC-dextran).

Animals