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Biomedical subjects

P A Tresco

Publications and source records attributed to P A Tresco.

At least 19 recordsLinked to original sources
PubMed2001

A novel surfactant-based immobilization method for varying substrate-bound fibronectin.

Most biomaterials can be rendered adhesive for anchorage-dependent cells by adsorption of serum, isolated extracellular matrix proteins, or immobilization of peptide sequences. However, difficulties are frequently encountered in characterizing the adsorbed layer due to conformational changes in the molecules following adsorption and interference from nonspecifically adsorbed molecules. In this study, we have investigated a technique for covalently immobilizing fibronectin to the PEO-containing triblock copolymer Pluronic F108 ("F108"). We have compared this technique to solution adsorption of fibronectin for its ability to provide controlled variation of bound fibronectin and regulation of fibroblast behavior. Both simple adsorption and covalent immobilization were effective for varying substrate-bound fibronectin. However, adsorption of fibronectin did not effectively regulate fibroblast attachment or spreading in either serum-free or serum-containing media. Fibroblast attachment, spreading, cytoskeletal organization, and proliferation were effectively regulated in response to fibronectin immobilized to F108. Furthermore, F108-treated surfaces without immobilized fibronectin did not support nonspecific fibroblast attachment, even in the presence of serum-containing medium. Fibroblasts were observed to only proliferate on surfaces with high levels of immobilized fibronectin that supported extensive cell spreading and cytoskeletal organization. In summary, covalent immobilization of fibronectin to F108 provided controlled regulation of fibroblast behavior without interference from nonspecific protein adsorption, even in the presence of serum-containing medium.

3T3 Cells↗
PubMed2001

Surfactant-immobilized fibronectin enhances bioactivity and regulates sensory neurite outgrowth.

A PEO-containing surface coating was investigated as a means to control neurite outgrowth in the presence of serum. Various ratios of end-group-activated tri-block copolymer Pluronic F108 were used to immobilize the extracellular matrix protein fibronectin (FN). Primary cultures of dorsal root ganglion neurons were cultured on F108-immobilized FN or, as a control, on FN adsorbed from solution directly to polystyrene. Although FN surface concentration could be controlled in a dose-dependent manner by either technique, dose-dependent control of neuronal behaviors was best achieved on F108-immobilized FN. This effect was similar regardless of the presence of serum in the culture medium. F108-immobilized FN supported twofold greater maximal neurite outgrowth than did directly adsorbed FN. Furthermore, at similar surface concentrations, F108-FN was significantly more active in promoting neurite outgrowth. Polypropylene filament bundles treated with F108-immobilized FN supported robust outgrowth from explants of dorsal root ganglia, demonstrating the utility of the surface coating on clinically relevant materials with more complex shapes. The ability to control neuronal behaviors in a serum-resistant manner, coupled with enhanced biologic activity, demonstrates the potential for surfactant-based immobilization as a method for generating biointeractive materials for tissue engineering.

Adsorption↗
PubMed2001

Substrate-bound human recombinant L1 selectively promotes neuronal attachment and outgrowth in the presence of astrocytes and fibroblasts.

Axonal pathfinding is a complex process that is mediated through cell-matrix and cell-cell interactions. A large number of studies have demonstrated that ECM and ECM-derived proteins and peptides are potent promoters of neurite outgrowth, however much less attention is given to the fact that these same ligands also elicit responses in a wide variety of non-neuronal cell types. We examined the use of a substrate-bound recombinant form of human L1, an integral membrane protein, as a ligand for bridging materials for repairing the CNS by studying its effectiveness in promoting specific responses of neuronal cells in the presence of astrocytes and fibroblasts. L1, a cell adhesion molecule expressed in the developing CNS and PNS, has strong neurite promoting activity, and contributes to axonal guidance and axonal fasciculation during development. In this study, substrates treated with L1-Fc were compared to subtrates treated with fibronectin and poly-lysine (PDL) with respect to their interaction with a variety of cell types, including three types of neurons (DRG neurons, cerebellar granule neurons, and hippocampal neurons), astrocytes, dermal fibroblasts, and meningeal cells. L1-Fc-treated substrates supported significantly higher levels of neurite outgrowth relative to fibronectin and PDL, while inhibiting the attachment of astrocytes, meningeal cells, and fibroblasts. We also show that neuronal cells attach to and extend neurites on 30 microm diameter L1-Fc-treated filaments as an example of a potentially useful bridging substrate. The high level of biological specificity displayed by surface-bound L1, along with the fact that it is a potent promoter of neurite outgrowth, is normally expressed on axons and regulates axonal fasciculation during normal development bodes well for its use on bridging materials for the repair of the CNS, and suggests that cell adhesion molecules, in general, may be useful for biomaterial modification. Moreover, small diameter filaments coated with L1-Fc may function in an analogous way to pioneering axons that guide the growth of axons to distal targets during development.

Animals↗
PubMed2001

Characterization of rat meningeal cultures on materials of differing surface chemistry.

To better understand the interactions of cells derived from meningeal tissues with the surfaces of devices used for the treatment of central nervous system disorders, the behavior of primary postnatal day 1 rat meningeal cultures was evaluated on biomaterials of differing surface chemistry. Meningeal cultures in serum containing media were analyzed for attachment, spread cell area, proliferation, the production of extracellular matrix (ECM), and neuronal outgrowth. In general, both cell attachment as well as cell spread area decreased with increasing substrate hydrophobicity, whereas cell division as indicated by BrdU incorporation and time to confluence, was lower on the most hydrophobic materials. We suggest that such differences immediately after cell seeding were most likely mediated by differences in surface adsorption of proteins. In longer-term experiments, most of the materials were colonized by meningeal cultures irrespective of surface chemistry, and all cultures were equally inhibitory to neuronal outgrowth suggesting that over time, cells can modify the substrate perhaps by secretion of extracellular matrix molecule proteins. Our data suggests that cell type-specific differences in response to different biomaterials may play an important role in determining the ultimate nature and composition of the CNS at the host-biomaterial interface.

Animals↗
PubMed2000

Cellular transplants as sources for therapeutic agents.

Soluble factors normally produced by cells of the human body are of increasing importance as potential therapeutic agents. Although considerable progress has been made in understanding the etiology and pathogenesis of disease, in developing animal models and newer experimental therapeutics, few discoveries have been translated into clinically effective ways of delivering the multiple therapeutic agents obtained from living mammalian cells. This review examines the use of transplanted cells as alternatives to conventional delivery systems to deliver a variety of protein based therapeutic agents. The chapter begins with a set of questions to establish the complexity and challenges of this form of drug delivery. The following section focuses the discussion on our understanding of genetic engineering, tissue engineering, and some areas of developmental biology as they relate to the development of this nascent field. Much of the discussion has a neuro/endocrine emphasis. The chapter ends by listing the basic ingredients needed to push the use of transplanted cells toward medical practice and some general comments about future developments.

Animals↗
PubMed2000

Technology of mammalian cell encapsulation.

Entrapment of mammalian cells in physical membranes has been practiced since the early 1950s when it was originally introduced as a basic research tool. The method has since been developed based on the promise of its therapeutic usefulness in tissue transplantation. Encapsulation physically isolates a cell mass from an outside environment and aims to maintain normal cellular physiology within a desired permeability barrier. Numerous encapsulation techniques have been developed over the years. These techniques are generally classified as microencapsulation (involving small spherical vehicles and conformally coated tissues) and macroencapsulation (involving larger flat-sheet and hollow-fiber membranes). This review is intended to summarize techniques of cell encapsulation as well as methods for evaluating the performance of encapsulated cells. The techniques reviewed include microencapsulation with polyelectrolyte complexation emphasizing alginate-polylysine capsules, thermoreversible gelation with agarose as a prototype system, interfacial precipitation and interfacial polymerization, as well as the technology of flat sheet and hollow fiber-based macroencapsulation. Four aspects of encapsulated cells that are critical for the success of the technology, namely the capsule permeability, mechanical properties, immune protection and biocompatibility, have been singled out and methods to evaluate these properties were summarized. Finally, speculations regarding future directions of cell encapsulation research and device development are included from the authors' perspective.

Alginates↗
PubMed2000

Relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for anchorage-dependent cells on model surfaces.

Many research and commercial applications use a synthetic substrate which is seeded with cells in a serum-containing medium. The surface properties of the material influence the composition of the adsorbed protein layer, which subsequently regulates a variety of cell behaviors such as attachment, spreading, proliferation, migration, and differentiation. In this study, we examined the relationships among cell attachment, spreading, cytoskeletal organization, and migration rate for MC3T3-E1 osteoblasts on glass surfaces modified with -SO(x), -NH(2), -N(+)(CH(3))(3), -SH, and -CH(3) terminal silanes. We also studied the relationship between cell spread area and migration rate for a variety of anchorage-dependent cell types on a model polymeric biomaterial, poly(acrylonitrile-vinylchloride). Our results indicated that MC3T3-E1 osteoblast behavior was surface chemistry dependent, and varied with individual functional groups rather than general surface properties such as wettability. In addition, cell migration rate was inversely related to cell spread area for MC3T3-E1 osteoblasts on a variety of silane-modified surfaces as well as for different anchorage-dependent cell types on a model polymeric biomaterial. Furthermore, the data revealed significant differences in migration rate among different cell types on a common polymeric substrate, suggesting that cell type-specific differences must be considered when using, selecting, or designing a substrate for research and therapeutic applications.

3T3 Cells↗
PubMed2000

Chronic recording of regenerating VIIIth nerve axons with a sieve electrode.

A micromachined silicon substrate sieve electrode was implanted within transected toadfish (Opsanus tau) otolith nerves. High fidelity, single unit neural activity was recorded from seven alert and unrestrained fish 30 to 60 days after implantation. Fibrous coatings of genetically engineered bioactive protein polymers and nerve guide tubes increased the number of axons regenerating through the electrode pores when compared with controls. Sieve electrodes have potential as permanent interfaces to the nervous system and to bridge missing connections between severed or damaged nerves and muscles. Recorded impulses might also be amplified and used to control prosthetic devices.

Animals↗
PubMed2000

Fibronectin immobilized by a novel surface treatment regulates fibroblast attachment and spreading.

In order to understand the influence of cell-adhesive molecules on anchorage-dependent cell behavior on biomaterial surfaces, a model system is required where these molecules can be applied to surfaces with controlled surface ligand density and resistance to the adsorption of additional proteins present in the medium. This study asked whether fibronectin could be immobilized in a controlled manner to a hydrophobic surface with a chemically modified triblock surfactant. ELISA studies indicated that variation of the soluble fibronectin concentration used for immobilization could be used to control the amount of fibronectin immobilized to the surface. Furthermore, fibroblasts seeded on these surfaces in 10% serum-containing medium attached and spread as a function of the amount of immobilized fibronectin. Surfaces treated with unmodified surfactant did not support cell attachment, suggesting that cell attachment and spreading were primarily regulated by the immobilized fibronectin with minimal interference from adsorption of serum proteins. Together, these results suggest that covalent immobilization to Pluronic F108 provides a method for studying cellular responses to cell adhesive proteins with little interference from competing adsorbates, even in the presence of complex biological fluids such as serum. This technique may be applicable to a variety of existing hydrophobic biomedical polymers as a basic science tool as well as for influencing cell behavior at implant interfaces.

Animals↗
PubMed1999

Characterization of cortical astrocytes on materials of differing surface chemistry.

The behavior of cortical astrocytes was evaluated on a number of medically relevant materials of differing physicochemical properties. This study describes cell attachment, DNA synthesis, production of extracellular matrix (ECM) proteins, and neuronal interactions of perinatal rat astrocytes in vitro. The number of attached astrocytes initially differed among the materials, decreasing with increasing material hydrophobicity. In contrast, the rate of DNA synthesis increased with increasing material hydrophobicity. With the exception of only one material, astrocytes reached confluence by 12 days in culture on all the materials tested. Furthermore, the expression of characteristic ECM proteins and the fundamental ability of astrocytes to support neuronal attachment and growth was qualitatively identical between populations of astrocytes on different materials. The ability of astrocytes to colonize different surfaces initially was mediated via adsorbed serum proteins, as reducing the capacity of a model surface to adsorb proteins inhibited astrocyte colonization for up to 2 weeks in culture. We propose that astrocytes are relatively insensitive to differences in surface chemistries so long as the proteins necessary for cellular attachment are capable of adsorbing to the material to some extent. It seems likely that the ability of astrocytes to produce and remodel a matrix creates a surface environment that eventually becomes similar regardless of the surface chemistry of the underlying material.

Animals↗
PubMed1999

Surface modification for controlled studies of cell-ligand interactions.

This work describes a method for coupling cell adhesion peptides to hydrophobic materials for the purpose of controlling surface peptide density while simultaneously preventing nonspecific protein adsorption. PEO/PPO/PEO triblock copolymers (Pluronic F108) were equipped with terminal pyridyl disulfide functionalities and used to tether RGD containing peptides to polystyrene (PS). The density of F108 on PS was 1.4 E5 +/- 2.12 E1 molecules/microm2. XPS and ToF SIMS indicated that the F108 coating was homogeneous and that the unmodified and activated F108 distributed evenly on PS. By mixing unmodified F108 with PDS-activated F108 prior to adsorption, it was possible to vary peptide density between 0 and 8.7 E4 +/- 2.66 E3 peptides/microm2, while otherwise, maintaining consistent surface properties. GRGDSY grafted PS supported cell attachment, spreading, and development of cytoskeletal structure, all of which were found to increase with increasing peptide density. Cell proliferation followed this same trend, however, maximal growth occurred at a submaximal peptide density. Cell aspect ratio varied in a biphasic manner with GRGDSY density. F108 coated PS and GRGESY grafted PS were inert to cell adhesion. Cells released from GRGDSY grafted PS upon addition of either a reducing agent or free GRGDSY, which indicates that cell-substrate interactions were mediated solely by the tethered peptides.

3T3 Cells↗
PubMed1998

Extended local access fibers: adjustable treatment of deep sites in the brain.

We describe a minimally invasive method for administering diffusible substances to computer-targeted regions of the primate brain. Treatments enter the brain by diffusion or under pressure from the portion of a curvilinear implant that lies within the region of interest. During the implantation surgery, a guide-tube cannula is passed through a cranial burr-hole and concentric, telescoping needles are extended from the cannula in sequence to trace a pre-planned course through brain tissue. After the leading end of the longest needle emerges through another burr-hole, the surgeon fastens a hollow dialysis fiber to its tip and draws the fiber into the brain by retracting the telescoping device in an orderly sequence. The surgery affects brain tissue only along the course of the fiber, causing about the same acute damage as the stereotactic introduction of an ordinary straight needle. The 'extended local access fiber' is relatively soft, flexible, and biocompatible. It remains permanently in the brain, with its semipermeable portion lodged in the target region. Leading and trailing portions are accessible from outside the brain. Experiments indicate a broad range of possible trajectories and confirm that substances delivered by access fiber can have physiological effects even 10 weeks after implantation.

Animals↗
PubMed1998

Relative importance of surface wettability and charged functional groups on NIH 3T3 fibroblast attachment, spreading, and cytoskeletal organization.

Understanding the relationships between material surface properties, adsorbed proteins, and cellular responses is essential to designing optimal material surfaces for implantation and tissue engineering. In this study, we have prepared model surfaces with different functional groups to provide a range of surface wettability and charge. The cellular responses of attachment, spreading, and cytoskeletal organization have been studied following preadsorption of these surfaces with dilute serum, specific serum proteins, and individual components of the extracellular matrix. When preadsorbed with dilute serum, cell attachment, spreading, and cytoskeletal organization were significantly greater on hydrophilic surfaces relative to hydrophobic surfaces. Among the hydrophilic surfaces, differences in charge and wettability influenced cell attachment but not cell area, shape, or cytoskeletal organization. Moderately hydrophilic surfaces (20-40 degree water contact angle) promoted the highest levels of cell attachment. Preadsorption of the model surfaces with bovine serum albumin (BSA) resulted in a pattern of cell attachment very similar to that observed following preadsorption with dilute serum, suggesting an important role for BSA in regulating cell attachment to biomaterials exposed to complex biological media.

3T3 Cells↗
PubMed1998

A novel method for surface modification to promote cell attachment to hydrophobic substrates.

The ability to study and regulate cell behavior at a biomaterial interface requires strict control over material surface chemistry. Perhaps the greatest challenge to researchers working in this area is preventing the fouling of a given surface due to uncontrolled protein adsorption. This work describes a method for coupling peptides to hydrophobic materials for the purpose of simultaneously preventing nonspecific protein adsorption and controlling cell adhesion. A hexapeptide containing the ubiquitous RGD cell-adhesion motif was coupled to polystyrene (PS) via a polyethylene oxide (PEO) tether in the form of a modified PEO/PPO/PEO triblock copolymer. Triblocks were adsorbed onto PS at a density of 3.3 +/- (5.14 x 10(-4)) mg/m2 (1.4 x 10(5) +/- 2.12 x 10(1) molecules/microm2), which was determined by isotope 125I labeling. The peptide, GRGDSY, was activated at the N terminus with N-Succinimidyl 3-(2-pyridyldithio) propionate and coupled to immobilized triblocks where the terminal hydroxyls had been converted to sulfhydryl groups. Surface peptide density was measured by amino acid analysis and found to be 1.4 x 10(4) +/- 0.47 x 10(4) molecules/microm2. PS modified with PEO/PPO/PEO copolymers alone was found to be inert to cell adhesion both in the presence of serum proteins and when exposed to activated RGD peptide. In contrast, PS conjugated with RGD via endgroup-activated PEO/PPO/PEO copolymers supported cell adhesion and spreading. The surface coupling scheme reported here should prove valuable for studying cell-ligand interactions under simplified and highly controlled conditions.

3T3 Cells↗
PubMed1998

Three-dimensional reconstruction of the membranous vestibular labyrinth in the toadfish, Opsanus tau.

Membranous vestibular labyrinths from the oyster toadfish, Opsanus tau, were fixed, dissected from the animal, stained, and embedded in rectangular blocks of clear histological resin. Photomicrographs of complete embedded labyrinths were taken from six orthogonal directions and used to construct three-dimensional (3D) geometrical models of the semicircular canals, ampullae, utricular vestibule and common crus. Membraneous ducts and ampullae were modeled using a set of cross-sectional elliptical curves laced together to generate curved tubular models of each structure. The ensemble of these curved tubes was used to generate a complete 3D reconstruction of the outside surface of the membranous labyrinth. When viewed from six orthogonal directions, reconstructions closely matched the embedded tissue. Dimensions of the reconstruction and histological sections were compared to measurements of fresh tissue taken from the same animals prior to fixation and used to correct the reconstructions for tissue shrinkage. Results provide estimates of the endolymphatic volumes, local cross-sectional areas and elliptical eccentricities as well as 3D orientations of the geometric canal planes relative to the skull. Ten micrometer histological sections of the material were also prepared to measure wall thickness in various regions of the labyrinth.

Animals↗
PubMed1998

Skeletal myogenesis on elastomeric substrates: implications for tissue engineering.

Studies geared towards understanding the interaction between skeletal muscle and biomaterials may provide useful information for the development of various emerging technologies, ranging from novel delivery vehicles for genetically modified cells to fully functional skeletal muscle tissue. To determine the utility of elastomeric materials as substrates for such applications, we asked whether skeletal myogenesis would be supported on a commercially available polyurethane, Tecoflex SG-80A. G8 skeletal myoblasts were cultured on Tecoflex two-dimensional solid thin films fabricated by a spin-casting method. Myoblasts attached, proliferated, displayed migratory activity and differentiated into multinucleated myotubes which expressed myosin heavy chain on solid thin films indicating that Tecoflex SG-80A was permissive for skeletal myogenesis. Porous three-dimensional (3-D) cell scaffolds were fabricated in a variety of shapes, thicknesses, and porosities by an immersion precipitation method, and where subsequently characterized with microscopic and mechanical methods. Mechanical analysis revealed that the constructs were elastomeric, recovering their original length following 100% elongation. The 3-D substrates were seeded with muscle precursors to determine if muscle differentiation could be obtained within the porous network of the fabricated constructs. Following several weeks in culture, histological studies revealed the presence of multinucleated myotubes within the elastomeric material. In addition, immunohistochemical analysis indicated that the myotubes expressed the myosin heavy chain protein suggesting that the myotubes had reached a state of terminal differentiation. Together the results of the study suggest that it is indeed feasible to engineer bioartificial systems consisting of skeletal muscle cultivated on a 3-D elastomeric substrate.

Animals↗
PubMed1996

A diffusible coupling signal from the transplanted suprachiasmatic nucleus controlling circadian locomotor rhythms.

The mammalian suprachiasmatic nuclei (SCN) transmit signals to the rest of the brain, organizing circadian rhythms throughout the body. Transplants of the SCN restore circadian activity rhythms to animals whose own SCN have been ablated. The nature of the coupling signal from the grafted SCN to the host brain is not known, although it has been presumed that functional recovery requires re-establishment of appropriate synaptic connections. We have isolated SCN tissue from hamsters within a semipermeable polymeric capsule before transplantation, thereby preventing neural outgrowth but allowing diffusion of humoral signals. Here we show that the transplanted SCN, like neural pacemakers of Drosophila and silkmoths, can sustain circadian activity rhythms by means of a diffusible signal.

Animals↗