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WO1992000047A1 - Procede de regulation de la croissance cellulaire sur des surfaces - Google Patents

Procede de regulation de la croissance cellulaire sur des surfaces Download PDF

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Publication number
WO1992000047A1
WO1992000047A1 PCT/US1991/004466 US9104466W WO9200047A1 WO 1992000047 A1 WO1992000047 A1 WO 1992000047A1 US 9104466 W US9104466 W US 9104466W WO 9200047 A1 WO9200047 A1 WO 9200047A1
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WIPO (PCT)
Prior art keywords
molecules
adhesion
functional group
group
growth
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PCT/US1991/004466
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English (en)
Inventor
Chaim N. Sukenikk
Lloyd A. Culp
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Case Western Reserve University
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Publication of WO1992000047A1 publication Critical patent/WO1992000047A1/fr

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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y30/00—Nanotechnology for materials or surface science, e.g. nanocomposites
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/28—Materials for coating prostheses
    • A61L27/34—Macromolecular materials
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/38—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix containing added animal cells
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin

Definitions

  • the present invention relates in general to cell growth and, in particular, to a method for controlling cell growth on a surface utilizing a molecular monolayer.
  • a metallic implantable device such as a hip implant with its surface covered with tiny projections or posts for tissue ingrowth, is described in U.S. Patent No. 4,608,052, which is also incorporated herein by reference.
  • these techniques suffer from the fact that the body may recognize the metal as a foreign material and produce a fibrous layer between the body and the implant, preventing a close knit between the body and the implant.
  • Another effort to deal with this problem has been to coat the implant with a bone-like calcium phosphate crystal called hydroxyapatite, which the body may accept as a non-foreign bone material.
  • hydroxyapatite is useful, it is useful only in connection with bone-forming cells.
  • fibroblasts and neuroblasts are the preselected cell types, and the growth of each type is controlled.
  • cell growth means cell survival, cell division, and/or cell differentiation.
  • the process comprises first coating the surface with a molecular monolayer and providing a preselected functional group at the distal end thereof. A layer of plasma fibronectin or other adhesion mediating molecule is then coated onto the molecular monolay er.
  • the substrate thus prepared will affect and control th growth characteristics of different cell types in contac with that surface. As a result, growth of certain types o cells which would facilitate tissue ingrowth and knittin between implant and body can be enhanced, and growth of othe types of cells on the surface can be repressed or inhibited.
  • a cell growth surface is a surface upon which cell growth ma take place, and includes a glass slide, a petri dish, a 24- well dish, and an industrial bioreactor with beads, baffles and/or stirrers therein.
  • cell culture may be grown on surfaces in a Corning Pyrex Slo Speed Stirring Vessel, #26501-1L, containing therein Kontes Cytocarriers.
  • Implantable devices include devices implant- able in humans as well as devices implantable in animals. As used in this specification and the claims herein, adhering includes both active and passive attachment.
  • the present invention finds utility (a) in the field of body implants and prosthetics, particularly implant- able devices made of titanium, (b) in applications involving bio-repulsive surfaces for implants and moving parts of prosthetics, as well as more controlled bio-adhesive surfaces for the structure of the prosthetic device, and also (c) in the field of cell and tissue growth,* where containers and laboratory dishes and glassware with preselected surface characteristics can control, enhance, repress, and otherwise mediate growth of preselected cell types and cultures.
  • Surface treatments that enhance the rate of cell attachment and growth would be a major benefit to both research labora- tories and to the scaled-up production of specific cell lines and cell-derived materials- Many aspects of the foregoing discussion and invention are disclosed in Lewandowska, K.
  • Titanium is used increasingly as an implant materi- al. Among other reasons, its mechanical properties are closer to those of bone than are stainless steel and cobalt- chromium alloys. Coatings or surface alterations that promote cell attachment and regulate physiological response would make titanium even more useful.
  • Implants made of metals other than titanium could also be coated using reason- ing and procedures similar to those described herein to control cell attachment and regulate cell-type specific physiological response.
  • Thin organic, molecular monolayer films offer an excellent method for the modification of surface properties.
  • a high level of molecular monolayer uniformity can usually be achieved using a carbon chain at least 14 carbons long excluding the functional end group. However, somewhat shorter carbon chains may be successful in this application. Carbon chains containing 22 carbon atoms have been success- fully prepared in other applications, and it is believed that carbon chains of similar length, or longer, may be used herein.
  • the carbon chain is typically polymethylene to assure sufficient chain flexibility for assembly and packing-
  • polymethylene chains in this application can toler- ate, and are meant to include, the incorporation of double bonds, an aromatic ring, a limited number of hetero-atoms, and/or halogenated substituents or segments.
  • Self-assembly of SiCl ⁇ -terminated long-chain amphiphiles forms well-ordered, siloxy-anchored, crosslinked monolayers, as described in U.S. Patent No. 4,539,061 to Sagiv, the contents of which are hereby incorporated by reference herein.
  • Sagiv does not teach any metho of consistently preparing a titanium surface so that it will accept a molecular monolayer.
  • a clean titanium surface, unprepared in accordance with the present invention, will generally not accept or bond to a molecular monolayer as described in Sagiv.
  • the present disclosure teaches a solu- tion to this problem, which comprises increasing the number of hydroxy groups available for reaction on the metallic surface by maintaining the metallic surface in contact with boiling water for a sufficient period of time, or with water at a temperature of more than 40 degrees Centigrade with sonication for a sufficient period of time.
  • the modification of titanium surfaces with cova- lently-attached, self-assembled monolayers offers many advantages.
  • the coating process involves dipping the surface being treated into a dilute, homogeneous solution of surfactant in an organic solvent, it is versatile and can be applied to materials and • implants of almost any configura- tion. Coating of already fabricated implants and prostheses would thus be readily achieved. Since the monolayer film so completely isolates the substratum from the outside environ- ment, it also permits the creation of surfaces with specific properties on various bulk materials. Finally, the ease with which such surfaces can be transformed by conventional organic chemistry allows the creation of surfaces with the functionality needed to impart desirable chemical and physi- cal properties. The stability, uniformity, and manipulabili- ty of these surfaces should all combine to make them useful in the design of new biomaterials.
  • any oxide or hydroxide- bearing surface similar to glass or titanium may be expected to undergo chemistry and biochemistry similar to that de- scribed herein.
  • molecular monolayers may be applied to a wide range of surfaces.
  • Various functional groups have been incorporated into the surface of these very uniform molecular monolayer assemblies. See Balachander, N., and Sukenik, CN. , "Func- tionalized Siloxy-Anchored Monolayers With Exposed Amino, Azido, or Cyano Groups," Tetrahedron Letters, 29:5593-5594 (1988), the contents of which are incorporated by reference herein.
  • a surface to which is attached a molecular monolayer having a preselected functional group at its distal end is referred to as a "derivatized surface.”
  • the ability of these monolayers to effectively isolate their substrate is clear. Since not all functional groups can coexist with the SiClg group needed to anchor the monolayer, surfactants or monolayer precursor molecules containing a chemically modifiable group that can coexist with the SiClg group have been developed. Given these materials and the stability of the siloxy-bound monolayer, in situ generation of yet additional functionality can be achieved.
  • Adhesion-mediating molecules include several proteins that have cell-type-specific receptors for selected cell populations.
  • Fibronectin as an extracellular matrix glycoprotein, is an adhesion-mediating molecule that mediates adhesion of many mesenchymal and some non-mesenchymal cells to their collagen environment. This occurs by the binding to fibronectin of (a) glycoprotein receptor complexes on the cell surface called "integrins," as well as of (b) heparan sulfate proteoglycans on the cell surface. This facilitates the complete physiological response from some cells. Laminin is also an adhesion-mediating molecule.
  • Mouse Balb/c 3T3 cells are an excellent model of fibroblasts that come from many tissues of both human and non-human animal species.
  • Platt neuroblastoma cells are an excellent model for the differen- tiation processes of some neuron populations that come from human and non-human species.
  • the adhesion-mediating pro- Waits of mouse Balb/c 3T3 cells are essentially identical to those of normal (non-malignant) fibroblast cells.
  • the adhesion-mediating processes of Platt neuroblastoma cells are essentially identical to those of normal (non-malignant) neuron-derived cells.
  • FIG. 1 illustrates a molecular model of derivatized substrate
  • FIG. 2 illustrates the binding of plasma fibronectin to substrata
  • FIG. 3 illustrates the quantitation of cell attach- ment on substrata
  • FIG. 4 illustrates the quantitation of neurites on substrata
  • FIG. 5 illustrates a molecular model of monolayer functionalized surfaces on glass and on titanium
  • FIG. 6 illustrates the relative degree of adsorption of plasma fibronectin to glass and titanium surfaces.
  • Plasma fibronectin was adsorbed onto glass surfaces derivatized with an alkyl chain and six chemical end groups interfacing with the bound plasma fibronectin.
  • the response of fibroblasts (Balb/c 3T3 cells) and human neuron-derived (Platt neuroblastoma) cells adhering to the plasma fibro- nectin was examined. Using new derivatization protocols, the following surfaces were tested in order of increasing polari- ty: [CH 3 ], [C-C], [Br], [CN], [Diol], [COOH], and underivatized glass [SiOH].
  • these experiments demonstrate that different chemical end groups on the substratum modulate, control, enhance, repress, and/or inhibit functions for cell adhesion, growth, and their specialized differentiation functions, principally by affecting the conformation of these molecules rather than the amounts bound. Furthermore, these experiments confirm multiple-receptor interactions with the fibronectin molecules in cell-type-specific adhesion pat- terns.
  • BSA bovine serum albumin
  • FIG. 1 illustrates glass coverslips derivatized 0 by the attachment of a functionalized 14-carbon aliphatic 1 chain to the surface-available silicon atoms.
  • a 2 siloxane network covalently anchors an array of hydrocarbon 3 chains terminating with one of the end groups [X] interfacing 4 the medium.
  • [X] is the active component in the binding 5 reactions of fibronectin.
  • Deposition of the self-assembled monolayer films 9 was achieved by dipping glass coverslips, cleaned by an Argon 0 plasma, into 20 mM solutions of the SiCl 3 derivative in 1 dicyclohexyl for 2-5 minutes, achieving maximal derivati- 2 zation as ascertained below.
  • [CN] and [Br] have water contact angles of 74 and 81 , respectively, and show the expected ESCA signals for the heteroatom ([CN] N at 403 eV; [Br] Br at 72 eV, uncorrected for shift caused by insulator substrate).
  • [CN] on silicon ATR prisms have an infrared absorption at 2,247 cm " .
  • the [Diol] and [COOH] surfaces, derived from the hydrophobic [C C] monolayers, were hydrophilic (water contact angles of 30 and 52 , respective- ly).
  • Adhesion Assays EGTA-detached 3T3 (10 x 10 4 ) or Platt (5 x 10 4 ) cells were inoculated into wells containing adhesion medium and plasma fibronectin-coated, derivatized glass coverslips. To quantitate attachment, cells had been previously radio-
  • 3T3 cells spreading for 4 hours were fixed with 3.7% formaldehyde in PBS for 20 minutes and then treated as previously described (Laterra et al., J. Cell Biol. , 96:112-123 (1983)) to bind rhodamine-phalloidin to the F-actin-containing networks.
  • Stained coverslips were inverted into 50% glycerol:PBS and evaluated in the Nikon Diaphot microscope with fluorescence 4 illumination (photographed under an xlOO objective with Kodak 2475 recording film) .
  • FIG. 1 Plasma fibronectin binding to derivatized substrate (FIG. 1) was tested by ELISA.
  • FIG. 2 illustrates plasma fibronectin (pFN) binding to substrata.
  • BSA bovine serum albumin
  • plasma fibronectin (2.5 ug/ml) was mixed with an excess of bovine albumin (17.5 ug/ml) prior to adsorption of the mixture to surfaces (FIG. 2)
  • plasma fibronectin still bound effectively and competitively achieved a concentration on the substratum similar to that of plasma fibronectin alone, indicating the effectiveness of plasma fibronectin interaction with all surface end groups in competition with albumin.
  • Two excep- tions were noted with the [CH ] and [COOH] substrata with a smaller amount of plasma fibronectin bound. (However, this small reduction could not be an explanation for altered cell responses as shown below) .
  • cytoplasmic spreading and differentiation of cells were significantly different among substrata.
  • Reorganization of microfilaments (F-actin) into stress fibers by fibroblasts on fibronectin requires complex reactions, including trans- membrane signaling from fibronectin to both heparan sulfate proteoglycans (Laterra et al., J. Cell Biol. 96:112-123 (1983) and the glycoprotein integrin (Tamkun et al., Cell , 46:271-282 (1986) on the cell surface. Burridge et al., Annu. Rev. Cell Biol. 4:487-525 (1988).
  • stress fibers and focal contacts on the substratum are a diagnostic indicator of the complete response of fibroblasts permitting subsequent movement, cell division, and expression of genes linked to anchorage dependence (Dike and Farmer, Proc. Natl . Acad. Sci . U.S.A. , 85:6792-6796 (1988)).
  • F-actin cytoskeletal reorganization in adherent 3T3 cells 3T3 cells were detached from stock cultures and washed by repeated resuspension/centrifugation.
  • Rhodamine-phalloidin stains extensive stress fibers formed on plasma fibronectin-coated [SiOH].
  • cells on highly polar [COOH] and [Diol] surfaces had greatly reduced F-actin organization with linear bundles of limited distance and lacking the extensive pattern shown with respect to [SiOH].
  • a similar pattern was observed on [CN] surfaces.
  • Some thin fibers could be identified in approximately one-third of the cells on both [COOH] and [CN] substrata.
  • Hydrophobic substrata represented by [CH ] yielded a stress fiber pattern similar to the [SiOH] control in one subpopulation of cells and some thinner fibers evident in a second subpopulation of cells.
  • the results of the inhibitor study confirmed the differing binding relationships of plasma fibronectin on derivatized substrata with the integrin complex as the cell surface. Since cells were treated uniformly in this paradigm, these results support the belief that plasma fibronectin on these substrata has differ- ing conformations with varying interactions with cell surface receptors (such as integrins and heparan sulfate proteo- glycans) .
  • neuritogenesis was tested on derivatized substrata to determine whether fibronectin conformational changes generate all-or-none or intermediate responses from such cells.
  • neuritogenesis of Platt neuroblasto- ma cells on substrata Platt human neuroblastoma cells were detached from stock cultures by EGTA treatment, as described
  • Platt neuroblastoma cells were treated as described above with regard to neuritogenesis of Platt neuroblastoma cells on substrata.
  • Neurite-bearing cells were enumerated as defined by Mugnai et al , J. Cell Biol. , 106:931-943 (1988) and their percentage in the total adherent cell population determined.
  • For Platt there are marked differences in the percentage of neurite-bearing cells on these substrata, as seen in FIG. 4.
  • fibronectin functions can be modulated by chemical end groups of the inert substratum to which fibronectin is bound.
  • Substrata of all six chemical groups adsorbed the same amounts of fibronectin when compared with underivatized glass, including a case in which plasma fibronectin competes with an excess of albumin for binding (i.e., an 8:1 mass excess and a 28:1 molar excess). Since a diverse series of end groups were used, the binding of fibronectin must occur through a multi- plicity of amino acid side-chain interactions with substrata, including hydrogen bonding, van der Waals interaction, and ionic interactions, any one of which may be sufficient for binding.
  • fibronectin substrata to facilitate attachment processes only (including the glycoprotein integrin class, the heparan sulfate proteoglycans, and the highly sialylated gangliosides) . These data indicate that at least one of the binding domains along substratum-bound fibronectin molecules is available for interactions with one or more of these surface molecules in all cases.
  • cytoplasmic spreading and differentiation require transmembrane signaling from surface receptors that bind coordinately to fibronectin and to cytosolic elements within the cell.
  • F-actin reorganization in the 3T3 cells was reasonably effective on the hydrophobic sub- strata, while these substrata were the poorest for neurite formation of neuroblastoma cells.
  • the [Br] substratum yielded an excellent stress fiber pattern in 3T3 cells, but was poor for eliciting neurites from the neuroblastoma cells.
  • the dimeric fibronectin molecule exhibits complex binding properties as it interacts with inert substrata containing various end groups. These end groups can modulate the functions of fibronectins during their reaction with a multiplicity of cell surface receptors.
  • This level of regulation and control of adhesion-promoting proteins and the cells adhering thereto is important with regard to the effectiveness of biomaterial interactions with differing biological systems, such as implants in a body. In this regard, differing cell and tissue types from the body or animal are predicted to respond differently, based on the parameters observed in this study.
  • titanium has been modified by covalent attachment of an organic molecular monolayer an- chored by a siloxane network.
  • the titanium surface often requires enhancement prior to attachment of the monolayer.
  • This monolayer coating completely covers the metal and allows controlled modification of surface properties by modification of the exposed chemical end groups of the monolayer-forming surfactant.
  • glass and titanium are derivatized with the same chemical end groups and coated with plasma fibronectin, and preselected cell types are adhered thereto, the responses are cell-type-specific, as discussed above, and are indepen- dent of the character of the substrate as glass or titanium.
  • Identical surfaces are obtained on the glass and titanium; only the monolayer coating interacts with the environment. Surfaces bearing each of four different chemical end groups were used; see FIG. 5.
  • the surfactant was added to dicyclohexyl under inert atmosphere and transferred to the bench top. All surfactant solutions were used within 1 to 3 hours after their preparation. Monolayers were prepared by holding the substrate (glass or titanium) with Teflon- coated tweezers and immersing it into a 10 mL beaker contain- ing the surfactant solution and a magnetic stirrer. The substrate is quickly withdrawn after 2 to 15 minutes, washed twice with CHC1 3 and water, and Soxhletted with hot CHC1 3 or 1:1 v/v CHCl 3 /EtOH for 15 minutes.
  • X-Ray Photoelectron Spectroscopy XPS measurements were carried out on a PHI-Unicam Perkin Elmer instrument. Analyses were done using Mg K -_7 lines at a pressure of 10 -9 torr with a take-off angle of 45 degrees. Survey spectra were recoded on a 1 mm spot, with 150 eV pass energy, 200 W electron beam power, and an acqui- sition time of 7 minutes. Multiplex spectra of the individ- ual elements were carried out on a 1 mm spot, with 50 eV pass energy and a 30-minute acquisition time. Peak positions are referenced to the C Is peak at 285 eV.
  • Platt neuroblastoma cells were grown in stock culture in Dulbecco's modified Eagle's medium (DMEM) supple- mented with 5% newborn calf serum and antibiotics. These cells make neurites constitutively in serum-containing or protein-free media on plasma fibronectin-adsorbed tissue culture substrata. Dulbecco's medium supplemented only with 250 ug/mL heat-treated bovine albumin, referred to as "adhe- sion medium,” is used for all animal cell adhesion experi- ments. Cells were treated as described in Example I.
  • Phase contrast microscopy required 5% glutaralde- hyde fixation (in PBS) of cells and examination of glass coverslips under a Nikon Diaphot microscope using Kodak 2415 film. Cells fixed on titanium were examined and photographed under epi-illumination in a Zeiss photo-microscope, using the same film.
  • coverslips were treated as described in Example I. Briefly, they were fixed in a 2% paraformaldehyde/2% glutaraldehyde mixture in 2X DMEM, dehydrated with increasing concentrations of absolute ethanol-water, critical point-dried in liquid CO2, and sputter-coated with gold-palladium (Technics Hummer V). Coverslips were examined on a JEOL 840 SEM (tilt angle 35 degrees) and photographed with Polaroid 55 positive- negative film.
  • the difference between advancing and receding contact angles (hysteresis) for a given surface is related, among other things, to the heterogeneity of the surface.
  • the [CH 3 ] and surfaces (FIG. 5) have advancing water contact angles of 110° and 105°, respectively, and are both hydrophobic and oleophobic. One or the other of these surfaces served as the hydrophobic test surface in each of the experiments below.
  • the diol-terminated monolayer and the bare glass and titanium are all hydrophilic.
  • the [Br] surface has an advancing contact angle of 82 degrees for both substrates, and is of intermediate hydrophobicity.
  • the somewhat greater spread in contact angle values and the greater hysteresis for the titanium surfaces reflects greater surface heterogeneity and is consistent with the difference in texture and surface roughness seen in the scanning elec- tron microscopy (SEM) of these surfaces in the animal cell adhesion study (vide infra).
  • SEM scanning elec- tron microscopy
  • XPS was used to verify the elemental composition of the monolayer.
  • Their hydrocarbon packing was determined using the integrated intensities of the C Is peak. Since glass is insulating, the peak positions were adjusted by fixing the C Is peak at 285 eV. All surfaces showed the expected carbon peak and the [Br] surfaces showed the ex- pected peak at 70.2 eV.
  • the integrated peak intensities were consistent with comparable monolayer packing on both glass and titanium substrata and comparable packing density among the various monolayers.
  • coverslips in wells were rinsed with PBS and adsorbed fibronectin tested in an ELISA assay as described in Materials and Methods. Standard errors of multiple determinations are shown with the error bars.
  • FIG. 6 where a super-saturating amount of plasma fibronectin (20 ug/mL) was incubated with either glass or titanium coverslips for 1 hour, comparable amounts of fibronectin bound to both underivatized glass or titanium surfaces, as well as to the three classes of derivatized glass or titanium surfaces.
  • albumin adsorption blocked the substratum from plasma fibronectin binding and provided a negative control substratum to evaluate cell responses.
  • neuronal cells require several different receptors to interact with different binding domains of fibronectin, this cell system is particularly sensitive to conformational changes that may occur upon fibronectin binding to various derivatized substrata.
  • Cell attachment on all surfaces was comparable, but spreading and neurite responses mediated by transmembrane signaling processes were quite different.
  • neuroblastoma cells became bipolar; some cells were extending, short, neurite-like pro- Waits, while some cells were extending, long, linear, thin neurites.
  • plasma fibronectin binds comparably to derivatized glass or titanium surfaces; fibronectin binding is not limiting cell response. However, this binding was only tested in homogeneous solutions of plasma fibronectin.
  • these results with animal cell adhesion responses verify that the chemical end groups facing the medium, and therefore interacting directly with fibronectin molecules bound to the surface, alter the conformation of fibronectin molecules in ways that lead to differing cell surface receptor responses from cells. Therefore derivati- zation of biomaterials can be used to manipulate the short term (and possibly long term) responses from select animal cell types.

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Abstract

Procédé de sélection de types de cellules se développant sur une surface, tel qu'un dispositif implantable ou une surface de croissance cellulaire. Le dispositif implantable peut avoir une surface en titane. Le procédé consiste à fixer une monocouche moléculaire à la surface de la structure. La monocouche comporte un groupe fonctionnel au niveau de son extrémité distale. Les groupes fonctionnels possibles sont CH3, CH=CH2, Br, CN, COOH, et CHOHCH2OH. On enduit la monocouche d'une molécule enduisant une adhérence telle que la fibronectine. Les cellules viennent alors au contact du revêtement. Le caractère du groupe fonctionnel affecte les caractéristiques de croissance de la cellule d'adhérence ou de contact, indépendamment de la nature de la structure sous-jacente. L'invention concerne également un procédé de préparation d'une surface métallique telle que du titane en vue de l'application d'une monocouche moléculaire. On place la surface dans de l'eau chaude (40 à 50 °C) pendant quatre heures avec sonication, ou dans de l'eau bouillante pendant 8 heures sans sonication.
PCT/US1991/004466 1990-06-22 1991-06-20 Procede de regulation de la croissance cellulaire sur des surfaces WO1992000047A1 (fr)

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WO1998052619A3 (fr) * 1997-05-22 1999-03-18 Merck Patent Gmbh Implants recouverts de peptides et procede permettant de les preparer
WO1999026674A3 (fr) * 1997-11-24 1999-09-16 Herbert P Jennissen Procede pour immobiliser des molecules mediatrices sur des materiaux d'implants inorganiques et metalliques
WO2002009788A1 (fr) * 2000-08-01 2002-02-07 Jennissen Herbert P Procede de production de surfaces d'implants bioactives
WO2003045461A1 (fr) * 2001-11-23 2003-06-05 Feg Textiltechnik Forschungs- Und Entwicklungsgesellschaft Mbh Produit textile a surface modifiee et procede de modification de surface correspondant
EP1338292A1 (fr) * 2002-02-20 2003-08-27 NGK Spark Plug Co. Ltd. Biomatériau osteoconducteur, et procédé de préparation
WO2004100926A3 (fr) * 2003-05-13 2005-02-24 Medtronic Inc Materiaux pouvant etre traites a l'humidite pour la delivrance d'agents, procedes, et dispositifs medicaux
CN115369105A (zh) * 2022-09-27 2022-11-22 同济大学 一种藻细胞固定方法和应用

Citations (2)

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