WO2000027444A1

WO2000027444A1 - Functionalised polymers for tissue engineering

Info

Publication number: WO2000027444A1
Application number: PCT/GB1999/003727
Authority: WO
Inventors: Jöns Gunnar HILBORN; Peter Frey
Original assignee: Ecole Polytechnique Federale De Lausanne; Centre Hospitalier Universitaire Vaudois; Kiddle, Simon
Priority date: 1998-11-09
Filing date: 1999-11-09
Publication date: 2000-05-18
Also published as: EP1128853A1; AU1061000A; GB9824562D0

Abstract

Methods for functionalising polymers for tissue engineering are disclosed in which a polymer matrix is activated to bind to biological materials such as cells by covalently bonding a hydrophilic polymer to carboxy groups of a polymer matrix. The hydrophilic polymer comprises a ligand group or ligand group precursor which can bind to an extracellular matrix protein, thereby allowing cells to adhere and proliferate on the polymer matrix.

Description

FUNCTIONALISED POLYMERS FOR TISSUE ENGINEERING

Field of the Invention The present invention relates to materials and methods for functionalising polymers, and in particular for producing a polymer matrix having a surface, comprising ligand groups which are capable of interacting with a biological material such as cells. These polymer matrices are useful in the production of tissue engineered products.

Background of the Invention

Worldwide millions of patients suffer from tissue loss or organ failure per year. Today, solutions to restore organ structure and function are based on the principle "robbing Peter to pay Paul" (Patrick et al, 1998), that is, defects in tissue resulting from congenital abnormalities, disease or trauma are repaired by transferring healthy donor tissue to the site of the tissue defect. These solutions have resulted in enormous improvements in patient survival and increases quality of life.

It is estimated that in the United States alone, the cost of treating organ failure and tissue loss was more than $400 billion per annum (Lipkin, 1995) . In addition, as life expectancy continues to rise, so will the need for tissue and organ treatments. All of these factors mean that the demand for replacement organs is today very high and will continue to rise. However, this demand is balanced by the scarcity of donors, the problem of foreign body rejection, the requirement for intensive patient care and the high cost of transplantation operations. Therefore, tissue engineered products are expected to have an enormous impact on human welfare in the future. The importance of tissue engineered products can be appreciated by considering what happens during wound healing. Upon injury to the extracellular matrix, inflammatory response cells remove damaged -tissue using both intracellular and extracellular degradation. The inflammation response also involves the migration of cells to the wound site to assist the healing process. Two mechanisms are involved in the replacement of dead tissue with living, regeneration and repair. Repair involves the replacement of damaged tissue with granulation tissue which is able to retract the wound space resulting in formulation of scar tissue. While retraction is of great importance to avoid infection and maintain homeostasis, it often results in severe scarring and organ malfunction. Conversely, regeneration replaces damaged tissue with functional tissue having the same or similar function and is generally the outcome when injury has occurred in tissue composed of labile or stabile cells .

The ability of different species to regenerate organs varies widely. For example, with assistance an immature frog can regenerate entire amputated limbs provided it has not yet undergone metamorphosis (Wallace, 1981) . However, mammals do not have this capability and instead non physiological connective tissue forms in the wounded area leading to scar tissue. On the other hand, if cultured autologous cells from a particular organ are implanted into a partly removed organ, the surrounding tissue can induce the synthesis of new tissue or transform the implant to physiological tissue, in situations where spontaneous regeneration would not normally occur. Most mammalian cells must be in contact with a tissue structure, such as the extracellular matrix, in order to survive. Tissue engineered materials are typically made to mimic this situation by harvesting cells from a specific organ or tissue type, expanding the cells in vi tro and seeding them onto a suitable scaffold material to provide the tissue engineered material for transplantation. A number of requirements are placed such scaffolds and meeting these requirements is a key step in the successful production of tissue implants. Firstly, the scaffold or template carrying the cells needs to provide temporary mechanical support for the cells and needs to simulate the natural extracellular matrix. In addition, the cells must adhere to the scaffold and function normally on it. The scaffold must also have the right surface chemistry to guide and reorganise the cell mass, e.g. by immobilising proteins such as those found in the extracellular matrix or specific peptide sequences on the scaffold to interact with cells. Finally, in some cases, after the support has provided temporary mechanical support for the cells, it should be readily absorbed by the body, leaving no residues and producing no toxic degradation products.

In the prior art, scaffolds have been made out of extracellular matrix components, such as collagen, laminin or fibronectin. These materials have excellent cell adhesion properties, biodegradability and biocompatibility, but suffer from the disadvantages that they cannot be freely or reproducibly processed into stable three dimensional shapes without losing their biocompatibility and that they have poor mechanical strength. Scaffolds have also been made out of thermoplastic biodegradable polymers, such as polylactic acids, polyglycolic acid or their copolymers . These materials have excellent strength and ductility and can readily be made into various shapes or products, but have hydrophobic surfaces which lack functional groups capable of interacting with cells.

In order to overcome these problems, there" have been many attempts in the prior art to synthesize new polymers which combine biodegradability and surface reactivity with good mechanical properties. WO94/09760 discloses method of synthesizing polymers from a mixture of hydroxy acids and amino acids, the side chains of the amino acids providing functional groups to which cells can be coupled. The application exemplifies the functionalisation of poly (lactic acid-co-lysine) with an arginine-glycine-aspartic acid (RGD) sequence coupled using 1, 1-carbonyldiimidazole (CDI) in the bulk. However, this method provides a polymer which is chemically different from PLA and, in practice, the mechanical properties of the polymer have been shown to be inadequate.

Other approaches have focussed on the activation of polymer surfaces, either by exposing polymers to radiation or by treating them chemically, in an attempt to render their surfaces reactive towards proteins or cell adhesion. However, approaches employing UV radiation, electron beams, gamma radiation or plasma to treat polymer surfaces suffer from a lack of molecular specificity and are often accompanied by undesirable alterations to the composition or strength of the base polymer. By way of example, WO97/05193 discloses processes for activating the surface of copolymers of lactic acid and e-caprolactone by treatment with strong acid or base, cold plasma or electromagnetic radiation. In a further approach, glutaraldehyde has been used to activate the surface of poly (D, L-lactide-co-glycolide) , allowing the subsequent coupling of recombinant hirudin (r-Hir) to improve haemocompatibility (Seifert et al, 1997) . However, this reaction has the drawback that glutaraldehyde used in the activation step -is toxic. Another prior art approach employs a two step process in which aqueous base is used to activate the surface of poly (L-lactic acid), producing carboxyl groups which were then be coupled to RGD peptide to provide a site for cell attachment (Yamoaka et al, 1996) .

W097/46267 discloses a method of rendering the surface of a hydrophobic biodegradable polymer more hydrophilic by physically adsorbing a polymeric surfactant on the surface of the hydrophobic, cross-linking the surfactant molecules to each other and reversibly attaching a bioactive species to this hydrophilic layer using degradable chemical linkages. The aim of this approach is to provide a polymeric scaffold which is readily degradable in use. However, the use of physisorption leads to the risk that the bioactive species or the surfactant layer can become detached from the polymer matrix, a particular problem in in vivo use.

Summary of the Invention

Broadly, the present invention relates to a method of functionalising a polymer matrix to provide ligand groups on the surface of the polymer which are capable of binding to biological materials such as cells. In this method, a functionalising reagent directly reacts with carboxy groups in the polymer to produce ligand groups or ligand group precursors on the polymer surface.

Thus, in contrast to the prior art methods, the present invention does not rely on new polymer synthesis, the activation of the polymer surface by exposing the polymer to radiation or harsh chemical reagents, or the use of physical adsorption to introduce a cell reactive layer to approach the problem of introducing functional groups into the polymer. Instead, the method employs a functionalising reagent and the inherent reactivity of the polymer backbone or side groups to produce functional groups which can interact with biological materials such as peptides, proteins or cells.

Accordingly, in a first aspect, the present invention provides a method for producing a polymer matrix having a surface comprising ligand groups which are capable of interacting with a biological material, the method comprising reacting a carboxy-group containing polymer matrix with a functionalising reagent which is a hydrophilic polymer comprising a ligand group or a ligand group precursor, wherein the reaction is carried out in a solvent in which the polymer matrix is insoluble and the functionalising reagent is soluble and the functionalising reagent reacts and covalently bonds to the carboxy groups on the surface of the polymer matrix.

As the reaction is carried out in a solvent in which the matrix polymer is insoluble, this helps to avoid shape changes by swelling and minimises solvent residues in the matrix and promotes the reaction at the surface of the polymer matrix.

In a preferred embodiment, the polymer matrix is reacted with a functionalising reagent, such a hydrophilic polymer, represented by the general formula R-YX, wherein R comprises the ligand group or a ligand precursor and Y is a functional group which is capable of reacting with carbonyl groups of the polymer in the general reaction:

a r x po ymer n so u on

In this reaction scheme, when the reaction is carried out in an aprotic solvent, R-YX is ROH, RCOOH, RNH₂, RCOOR, RCOOCOR, RSH or another species that reacts with the carboxy groups of esters, carbonates or anhydrides. Alternatively, when the reaction is carried out in an aqueous solvent, R-YX is RNH₂ or another species which is significantly more nucleophilic than water.

Thus, the present invention provides a method of activating the surface of polymers which would otherwise be relatively unreactive with biological materials, and helps to avoid the prior art problem of weakening the bulk properties of the polymer by treatment with radiation or the use of toxic reagents. The use of a functionalising reagent is also different to the chemical activation of the prior art relying on a two step reaction in which the polymer surface is initially activated, e.g. by hydrolysis with aqueous base, as the functionalising reagent reacts with the polymer and introduces the ligand or ligand precursor in the same step. The carboxy groups may be present in the backbone and/or side chains of the polymer. Examples of polymers having suitable carboxy groups include polyesters, polycarbonates or polyanhydrides . The method is applicable to the treatment of polymers which are non degradable or degradable in vivo. Preferred degradable polymers include polyacrylic acid, polylactic acid (PLA), polyglycolic acid, polyglycolic acid-lactic acid (PGLA) copolymers or mixtures of polylactic acid and polyglycolic acid. Non degradable polymers such as polyethyleneterephtalate (PET), polycarbonate (PC) or polymethylmethacrylate (PMMA) may serve as temporary cell carriers or permanent implants.

The method may also comprise the step of casting or shaping the polymer into a form suitable for the implant or cell support. Preferably, the moulding or casting step is carried out before attachment of the biological material or the activating reaction. The implant may be to replace tissue or organs. Examples of implants produced according to the present invention are discussed in more detail below.

The functionalising reagent is preferably a hydrophilic polymer selected from one or more of polyacrylic acid, polymethylacrylic acid, polyvinylalcohol, polyethyleneimine, polyvinylamine, dextrane or polyethyleneoxide .

In a further aspect, the present invention provides a method of producing a polymer matrix including cells immobilised on the surface of the matrix, the method comprising: having reacted the polymer matrix comprising carboxy groups with a hydrophilic polymer so that the hydrophilic polymer covalently bonds to the surface of the polymer matrix via the carboxy groups, the hydrophilic polymer comprising ligand groups which are capable of binding to cells, wherein the ligand groups are an extracellular matrix protein capable of adhering to cells or a fragment thereof which retains the property of adhering to the cells ; the further step of contacting the polymer matrix with cells so that the cells adhere to the ligand groups.

The method therefore contacts the biological material with the polymeric scaffold with the biological material under conditions in which the biological material binds or covalently attaches to the ligand groups on the polymeric scaffold. This is often referred to as seeding the polymeric scaffold, and may involve the further step of checking viability of the cells after the immobilisation reaction is completed.

In a further aspect, the present invention provides a polymer matrix produced by one of the above methods.

In a further aspect, the present invention provides a tissue engineered product comprising: a polymer matrix comprising an carboxy-group containing polymer, the polymer matrix having a surface to which hydrophilic polymer molecules are covalently bonded to the carboxy groups of the polymer; the hydrophilic polymer comprising ligand groups for binding to cells, wherein the ligand groups are an extracellular matrix protein or a fragment thereof which retains the property of adhering to the cells; cells adhered to the polymer matrix via the ligand groups . In further aspects, the present invention provides polymer matrices produced by the above methods for use in methods of medical treatment, and the use of such polymer matrices for the preparation of a polymeric implant for transplantation into a patient, wherein the polymer matrix is contacted with the biological mat-erial under conditions in which the biological material adheres or covalently binds to the ligand groups on the hydrophilic polymer .

By way of example, embodiments of the present invention will now be described in more detail below.

Detailed Description Polymers:

There are a range of polymers known in the art that include carboxy (-COO-) groups, present in either the polymer backbone or side chains, that can be treated to provide ligand groups or ligand group precursors on the polymer surface suitable for binding or linking to biological materials. These include polyesters, polycarbonates and polyanhydrides, e.g. polyglycolic acid, polylactic acid (PLA) , polyglycolic acid-lactic acid (PGLA) copolymers or mixtures of polylactic acid and polyglycolic acid, polyethyleneterphtalate (PET) , polycarbonate (PC) or polymethylmethacrylate (PMMA) . Some of these polymers (e.g. PET, PC or PMMA) are stable in vivo and can be used as temporary cell carriers or permanent implants. However, in general, the use of biodegradable polymers is preferred.

Polymer Processing:

Techniques such as solvent casting and compression moulding are well known in the art. Solvent casting can be used to obtain thin polymer films, and can be controlled by selection of the solvent, the concentration of the polymer solution, the molecular weight of the polymer, the casting surface and the solvent evaporation rate. Compression moulding is used to form polymeric shapes by compressing finely ground polymer powder, varying the contact surface, pressure, temperature, cooling rate, and the molecular weight and polymer powder size. Other techniques such as injection moulding, fibre extrusion, fibre weaving can also be employed to produce the polymeric object used in the production of the implant .

Functionalising Reagents :

The functionalising reagent is preferably a hydrophilic polymer capable of reacting with the polymer matrix.

Suitable hydrophilic polymer include poly (acrylic acid), poly (methylacrylic acid), polyvinylalcohol, polyethyleneimine, polyvinylamine, dextrane, polyethyleneoxide .

Without wishing to be bound by any particular theory, the inventors believe that when the hydrophilic polymers covalently bond at one or more points to the polymer matrix, they form a "brush layer" that extends out from the surface into the aqueous solution. In view of the positive interaction with water and the high molecular mobility of the hydrophilic polymers, they efficiently shield the immobilised biological materials attached to the ligand groups from direct and potentially detrimental contact with the hydrophobic polymer matrix. This shielding gap tends to be maintained as any compression of the polymer brushes requires the expulsion of adsorbed water and a reduction of molecular mobility, and consequently a large entropic loss. The spacing effect provided by the polymer brushes is typically observed for hydrophilic oligomers having molecular weights of 1000 g/mol or more, and becomes more pronounced for high molecular weight hydrophilic polymers having molecular weights in the range of 10,000 to 1,000,000 g/mol. For best results, the hydrophilic polymer brushes are preferably tethered or anchored on a surface with one end of the chain free and as densely grafted as possible.

It has been shown that in the development of bioactive polymer surfaces, it is important to be able to control and retain the conformation of immobilised ligand groups as this has an effect on the surface/cell interaction (see Massai et al, 1991, and Juliano et al, 1993) . For instance, protein adsorption or immobilisation directly onto solid surfaces is often accompanied by comformational changes or significant loss in activity (see Juliano et al, supra , and Andrade et al ,1996) . In the present invention, this problem is ameliorated by using the hydrophilic polymers to increase the distance between the biological material and the polymer surface, and so reduce any deleterious effect caused by the close proximity of the group to the polymer surface.

The compatibility of the functionalised polymer matrix with biological materials such as cells is further enhanced by the comparatively high density of ligand groups that it is possible to associate with the polymer matrix using the methods of the invention. The functionalisation of the surface layer of the matrix can be determined by reacting the ligand groups with amino groups (or another element not present in the base polymer matrix) , and using X-ray photoelectron spectroscopy (XPS) to measure the nitrogen content of the functionalised surface. Typically, prior art chemical treatment methods lead to a nitrogen content of between 4.0 and 4.5%. In contrast, the present invention achieves surface nitrogen contents of greater than 7%, more preferably greater than 10%, more preferably greater than 15%, more preferably greater than 20% and even greater than 30%. This provides surfaces having a high degree of compatibility with biological mat-erials adhered or attached to the polymer matrix, especially cells.

In general, the functionalising reagents can be represented by the general formula R-YX in which R represents a ligand group or a ligand group precursor, optionally including a spacer group (see below) . Y is generally a nucleophilic group which is capable of attacking carboxy groups in the polymer.

Thus, by way of example, R-YX can be R'OCOR", R'OH, R'NH₂, HOCOR" as shown in the following table:

Preferably, the ligand or ligand precursor are extracellular matrix proteins or fragments thereof which are capable of promoting cell adhesion to the polymer matrix. Preferred extracellular matrix adhesion proteins include collagen (especially types I, III, IV), fibronectin and laminin. Alternatively, a cell adhesive peptide sequences such as RGD or YIGSR can be employed. Unlike the physical adsorption described in W097/46267, the covalent attachment of the functionalising reagent has the benefit that it is unlikely to become detached from the polymer matrix under the conditions encountered in use.

In some embodiments, the ligand group is one member of a specific binding pair and the other member of the specific binding pair is associated with the biological material to be immobilised on the polymer surface. The term "specific binding pair" is used to describe a pair of molecules comprising a specific binding member (sbm) and a binding partner (bp) which have particular specificity for each other and which under normal conditions bind to each other in preference to binding to other molecules. The term is also applicable where either or both of the specific binding member and binding partner comprise the binding part of a larger molecule, and to the use of active portions of one or both specific binding pair members, e.g. active portions which substantially retain the binding activity and specificity of the complete binding pair member.

Examples of specific binding pairs include ligands and receptors, enzymes and substrates, and antigens and antibodies. Typically, the interaction between specific binding pair members is reversible.

In other embodiments, the ligand group or ligand precursor can comprise a functional group such as a carboxylic acid, alcohol or amine groups, that is susceptible to traditional immobilisation reactions (Protein Immobilisation, Fundamentals and Applications, Ed. RF Taylor, Marcel Dekker Inc, NY, 1991) . Thus, ligand groups may be reacted directly with the biological material so that they become covalently bound to the polymer. Alternatively, the ligand precursor can be reacted to form a ligand group which can be covalently bound to a biological material or to produce a ligand group which is one member of a specific binding pair.

Implants : The functionalised polymer matrices of the invention can be linked to biological materials to provide tissue engineered products such as implants. These products may be to replace tissue or organs in humans or animals. It is possible to produce implants based on cultured cells from any part of the human body. Practically, however, the tissue engineered products are most readily made using epithelial cells (urothelial, keratinocyte) , endothelial cells (vascular endothelium) , nerve cells, muscle, bone and cartilage cells, optimally of autologous origin. Further examples include hormone or mediator cells which can be cultured and retransferred as functional replacements of diseased organs. In this case, if non-autologous cells are used, it may be advantageous to encapsulate the cells to prevent rejection by the patient receiving the implant.

Materials and Methods

Polymer modification: The modifications were carried out by transesterification, ester interchange, acidolysis, alcoholysis or aminolysis using the general reaction described above, employing polyacrylic acid, polyvinylic acid or polyethyleneamine as the hydrophilic polymers.

XPS determinations: For the determination of surface chemical composition, XPS measurements were recorded on a PHI 5500 system equipped with hemispherical analyser and a non chromatised Mg Kα X-ray source having a pass energy of 1253.6 eV. The analysis was carried out under UHV conditions (10^~9 Torr) on an area of 0.12 mm². Spectra were taken at 45°. Urothelial cells: These were obtained by scraping mucosal surface from the submucosal layer under sterile conditions and placing the cells obtained in serum free keratinocyte growth medium containing 5ng/ml epidermal growth factor and 50ng/ml bovine pituitary extract. The cells were incubated at 37°C with 5% carbon dioxide in an humidified atmosphere. For cell passage, cultures were incubated for 5 minutes in 0.05% trypsin/lmM EDTA and then trypsin inhibitor was added (Cilento et al, 1994).

Smooth muscle cells: Fragments of muscular layer were dissected and detached from the lamina. The muscle layer was minced into small pieces. Each piece was placed in a well with Dulbecco' s modified Eagle' s medium with new born calf serum at 10%. Correct explanted exhibits cells emanating around the tissue fragments. The medium was changed twice a week until confluence of cells was achieved. Cells were typsinized and passaged (Baskin et al, 1993) .

Cell adhesion: The attachment of urothelial cells and bladder muscle cells to the activated polymer surface was assessed by linking the ligand groups to vitrogen (Collagen Corp. Palo Alto, USA) , a mixture of bovine collagen type 1 and type 3. This provides a good test of the effectiveness of the technique as these cells do not adhere nor proliferate on the corresponding unmodified surfaces. The functionalised polymer matrices were reacted with collagen using N-ethyl-N' - (3- dimethylaminopropyl) carbodiimide hydrochloride (EDC) as coupling agent (Lloyd and Burns, J. Polym. Sci. Polym. Chem. Ed. , 17:3459, 1979) . Example 1 :

10% polyacrylic acid (PAA) (MW 5000) in dry dimethylpropylurea (DMPU) with 1% titaniumisopropoxide (Ti(OPr¹)₃) as catalyst was allowed to react with the surface of polyethyleneterephtalate (PET) for 2 minutes. The hydrophobic PET surface turned hydrophilic as shown by water immersion, even after prolonged extraction in water. The presence of PAA was further demonstrated by X-ray photoelectron spectroscopy (XPS) where the carbon to oxygen atomic ratio (C/O) changed from 2.5 to close to 1.5 after grafting.

The grafted surface was then reacted with collagen as described above.

Example 2 : 10% PVA (polyvinylalcohol) in DMPU with 400ppm titanium isopropoxide was allowed to react with the surface of PET for 2 minutes. The hydrophobic PET surface turned hydrophilic as shown by water immersion indicating the successful immobilisation of the hydrophilic polymer (PVA) even after prolonged extraction in water. The presence of PVA was further demonstrated by XPS (C/O ratio close to 1.5).

The grafted hydroxyl groups were activated with cyanogen bromide (5% in CH₂C1₂ at 0°C, 14 hours), washed with CH₂C1₂ and dried. The dried surface was immediately immersed in NaHC0₃ buffer (0.1M, pH8.3) with NaCl (0.5M) containing lOmg collagen/ml at 4°C for 4 hours. The functionalised collagen surface was washed with water.

Example 3 :

10% polyethyleneimine (PEA) in water was allowed to react with the surface polylactic acid for 30 minutes at 50^°C. The surface became hydrophilic and was washed extensively with water. The presence of PEA was determined by XPS using the nitrogen as signal. Protein immobilisation on this surface was carried out with EDC and an acid containing ligand as described in the literature.

Example 4:

10% tryptophane in a basic water solution was allow to react with PLA surface for 30 minutes at 50°C. The surface turned hydrophilic and remained hydrophilic after extensive water washing confirming that the hydrophilic polymer was covalently linked and not merely physically adsorbed on the surface of the polymer matrix. The presence of surface attached tryptophane was verified by XPS using the nitrogen as signal and by a weak absorption at 240-300nm shown by ultraviolet spectroscopy. The acid groups of tryptophane were activated to collagen using EDC as coupling agent (Lloyd and Burns, supra ) .

Example 5 :

10% of peptide YIGSR in NaHC0₃ buffer (0.1M, pH 8.3) with NaCl (0.5M) was allowed to react with PLA surface for 1 hour at 50°C. The polymer was extensively washed with distilled water and dried. The presence of YIGSR was by XPS using the nitrogen as signal. The acid groups of YIGSR were reacted as before with collagen using EDC. These substrates left in distilled water were sterilized by gamma-irradiation (12-18 hours, 18 KGrey) . Populations of urothelial cells and bladder muscle cells can be expanded on any of the above mentioned collagen functionalised polymeric scaffolds in a serum free medium to after 7 days culture obtain a monolayer structure. The cells on the matrices had the same morphology appearance as cells cultivated in a flask. Untreated references (controls) showed no capability of acting as scaffold support. Apart from collagen type 1 and type 3, other extracellular matrix proteins such as collagen type 4, laminin and fibronectin could be covalently grafted on the surface functionalised polymers.

Example 6 :

In this example, the nitrogen content of vitrogen immobilised on PAA-grafted surfaces determined. Following surface activation for 1 hour by EDC in with buffer solution (BupH MES from Pierce), pH 4.7, containing 5 mg/ml EDC at 4°C, the activated surface was wahsed 3 times with PBS pH 7.4 to eliminate the molecules of EDC that did not react. Vitrogen was then immobilised by contacting the activated polymer matrix with vitrogen (3 mg/ml) diluted in BupH MES buffer solution to obtain a 0.5 mg/ml vitrogen solution.

Using XPS as described in Materials and Methods, the % nitrogen content of the surface layer was determined after various reaction times:

2h30: 7.7 % N

5h30: 13.1 % N

24h00: 32.2 % N

This shows that the methods disclosed herein increase the capacity of binding significantly due to the distribution of functional groups throughout the interphase.

Example 7 : In further comparative experiment, a polyethylene terephatalete surface was hydrolyzed with 2-0% sodium hydroxide solution for 2 hours at 50°C and 4h at 40°C. The modified films were washed with deionised water. The activation of modified films was carried with 0.2g N- ethyl-N' - (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) in the presence of 0.015 g Vitrogen (collagen types I and III, Collagen Corp., Palo Alto, US) in 2.3ml distilled water allowing for collagen immobilization at 4°C overnight. Immobilised films were washed with PBS two times and with deionised water and dried. A poly (acrylic acid) grafted brush surface was treated using the same conditions for activation and immobilization. ESCA confirmed the above reported differences .

The surfaces were rinsed with water and stored in water, radiation sterilised (18KGrey) and surfaces analysis was carried out using XPS at 10^~9 mbar with a PHI hemispherical analyser and MgK_a radiation.

The prior art treatment as described in WO97/05193 at best gave protein concentrations upon immobilization which were less than 5% nitrogen by XPS. This was reflected in the fact that human urothelial and smooth muscle cells adhere and proliferate poorly.

In contrast, for polymer matrices functionalised according to the invention with hydrophilic polymers significantly higher loadings of proteins could be obtained using the same immobilisation conditions and nitrogen concentration as measured by XPS up to 32% were obtained. When concentrations of 15% nitrogen and above were obtained both cell types adhere and proliferate to reach confluence on substrates in which the polymer matrix was immobilised with collagen ligand binding groups .

References :

The references cited herein are all incorporated by reference .

Patrick et al, in Frontiers in Tissue Engineering, Elsevier, New York, USA, Chapter 1, 1998. -

Lipkin, Science News, 148:24-26, 1995.

Wallace, Vertebrate Limb Regeneration, Wiley, New York, USA, 1981.

WO94/09760 (Massachussets Institute of Technology) .

WO97/05193 (Sanitaria Scalageri Spa) .

W097/46267 (Gore Enterprise Holdings, Inc) .

Scifert et al, Biomaterials, 18 (22) : 1495-502, 1997.

Yamoaka et al, Adv. Biomater. Biomed. Eng. Drug Delivery

Syst., 5th edition, Eds. Ogala et al, Tokyo, Japan, 1996.

Cilento et al, Journal Urol . 153:665-670, 1994.

Baskin et al, Journal Urol. 149, 190-197, 1993.

Massai et al, J. Biomed. Mater. Res., 25:223-242, 1991.

Juliano et al, J. Biomed. Mater. Res ., 27 : 1103-1113, 1993.

Andrade et al, "Proteins at interfaces: Principles, problems and potential" (pages 19-55), in Interfacial Phenomena and Bioproducts, Ed. Brash, Marcel Dekker, Inc, New York, 1996.

Claims

Claims :

1. A method for producing a polymer matrix having a surface comprising ligand groups which are capable of interacting with a biological material, the method comprising reacting a carboxy-group containing polymer matrix with a functionalising reagent which- is a hydrophilic polymer comprising a ligand group or a ligand group precursor, wherein the reaction is carried out in a solvent in which the polymer matrix is insoluble and the functionalising reagent is soluble and the functionalising reagent reacts and covalently bonds to the carboxy groups on the surface of the polymer matrix.

2. The method of claim 1, wherein the carboxy-group containing polymer is a polyester, polyanhydride or polycarbonate .

3. The method of claim 1 or claim 2, wherein the hydrophilic polymer is polyacrylic acid, polymethylacrylic acid, polyvinylalcohol, polyethyleneimine, polyvinylamine, dextrane or polyethyleneoxide .

4. The method of any one of claims 1 to 3, wherein the hydrophilic polymer covalently bonds to carboxy groups which are part of the backbone of the polymer matrix.

5. The method of any one of the preceding claims, wherein the nitrogen content of the surface of the polymer matrix in which the ligand groups have reacted with a polypeptide is greater than 10% as determined using XPS.

6. The method of claim 5, wherein the nitrogen content of the surface is greater than 15%.

7. The method of any one of the preceding claims, wherein the reaction is carried out in an aprotic solvent and the hydrophilic polymer is represented by the general formula R-YX, where R-YX is ROH, RCOOH, RNH₂, RCOOR, RCOOCOR, RSH or another species that reacts- with the carboxy groups of polymer matrix.

8. The method of any one of claims 1 to 6, wherein the reaction is carried out in an aqueous solvent and the hydrophilic polymer is represented by the general formula R-YX and, R-YX is RNH₂ or another species which is more nucleophilic than water and reacts with the carboxy groups of polymer matrix.

9. The method of any one of the preceding claims, wherein the polymer matrix is formed from a polymer which is degradable in vivo .

10. The method of claim 5, wherein the polymer is selected from polylactic acid (PLA), polyglycolic acid, polyglycolic acid-lactic acid (PGLA) copolymers or mixtures of polylactic acid and polyglycolic acid.

11. The method of any one of claims 1 to 8, wherein the polymer matrix is formed from a polymer which is stable in vivo.

12. The method of claim 11, wherein the polymer is selected from polyethyleneterephtalate (PET), polycarbonate (PC) or polymethylmethacrylate (PMMA).

13. The method of any one of the preceding claims, further comprising the step of reacting the ligand precursor to produce a ligand group.

14. The method of any one of the preceding claims, wherein the ligand group is one member of a specific binding pair for binding to the other member of the specific binding pair associated with the biological material.

15. The method of any one of claims 1 to 13, wherein the ligand group comprises a functional group which is capable of covalent attachment to the biological material.

16. The method of any one of claims 1 to 13, further comprising the step of reacting the ligand groups so that they covalently bond to a polypeptide or peptide fragment.

17. The method of claim 16, wherein the polypeptide is a an extracellular matrix protein capable of adhering to cells or a fragment thereof which retains the property of adhering to the cells.

18. The method of claim 17, wherein the extracellular matrix protein is collagen, fibronectin, laminin or a cell adhesion peptide sequence such as RGD or YIGSR.

19. The method of any one of the preceding claims, further comprising the step of adhering cells to the polymer matrix.

20. The method of claim 16, wherein the cells are epithelial cells, endothelial cells, nerve cells, muscle, bone and cartilage cells.

21. The method of claim 19 or claim 20, further comprising culturing the cells adhered to the matrix.

22. A polymer matrix produced by the method of any one of claims 1 to 21.

23. A method of producing a polymer matrix having cells immobilised on the surface of the matrix, the method comprising: having reacted the polymer matrix with a hydrophilic polymer so that the hydrophilic polymer covalently bonds to the surface of the polymer matrix via carboxy groups of the polymer matrix, the hydrophilic polymer comprising ligand groups which are capable of binding to cells, wherein the ligand groups are an extracellular matrix protein capable of adhering to cells or a fragment thereof which retains the property of adhering to the cells; the further step of contacting the polymer matrix with cells so that the cells adhere to the ligand groups.

24. A tissue engineered product comprising: a polymer matrix comprising an carboxy group containing polymer, the polymer matrix having a surface to which hydrophilic polymer molecules are covalently bonded to the carboxy groups of the polymer; the hydrophilic polymer comprising ligand groups for binding to cells, wherein the ligand groups are an extracellular matrix protein or a fragment thereof which retains the property of adhering to the cells; cells adhered to the polymer matrix via the ligand groups.

25. A polymer matrix having a biological material immobilised thereon produced by the method of claim 24.

26. A polymer matrix produced by the method of claim 24 for use in a method of medical treatment.

27. Use of a polymer matrix produced by the method of any one of claims 1 to 21 for the preparation of a polymeric implant for transplantation into a patient, wherein the polymer matrix is contacted with the biological material under conditions in which the biological material adheres or covalently binds to the ligand groups on the hydrophilic polymer.