WO2009002246A1

WO2009002246A1 - Stabilised porous structures, process for production thereof and use thereof

Info

Publication number: WO2009002246A1
Application number: PCT/SE2008/000407
Authority: WO
Inventors: Kjell Nilsson
Original assignee: Celltrix Ab
Priority date: 2007-06-26
Filing date: 2008-06-19
Publication date: 2008-12-31

Abstract

The present invention relates to a process of stabilising porous structures. The method comprises two or more consecutive reaction steps in which different target groups in the porous structures are allowed to react with cross-linking reagents.

Description

STABILISED POROUS STRUCTURES, PROCESS FOR PRODUCTION THEREOF AND USE THEREOF

Technical Field

The present invention relates to a process for producing a stabilised porous structure in the form of spherical particles or a three-dimensional structure, and a stabilised porous structure produced by the process. The invention further relates to use of the stabilised porous structure. Technical Background

The majority of animal cells are surface dependent, that is they must be attached to a surface to be able to survive and/or multiply. Traditionally this surface has been the inside of glass or plastic bottles. This has made it very difficult to culture cells on a large scale or implant the cells. The size of the cells is 5-20 μm.

Microcarriers are small particles, 0.2 mm diameter, to which the cells can adhere and multiply (van Wezel, A.L. Nature 216 (1967) 64-65 Growth of cell strains and primary cells on microcarriers in homogeneous culture). These particles have to a certain degree facilitated the culture of surface- dependant cells on a large scale.

The commonest type of microcarriers is spherical carriers made of dextran and modified by derivatisation with positive groups. As a result, the cells adhere to the carriers. Another technique of making cells adhere is to either to make the carriers of gelatine or to bind gelatine to the surface of dextran particles. Gelatine is made of collagen which is the substance to which cells normally adhere. The carriers that are currently available for cells are not optimal in every respect. These carriers are often homogeneous, that is the cells can only adhere to/grow on the surface of them. This means that the available surface for cell adhesion/cell growth will be restricted to the surface of the carriers. Moreover the cells can only adhere/grow in two dimensions, in contrast to the three-dimensions that is the normal condition in vitro. A further restriction of the prior art systems is that in the case of using the carriers when culturing in culture vessels the cells will be damaged by the forces originating from the agitating system. This has previously been remedied to a certain extent by producing particles with a large number of encased cavities by an emulsifying process (Kjell Nilsson and Klaus Mosbach, Swedish patent 8504764-5, Macroporous particles, process for production thereof and use thereof). The document describes how particles with a large number of encased cavities can be prepared by adding to an aqueous solution of the matrix material a solid, liquid or gaseous cavity-forming compound. After preparation of the particles by dispersion in a water-insoluble dispersing agent, the matrix is made water- insoluble by cooling, covalent cross-linking or polymerisation. The cavity- forming compound is removed to provide the encased cavities.

The field of application of the particles involves the use as ion exchangers, gel filter media, chromatography media and microcarriers in cell culture. The matrix is made of protein, polysaccharide or polyacrylamide.

The invention of Swedish patent 8504764-5 resulted in particles where some of the cavities of the particles were available for cell adhesion/cell growth. However, it has been found that the particles obtained were not optimal in certain respects. It would be optimal if all cavities were joined so that a continuous porous phase and a continuous matrix phase were obtained. By the invention of Swedish patent 0201779-6, this state was achieved by a combination of emulsifier and solvent.

This state made it possible to produce both particles and other three- dimensional shapes.

The stability of macroporous structures produced according to one of the above patents is most important for their potential fields of application. If the structures are to be sterilised by heating to 120⁰C, for instance in autoclaving, the thermal stability must be so high that the three-dimensional structure is maintained. Another field of application where the stability is highly important is in exposure to biological systems, for instance in implantation. The structures are usually stabilised chemically by bi- or polyfunctional reagents being allowed to react with functional groups on the matrix material. Synthetic polymers are usually stabilised by reaction with functional reactive monomers, dimers and/or polymers. Thus, a problem of structures according to prior art technique is that their stability cannot be adjusted to the field of application. Summary of the Invention

The object of the present invention therefore is to provide structures the stability of which can be varied and controlled.

This object is achieved by introducing cross-linking systems in the structures.

According to one aspect, the present invention relates to a process of stabilising porous structures, the process comprising two or more consecutive reaction steps in which different target groups in the porous structures are allowed to react with cross-linking reagents.

According to another aspect, the invention relates to a stabilised porous structure produced by various target groups in the porous structure being allowed to react with two or more consecutive cross-linking reagents. According to yet another aspect, the invention relates to use of the stabilised porous structure as carrier of cells.

According to another aspect, the invention relates to use of the stabilised porous structure for culture of artificial skin, artificial organs, fatty tissue and blood vessels. According to a further aspect, the invention relates to use of the stabilised porous material as an implant.

According to one more aspect, the invention relates to a method for implantation of a stabilised porous material as stated above as carrier of cells in an individual for production of substances, comprising implanting said stabilised porous material in the individual and then allowing the cells on the stabilised, porous material to produce said substances.

Furthermore the invention relates to a method of improving in vivo healing of damaged tissue. Detailed Description of the Invention The porous structures according to the present invention are stabilised by consecutive reactions with cross-linking reagents. Examples of cross- linking reagents are stated in Table 1. This table is not complete but is only shown as an example. A person skilled in the art can easily add further cross- linking reagents.

The reaction conditions in which the reactions are performed also influence which target groups can react. The reaction at pH >12 results in hydroxyl groups being reactive. Table 1

Porous structures containing carboxyl groups can, for instance in the first step, react with a cross-linking reagent activating these groups. An example of such a cross-linking reagent is EDC. An activated carboxyl group can then react with a neighbouring amino group and create cross-linking. It is also possible to let the activated carboxyl groups react with a diamine. This increases the number of amino groups which can react with cross-linking reagents having amino groups as target group.

In the second step, amino groups can be allowed to react with a suitable cross-linking reagent, for instance diisocyanates.

Hydroxyl groups of the porous structure can be used for cross-

¹ 1 -ethy|-3-(3-dimethylam inopropyl)carbodiim ide linking with, for instance, epichlorohydrin. By blocking a reaction with amine, diamine or polyamine, a number of amino groups are introduced, which can be used in reaction step 2.

Porous structures that are formed by polymerisation can be stabilised by introduction of monomers with reactive target groups, for instance carboxyl or amino groups. The increased stability is then obtained by treatment with cross-linking agents.

In an embodiment of the invention, the stabilised porous material is used for culture of artificial skin, artificial organs, fatty tissue and blood vessels.

The stabilised porous material according to the present invention can be used both as carrier of cells in cell culture and as carrier of existing cells for production of a desirable substance before and/or after implantation in an individual. The cells can be either the individual's own cells or cells from another source (characteristic of the species or foreign to the species). In some cases, the cells as such can be the desirable product, for instance precursors of adipocytes (preadipocytes) on the carrier which after implantation can multiply and then be converted into adipocytes (adipose cells). One field of application is for instance plastic surgery. It is also possible to implant the produced porous structures according to the invention in a human body without addition of cells. After implantation, the neighbouring cells in the human body will migrate into and colonise the structure. If the stabilised structure is biologically degradable and is dissolved, the colonising cells will have formed a structure corresponding to the implant when the porous structure has been dissolved. It is also possible to stabilise the structure to such a degree that the structure will not be dissolved.

The stabilisation of the material determines the time it takes to dissolve the structure in the human body. Thus it is possible to control the dissolution for the intended application. An example of this is in plastic surgery where carriers according to the invention without accompanying cells are injected at the site of a wrinkle. The cells surrounding the carriers migrate toward the carriers and colonise them. Gradually, as the carriers are being dissolved by surrounding enzymes, the migrated cells occupy the site of the wrinkle. This results in the wrinkle being smoothed out.

Yet another example is to use the material with adhering cells when testing drugs. By measuring variables which reflect the state of the adhered cells, predictions can be made about the effectiveness/toxicity of the potential drug.

Another example is skin cells on the carrier which can be used for treating different types of injuries to the skin.

Another example is myoblasts (muscle cells), which can be used in treatment of, for instance, cardiac infarction. One more example is hepatocytes (liver cells) which can be used to render toxic substances in liver lesions harmless. Also more complex structures, such as islets of Langerhans, can be attached to and/or in the porous carrier. Islets of Langerhans are composed of a plurality of different cell types and constitute the system that regulates the blood sugar level. These islets are considerably larger and require a pore size of the carrier of 50-200 μm.

The stabilised porous structures according to the invention can be used in improvement of in vivo healing of damaged tissue, in which cells are introduced on the structure, after which the structure is implanted in a site having damaged tissue.

As an alternative, the structure can be implanted in a site having damaged tissue without previous introduction of cells, after which the individual's own cells are allowed to proliferate on the structure.

By the term "substance" as used herein is meant the substances which can be produced by different cells or microorganisms, for instance antibiotics, pharmaceutical substances, such as dopamine which is a key substance in Parkinson's disease, and various interferons which are active substances in treatment of cancer.

By the term "porous" as used herein is meant that the material has pores of the size in which the cells can grow.

The degradability of the biocompatible porous material according to the invention is determined by the degree of stabilisation of the material. An agent can be added to improve or change the adhesion of cells to said stabilised porous material during production, or the agent can be chemically bonded to the polymer or be added later. Agents that affect the adhesion of cells can be either single molecules or proteins. Examples of the former are positively or negatively charged substances, such as hexamethylene diamine and aminocaprioc acid. Also uncharged structures, such as fatty acids, can be attached to the matrix. Examples of more complex structures are peptides containing the amino acid sequence arginine-glycine- asparagine or derivatives thereof. This sequence promotes the adhesion of cells to the carrier. Examples of proteins are fibronectin and laminin. Also non-defined mixtures of proteins (obtained by extraction of tissues), such as ECM (extracellular matrix), can be used.

To prevent rejection, the stabilised structures with adhering cells can be encapsulated with another material which serves to prevent cells and proteins of the human body from recognising or reacting with the adhered cells. This material can be a polysaccaride or polymer. Thus the material functions as a form of mechanical barrier against the proteins and cells of the human body.

To obtain a shape suitable for the specific application, the stabilised structure can be formed into various three-dimensional structures according to prior art methods. Thus spherical particles can be produced by an emulsifying process and membranes can be made by casting on/between plates. Special structures can be cast in specially made moulds, for instance ears. Finishing by mechanical methods can also be used to provide the final structure.

The stabilised porous material is produced so that the cells are present both in side the continuous pore structure and outside the stabilised porous material. This results in the material being optimally utilised.

In this description, the expression "carrier of cells" is intended to comprise carriers that can be used in culture of various cells and carriers that can be used for cells to provide production of desirable substances. The expression "carrier of cells" also comprises medical implants to be implanted in the human body. A surprising effect in production of the stabilised porous material according to the invention is that the stability of the obtained material can be varied within wide ranges. Examples Example 1

Stabilisation of spherical gelatine particles - amino groups as target group

20 g porous gelatine particles is mixed with 500 ml 0.05 M phosphate buffer, pH 8. 2 ml hexamethylene diisocyanate is added and the reaction is allowed to proceed for 120 min. Example 2

Stabilisation of spherical gelatine particles - carboxyl groups as target group 20 g porous gelatine particles is mixed with 300 ml acetone containing 20% water. 3 ml diisopropylcarbodiimide is added and the reaction is allowed to proceed over night. Example 3

Stabilisation of spherical gelatine particles - carboxyl and amino groups as target group

20 g porous gelatine particles is mixed with 300 ml acetone containing 20% water. 3 ml diisopropylcarbodiimide is added and the reaction is allowed to proceed over night.

The particles are washed with acetone to remove excess of diisopropylcarbodiimide. 200 ml 0.05 M phosphate buffer, pH 8, and 3 ml hexamethylene diisocyanate are added and the reaction is allowed to proceed for 60 min. The degree of stability is compared by determining the volume provided by one gram of dry particles after heating them to 12O⁰C by autoclaving, Table 2. , Table 2

Another technique of comparing the stability is to treat the gelatine particles with proteolytic enzyme which cleaves the structure.

To this end, 100 mg material was mixed with phosphate buffer and autoclaved for 20 min. 1 ml collagenase (0.4 mg/m, 0.4 U/mg) was added and the mixture was incubated at 37°C. The time of dissolution of the particles was measured, Table 3. Table 3

It is apparent from the tables that a significantly increased stability is obtained by using two consecutive cross-linking reactions. Table 3 shows the unexpected result that the combination of two consecutive cross-linking reactions provides a stability that is greater than the sum of the individual reactions.

Claims

1. A process of stabilising porous structures, the process comprising two or more consecutive reaction steps in which different target groups in the porous structures are allowed to react with cross-linking reagents, wherein the porous structures are synthetic polymers or proteins.

2. A process as claimed in claim 1 , in which the target groups are selected from hydroxyl groups, amino groups and/or carboxyl groups.

3. A process as claimed in any one of the preceding claims, in which the cross-linking reagents are selected from di- or polyfunctional chemically reactive compounds.

4. A process as claimed in any one of the preceding claims, further comprising addition of an agent to change the adhesion of cells to the porous structures.

5. A process as claimed in claim 4, in which the agent contains single molecules and/or proteins.

6. A process as claimed in claim 4 or 5, in which the agent comprices hexamethylene diamine, aminocaproic acid, fatty acids, fibronectin, laminin or peptides containing the amino acid sequence arginine-glycine-asparagine.

7. A process as claimed in any one of the preceding claims, in which the stabilised porous structure, together with cells introduced on the structure, is encapsulated with a polysaccharide or polymer.

8. A stabilised porous structure of synthetic polymers or proteins produced by target groups in the porous structure being allowed to react with two or more consecutive cross-linking reagents.

9. A stabilised porous structure as claimed in claim 8, in which the target groups are hydroxyl groups, amino groups and/or carboxyl groups.

10. A stabilised porous structure as claimed in claim 8 or 9, in which the cross-linking reagents are selected from di- or polyfunctional chemically reactive compounds.

11. A stabilised porous structure as claimed in any one of claims 8-10, in which the structure is of spherical shape.

12. A stabilised porous structure as claimed in any one of claims 8-11 , in which the structure has the form of a membrane.

13. Use of a stabilised porous structure as claimed in any one of Claims 8-12 as carrier of cells.

14. Use of a stabilised porous structure as claimed in any one of claims 8-12 for culture of artificial skin, artificial organs, fatty tissue and blood vessels.

15. Use of a stabilised porous structure as claimed in any one of claims 8-12 as an implant.

16. A method for implantation of a biocompatible stabilised porous structure as claimed in any one of claims 8-12 as carrier of cells in an individual for production of substances, comprising introduction of cells on the structure and implantation of the structure in the individual, after which the cells adhering to the stabilised porous structure produce said substances.

17. A method for implantation of a biocompatible stabilised porous structure as claimed in any one of claims 8-12, in which the structure is implanted in the body of an individual followed by migration of surrounding cells to the structure and colonisation thereof.

18. A method of improving in vivo healing of damaged tissue, comprising introduction of cells on a biocompatible stabilised porous structure as claimed in any one of claims 8-12 and implantation of the structure in a site having damaged tissue.

19. A method of improving in vivo healing of damaged tissue, comprising implantation of a biocompatible stabilised porous structure as claimed in any one of claims 8-12 in a site having damaged tissue, after which the individual's own cells are allowed to proliferate on the structure.