WO1999064563A1

WO1999064563A1 - Polymer carrier for cultivation of keratinocytes

Info

Publication number: WO1999064563A1
Application number: PCT/CZ1999/000017
Authority: WO
Inventors: Jiří LABSKÝ; Jir^¿í VACÍK; Karel Smetana; Barbora DVOR^¿ÁNKOVÁ
Original assignee: Ústav Makromolekulární Chemie Akademie Věd České Republiky; Univerzita Karlova; 1. Lékařská Fakulta Univerzity Karlovy; 3. Lékařská Fakulta Univerzity Karlovy
Priority date: 1998-06-10
Filing date: 1999-06-09
Publication date: 1999-12-16
Also published as: AU4029399A

Abstract

A polymer carrier for keratinocyte cultivation on biologically active polymer bases, prepared by radical polymerization of a polymerization mixture containing 1-95 wt.% of a radical-polymerizable monomer, 0.0-10 wt.% of a crosslinker, 0.1-5 wt.% of an initiator, 0.0-60 wt.% of a solvent, 0.0-50 wt.% of a polymerizable derivative of a sterically hindered amine, 0.0-60 wt.% of a polymerizable saccharide derivative, and polymerizable derivatives of reactive φ-acryloyl- or methacryloyl amino acids, which can be used for additional modification of polymer carriers with appropriate saccharide or sterically hindered amine derivatives. A hydrophilic polymer carrier with no bonded polymerizable saccharide or sterically hindered amine derivatives can be activated by sorption of specific derivatives of biologically active substances on the carrier surface.

Description

Polymer carrier for cultivation of keratinocytes

Technical Field

The invention relates to the polymer carrier for cultivation of keratinocytes on biologically active polymer bases.

Background Art: Keratinocytes are commonly utilized for covering large skin defects such as burns, trophic-ulcers, and bed-sores. A small piece of skin is taken from a patient (about 3 sq. cm), from which keratinocytes are isolated enzymatically. These are then cultivated under the conditions of tissue cultures together with mouse 3-T-3 fibroblasts (so-called feeder cells), in which proliferation was stopped by γ-irradiation or chemically. Human keratinocytes cannot adhere to the cultivation vessel without these feeder cells. After extinction of feeder cells and proliferation of keratinocytes, the latter are enzymatically released from the bottom of the cultivation vessel, attached to a greasy tulle and transferred to skin defects (Green et al., Proc. Natl. Acad. Sci. USA 76, 5665- 5668, 1979). Using this procedure, large areas of the damaged skin can be covered from a relatively small biopsy.. But the transfer proper of cells onto a greasy tulle is technically demanding and is frequently the cause of failure. Therefore a technology was prepared issuing from the Green method, which makes it possible to cultivate keratinocytes directly on polymer carriers and transplant them directly with the carriers onto the wound area (Vacik et al., Czech Patent 281 269, 1996; Dvofankova et al., Folia Biol., Praha, 42, 83-86, 1996; Smetana et al, J. Mater. Sci. Mater. Med. 8, 587-590, 1997; Dvofankova et al., Biomaterials, V. 19 , 141-146 (1998).

Polymer carriers are prepared by radical polymerization under the conditions common for these types of polymerizations. The prepared carriers are additionally purified by washing out perfectly the residues of unreacted monomers or the used solvent. So far, polymer carriers of poly(HEMA) material, prepared by radical polymerization of 2-hydroxyethyl methacrylate (HEMA), have been used. Before application of cells, it is necessary to preincubate the carrier for 24 h in a medium containing 25 % of bovine serum. The keratinocyte cultivation proceeds in the presence of feeder cells under the conditions of tissue cultures, hence together with mouse 3-T-3 fibroblasts, the proliferation of which was stopped by γ-irradiation or chemically. The procedure also complicates the whole transplantation process continuing to be connected with necessary preincubation and the presence of feeder cells.

A significant shortcoming is also the fact that the presence of feeder cells from the cultivation system loads the patient's immunological system and that the carrier prepared in this way does not possess properties which would suppress the formation of free radicals or reactive oxygen products, which makes the healing of the affected tissue difficult.

Summary of Invention

The substance of the invention, which eliminates the above-mentioned shortcomings, is the polymer carrier for cultivation of keratinocytes prepared by radical polymerization of a polymerization mixture which contains 1-95 wt.-% of radical- polymerizable monomers, 0.0-10 wt.-% of a crosslinker, 0.0-10 wt.-% of an initiator, 0.0-60 wt.-% of a solvent, 0.0-60 % of polymerizable saccharide or disaccharide derivatives, 0.0-50 wt.-% of polymerizable α-amino acid derivatives or their reactive derivatives and 0.0-50 wt.-% of polymerizable derivatives of sterically hindered amine of general formula

where W is -CH(X)- or -CH(X)CH - and X is a radical-polymerizable group.

Keratinocytes can be cultivated on such bases prepared in this way without any prior modification.

The object of the invention is further developed by pointing to suitable compounds and their combinations and procedures. The polymer carrier prepared in the absence of polymerizable saccharide derivatives is further conditioned by adsoφtion of biologically active saccharides selected from the group of polysaccharides heparin (A), heparan sulfate (B), hyaluronic acid (C), further monosaccharides conjugated with albumin or with a polymer carrier such as glucuronic acid (D), β-D-galactose (E), β(α)-D-N-acetylgalactosamine (F), β(α)-D-glucosamine (G), β-D-mannose (H), where the adsorption proceeds at a concentration of 10-500 ig/ml of PBS (phosphate-buffered saline) at temperatures 4-37° C for 1-12 h.

The invention is based on the new finding that with certain properties of the polymer carrier, it is possible to cultivate keratinocytes in the absence of auxiliary cells on polymers with biologically active polysaccharides, neoglycoproteins and neoglycoligands adsorbed on a synthetic polymer carrier. In further applications of the polymer carrier as an optimum cover for transplanted cells, the presence of chemically bonded sterically hindered amine derivatives plays an important role. These amines preferentially react with oxygen and its reduced derivatives, such as superoxide, hydroxy radical, hydrogen peroxide, dialkyl peroxides, alkylhydroperoxides, and hence prevent the destructive oxidation of the living tissue. Thus, in contact with the living tissue, they have the ability to liquidate, in a pronounced way, reactive oxygen products and to contribute to acceleration of healing of damaged tissues. In polysaccharide adsoφtion, they are used in a native form or with bonded biotin, which facilitates their adsoφtion. Monosaccharides are used in the form of neoglycoproteins of the general structure: monosaccharide - bovine serum albumin with or without biotin (Lee and Lee in Lectins and Glycobiology, H.-J. Gabius and S. Gabius, Eds, Springer Laboratory, Berlin 1993, pp 9-22; Gabius et al., Histol. Histopathol. 8, 369-383, 1993) or in the form of neoglycoligands: monosaccharide - poly[N-(2- hydroxyethyl)acrylamide] with or without biotin (Bovin in Lectins and Glycobiology, H.-J. Gabius and S. Gabius, Eds, Springer Laboratory, Berlin 1993, pp 23-28; Bovin et al., Chem. Soc. Rev. 24, 413-321, 1995).

The given polysaccharides or monosaccharide derivatives are immobilized on the polymer carrier surface by adsoφtion of individual substances or their combinations (e.g., B:F 1 :1, B:F:H 2:1 :1). It is proceeded as follows. The polymer carrier is incubated, under sterile conditions, with a polysaccharide (A-C), neoglycoprotein or neoglyco ligand (D-H) or their mixture in concentration of 10-500 μg/ 1 ml PBS (phosphate-buffered physiological saline) for 1-12 h. An excess solution is removed, the carrier is carefully rinsed with a cultivation medium and keratinocytes are applied at a density of 1-5 x 10⁶ per 50 cm² area. Cells are cultivated at 37° C in the atmosphere of

3.3 % of CO₂. In this way, both the keratinocytes from an enzymatically released, fine dermo-epidermal graft taken from the patient and those from frozen cells can be cultivated.

The proliferated keratinocytes on the polymer carrier can be transferred onto a skin defect and used for its covering. At that, the carrier with cells is oriented in such a way that the cells are applied onto the wound area and the polymer carrier forms an optimum cover of transplanted cells. In this way, both autologous (patient's own cells) cells, forming a permanent cover, and allogenic cells (of another human), which pronouncedly stimulate the healing, can be applied. If the polymer carrier is prepared using polymerizable saccharide derivatives, the preincubation of the carrier proper with polysaccharides, neoglycoproteins or neoglycoligands can be obviated. In addition, if covalently bonded onto a polymer carrier, the concentration of saccharides can be exactly determined. Further procedure of cultivation is retained.

By eliminating feeder cells from the cultivation system, the immunological loading of the patient is lower and the process of keratinocyte transplantation is simpler and more effective.

Examples

Example 1

A mixture of 100 g of 2-hydroxyethyl methacrylate, 0.4 g of ethyiene dimethacrylate,

1.4 g of 2-hydroxy-2-methylpropiophenone, and 6 g of 2,2,6,6-tetramethylpiperidin-4-yl methacrylate was polymerized on a polypropylene film by 10 min irradiation with a series of 175-W UV lamps from a distance of 18 mm. A foil 1 mm thick was formed, which was extracted with a mixture of ethanol and water (1 :1) for 48 h. The foil can be water-swollen to the water content of 36 %. Example 2

A mixture of 80 g of 2-hydroxyethyl methacrylate, 5 g of N-(2,2,6,6- tetramethylpiperidin-4-yl)methacrylamide, 0.6 g of ethyiene dimethacrylate, 0.5 g of 2,2'-azobis(2-methylpropionitrile) is dosed, after bubbling with argon (10 min), under an inert atmosphere into molds suitable for preparation of films (thickness 1.3 mm) where it is polymerized at 70° C for 12 h. The resulting film is washed with 30% ethanol and water.

Example 3

A mixture of 40 g of l-vinylpyrrolidin-2-one, 40 g of 2-acetoxyethyl methacrylate, 0.75 g of l,r-divinyl-3,3'-(ethane-l,l-diyl)di(pyrrolidin-2-one), 15 ml of glycerol, 2 g of 2- deoxy-2-methacryloylamino-D-galactopyranose, 2.5 g of 1,2,2,6,6- pentamethylpiperidin-4-yl methacrylate and 0.50 g of dimethyl 2,2'-azobis(2- methylpropionate) was bubbled with argon (10 min) and filled into molds for preparation of foils (foil thickness 1.1 mm) and polymerized for 12 h at 71° C. The foils were washed with 30 % ethanol and finally swollen in distilled water.

Example 4 A mixture of 60 g of 2-hydroxyethyl methacrylate, 20 g of 2-(2-hydroxyethoxy)ethyl methacrylate, 3 g of 2-methacryloyloxyethyl 2,2,5,5-tetramethyl-lH-2,5-dihydropyrrole- 3-carboxylate, 0.5 g of ethyiene dimethacrylate, 3.5 g of 2-deoxy-2-{[6- (methacryloylamino) hexanoyl] amino }-D-glucopyranose, 0.5 g of 2,2'- azobis(propionitrile), 20 ml of poly(ethylene glycol) 300 (Macrogolum 300), after bubbling with argon (12 min) is dosed in an inert atmosphere into moulds for preparation of foils (thickness 1.6 mm) and polymerized at 72° C for 11 h. The copolymer was extracted at 25° C with a mixture of 30 % ethanol for 72 h. The resulting foils were swollen in distilled water. Example 5

A mixture of 40 g of 1 -vinyl -2-pyrrolidone, 30 g of 2-acetoxyethyl methacrylate, 0.75 g of l,r-divinyl-3,3-'(ethane-l,l-diyl)di(pyrrolidin-2-one), 3.5 g of 4-nitrophenyl 10-(methacryloylamino)decanoate, 0.2 g of dimethyl 2,2'-azobis(2 -methylpropionate). After bubbling argon for 10 min, the reaction mixture was filled into moulds for foil preparation (1.6 mm thickness) and polymerized for 12 h at 71° C. The reaction with a 25-fold molar amount of D-glucosamine was performed after swelling the foil in dimethyl sulf oxide (2 days, laboratory temperature). After finishing the reaction, the foil was swollen in a 3 % solution of ammonia and finally in distilled water.

Example 6

A mixture of 100 g of 2-hydroxyethyl methacrylate, 0.4 g of ethyiene dimethacrylate, 2 g of 2-deoxy-2-methacryloylamino-D-galactopyranose, and 0.14 g of dimethyl 2,2'- azobis(2-methylpropionate) was polymerized in polymerization forms (foil thickness 2 mm) at 68° C for 12 h. The foil formed was extracted with a mixture of ethanol and water (1:1) for 48 h. The foil can be swollen in water to the water content of 36 %.

Foils from a mixture of HEMA and 2-(2-hydroxyethoxy)ethyl methacrylate were prepared analogously.

Example 7

A mixture of 40 g of l-vinylpyrrolidin-2-one, 40 g of 2-acetoxyethyl methacrylate, 0.75 g l,r-3,3'-(ethan-l,l-diyl)di(pyrrolidin-2-one), 0.15 g of dimethyl 2,2'-azobis(2- methylpropionate) was bubbled with argon (10 min), and filled into moulds for foil preparation (1.5 mm thickness) and polymerized for 12 h at 71° C. The foils were washed with 30 % ethanol and finally swollen in distilled water.

Example 8 The carrier prepared according to Examples 6 or 7 in the form of a disk, square or net 25-100 in diameter was placed on the bottom of a cultivation vessel and preincubated with polysaccharides or neoglycoligands (A-H) alone or in combination (e.g., B:F 1:1, B:F:H 2:1:1) in concentration 10-500 g/ml PBS for 1-12 h. Human keratinocytes were applied onto a carrier in density of 4 x 10 /cm . The cells were cultivated at 37° C in the atmosphere of 3.3 % CO₂. Keratinocytes for cultivation are obtained by taking from a patient a fine dermo-epidermal graft of 0.2-0.3 mm thickness, from which keratinocytes were obtained by treatment with trypsin.

The cultivation medium contained: 1) Eagle-H MEM with nonessential amino acids and sodium pyruvate (Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague)

2) glutamine (Institute for Serums and Vaccination Compounds, Prague), 0.3 mg/ml

3) 10 % bovine serum (ZVOS Hustopece)

4) hydrocortison (Spofa Comp., Prague), 0.5 μmol/ml 5) penicillin (200 units/ml), BIOTIKA, Slovak Republic

6) gentamicin (0.16 mg/ml), lek. Slovenia

7) insulin (Actrapid MC NOVO, Denmark), 0.12 I.U/ml

8) choleratoxin (Sigma, Prague), 10^"'° M

9) epidermal growth factor (Sigma, Prague), 10 ng/ml.

Example 9

Evaluation of polymer carriers

Orientation experiments with keratinocyte cultivation were performed on various polymer carriers, whose composition is given in the following table. The carriers were prepared by radical polymerization using thermal or light initiators.

The polymerization using a thermal initiator was carried out after mixing and bubbling the polymerization mixture (argon, 10 min) between polypropylene plates at 70° C for 12 h (foil thickness 1.2 mm). The foils were extracted with 30 % ethanol for 3 days and with distilled water for 3 days.

Polymerization using UV initiation was performed in such a way that the polymerization mixture, after mixing, was poured onto a polyester base, where it was irradiated with 175-W UV lamps from a distance of 15 cm for 12 min. The foils were washed with 30 % ethanol (2 days) and with distilled water (3 days).

The composition of polymerization charges for preparation of various polymer carriers is given in the following table 1.

a. The hydrophilic monomers used:

1. 2-hydroxyethyl methacrylate

2. (2-hydroxyethoxy)ethyl methacrylate

3. l-vinylpyrrolidin-2-one

4. 2-acetoxyethyl methacrylate 5. methacrylic acid

b. Crosslinkers:

11. ethyiene dimethacrylate

12. l,r-(ethane-l,l-diyl)di(pyrrolidin-2-one)

c. Polymerizable saccharides:

A. 2-deoxy-2- { [6-(methacryloylamino)hexanoyl] amino } -D-glucopyranose

B. 2-deoxy-2-methacryloylamino-D-galactopyranose

C. 2-methacryloyloxyethyl 2-acetamino-2-deoxy-D-glucopyranoside D. 6-deoxy-6-methacryloylamino-D-glucopyranose d. Polymerizable sterically hindered amines:

I. 4-methacryloylamino-2,2,6,6-tetramethylpiperidine

II. (l,2,2,6,6-pentamethylpiperidin-4-yl) methacrylate III. N-(2,2,5,5-tetramethylpyrrolidin-3-yl)methacrylamide

IV. 1 ,2,2,6,6-pentamethylpiperidin-4-yl 6-(methacryloylamino)hexanoate

e. Polymerization type - initiator used

- thermal polymerization: initiator dimethyl 2,2'-azobis(2-methylpropionate) - UV polymerization initiator 2-hydroxy-2-methylpropiophenone

Solvent: glycerol (denoted G and amount in wt.-% given) Poly(ethylene glycol 300 (Macrogolum 300) (denoted M and amount in wt.-% given)

Table 1.

Remarks: E.N. Number of Experiment, Cross.: Crosslinker

Polymerisation: T-thermal, L-light, S-solvent

Note: the numbers given in the table are weight % relative to the polymerization mixtures.

f. Evaluation

The polymers prepared under the experiment Nos. 1-3 are standard polymer carriers which served as reference polymer bases. Human keratinocytes grow on these polymer bases after preincubation with bovine serum in the presence of mouse fibroblasts or after adsoφtion of bioactive saccharides in the absence of mouse fibroblasts.

Adhesion of human keratinocytes to polymer bases under the experiment Nos. 4- 7 is markedly better in comparison with reference bases (1-3). However, also in this case, activation of the base using sugars is necessary. The results of cultivation of human keratinocytes on polymer bases Nos. 8-11 are very good. Keratinocytes adhere and grow without prior preincubation with bioactive polysaccharides. Base No. 10 appears the best. The results of cultivation on bases Nos. 12 and 13 were very close to cultivation results on base No. 10.

Industrial Applicability

The polymer carrier for cultivation of keratinocytes on biologically active polymer bases can be utilized primarily for covering and, at the same time, treatment of large skin defects such as burns, trophic-ulcers and bed-sores.

Claims

A polymer carrier for keratinocyte cultivation prepared by radical polymerization of a polymerization mixture containing 1-95 wt.-% of polymerizable monomers, 0.0-10 % wt.-% of a crosslinker, 0.0-10 wt.-% of an initiator, 0.0-60 wt.-% of a solvent, 0.0-60 wt.-% of polymerizable saccharide or disaccharide derivatives, 0.0-50 wt.-% of polymerizable sterically hindered amine derivatives, 0.0-30 wt.-% of polymerizable α-amino acid derivatives or their reactive derivatives.

A hydrophilic polymer carrier for keratinocyte cultivation as claimed in claim 1, wherein the polymerization mixture contains 1-95 wt.-% of monomers, individual or in combination, selected from the group: acrylic and methacrylic acids, alkyl acrylates and methacrylates hydroxyalkyl acrylates and methacrylates, (alkyloxy)alkyl acrylates and methacrylates, (acyloxy)alkyl acrylates and methacrylates, acryl- and methacrylamides, (substituted alkyl)acrylamides and -methacrylamides, (hydroxyalkyl)acrylamides and - methacrylamides, 1 -vinyllactams, diacetonacrylamide (l,l-dimethyl-3- oxobutyl)acrylamide . A polymer carrier for keratinocyte cultivation prepared as claimed in claims 1 and 2 wherein the polymerization mixture contains 0.0-10 wt.-%, individual or in combination, of substances selected from the group: 1 , 1 '-divinyl-3 ,3 '-(ethane- 1 , 1 -diyl)di(pyrrolidin-2-one)

ethyiene diacrylate, ethyiene dimethacrylate

R R

H₂C=C-CO-0-CH₂CH_rO-CO-C=CH₂ R = H, CH₃

α-acryloyl-ω-acryloyloxypoly(oxyethylene) α-methacryloyl-ω-methacryloyloxypoly(oxyethylene)

R = H, CH,

where m is 2-20.

4. A polymer carrier for keratinocyte cultivation prepared as claimed in claims 1-3 wherein it contains 0.1-10 wt.-% of substances, individual or in combination, selected from the group of substances, which undergo thermal decomposition thereby enabling a polymerization reaction, such as azodinitriles, azodiesters, dialkylperoxides, diacylperoxides, or systems capable of initiating polymerization on irradiation with UV light or daylight such as benzoin derivatives and α-diketones (camphorquinone) in a mixture with tertiary amines.

5. A polymer carrier for keratinocyte preparation as claimed in claims 1-4 wherein it contains a solvent in an amount of 0.0-60 wt.-% and individually or in combination: ethyiene glycol, di- and oligoethylene glycols, glycerol, ethyiene glycol and diethylene glycol mono- and diethers, 1 -methylpyrrolidin-2-one, dimethylformamide, dimethylacetamide, dimethylsulfoxide.

6. A polymer carrier for keratinocyte preparation as claimed in claims 1-5 wherein polymerizable saccharide derivatives in amounts 0.0-60 wt.-% contain, individually or in combination polymerizable saccharide or disaccharide derivatives selected from the group:

2-deoxy-2- { [(n+ 1 )-(acryloylamino)alkanoyl] amino } -D-glucopyranose 2-deoxy-2- { [(n+ 1 )-(methacryloylamino)alkanoyl] amino } -D-glucopyranose 2-deoxy-2-{[6-(methacryloylamino)hexanoyl]amino}-D-glucopyranose for n = 5, R = CH₃

R₇ is

n is 1-10

2-deoxy-2- { [(n+ 1 )-(acryloylamino)alkanoyl] amino } -D-galactopyranose 2-deoxy-2- { [(n+ 1 )-(methacryloylamino)alkanoyl] amino } -D-galactopyranose 2-deoxy-2- { [6-(methacryloylamino)hexanoyl] amino } -D-galactopyranose for n = 5, R = CH₃

R - H, CH₃, n is 1-10

2-deoxy-2- { [(n+ 1 )-(acryloylamino)alkanoyl]amino } -D-mannopyranose 2-deoxy-2- { [(n+ 1 )-(methacryloylamino)alkanoyl]amino } -D-mannopyranose 2-deoxy-2- { [6-(methacryloylamino)hexanoyl] amino } -D-mannopyranose for n = 5, R = CH₃

H₂C =C -CO NH -[-CH₂-^CO - R = H, CH,

= 1-10, R₇ is

2-(methacryloyloxy)ethyl 2-acetamido-2-deoxy-D-glucopyranoside (for n = 1)

n can be 1-3

6-deoxy-6-methacryloylamino-D-glucopyranose

4-O-(β-D-galactopyranosyl)-N-(4-vinylbenzoyl)( 1 ->4)-β-D- glucopyranosylamine

2-acetamido-4-O-(2-acetamido-2-deoxy-β-D-glucopyranosyl)-( 1 → 4)-2-deoxy- N-(4-vinylbenzoyl)-β-D-glucopyranosylamine

n-(acryloylamino)alkyl O-(β-D-galactopyranosyl)(l— > )-2-acetamido-2-deoxy- β-D-glucopyranoside, n = 2-10 n-(methacryloylamino)alkyl O-(β-D-galactopyranosyl)(l— »4)-2- acetamido-2- deoxy-β-D-glucopyranoside, n = 2-10

2-(acryloylamino)ethyl O-(β-D-galactopyranosyl)(l-»4)-2-acetamido-2-deoxy- β-D-glucopyranoside, n = 2, R = H

R = H, CH₃

A polymer carrier for keratinocyte cultivation as claimed in claims 1-6 wherein it contains, individually or in combination, 0.0-50 wt.-% of the compound selected from the group of polymerizable sterically hindered amine derivatives of general formula

where W is -CH(X)- or -CH(X)CH₂- and X is a radical-polymerizable group

R R

I R I

— 0-COC=CH₂ — NHCOC=CH₂ -O-CO-^CHj -NHCOC=CH,

R

NHCOCH; NHCOC=CH„ NHCO +-f< I CH ^■NHCOC=CH₂ R= H, CH

nis 1-10, mis 1 or 2

R

-COO-C^CH -O- CO-C=CH,

R is H, CH₃

and where R_\ is H or alkyl C C₄, Ri is H or alkyl C-C₄, OH an oxygen radical formed by additional oxidation, the substances being selected from the group including:

N-(2,2,5,5-tetramethylpyrrolidin-3-yl)acrylamide

N-(2,2,5,5-tetramethylpyrrolidin-3-yl)methacrylamide

where R^ is H or alkyl Cι-C₄, OH or an oxygen radical formed by additional oxidation ( 1 -R -2,2,6,6-tetramethylpiperidin-4-yl) acrylate

(1 -R_ό-2,2,6,6-tetramethylpiperidin-4-yl) methacrylate

where Rβ is H or alkyl Cι-C₄, OH or oxygen radical formed by additional oxidation

(l-R₆-2,2,6,6-tetramethylpiperidin-4-yl) (n+l)-(acryloylamino)alkanoate

( 1 -R₆-2,2,6,6-tetramethylpiperidin-4-yl) (n+ 1 )-(methacryloylamino)alkanoate of general formula

where R_ό is H or alkyl C C₄, OH or oxygen radical formed by additional oxidation

2-(acryloyloxy)ethyl l-R₆-2,2,6,6-tetramethylpiperidine-4-carboxylate 2-(methacryloyloxy)ethyl l-R -2,2,6,6-tetramethylpiperidine-4-carboxylate

where R₆ is H or alkyl Cι-C₄, OH or oxygen radical formed by additional oxidation

N-(l-R -2,2,6,6-tetramethylpiperidin-4-yl)acrylamide

N-( 1 -R₆-2,2,6,6-tetramethylpiperidin-4-yl)methacrylamide

where R₆ is H or alkyl C_t-C_t, OH or oxygen radical formed by additional oxidation

N- { (m+ 1 )-oxo-(m+ 1 )- [( 1 -R₆-2,2,6,6-tetramethylpiperidin-4- yl)amino] alkyl } acrylamide

N-{(m+l)-oxo-(m+l)-[(l-R_ό-2,2,6,6-tetramethylpiperidin-4-yl)amino]alkyl} methacrylamide of general formula:

where R_ό is H or alkyl Cι-C₄, OH or oxygen radical formed by additional oxidation

N-(l-R₆-2,2,5,5-tetramethylpyrrolidin-3-yl)acrylamide N-(l-R₆-2,2,5,5-tetramethylpyrrolidin-3-yl)methacrylamide

where R_ό is H or alkyl Cι-C , OH or oxygen radical formed by additional oxidation

N- { (n+ 1 )-oxo-(n+ 1 )- [( 1 -R₆-2 ,2 ,5 , 5 -tetramethylpyrrolidin-3 -yl) amino] alkyl } acrylamide

N- { (n+ 1 )-oxo-(n+ 1 )-[( 1 -R₆-2,2,5 , 5 -tetramethylpyrrolidin-3 -yl)amino] alkyl } methacrylamide

where R_ό is H or alkyl CrC₄, OH or oxygen radical formed by additional oxidation.

A polymer carrier prepared as claimed in claims 1-7 wherein the polymerization mixture contains 0.0-30 wt.-% of polymerizable reactive ω- amino acid derivatives, which are modified in the prepared polymer by the reaction with an appropriate amino derivative of a saccharide or disaccharide, the appropriate ω-amino acid derivatives being: a) activated esters of co-(acryloylamino)alkanoic and ω-

(methacryloylamino)alkanoic acids of general formula

R

H₂C =C C0₂-[CH n-°2-*1 R = H, CH,

R H₂C =C -C0₂-j-NHCH₂CO m — R1

where n = 1-12, m = 2,3, RI are esters of 4-nitrophenol, pentachlorophenol, N-hydroxysuccinimide, N-hydroxyphthalimide

b) Polymerizable ω-amino acid derivatives, which are activate for the reaction with amino sugars with dicyclohexylcarbodiimide.where RI is H, the appropriate amino sugar derivatives being

2-[(n+l)-(aminoalkanoyl)amino-2-deoxyglucopyranose for n = 1-12

2-[(n+l)-(aminoalkanoyl)amino-2-deoxygalactopyranose for n= 1-12

2-[(n+l)-(aminoalkanoyl)amino-2-deoxymannopyranose for n = 1-12

n-aminoalkyl 2-acetamido-2-deoxy-^β-D-glucopyranoside, n = 2-10

n-aminoalkyl β-D-galactopyranosyl( 1 - 4)-2-acetamido-2-deoxy-β-D- glucopyranoside, x = 2-10

A polymer carrier for keratinocyte cultivation prepared as claimed in claims 1-8 wherein the reactive ω-amino acid derivatives are additionally modified in the prepared polymer by the reaction with an appropriate amino derivative of a sterically hindered amine, the appropriate sterically hindered amine derivatives being:

4-amino- 1 -R₆-2,2,6,6-tetramethylpiperidine

where R_ό is H, alkyl Cι-C₄, OH or O radical

4-amino- 1 -R_ό-2,2,5,5-tetramethylpyrrolidine

where R₆ is H, alkyl CpC₄, OH or O radical

(n+ 1 )-amino-N-( 1 -Rό-2,2,6,6-tetramethylpiperidin-4-yl)alkanamide

where R₆ is H, alkyl Cι-C₄, OH or O radical

l-R_ό-2,2,6,6-tetramethylpiperidin-4-yl (n+l)-aminoalkanoate

where R_ό is H, alkyl Cj-C₄, OH or O radical

(x+l)-amino-N-(l-R >6 -2,2,5,5-tetramethylpyrrolidin-3-yl)alkanamide

where R_ό is H, alkyl Cι-C₄, OH or O radical

l-R_ό-2,2,5,5-tetramethylpyrrolidin-3-yl (n+l)-aminoalkanoate

where R_ό is H, alkyl Cι-C₄, OH or O radical.

10. A polymer carrier for keratinocyte preparation claimed in claims 1-9 wherein it is prepared by conditioning of the polymer carrier by adsorption of biologically active saccharides selected from the group of polysaccharides heparin, heparan sulfate, hyaluronic acid, monosaccharides conjugated with albumin or polymer carrier of glucuronic acid, β-D-galactose, β(α)-D-N-acetylgalactosamine, β(α)- D-N-acetylglucosamine, α-D-mannose, where the adsoφtion proceeds at concentrations 10-500 μg/ml PBS at 4-37° C for 1-12 h.