CS274204B1 - Cultivation support plate or implant - Google Patents

Abstract

The invention concerns a cultivation support plate or implant. The essence of the invention is the use of polymers and copolymers of acyloxyalkyl(meth)acrylates, containing units of general formula I, where R<1> = H or CH3; R<2> = (CH2)n-O-3, n = 2-4, or (CH2CH2-O-)n, n = 1-3; R<3> = alkyl containing 1-4 carbon atoms, whereas the carbon in the alpha-position contains, in view of carbonyl, two hydrogen atoms, as a cultivation support plate for cultivating animal or human cells, or as an implant.<IMAGE>

Description

The invention relates to the use of polymers and copolymers of aeyloxyalkyl (meth) acrylates as a culture support for the cultivation of animal or human cells, optionally as implants.

The materials used to date for the growth of animal or human cells or implants are conventional hydroxyalkyl methacrylate type polymers, for example poly (2-hydroxyethyl methacrylate) or polyEEMA-collagen composite materials.

It has now been shown that acyloxyalkyl (meth) acrylate polymers or copolymers have completely exceptional properties.

The object of the invention is to use polymers and copolymers of acyloxyalkyl (meth) acrylates containing structural units of the general formula I

R

The carbon atom in the alpha position relative to the carbonyl contains two hydrogen atoms as culture supports for the cultivation of animal or human cells or implants.

These polymers or copolymers allow the attachment and growth of animal or human cells on their surface. These are mainly cell types that require attachment to a solid culture medium for growth, for example, primoculture and diploid cells of mesenchymal origin (fibroblasts, chondrocytes, osteoblasts, myoblasts) or epithelial or endothelial-type cells. They are therefore suitable for use in the field of biology and biotechnology in the cultivation of animal or human cells in vitro. Another field is medicine, where new polymers or copolymers can find application as allopastic or prosthetic materials. Also, various fields of technology or industry can utilize said polymeric materials because of their other properties, such as optical, mechanical, electroinsulating, and the like.

Polymers and copolymers suitable for use in the present invention are prepared by the free-radical polymerization or copolymerization of the respective monomers prepared in a manner known per se by acylation of the corresponding hydroxyalkyl methacrylates or acrylates, for example by treatment with an acid chloride of formula II

r ³ -CO-O-CO-R ³ (II) or an acid anhydride of formula III (III),

using a conventional radical polymerization technique, i.e., block, solution, emulsion or suspension (pearl).

Acetoxyethyl methacrylate (AEMA) based polymers or copolymers are currently the most readily available, since its preparation can be based on commercially available 2-hydroxyethyl methacrylate (HEMA). The monomeric acetoxyethyl methacrylate (ΔΕΜΑ) is readily polymerized in the presence of suitable free radical polymerization initiators, for example dibenzoyl peroxide, diisopropyl percarbonate, tert. butyl peroctoate and the like: the polymerization or copolymerization may be carried out according to the intended application of the resulting polymer product, for example by block, solution or suspension polymerization techniques.

The most proven are the copolymers of acetoxyethyl methacrylate and 2-hydroxyethyl methacrylate, which have been tested in the form of a culture pad in tissue cultures and as implants in an experimental animal organism. These routes confirmed the excellent biological tolerance of the new type of polymeric materials.

The following are examples which illustrate the nature of the invention.

CS 274 204 B1

Example 1

The monomeric acetoxyethyl methacrylate (AEMA) was prepared by treatment of acetic anhydride (102.5 g - ¹ mol) with 2-hydroxyethyl methacrylate (HEMA) (130 g - 1 mol) from Rohm (Germany) containing 200 ppm hydroquinone stabilizer. This reaction was acid catalyzed with concentrated sulfuric acid (0.5 mL). The reaction mixture in the flask was first cooled (exothermic) by standing on ice, then allowed to react freely at room temperature for 48 hours. The crude acetoxyethyl methacrylate monomer was stripped of water-soluble impurities by shaking it with distilled water (3x00 ml) and finally, after drying over anhydrous sodium sulfate, distilled under vacuum (t 44 ° C at 24 Pa) with the addition of tert-butylpyrocatechin as polymerization inhibitor.

Solution polymerization in ethylene glycol monomethyl ether (methyl cellosolve from Fischer-Chemie, Germany) produced a number of soluble samples of EEMA / AEMA copolymers as well as both homopolymers. The composition of the starting monomer mixture is shown in the following table:

HEMA (g) 10.0 9.5 9.0 6.5 8.0 7.5 7.0 Ó, 5 6.0 5.5 AEMA (g) 0 0.5 1.0 1.5 2,0 2.5 3.0 3.5 4.0 4,5 continued table

HEMA (g) 5.0 4.0 3.0 2,0 1.0 0 AEMA (g) 5.0 6.0 7.0 8.0 9.0 10.0

The concentration of monomer (or mixture of monomers) in the solution was 10 g / 100 ml, i.e. the final volume was adjusted to 100 ml with solvent. 0.25% dibenzoyl peroxide, based on the weight of the monomers, was used as the initiator of the free radical polymerization. The mixture, placed in thick glass ampoules of 110 ml, was bubbled with pure nitrogen for 2 minutes and the ampoules then sealed by capillary portions.

The polymerization itself proceeded by heating the ampoule in a water bath at 80 ° C for 15 hours, at which time about 80-85% conversion was achieved. After cooling, the contents of the ampoule were precipitated in one liter of distilled water and the polymeric products were further extracted in 500 ml of fresh distilled water (3 times total wash water exchange, after 4 hours of extraction at 20-22 ° C). The polymer products were then dried under vacuum at room temperature and stored in well sealed dust bags.

Stock solutions of 5% by weight were prepared by dissolving samples of the copolymers and both homopolymers in pure methyl cellosolv. and these were then used to prepare culture plates for tissue culture testing. The procedure was as follows:

Plastic petri dishes for tissue cultures of Koh60 mm (Koh-i-nor Dalečín) were coated inside with the tested polymeric material; the solution was poured into a dish, the excess was discarded and the adhering layer was then dried in a dust-free box at room temperature for 2 days. In this way, thin films of said polymeric materials were prepared which adhered adherently to the backing material (polystyrene). Petri dishes were then sealed in double polyethylene foil and exposed to the sterilizing effect of ionizing radiation (LINAC, Tesla Praha) for a total dose of 2.5 Mrad.

Biological tests on tissue cultures of chicken myohlasts confirmed the suitability of said polymeric materials as culture substrates. Even the low acetoxyethyl methacrylate content of the 2-hydroxyethyl methacrylate copolymer caused these mesenchymal cells to adhere well to the support and further to grow and proliferate; formed a monolayer over 8 days of cultivation in minimal Eagle's medium with 0% fetal calf serum (one culture medium change). This shows an excellent biological tolerance of the prepared copolymers based on acetoxyethyl methacrylate. The 2-hydroxyethyl methacrylate homopolymer itself, on its hydrated surface, does not allow the cells to adhere, and hence their further growth, even though it is not itself cytotoxic. It is a sorption mechanism of anchoring certain protein components

CS 274 204 B1 from the serum used, which cells require for adhesion and foreign material and for their further growth. This corresponds to a certain free energy value of the polymer / water surface (^ sw) (calculated from the measured values of the contact angles of air bubbles and octane drops on the surface of the material in water): see comparison of ^? Sw (10 "^ Joule / m ^) poly-2-hydroxyethyl methacrylate (polyHEMA) alone: 0.057 and for HEMA / AEMA copolymers containing acetoxyethyl methacrylate: 5% by weight: 0.8, 10% by weight: 2.5, 50% by weight: 3.7, 80% wt .: 7.9, 90 wt%: 13.2; polyAEMA alone: 14.1. For comparison, ^w values for regenerated cellulose (cellophane): 0.19 and for polyethylene: 42 (see E. Brynda, N.A. Cepalova,

M, gallery: Equilibrium adsoption of human serum albumins and human fibrinogen on hydrophobic and hydrophilic surfaces. J. Biomed. Mater. Res. 18, 685-693 (1964).

It should be added here that only certain free energy values of the polymer / water interfaces (sw values) make the materials suitable for sustained adsorption of growth factors (certain serum proteins) in the intact, i.e. native, state. Highly hydrophilic (low sw) materials, such as polyHEMA or cellophane, virtually do not allow irreversible adsorption of proteins, while highly hydrophobic (high sw) materials, such as the untreated polystyrene or polyethylene surface, adsorb the proteins so tightly that denaturation and loss of their biological activity. Normal cells do not direct and grow on any of these materials. Polymers and copolymers of acyloxyalkyl methacrylates or corresponding acrylates are those whose surface energy and the corresponding free energy at the interface with water ( ^sw values) guarantee the uptake of growth factors from the serum intact. They are therefore particularly suitable as a culture support for the cultivation of animal or human cells in biological laboratories or in industrial biotechnology: for all cell types that require attachment to a solid surface to grow.

Example 2

Samples of 2-hydroxyethyl methacrylate / acetoxyethyl methacrylate copolymer having a foil size of 150 x 150 x 1 mm were prepared from the monomeric acetoxyethyl methacrylate prepared as described in Example 1. Round discs / 10 mm were punched out and implanted into the experimental animal (Wistar rats)

In addition to the respective monomers, the starting polymerization mixture also contained 0.5 wt. % of ethylene dimethacrylate sulfide and 0.15 wt. N, N * -azobisisobutyronitrile as polymerization initiator, based on the polymer mass. Here, 30% by volume of pure diacetin, based on the total volume of the polymerization mixture, was used as the inert diluent.

Glass molds and a silicone rubber frame of suitable dimensions were used as molds and were clamped together by clamps. The actual polymerization of the vented monomer mixture was carried out at 60 ° C for 20 hours by heating the mold in an aqueous thermostat.

Samples of copolymers containing 25, 50 and 75 wt. AEMA (75, 50 and 25 wt% ΞΕΜΑ) as well as gel samples of polyHEMA and polyAEMA alone. The ellies were first extracted in boiling distilled water for 48 hours. A total of 5 x 1 liter of water was used per film. Prior to implantation into the popliteal area, all samples were sterilized by autoclaving in isotonic sodium chloride solution (0.9%) for 30 minutes at 120 ° C. Implant specimens were collected at 1.3, 6, and 12 months in the host and, after fixation in 10% formalin solution, were subjected to the usual histological examination, along with adjacent tissues and connective tissue.

Implant test results confirmed excellent biological tolerance of sulfur gels based on AEMA polymers and HEMA / AEMA copolymers as compared to conventional polyHEMA gel. While the polyHEMA hydrogel is also well tolerated by living organisms, it is encapsulated over time by fibrosis tissue that does not adhere to the implanted material, and the implant body can be effortlessly removed from the capsule. The situation is different in the case of AEMA copolymers and homopolymers: here the fibrosis cap fits tightly to the implanted material and also adheres strongly to it - the implant bodies could not be removed without damage.

Also, the tendency to deposit calcium salts (caldification) into the interior of the implant is strongly suppressed in the case of HEMA / AEMA copolymers and polyAEMA gel alone with increasing obGS 274 204 B1 by the AEMA range; samples with 50 wt. AEMA and above do not calculate at all.

Based on these experiments, it can be assumed that cross-linked AEMA-based gels and potentially other types of polymeric acyloxyalkyl methacrylates or corresponding acrylates may find important applications in human medicine, such as alloplastic and prosthetic materials.

Unlike conventional polyHEMA-based materials, these new materials have a number of advantageous properties: they are elastic even in the anhydrous state and at normal temperatures (their Tg is below freezing), are less prone to calcifications (even in the form of HEMA copolymers) and tight they adhere to the tissues, which improves the fixation of the implanted prosthesis in a certain locality of the body and thus its functionality.

Claims (1)

Use of polymers and copolymers of acyloxyalkyl (meth) acrylates containing structural units of formula (I) -gh ₂ -c (I) COOR ² -OOR ³ , wherein R ¹ = H- or CH 2 -; R = ^a. (CH _{2) n} -O-, n = 2-4, or (CH ₂ OH ₂ -O-) _n, n = 1-3; R ^{@ 1} = alkyl containing 1-4 carbon atoms, the carbon in the alpha position relative to the carbonyl contains two hydrogen atoms, as culture supports for the cultivation of animal or human cells, and optionally as implants.