Biomedical polymer material for tissue repair and engineering

The invention relates to a biomedical polymer material for tissue repair and engineering according to the preamble of claim 1.

The number of patients suffering from tissue or organ failure is increasing, while the supply of autogenous tissues and organs for transplantation is limited. This calls for new modalities to treat these problems. Potentially, "artificial" tissues and organs might be used instead of autogenous ones, providing their biological functionality approximates that of the original tissues and organs. One of the routes which might lead to such tissue substitutes is tissue engineering where a construct consisting of a suitable scaffold seeded with autogenous cells is implanted in place of damaged or malfunctioning tissues and organs. An important component in reaching such a goal is an availability of a suitable scaffold for cells.

Among candidate biomaterials for scaffolds bioresorbable polymers of natural or synthetic origin and/or ceramics play an important role. The type of biomaterial for scaffolds depends on the type of tissue to be repaired, i.e. potentially different materials may be needed for the repair of hard and soft tissues. It is generally appreciated that scaffolds for tissue engineering should support attachment, spreading and proliferation of cells. It should allow for the production and maintenance of the extracellular matrix. The scaffold should be microporous with interconnecting pores of suitable size to allow for the ingrowth of cells, blood vessels and tissues. It should be produced from biocompatible and bioresorbable/biodegradable materials, to allow for the gradual replacement of the scaffold matrix with newly formed tissue. Optimally, the scaffold's resorption time should match the rate at which the new tissue is formed. Scaffold's mechanical properties should ensure its functionality.

Among candidate bioresorbable polymers for scaffolds polyhydroxyacids are most frequently used. Yet another group of polymers with potential application for tissue engineering are biodegradable polyurethanes. These polymers can be synthesized as hydrophilic, hydrophobic or amphiphilic depending on the intended application. Their mechanical properties and rates of degradation can be well controlled and the interaction with cells and tissues can be modulated.

Some polyurethanes for example can be processed into implantable devices such as microporous membranes and 3-D porous scaffolds which are tolerated by the living tissues. The drawbacks of these devices and especially in case they are used for tissue repair and regeneration are associated with the fact that their interaction with cells and tissues is far from optimal, i.e. their potential to stimulate cell attachment, growth and proliferation is limited.

On this point, the invention intends to provide remedial measures. The invention is based on the objective of providing a biomedical polymer material for implantable devices and particularly for tissue repair and engineering implants. Their interaction with biological cells and tissues is dramatically enhanced.

The invention solves the posed problem with a biomedical polymer material for tissue repair and engineering device that displays the features of claim 1.

The advantages achieved by the invention are essentially to be seen in the fact that due to the biomedical polymer material for tissue repair and engineering the interaction with cells is enhanced and the growth of cells is affected.

The biomedical polymer materials according to the invention do not invoke teratogenic, embryotoxic, mutagenic, carcinogenic, allergenic and immunotoxic effects and do not harmfully affect the functions of the endocrine system, i.e. they fulfill the requirements for materials for implantable devices.

Said biomedical polymer materials may be chosen from the group of polyurethanes, polyamides, polyamines, polyesters, polyurethaneacrylates and polyacrylates.

The compounds to be incorporated into said polymer material may be chosen from the group of dolichols or prenols, preferably a polyprenol.

The term prenol is a contracted name for isoprenoid alcohol with the formula:

H-[cH ₂ —

Plant polyprenols are reported to be pharmacologically active. Polyprenols also seem to affect the growth of cells in culture. Alpha-saturated polyprenols induces phenotypic changes in Ehrlich ascites tumor (EAT) cells. EAT cells attach to glass and spread on it but grow in an overlapping pattern.

It is suggested that polyprenols may represent a class of compounds which by interference with the biosynthesis of plasma membrane constituents influence surface properties of EAT cells and induce spreading.

The isoprene chain of said compounds preferably contains at least 4 isoprene units.

Surprisingly it has been found that by incorporation of long-chain isoprenoid alcohols the results obtained with the above described known devices can be considerably improved. The term "long-chain" means the presence of at least 4 isoprenoid units in the molecule; prenol 10 has a total of 10 C atoms and prenol 11 has a total of 11 C atoms.

Said polyprenol may be a natural polyprenol, preferably a plant polyprenol.

Said polymer materials may be either biodegradable or alternatively essentially non- degradable. Further said polymer material may be porous, preferably with interconnected pores. These pores may on one hand have a size larger than of 0,01 μm, preferably larger than 1 μm and on the other hand a size smaller than 1200 μm, preferably smaller than 700 μm.

In a special embodiment said polymer material is essentially non-porous and hydrophilic. In another embodiment said polymer material is essentially non-porous and amphiphilic.

Said polymer material may have the shape of a microporous membrane, preferably comprising two distinct layers, a top layer and a bottom layer. Said top layer may have a pore size in the range of 0,005 - 1 ,000 μm and said bottom layer may have a pore size in the range of 30 - 50 μm, preferably 0,1 - 0,2 μm.

In a further embodiment said polymer material may have the shape of a three- dimensional porous scaffold. In particular the impregnation of a microporous biodegradable polyurethane scaffold for tissue engineering with long-chain polyprenols (mixture of prenol 10 and act 11 isolated from Magnolia cobus) has shown beneficial

effects on their interaction with chondrocytes. The scaffolds used were designed as and "artificial periosteum" for the repair of articular cartilage defects. The pores of said scaffold may have a size of 10 - 1200 μm, preferably of 100 - 400 μm. Alternatively the pores of said scaffold may have a size of 1 - 100 μm, preferably of 3 - 20 μm. Typically the pore size is 5 μm.

In a further embodiment said physical incorporation of said compounds into said polymer material is obtained by spraying, brushing or impregnation of the surface of said polymer material with a solution of said compounds. These compounds, e.g. a polyprenol can be deposited in the scaffolds by impregnation, for example. The versatile chemistry of polyurethanes also allows incorporating polyprenols in the polyurethane backbone chain or as side chains upon synthesis.

The biomedical polymer materials according to the invention may purposefully be used as an artificial skin, as a bone substitute or as artificial cartilage.

Manufacture of biodegradable polyurethane membranes

The biodegradable linear polyurethane used for the preparation of the microporous membranes was synthesized in a two-step bulk polymerization. Monomers used were aliphatic hexamethylene diisocyanate (HMDI), poly(ε-caprolactone) diol (PCL) with a molecular weight of 530 and isosorbide diol (1 ,4:3,6-dianhydro-D-sorbitol) (Iso) chain extender. Dibutyltin dilaurate (DBDL) was used as catalyst.

The microporous membranes were prepared from the polymer solution in the mixture of dimethylsulfoxide (DMSO) and acetone using a phase-inverse process. The membranes were formed on PTFE-coated rollers (diameter 30 mm, length of 150 mm) using water as a precipitant. Deposition of 30 layers of the polyurethane solution allowed obtaining microporous membranes with satisfactory mechanical properties. The membranes were rinsed in a mixture of water and ethanol (80:20 vol/vol%) and subsequently dried a vacuum oven at 5O ⁰ C. Circular samples with a diameter of 14 mm were cut from the membranes and incubated at room temperature for 10 days in a mixture of polyprenols in hexane (2.65 vol-vol%). The membranes after incubation were dried at 5O ⁰ C to a constant weight in a vacuum oven, fixed between PTFE rings,

packed in double pouches, sterilized with a cold-cycle ETO process and next evacuated again at 5O ⁰ C and 4x1 (H mbar for 5 hours.

Isolation of polyprenols

Long-chain polyprenols (a mixture of prenol-10 and prenol-11) were isolated from leaves of Magnolia cobus. Dried lives (200 mg) were homogenized at high speed for 1 min in acetone-hexane 1 :1 v/v mixture using an Ultra-Turrax T25 mixer. Next the extract was subjected to alkaline hydrolysis. Analytical separation of polyprenols was performed by TLC on Silica gel plates in ethyl acetate:toluene 5:95 v/v mixture and on RP-18 plates in acetone. Spots of lipids were detected with iodine vapor and identified with standards. The unsaponifiable lipids were chromatographed on a Silica Gel 60 column and eluted with hexane containing increasing concentration of diethyl ether (0- 18%). The course of elution was monitored by TLC. A semiquantitative determination of polyprenols was performed by comparing the size and intensity of the spot detected on an adsorption chromatography with that of a known amount of a standard substance. All organic solvents used for extraction and chromatography were Silica gel TLC plates and R18-plates with concentrating zone and silica gel for column chromatography. After the isolation polyprenols gave single spots on Silica Gel G TLC plates in ethyl acetate/toluene 5:95 v/v mixture and on RP-18 HP TLC plates in acetone.

Characterization of polyurethane membranes and polyprenol

A) Thermal analysis

A Perkin-Elmer (Norwalk, CONN) differential scanning calorimeter (Pyris DSC-1) calibrated with indium was used to evaluate thermal properties of the polyurethane membranes and polyprenols. The weight of polymer samples was 5 to 8 mg, and polyprenol 5 to 6 mg. The samples were scanned at a heating rate of 10°C/min under dry, oxygen free nitrogen flowing at a rate of 50 to 60 ml/min. The samples were scanned from 15 to 12O ⁰ C.

B) Infrared Spectroscopy

Infrared spectra of the polyurethane, the polyprenol and the polyurethane membranes impregnated with polyprenol were recorded in a transmission and reflection modes

using a Fourier-Transform Perkin Elmer 2000 FT-IR spectrometer. An attenuated total reflection (ATR) unit was fitted with KRS-5 crystal (45° entrance angle). Thirty scans were taken for each sample.

C) Scanning electron microscopy of polyurethane scaffolds

A Hitachi model S-4100 field emission scanning electron microscope operated at 2.0 kV was used to observe the polyurethane samples sputtered with a 5 nm thick platinum layer.

D) Scanning electron microscopy of chondrocytes on polyurethane scaffolds Transmission electron microscope, operated at an accelerating voltage of 80 kV was used to observe chondrocytes on polyurethane scaffolds. Samples were sputtered with gold layer.

E) Chondrocyte isolation

Under aseptic conditions the articular-epiphyseal cartilage fragments were collected from bones of legs of 5 days old inbred LEW rats and digested in 0.25% collagenase, 0.05% DNase and tosyl-L-lysine chloromethyl ketone (TLCK) for 36 hours. Released chondrocytes after filtration were centrifuged at 200-300 G for 7 min and next seeded onto scaffolds kept in 24-well plate. There were 250,000 cells in 1 ml of medium seeded onto each scaffold. The cells were cultured at 37 ⁰ C for 2 and 5 weeks in DMEM-F12 containing 10% calf serum (50 μg/ml) and antibiotics (Penicillin 10000 lU/ml, Streptomycin 10000 μg/ml and Aphotericin B 25 μg/ml).

F) Cell morphology

At the end of the experimental period, the scaffolds with cells were rinsed 3 times with PBS and than were fixed in a mixture 2.5% αlutaraldehvde. postfixed in 1 % osmium teroxide, dehydrated for 10 min in ethanol with concentrations of 50%, 70%, 80%, 90%, 96% and 100% and additionally for 30 min in 100% ethanol. Next, samples were dried in a critical point dryer, sputtered with gold layer and observed under the scanning electron microscope (JEOL JEM 1200EX).

The invention and additional configurations of the invention are explained in even more detail with reference to the partially schematic illustration of several embodiments.

Shown are:

Fig. 1. The chemical structures of A. (Poly)prenol; B. lsoprene unit; C. Dolichol.

Fig. 2. Scanning electron microscopy image of the surface of the microporous polyurethane membranes which was seeded with cells. Fig. 3. DSC thermograms of the polyprenol samples. Fig. 4. Infrared spectra of: Polyurethane membrane; Polyprenols; and Polyurethane membranes impregnated with polyprenols. Fig. 5. Scanning electron micrographs of rat chondrocytes growing on the polyurethane membrane not modified with polyprenols at 2 weeks of cell culture. Fig. 6. Scanning electron micrographs of rat chondrocytes growing on the polyurethane membranes modified with polyprenols at 2 weeks of cell culture. Fig. 7. Scanning electron micrographs of rat chondrocytes growing on the polyurethane membranes not modified with polyprenols at 5 weeks of cell culture; and. Fig. 8. Scanning electron micrographs of rat chondrocytes growing on the polyurethane membranes modified with polyprenols at 5 weeks of cell culture.

Fig. 1 shows the chemical structure of two different isoprenols: (poly)prenol (A) and dolichol (C) as well as the chemical structure of an isoprene unit (B) present in the backbone of both isoprenols. Polyisoprenols - poly-c/s-prenols is a general term which includes polyprenols and dolichols. Polyisoprenols are natural products, derivatives of the C5 isoprene unit, each containing one double bond. Polyprenols (A) represent a subgroup of prenols in which n is greater than 4. A polyprenol amphiphilic molecule consists of a hydroxyl group (a hydrophilic part), and a long unsaturated isoprene chain (the repeating isoprene residues) mainly of poly-cis configuration (a hydrophobic part). Plant poly-cis prenols with the structure ωtxcyOH (where, ω is an isoprene residue farthest from the hydroxyl group, t is a trans-isoprene residue, c is a cis-isoprene

residue and -OH is the hydroxyl group), contain two or three internal trans isoprene units (B). In dolichols (C), the double bond in the α - residue is hydrogenated, and this distinguishes them from the polyprenols with a double bond in the α - residue.

Fig. 2 shows a scanning electron microscopy image of the surface of the microporous polyurethane membranes which was seeded with cells. Scale bar represents 100 μm. The elastomeric membrane had interconnected pores with an average size in the range of 5 - 100 μm.

Fig. 3 shows the DSC thermogram of the mixture of long-chain polyprenols (decaprenol and undecaprenol). The thermogram shows two thermal transitions at temperatures in the range of 35.5 to 39.7 ⁰ C. The melting peak temperature at 36.4 ⁰ C also called pretransition corresponds to the crystal-crystal transition. The high-energy melting endotherm at 38.O ⁰ C with δH = 3.55 J/g corresponds to the gel-to-liquid crystal transition or the solid-to-mesophase transition.

Fig. 4 shows typical infrared spectra of the polyurethane membrane (A), the polyprenol (B) and the polyurethane membrane impregnated with polyprenol (C). The spectrum of the polyurethane shows characteristic IR bands. The peak at 1681 and 1533 cm" ¹ are typical for v(C=O) (amide I) and δ(NH) with v(CO-N) (amide II) while the peak at 1724 cm" ¹ is typical for ester v(C=O) present in the polyurethane. In the infrared spectrum of the polyprenol there were the absorption bands at 2962, 1449 and 1376 cm ^"1 which were assigned to the methylene -CH3 groups and the weak peaks in the range of 1667-

1580 cm" ¹ assigned to the -C=C- group. Both groups are specific for the polyprenol. Infrared spectrum of polyurethane membranes impregnated with polyprenols shows one absorption band at 1376 cm ^"1 assigned to methylene group originating exclusively from polyprenols. The presence of this adsorption band confirmed the successful impregnation of polymeric membranes with polyprenols. The other adsorption bands found in the spectrum of the polyprenols are not present in the spectrum of the polyurethane scaffolds impregnated with polyprenols. These bands are masked by strong adsorption bands originating from the chemical groups of the polyurethane.

Figs. 5 - 8 show cell growth on the polyurethane membranes of unmodified membranes and polyprenol-modified membranes. Scale bars represent 10 μm.

Throughout the whole culture period of 5 weeks the cells grew into the scaffold and maintained the round shape characteristic for chondrocyte-like-morphology. During the first 2 weeks of culturing the number of cells was comparable for nonmodified and modified membranes. The cells attached firmly to the membrane surface, grew deeply into the pores and deposited fibrillar extracellular matrix.

Fig. 5 shows chondrocytes growing into the pores of the nonmodified membrane at 2 weeks. The cells with diameters of about 10 μm maintain round shape. The surface of the cells is rough with grainy texture. The chondrocytes growing into the porous membranes modified with long-chain polyprenols at 2 weeks are shown in Fig. 6. Chondrocytes also in this case maintain spherical shape. Frequently, a few cells invaded the same pore. In Fig. 6 chondrocytes with diameters of approximately 5 μm are in direct contact with pore walls. Chondrocytes shown in Fig. 6 contact the surface of pores via abundant fibrillar matrix. During the next weeks of culturing the number of chondrocytes increased, in both nonmodified membranes and membranes modified with prenols. The cells produced a three-dimensional fibrillar network.

Fig. 7 shows the morphology of a chondrocyte with a diameter of about 15 μm growing on the nonmodified membrane at 5 weeks. The cell is attached to the membrane surface via a fibrillar matrix. Fig. 8 shows a large number of chondrocytes in the membrane modified with polyprenols at 5 weeks of cell culture. The morphology of cells communicating with each other and firmly attached to the pore walls via podia and fibrillar extracellular matrix.

Microporous membranes from the polyprenol-modified biodegradable polyurethanes according to the invention support attachment and growth of rat chondrocytes. The cells invaded the pores of the membranes, maintained round morphology and produced abundant fibrillar extracellular matrix resembling the network formed by chondrocytes in vivo. Impregnation of the membranes with biologically active amphiphilic polyprenols with the poly-c/s configuration of the isoprenoid chain seems to facilitate the cell - material interaction.

Example 1

An "artificial skin" construct for the treatment of full-thickness skin wounds was produced from experimental biocompatible, biodegradable polyurethane. The construct consisted of two layers, i.e. the "dermis" layer and the "epidermis" layer. The porous "dermis" layer was produced from the polyurethane synthesized using lysine triisocyanate, poly(ε-caprolactone diol), poly(ethylene oxide diol), polyprenol extracted from Aloe vera and 1 ,4-butane diol chain extender. The porous "epidermis" layer was produced form poly(ε-caprolactone diol), poly(ethylene oxide diol), and 1 ,4-butane diol chain extender. The bilayer "artificial skin" used to treat full-thickness skin wounds in minipigs facilitated wound healing without scar formation.

Example 2

Porous 3-D scaffold was produced from isosorbide-based biodegradable polyurethane using salt-leaching-phase inverse process. The scaffold was impregnated with long- chain polyprenols (a mixture of prenol-10 and prenol-11) isolated from leaves of Magnolia cobus. The impregnated scaffold was sterilized using a cold-cycle ETO technique and seeded with human chondrocytes. The cells attached well to scaffold's surface, have ingrown its porous structure, maintained the round shape characteristic of chondroblastic morphology and produced abundant extracellular matrix. The total amount of DNA, proteins and proteoglycans increased with the time of the experiment up to 9 weeks.

Example 3

Biodegradable polyurethane was synthesized from hexamethylene diisocyanate, polyol based on adipic acid and ethylene diol with MW 750 dalton and 1 ,4-butane diol chain extender. The synthesis was carried out in the presence of long-chain polyprenols mixture extracted from the leaves of Ginkgo biloba. The polymer was processed into porous 3-D scaffolds using a salt-leaching technique. The scaffold with a potential application for cancellous bone graft substitutes supported attachment and proliferation of sheep osteoblasts.