Polymer for tissue engineering

Abstract

The invention relates to a polymer for tissue engineering from biodegradable polyphosphazenes, having photopolymerizable side groups, wherein the side groups of the polyphosphazenes are formed exclusively from amino acids and/or amino acid derivatives, which are bonded to the backbone of the polyphosphazene via the amino group of the amino acid and to a spacer attached to the acid group with a carbon chain of length m, which has a vinyl group at the free end, wherein m=0 to m=10.

Claims

1. A polymer for tissue engineering, comprising: biodegradable polyphosphazenes, having photopolymerized side groups, wherein the side groups of the polyphosphazenes are formed exclusively from amino acids and/or amino acid derivatives, each having a vinyl group at the free end prior to crosslinking of the side groups and which are bonded to the backbone of the polyphosphazene via the amino group of the amino acid and have a spacer on the acid group, which carbon chain is present with the length m=0 to m=10, which has said vinyl group at the free end, wherein said side groups are crosslinked via said vinyl groups at the free ends of said side groups, wherein each polyphosphazene of the polymer, prior to crosslinking of said side groups, conform to the structural formula ##STR00002## wherein X.sub.1 stands for NH and X.sub.2 stands for O, S or NH, and R.sub.1 is the side chain of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serynyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutainyl, aspartoyl, Glutaoyl, lysinyl, arginine or histidinyl, and m=0 to 10 and n=3 to 1000.

2. A polymer as claimed in claim 1, wherein said side groups prior to crosslinking of said side groups are formed from amino acid vinyl ester or amino acid allyl ester.

3. A polymer as claimed in claim 1, wherein said vinyl groups of said side groups prior to crosslinking are joined by thiol compounds when performing crosslinking of said side groups, said thiol compounds having at least two thiol groups.

4. A polymer as claimed in claim 3, wherein, when performing crosslinking of said side groups, adipic acid divinyl esters are cross-linked to said vinyl groups, which are present prior to crosslinking of said side groups, with said thiol compounds with said at least two thiol groups.

5. A polymer as claimed in claim 3, wherein said thiol compound has three thiol groups.

6. A polymer as claimed in claim 1, wherein the polymer has homogeneously distributed leachable porogen included in its polymer matrix.

7. A polymer as claimed in claim 1, wherein the polymer is a three-dimensional scaffold of a desired form or shape by bringing the vinyl capped polyphosphazenes into the desired form or shape and performing photopolymerization or photo-cross-linking.

8. A polymer as claimed in claim 7, wherein said three-dimensional scaffold composed of said photopolymerized or photo-cross-linked polymer is rigid and porous.

9. A polymer for tissue engineering, comprising: biodegradable polyphosphazenes, having photopolymerized side groups, wherein the side groups of the polyphosphazenes are formed exclusively from amino acids and/or amino acid derivatives, each having a vinyl group at the free end prior to crosslinking of the side groups and which are bonded to the backbone of the polyphosphazene via the amino group of the amino acid and have a spacer on the acid group, which carbon chain is present with the length m=0 to m=10, which has said vinyl group at the free end, wherein said side groups are crosslinked via said vinyl groups at the free ends of said side groups, wherein said vinyl groups of said side groups prior to crosslinking are joined by thiol compounds when performing crosslinking of said side groups, said thiol compounds having at least two thiol groups, and wherein, when performing crosslinking of said side groups, acid divinyl esters are cross-linked to said vinyl groups, which are present prior to crosslinking of said side groups, with said thiol compounds with said at least two thiol groups.

10. A polymer as claimed in claim 9, wherein each polyphosphazene of the polymer, prior to photopolymerization or photo-cross-linking conform to the structural formula ##STR00003## wherein X.sub.1 stands for NH and X.sub.2 stands for O, S or NH, and R.sub.1 is the side chain of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serynyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutainyl, aspartoyl, Glutaoyl, lysinyl, arginine or histidinyl, and m=0 to 10 and n=3 to 1000.

11. A polymer as claimed in claim 9, wherein said side groups, prior to crosslinking of said side groups, are formed from amino acid vinyl ester or amino acid allyl ester.

12. A polymer as claimed in claim 9, wherein said vinyl groups, which are present prior to crosslinking of said side groups, are joined by thiol compounds when performing crosslinking of said side groups, said thiol compounds having at least two thiol groups.

13. A polymer as claimed in claim 12, wherein said polymer comprises adipic acid divinyl esters, said adipic acid divinyl esters are cross-linked to said vinyl groups, which are present prior to crosslinking of said side groups, with said thiol compounds with said at least two thiol groups when performing crosslinking of said side groups.

14. A polymer as claimed in claim 12, wherein said thiol compound has three thiol groups.

15. A polymer as claimed in claim 8, wherein pores are formed by washing out a homogeneously distributed leachable porogen which is included in the polymer matrix of the photo-polymerized polymer.

16. A polymer as claimed in claim 9, wherein said thiol compound is a trimethylolpropane tris (3-mercaptopropionate).

17. A polymer as claimed in claim 6, wherein said porogen is a salt.

18. A polymer as claimed in claim 1, wherein the polymer is dimensionally stable.

19. A polymer as claimed in claim 9, wherein said polymer comprises additional molecules, said molecules are covalently bonded to a thiol group of said thiol compounds with said at least two thiol groups.

20. A polymer for tissue engineering, comprising: biodegradable polyphosphazenes, having photopolymerized side groups, wherein the side groups of the polyphosphazenes are formed exclusively from amino acids and/or amino acid derivatives, each having a vinyl group at the free end prior to crosslinking of the side groups and which are bonded to the backbone of the polyphosphazene via the amino group of the amino acid and have a spacer on the acid group, which carbon chain is present with the length m=0 to m=10, which has said vinyl group at the free end, wherein said side groups are crosslinked via said vinyl groups at the free ends of said side groups, wherein the polymer is a three-dimensional scaffold of a desired form or shape by bringing the vinyl capped polyphosphazenes into the desired form or shape and performing photopolymerization or photo-cross-linking, and wherein each polyphosphazene of the polymer, prior to photopolymerization or photo-cross-linking conform to the structural formula ##STR00004## wherein X.sub.1 stands for NH and X.sub.2 stands for O, S or NH, and R.sub.1 is the side chain of alanyl, valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serynyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutainyl, aspartoyl, Glutaoyl, lysinyl, arginine or histidinyl, and m=0 to 10 and n=3 to 1000.

Description

(1) The invention is illustrated by means of a manufacturing example and a drawing:

(2) FIG. 1: Shows the reaction scheme for the preparation of an exemplary polyphosphazene, having polymerizable side groups according to the invention.

(3) To prepare a polyphosphazene according to the invention, a macromolecular substitution of polydichlorophosphazene was carried out, synthesized by a living polymerization of trichlorophosphoranimine. 24.5 mg of PCl.sub.5 (0.12 mmol, 1 eq.) and 0.66 g of Cl.sub.3PNSiMe.sub.3 (2.94 mmol, 25 eq.) were dissolved in 10 ml of anhydrous CH.sub.2Cl.sub.2 in the glove box and stirred at room temperature for 16 hours. The solvent was removed under reduced pressure, and the resulting polydichlorophosphazene was used without further purification.

(4) Yield quantitative: .sup.31P-NMR (CDC13): =18.16 ppm.

(5) The macromolecular substitution of polydichlorophosphazene was carried out according to the method showed in FIG. 1, the polymer obtained with 1 side groups of glycine allyl esters.

(6) First, 1.52 g of 2-(tert-butoxycarbonylamino) acetate (7.06 mmol, 2.4 eq) in trifluoroethanoic acid (TFA)/CH.sub.2Cl.sub.2 () was deprotected for 6 hours. The solvents were carefully removed under vacuum to obtain allyl-2-aminoacetate. Allyl-2-aminoacetate was dissolved in anhydrous THF and a large excess of NEt3 was added to neutralize TFA residues. Polydichlorophosphazene (0.66 g, 2.94 mmol, 1 eq.), dissolved in anhydrous THF, was then added to the solution of allyl-2-aminoacetate. The reaction was stirred at room temperature for 24 hours. Precipitated salt was removed by filtration and the reaction mixture was concentrated under vacuum. The polymer was purified by precipitation from THF in cooled diethyl ether. The polymer was then dissolved in ethyl acetate and further washed with H.sub.2O and a brine and dried over MgSO.sub.4. The solvent was removed under vacuum and the product was further dried under high vacuum to obtain the polymer 1 as a yellowish highly viscous product.

(7) Yield: 0.66 g (80%). .sup.1H-NMR (CDCl.sub.3): =3.75 (br, 2H), 4.55 (br, 2H), 5.19 to 5.31 (br, m, 2H), 5.84 to 5.93 (br, m, 1H) ppm. .sup.31P-NMR (CDCl.sub.3): =1.73 ppm. .sup.13C-NMR (CDCl.sub.3): =42.8 (NHCH.sub.2), 65.5 (OCH.sub.2), 118.4 (CH.sub.2), 132.2 (CH), 172.5 (CO) ppm. FTIR (solid): v.sub.max=3341 (NH), 2938 (CH), 1737 (CO), 1650 (CC), 1188 (PN) cm.sup.1.

(8) In order to obtain a porous, three-dimensional scaffold based on glycine, the polymer 1 was cross-linked by thiol-ene-photopolymerization with thiol trimethylolpropane tris (3-mercaptopropionate), hereinafter referred to as tri-thiol. The photopolymerization of the allyl groups of the polymer 1 and the tri-thiol was carried out in small amounts (about 1 wt %) as a photo-initiator at room temperature in the presence of a porogen in CHCl.sub.3 with 2,2-dimethoxy-2-phenylacetophenone (DMPA). In a glass vial, polymer 1 (90.0 mg, 0.33 mmol, 1 eq.) and 1 mg were dissolved in 1 ml of CHCl.sub.3. Then, 0.5 ml of polyethylene glycol with a nominal molecular weight of 200 g/mol (PEG-200), tri-thiol (72 l, 87.6 mg, 0.22 mmol, 0.67 eq) and NaCl as porogen (c. 4.2 g, 75 wt % of the reaction mixture) was added to obtain a homogeneous mixture with fully dispersed NaCl particles.

(9) The mixture was exposed to ultraviolet light in a UV reactor for 1.5 hours.

(10) The material was removed from the vial and repeatedly washed into a H.sub.2O excess to wash out the salt and the PEG-200. The scaffolds were purified by soxhlet extraction, using EtOH for 16 hours, and dried under vacuum to obtain a polymer 2 as a porous pellet. The solidification of the reaction mixture showed a successful formation of the cross-linked polymer network around the porogen.

(11) Result: .sup.31P-NMR (solid): =7.7 ppm. .sup.13C-NMR (solid): =7.6 (CH.sub.3), 26.8 (CH.sub.2), 43.8 (NHCH.sub.2), 65.1 (OCH.sub.2), 172.1 (CO) ppm. FTIR (solid): v.sub.max=3342 (NH), 2926 (CH), 1729 (CO), 1188 (PN) cm.sup.1. Elementary analysis: calculated, C, 44.57%; H, 6.23%; N, 7.80%; S, 11.90%; P, 5.75%. found, C, 43.97%; H, 6.21%; N, 7.08%; S, 11.23%; P, 5.47%.

(12) The polymer 1 was also mixed with a commercially available adipic acid vinyl ester (VE) in various proportions to change the degradation rates of the obtained scaffolds. The conditions for the thiol-ene-cross-linking reaction were similar to the thiol-ene-cross-linking reaction of the polymer 1 in an adjustment of the molar ratio of the alkene groups to the thiol groups (1/1).

(13) For a polymer 3, 27 wt % of the polymer 1 was mixed with 53 wt % of tri-thiol and 20 wt % of deionized water and subjected to a thiol-ene-cross-linking reaction.

(14) Result: FTIR (solid): v.sub.max=3353 (NH), 2930 (CH), 1728 (CO), 1184 (PN) cm-1.

(15) Analysis: calculated, C, 47.91%; H, 6.50%; N, 4.19%; S, 12.79%; P, 3.09%. found, C, 47.50%; H, 6.54%; N, 4.17%; S, 12.36%; P, 3.25%.

(16) The degradation studies were carried out in deionized H.sub.2O at 37 C. for 12 weeks. Samples containing 30 mg of Polymers 2 and 3 were placed in sealed vials and incubated in 2 ml of H.sub.2O. A data analysis was carried out three times at appropriate intervals over the examination period.

(17) The samples were dried in a vacuum oven at 40 C. until the weight was constant. The mass loss was determined gravimetrically, wherein the respective established average value of the mass losses as a percentage compared to the initial weight of the degradation sample are shown in the following table.

(18) TABLE-US-00001 Loss of Weight [in %] after 10 Days 20 Days 42 Days 56 Days 90 Days 120 Days Polymer 2 5 16 33 60 62 85 Polymer 3 0 0 7 7 20 26

(19) The table shows that the polymer 2, which had the highest proportion of polyphosphazenes exhibited a pronounced degradation profile in a neutral aqueous solution at 37 C. As a result of the hydrolytic degradation of the polyphosphazene backbone, the network bonds of the cross-linked organic mass are separated, which leads to an improvement in the overall biodegradation rates of the scaffold. With the decrease of the polyphosphazene content and the increase of the adipic acid vinyl ester, the degradation rate is considerably reduced which is attributed to the increased hydrophobicity.

(20) Due to the preferred application of the three-dimensional scaffolds for tissue engineering according to the invention, the cytotoxicity of these scaffolds was investigated in conjunction with primary epithelial cells and stem cells (ASC) obtained from adipose tissue, whereby no cytotoxicity could be detected for cells in a medium previously treated with a polymer 2, which has a particularly high proportion of polyphosphazenes. Furthermore, preliminary studies also showed no cytotoxicity of polymer 2 in a cell culture medium at 37 C. for a period of 42 days in which 33% of the polymer had already been degraded, suggesting the non-toxicity of degradation products or their intermediates.