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    • 专利摘要:
    A polymer structure for tissue engineering based on a biodegradable polyphosphazene with photopolymerizable side groups is described. In order to provide favorable conditions for biodegradation and for tissue engineering well suited three-dimensional frameworks, it is proposed that the polyphosphazene forms a crosslinkable structure according to the structural formula, wherein X1, X2 are NH, O or S, R1 is an alanyl, Valinyl, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryptophanyl, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutainyl, aspartoyl, glutoyl, lysinyl, argininyl and histidinyl, and where m = 0 to 10 and n = 3 to 1000.
    公开号:AT515955A1
    申请号:T50416/2014
    申请日:2014-06-16
    公开日:2016-01-15
    发明作者:Ian Teasdale；Sandra Wilfert；Tamara Aigner；Aiztiber Iturmendi；Oliver Brüggemann；Klaus Schröder；Gbenga Olawale；Florian Hildner；De Llobet Maria Rigau
    申请人:Universität Linz；
    IPC主号:
    专利说明:

    The invention relates to a polymer structure for tissue growth based on a biodegradable polyphosphazene with photopolymerizable side groups and to a three-dimensional framework for tissue engineering using this polymer structure.
    Advanced materials for biomedical applications, such as implants, controlled release drug delivery systems, or tissue engineered scaffolds, are based on biodegradable polymers. For polymers that can be successfully used as templates for tissue engineering, certain biological and physical properties, such as biocompatibility, specific mechanical properties, customizable degradation rates and non-toxic degradation products, as well as a morphology that promotes tissue growth are required. For this field of application, PoSy (organo) phosphazenes are of particular interest because of their very different properties depending on the substituents of the side groups, but above all the possibility of changing the rate of degradation of the inorganic backbone. In addition, the degradation products form Unlike acidic degradation products of many other biodegradable polymers used in biomedicine, a nearly neutral, pH-buffered mixture of phosphates and ammonia, so unwanted side effects, such as tissue irritation and inflammatory or aller¬gische reactions, largely absent.
    In order to adjust the rate of degradation of polyphosphazenes, it is known (US Pat. No. 6,077,918) to provide, on the one hand, hydrophobic side groups, such as p-methylphenoxy and other aromatic groups, and, on the other hand, hydrolytic side groups, such as amino acid alkyl esters. Thus, for example, the hydrophobicity of an ethyl glycinate-substituted polyphosphazene is increased by adding p-methylphenoxy as a carbon substituent, while the ethylgiycinate side group makes it possible to biodegrade it to harmless degradation products in an aqueous environment. A disadvantage, however, is that the mechanical properties required for use in the field of tissue engineering can only be achieved with an additional polymer which is not based on a polyphosphazene.
    In order to provide highly porous, three-dimensional, biodegradable polymer structures based on polyphosphazenes for the cultivation of skeletal tissues, it is also known (US Pat. No. 6,235,061) to use polyphosphazen having hydrolytically unstable side groups, such as glycosyl, glycinyl, glyceryl, To substitute imidazolyl or ethoxy groups. The high porosity is made possible by salts, preferably sodium salts, which are admixed with the polymer dissolved in an organic solvent, preferably tetrahydrofuran (THF), in order to be redissolved from the cured polymer after evaporation of the solvent, so that an open-pore Polymer matrix is obtained with evenly distributed over the volume of pores. A disadvantage, however, is that the mechanical properties of these known polymer structures can not meet the higher requirements for use as an implant.
    The invention is thus based on the object of designing a polymer structure for tissue growths of the type described above in such a way that not only advantageous conditions for adjustable biodegradation are created, but also the prerequisites are created with the aid of these polymer structures a simple way for tissue engineering to create well-suited, dreidi¬mensionale scaffolds that can also meet higher mechanical requirements.
    Starting from a polymer structure of the kind described in the introduction, the invention achieves the stated object in that the polyphosphazene has a crosslinkable structure according to the structural formula
    where Χ-ι, X2 stands for NH, O or S,
    Ri is selected from alanyl, valinyi, leucinyl, isoleucinyl, prolinyl, phenylalaninyl, tryp-tophanyi, methioninyl, glycinyl, serinyl, threoninyl, cysteinyl, tyrosinyl, asparaginyl, glutainyl, aspartoyl, glutoyl, lysinyl, argininyl and histidine's comprehensive group and where m = 0 to 10 and n = 3 to 1000.
    Because of the side groups of an amino acid vinyl ester or an allyl amino acid ester, the comparatively short-chain polyphosphazene can be subjected to photopolymerization with the result of pronounced cross-linking with covalent bonds, so that all prerequisites for producing a three-dimensional, cross-linked polymer skeleton are met The incorporation of glycine spacers adjacent to the backbone implies, in particular, an acceleration of the hydrolytic degradability of the polyphosphazene backbone, with the two reactive carbon-carbon backbones leading to Double bonds per repetition unit offer advantageous conditions for the subsequent photochemical crosslinking and functionalization of the polymer.
    Three-dimensional frameworks for tissue engineering can be produced particularly advantageously on the basis of polyphosphazenes substituted with amino acid vinyl esters or amino acid allyl esters if the polyphosphazene is photopolymerized or photocrosslinked. For the photoreaction, a thiol group can be employed, with the use of a corresponding variety of multifunctional thiols with different spacers allowing the adaptation of the respective properties to the given requirements and the control of the hydrophilic and hydrophobic behavior of the finished framework. Particularly advantageous proportions could be ensured in this context if the thiol group used is a thioltrimethylolpropane tris (3-mercaptopropionate). Functionalization via thiol-ene addition allows covalent attachment of the various molecules to a thiol group. Thus, for example, the biodegradability of the poly mergerüsts by the polymerization of the polyphosphazene with thiol groups and a divinyl adipate due to the hydrophobicity of this ester are permanently affected. The properties of the three-dimensional, cross-linked Ge¬rüsts can thus be easily adjusted, with only the synthesis of a single poly (organo) phosphazene is required.
    The requisite porosity of the three-dimensional framework for tissue engineering may be obtained in various ways in a manner known per se, e.g. B. by stereo lithography or foaming by a propellant gas can be achieved. However, particularly simple production conditions result if the crosslinking component is admixed with a porogen, preferably a salt, which is leached out of the polymer after polymerization and leaves a coherent pore system.
    To prepare a polyphosphazene according to the invention, a macromolecular substitution of polydichlorophosphazene was carried out, which was synthesized via a living polymerization of trichlorophosphoranimine, in the glove box 24.5 mg of PCI5 (0.12 mmol, 1 eq.) And 0, 66 g of Ci3P = N-SiMe3 (2.94 mmol, 25 eq.) Dissolved in 10 ml of anhydrous GH2Ci2 and stirred for 16 hours at room temperature. The solvent was removed under reduced pressure and the resulting dichlorophorophosphazene used without further purification.
    Yield quantitatively: 31P-NMR (CDC! 3): δ = -18.16 ppm.
    The macromolecular substitution of poly (dichlorophosphazene) was carried out according to the procedure outlined below, wherein the polymer obtained had 1 side groups of glycine and allyl ester.
    First, 1.52 g of 2- {tert-butoxycarbonylamino) -acetate (7.06 mmol, 2.4 eq.) In trifuoroic acid (TFA) / CH 2 Cl 2 (1/3) was deprotected for 6 hours. The solvents were carefully removed under vacuum to obtain ailyl 2-aminoacetate. Allyl -2-aminoacetate was dissolved in anhydrous THF and a large excess of NEt3 added to neutralize TFA residues. Poly-dichlorophosphazene (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 concentrated under vacuum. The polymer was purified by precipitation from THF in chilled diethyl ether. The polymer was then dissolved in ethyl acetate and washed further with H 2 O and brine, and dried over MgSC> 4. The solvent was removed under vacuum and the product dried under high vacuum wei¬ter to obtain the polymer 1 as a yellowish highly viscous product.
    Yield; 0.66 g (80%). 1H ~ NMR (CDCl3): δ - 3.75 (br, 2H), 4.55 (br, 2H), 5.19 to 5.31 (br, m, 2H), 5.84 to 5.93 (br , m, 1H) ppm31 P-NMR (GDGR): δ - 1.73 ppm.13G-NMR (CDCis): δ = 42.8 (NH-CH2), 65.5 (OCH2), 118.4 ( -CH2), 132.2 (-CH-), 172.5 (CO) ppm. FTIR (fixed): vmax - 3341 (NH), 2938 (GH), 1737 (C = 0), 1650 (G = C), 1188 (P = N) cm'1.
    In order to obtain a porous, three-dimensional glycine-based scaffold, the polymer 1 was crosslinked by thiol-ene photopolymerization with the aid of thiol-trimethylolpropane tris (3 "mercaptopropionate) (trithiol). Photopolymerization of the allyl groups of polymer 1 and trithiol was carried out at room temperature in the presence of a porogen in CHCl 3 with 2,2-dimethoxy-2-phenylacetophenone (DMPA) as a photoinitiator in small quantities (about 1% by weight). In a glass vial, polymer 1 (90.0 mg, 0.33 mmol, 1 eq.) And 1 mg in 1 mL CHCl 3 were dissolved. Then 0.5 ml Polyethyienglyco! with a nominal molecular weight of 200 g / moi (PEG-200), trithiol (72 μΙ, 87.6 mg, 0.22 mmol, 0.67 eq.) and NaCl as the porogen (about 4.2 g, 75 wt The mixture was exposed to ultraviolet light for 1.5 hours in a UV reactor. The material was removed from the vial and washed out to remove the salt of the PEG-200 repeatedly placed in an H 2 O excess. The scaffolds were cleaned by Soxhlet extraction using EtOH for 16 hours and dried under vacuum to obtain a polymer 2 as a porous pellet. Solidification of the reaction mixture showed successful formation of the cross-linked polymer network around the porogen.
    Result: 31 P-NMR (solid): δ = 7.7 ppm. 13 C-NMR (solid): δ-7.6 (CH 3), 26.8 (CH 2), 43.8 (NH-GH 2), 65.1 (OCH 2), 172.1 (C-O) ppm. FTIR (fixed): vmax = 3342 (NH), 2926 (GH), 1729 (C = 0), 1188 (P = N) cm " 1. Elemental analysis: 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%.
    Polymer 1 was also blended with a commercially available adipic di-vinyl ester (VE) in various proportions to alter the degradation rate of the scaffolds obtained. The conditions for the thioi-ene crosslinking reaction were similar to the thioi-ene cross-linking reaction of polymer 1 for an adjustment of the Mo ratio from the Aiken groups to the thio groups (1/1). For a polymer 3, 27 wt % of polymer 1 was mixed with 53% by weight of trithiol and 20% by weight of VE and subjected to a thioi-ene cross-linking reaction.
    Result: FTIR (solid): vmax = 3353 (NH), 2930 (CH), 1728 (C = 0), 1184 (P = N) cm'1. 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%.
    The degradation studies were performed in deionized H 2 O at 37 ° G for 12 weeks. Samples containing 30 mg of polymers 2-5 were placed in sealed vials and incubated in 2 ml of H 2 O. A data analysis was taken in each case three times at appropriate time intervals over the investigation period. The samples were dried in a vacuum oven at 40 ° C until the weight was constant. The mass loss was determined gravimetrically, with the respectively determined average value of the mass vereses being indicated as a percentage in comparison to the initial weight of the degradation sample in the table below.
    The table shows that Polymer 2, which had the highest level of polyphosphazenene, showed 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 crosslinked organic mass are separated, resulting in an improvement in the overall degradation rate of the backbone. With the decrease in the polyphosphazene content and the increase in the divinyl adipate, the rate of degradation is significantly reduced, which is attributed to the increased hydrophobicity.
    Because of the preferred use of the three-dimensional tissue engineered scaffolds of the present invention, the cytotoxicity of these scaffolds has been studied in conjunction with primary epitic and adipose tissue-derived (ASC) strains which failed to detect cell cytotoxicity in a medium that had previously been treated with a polymer 2 that had previously been purified 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 degraded, indicating the non-toxicity of the degradation products or their intermediates.
    权利要求:
    Claims (5)
    [1]
    1. Polymer structure for tissue engineering based on a biodegradable polyphosphazene with photopolymerizable side groups, characterized gekenn¬zeichnet that the polyphosphazene a crosslinkable structure according to the structural formula

    wherein Xi, X2 are NH, O or S, Ri is an Aianyi, Vaiinyl, Leucinyi, Isoieucinyl, Proiinyl, Phenyialaninyi, Tryp-tophanyi, Methioninyi, Giycinyi, Serinyi, Threoninyl, Cysteinyl, Tyrosinyi, Asparagi-nyl, Giutaiiiyl, Aspartoyi, Giutaoyi, Lysinyi, Argininyi and Histidinyi, and where m = 0 to 10 and n = 3 to 1000.
    [2]
    2. Three-dimensional scaffold for the tissue growth with a polymer structure according to claim 1, characterized in that the polyphosphazene is photopolymerized or photocrosslinked.
    [3]
    3. Three-dimensional scaffold according to claim 2, characterized in that a thiol group is used for the photoreaction.
    [4]
    4. Three-dimensional scaffold according to claim 2 or 3, characterized in that the polyphosphazene is photopolymerized together with thiol groups and a niyl adipate.
    [5]
    5. Three-dimensional framework according to one of claims 2 to 4, gekennzeich¬ by pores leached from the polymer matrix porogen.
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    WO2015192158A1|2015-12-23|
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    引用文献:
    公开号 | 申请日 | 公开日 | 申请人 | 专利标题
    WO1995028150A1|1994-04-15|1995-10-26|The Penn State Research Foundation|Photocrosslinkable polyphosphazenes and their use as microencapsulation materials|
    WO1995032736A1|1994-05-31|1995-12-07|The Penn State Research Foundation|Immobilization of biologically active materials and diagnostic agents in cross-linked poly|
    WO2007056316A2|2005-11-04|2007-05-18|University Of Virginia Patent Foundation|Injectable physiological temperature setting cement composites for spinal fusion and related method thereof|
    WO2008153278A1|2007-06-14|2008-12-18|Korea Institute Of Science And Technology|Phosphazene hydrogels with chemical corss -link, preparation method thereof and use thereof|
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    US8586020B2|2011-06-30|2013-11-19|Korea Institute Of Science And Technology|Poly composition for biomaterials|WO2021259848A1|2020-06-26|2021-12-30|Covestro Deutschland Ag|Polyurethanes based on side chain diol- and diaminopolyphosphazenes and their hydrogels|
    WO2021259847A1|2020-06-26|2021-12-30|Covestro Deutschland Ag|Polyurethanes based on terminal diol- and diaminopolyphosphazenes and their hydrogels|
    CN111909381B|2020-09-11|2021-06-15|东部超导科技（苏州）有限公司|Ultraviolet crosslinking polyphosphazene, preparation method thereof, composite coating low-temperature measurement optical fiber and preparation method thereof|
    法律状态:
    2019-07-15| PC| Change of the owner|Owner name: NP LIFE SCIENCE TECHNOLOGIES KG, AT Effective date: 20190611 Owner name: BIOMED-ZET LIFE SCIENCE GMBH, AT Effective date: 20190611 Owner name: TRANSFERCENTER FUER KUNSTSTOFFTECHNIK GMBH, AT Effective date: 20190611 |
    优先权:
    申请号 | 申请日 | 专利标题
    ATA50416/2014A|AT515955B1|2014-06-16|2014-06-16|Polymer structure and three-dimensional framework for tissue engineering|ATA50416/2014A| AT515955B1|2014-06-16|2014-06-16|Polymer structure and three-dimensional framework for tissue engineering|
    CA2951728A| CA2951728A1|2014-06-16|2015-06-16|Polymer for tissue engineering|
    EP15744854.9A| EP3180390B1|2014-06-16|2015-06-16|Polymer for tissue engineering|
    US15/319,339| US10815340B2|2014-06-16|2015-06-16|Polymer for tissue engineering|
    PCT/AT2015/050151| WO2015192158A1|2014-06-16|2015-06-16|Polymer for tissue engineering|
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