Collagen based materials

Abstract

There are provided collagen based polymeric materials comprising collagen molecules and/or collagen derived molecules which have been functionalized by the addition of one or more ethylenically unsaturated moieties and which have been cross-linked via said moieties. Also provided are collagen based compositions and methods of producing collagen based polymeric materials.

Claims

1. A method of producing a collagen based polymeric material wherein the method comprises cross linking functionalised collagen and/or collagen derived molecules having one or more ethylenically unsaturated moieties, wherein the one or more ethylenically unsaturated moieties includes 4-vinylbenzyl chloride (4VBC).

2. A method according to claim 1, wherein the method comprises the steps of: (a) Functionalising collagen and/or collagen derived molecules by the addition of one or more moieties comprising a vinyl group; and (b) Crosslinking of the functionalised collagen and/or collagen derived molecules of (a).

3. A collagen based composition comprising a functionalised collagen and/or collagen derived molecule, wherein the collagen and/or collagen derived molecule is functionalised by addition of one or more ethylenically unsaturated moieties wherein the one or more ethylenically unsaturated moieties includes 4-vinylbenzyl chloride (4VBC).

4. A method of producing a collagen based polymeric material according to claim 1, wherein the material is produced in the form of one or more fibres and/or one or more filaments and/or one or more films and/or one or more gels and wherein the method comprises the steps of: (a) Functionalising collagen and/or collagen derived molecules with the one or more ethylenically unsaturated moieties; (b) Combining the functionalised collagen and/or collagen derived molecules with a vehicle; and (c) Wet spinning the functionalised collagen and/or collagen derived molecules.

5. A method according to claim 1, wherein the material is produced in the form of one or more fibres and/or one or more filaments and/or one or more films and/or one or more gels and wherein the method comprises the steps of: (a) Functionalising collagen and/or collagen derived molecules with the one or more ethylenically unsaturated moieties; (b) Combining the functionalised collagen and/or collagen derived molecules with a vehicle; and (c) Wet spinning the functionalised collagen and/or collagen derived molecules; and wherein a photoinitiator is added during or after steps (b) and/or (c).

6. A method according to claim 4, wherein the formed collagen fibres and/or filaments have a diameter of between about 0.01 m and 50 m.

7. A method according to claim 1, further comprising: forming a web, a tow, a yarn, and/or a nonwoven of functionalised collagen fibres and/or filaments and/or films, wherein a pore size of the web, the tow, the yarn, and/or the nonwoven of functionalised collagen fibres and/or filaments and/or films is between about 5 m and 350 m.

8. A method according to claim 1, wherein the material or composition comprises functionalised collagen which has a preserved triple helical structure.

9. A method according to claim 1, wherein [4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone] (Irgacure 2959) is used as a photoinitiator for cross-linking.

10. A method according to claim 1, wherein a degree of functionalisation of the collagen is controllable and is variable between 5 mol % and 100 mol %.

11. A method according to claim 1, wherein a compressive modulus (E) of the material is between 50 kPa and 300 kPa.

12. A method according to claim 1, wherein a swelling ratio of the material is higher than at least 100 wt %.

13. A method according to claim 1, wherein a degradation time of the material is higher than at least 3 days and wherein a denaturation temperature of the material is between about 60 C. and 90 C.

14. A method according to claim 1, wherein the one or more ethylenically unsaturated moieties includes a combination of 4-vinylbenzyl chloride (4VBC) and glycidyl methacrylate (GMA).

15. A method according to claim 1, wherein the one or more ethylenically unsaturated moieties includes a combination of 4-vinylbenzyl chloride (4VBC) and methacrylic anhydride (MA).

16. A method according to claim 5, wherein the photoinitiator is [4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone] (Irgacure 2959).

Description

(1) The present invention will now be further described by way of example with reference to the accompanying figures in which:

(2) FIG. 1 shows compression modulus of collagen hydrogels;

(3) FIG. 2 shows an SEM picture of hydrogel CRT-GMA50 in hydrated conditions;

(4) FIG. 3 shows water uptake of collagen hydrogels;

(5) FIG. 4 shows a wide angle x-ray scattering spectra of dry collagen materials;

(6) FIG. 5 shows a L929 cell morphology picture following culture on exemplarily GMA-based hydrogel extract;

(7) FIG. 6 shows a L929 cell morphology picture following culture on cell culture medium;

(8) FIG. 7 shows a L929 cell morphology picture of vital cells cultured in contact with the exemplarily GMA-based hydrogel after 24 hours; and

(9) FIG. 8 shows an LDH assay of cells cultured on sample extracts of GMA-based hydrogel.

EXAMPLES

(10) Isolation of Type I Collagen

(11) Type I collagen was isolated from rat tail tendons. Frozen rat tails were thawed in ethanol for 20 minutes. Individual tendons were pulled from the tendon sheath, minced, and placed in 17.4 M acetic acid solution at 4 C. for 72 hours. After 72 hours, the mixture was centrifuged at 20, 000 rpm for 1 hour. The pellet was discarded and the crude collagen solution was neutralised with 0.1 M NaOH. The solution was stirred overnight (16-24 hours) at 4 C. before a further centrifugation step at 10,000 rpm for 45 minutes at 4 C. The supernatant was removed and an equal volume of 17.4 M acetic acid was added to re-solubilise the collagen pellet. The mixture was freeze-dried to obtain the collagen.

Example 1

(12) Synthesis of Glycidyl Methacrylate (GMA)-Functionalised Collagen (Collagen Based Composition)

(13) 0.25 wt % of the isolated type I collagen was stirred in 10 mM HCl solution at 25 C. for 4 hours until a clear solution was obtained. The solution was neutralised to pH 7.4 with NaOH]. Glycidyl methacrylate (GMA) was added in a 10, 25, 50, or 75 molar excess with respect to the collagen lysines along with an equimolar monomer amount of triethylamine (TEA). 1.0 wt % Tween-20 was added to increase monomer miscibility. The reaction was allowed to proceed for 24 hours at 25 C. After 24 hours, the mixture was precipitated in a 10-15 volume excess of ethanol and stirred for 48 hours at 25 C. Ethanol-precipitated functionalised collagen was recovered by centrifugation at 10,000 rpm for 60 minutes at 4 C. The pellet was air dried.

(14) Preparation of Photo-Crosslinked Glycidyl Methacrylate (GMA)-Functionalised Collagen Hydrogel (Collagen Based Polymeric Material)

(15) GMA-functionalised collagen was stirred in PBS solution containing 1 wt % Irgacure 2959 [4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone] (12959) at 4 C. for 48 hours. The PBS-Irgacure solution was prepared by stirring at 50 C. in the dark until a clear solution was obtained. The resulting solution was then poured into a Petri dish and incubated in a vacuum dessicator (25 C., up to 20 minutes) to remove air bubbles. Crosslinking was achieved by UV irradiation using a Spectroline UV lamp] at 365 nm and an intensity of 9 mW/cm.sup.2 for 20 minutes on each side of the Petri dish. The formed hydrogel was washed in distilled water and PBS to remove unreacted compounds for at least 48 hours.

Example 2

(16) Synthesis of 4-Vinylbenzyl Chloride (4VBC)-Functionalised Collagen (Collagen Based Composition)

(17) 4-vinylbenzyl chloride (4VBC)-functionalised collagen was prepared by the same method outlined for example 1.

(18) Preparation of Photo-Crosslinked 4-Vinylbenzyl Chloride (4VBC)-Functionalised Collagen Hydrogel (Collagen Based Polymeric Material)

(19) 4VBC-functionalised collagen was stirred in 10 mM HCl solution containing 1 wt % Irgacure 2959 [4-(2-hydroxyethoxy)phenyl-(2-hydroxy-2-propyl)ketone] (I2959) at 4 C. for 48 hours. The resulting solution was then poured into a Petri dish and incubated in a vacuum desiccator (25 C., up to 20 minutes) to remove air bubbles. Crosslinking was achieved by UV irradiation using a Spectroline UV lamp at 365 nm and an intensity of 9 mW/cm.sup.2 for 20 minutes on each side of the Petri dish. The formed hydrogel was washed in distilled water and PBS to remove unreacted compounds for at least 48 hours.

Example 3

(20) Synthesis of Methacrylic Anhydride (MA)-Functionalised Collagen

(21) Methacrylic anhydride (MA)-functionalised collagen was prepared by the same method outlined in Example 1.

(22) Preparation of Photo-Crosslinked Methacrylic Anhydride (MA)-Functionalised Collagen Hydrogel (Collagen Based Polymeric Material)

(23) The photo-crosslinked methacrylic anhydride (MA)-functionalised collagen hydrogel was prepared by the same method outlined in Example 1.

(24) Hydrogel Testing

(25) Resulting hydrogels were tested as for swelling behaviour and mechanical properties together with a commercially-available wound dressing product, Aquacel.

(26) Aquacel is a sodium carboxymethylcellulose-based wound dressing manufactured by Convatec. It is produced as a textile fibre and is available both as a ribbon for packing cavities, and as a flat non-woven pad for application to larger open wounds.

(27) Chemical Characterisation of Hydrogels

(28) Degree of functionalisation (F) of collagen lysines was determined by 2,4,6-trinitrobenzenesulfonic acid (TNBS) colorimetric assay. 11 mg of the dry sample was mixed with 1 mL of 4 wt % NaHCO.sub.3 pH 8.5 and 1 mL of 0.5 wt % TNBS solution at 40 C. under mild shaking. After 4 hours, 3 mL of 6 M HCl solution was added and the mixture was heated to 60 C. Samples were cooled and extracted 3 times with anhydrous ethyl ether to remove any unreacted TNBS. A reference sample was prepared in the same way except that the HCl solution was added before the addition of TNBS. The content of free amino groups and F were calculated as follows:

(29) $\frac{\mathrm{moles}\left(\mathrm{Lys}\right)}{g\left(\mathrm{collagen}\right)}=\frac{2\mathrm{Abs}\left(346\mathrm{nm}\right)0.02}{1.4{10}^{4}bx}$$\mathrm{and}F=1-\frac{{\mathrm{moles}\left(\mathrm{Lys}\right)}_{\mathrm{Funct}.Math.\mathrm{Collagen}}}{{\mathrm{moles}\left(\mathrm{Lys}\right)}_{\mathrm{Collagen}}}$
wherein Abs(346 nm) is the absorbance value at 346 nm, 1.410.sup.4 is the molar absorption coefficient for 2,4,6-trinitrophenyl lysine in L/mol cm.sup.1, b is the cell path length (1 cm), x is the sample weight in grams, and moles((Lys).sub.Funct.Collagen and moles((Lys).sub.Collagen represent the lysine molar content of functionalised and native collagen respectively.

(30) Collagen functionalisation was also investigated by .sup.1H-NMR. 10 mg of dry sample was dissolved in 1 mL deuterium oxide and the .sup.1H-NMR spectra was recorded on a Bruker Avance spectrophotometer (55 MHz).

(31) Raman spectra were also obtained to investigate the collagen functionalisation. A Renishaw microscope with 2 exciting lasers (HeNe at 663 nm, IR diode laser at 780 nm) was used to obtain Raman spectra of dry samples (up to 100 mg).

(32) Attenuated Total Reflectance Fourier-Transform Infrared (ATR FT-IR) was also carried out on dry samples (up to 100 mg) using a Perkin-Elmer Spectrum BX spotlight spectrophotometer with diamond ATR attachment. Scans were conducted from 4000 to 600 cm.sup.1 with 64 repetitions averaged for each spectrum. Resolution was 4 cm.sup.1 and interval scanning was 2 cm.sup.1.

(33) Collagen Conformation

(34) The conformation of crosslinked collagen was investigated using circular dichroism (CD). CD spectra were obtained with a Jasco J-715 spectropolarimeter. Samples had a concentration of 0.2 mg/mL in 10 mM HCl. CD spectra were acquired with a 2 nm band width and 20 nm/min scanning speed. Solutions were measured in a quartz cell with a pathlength of 1.0 mm. Temperature ramp measurements at a fixed wavelength of 221 nm were conducted between 20 and 60 C. with a 20 C./hour heating rate. The denaturation temperature (T.sub.d) was determined as the mid-point of thermal-transition.

(35) Protein conformation of photo-crosslinked collagen networks was investigated by Wide Angle X-ray Scattering (WAXS). WAXS measurements were carried out on dry samples with a Bruker D8 Discover (40 kV, 30 mA, 0.154 nm, X-ray wavelength A=0.154 nm). The detector was set at a distance of 15 cm covering 2 from 5 to 40. The collimator was 2.0 mm and the exposure time was 50 s per frame. WAXS measurements were coupled with Differential Scanning calorimetry (DSC) in order to investigate the thermal denaturation of collagen samples (TA Instruments Thermal Analysis 2000 System and 910 Differential Scanning calorimeter cell base). DSC temperature scans were conducted over a 10 to 200 C. temperature range at a 10 C./min heating rate. 10-15 mg samples were used for each measurement. The DSC cell was calibrated using indium with 20 C./min heating rate under 50 cm.sup.3/min nitrogen atmosphere.

(36) Scanning Electron Microscopy

(37) Sample morphological investigations were carried out via SEM and EDS (JEOL SM-35, cool stage) in fully hydrated hydrogels in order to identify any structural or morphological feature.

(38) Swelling Tests

(39) 2-5 mg of dry sample was placed in 1 mL distilled water/PBS at pH 7.4 and 37 C. under mild shaking (following method of Roger P. Brown, Handbook of Polymer Testing: Physical Methods). Upon equilibrium with water, water uptake (WU) was calculated according to the following equation:

(40) $\mathrm{WU}\left(\%\right)=\frac{W_s-W_d}{W_d}100$
wherein W.sub.s and W.sub.d are swollen and dry samples respectively. Swollen samples were blotted on blotting paper prior to measurement of W.sub.s.
Compression Tests

(41) Water-equilibrated hydrogel discs (0.8 cm) were compressed at room temperature with a compression rate of 3 mm.Math.min.sup.1 (Instron 5544 UTM, ASTM standards). A 500 N load cell was operated up to sample break. The maximal compressive stress (.sub.max) and compression at break (.sub.b) were recorded, so that the compressive modulus (E) was calculated by fitting the linear region of the stress-strain curve. Four replicas were employed for each composition and results expressed as averagestandard deviation.

(42) Extract Cytotoxicity Assays

(43) Cytotoxicity assays were conducted with L929 mouse fibroblasts and -sterilised photocrosslinked samples by the EN DIN ISO standard 10993-5. 0.1 mg of dry -sterilised sample was incubated in 1 mL cell culture medium (Dulbecco's Modified Eagle Medium) at 37 C. for 72 hours. Sample extract was collected and added to 80% confluent L929 cells. Dimethyl sulfoxide (DMSO) and virkon were used as negative controls. Double strength media was used as a positive control. Cell morphology was investigated after 48 hours cell culture using a transmitted light microscope in phase contrast mode (Zeiss, Germany).

(44) Quantitative analysis of cell viability was carried out via LDH assay.

(45) Properties of functionalised collagen and collagen based hydrogels were assessed and results are presented in Tables 1 and 2 which follow.

(46) TABLE-US-00001 TABLE 1 Chemical and Structural Properties of Functionalised Collagens. Samples are coded as: XXXX YY. Whereby XXXX identifies the type of system, either GMA- or 4VBC based, while YY indicates the molar ratio of monomer with respect to the molar content in non-functionalized collagen Denaturation Yield/ Free Lysine Total Vinyl Temperature Sample wt % Content/mol g.sup.1 Content/mol g.sup.1 (T.sub.d)/ C. 4VBC 10 251 43 42 4 35 4VBC 25 86 255 14 84 3 37 4VBC 50 83 246 13 99 1 4 VBC 75 240 49 132 49 30 GMA 10 293 83 79 83 37 GMA 25 85 175 21 96 22 39 GMA 50 81 172 16 154 14 37 GMA 75 145 1 228 1 35 MA 25 65 25 2

(47) TABLE-US-00002 TABLE 2 Thermo-Mechanical Properties of Collagen-Based Hydrogels. Samples are coded as CRT-XXXX YY, XXXX and YY have the same meaning as reported in table 1, while CRT is used here to identify the sample as collagen hydrogel instead of functionalized collagen. Shrinking Com- Com- Com- Temper- Equilibrium pressive pressive pression ature/ Water Modulus/ Stress at at Sample C. Uptake/wt % kPa Break/kPa Break/kPa Collagen 67 7 2863 404 CRT- 2770 123 247 103 35 19 41 6 4VBC 25 CRT- 2980 660 226 33 36 13 35 2 4VBC 50 CRT- 64 2 1956 316 GMA 10 CRT- 79 3 1230 179 136 57 28 6 73 3 GMA 25 CRT- 105 1 707 31 51 35 12 10 53 13 GMA 50

(48) Properties and characteristics of the functionalised collagens and collagen based hydrogels are further illustrated by the accompanying figures which also show a comparison with a commercially-available wound dressing product, Aquacel.

(49) FIG. 1 shows compression modulus of collagen hydrogels (CRT-4VBC25, CRT-4VBC50, CRT-GMA25, CRT-GMA50) and Aquacel, as commercially available wound dressing reference. FIG. 2 shows an SEM picture of hydrogel CRT-GMA50 in hydrated conditions. FIG. 3 shows water uptake of collagen hydrogels and Aquacel and FIG. 4 shows a wide angle x-ray scattering spectra of dry collagen materials. In FIG. 4 there is shown wide-angle x-ray scattering of in-house isolated collagen from rat-tail (gray), photo-crosslinked GMA-functionalised collagen (solid black) and photo-crosslinked 4VBC-functionalised collagen (dashed back).

(50) FIGS. 5, 6 and 7 show a L929 cell morphology picture following culture on exemplarily GMA-based hydrogel extract (FIG. 5) and cell culture medium (FIG. 6); vital cells cultured in contact with the hydrogel after 24 hours (FIG. 7). FIG. 8 shows LDH assay of cells cultured on sample extracts of GMA-based hydrogel.

(51) Attention is directed to all papers and documents which are filed concurrently with or previous to this specification in connection with this application and which are open to public inspection with this specification, and the contents of all such papers and documents are incorporated herein by reference.

(52) All of the features disclosed in this specification (including any accompanying claims, abstract and drawings), and/or all of the steps of any method or process so disclosed, may be combined in any combination, except combinations where at least some of such features and/or steps are mutually exclusive.

(53) Each feature disclosed in this specification (including any accompanying claims, abstract and drawings) may be replaced by alternative features serving the same, equivalent or similar purpose, unless expressly stated otherwise. Thus, unless expressly stated otherwise, each feature disclosed is one example only of a generic series of equivalent or similar features.

(54) The invention is not restricted to the details of the foregoing embodiment(s). The invention extends to any novel one, or any novel combination, of the features disclosed in this specification (including any accompanying claims, abstract and drawings), or to any novel one, or any novel combination, of the steps of any method or process so disclosed.