Branched-Blocked Copolymer Photo-Crosslinker Functionalized with Photoreactive Groups and Its Use for Shaping Degradable Photo-Crosslinked Elastomers Suitable for Medical and Tissue-Engineering Applications

Abstract

The present invention relates to novel degradable branched-blockcopolymers, comprising a star-shaped copolymer central core or a linear copolymer central core, functionalized with photoreactive groups chosen among aryl-azide, (meth)acrylate or thiol groups. The present invention also relates to the use of these degradable branched-block copolymers as photo-crosslinkers to provide degradable photo-crosslinked elastomers as biomaterials suitable for medical and tissue engineering applications. A method for preparing a degradable photo-crosslinked polymer, preferably a degradable photo-crosslinked elastomer, starting from the branched-block copolymer of the invention via a shaping process and an irradiation step is also provided.

Claims

1. A degradable branched-block copolymer comprising a polyether central core having n arms and degradable polymer chains extending from each arm of the polyether central core, the polyether central core being a star core or a linear core, each degradable polymer chain being constituted by l monomer unit(s) of a degradable polymer, wherein each degradable polymer chain is identical and functionalized at its extremity by a photoreactive group chosen among an aryl-azide derivative, a (meth)acrylate group or a thiol group, said degradable branched-block copolymer being illustrated by the following schema: ##STR00024## wherein - - - is the monomer unit of the degradable polymer constituting the degradable polymer chain, G is the photoreactive group, n is an integer between 4 and 32, and ##STR00025##  is ##STR00026## wherein ##STR00027##  is the star polyether central core, and is the monomer unit corresponding to the polyether core, and m is comprised between 4 and 400 and l is comprised between 4 and 1500, or ##STR00028##  is ##STR00029## wherein is the monomer unit which forms the linear polyether central core, and R is a multivalent branched functional group comprising a number n/2 of terminal functions or atoms selected among oxygen atom or NH group, each of this terminal function being linked to one polymer chain and m is comprised between 4 and 600 units and l is comprised between 2 and 400.

2. The degradable branched-block copolymer according to claim 1, wherein the polyether of the central core is chosen among polyethylene glycol (PEG), poloxamer or poloxamine.

3. The degradable branched-block copolymer according to claim 1, wherein the polyether core is a linear core and n is an integer between 4 and 16.

4. The degradable branched-block copolymer according to claim 1, wherein the polyether core is a star central core, the resulting star copolymer being illustrated by the following schema: ##STR00030## wherein is the monomer unit corresponding to the polyether core, m being comprised between 4 and 400, - - - is the monomer unit of the degradable polymer, l being comprised between 4 and 1500, G is the photoreactive group, and n is an integer of at least 4.

5. The degradable branched-block copolymer according to claim 4, wherein the polyether star central core has 4, 6 or 8 arms.

6. The degradable branched-block copolymer according to claim 1, wherein the degradable polymer of the degradable polymer chains is selected from the group constituted by a polyester, a polycarbonate, and mixtures thereof.

7. The degradable branched-block copolymer according to claim 6, wherein the polyester is selected from the group consisting of poly(lactide) (PLA), poly(ε-caprolactone) (PCL), polyhydroxybutyrate (PHB), polyhydroxybutyrate-co-hydroxyvalerate (PHBV), polyglycolic acid (PGA), poly(3-hydroxyvalerate), polydioxanone and mixtures thereof.

8. The degradable branched-block copolymer according to claim 1 wherein the photo-reactive group is an aryl-azide derivative.

9. The degradable branched-block copolymer according to claim 1 wherein the photo-reactive group is a (meth)acrylate or a thiol group, preferably a (meth)acrylate group.

10. The degradable branched-block copolymer according to claim 9, functionalized with a (meth)acrylate or a thiol group, wherein the ratio m/l is strictly superior to 0 and lower than or equal to 3 when the polyether core is a star core, and the ratio m/(n*l) is strictly superior to 0 and lower than or equal to 1 when the polyether core is a linear core.

11. A method for photo-crosslinking a polymer, comprising using the degradable branched-block polymer as defined in claim 1 as a photo-crosslinker agent.

12. The method according to claim 11, wherein a prepolymer is photo-crosslinked with the degradable branched-block copolymer of claim 1.

13. The method according to claim 12, wherein the degradable branched-block copolymer is functionalized with an aryl-azide derivative and the prepolymer is a non-functionalized prepolymer comprising CH-bonds.

14. The method according to claim 12, wherein the degradable branched-block copolymer is functionalized with a (meth)acrylate or a thiol group and the prepolymer has a molecular weight lower than 50 000 g/mol and is functionalized with a photo-crosslinkable group.

15. The method according to claim 11 for preparing a degradable polymeric biomaterial suitable for medical and soft engineering applications or in medical reconstruction system.

16. Method for preparing a degradable photo-crosslinked polymer, said method comprising the steps of: (a) preparing a solution or a solid blend comprising the degradable branched-block copolymer as defined in 1, and optionally a prepolymer, (b) performing a shaping process on the solution or the solid blend resulting from step (a) to provide a shaped object, (c) irradiating the shaped object resulting from step (b) under light, (d) recovering the degradable photo-crosslinked polymer.

17. The method according to claim 16, wherein the shaping process is chosen among extrusion, film coating, film spraying, film casting, electrospray, electrospinning, or 3D printing technologies.

18. The method according to claim 16, wherein step (b) and step (c) are achieved simultaneously.

19. The degradable blanched-block copolymer according to claim 1, wherein the polyether core is a linear core and n is equal to 4, 8 or 16.

20. The method according to claim 11 for preparing an elastomeric biomaterial, suitable for catheters, drains, fixation devices, dressings, films, patch, implant or scaffolds.

21. The method according to claim 16, wherein the degradable photo-crosslinked polymer is a degradable crosslinked elastomer in the form of a film, threads, fibers, tubes, mesh or mats.

Description

DESCRIPTION OF THE DRAWINGS

[0145] FIG. 1: Scheme of the design of elastic micro-fibrous scaffold based on the multifunctional aryl-azide star block copolymer photo-crosslinker of the present invention.

[0146] FIG. 2: (a) Synthetic scheme of the degradable copolymer photo-crosslinker PEG.sub.8arm10k-PLA.sub.94-fN3 (s-PLA-fN3); (b) .sup.1H NMR spectra of PEG.sub.8arm10k-PLA.sub.94-fN3 (s-PLA-fN.sub.3); (c) SEC analysis of (1) PEG.sub.8arm10k-PLA.sub.94 and SEC analysis of (2) PEG.sub.8arm10k-PLA.sub.94-fN3. UV detector (270 nm.sup.−1).

[0147] FIG. 3: Influence of the type of UV-bulb (MB vs. MHB) used to generate the elastomers evaluated by the gel fraction analysis.

[0148] FIG. 4: FTIR analysis of scaffolds before and after UV irradiation at different time with two different UV-bulb: (a) Metal Halide Bulb and (b) Mercury Bulb. The photo-activation of aromatic bis(aryl-azide) generating nitrene was showed by the loss of the characteristic azide IR band located at 2110 cm-1.

[0149] FIG. 5: Crosslinking kinetics of the elastomers PLA.sub.50PLU(50-200)/s-PLA-fN3 evaluated by gel fraction. (a) PLA.sub.50PLU50/s-PLA-fN3; (b) PLA.sub.50PLU100/s-PLA-fN3; (c) PLA.sub.50PLU200/s-PLA-fN3; (d) Crosslinking kinetics of the elastic microfibers scaffolds PLA.sub.50PLU (50-200)/s-PLA-fN3 evaluated by gel fraction. (Data are expressed as means±SD and correspond to measurements with n=3).

[0150] FIG. 6: Gel fraction as a function of the nature of the aryl-azide photo-crosslinker (s-PLA-fN3 at 10, 25 and 50 wt % vs. BA at 5 wt %) and the overall content of aryl-azide groups in the blend (n(N3)) (10 minutes irradiation time).

[0151] FIG. 7: SEM images, microfiber diameter distributions of the different scaffolds based on PLA.sub.50-PLU200/s-PLA and PLA.sub.50-PLU200/s-PLA-fN3 (before and after 2 min of UV curing). Scale bar of SEM is 30 μm. (a) PLA.sub.50-PLU200/s-PLA 90/10; (b) PLA.sub.50-PLU200/s-PLA-fN3 90/10 uncured; (c) PLA.sub.50-PLU200/s-PLA-fN3 90/10 UV-cured; (d) PLA.sub.50-PLU200/s-PLA 75/25; (e) PLA.sub.50-PLU200/s-PLA-fN3 75/25 uncured; (f) PLA.sub.50-PLU200/s-PLA-fN3 75/25 UV-cured; (g) PLA.sub.50-PLU200/s-PLA 50/50; (h) PLA.sub.50-PLU200/s-PLA-fN3 50/50 uncured; (i) PLA.sub.50-PLU200/s-PLA-fN3 50/50 UV-cured.

[0152] FIG. 8: Mechanical tensile properties of UV-cured fibrous scaffolds based on PLA.sub.50-PLU200/s-PLA-fN3 in hydrated and dry state at 37° C.

[0153] FIG. 9: Water uptake of fibrous scaffolds based on PLA.sub.50-PLU200/s-PLA or PLA.sub.50-PLU200/s-PLA-fN3 at different ratios.

[0154] FIG. 10: Evaluation of scaffold degradation (a) remaining mass during degradation time for fibrous scaffolds in PBS at 37° C. Data correspond to measurement in triplicate; (b) SEM images of PLA.sub.50-PLU200/s-PLA or PLA.sub.50-PLU200/s-PLA-fN3 50/50 over degradation time in PBS at 37° C., magnifications ×5000. (Data are expressed as means±SD and correspond to measurements with n=3).

[0155] FIG. 11: Cytotoxicity assessed on L929 cells after treatment with extracts of fibrous scaffolds based on PLA.sub.50-PLU200/s-PLA or PLA.sub.50-PLU200/s-PLA-fN3 at different ratios for 24 h. (Data are expressed as means±SD and correspond to measurements with n=9 per condition).

[0156] FIG. 12: Stress-strain curves for films based on PEG(s-PLA-MC Mn=50 000 g.Math.mol-1 (a) films prepared by hot melt press after UV irradiation, analyses carried out at 37° C., at 10, 50 and 100% strain (10 cycles); (b) comparison of films prepared by hot melt press after UV irradiation versus without UV irradiation, analyses carried out at 37° C., at 100% strain (10 cycles).

[0157] FIG. 13: Stress-strain curves (at 37° C.) for elastomers based on s-PLA-MC Mn=50 000 g.Math.mol-1 in the presence or absence of photo-initiator (PI, in this case 2,2 dimethoxy-2-phenylacetophenone).

[0158] FIG. 14: Degradation of s-PLA-50-MC-based films by solvent evaporation with respect of a) remaining weight, b) remaining crosslinking and c) water uptake.

[0159] FIG. 15: Gel fraction (%) of fibrous scaffold based on PLA.sub.50-PLU200/s-PLA-fN.sub.3 75/25 (left) or PLA.sub.50-PLU200/s-PLA-fN.sub.3 50/50 (right) with in-process-UV-curing or post-process-UV-curing.

[0160] FIG. 16: Cyclic tensile tests for a 15% elongation of fibrous scaffold based on (a) PLA-Pluronic-PLA/s-PLA-fN.sub.3 75/25 and (b) PLA-Pluronic-PLA/s-PLA-fN.sub.3 50/50, both obtained after post-treatment UV curing. The analyses are carried out at a temperature of 37° C. over 10 cycles.

[0161] FIG. 17: SEM image of a fibrous scaffold based on s-PLA-100-MC (100 000 g.Math.mol-1).

[0162] FIG. 18: Photo of porous materials obtained by 3D printing from s-PLA-50-MC (50 kg.Math.mol-1).

[0163] FIG. 19: Photo of materials obtained by 3D printing from s-PLA-50-MC (50 kg.Math.mol-1).

[0164] FIG. 20: Photo of cube obtained by 3D printing from s-PLA-50-MC (50 kg.Math.mol-1).

[0165] The present invention is illustrated by the following examples.

EXAMPLES

1. Materials and Methods

1.1 Materials

[0166] D,L-lactide and L-lactide were purchased from Purac (Lyon, France). 8-arm Poly(ethylene glycol) (tripentaerythritol) (PEG.sub.8arm10k, Mw=10 000 g.Math.mol-1) was purchased from JenKem Technology Co., Ltd (Beijing, China). Poloxamer (Pluronic@F127, Mw=12 600 g.Math.mol-1), tin(II) 2-ethylhexanoate (Sn(Oct).sub.2, 95%), dichloromethane (DCM), diethylether (Et.sub.2O), N,N-dicyclohexyl-carbodiimide (DCC), 4-(dimethylamin)pyridine (DMAP) and N,N-dimethylformamide (DMF), tetrahydrofuran (THF) were purchased from Sigma-Aldrich (St Quentin Fallavier, France). 2,6-Bis(4-azidobenzylidene)-4-methylcyclohexanone (BA) and 4-azidobenzoic acid were bought from TCl (Paris, Europe). All chemicals were used without further purification with exception of DCM and DCC. DCM was dried over calcium hybrid and freshly distillated before use. DCC was solubilized in anhydrous DCM with MgSO.sub.4, stirred during 6 hours, then filtered and dried before use.

1.2 Characterization

[0167] FT-IR

[0168] FT-IR spectra of polymer films were recorded with a Perkin Elmer Spectrum 100 spectrometer.

[0169] TGA

[0170] TGA analyses were recorded under nitrogen atmosphere with a Perkin Elmer TGA 6. Sample are maintained at 30° C. for 1 minute and then, heated to 300° C. at a rate of 10° C..Math.min.sup.−1.

[0171] SEC

[0172] Average molecular weights (Mn) and dispersities (Ð) were determined by size exclusion chromatography (SEC) Shimadzu using two mixed medium columns PLgel 5 μm MIXED-C (300×7.8 mm), Shimadzu RI detector 20-A and Shimadzu UV detector SPD-20A (370 nm.sup.−1) (40° C. thermostatic analysis cells). Tetrahydrofuran (THF) was the mobile phase with 1 mL.Math.min.sup.−1 flow at 30° C. (column temperature). Polymer was dissolved in THF to reach 10 mg.Math.mL.sup.−1 concentration; afterwards, solution was filtered through a 0.45 μm Millipore filter before injection. Mn and Ð were expressed according to calibration using polystyrene standards.

[0173] NMR Spectra

[0174] .sup.1H NMR spectra were recorded from an AMX Brucker spectrometer operating at 300 MHz at room temperature. The solvent used was deutered chloroform and DMSO-d6. The chemical shift was expressed in ppm with respect to tetramethylsilane (TMS).

[0175] Thermal properties of the polymers were analysed by differential scanning calorimetry (DSC) from a Perkin Elmer Instrument DSC 6000 Thermal Analyzer characterized of the different polymers. It was carried out under nitrogen. Samples were heated to 100° C. (10° C..Math.min.sup.−1), then cooled to −50° C. (10° C..Math.min.sup.−1), before a second heating ramp to 120° C. (5° C..Math.min.sup.−1). Samples based on PEG.sub.8arm10k-PLA.sub.94 were heated to 180° C. (10° C..Math.min.sup.−1), then cooled to −50° C. (10° C..Math.min-1), before a second heating ramp to 180° C. (5° C..Math.min.sup.−1). Glass transition temperature (Tg) was measured on the second heating ramp.

[0176] Morphology of the samples was examined with a Hitachi S4800 Scanning electron microscope (Technology platform of IEM Laboratory of the Balard Chemistry pole) with an acceleration voltage of 2 kV and at magnifications ×500, ×1000 and ×5000 times with 3 images at each magnification.

1.3 Synthesis of Copolymers

[0177] Triblock copolymer PLA.sub.50-Pluronic®-PLA.sub.50 (prepolymer PLA.sub.50PLU), PEG.sub.8arm10k-PLA.sub.50 (50% L-Lactic units and 50% D-Lactic units), PEG.sub.8arm10k-PLA.sub.94 (94% L-Lactic units and 6% D-Lactic units) (star copolymer non functionalized, s-PLA) were synthesized by ring-opening polymerization (ROP) as described in a previous work of the inventors (Leroy, A. et al, Mater. Sci. Eng. C. 33 (2013) 4133-4139).

[0178] Pla.sub.50-Pluronic®-Pla.sub.50 (Prepolymer)

[0179] For PLA.sub.50PLU, three molecular weights were targeted: 50 000, 100 000 and 200 000 g.Math.mol.sup.−1, with the corresponding copolymers being noted as PLA.sub.50PLU50, PLA.sub.50PLU100 and PLA.sub.50PLU200, respectively.

[0180] For this, determined amounts of D.L-lactide, L-lactide and Pluronic®F127 were introduced in a flask, to which Sn(Oct).sub.2 was then added (0.1 mol % with respect to .sub.D, L-lactide units). Argon-vacuum cycles were applied before sealing the flask under vacuum. ROP was carried out in an oven at 130° C. for 5 days under constant stirring. Afterwards, the mixture was dissolved in DCM and precipitated in cold Et.sub.2O. The final triblock copolymer was dried under reduced pressure to constant mass.

[0181] .sup.1H NMR (300 MHz; CDCl.sub.3): δ (ppm)=5.1 (q, 1H, CO—CH—(CH.sub.3)—O), 3.6 (s, 4H, CH.sub.2—CH.sub.2—O), 3.5 (m, 2H, CH(CH.sub.3)—CH.sub.2—O), 3.4 (m, 1H, CH(CH.sub.3)—CH.sub.2—O), 1.5 (m, 3H, CO—CH(CH.sub.3)—O), 1.1 (m, 3H, CH(CH.sub.3)—CH.sub.2—O).

[0182] The copolymer molecular weight was determined using the equations (1) and (2) acknowledging a molecular mass of 72 g.Math.mol.sup.−1 for the lactic unit.

DP.sub.PLA=DP.sub.PEG*(I5.1PLA peak integration/I3.6PEG peak integration)  (1)

Mn=2*(DP.sub.PLA*72)+Mn.sub.Pluronic F127  (2)

PEG.sub.8arm10k-PLA.sub.94 20 000 g/mol (s-PLA-20), PEG.sub.8arm10k-PLA.sub.50 25 000 g/mol (s-PLA-25), PEG.sub.8arm10k-PLA.sub.50 50 000 g/mol (s-PLA-50) and PEG.sub.8arm10k-PLA.sub.50 100 000 g/mol (s-PLA-100) (non functionalized)

[0183] For PEG.sub.8arm10k-PLA.sub.94 an overall molecular weight of 20 000 g.Math.mol.sup.−1 was targeted.

[0184] For PEG.sub.8arm10k-PLA.sub.50 an overall molecular weight of 25 000 g.Math.mol.sup.−1 or 50 000 g.Math.mol.sup.−1 or 100 000 g.Math.mol.sup.−1 was targeted.

[0185] For this, determined amounts of D.L-lactide, L-lactide and PEG.sub.8arm10k were introduced in a flask, to which Sn(Oct).sub.2 was then added (0.1 mol % with respect to .sub.D,L-lactide units). Argon-vacuum cycles were applied before sealing the flask under vacuum. ROP was carried out in an oven at 130° C. for 5 days under constant stirring. Afterwards, the mixture was dissolved in DCM and precipitated in cold Et.sub.2O. The final star copolymer was dried under reduced pressure to constant mass. A low dispersity of 1.1 was determined by SEC analysis.

[0186] PEG.sub.8arm 10k-PLA.sub.94:

[0187] .sup.1H NMR (300 MHz; CDCl.sub.3): δ (ppm)=5.1 (q, 1H, CO—CH—(CH.sub.3)—O), 4.3 (m, 2H, O—CH.sub.2—C—CH.sub.2—O), 3.6 (s, 4H, CH.sub.2—CH.sub.2—O), 3.3 (m, 2H, O—CH.sub.2—C—CH.sub.2—O), 1.5 (t, 3H, CO—CH—(CH.sub.3)—O).

[0188] The star copolymer molecular weight was determined using equations (1) and (3)

Mn=8*(DP.sub.PLA*72)+Mn.sub.pEG8arm10k  (3)

1.4 Synthesis of the aryl-azide-Functionalized PEG.SUB.8arm.10k-PLA.SUB.94 .(s-PLA-fN.SUB.3.) (FIG. 2a)

[0189] The 8-armed star copolymer PEG.sub.8arm10k-PLA.sub.94 (Mn.sub.theo=20 kg.Math.mol.sup.−1) was solubilized in freshly distilled DCM (20% w/v). Determined amounts of 4-azido benzoic acid (2.5 eq./OH group), DCC (2.5 eq./OH group) and DMAP (2.5 eq./OH group) were added. The mixture was heated at 45° C. for 6 days under stirring in the dark. The reaction medium was filtered and washed (three times) by an aqueous solution of Na.sub.2CO.sub.3 then dried with MgSO.sub.4. The copolymer solution was precipitated in cold diethyl ether in the dark. The aryl-azide functionnal PEG.sub.8arm10k-PLA.sub.94 (s-PLA-fN.sub.3) was dried under reduced pressure to constant mass. The yield of functionalization was determined by comparing the integration of the aryl-azide characteristic signal at 8.0 and the integration of proton resonance at 4.2 ppm.

[0190] .sup.1H NMR (300 MHz; DMSO-d6): δ (ppm)=8.0 (d, 2H aromatic ring, CH═CH—C—N.sub.3), 7.3 (d, 2H aromatic ring, CH═CH—N.sub.3), 5.1 (q, 1H, CO—CH—(CH.sub.3)—O), 4.3 (m, 2H, O—CH.sub.2—C—CH.sub.2—O), 3.6 (s, 4H, CH.sub.2—CH.sub.2—O), 3.3 (m, 2H, O—CH.sub.2—C—CH.sub.2—O), 1.5 (t, 3H, CO—CH—(CH.sub.3)—O). (FIG. 2b).

[0191] Experimental molecular weight calculated from the .sup.1H NMR spectra (Mn=18 600 g/mol) and dispersity of Ð=1.1 determined by SEC analysis showed that no degradation of the s-PLA copolymer occurred during the synthesis.

[0192] The grafting of 4-azidobenzoic acid onto s-PLA chain-ends was further confirmed by SEC analyses. After functionalization, a UV signal characteristic of aryl-azide groups (270 nm.sup.−1) was visible at a retention time corresponding to the refractive index signal of the star copolymer (FIG. 2-c-2). This was not the case for the starting s-PLA copolymer (FIG. 2-c-1).

[0193] These results confirmed the successful chain-end functionalization of s-PLA with aryl-azide moieties, yielding the expected multi(aryl-azide) macromolecular photo-crosslinker s-PLA-fN.sub.3.

1.5 Synthesis of the methacrylate-Functionalized PEG.SUB.8 arm.10k-PLA.SUB.50 .25 000 (S-PLA-25-MC), PEG.SUB.8arm.10k-PLA.SUB.50 .50 000 (s-PLA-50-MC) and PEG.SUB.8arm.10k-PLA.SUB.50 .100 000 (S-PLA-100-MC)

[0194] The 8-armed star copolymer PEG.sub.8arm10k-PLA.sub.50 (Mn.sub.theo=25 kg.Math.mol.sup.−1), PEG.sub.8arm10k-PLA.sub.50 (Mn.sub.theo=50 kg.Math.mol.sup.−1) or PEG.sub.8arm10k-PLA.sub.50 (Mn.sub.theo=100 kg.Math.mol.sup.−1) was solubilized in freshly distilled DCM (20% w/v). Triethylamine (5 eq./OH group) was added and the resulted mixture was cold to 0° C. Methacryloyl chloride (5 eq./OH group) was added with a casting ampoule, under stirring at 0° C. Once the addition is completed, the mixture was stirred at room temperature for 72 h in dark. Then, the product was filtered and then precipitated in cold diethyl ether. The methacrylate-functionalized PEG.sub.8arm10k-PLA.sub.50 (s-PLA-25-MC), PEG.sub.8arm10k-PLA.sub.50 (s-PLA-50-MC) or PEG.sub.8arm10k-PLA.sub.50 (s-PLA-100-MC) was solubilized in DCM and washed with basic aqueous phase, in the dark. The organic layer was concentrated under vacuum pressure to afford a concentrated solution which was precipitated in cold diethyl ether. The recovered product was then dried under reduced pressure.

[0195] The yield of functionalization was determined by NMR (95% of functionalization) s-PLA-25-MC:

[0196] .sup.1H NMR (300 MHz; CDCl.sub.3) δ (ppm)=6.2 (d, 1H, CO—C(CH.sub.3)═CH.sub.2), 5.6 (d, 1H, CO—C(CH.sub.3)═CH.sub.2), 5.1 (q, 1H, CO—CH—(CH.sub.3)—O), 4.3 (m, 2H, C—CH.sub.2—O), 3.6 (s, 4H, CH.sub.2—CH.sub.2—O), 3.3 (O—CH.sub.2—C—CH.sub.2—O), 2.0 (s, 3H, CO—C(CH.sub.3)═CH.sub.2), 1.5 (t, 3H, CO—CH—(CH)—O).

1.6 Synthesis of the acrylate-Functionalized PEG.SUB.8arm.10k-PLA.SUB.50 .(s-PLA-A)

[0197] The 8-armed star copolymer PEG.sub.8arm10k-PLA.sub.50 (Mn.sub.theo=25 kg.Math.mol.sup.−1) was solubilized in freshly distilled DCM (20% w/v). Triethylamine (15 eq./OH group) was added and the resulted mixture was cold to 0° C. Acryloyl chloride (15 eq./OH group) was added with a casting ampoule, under stirring at 0° C. Once the addition is completed, the mixture was heated at 45° C. for 72 h in dark. Then, the product was filtered and then precipitated in cold diethyl ether. The acrylate-functionalized PEG.sub.8arm10k-PLA.sub.50 (s-PLA-A) was solubilized in DCM and washed with basic aqueous phase, in the dark. The organic layer was concentrated under vacuum pressure, in dark at room temperature, to afford a concentrated solution which was precipitated in cold diethyl ether. The recovered product was then dried under reduced pressure.

1.7 Shaping of the Polymers and Photo-Crosslinking

[0198] Films by Solvent Evaporation

[0199] For elastomers crosslinked with 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone (BA), PLA.sub.50PLU copolymers with defined molecular weights were stirred in DCM with 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone (BA) (2-5 wt % of the polymer).

[0200] For elastomers crosslinked with s-PLA-fN.sub.3, PLA.sub.50PLU copolymers with defined molecular weights were mixed with s-PLA-fN.sub.3 at different weight ratios (10, 25 and 50 wt %) and stirred in DCM.

[0201] For control, the same protocol was followed by replacing s-PLA-fN.sub.3 by the non-functional s-PLA.

[0202] For elastomer obtained starting from s-PLA-50-MC only (without other prepolymer), s-PLA-50-MC was dissolved and stirred in DCM. Photo-initiator 2,2-dimethoxy-2-phenylacetophenone (PI) can be added (at 2 wt % of the copolymer)

[0203] Solutions were dried out in an aluminum mold to obtain thin films. Films were stored in a dark place for 24 h. The resulting films were further dried under vacuum for 24 h.

[0204] Films by Press

[0205] The press was heated at 155° C. Then, the copolymer s-PLA-50-MC is in powder form is deposited on Teflon paper and heated to 155° C. and a pressure of 5-6 bar is applied for 10 minutes. After this step, the film of a few micrometers is placed in the freezer for 5 minutes.

[0206] Microfibers-Based Tissues by Electrospinning Process

[0207] Electrospun Polymer Solutions

[0208] Polymer blends PLA.sub.50PLU and s-PLA-fN.sub.3 or s-PLA (90/10, 75/25 and 50/50 w/w noted 90/10, 75/25 an 50/50 in the rest of the text, respectively) were dissolved in DCM/DMF (50/50 v/v)[40]. Blend concentrations were chosen to produce fibers without beads (90/10: 14 wt %, 75/25: 18 wt %, 50/50: 22 wt %). All mixtures were mechanically stirred at room temperature overnight, until total dissolution.

[0209] The copolymer, s-PLA-50-MC or s-PLA-100MC was dissolved in a DCM/DMF solution (70/30 v/v) at a concentration of 35% by weight for s-PLA-100-MC and 40% by weight fors-PLA-50-MC. The polymer solution was mechanically mixed at room temperature overnight until it is completely dissolved.

[0210] Electrospinning Process

[0211] Electrospinning process was carried out with a horizontal syringe pump device. A high voltage power supply was set at 12-15 kV. Polymer solutions filled a 10 mL syringe with a 21-gauge needle (inner diameter 0.82 mm). Feed rate (1.8 mL/h for s-PLA-50-MC and s-PLA-100-MC and 2.1 mL/h the others polymers) was controlled with the syringe pump (Fresenius Vial Program 2 IEC). The collector was a square aluminum foil and located 15 cm from the needle tip. Experiments were performed at room temperature. The fibrous scaffold was collected after 40 minutes of electrospinning. It was dried overnight before further experiments.

[0212] A step of UV curing of the fibers is optionally achieved during the electrospinning process using UV LEDs. The UV curing is performed throughout electrospinning process.

[0213] The LEDs (365 and 385 nm) from the DYMAX QX4 controller are located at a distance of 8 cm from the collector. The LEDs have an intensity between 14 W.Math.cm.sup.−2 and 19 W.Math.cm.sup.−2. The ACCU-CAL 50-LED radiometer is used to measure the UV dose received by the samples.

[0214] Said step of UV curing of the fibers can also be achieved after the electrospinning process, also for a time of 2 min.

[0215] Photo-Crosslinking of Films

[0216] Films were irradiated under UV light (mercury or metal halide bulb) under inert atmosphere for different times (1 min<t<20 min) with a Dymax PC-2000 system (75 mW.Math.cm.sup.−2). For sake of clarity, in the rest of the text a 10 minutes irradiation time corresponds to 5 minutes of irradiation per side of the film. The distance measured between the bulb and samples was 13.5 cm. Intensity of radiation doses was evaluated using ACCU-CAL™ 50 system. Later, elastomer films were cut, weighed and put in DCM (10 mL). After three washes, the insoluble crosslinked parts were removed from DCM and dried under vacuum during 24 h. Finally, samples were weighed to determine the gel fraction according to equation (4) below.

[0217] Photo-Crosslinking of Fibrous Scaffold

[0218] To guaranty low temperature inside the enclosure and maintain the morphology of the fibers, fibrous scaffolds were irradiated under UV light (mercury bulb) and inert atmosphere for 2 seconds at a frequency of 0.5 Hz. The sequential flashes were applied for determined periods using a Dymax PC-2000 system (75 mW.Math.cm.sup.−2). The distance, the intensity of irradiation and the gel fraction were measured using the protocol described for films.

3D Materials by Stereolithography

Synthesis of Copolymer Solution

[0219] The copolymer s-PLA-50-MC was dissolved in ethyl lactate at a concentration of 400 g/L. The photoinitiator Omnirad RPO-L was added to this solution at a concentration of 2% by weight. The resulting mixture was then mechanically stirred for 24 h.

[0220] Shaping Process

[0221] The desired structure is modeled by the OnShape software, then printed using the Phrozen Shuffle 3D printer. The polymer solution is irradiated layer by layer (2 min for 50 μm) using 405 nm (50 Watts) LEDs.

[0222] At the end of the printing process, the object undergoes a post-curing step using a FormLab-Form Cure: wavelength 405 nm, irradiation on both sides 5 min, at 45° C.

[0223] Gel fraction (=Crosslinking yield)

[00001]$\begin{array}{cc}\mathrm{Gel}\mathrm{fraction}\left(\%\right)=\left(\mathrm{Weight}\mathrm{of}\mathrm{insoluble}\mathrm{cross}-\mathrm{linked}\mathrm{parts}/\mathrm{Weight}\mathrm{of}\mathrm{initial}\mathrm{sample}\right)*100& \left(4\right)\end{array}$

[0224] The gel fraction percentage value allows to evaluate the efficiency of the tested photo-crosslinker. The higher the gel fraction value, the more effective the photo-crosslinker is.

[0225] 1.8 Mechanical Properties

[0226] Tensile mechanical tests were carried out on micro-fibers scaffold samples. Samples were cut (30×10 mm) and thickness was measured with a micrometer. Scaffolds were analyzed in triplicate at 37° C. (dry and hydrated state) with an Instron 3344 with a deformation rate of 10 mm/min. Young modulus (E, MPa), stress at yield (σ.sub.y, MPa), strain at yield (ε.sub.y,%), stress at break (σ.sub.break, MPa) strain at break (ε.sub.break,%) were expressed as the mean value of the three measurement.

1.9 Degradation Study of Fibrous Materials

[0227] Fibrous tests samples were cut (10×10 mm), weighed (Wi=initial weight) and placed in 5 mL of phosphate buffered saline (PBS) (pH 7.4) at a constant temperature (37° C.) under stirring. At different time points, fibrous materials were removed from PBS, weighed (Ww=weight of the wet samples), then dried to constant mass (Wx=weight dry after x time in PBS). The remaining mass of the samples was calculated from equation (5).

Remaining mass(%)=(1−((Wi−Wx)/Wi))*100  (5)

[0228] Water uptake was determined from equation (6)

Water uptake(%)=((Ww−Wi)/Wi)*100  (6)

1.10 Degradation Study of Films

[0229] The degradation of s-PLA-50-MC-based films was studied for one month. The films (L=2 mm and l=0.5 mm) were weighed (Wi=Initial mass) then introduced into a PBS solution (pH=7.4) and agitated at 37° C. At different times (3, 8, 15, 15, 22 and 30 days), the films are recovered, weighed (Ww=Wet Mass) and dried for 24 hours. The films are then weighed again (Wd=Dry mass) and introduced into a DCM solution. After three washes, the samples are dried overnight and weighed (Wcd=cross-linked dry mass). Thus, during this degradation, the conservation of the mass of the material, the absorption of water and the conservation of chemical bridges are evaluated according to the following respective equations:

[00002]$\begin{array}{cc}\mathrm{Remaining}\mathrm{mass}\left(\%\right)=\left(1-\left(\left(\mathrm{Wi}-\mathrm{Wd}\right)/\mathrm{Wi}\right)\right)*100& \left(5\right)\end{array}$$\begin{array}{cc}\mathrm{Water}\mathrm{uptake}\left(\%\right)=\left(\left(\mathrm{Ww}-\mathrm{Wi}\right)/\mathrm{Wi}\right)*100& \left(6\right)\end{array}$$\begin{array}{cc}\mathrm{Remaining}\mathrm{chemical}\mathrm{bridges}\left(\%\right)=\left(1-\left(\mathrm{gel}\mathrm{fraction}\left(i\right)-\mathrm{gel}\mathrm{fraction}\left(m\right)\right)/\mathrm{gel}\mathrm{fraction}\left(i\right)\right)*100& \left(7\right)\end{array}$

[0230] where gel fraction (i) is the initial gel fraction and gel fraction (m) is the fraction at different times. As a reminder,

[00003]$\begin{array}{cc}\mathrm{gel}\mathrm{fraction}\left(\%\right)=\left(\frac{\mathrm{Wcd}}{\mathrm{Wi}}\right)*100& \left(8\right)\end{array}$

1.11 Cytotoxicity Assay

[0231] Cells and control polymer films were chosen in accordance with ISO 10993-5 guidelines. Mouse fibroblasts L929 cells (ECACC 85011425) were maintained in DMEM high glucose supplemented with 5% Fetal Bovine Serum (FBS), 2 mM L-glutamine and 1% penicillin/streptomycin and cultured at 37° C. and 5% CO2. Cells were tested to be free of mycoplasms. Negative (RM-C High Density Polyethylene noted C−) and positive (RM-B 0.25% Zinc DiButyldithioCarbamate (ZDBC) polyurethane noted C+) control films were purchased from Hatano Research Institute (Ochiai 729-5, Hadanoshi, Kanagawa 257, Japan). Cytotoxicity was assessed on extracts. First, extractions were carried out at 0.1 g per mL for 72 h at 37° C. under sterile conditions on complete growth medium following ISO 10993-12 recommendations. L929 cells were seeded at 15.10.sup.3 cells per well in a 96-well plate and allowed to attach overnight. The culture medium was then removed and discarded from the cultures and an aliquot of the fibers extract was added into each well. Aliquots of the blank, negative and positive controls were added into additional replicate wells (n=9). After 24 h incubation under appropriate atmosphere, extract's cytotoxicity was assessed by Lactate Dehydrogenase (LDH) assay (Pierce), according to the manufacturer's instruction. Briefly, medium from well was transferred to a new plate and mixed with LDH Reaction Mixture. After 30 minutes of incubation at room temperature, absorbances at 490 nm and 680 nm were measured using a CLARIOstar@ microplate-reader (BMG LABTECH's) to determine LDH activity.

[0232] The percentage of cytotoxicity were calculated from equation (7)

Cytotoxicity(%)=(((sample LDH activity)−LDH−)/(LDH+“−”LDH−))*100  (9)

[0233] Where “LDH−” represents Spontaneous LDH Release Control (water-treated) and “LDH+” Maximum LDH Release Control activity obtained after cell lysis.

2. Results and Discussion

2.1 Evaluation of bis(aryl-azide) from the Prior Art as Photo-Crosslinker

[0234] In order to prepare degradable elastomeric biomaterials starting from non-functional polyesters, we first focused on the triblock PLA.sub.50-Pluronic®-PLA.sub.50 (PLA.sub.50PLU). Targeted and experimental molecular weights (50 000, 100 000 and 200 000 g.Math.mol.sup.−1) were in agreements based on .sup.1H NMR spectra. Dispersities between 1.5 and 1.8 were determined by SEC analysis, which is in agreement with values classically obtained for the ROP of high molecular weight polyesters.

These Copolymers were Further Used to Evaluate the Real Potential of 2,6-bis(azidobenzylidene)-4-methylcyclohexanone (BA) from the Prior Art as Photo-Crosslinker

[0235] The three different triblock copolymers PLA.sub.50PLU50, PLA.sub.50PLU100 and PLA.sub.50PLU200 were mixed with BA, at different concentration of BA (2 wt % and 5 wt %). Gel fractions results (FIG. 3) showed low crosslinking efficiency using BA as photo-crosslinker (gel fraction <15%) despite a proven activation of aryl-azide. This was evidenced through the disappearance of the band at 2100 nm.sup.−1, which is characteristic of the azide group (FIG. 4).

[0236] This lack of crosslinking despite aryl-azide photoactivation was attributed to the formation of azo-dimers and termination reactions that do not allow crosslinking. Furthermore, molecular weight of the prepolymer PLA.sub.50PLU copolymer did not influence significantly the crosslinking efficiency compared to nature of the UV-bulb used (metal halide bulb versus mercury bulb) and BA concentration. As expected, gel fraction increased with mercury bulb and higher BA concentration (5% wt).

[0237] Taking into account these results, we hypothesized that the limited functionality of BA (2 aryl-azide groups) associated to the direct proximity of the reactive groups on this small organic molecule could explain the poor outcome of BA-based crosslinking.

2.2 Degradable Elastomers Photo-Crosslinked by s-PLA-fN3 Shaped as Films

[0238] Influence of the PLA.sub.50PLU Prepolymer Molecular Weight and the Content of s-PLA-fN3 on the Crosslinking Efficiency

[0239] To evaluate the potential of s-PLA-fN3 for the preparation of degradable elastomeric biomaterials, we first focused on the influence of the PLA.sub.50PLU molecular weight and the content of s-PLA-fN3 on the crosslinking efficiency. Based on the study carried out on bis(aryl-azide) photo-crosslinker, films having a thickness of 20 μm were prepared from PLA.sub.50PLU(50-200)/s-PLA-fN3 blends at various compositions (90/10, 75/25 and 50/50 w/w) prior to irradiation under UV-light for period 10 minutes (5 minutes for each side). Results are summarized in FIG. 5.

[0240] As expected, the initial content of s-PLA-fN3 in the mixture had a strong influence on the crosslinking efficiency with gel fractions around 15%, 35% and 55% when s-PLA-fN3 ratios varied from 10 wt %, 25 wt % and to 50 wt %, respectively. On the opposite, the molecular weight of the PLA.sub.50PLU did not show any significant impact on the crosslinking efficiency. For a defined weight ratio of PLA.sub.50PLU (50-200)/s-PLA-fN3 gel fractions were similar whatever the PLA.sub.50PLU molecular weight. At the temperature of UV crosslinking, chain mobility is higher for PLA.sub.50PLU50 compared to PLA.sub.50PLU200 but this higher mobility does not seem to significantly impact the crosslinking efficiency. Only at a 50/50 ratios, a slightly lower gel fraction was obtained for the PLA.sub.50PLU50 compared to PLA.sub.50PLU100 or PLA.sub.50PLU200. This result might be due to a lower chain entanglement combined with higher chain mobility that partly prevent reaction between the active nitrene species and the polymeric chains.

[0241] Kinetics of the Photo-Crosslinking

[0242] Kinetics of the photo-crosslinking were then followed over a 10 minutes period of time (FIG. 5). After 2 minutes of UV-irradiation, the maximum gel fraction was already reached for most PLA.sub.50PLU/s-PLA-fN3 blends, which confirmed that aryl-azide photo-crosslinking is a very fast process, whatever the molecular weight of the PLA.sub.50PLU copolymer.

[0243] Comparison of the BA (Prior Art) and s-PLA-fN3 Efficiencies as Photo-Crosslinkers

[0244] Finally, the crosslinking efficiency of molecular bis(aryl-azide) photo-crosslinker BA and macromolecular multi(aryl-azide) photo-crosslinker s-PLA-fN3 with respect to the overall aryl-azide groups concentration in the blends were compared (FIG. 6).

[0245] It is to note that the concentration of aryl-azide groups was higher in PLA.sub.50PLU (50-200)-BA5 mixtures (5 wt % of BA, 11 μmol) than in all PLA.sub.50PLU/s-PLA-fN3 blends even when the highest concentration of s-PLA-fN3 (50 wt %, 8 μmol) was used. However, gel fractions obtained were higher with macromolecular 8-branched star photo-crosslinker than BA, even for the lowest content of s-PLA-fN3 (10 wt %, ca. 2 μmol), which corresponds to 5.5 times less photo-reactive moieties compared to 5 wt % of BA.

[0246] As expected, with 8 aryl-azide groups present on the s-PLA-fN3 star macromolecular photo-crosslinker, active nitrene species have more probability to be in contact with the PLA.sub.50PLU polymeric chain and to act as a crosslinking agent than the bi-functional BA. Moreover, reducing the mobility of the cross-linking agent due to its macromolecular nature and expected chains entanglement may also explain this enhanced efficiency of crosslinking.

2.3 Micro-Scale Scaffolds Using Aryl-Azide Star-Shaped s-PLA-fN3 as Photo-Crosslinker

[0247] Based on the results obtained on films PLA.sub.50PLU(50-200) that demonstrated a high potential of s-PLA-fN3 as photo-crosslinker, the next step was to evaluate the transferability of this approach into the electrospinning process to produce elastomeric and degradable scaffolds based on photo-crosslinked fibers. Having shown that the molecular weight of the PLA.sub.50PLU copolymer does not influence the outcome, this next study was limited to PLA.sub.50PLU200 that proved to be easily electrospun. The same ratios of PLA.sub.50-PLU200/s-PLA-fN3 (90/10, 75/25 and 50/50) were produced as described in the experimental section. Resulting scaffolds had a thickness of nearly 250 μm. To guaranty low temperature inside the enclosure (see experimental section and FIG. 6 for more details) and maintain the morphology of the fibers, fibrous scaffolds were irradiated under UV light (mercury bulb) and inert atmosphere for 2 seconds at a frequency of 0.5 Hz. Various parameters have been investigated and are discussed in the following sections.

[0248] Fibers Morphology

[0249] Fibers morphology was analyzed by SEM an typical images are shown in FIG. 7. For a defined ratio, no difference in fiber diameter distribution was noticed between fibers based on s-PLA or s-PLA-fN3 even after UV-curing. In brief, all fiber diameters were in the range of 1 to 2 μm. The lowest fibers diameter (1.2 μm) was obtained with PLA.sub.50-PLU200/s-PLA-fN3 90/10 blends and increased with the content of s-PLA-fN3 with fiber diameters of 1.65-1.98 μm and 1.74-2.13 μm for 75/25 and 50/50 blends, respectively. However, fiber distribution was more heterogeneous for the latter. It might be due to a non-total solvent evaporation that cause flatten fibers leading to interconnected fibers.

[0250] In-Situ Photo-Crosslinking Evaluation

[0251] In order to determine optimal UV-curing time to obtain an elastic micro-fibers scaffold, crosslinking study was conducted. Fibrous scaffolds based on PLA.sub.50PLU200/s-PLA-fN3 under UV light (mercury bulb) and inert atmosphere for 2 seconds at a frequency of 0.5 Hz. After 2 minutes of UV-irradiation, the gel fraction obtained was maximal (20-25%) (FIG. 5-d). This irradiation time was therefore selected for the rest of the studies. Values of gel fraction were lower for fibrous scaffolds (20-25%) than that of 20 μm films (15-65%). This may be due to both the thickness, ca. 250 μm, and opaque nature of the highly porous scaffolds, which may restrict UV penetration to few microns at the surface of the scaffolds. Considering UV barrier properties of aryl-azide compounds combined with s-PLA-fN3 polymer crystallinity, UV light might photo-cured fibers only on surface (few micrometers). Hence, no significant difference between fibrous scaffolds regardless of s-PLA-fN3 concentrations was noticed.

[0252] Mechanical Properties A major challenge in the field of synthetic resorbable materials, dedicated to soft tissue reconstruction, is to ensure the mechanical properties preservation of the biomaterial/host tissues complex over degradation and healing processes. Therefore, PLA.sub.50-PLU200/s-PLA-fN3 mechanical behaviors were evaluated under dry and hydrated state at 37° C. (Table 1).

TABLE-US-00001 TABLE 1 Elastic microfibers scaffolds (FS) mechanical properties in the dry and hydrated state at 37° C. Young's modulus (E), ultimate stress (σ.sub.break), ultimate strain (ε.sub.break), and elastic limit (ε.sub.y). (Data are expressed as means ± SD and correspond to measurements with n = 3). Dry state Hydrated state Fibrous E ε.sub.y σ.sub.break ε.sub.break E ε.sub.y σ.sub.break ε.sub.break scaffolds blends (MPa) (%) (MPa) (%) (MPa) (%) (MPa) (%) PLA50PLU200/ 0.7 ± 0.1 12 ± 1  0.6 ± 0.1 174 ± 26 15.9 ± 1.5  5 ± 1 1.7 ± 0.2 117 ± 14 S-PLA-fN3 90/10 PLA50PLU200/ 0.3 ± 0.1 182 ± 4  1.4 ± 0.3 333 ± 68 10.6 ± 1.0  3 ± 0 1.4 ± 0.1 176 ± 16 S-PLA-fN3 75/25 PLA50PLU200/ 0.2 ± 0.0 115 ± 10  0.6 ± 0.0 257 ± 32 6.6 ± 2.3 3 ± 0 0.9 ± 0.2  89 ± 21 S-PLA-fN3 50/50 PLA50PLU200/ 11.6 ± 2.5  3 ± 1 1.0 ± 0.1 120 ± 13 18.4 ± 4.5  3 ± 1 1.7 ± 0.4 101 ± 14 S-PLA 90/10 PLA50PLU200/ 29.3 ± 1.3  1 ± 0 2.1 ± 0.3 171 ± 28 34.2 ± 14.6 1.7 ± 1.sup.  1.9 ± 0.3 97 ± 6 S-PLA 75/25 PLA50PLU200/ 2.4 ± 0.9 7 ± 2 1.2 ± 0.2 146 ± 11 5.6 ± 0.3 3 ± 1 0.7 ± 0.0 93 ± 9 S-PLA 50/50

[0253] In a dry state at 37° C., non UV-cured fibrous scaffolds based on PLA.sub.50PLU200/s-PLAthe 75/25 ratio had the lower deformability with a high Young modulus (E=29.3 MPa) and a low elastic limit (ε.sub.y=1.3%). The 50/50 ratio on the opposite was the most deformable material (E=2.4 MPa and ε.sub.y=7.3%). Fiber diameters in the observed range (ca. 1-2 μm) did not influence mechanical properties. On the other hand, in a dry state at 37° C., UV-cured fibrous scaffolds based on PLA.sub.50PLU200/s-PLA-fN3 showed higher elastic properties (E=0.22-0.68 MPa and ε.sub.y=12-182%) than non UV-cured fibrous scaffolds (E=2.44-29.3 MPa and ε.sub.y=1.3-7.3%). A remarkable increase of elastic limit was therefore obtained thanks to the fibers crosslinking with quasi-linear stress-strain curves (FIG. 7). It yielded scaffolds with lower ultimate stress (0.58-1.38 MPa for crosslinked FS vs. 1.01-2.01 MPa for the non-crosslinked) and much higher ultimate strain (174-333% vs. 120-146%). As expected, FS PLA.sub.50PLU200/s-PLA-fN3 75/25 and 50/50 showed higher elastic properties (E=0.22-0.34 MPa; ε.sub.y=115-182%) than 90/10 (E=0.68 MPa; ε.sub.y=12%) confirming the interest of using the star-shaped macromolecular s-PLA-fN3 photo-crosslinker. It is to note that for similar crosslinking efficiencies (FIG. 5-d), highest elasticity and ultimate stress were reached with FS PLA.sub.50PLU200/s-PLA-fN3 75/25. It may be explained by the combination of efficient crosslinking and good balance between long and short polymer chains.

[0254] In the hydrated state at 37° C., Young's modulus and ultimate stress of fibrous scaffolds were always higher than in dry state, whereas elastic limit and ultimate strain were lower than in dry state (FIG. 8). This may appear counterintuitive considering the well-known plasticizing effect of water However, these results could be assigned to microphase separation phenomena that have recently been reported in literature for PLA-b-PEG-b-PLA copolymers. In more details, PEG blocks (more flexible, lower transition temperature) have an initial role of plasticizer for the blend, but PEG segments a prone to migration upon water uptake, which results microphase separation and stiffening. In our case, due to the core-shell structure of the crosslinked fibers (crosslinked shell, uncrosslinked core see degradation), this phenomenon may overshadow the impact of the crosslinking in the hydrated state.

[0255] Degradation

[0256] Scaffolds degradation was followed over 1 month (FIG. 10-a). As expected, non-crosslinked fibrous scaffolds showed a faster degradation (remaining mass from 65% to 85%) compared to their crosslinked counterparts (remaining mass from 90% to 95%). Only FS with high content of PLA.sub.50-PLU200 (90/10) exhibited similar degradation profiles with almost no degradation over 1 month (weight loss <2%). It was also observed that the higher star copolymer (s-PLA and s-PLA-fN3) content, the faster the weight loss. This is due to the hydrophilic segments of PEG that favor water uptake (FIG. 9) of FS (150-300%), which promotes their hydrolytic degradation. Interestingly, degradation profiles of all crosslinked fibers were quasi-linear as expected for chemically crosslinked elastomers. Another difference between crosslinked and non-crosslinked fibers was the additional erosion observed for the latter. This phenomenon is illustrated by the SEM pictures presented in FIG. 10b. The absence of erosion upon degradation for the FS PLA.sub.50-PLU200/s-PLA-fN3 50/50 despite weight loss (10% after 1 month) partly confirms the core-shell structure. In fact, crosslinked networks are known to maintain their 3D shape over degradation, which is observed here. While non- or less-crosslinked core chains degrade, their diffusion through the crosslinked shell is impeded, which results in a slower weight loss. Thus, UV-curing of the electropsun fibrous scaffolds allows one to modulate the degradation profile and may be useful to fit the properties of the scaffolds in the frame of soft-tissue engineering applications.

[0257] Cytocompatibility Study

[0258] Finally, following the mechanical and degradation studies of the fibrous scaffolds, one last mandatory step to validate their potential for use with cells is the validation of their cytocompatibility. The different copolymers PLA, PluronicF127 and PEG have already been approved by FDA. However, residual unreacted s-PLA-fN3 inside fibers may leach out from the fibers. For this reason, the cytotoxicity of the scaffolds was assessed on extracts following ISO 10993-12 recommendations. The extracts from scaffolds, C− and C+ were added on L929 fibroblasts seeded into wells and cytotoxicity was evaluated over a 24 hours period.

[0259] Only extracts from positive control films (C+) gave around 45-50% of cytotoxicity on L929 cells. Results (summarized on FIG. 11) show the absence of cytotoxicity of the extracts in contact with L929 cells even with extracts from the scaffolds containing the highest s-PLA-fN3 concentration (50/50). Thus, this preliminary assay confirmed the potential of the proposed degradable elastomeric biomaterials for cell-contacting applications, whose cytocompatibility will be further investigated in future dedicated work.

2.4 Versatility of s-PLA-fN.SUB.3 .as Photo-Crosslinker

[0260] In order to highlight the broad applicability of the proposed strategy and the versatility of the multi(aryl-azide) s-PLA-fN.sub.3 as a crosslinker, non-functional polymers with high molecular weight were selected among various families including polyesters (PLA.sub.50), polyethers (PEO) and poly(methacrylate)s (PMMA). Gel fractions in the range 45 to 70% (Table 2) confirmed that crosslinking can be obtained whatever the polymer nature and despite high molecular weights.

TABLE-US-00002 TABLE 2 Influence of the nature of the polymer on the crosslinking efficiency evaluated by gel fraction analyses (s-PLA-fN.sub.3 used as the crosslinker, 20 μm thick films, mercury bulb, 5 minutes UV-irradiation per side). (Data are expressed as means ±SD and correspond to measurements with n = 3). Molecular weight % wt of n(N.sub.3) in the Polymers (g .Math. mol.sup.−1) s-PLA-fN.sub.3 film (μmol) Gel fraction (%) PLA.sub.50PLU 200 000 50 8 54 ± 4 PLA.sub.50 200 000 50 8 53 ± 5 PEO 300 000 50 8 73 ± 2 PMMA 350 000 50 8 45 ± 3

2.5 Degradable Elastomers Photo-Crosslinked by s-PLA-MA Shaped as Films

[0261] The methacrylate-functionalized star copolymer s-PLA-MA was shaped as films using press or by means of solvent evaporation according to the methods described above. The films were then irradiated with UV light as described in point 1.7.

[0262] Gel Fractions

[0263] The gel fractions, calculated according to equation (4), of the crosslinked elastomers films are summarized in Table 3 (see also FIGS. 12 and 13).

TABLE-US-00003 TABLE 3 Gel fraction of the crosslinked elastomer films Elastomer film Gel fraction (%) s-PLA-MC (by press) 78 ± 4 s-PLA-MC (by solvent evaporation) 92 ± 1

[0264] Degradation

[0265] The degradation of s-PLA-50-MC (50 000 g/mol) based films made by solvent evaporation is illustrated on FIG. 14.

[0266] The remaining mass of non-functional block copolymer s-PLA decreased and reached 80% after 1 month of hydrolytic degradation (FIG. 14 a)—dotted line). On the contrary, no degradation occurred for s-PLA-50-MC after 1 month in terms of remaining weight and crosslinking (FIG. 14.a) (full line)—b)). Thus, the degradation process was slowed down by introducing covalent bonds inside polymer matrix.

[0267] Moreover, s-PLA-50-MC showed partial water uptake (80-85%) and its material structure was preserved in water (FIG. 14 c)).

2.6 Micro-Fibers-Based Tissues by Electrospinning Process

[0268] 2.6.1 Micro-Fibers-Based Tissues Using Aryl-Azide Star-Shaped s-PLA-fN3 as Photo-Crosslinker and PLA.sub.50-PLU200

[0269] UV Curing Step

[0270] In process-UV-curing allowed an increase of the gel fraction of the fibrous scaffold compared to post process-UV curing from 23% to 52% for the fibrous scaffold PLA.sub.50-PLU/s-PLA-fN.sub.3 75/25 and from 22% to 77% for the fibrous scaffold PLA.sub.50-PLU/s-PLA-fN.sub.3 50/50 (see FIG. 15). The UV-curing of the fibrous scaffolds in thickness prevented the UV barrier of the aryl azide reactive groups allowing higher covalent bonds formation inside fibrous scaffolds.

[0271] Mechanical Properties

[0272] From the mechanical study, only the fibrous scaffolds based PLA.sub.50-PLU/PEG.sub.s8-PLA-fN3 with the ratios 75/25 and 50/50 exhibited rubber-like behavior. Thus, the ability of those elastomeric fibrous scaffolds to deform reversibly without loss of energy has been investigated through the cyclic stress-strain curves (see FIG. 16).

[0273] Both the photo-crosslinked FS PLA.sub.50-PLU200/PEG.sub.s8-PLA-fN.sub.3 showed mechanical conservation over cyclic loads under 15% of deformation for both fibrous scaffolds.

2.6.2 Micro-Fibers-Based Tissues Using s-PLA-MC 100

[0274] Fibers Morphology

[0275] The fibrous scaffolds based on s-PLA-MC (100 000 g.Math.mol.sup.1) had micrometer fibers (2.8±0.3 μm) that is suitable for tissue engineering applications (see FIG. 17).

2.7 3D Materials by Stereolithography

[0276] Different materials were obtained from stereolithography process using s-PLA-50-MC polymer and are summarized in FIGS. 18 to 20. From our study, we were able to produce materials with various porous diameters (d=1 mm—FIG. 18|d=4 mm and d=7 mm—FIG. 19). As shown in FIG. 20, 3D material at millimeter scale could be obtained with multi(methacrylate) block copolymer s-PLA-50-MC.