Patent ID: 12252589

The present invention is illustrated by the following examples.

EXAMPLES

1. Materials and Methods

1.1 Materials

D,L-lactide and L-lactide were purchased from Purac (Lyon, France). 8-arm Poly(ethylene glycol) (tripentaerythritol) (PEG8arm10k, Mw=10 000 g·mol-1) was purchased from JenKem Technology Co., Ltd (Beijing, China). Poloxamer (Pluronic@F127, Mw=12 600 g·mol-1), tin(II) 2-ethylhexanoate (Sn(Oct)2, 95%), dichloromethane (DCM), diethylether (Et2O), 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 MgSO4, stirred during 6 hours, then filtered and dried before use.

1.2 Characterization

FT-IR

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

TGA

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.·min−1.

SEC

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−1) (40° C. thermostatic analysis cells). Tetrahydrofuran (THF) was the mobile phase with 1 mL·min−1flow at 30° C. (column temperature). Polymer was dissolved in THF to reach 10 mg·mL−1concentration; afterwards, solution was filtered through a 0.45 μm Millipore filter before injection.Mnand Ð were expressed according to calibration using polystyrene standards.

NMR Spectra

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).

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.·min−1), then cooled to −50° C. (10° C.·min−1), before a second heating ramp to 120° C. (5° C.·min−1). Samples based on PEG8arm10k-PLA94were heated to 180° C. (10° C.·min−1), then cooled to −50° C. (10° C.·min-1), before a second heating ramp to 180° C. (5° C.·min−1). Glass transition temperature (Tg) was measured on the second heating ramp.

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

Triblock copolymer PLA50-Pluronic®-PLA50(prepolymer PLA50PLU), PEG8arm10k-PLA50(50% L-Lactic units and 50% D-Lactic units), PEG8arm10k-PLA94(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).

Pla50-Pluronic®-Pla50(Prepolymer)

For PLA50PLU, three molecular weights were targeted: 50 000, 100 000 and 200 000 g·mol−1, with the corresponding copolymers being noted as PLA50PLU50, PLA50PLU100 and PLA50PLU200, respectively.

For this, determined amounts ofD.L-lactide,L-lactide and Pluronic®F127 were introduced in a flask, to which Sn(Oct)2was then added (0.1 mol % with respect toD, 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 Et2O. The final triblock copolymer was dried under reduced pressure to constant mass.

1H NMR (300 MHz; CDCl3): δ (ppm)=5.1 (q, 1H, CO—CH—(CH3)—O), 3.6 (s, 4H, CH2—CH2—O), 3.5 (m, 2H, CH(CH3)—CH2—O), 3.4 (m, 1H, CH(CH3)—CH2—O), 1.5 (m, 3H, CO—CH(CH3)—O), 1.1 (m, 3H, CH(CH3)—CH2—O).

The copolymer molecular weight was determined using the equations (1) and (2) acknowledging a molecular mass of 72 g·mol−1for the lactic unit.
DPPLA=DPPEG*(I5.1PLA peak integration/I3.6PEG peak integration)  (1)
Mn=2*(DPPLA*72)+MnPluronic F127(2)
PEG8arm10k-PLA9420 000 g/mol (s-PLA-20), PEG8arm10k-PLA5025 000 g/mol (s-PLA-25), PEG8arm10k-PLA5050 000 g/mol (s-PLA-50) and PEG8arm10k-PLA50100 000 g/mol (s-PLA-100) (non functionalized)

For PEG8arm10k-PLA94an overall molecular weight of 20 000 g·mol−1was targeted.

For PEG8arm10k-PLA50an overall molecular weight of 25 000 g·mol−1or 50 000 g·mol−1or 100 000 g·mol−1was targeted.

For this, determined amounts ofD.L-lactide,L-lactide and PEG8arm10k were introduced in a flask, to which Sn(Oct)2was then added (0.1 mol % with respect toD,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 Et2O. The final star copolymer was dried under reduced pressure to constant mass. A low dispersity of 1.1 was determined by SEC analysis.

PEG8arm10k-PLA94:

1H NMR (300 MHz; CDCl3): δ (ppm)=5.1 (q, 1H, CO—CH—(CH3)—O), 4.3 (m, 2H, O—CH2—C—CH2—O), 3.6 (s, 4H, CH2—CH2—O), 3.3 (m, 2H, O—CH2—C—CH2—O), 1.5 (t, 3H, CO—CH—(CH3)—O).

The star copolymer molecular weight was determined using equations (1) and (3)
Mn=8*(DPPLA*72)+MnpEG8arm10k(3)

1.4 Synthesis of the aryl-azide-Functionalized PEG8arm10k-PLA94(s-PLA-fN3) (FIG.2a)

The 8-armed star copolymer PEG8arm10k-PLA94(Mntheo=20 kg·mol−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 Na2CO3then dried with MgSO4. The copolymer solution was precipitated in cold diethyl ether in the dark. The aryl-azide functionnal PEG8arm10k-PLA94(s-PLA-fN3) 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.

1H NMR (300 MHz; DMSO-d6): δ (ppm)=8.0 (d, 2H aromatic ring, CH═CH—C—N3), 7.3 (d, 2H aromatic ring, CH═CH—N3), 5.1 (q, 1H, CO—CH—(CH3)—O), 4.3 (m, 2H, O—CH2—C—CH2—O), 3.6 (s, 4H, CH2—CH2—O), 3.3 (m, 2H, O—CH2—C—CH2—O), 1.5 (t, 3H, CO—CH—(CH3)—O). (FIG.2b).

Experimental molecular weight calculated from the1H 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.

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−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).

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-fN3.

1.5 Synthesis of the methacrylate-Functionalized PEG8 arm10k-PLA5025 000 (S-PLA-25-MC), PEG8arm10k-PLA5050 000 (s-PLA-50-MC) and PEG8arm10k-PLA50100 000 (S-PLA-100-MC)

The 8-armed star copolymer PEG8arm10k-PLA50(Mntheo=25 kg·mol−1), PEG8arm10k-PLA50(Mntheo=50 kg·mol−1) or PEG8arm10k-PLA50(Mntheo=100 kg·mol−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 PEG8arm10k-PLA50(s-PLA-25-MC), PEG8arm10k-PLA50(s-PLA-50-MC) or PEG8arm10k-PLA50(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.

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

1H NMR (300 MHz; CDCl3) δ (ppm)=6.2 (d, 1H, CO—C(CH3)═CH2), 5.6 (d, 1H, CO—C(CH3)═CH2), 5.1 (q, 1H, CO—CH—(CH3)—O), 4.3 (m, 2H, C—CH2—O), 3.6 (s, 4H, CH2—CH2—O), 3.3 (O—CH2—C—CH2—O), 2.0 (s, 3H, CO—C(CH3)═CH2), 1.5 (t, 3H, CO—CH—(CH)—O).

1.6 Synthesis of the acrylate-Functionalized PEG8arm10k-PLA50(s-PLA-A)

The 8-armed star copolymer PEG8arm10k-PLA50(Mntheo=25 kg·mol−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 PEG8arm10k-PLA50(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

Films by Solvent Evaporation

For elastomers crosslinked with 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone (BA), PLA50PLU copolymers with defined molecular weights were stirred in DCM with 2,6-bis(4-azidobenzylidene)-4-methylcyclohexanone (BA) (2-5 wt % of the polymer).

For elastomers crosslinked with s-PLA-fN3, PLA50PLU copolymers with defined molecular weights were mixed with s-PLA-fN3at different weight ratios (10, 25 and 50 wt %) and stirred in DCM.

For control, the same protocol was followed by replacing s-PLA-fN3by the non-functional s-PLA.

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)

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.

Films by Press

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.

Microfibers-Based Tissues by Electrospinning Process

Electrospun Polymer Solutions

Polymer blends PLA50PLU and s-PLA-fN3or 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.

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.

Electrospinning Process

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.

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.

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·cm−2and 19 W·cm−2. The ACCU-CAL 50-LED radiometer is used to measure the UV dose received by the samples.

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

Photo-Crosslinking of Films

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·cm−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.

Photo-Crosslinking of Fibrous Scaffold

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·cm−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

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.

Shaping Process

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.

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.

Gel fraction (=Crosslinking yield)

Gel⁢fraction⁢(%)=(Weight⁢of⁢insoluble⁢cross-linked⁢parts/Weight⁢of⁢initial⁢sample)*100(4)

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.

1.8 Mechanical Properties

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 (σy, MPa), strain at yield (εy,%), stress at break (σbreak, MPa) strain at break (εbreak,%) were expressed as the mean value of the three measurement.

1.9 Degradation Study of Fibrous Materials

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)

Water uptake was determined from equation (6)
Water uptake(%)=((Ww−Wi)/Wi)*100  (6)

1.10 Degradation Study of Films

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:

Remaining⁢mass⁢(%)=(1-((Wi-Wd)/Wi))*100(5)Water⁢uptake⁢(%)=((Ww-Wi)/Wi)*100(6)Remaining⁢chemical⁢bridges⁢(%)=(1-(gel⁢fraction(i)-gel⁢fraction⁢(m))/gel⁢fraction⁢(i))*100(7)

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

gel⁢fraction(%)=(WcdWi)*100(8)

1.11 Cytotoxicity Assay

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 15.103cells 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.

The percentage of cytotoxicity were calculated from equation (7)
Cytotoxicity(%)=(((sample LDH activity)−LDH−)/(LDH+“−”LDH−))*100  (9)

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

In order to prepare degradable elastomeric biomaterials starting from non-functional polyesters, we first focused on the triblock PLA50-Pluronic®-PLA50(PLA50PLU). Targeted and experimental molecular weights (50 000, 100 000 and 200 000 g·mol−1) were in agreements based on1H 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

The three different triblock copolymers PLA50PLU50, PLA50PLU100 and PLA50PLU200 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−1, which is characteristic of the azide group (FIG.4).

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 PLA50PLU 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).

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

Influence of the PLA50PLU Prepolymer Molecular Weight and the Content of s-PLA-fN3 on the Crosslinking Efficiency

To evaluate the potential of s-PLA-fN3 for the preparation of degradable elastomeric biomaterials, we first focused on the influence of the PLA50PLU 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 PLA50PLU(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 inFIG.5.

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 PLA50PLU did not show any significant impact on the crosslinking efficiency. For a defined weight ratio of PLA50PLU (50-200)/s-PLA-fN3 gel fractions were similar whatever the PLA50PLU molecular weight. At the temperature of UV crosslinking, chain mobility is higher for PLA50PLU50 compared to PLA50PLU200 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 PLA50PLU50 compared to PLA50PLU100 or PLA50PLU200. 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.

Kinetics of the Photo-Crosslinking

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 PLA50PLU/s-PLA-fN3 blends, which confirmed that aryl-azide photo-crosslinking is a very fast process, whatever the molecular weight of the PLA50PLU copolymer.

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

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).

It is to note that the concentration of aryl-azide groups was higher in PLA50PLU (50-200)-BA5 mixtures (5 wt % of BA, 11 μmol) than in all PLA50PLU/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.

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 PLA50PLU 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

Based on the results obtained on films PLA50PLU(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 PLA50PLU copolymer does not influence the outcome, this next study was limited to PLA50PLU200 that proved to be easily electrospun. The same ratios of PLA50-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 andFIG.6for 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.

Fibers Morphology

Fibers morphology was analyzed by SEM an typical images are shown inFIG.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 PLA50-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.

In-Situ Photo-Crosslinking Evaluation

In order to determine optimal UV-curing time to obtain an elastic micro-fibers scaffold, crosslinking study was conducted. Fibrous scaffolds based on PLA50PLU200/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.

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, PLA50-PLU200/s-PLA-fN3 mechanical behaviors were evaluated under dry and hydrated state at 37° C. (Table 1).

TABLE 1Elastic microfibers scaffolds (FS) mechanical properties in the dry and hydrated state at37° C. Young's modulus (E), ultimate stress (σbreak), ultimate strain (εbreak), andelastic limit (εy). (Data are expressed as means ± SD and correspond to measurements with n = 3).Dry stateHydrated stateFibrousEεyσbreakεbreakEεyσbreakεbreakscaffolds blends(MPa)(%)(MPa)(%)(MPa)(%)(MPa)(%)PLA50PLU200/0.7 ± 0.112 ± 10.6 ± 0.1174 ± 2615.9 ± 1.55 ± 11.7 ± 0.2117 ± 14S-PLA-fN3 90/10PLA50PLU200/0.3 ± 0.1182 ± 41.4 ± 0.3333 ± 6810.6 ± 1.03 ± 01.4 ± 0.1176 ± 16S-PLA-fN3 75/25PLA50PLU200/0.2 ± 0.0115 ± 100.6 ± 0.0257 ± 326.6 ± 2.33 ± 00.9 ± 0.289 ± 21S-PLA-fN3 50/50PLA50PLU200/11.6 ± 2.53 ± 11.0 ± 0.1120 ± 1318.4 ± 4.53 ± 11.7 ± 0.4101 ± 14S-PLA 90/10PLA50PLU200/29.3 ± 1.31 ± 02.1 ± 0.3171 ± 2834.2 ± 14.61.7 ± 11.9 ± 0.397 ± 6S-PLA 75/25PLA50PLU200/2.4 ± 0.97 ± 21.2 ± 0.2146 ± 115.6 ± 0.33 ± 10.7 ± 0.093 ± 9S-PLA 50/50

In a dry state at 37° C., non UV-cured fibrous scaffolds based on PLA50PLU200/s-PLAthe 75/25 ratio had the lower deformability with a high Young modulus (E=29.3 MPa) and a low elastic limit (εy=1.3%). The 50/50 ratio on the opposite was the most deformable material (E=2.4 MPa and εy=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 PLA50PLU200/s-PLA-fN3 showed higher elastic properties (E=0.22-0.68 MPa and εy=12-182%) than non UV-cured fibrous scaffolds (E=2.44-29.3 MPa and εy=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 PLA50PLU200/s-PLA-fN3 75/25 and 50/50 showed higher elastic properties (E=0.22-0.34 MPa; εy=115-182%) than 90/10 (E=0.68 MPa; εy=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 PLA50PLU200/s-PLA-fN3 75/25. It may be explained by the combination of efficient crosslinking and good balance between long and short polymer chains.

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.

Degradation

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 PLA50-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 inFIG.10b. The absence of erosion upon degradation for the FS PLA50-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.

Cytocompatibility Study

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.

Only extracts from positive control films (C+) gave around 45-50% of cytotoxicity on L929 cells. Results (summarized onFIG.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-fN3as Photo-Crosslinker

In order to highlight the broad applicability of the proposed strategy and the versatility of the multi(aryl-azide) s-PLA-fN3as a crosslinker, non-functional polymers with high molecular weight were selected among various families including polyesters (PLA50), 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 2Influence of the nature of the polymer on the crosslinking efficiency evaluated by gelfraction analyses (s-PLA-fN3used as the crosslinker, 20 μm thick films, mercury bulb, 5minutes UV-irradiation per side). (Data are expressed as means ±SD and correspond tomeasurements with n = 3).Molecular weight% wt ofn(N3) in thePolymers(g · mol−1)s-PLA-fN3film (μmol)Gel fraction (%)PLA50PLU200 00050854 ± 4PLA50200 00050853 ± 5PEO300 00050873 ± 2PMMA350 00050845 ± 3

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

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.

Gel Fractions

The gel fractions, calculated according to equation (4), of the crosslinked elastomers films are summarized in Table 3 (see alsoFIGS.12and13).

TABLE 3Gel fraction of the crosslinked elastomer filmsElastomer filmGel fraction (%)s-PLA-MC (by press)78 ± 4s-PLA-MC (by solvent evaporation)92 ± 1
Degradation

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

The remaining mass of non-functional block copolymer s-PLA decreased and reached 80% after 1 month of hydrolytic degradation (FIG.14a)—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.

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

2.6 Micro-Fibers-Based Tissues by Electrospinning Process

2.6.1 Micro-Fibers-Based Tissues Using Aryl-Azide Star-Shaped s-PLA-fN3 as Photo-Crosslinker and PLA50-PLU200

UV Curing Step

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 PLA50-PLU/s-PLA-fN375/25 and from 22% to 77% for the fibrous scaffold PLA50-PLU/s-PLA-fN350/50 (seeFIG.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.

Mechanical Properties

From the mechanical study, only the fibrous scaffolds based PLA50-PLU/PEGs8-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 (seeFIG.16).

Both the photo-crosslinked FS PLA50-PLU200/PEGs8-PLA-fN3showed 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

Fibers Morphology

The fibrous scaffolds based on s-PLA-MC (100 000 g·mol1) had micrometer fibers (2.8±0.3 μm) that is suitable for tissue engineering applications (seeFIG.17).

2.7 3D Materials by Stereolithography

Different materials were obtained from stereolithography process using s-PLA-50-MC polymer and are summarized inFIGS.18to20. 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 inFIG.20, 3D material at millimeter scale could be obtained with multi(methacrylate) block copolymer s-PLA-50-MC.