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4D polycarbonates via stereolithography as scaffolds for soft tissue repair
Methods . Instrumentation . All starting reagents were commercially available, purchased from Sigma–Aldrich (unless otherwise stated), and used without purification. Solvents were of ACS grade or higher. NMR spectra (400?MHz for 1 H and 125?MHz for 13 C) were recorded on a Bruker 400 spectrometer and processed using MestReNova v9.0.1 (Mestrelab Research, S.L., Santiago de Compostela, Spain). Chemical shifts were referenced to residual solvent peaks at δ ?=?7.26 ppm ( 1 H) and δ ?=?77.16 ppm ( 13 C) for CDCl 3 and δ ?=?2.50 for ( 1 H) and δ ?=?39.52 ppm ( 13 C) for d 6 -DMSO. Size exclusion chromatography (SEC) was performed using an Agilent 1260 Infinity II Multi-Detector GPC/SEC System equipped with both RI and ultraviolet (UV) detectors ( λ ?=?309?nm); PLGel 3?μm (50?×?7.5?mm) guard column and two PLGel 5?μm (300?×?7.5?mm) mixed-C columns with CHCl 3 with 5?mM triethylamine as the eluent (flow rate 1?mL/min, 50?°C) were used for separation. A 12-point calibration was developed using poly(methyl methacrylate) standards (PMMA, Easivial PM, Agilent) and applied for determination of molecular weights and dispersity ( ? M ). An Anton Paar rheometer (Anton Paar USA Inc, Ashland, VA, USA) fitted with a detachable photoillumination system with two parallel plates (10?mm disposable aluminum hollow shaft plate, Anton Paar) was used for rheology studies using RheoCompass software (v1.20.496). Uniaxial tensile testing was performed using a Testometric MCT-350 equipped with a 100?kgf load cell (Testometric Company Ltd, Rochdale, United Kingdom) and manual tension clamps. Dynamic mechanical analysis was performed using a Mettler-Toledo TT-DMA system (Mettler-Toledo AG, Schwerzenbach, Switzerland) fitted with an immersed static water bath with external recirculating heater system, and samples analyzed using Mettler-Toledo STARe v.10.00 software. 3D printing scaffolds and templates were processed using Solidworks 2019 (Dassault Systemes, Vélizy-Villacoublay, France) and printed using a custom digital light processing system that has been previously reported 59 . Micro-computed tomography analysis was performed using a Skyscan 1172 Micro-CT (e2v technologies plc, Chelmsford, UK) using CT-analyser software V1.15.4.0 (CTAn) (Bruker Micro-CT, Belgium) at an isotropic pixel size of 7-13 μm, a camera exposure time of 500?ms, a rotation step of 0.4°, frame averaging of 5 and medium filtering with a flat field correction. Image reconstruction was performed using a NRecon 1.6.2 (SkyScan, e2v technologies plc, Chelmsford, UK). Synthesis of TMPAC monomer 60 . Trimethylolpropane allyl ether (100.0?g, 573.7?mmol) was added to a round bottom flask with 200?mL tetrahydrofuran (THF), and cooled to 0?°C for 1?h. Ethyl chloroformate (124.5?g, 1.1?mol) was added as a single volume to the solution and allowed to again cool to 0?°C for 15?min. Triethylamine (116.2?g, 1.1?mol) was added dropwise over the course of 1?h, at which time the solution was allowed to slowly return to ambient temperature over a 12?h period. The precipitate was filtered off and the solute concentrated to a slightly yellow oil and dissolved in ethyl acetate. The organic layer was washed twice with 1?M HCl and once with brine, and concentrated to a colorless, slightly viscous oil. The oil was distilled to achieve TMPAC (98.8?g, 493.8?mmol, 86% yield). Characterization matched previously reported materials 60 . 1 H NMR (CDCl 3 , 400?MHz): δ ?=?0.94 (t, 3 J H–H ?=?7.6?Hz), 1.55 (q, 2 H, 3 J H–H ?=?7.6?Hz), 3.47 (s, 2 H), 3.88–4.05 (m, 2 H), 4.23 (d, 3 J H–H ?=?10.1?Hz, 2 H), 4.52 (d, 3 J H–H ?=?10.1?Hz, 2 H), 5.21–5.42 (m, 2 H), 5.78–5.90 (m, 1 H) ppm. 13 C NMR (CDCl 3 , 125?MHz): δ ?=?148.59 (C?=?O), 134.02 (CH), 117.49 (CH 2 ), 72.81 (CH 2 ), 72.42 (CH 2 ), 68.24 (CH 2 ), 35.45 (C), 23.31 (CH 2 ), 7.37 (CH 3 ). Synthesis of NTC monomer 61 . Pentaerythritol (40.9?g, 300.6?mmol) was added to a round bottom flask containing deionized water (500?mL), and was subsequently heated to 80?°C with stirring. Once the solids had dissolved, the colorless mixture was then cooled to 20?°C, at which time concentrated HCl (~500??L, ~2 drops) was added, followed by 5-norbornene-2-carboxaldehyde (30.5?g, 253.8?mmol). The mixture was then stirred for 8?h, and the resulting orange precipitate was isolated using vacuum filtration before being recrystallized from hot toluene/isopropyl alcohol (80/20) as white crystals to yield 2-norbornene-5,5-bis(hydroxymethyl)-1,3-dioxane (NHD). NHD (17.0?g, 71.0?mmol) was dissolved in THF (400?mL) in a round bottom flask and cooled to 0?°C, at which point ethyl chloroformate (20.4?mL, 212?mmol) was added as a single volume, followed by dropwise addition of triethylamine (29.5?mL, 212?mmol) 1?h while maintaining a 0?°C temperature. Upon completing the addition, the reaction was allowed to come to 20?°C and was allowed to stir for 12?h, after which the precipitate was filtered and concentrated to yield white crystals. The white crystals were recrystallized in hot cyclohexane/THF (90/10) (15.4?g, 58.7?mmol, 71%) as the NTC monomer. Characterization matched previously reported materials 61 , 62 . 1 H NMR (DMSO- d 6 , 400?MHz): δ ?=?6.17 (m, 1 H, 3 J H–H ?=?5.7, 3.0?Hz), 5.93 (m, 1H, 3 J H–H ?=?5.7, 2.8?Hz), 4.51 (s, 2 H), 4.06 (s, 2 H), 3.89–3.83 (m, 3 H), 3.61–3.58 (m, 2 H), 2.85 (s, 1 H), 2.78 (s, 1 H), 2.22 (ddd, 1 H, 3 J H–H ?=?12.8, 8.6, 3.9?Hz), 1.75 (m, 1H, 3 J H–H ?=?12.8, 9.3, 3.8?Hz), 1.31–1.17 (m, 2 H), 0.74 (m, 1 H, 3 J H–H ?=?11.9, 4.1, 2.6?Hz). 13 C NMR (DMSO- d 6 , 125?MHz): δ ?=?148.52 (C?=?O), 138.00 (CH), 132.48 (CH), 107.38 (CH), 71.49 (CH 2 ), 70.37 (CH 2 ), 68.91 (CH 2 ), 49.28 (CH 2 ), 43.57 (CH), 42.20 (CH), 31.53 (CH), 28.34 (CH 2 ). Synthesis of aliphatic polycarbonate . Ring opening polymerization of the cyclic monomers was used to obtain oligomers. To an open round bottom flask, CHCl 3 and cyclic monomer(s) were added followed by 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU). For PolyTMPAC, TMPAC (100?g, 500.0?mmol) was dissolved in 100?mL CHCl 3 . DBU (1.44?g, 9.5?mmol) and water (150??L, 8.3?mmol) were added as a single unit. The resulting solution was stirred for 24?h at 20?°C, after which the DBU was quenched with the addition of Amberlyst A15 H + acidic resin, precipitated into ice cold hexanes, and was then filtered through a silica plug in ethyl acetate. The solution was concentrated in vacuo to yield a viscous, colorless liquid (96.2?g, 96%). 100% TMPAC . 1 H NMR (DMSO- d 6 , 400?MHz,): δ ?=?0.82 (t, 3 J H–H ?=?7.6?Hz, 3H), 1.45 (d, 3 J H–H ?=?9.4?Hz, 2H), 3.32 (s, 2H), 3.87 (dd, 3 J H–H ?=?5.4?Hz 3 J H–H ?=?1.8?Hz, 2H), 4.04–4.21 (m, 4H), 5.11–5.32 (m, 2H), 5.79–5.93 (m, 1H), 6.88 (s, 1H). 13 C NMR (DMSO- d 6 , 125?MHz;): δ ?=?155.15 (C = O), 134.67 (CH), 116.73 (CH 2 ), 72.27 (CH 2 ), 69.46 (CH 2 ), 67.78 (CH 2 ), 41.87 (C), 22.58 (CH 2 ), 7.45 (CH 3 ). SEC (RI detection, CHCl 3 ) M n : 2.1?kDa, ? M ?=?1.2. 75% TMPAC/25% NTC . 1 H NMR (400?MHz, CDCl 3 ): δ 6.15 (m, 1H), 5.93 (m, 1H), 5.83 (m, 3H), 5.26 (m, 6H), 4.41 (m, 2H), 4.10 (m, 12H), 3.40–4.00 (m, 13H), 3.32 (s, 6H), 2.93 (m, 2H), 2.30 (m, 1H), 1.81 (m, 1H), 1.49 (q, 3 J H–H ?=?9.4?Hz, 6H), 1.44–1.15 (m, 2H), 0.89 (m, 11H). 13 C NMR (400?MHz, CDCl 3 ): 155.14 (C = O), 134.68 (CH), 132.92 (CH), 116.73 (CH 2 ), 106.93 (CH), 72.27 (CH 2 ), 69.46 (CH 2 ), 67.78 (CH 2 ), 49.26 (CH 2 ), 43.60 (CH), 41.87 (CH), 37.30 (C), 28.38 (C), 22.59 (CH 2 ), 7.46 (CH 3 ). SEC (RI detection, CHCl 3 ) M n ?=?2.0?kg·mol ?1 , ? M ?=?1.31 50% TMPAC/50% NTC . 1 H NMR (400?MHz, CDCl 3 ): δ 6.14 (m, 1H), 5.92 (m, 1H), 5.83 (m, 1H), 5.25 (m, 2H), 4.41 (m, 2H), 4.10 (m, 4H), 3.40–4.00 (m, 10H), 3.32 (s, 2H), 2.93 (m, 2H), 2.30 (m, 1H), 1.81 (m, 1H), 1.49 (q, 3 J H–H ?=?9.4?Hz, 2H), 1.44–1.15 (m, 2H), 0.89 (m, 5H). 13 C NMR (400?MHz, CDCl 3 ): 154.87 (C = O), 137.71(CH), 134.68 (CH), 132.66 (CH), 116.73 (CH 2 ), 106.93 (CH), 72.27 (CH 2 ), 69.46 (CH 2 ), 67.78 (CH 2 ), 49.26 (CH 2 ), 43.60 (CH), 41.87 (CH), 37.30 (C), 28.38 (C), 22.59 (CH 2 ), 7.46 (CH 3 ). SEC (RI detection, CHCl 3 ) M n ?=?2.5?kg·mol ?1 , ? M ?=?1.37. 25% TMPAC/75% NTC . 1 H NMR (400?MHz, CDCl 3 ): δ 6.16 (m, 3H), 5.93 (m, 3H), 5.83 (m, 1H), 5.27 (m, 2H), 4.41 (m, 6H), 4.10 (m, 4H), 3.40–4.00 (m, 22H), 3.32 (s, 2H), 2.93 (m, 6H), 2.30 (m, 3H), 1.81 (m, 3H), 1.49 (q, 3 J H–H ?=?9.4?Hz, 2H), 1.44–1.15 (m, 6H), 0.89 (m, 6H). 13 C NMR (400?MHz, CDCl 3 ): 154.87 (C = O), 137.71(CH), 134.68 (CH), 132.66 (CH), 116.73 (CH 2 ), 106.93 (CH), 72.27 (CH 2 ), 69.46 (CH 2 ), 67.78 (CH 2 ), 49.26 (CH 2 ), 43.60 (CH), 41.87 (CH), 37.30 (C), 28.38 (C), 22.59 (CH 2 ), 7.46 (CH 3 ). SEC (RI detection, CHCl 3 ) M n ?=?2.5?kg·mol ?1 , ? M ?=?1.34. 100% NTC . 1 H NMR (400?MHz, CDCl 3 ): δ 6.14 (m, 1H), 5.92 (m, 1H), 4.42 (m, 2H), 3.40–4.00 (m, 7H), 2.80–2.93 (m, 2H), 2.30 (m, 1H), 1.81 (m, 1H), 1.44–1.15 (m, 2H), 0.83 (m, 1H). 13 C NMR (400?MHz, CDCl 3 ): 154.62 (C = O), 137.08 (CH), 132.68 (CH), 106.84 (CH), 72.04 (CH 2 ), 69.49 (CH 2 ), 67.00 (CH 2 ), 49.25 (CH 2 ), 43.61 (CH), 42.22 (CH), 37.30 (C), 28.38 (CH 2 ). SEC (RI detection, CHCl 3 ) M n ?=?2.6?kg·mol ?1 , ? M ?=?1.37. Synthesis of aliphatic poly(carbonate urethane) . In a representative synthesis of the poly((TMPAC- co- hexamethylene diurethane), PolyTMPAC (2?kDa, 5.0?g, 2.5?mmol) was dissolved in a round bottom flask containing dry THF at 60?°C under N 2 , to which hexamethylene diisocyanate (HDI) (1.0?g, 6.0?mmol) was added. The mixture was allowed to stir for 48?h, during which time the viscosity visually increased dramatically. At 48?h, the temperature was increased to 80?°C and allowed to stir for 12?h, at which time the entire solution was added to 50?mL MeOH. The solution was concentrated, washed with 1?M HCl twice and once with saturated brine solution, and collected as a highly viscous, transparent oil (5.94?g, 99%). 1 H NMR (400?MHz, CDCl 3 ): δ 5.77 (m, 1H), 5.17 (m, 2H), 4.01 (s, 4H), 3.85 (m, 2H), 3.24 (m, 2.5H), 3.06 (m, 0.5H), 1.54(m, 0.5H), 1.14–1.45 (m, 4H), 0.87 (t, 3 J H-H ?=?7.6?Hz, 3H). 13 C NMR (400?MHz, CDCl 3 ): δ 155.15 (C = O), 134.67 (CH), 116.73 (CH 2 ), 72.27 (CH 2 ), 69.46 (CH 2 ), 67.78 (CH 2 ), 41.87 (C), 40.82 (CH 2 ), 29.88 (CH 2 ), 26.17(CH 2 ), 22.58 (CH 2 ), 7.45 (CH 3 ). SEC (RI detection, CHCl 3 ) M n ?=?3.5?kg·mol ?1 , ? M ?=?1.81. Synthesis of isophorone di(allyl urethane) . Isophorone diisocyanate (10.00?g, 0.045 moles) was added by canula transfer to a round bottom flask (dried 120?°C overnight and sealed) followed by dry THF (40?mL). Freshly distilled allyl alcohol (5.54?g, 0.095 moles), stored over molecular sieves, was added dropwise to the solution while stirring at 300?rpm. Upon complete transfer of the allyl alcohol, the reaction was heated to 50?°C and held isothermally for 24?h, at which point residual diisocyanate was quenched with water (at 50?°C). Crude urethane was obtained after dissolving the reaction mixture in ethyl acetate, washing with 1?M HCl (3 washes) and brine (1 wash) and concentrating the product. A viscous clear oil was collected after column chromatography (25:75 EtOAc:Hexane) and concentrated in vacuo to yield a colorless oil (1.2?g, 3.5?mmol, 7.8 %). 1 H NMR (400?MHz, CDCl 3 ): δ 5.85–5.94 (m, 2H), 5.18–5.30 (m, 4H), 4.86 (m, 1H), 4.53–4.55 (m, 5H), 3.79–3.82 (m, 1H), 2.91-2.92 (d, 3 J H–H ?=?3.0?Hz, 2H), 1.67–1.74 (t, 3 J H–H ?=?9.0?Hz, 2H), 1.17–1.21 (m, 2H), 0.83–1.05 (m, 11H), 13 C NMR (400?MHz, CDCl 3 ): 156.69 (C = O), 155.51 (C = O), 132.94 (CH), 117.72 (CH 2 ), 65.59 (CH 2 ), 54.89 (CH 2 ), 47.05 (CH), 45.62 (CH 2 ), 44.66 (CH 2 ), 41.85 (CH 2 ), 36.41 (C), 35.04 (CH 3 ), 31.86 (C), 29.70(CH 3 ), 27.63(CH 3 ), 23.24(CH 3 ). Mass spectrometry (ESI); m/z ?=?338.22 (M + ). Elem. anal. Calcd for C 18 H 30 N 2 O 4 : C, 63.88; H, 8.93; N, 8.28%. Found: C, 63.81; H, 8.77; N, 8.24%. Formulation of poly(TMPAC) resins . Oligomer, reactive diluents and other diluents were added to a vial, along with the 4 arm tetrathiol (pentaerythritol tetrakis(3-mercaptopropionate) (PETMP)). After mixing, photointiator and photoinhibitor were added. As an example, the polyTMPAC resin consisted of isophorone di(allyl urethane) reactive diluent (13.78?g, 40.7?mmol), polyTMPAC (15.28?g, 7.6?mmol), 1,3,5-triallyl-1,3,5-triazine-2,4,6(1H,3H,5H)-trione as a second reactive diluent species (14.65?g, 58.7?mmol), PETMP (24.41?g, 53.2?mmol), and propylene carbonate as an unreactive diluent (16.54?g, 162.1?mmol) mixed together for 8?h at ambient temperature. To this was added Irgacure 819 (photoinitiator, 0.82?g, 1?wt%), and paprika extract (photoinhibitor, 0.50?g, 0.75?wt%) in a dark room with little ambient light, followed by 1?h of stirring. After homogenization of the resin, the resin was placed in a brown glass container and stored at room temperature in the dark. Spectroscopic analysis of thiol-ene crosslinking . Conversion of alkenes in the oligomeric and monomeric reactive components by reactions with the thiols in PETMP with 1% wt photoinitiator and no inhibitor were performed to study crosslinking kinetics. Experiments were performed in 0.5?mL CDCl 3 at ambient conditions, exposed to λ ?=?340–430?nm light for discrete timepoints prior to storage in brown glass vials. Photorheology . Crosslinking kinetics of resin samples were examined by measuring the dampening or phase ratio (tan δ ), storage moduli, loss moduli, complex viscosity, and film thickness to determine the gelation time(s) 63 . Resin samples were subjected to oscillatory shearing between two parallel plates (500??m gap), one made of glass and transparent, at 1?Hz for 50?sec without irradiation, at which time the resins were irradiated with λ ?=?430–520?nm light and measurements were taken every 0.2?s over the course of 2?min. The inflection points of the moduli plots (storage and loss), and the peak tan δ values, were used to determine the time to gelation of the resin. Sample shrinkage was determined by measuring the gap between the plates at the same sampling rate as the other metrics. Mechanical testing . Printed modified ASTM Type IV dogbones were examined in uniaxial tensile testing at ambient moisture and temperature. Samples were placed in the tension clamps and allowed to vibrationally equilibrate for 10?min, at which point each sample was extended at 5?mm·min ?1 until failure (defined as total sample failure). Seven samples were run per composition. Dynamic mechanical analysis . Rectangular dynamic mechanical analysis (DMA) samples were prepared via 3D printing sample bars (2.0?×?0.5?×?0.2?cm). Samples were analyzed in tension mode using the standard autotension mode, with a testing frequency of 1?Hz, a preload force of 1?N, and a static force of 0.1?N. Thermal sweeps were conducted at 2?°C·min ?1 . Samples were equilibrated at ?30?°C for 5?min and were heated to 200?°C at a rate of 10?°C·min ?1 . The peak ratio between the loss and storage moduli ( E” / E’ , tan δ ) was defined as the T g . Relaxation kinetics studies of the printed scaffolds were conducted using submerged samples at 37?°C in phosphate buffered saline (PBS) solution, in compression mode. Printed porous scaffolds (1?cm 3 ) were placed in compression and deformed 10??m, 1?Hz with a preload of 0.1?N at ambient conditions (not submerged) for ~60?sec. At this time, the scaffold was then immersed in the PBS solution and held isothermally at 37?°C for 60?min. Storage moduli and tan δ values were recorded as a function of time to determine the behavior of the polymer during initial submersion/introduction to biologically mimicking conditions. Expansion forces were measured using the same method in creep mode. Shape memory experiments were performed using the same porous scaffolds in compression mode. The samples were equilibrated at 60?°C for 1?h, deformed by ? 30% (load dependent deformation) and cooled to ?20?°C. Once the sample was isothermal with the cooled chamber, the load was removed, and the sample expansion was monitored as a function of force and displacement of the compression clamp as the sample was heated to 60?°C at 10?°C·min ?1 . Testing was performed in triplicate. 3D printing . Scaffolds based upon previously reported geometries were printed from polycarbonate resins using optimized conditions (dependent upon composition) 63 , 64 . Resins were added in 10?mL quantities to the resin tray, allowing for complete and even coverage of the printer optical window on the surface of the printing plate. Porous scaffolds were printed by exposing resins to λ ?=?405?nm light using a custom-built digital light processing unit and printing parameters were individually determined for each resin composition through optimization of irradiance, irradiation time, resulting film thickness, and semi-quantified feature resolution (percentage of theoretical resolution), and were further optimized in the printing vat as necessary 59 . The z-stage step transition was set to 100??m, and each slice was exposed for 6?s, on average. Print resolution was determined through image analysis (Image J) of the theoretical structure, and pore size analysis using microscopy from the printed structure. The final structures are rinsed with acetone to remove residual resin and photoinhibitor, as denoted by colour removal. Degradation analysis . Porous scaffolds and nonporous scaffolds were immersed in degradation solution, following previously established protocols for static degradation analysis 17 . For dynamic degradation studies, films were tested using the DMA in tension mode with the autotensioner, loaded with a 0.1?N preload and 10?Hz oscillation. Films were immersed in 5?M NaOH solution at 37?°C for the duration of testing. Samples were tested until failure, with the phase ratio and the storage moduli recorded over the course of the study. For in vivo degradation, samples were removed from subcutaneous tissue and sterilized using EtOH. Tissue was removed and scaffolds were extracted with hexane or methanol over a 48?h period, after which the extracted solutions were concentrated down and dissolved in either CDCl 3 or DMSO- d6 . Scaffold swelling ratio was determined by Eq.? 1 : $${\rm{Swelling}}\; {\rm{ratio}}=\frac{({m}_{{\rm{f}}}-{m}_{{\rm{i}}})}{{m}_{{\rm{i}}}}$$ (1) where m i is the original mass of the scaffold (dry) and m f is the mass of the scaffold after swelling (but blotted dry to remove droplets or excess solvent). The crosslink density, and therefore the remaining mass of the material, was determined by Eq.? 2 : $${\rm{Gel}}\; {\rm{fraction}}( \% )=\frac{{m}_{{\rm{f}}}}{{m}_{{\rm{i}}}}$$ (2) where m f is the final scaffold mass (dry) and m i is the original scaffold mass (dry). Printed void filling . A hexagonal void was produced in Solidworks, and the cross-sectional area was varied to produce irregular voids, one which is sharply irregular and the other possessing rounded edges. The voids were printed and used for studying void-filling behavior, using cross-sectional area of the void and the printed scaffold (cube) to determine void filling as a qualitative function of shape. Expansion forces in alginate gels . Alginate was dissolved in water at a concentration of 10?mg·mL ?1 , to which was added 5?mL of calcium chloride dihydrate (0.1?mg·mL ?1 ). The two components were mixed until gelation, and 10?mL of H 2 O was added as the gels were incubated at 37?°C overnight. Gel mechanical properties were matched adipose and glandular tissue using literature protocol 42 . Gels were cut with an eye-shaped opening, in the same manner as a lumpectomy surgery. Cubic scaffolds were shape fixed at 60% strain and inserted into the opening, where void filling and gel deformation were examined optically using the same cross-sectional analysis described for the “Printed Void Filling” section. The shape fixation behavior of the scaffold was further examined upon removal of the scaffold from the gel, and the shape recovery efficiency compared with the void-filling behavior, as well as the deformation of the alginate. The thin-walled computational molds previously described were then examined using determined loading forces and compared with the deformation found in alginate gels. An interior force of 1?N was initially applied uniformly to the interior (cut) surface of the gel in the same manner as the scaffold would be in contact and expand. The force was then scaled until deformation matched experimental results. Cytocompatibility and cellular analysis . Samples for cell culture studies ( n ?=?4) were prepared by spin coating a solution of 0.4?wt% polymer in CHCl 3 on a glass coverslip (1?min at 1000?rpm). Spin-coated glass coverslips were then sterilized by immersion in a 70% ethanol solution, fully dried before use, and placed into 12-well plates. NOR-10 (murine fibroblasts), Hs 792 (human fibroblasts), IC21 (murine macrophages), and D16 (murine adipocytes) cell lines were purchased from ATCC UK and cultured in DMEM (NOR-10 and Hs 792), RPMI-1640 (IC21), and DMEM/F12 (D16) media supplemented with 10% FBS (20% for NOR-10) and 1% pen/strep, at 37?°C and 5% CO 2 . 1% L-Alanyl-L-Glutamine was added in DMEM/F12 medium. MC3T3 (murine pre-osteoblasts) were purchased from Public Health England and cultured in Alpha Minimum Essential Medium with ribonucleosides, deoxyribonucleosides, 2?mM L-glutamine and 1?mM sodium pyruvate, but without ascorbic acid, 10% FBS and 1% pen/strep, at 37?°C and 5% CO 2 . Cell proliferation . Cell proliferation assays were performed on spin-coated glass slides by seeding the above cell lines ( n ?=?4, 2000 cells cm ?2 ) and measuring metabolic activity at selected timepoints (24?h, 3, 7, and 14 days of culture). Cell proliferation was evaluated by using a PrestoBlue? metabolic assay following the supplier’s instructions. Briefly, after removing the medium, 1?mL of PrestoBlue? solution (10% in cell culture medium) was added to each well, followed by incubation at 37?°C for 1–4?h. 100??L of solution was taken from each well and placed in triplicate into a 96-well plate. The fluorescence intensity (FI) was detected in a BMG Labtech Fluostar Omega Microplate Reader at wavelengths of 590?nm for excitation and 610?nm for emission. Cell spreading . Cells were seeded on spin-coated coverslips ( n ?=?4) at 4000 cells·cm ?2 . After 72?h, cells were fixed using a 4% paraformaldehyde solution for 10?min, permeabilized using 0.5% Triton X-100 in cytoskeleton stabilization (CS) buffer (0.1?M PIPES, 1?mM EGTA, and 4% (w/v) 8000?MW polyethylene glycol) at 37?°C for 10?min, rinsed thrice for 5?min each in CS buffer, and incubated in 0.1% sodium borohydride in PBS at ambient temperature for 10?min to quench aldehyde autofluorescence. Samples were then blocked in 5% donkey serum for 20?min at 37?°C and incubated overnight at 4?°C with mouse primary anti-vinculin antibody (Abcam, 1:100). Samples were then washed three times with 1% donkey serum for 5?min each, and then incubated with Alexa Fluor 647 Phalloidin for cytoskeleton staining (1:200) for 1?h followed by Alexa Fluor? IgG-594 secondary antibody (Invitrogen, donkey anti-mouse, 1:100). DAPI was used to stain the cell nuclei. Cells were imaged with a FV3000 Olympus confocal fluorescence microscope using 350, 594, and 633?nm excitation filters and a ×20 or ×40 objectives 65 . 3D cell experiments . 3D-printed scaffolds were sterilized by immersion in 70% ethanol, dried, and then placed in 24-well plates, and incubated for 24?h in cell culture medium at 37?°C, 5% CO 2 . The medium was then removed and cells (100,000 in 20?μL of medium) were seeded on top of the scaffolds ( n ?=?3) and incubated at 37?°C, 5% CO 2 for 3?h. After this time, 2?mL of culture medium was added and the cells were incubated again at 37?°C, 5% CO 2 for the selected timepoints (24?h, 3 days, 7 days). A live/dead assay (Invitrogen) was performed at each of the selected timepoints. Briefly, scaffolds were washed with PBS (3?×?2?mL) and incubated with a calcein/ethidium homodimer solution at 25?°C for 20?min, following the supplier’s instructions. Scaffolds were then washed with PBS (3?×?2?mL) and placed on a microscope slide for fluorescent imaging. Cells were imaged with a FV3000 Olympus confocal fluorescence microscope using 488?nm and 594?nm excitation filters and a ×4 air objective 65 . Image J was used for analysis. Surgical procedure . Experiments were performed in accordance with the European Commission Directive 2010/63/EU (European Convention for the Protection of Vertebrate Animals used for Experimental and Other Scientific Purposes) and the United Kingdom Home Office (Scientific Procedures) Act (1986) with project approval from the institutional animal welfare and ethical review body (AWERB). Anaesthesia was induced in adult male (8 weeks old) Sprague Dawley rats (200–300?g) with isofluorane (2–4%; Piramal Healthcare) in pure oxygen (BOC). Animals were placed prone onto a thermocoupled heating pad (TCAT 2-LV; Physitemp), and body temperature was maintained at 36.7?°C. The experimental material and control material (PLLA) were implanted over either the spinotrapezius or lateral aspect of the external obliques. Following an incision of ? 3?cm, the skin was separated from the muscle with large forceps, and any excess fat was removed. The implants were tunnelled under the skin and placed in direct contact with the muscle, at sites distal to the incision. The order of the implants was randomized but constrained so that each implant appeared in each location bilaterally at least once. The wounds were sealed with a subcuticular figure of 8 purse string suture with a set-back buried knot using 3-0 vicryl rapide suture (Ethicon). The surgical procedure was performed under the strictest of aseptic conditions with the aid of a nonsterile assistant. Post-surgical analgesia was administered, and rats were placed into clean cages with food and water ad libitum . Histological analysis . At 1-month and 2-month timepoints, samples were excised from the subcutaneous tissue and fixed with 4% paraformaldehyde for 24?h. After fixation, samples were washed with increasing percentages of ethanol (70–100%) for 30?min each, washed thrice with xylene, and embedded in paraffin wax blocks for sectioning. Slices (10–30??m thick) were cut using a Leica Biosystems microtome for histological analysis before being stained using hematoxylin and eosin stains or Masson’s Trichrome staining using protocols available through Sigma–Aldrich. Analysis was performed using light microscopy (Leica, ×4 and ×10 objectives) and image stitching was performed in ImageJ (NIH, Bethesda, MD). Brightfield images were analyzed and qualitatively assessed for general inflammation compared to PLLA control samples. Samples were also analyzed for a number of inflammatory cells utilizing a modified scoring system designed by the International Organization for Standardization (ISO 10993-6 Annex E). Scoring was based on a scale from 0 to 4 (0?=?none; 1?=?Rare, 1–5 Minimal; 2?=?5–10, Mild; 3?=?Heavy Infiltrate, Moderate; 4?=?Packed, Severe). Statistics and reproducibility . Statistical analysis of thermal, thermomechanical, and degradation results was performed using a standard one-way Student’s t -test, with probabilities of 0.05 used to assess the probability of differences between compositional behaviors. Specific reproduction numbers and experimental iterations are included in Figure captions and methodology text. Reporting summary . Further information on research design is available in the? Nature Research Reporting Summary linked to this article. .
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