4D polycarbonates via stereolithography as scaffolds for soft tissue repair

Abstract

3D printing has emerged as one of the most promising tools to overcome the processing and morphological limitations of traditional tissue engineering scaffold design. However, there is a need for improved minimally invasive, void-filling materials to provide mechanical support, biocompatibility, and surface erosion characteristics to ensure consistent tissue support during the healing process. Herein, soft, elastomeric aliphatic polycarbonate-based materials were designed to undergo photopolymerization into supportive soft tissue engineering scaffolds. The 4D nature of the printed scaffolds is manifested in their shape memory properties, which allows them to fill model soft tissue voids without deforming the surrounding material. In vivo, adipocyte lobules were found to infiltrate the surface-eroding scaffold within 2 months, and neovascularization was observed over the same time. Notably, reduced collagen capsule thickness indicates that these scaffolds are highly promising for adipose tissue engineering and repair.

Fig. 1. General reaction scheme for producing aliphatic polycarbonate-derived photopolymer resins.

Aliphatic polycarbonates were synthesized using organobase-catalyzed ROP of cyclic carbonate monomers, producing low viscosity prepolymer polycarbonates to which were added allyl- and norbornene-functionalized crosslinkers and multifunctional thiols (a) and combined with reactive diluents (R = hexamethylene and isophorone), a photoinitiator and photoinhibitor to produce a liquid resin (b).

Fig. 2. Materials formulation and 3D printing.

Gelation times, which correspond with photorheological phase transition behavior studies of resins (a) and conversion of alkene monomers to thioether-containing species via thiol-ene radical reactions with PETMP (b) irradiated at 340–430 nm at 10 mW·cm⁻² for 30 s for different resin compositions. Data are presented as mean values with error bars = standard deviation (n = 3). Porous scaffolds were reduced to 2D images and photocrosslinked in a digital light processing (DLP) 3D printing process (c) and were used to produce the same porous structures as the CAD renderings (pictograph of example prototoypes, scale bar = 2 mm (d) and micro-CT representative image, scale bar = 500 µm (e).

Fig. 3. Cellular response to polycarbonate-based materials.

Representative images of adipocytes (a, b) and fibroblasts (c, d) for PTMPTCX (a, c) and PNTCTX scaffolds (b, d). Confocal image of adipocytes on 3D PTMPTCX scaffolds at the top (e) and bottom (f) after 7 days proliferation. (Image a–d Scale bar = 10 µm; Image e, f Scale bar = 100 µm). Representative printed stair-step pyramidal-structure (with smooth glass-surface cast and alternating stair-step design), with corresponding images from both surfaces overlaid to display cell (pre-osteoblasts) migration after 7 days (g), displaying no differences between surface morphology and cellular proliferation. Representative images of cellular proliferation throughout PTMPTCTX foam with images taken at the top of the scaffold (where cells were seeded), bottom of the scaffold, and from the middle of the scaffold after the same time (h), inset µCT of foams. Scale bar = 500 µm.

Fig. 4. Thermomechanical properties of polycarbonate-based materials.

The relationship between T_g and NTC concentration in the printed polycarbonate materials as determined from phase transitions examined using DMA compression (a), stress-strain behavior for dogbones tested at 10 mm·min⁻¹ in uniaxial tensile mode (b), and representative cyclic compression behavior of printed porous PTMPCTX scaffolds in 37 °C PBS (c). Representative images of the PTMPTCX scaffold deformation at 25 °C are shown before loading (d), at 70% strain (e), and after the load is removed (f), with corresponding energy absorption for 100 cycles in alginate gels examined at 37 °C PBS, data are presented as mean values with error bars = standard deviation (n = 5) (g). Scale bar = 1 cm.

Fig. 5. Shape memory behavior.

Representative shape memory behavior for a printed porous polyNTC scaffold as it is transitioned from its original geometry (a) to a compressed state under loading (~50% strain, b), after which it is cooled to 25 °C and will retain its secondary shape after the deformation load is removed (c), and the return to the original geometry upon heating of the sample (d). The expansion forces of the PTMPTCX (e) and PNTCTX (f) using compression kinetic studies under in vitro conditions. In vitro void-filling behavior was further examined using compressed scaffolds (represented by PNTCTX here) in soft alginate molds, displaying shape fixation (g), void filling without deformation of the alginate (h), and shape fixation to the void shape even after removal of the scaffold (i); 3D-printed molds were further examined for void-filling efficiency and strain recovery (j). (All scale bars = 1 cm).

Fig. 6. Swelling and degradation behavior of 3D-printed materials.

Representative microscopy images of a printed PTMPTCX scaffold immersed in 5 M NaOH over 15 min, demonstrating surface-erosion behavior of the denoted strut (red circle) immediately upon immersion in the solution (a), at 5 min (b), 10 min (c), and 15 min (d). Representative curves of uniaxial testing of polycarbonate-derived material films immersed in 5 M hydrolytic degradation solution at 37 °C, with samples deformed 50 µm at 1 Hz until failure (e) and corresponding static gravimetric degradation analysis at the same conditions (f), along with in vitro strut erosion measurements from microscopic analysis of printed scaffolds immersed in 5 M NaOH at 37 °C (g). Post-implantation assessment of sample swelling (h) and gel fraction (i) were used to assess the extent and type of degradation of the samples after removal from tissue at discrete timepoints, with accompanying measurements of strut diameters from printed scaffolds ex vivo (j). Scale bar = 200 µm. Data are presented as mean values with error bars = standard deviation (n = 6).

Fig. 7. Histopathological analysis.

Representative histological images from PLLA control materials at 1 month (a) and 4 months (e) compared with PTMPTCX films at the same times (b, f). Masson’s Trichrome (c, d) and H&E (g, h) images of PTMPTCX (c, g) and PNTCTX (d, h) printed scaffolds after 4 months, respectively, with corresponding histological scoring and assessment. Scale bar = 200 µm.