Integration of 3D Printed and Micropatterned Polycaprolactone Scaffolds for Guidance of Oriented Collagenous Tissue Formation In Vivo

Abstract

Scaffold design incorporating multiscale cues for clinically relevant, aligned tissue regeneration has potential to improve structural and functional integrity of multitissue interfaces. The objective of this preclinical study is to develop poly(ε-caprolactone) (PCL) scaffolds with mesoscale and microscale architectural cues specific to human ligament progenitor cells and assess their ability to form aligned bone-ligament-cementum complexes in vivo. PCL scaffolds are designed to integrate a 3D printed bone region with a micropatterned PCL thin film consisting of grooved pillars. The patterned film region is seeded with human ligament cells, fibroblasts transduced with bone morphogenetic protein-7 genes seeded within the bone region, and a tooth dentin segment positioned on the ligament region prior to subcutaneous implantation into a murine model. Results indicate increased tissue alignment in vivo using micropatterned PCL films, compared to random-porous PCL. At week 6, 30 μm groove depth significantly enhances oriented collagen fiber thickness, overall cell alignment, and nuclear elongation relative to 10 μm groove depth. This study demonstrates for the first time that scaffolds with combined hierarchical mesoscale and microscale features can align cells in vivo for oral tissue repair with potential for improving the regenerative response of other bone-ligament complexes.

Keywords: 3D printing; cell alignment; micropatterning; periodontal ligaments (PDL); polycaprolactone (PCL).

Figure 1

Surface treatment of PCL films using aminolysis, hydrolysis, fibronectin coating, and combined treatment of hydrolysis and fibronectin assessed to determine conditions for increased hPDL cell attachment. Data shows mean percentage of ligament progenitor cells adhered to non-treated PCL films in vitro versus surface-treated PCL at 1 and 5 days post-seeding. (Error bars: ± SD; ** p<0.01; *** p<0.001, **** p<0.0001; # indicates p<0.05 relative to non-treated PCL film at each timepoint; n=3).

Figure 2

Micropatterned 2D PCL film design was used to determine effects of groove width on hPDL cell alignment in vitro. Films were patterned using three different molds (a) to embed grooved features onto the polymer surface (b, surface with 50um wide grooves, Nikon SMZ18 stereo microscope). DilC(12)-3 stained hPDL cells seeded on hydrolyzed and fibronectin treated PCL films with non-grooved (control) and grooved surfaces (c). White arrows indicate direction of grooves (scale bar=50um). Average orientation of hPDL cells on control (non-patterned), 50 um, 25 um, and 10 um grooved PCL films, where 0° indicates complete alignment with pattern and 90° indicates a cell that is perpendicular to a groove (d). Proportion of hPDL cells within a specific orientation angle for all PCL film groups, with 0–5° groups indicating highest alignment of cells within grooved microfeatures (e). (Error bars: ± SD; * p<0.05; ** p<0.01; **** p<0.0001).

Figure 3

Schematic design of the scaffold intergrating 3D printed and micropatterned regions for the bone-ligament oral complex. Anatomical features of the alveolar bone-periodontal ligament interphase present innately at the tooth root surface (a) were used to design a scaffold combining a 3D printed PCL region and a 3D patterned PCL film for the bone and PDL regions, respectively (b). A total of six groups (c) were tested in vivo by varying the geometry (width, W and depth, D of the grooves) of the PCL in the PDL region of the scaffold: (1) random-porous, salt-leached PCL sponge, (2) 400×400 um square pillars 250um in height, (3) square pillars with 60um wide and 30um deep grooves, (4) pillars with 60um wide and 10um deep grooves, (5) pillars with 15um wide, 30um deep and 15um wide, 10um deep (6) grooves. (d) An ex vivo mouse model was used to subcutaneously implant the combined scaffold to promote bone (B) and periodontal ligament formation, with a dentin chip (D) press-fit on top of the combined scaffold after cell-seeding the PDL region prior to implantation.

Figure 4

Formation of bone in the 3D printed scaffold region seeded with adenoviral BMP7 transduced gingival fibroblasts. Micro-computed tomography (uCT) of implanted scaffolds was used for bone volume (BV) and tissue mineral density (TMD) analysis of osseous tissue growth in vivo in the bone region of the scaffold (a) compared to the formation of tissue in the entire scaffold external to bone region at 3 and 6 weeks post-harvesting (b). (Error bars: ± SD; *** p<0.001, **** p<0.0001; n=5–6).

Figure 5

Histomorphological assessment of soft and mineralized tissue formation in the micropatterned PDL and 3D printed bone scaffold compartments, respectively. (a) H&E, Masson's trichrome, and DAPI (blue)/tubulin(green) staining was performed to assess bone (B) and tissue formation, collagen alignment, and cellular alignment in the region of the PCL film or sponge at 3 and 6 weeks (images shown are at 6 week timepoint only). Note the formation of fibers approaching the dentin surface and bone more distant in the bone region of the scaffold (near bottom of the H&E sections). (b) Formation of cementum-like tissue newly-deposited at the dentin (D) surface was observed on week 6 samples using H&E staining and immunohistochemical analysis for bone sialoprotein (BSP) positive expression. Scale bar = 100um.

Figure 6

Cell alignment and nuclear shape index assessed using immnufluorescence staining at 6 weeks. (a) DAPI (blue) and tubulin (green) shows increased cell alignment further from the pillar boundary in films with grooves compared to non-grooved pillars, and in films with deep grooves (30um) compared to more shallow grooves (10um). Scale bar = 50um. (b) Mean percentage of aligned cell nuclei (within 20° of preferred perpendicular orientation: 70°≤x≤110°) in vivo on PCL sponge, non-grooved pillars (0W, 0D), and grooved pillars (60 um and 15um wide (W) grooves) with varying groove depths (D) (10 um—red outline, and 30um—blue outline). (c) Mean nuclear shape index analysis indicating cellular elongation in vivo on all groups based on measure of nuclear circularity (C=4*π*area/perimeter²). (Error bars: ± SD; ** p<0.01; *** p<0.001, **** p<0.0001; # indicates p<0.05 relative to PCL sponge at each timepoint; n=5 in each group).

Figure 7

Collagen thickness at inter-pillar distances bordering grooved or non-grooved features used to determine oriented tissue formation at 6 weeks in vivo. (a) Masson's Trichrome stining of samples indicates increased collagen orientation perpendicular to dentin (D) chip at the pillar boundary in the presence of grooves (scale bar = 100um). (b) Mean thickness (um) of oriented collagen bundles in vivo at non-grooved (0W, 0D) and grooved (15–60um wide, 10–30um deep) pillar borders at 6 weeks based on Masson's Trichrome staining. (Error bars: ± SD; ** p<0.01; *** p<0.001, **** p<0.0001; n=5).