Biomimetic hybrid scaffolds for engineering human tooth-ligament interfaces

Abstract

A major clinical challenge in the reconstruction of large oral and craniofacial defects is the neogenesis of osseous and ligamentous interfacial structures. Currently, oral regenerative medicine strategies are unpredictable for repair of tooth-supporting tissues destroyed as a consequence of trauma, chronic infection or surgical resection. Here, we demonstrate multi-scale computational design and fabrication of composite hybrid polymeric scaffolds for targeted cell transplantation of genetically modified human cells for the formation of human tooth dentin-ligament-bone complexes in vivo. The newly-formed tissues demonstrate the interfacial generation of parallel- and obliquely-oriented fibers that grow and traverse within the polycaprolactone (PCL)-poly(glycolic acid) (PGA) designed constructs forming tooth cementum-like tissue, ligament, and bone structures. This approach offers potential for the clinical implementation of customized periodontal scaffolds that may enable regeneration of multi-tissue interfaces required for oral, dental and craniofacial engineering applications.

Figure 1

a) Schematic illustration of the 3-D wax printing system and dimension of hybrid scaffold shows polymeric architecture manufacturing. For the PDL interface, column-like structures were 0.8mm diameter and 0.3mm exposed heights and casted using PGA-HFIP solution. For the bone region of the hybrid scaffold, PCL-acetone solution was used for casting. PCL-acetone, pasted on the PCL-casted mold and PDL interface architectures were placed on it. b) After the acid-treatment of human tooth dentin slices, the complex with a polymer-casted hybrid scaffold and a dentin slice was assembled using fibrin gel with or without cells. The left is the 3-D designed hybrid scaffold and the right panel is the micro-CT scanned and 3-D reconstructed hybrid scaffold and a dentin slice. The scale bar: 50μm.

Figure 2. 3-D reconstructed colorized micro-CT images and hematoxylin and eosin (H&E) stained histology

The mineralized tissue (blue-colorized) was formed around the hybrid scaffolds and there was no ankylosis, bone fusion to the dentin surface (white-colorized). Red and blue dashed lined-boxes represent the PDL interface and bone regions, respectively. H&E stained histology slices showed PDL interfaces and bone region tissues to evaluate fibrous tissue orientation along the column-like structures in PDL interface, which were designed with perpendicular direction to the dentin surface. At 6 weeks, hPDL cell seeded specimens demonstrated perpendicular orientation to the dentin slices and along the column-like structures with limited evidence of cementum-like cells on the dentin surface. Yellow dash-line is the borderline of channel-type PDL architecture and black arrowed lines represent the fibrous cell/tissue directionality following the wall of PDL interface structure. Red triangles indicate the blood vessels and yellow triangles point cementum-like tissue layer or cell deposition for cementogenesis on the dentin surface. The scale bar: 50μm.

Figure 3. Quantitative analysis of micro-CT and histomorphometry for cementum-like tissue length

At 3 week time point, a) bone volume fraction (BVF) and b) bone mineral density (BMD) at 3 and 6 weeks were measured and analyzed statistically for bone regions of the hybrid scaffolds. a and b) In the aspects of BVF and BMD, there were statistically significant differences between the osteogenic factor and non-osteogenic factor groups in bone regions at 3 weeks (**p<0.01) and 6 weeks (*p<0.05 and **p<0.001). c) The cementum-like tissue length percentage represented of mineralized layer deposition on the dentin surface at 3 and 6 weeks. Full surface length of dentin slice, which faced to the PDL interface was measured and divided the measured the cementum-like tissue length (%). At 6 weeks, hPDL/BMP-7-hGF cell seeded group had statistically significant differences with no cell and hPDL cell seeded groups (*P=0.02433 and **p=0.00722). d) Qualitative results using H&E staining for the cementum-like tissue formation on the dentin surface. In 3 weeks, there was limited evidence of the mineralized layer formation but, in 6 weeks, significantly increased mineral deposition can be determined with the cementocyte-like cell embedded indicated by yellow triangles. For all statistical data analysis, two-tailed Kruskal-Wallis one-way ANOVA test and Mann-Whitney U-Test were utilized and data were mean ± standard error of mean (S.E.M.). scale bar: 50μm.

Figure 4. Cellularity and cell/tissue orientation in PDL interface using H&E staining and Immunofluorescence images

The fibrous tissue orientation was measured by semi-quantitative analysis with four different indexes; no cellularity, cellularity without fiber formation, cellularity with disorganized fiber, and cellularity with perpendicularly oriented fiber formation to the dentin surface. The H&E staining pictures are the representatives for these four indices. The percent number of the semi-quantitative analysis was calculated with total number of samples in each group/time point and the number of each observation index. Immunofluorescent images represent four typical examples of indexes with DAPI (blue) and tubulin (green) staining in cytoplasm. Scale bar: 50μm.

Figure 5. The reverse-engineered periodontal defect-fit scaffold modeling and the adaptation of customized designed scaffold on the root surface

a) The computer-aided design (CAD)-based software, NX5 was utilized to create PDL (red-colorized) and bone (blue-colorized) interfaces of the hybrid scaffold. The anatomical defect-fit scaffold had the perpendicular oriented PDL internal channel-structures and topological similarities of the periodontal defects. The Furcation defect design had separated two different parts with key (buccal)–lock (lingual) system to make easier assembling and implanting through the buccal-lingual penetration defect region. b–c) The red line was porcine mandible image with the customized scaffold and the blue line was the exposed periodontal defect site. b) The histogram represented the 99.9% adaptable scaffold to the root surface. The measured length was 3.00mm and scaffold was coated by 35% BaSO₄ solution. The yellow lines on the 2-dimensionally digitized slices represented the measured regions with 3.00mm length from the dentin (dental pulp side) to the middle of defect site. The abbreviations were that AB: alveolar bone, R: tooth root, and Sc: hybrid scaffold. c) The histogram was from 83.5% adaptable scaffold image. The concaved region of the red line can represent the gap distance (d^gap) between tooth root surface and PDL interface scaffold. d) Based on the method in Figure 5-c and d, total PDL interface length (d^total) and d^gap were linearly measured and the adaptation ratio was calculated in each layer, which had 3 different channel-type structures. There was no statistically significant difference (N.S.) among 6 different layers (p=0.1143) and the rage of adaptation was 83.3%<mean value of adaptation ratio<99.0% and data were mean ± standard deviation (S.D.). For the statistical analysis, the nonparametric Kruskal-Wallis one-way ANOVA test was used.