Design and fabrication of anatomically shaped human and rat tooth scaffolds by 3D bioprinting. Anatomic shape of the rat mandibular central incisor (A) and human mandibular first molar (B) were used for 3D reconstruction and bioprinting of a hybrid scaffold of poly-ϵ-caprolactone and hydroxyapatite, with 200-µm microstrands and interconnecting microchannels (diam., 200 µm), which serve as conduits for cell homing and angiogenesis (C,D). A blended cocktail of stromal-derived factor-1 (100 ng/mL) and bone morphogenetic protein-7 (100 ng/mL) was delivered in 2 mg/mL neutralized type I collagen solution and infused in scaffold microchannels for rat incisor scaffold (E) and human molar scaffold (F), followed by gelation.

Orthotopic tooth regeneration. (A) The rat mandibular incisor scaffold integrated with surrounding tissue, showing tissue ingrowth into scaffold microchannels and multiple tissue phenotypes, including the native alveolar bone (b), newly formed bone (nb), and a fibrous tissue interface reminiscent of the periodontal ligament (pdl). The newly formed bone (nb) showed ingrowth into microchannel openings and inter-staggered with scaffold microstrands (s). (B) Newly formed bone (nb) has bone trabeculae-like structures (arrows) and embedded osteocyte-like cells, immediately adjacent to a putative periodontal ligament (pdl) consisting of fibroblast-like cells and collagen buddle-like structures. (C) Newly formed bone (nb) is well-mineralized (von Kossa preparation), in contrast to the adjacent unmineralized, putative periodontal ligament (pdl). (D) Cells populated the scaffold’s microchannels even without growth-factor delivery. Remarkably, SDF1 and BMP7 delivery yielded substantial cell homing in microchannels (E). (F) Combined SDF1 and BMP7 delivery homed significantly more cells into microchannels than without growth-factor delivery (p < 0.01; N = 11). Angiogenesis took place in scaffolds’ microchannels without growth-factor delivery (G), but was more substantial with growth-factor delivery (H). (I) Combined SDF1 and BMP7 delivery elaborated significantly more blood vessels than without growth-factor delivery (p < 0.05; N = 11). (J) The numbers of recruited cells and blood vessels were quantified from 3 different locations along the entire root length of the rat mandibular incisor scaffold: the superior region of alveolar ridge and the inferior region of root apex, with a midpoint in between. s, scaffold; GF, growth factor(s). Scale: 100 µm.

In vivo orthotopic and ectopic implantation of anatomically shaped tooth scaffolds. (A) In vivo implantation of human mandibular molar scaffold into rat’s dorsum constitutes an ectopic model for tooth regeneration. (B) Harvest of human molar scaffold showing integration and tissue ingrowth. (C) Extraction of the right rat mandibular central incisor. (D) The extracted rat mandibular central incisor. (E) The fabricated rat mandibular central incisor scaffold. (F) Harvest of in vivo-implanted rat mandibular central incisor scaffold orthotopically in the extraction socket showing integration of the implanted scaffold. Scale: 5 mm.

Ectopic tooth regeneration. (A) In human mandibular molar scaffolds, cells populated scaffold microchannels without growth-factor delivery. (B) Combined SDF1 and BMP7 delivery induced substantial cell homing into microchannels. (C) Combined SDF1 and BMP7 delivery homed significantly more cells into the microchannels than without growth-factor delivery (p < 0.01; N = 11). (D) Combined SDF1 and BMP7 delivery elaborated significantly more blood vessels than without growth-factor delivery (p < 0.05; N = 11). (E,F) Mineral tissue in isolated areas in microchannels adjacent to blood vessels and abundant cells, and confirmed by von Kossa staining. (G) Tissue sections from coronal, middle, and two root portions of human molar scaffolds were quantified for cell density and angiogenesis. s, scaffold; GF, growth factor(s). Scale: 100 µm.