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Tissue Engineering of Complex Tooth Structures on Biodegradable Polymer Scaffolds

¹ Department of Cytokine Biology and Harvard-Forsyth Department of Oral Biology, The Forsyth Institute, Boston, MA 02115, USA;
² Department of Surgery, Massachusetts General Hospital and Harvard Medical School, Boston, MA, USA; and
³ Department of Oral and Maxillofacial Surgery, Nagoya University School of Medicine, Nagoya, Japan;

View larger version (129K): [in a new window]   Figure 1. Histology and immunohistochemistry of a 20-week implant. (A) Von Kossa stain for calcified mineralization in bioengineered tooth crown (50X magnification). Dark brown stain is positive for mineralized tissues. (B) A high-magnification (400X) photomicrograph of the Hertwig’s epithelial root sheath is shown, stained by the Von Kossa method to detect calcified mineralization. (C) High-magnification (200X) photomicrograph of cuspal region in bioengineered tooth crown. The tissue was stained by the Von Kossa method. (D) Hematoxylin and eosin (H&E) stain of a positive control porcine third molar cuspal region demonstrates morphology similar to that of the bioengineered tooth structure (200X). (E) BSP immunostain of 20-week bioengineered tooth crown (100X). Positive BSP expression is indicated by the arrow. (F) Negative pre-immune control immunostain for BSP in bioengineered tooth crown (100X). Abbreviations: d, dentin; od, odontoblasts; p, pulp; pd, predentin, hers, Hertwig’s epithelial root sheath.

View larger version (141K): [in a new window]   Figure 2. Histology and immunohistochemistry of a 25-week implant. (A) High-magnification photomicrograph (400X) of ameloblasts present in a bioengineered tooth crown on an H&E-stained section. (B) H&E stain of decalcified enamel from a positive control porcine third molar (200X). (C) Amelogenin expression (arrow) in decalcified enamel of a bioengineered tooth crown (200X). (D) Negative pre-immune control for amelogenin (200X). (E) Goldner’s stain of bioengineered tissues revealing enamel matrix (bright red) and bone and dentin (blue-green) (200X). (F) Collagen type I expression in odontoblasts and dentin in bioengineered tissues. Positive brown stain for collagen type I is indicated by the arrow (400X). (G) Negative pre-immune control for collagen type I (400X). (H,J) Bone sialoprotein (BSP) expression in odontoblasts (H) and dentin (J) in 25-week bioengineered tissues (400X). Positive brown stain for BSP is indicated by the arrows. (I,K) Negative pre-immune controls for BSP (400X). Abbreviations as in Fig. 1 and: e, decalcified enamel; am, ameloblast; od, odontoblast; p, pulp.

View larger version (158K): [in a new window]   Figure 3. Histology and immunohistochemistry of a 30-week implant. (A) H&E stain of dentin and enamel structures in a 30-week bioengineered tooth tissue (50X). (B) H&E stain of dentin, enamel, and putative cellular cementum in bioengineered tissue (400X). (C) H&E stain of enamel organ cells present in bioengineered tissues (400X). (D) H&E stain of enamel organ cells from a positive control porcine third molar tooth (400X). (E) Amelogenin expression in ameloblasts and enamel of 30-week bioengineered tissues. Positive brown stain is indicated by the arrows (400X). (F) Negative pre-immune control for amelogenin immunostaining (400X). (G) Bone sialoprotein (BSP) expression in dentin of 30-week bioengineered tissue (400X). Punctate expression pattern is indicated by arrows. (H) BSP expression in dentin of a positive control porcine third molar tooth (400X). Abbreviations as in Fig. 2 and: si, stratum intermedium; sr, stellate reticulum.

View larger version (51K): [in a new window]   Figure 4. Laser capture RT-PCR of dentin sialoprotein (DSPP) and ß-actin mRNA in bioengineered odontoblasts. Lane 1, no template negative control; Lane 2, porcine spleen tissue; Lane 3, positive control porcine third molar odontoblasts; Lane 4, tissue-engineered (TE) odontoblasts. The DSPP product was 223 bp, and the ß-actin product was 236 bp.