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RAPID COMMUNICATION |
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;
* corresponding authors, pyelick{at}forsyth.org, jbartlett{at}forsyth.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: tissue engineering • polymer scaffold • enamel • dentin
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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As tooth development progresses, dental mesenchyme differentiates into pulp and dentin, and the epithelial tissues of the enamel organ produce dental enamel. The cellular and molecular details of enamel, dentin, and cementum development have been characterized (Linde and Goldberg, 1993; Zeichner-David et al., 1995; Robey, 1996; Wu et al., 1996; Bartlett and Simmer, 1999; Grzesik et al., 2000; Jernvall and Thesleff, 2000; Saygin et al., 2000; Diekwisch, 2001; Saito et al., 2001).
To bioengineer teeth, we used a tissue engineering approach used by others to bioengineer small intestine successfully (Choi and Vacanti, 1997). Dissociated cells from tooth tissues were seeded onto biodegradable polymer scaffolds. The cell/polymer constructs were implanted into a suitable host such that a sufficient blood supply could support the growth of higher-ordered structures (Kim and Vacanti, 1999). Other approaches have been used to form partial tooth structures (Yamada et al., 1980; Yoshikawa and Kollar, 1981; Mina and Kollar, 1987; Slavkin et al., 1989; Thomas and Kollar, 1989). However, none of these approaches used dissociated tooth tissues.
Here we present histological, immunohistochemical, and molecular evidence demonstrating the successful bioengineering of complex tooth crowns closely resembling those of naturally developing teeth.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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We generated PLGA (Sigma-Aldrich, St. Louis, MO, USA) tooth scaffolds by packing PVS tooth molds to half-capacity with sodium chloride crystals (from 75 to 150 µm in diameter), then filling the remaining space with a 5% w/w 85:15 molar ratio PLGA solution in chloroform. Additional sodium chloride crystals were added to create a thick slush, and the mixture was lyophilized for 48 hrs. The scaffolds were placed in distilled water for 24 hrs to dissolve salt crystals.
Isolation and Dissociation of Porcine Third Molar Tooth Tissues
All experiments involving the use of animals were reviewed and approved by the IACUC at The Forsyth Institute. Third molar tooth buds were removed from six-month-old pig jaws and placed in 50 mL of Hanks’ balanced salt solution (HBSS) at 4°C. Enamel and pulp organ tissues were minced into 2- to 3-mm3 pieces in HBSS. Tissues were washed 5X in HBSS, minced into < 1 mm3 pieces, enzymatically treated (1.5 units of Vibrio alginolyticus collagenase and 12 units of Bacillus polymyxa dispase; Roche, Indianapolis, IN, USA) in HBSS for 25 min at 25°C, and gently dissociated by trituration. Cells were then washed in DMEM, pelleted, and counted. The largest organoid units remaining after treatment were 100-200 µm in diameter and consisted of approximately 20-30 cells. Typical cell yields were 1 x 107 cells per porcine tooth bud.
Tooth Scaffolds, Cell Seeding, and Implantation
Scaffolds were collagen-coated overnight at 4°C (1 mg/mL type I collagen/10 mM HCl), washed in phosphate-buffered saline (PBS) and DMEM, and seeded with 
2.0 x 106 cells in DMEM for 1 hr at 4°C. Laparotomies were performed on athymic rats, and scaffolds were implanted in the omentum as described (Choi and Vacanti, 1997).
Histology
After 20-30 wks, developing tooth tissues were excised, fixed in 4% formalin, embedded in paraffin, thin-sectioned, and decalcified in a solution consisting of 22.5% v/v formic acid and 10% w/v sodium citrate. Analyses included: hematoxylin and eosin (Stevens et al., 1990); Von Kossa (Stevens et al., 1990); and Goldner staining (Stevens et al., 1990).
Immunohistochemical Analysis
Immunohistochemical analysis was performed by means of the Vectastain ABC kit (Vector Laboratories, Inc., Burlingame, CA, USA). Antibodies included: affinity-purified rabbit anti-pig amelogenin polyclonal antibody (1:2000 dilution) (gift from J.P. Simmer, University of Texas, San Antonio); affinity-purified sheep anti-pig collagen (type I) (1:500 dilution), and rabbit anti-pig bone sialoprotein (BSP) polyclonal antibody (1:500 dilution) (gifts from J. Sodek, University of Toronto).
Laser-capture Microdissection and Reverse-transcription/Polymerase Chain-reaction (LCM, RT-PCR) Analysis
LCM RT-PCR analyses were performed with the use of a Leica laser-capture microscope model AS LMD. Approximately 200-500 odontoblast cells were dissected from paraffin-embedded sections of 25-week implant tissue, and positive control porcine third molar. Total RNA was isolated, reverse-transcribed (SuperscriptTM First-Strand Synthesis System for RT-PCR, Invitrogen, Carlsbad, CA, USA), and used as template in nested PCR reactions. PCR was performed (Platinum PCR Supermix, Invitrogen, Carlsbad, CA, USA) with the use of dentin sialophosphoprotein (DSPP) outer primers (forward primer 5'-CAGCCGCTGATTAATATTCCTAAA-3', reverse primer 5'- TAACATGGGACGTGCAGAAGAACT-3') and DSPP nested primers (forward primer 5'-ATGGGCCATTCCAGTTCCTC-3', reverse primer 5'-TGCCCACTCAGAGCCATTTC-3'). Reaction conditions for DSPP outer primers: 95°C for 2 min; 40 cycles at 95°C for 30 sec, 54.3°C for 30 sec, and 72°C for 30 sec to generate a 223-bp product. DSPP nested reactions were performed as described, except that the annealing temperature was 58°C. For ß-actin, outer primers (forward primer, 5'-GCGGGGCTACAGCTTCACCAC-3', reverse primer, 5'- ATCTCCTTCTGCATCCTGTCG-3'), and nested primers (forward primer, 5'- TGGACTTCGAGCAGGAGATGG-3', and reverse primer, 5'- CAGCACCGTGTTGGCGTAGAG-3') were used to generate a 236-bp product, under the same conditions as for the DSPP reactions.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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). Mineralized dentin (d) and pre-dentin layers (pd) were apparent, adjacent to odontoblast-like cells (od). Vascularized mesenchyme resembling that of pulp tissue (p) was also present (Figs. 1A, 1B, 1C). At higher magnification, putative odontoblasts (od) were visible lining the inner surface of a pre-dentin matrix (pd). Tissue closely resembling Hertwig’s root sheath epithelia (hers) was located adjacent to developing root tips (Fig. 1B). The bioengineered tooth structure (Fig. 1C) bore close resemblance to a tooth cusp from a porcine third molar (Fig. 1D).
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, arrow) immunostaining was observed in the dentin of the 20-week tissue-engineered implant, suggesting that mesenchymally derived dentin tissues were present in the 20-week tooth tissues. Controls treated with pre-immune serum exhibited no staining (Fig. 1F). Thus, by 20 wks, engineered tissues exhibited organized dentin and pulp structures.
Histological and Immunohistochemical Analyses of 25-week Implants
Histological analysis of 25-week implant tissue revealed tooth tissues with dimensions of approximately 2 by 2 mm. Tissue closely resembling decalcified enamel (e) was observed (Figs. 2A-2E) adjacent to the dentin (d), as is found in normally developing teeth (Fig. 2B). Amelogenin was immunodetected in the enamel matrix of the 25-week implant (Fig. 2C, arrow), while controls treated with pre-immune serum were negative (Fig. 2D). Numerous columnar cells possessing polarized nuclei that are characteristic of ameloblasts (am) were present adjacent to the enamel (Figs. 2A, 2E). Further evidence that this tissue was in fact mineralized enamel was demonstrated by a red Goldner stain (Fig. 2E), which stains dentin blue-green, and immature enamel bright red (Z. Skobe, personal communication).
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), similar to that observed in naturally formed porcine third molar tooth sections (Bronckers et al., 1993). In addition, tissue-engineered odontoblasts expressed BSP throughout the cytoplasm, and weaker expression was observed in the odontoblastic processes and dentin tubules (Figs. 2H, 2J, arrows), as was observed in natural porcine tooth tissues (Chen et al., 1993). Controls treated with pre-immune serum were negative (Figs. 2G, 2I, 2K). Thus, by 25 wks of development, bioengineered tooth tissues consisted of the two major mineralizing structures of the tooth, dentin and enamel.
Histological and Immunohistochemical Analyses of 30-week Implants
Histological analysis of 30-week post-implantation tissue demonstrated a layer of dentin surrounded by a thick layer of enamel (Fig. 3A). At higher magnification, putative cementoblasts (c) and cementum were present adjacent to the dentin (Fig. 3B), and rows of columnar ameloblast cells (am) with polarized nuclei were clearly visible (Fig. 3C). The ameloblasts in the bioengineered tissue were similar in appearance to those of porcine third molar teeth (Fig. 3D). The cellular tissue adjacent to the ameloblasts was morphologically similar to the stratum intermedium (si) which, in turn, adjoined cellular tissue resembling the stellate reticulum (sr) (Fig. 3C). This tissue organization is found in naturally developing enamel organs (Fig. 3D). The identity of the ameloblasts was confirmed by positive immunostaining for amelogenin. Amelogenin was present at the secretory ends of ameloblast cells and throughout the adjacent enamel matrix (Fig. 3E, arrow). No staining was observed in control sections (Fig. 3F). BSP was detected in the dentin tubules of both engineered (Fig. 3G) and positive control porcine third molars (Fig. 3H). Thus, at 30 wks, bioengineered tooth tissues contained enamel, dentin, and cementum-like mineralized tissues, and expressed appropriate tissue-identifying protein markers including type I collagen, BSP, and amelogenin.
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The expected 223-bp DSPP product was generated from control porcine third molar and tissue-engineered odontoblasts (Fig. 4, lanes 3 and 4). DSPP mRNA was not detected in the spleen (Fig. 4, lane 2). A 236-bp ß-actin product was detected in all tissues, confirming the integrity of isolated mRNAs (Fig. 4, lanes 2, 3, 4).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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In this report, we present morphological, histological, immunohistochemical, and LCM RT-PCR analyses characterizing bioengineered tooth tissues generated by seeding dissociated tooth bud cells onto biodegradable scaffolds. These analyses demonstrate the successful engineering of recognizable tooth structures exhibiting the cellular organization and presence of appropriate proteins found in natural teeth. The bioengineered teeth had well-defined pulp chambers, odontoblasts, pre-dentin, and dentin, and contained a morphologically correct enamel organ consisting of stellate reticulum, stratum intermedium, ameloblasts, and dental enamel. In addition, putative Hertwig’s root sheath epithelia were also present. At 20, 25, and 30 wks post-implantation, the bioengineered teeth had recognizable crown structures that exhibited distinct coronal and apical organization with recognizable cusps and root tips. A variety of other techniques has previously been used to generate similar tooth structures (Glasstone, 1967; Slavkin et al., 1969; Thesleff, 1976; Yamada et al., 1980; Yoshikawa and Kollar, 1981; Laine and Thesleff, 1986; Mina and Kollar, 1987; Slavkin et al., 1989; Thomas and Kollar, 1989). However, this report is the first to use dissociated tooth tissues seeded onto biodegradable scaffolds, a method that potentially provides the opportunity to generate teeth of pre-determined size and shape.
Bioengineered Tooth Structures Contain Dentin and Enamel-specific Proteins
Although dentin and bone are similarly composed of about 70% hydroxyapatite and 30% organic material (Linde and Goldberg, 1993), histological analyses demonstrate distinct cellular and morphological differences between these tissues. Individual bone-secreting osteoblasts exist as osteocytes in the bone matrix, giving it a trabecular appearance, while odontoblasts remain on the periphery of forming dentin, and extend cellular processes through the dentin matrix. Dentin tubules, and not osteoid bone, were clearly evident in 20-, 25-, and 30-week bioengineered tooth tissues (Figs. 1, 2, 3). In addition, columnar odontoblast-like cells were present adjacent to the forming dentin, with characteristic processes that radiated into and extended through the dentin tissues. These morphological features, along with positive immunostaining for protein markers collagen type I, BSP (Figs. 1, 2, 3), and DSPP expression (Fig. 4), strongly support the presence of odontoblasts and dentin in the bioengineered tooth tissues.
Mineralized enamel consists of greater than 95% mineral and is formed as an acellular tissue (LeFevre and Manly, 1932; Deakins and Volker, 1941). The enamel present in decalcified 25- and 30-week bioengineered tooth tissues closely resembled decalcified control porcine enamel and was immunoreactive with an amelogenin antibody, as were the columnar ameloblast-like cells adjacent to the enamel. By 30 wks, the bioengineered tooth tissues exhibited enamel covering cusps, as found in a natural tooth crown. This enamel was mature and virtually protein-free.
Our current tissue engineering efforts consistently generate small (2 by 2 mm) tooth structures using single-cell suspensions derived from both pig and rat tooth bud tissues. Notably, the bioengineered teeth are very small and do not conform to the size and shape of the scaffold. To address these issues, we are currently investigating dental epithelio-mesenchymal cell and cell-scaffold interactions in developing bioengineered tooth structures. Investigations into dental stem cell contribution to bioengineered tooth structures is currently a high priority.
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ACKNOWLEDGMENTS |
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Received June 19, 2002; Last revision July 30, 2002; Accepted August 6, 2002
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