Characterization of Cultured Rat Tooth Bud Cells
To determine the age at which rat tooth buds were optimal for tooth tissue bioengineering, we prepared cultured dental cell populations from 3- to 7-dpn rat tooth buds. At least 3 experiments with a minimum of 6 rat pups (48 molar tooth buds) were performed at each developmental stage. After 6 days in culture, the dental cells appeared to be heterogeneous, consisting of fibrous, mesenchymal-like cells and clusters of smaller, epithelial-like cells (Figs. 1A, 1B). Cultured 5-, 6-, and 7-dpn tooth bud cells appeared to be dying at 6 days, exhibiting numerous floating cells and low total cell counts. In contrast, 3- and 4-dpn tooth bud cell cultures appeared healthy and exhibited average cell count/tooth bud of (2.0 x 105) and (1.5 x 105), respectively. Since 4-dpn tooth bud cells appeared to be proliferating in culture while 3-dpn tooth bud cells did not, 4-dpn rat molar tooth bud cells were selected for use in all subsequent tooth-tissue-engineering experiments.
We used immunohistological analysis of cytokeratin expression to identify epithelial cells in mixed epithelial and mesenchymal dental cell cultures. Brightly fluorescing cytokeratin-positive dental epithelial cells were readily identifiable under UV illumination (Fig. 1D
, white arrows), while dental mesenchymal cells exhibited only background fluorescence. Positive control oral epithelium exhibited distinct cytokeratin immunoreactivity (Fig. 1C
). Six-day cultured dental cells were harvested and seeded onto either PGA or PLGA scaffolds for 1 hr at 37°C in a humidified, 5% CO2
environment. Light microscopic analysis of cell-seeded PGA and PLGA scaffolds revealed cells attached to both polymer scaffolds (Figs. 1E, 1F
Experimental and Control Implant Groups
Control groups C1-C3 consisted of: (C1) 7 non-dissociated 4-dpn molar tooth buds implanted as positive controls; (C2) 5 unseeded PGA scaffolds; and (C3) 5 unseeded PLGA scaffolds. Experimental groups E1 and E2 consisted of: (E1) 8 PGA scaffold implants seeded for 1 hr; and (E2) 8 PLGA scaffold implants seeded for 1 hr. Control and experimental implants were grown in the omenta of syngeneic adult rat hosts for 12 wks, as determined empirically by the detection of distinctly radio-opaque tissues in dental-cell-seeded scaffold implants.
Visual and Radiographic Analyses of Excised Implants
Histological Analysis of 12-week Implants
At 12 wks, experimental and control implants were excised and analyzed. By visual inspection, the implants appeared similar in color, size, and shape. Numerous experimental implants exhibited mineralized tissues protruding from the implant (Figs. 2A, 2B). Radiographic analyses of experimental implants revealed the presence of highly mineralized tissues (Figs. 3A', 3B'). Negative control, unseeded scaffold groups C2 and C3 contained no radio-opaque tissue (data not shown). A total of 7 out of 8 (88%) PGA and 4 out of 8 (50%) PLGA implants contained radio-opaque tissues.
We performed histological analyses to determine the cellular organization of mineralized implant tissues. All of the C1 control implants developed into accurately formed rat molar teeth containing identifiable dentin, enamel, and pulp (Figs. 3A, 3A'
), although cementum and periodontal ligament tissues were not definitively identifiable in these implants at 12 wks. Histological analyses of PGA and PLGA scaffold cell-seeded implants demonstrated the presence of dentin, enamel, and pulp tissues (Figs. 3B, 3B', and 3C, 3C'
, respectively). Hertwig’s epithelial root sheath structures formed on both types of scaffold material. Infiltrating lymphocytes were evident in some of the sectioned implants (Fig. 3C'
The mineralized tissues of control and experimental implant groups were examined with the use of Goldner’s stain (Bancroft and Gamble, 2002), which stains dentin and bone blue, newly formed enamel matrix red, and mature enamel matrix gray (Z. Skobe, personal communication). Intact tooth bud control implants exhibited blue-stained dentin, red-stained newly formed enamel, and gray-stained mature demineralized enamel (Figs. 3D, 3D'). Similarly, tooth tissues bioengineered on both PGA and PLGA scaffolds exhibited blue-stained dentin, and gray-stained mature enamel (Figs. 3E, 3E', and 3F, 3F', respectively). Tooth tissues generated on PGA scaffolds generally exhibited more mature enamel that stained gray with Goldner’s (Fig. 3E'), while PLGA cell-seeded scaffolds generated both immature and mature enamel that stained reddish to gray (Fig. 3F).
Immunohistochemical Analysis of Bioengineered Rat Tooth Tissues
We used immunohistochemical analysis to examine the expression of amelogenin in bioengineered enamel. Control intact tooth bud implants exhibited positive amelogenin expression in ameloblasts and in demineralized enamel (Fig. 4A, arrows), while pre-immune control tissues were negative (Fig. 4A'). Bioengineered enamel grown on both PGA and PLGA scaffolds exhibited positive staining for amelogenin (Figs. 4B, 4B', and 4C, 4C', respectively).