MATERIALS & METHODS
Animal Husbandry
Lewis rats were maintained, and surgeries were performed, in the Forsyth Institute Animal Facility, following National Institutes of Health (NIH) and ALAC regulations, IACUC protocol #01-009, and animal assurance #A3051-1.
Polymer Scaffold Fabrication
Rectangular scaffolds (1 x 5 x 5 mm) were fabricated from polyglycolic acid (PGA) and poly co-glycolide copolymer (PLGA) as previously described (Young et al., 2002). Briefly, PGA fiber mesh containing 3% w/w poly(L-lactic acid) was packed into molds in chloroform, lyophilized for 48 hrs, and sanitized with 75% ethanol. We generated PLGA tooth scaffolds by packing polyvinylsiloxane molds half full with NaCl crystals, filling the remaining space with a 5% w/w, 85:15 molar ratio PLGA solution in chloroform, lyophilizing for 48 hrs, leaching the scaffolds in distilled water for 24 hrs, and sanitizing in 75% ethanol.
Isolation, Culturing, and Seeding of Rat Tooth Bud Cells
Molar tooth buds were isolated from 3- to 7-dpn Lewis rat pups and minced into balanced salt solution (HBSS, Gibco BRL, Gaithersburg, MD, USA). Tooth bud tissues were digested with type I collagenase (0.66 mg/mL, Sigma-Aldrich, St. Louis, MO, USA) and Dispase I (0.33 mg/mL; Boehringer Mannheim, Indianapolis, IN, USA), dissociated by trituration, and washed 5x in 50% Dulbecco’s modified Eagle medium (DMEM, Gibco BRL, Gaithersburg, MD, USA) containing 10% fetal bovine serum (FBS), 5 mL Glutamax, 50 units/mL penicillin, 50 µg/mL streptomycin, 2.5 µg/mL ascorbic acid, and 50% F12 medium (Sigma-Aldrich Corp, St. Louis, MO, USA). Single-cell suspensions were generated by filtration through a Falcon 40-micron cell strainer, typically yielding 2.4 x 105 cells/tooth bud. Cells were re-suspended in DMEM/F12, plated into 75-cm2 (T75) culture flasks (Costar, Cambridge, MA, USA) at 2.5 x 105 cells/mL, and grown in 5% CO2 at 37°C until the cells reached confluence at 6 days. Cells were harvested by trypsinization (0.25% trypsin/EDTA; Gibco-Invitrogen Corp., Tulsa, OK, USA) for 10 min at 37°C, washed twice with the same medium, recounted, split into equal portions, and statically seeded onto PGA and PLGA scaffolds for 1 hr prior to implantation into the omenta of syngeneic Lewis rat hosts.
Immunohistochemical analysis of cytokeratin expression in cultured epithelial tooth bud cells was performed with the use of the monoclonal pan-cytokeratin antibody PCK-26 (Sigma-Aldrich, St. Louis, MO, USA), according to the manufacturer’s recommended protocol.
Omental Implant Procedure
Adult Lewis rats (Charles River Laboratories, Wilmington, MA, USA), aged 6–12 mos, were used as hosts for tooth tissue implants. Omental surgeries were performed as previously described (Young et al., 2002).
Analyses of Implant Tissues
Radiographic analyses were performed with the use of a Hewlett-Packard Faxitron (Model 43855 TO-2) and Kodak (Rochester, NY, USA) high-speed SO-253 holographic film at 40 Kv and 3 mA for 30 min at a focal distance of 40 cm. After visual and radiographic inspections, implants were fixed in 5% formalin for 24 hrs, decalcified, embedded in paraffin, sectioned at 6-micron intervals, and stained with hematoxylin and eosin (H&E) or Goldner’s trichrome. Immunohistochemical analyses were performed using a polyclonal amelogenin antibody as previously described (Young et al., 2002). Sectioned and stained specimens were examined with the use of a Leica DMRE compound microscope and digital Zeis Axiocam digital camera (Stuttgart, Germany).
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