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Bioengineered Dental Tissues Grown in the Rat Jaw
S.E. Duailibi1, ,
M.T. Duailibi1,,
W. Zhang2,
R. Asrican3,
J.P. Vacanti4 and
P.C. Yelick2,*
1 University Federal of São Paulo, Department of Plastic Surgery, UNIFESP-CINTERGEN, Interdisciplinary Center of Gene Therapy, São Paulo, Brazil;
2 Division of Craniofacial and Molecular Genetics, Department of Oral and Maxillofacial Pathology, Tufts University, Boston, MA 02111, USA;
3 Department of Cytokine Biology, The Forsyth Institute, and Department of Developmental Biology, Harvard School of Dental Medicine,
4 Laboratory for Tissue Engineering and Organ Fabrication, Massachusetts General Hospital and Department of Surgery, and Harvard Medical School, Boston, MA, USA
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Figure 1. Dental cell proliferation in culture. (A) Analysis of cell number at day 0, and after 10 days in culture, revealed that enzyme concentrations of 0.4 mg/mL collagenase and 0.2 mg/mL dispase resulted in the highest cell yields. (B) Growth curves for digested tooth bud cell preparations confirmed that tooth buds digested with 0.4 mg/mL type I collagenase and 0.2 mg/mL dispase I exhibited the highest cell number after 10 days in culture. (C) Digital images of cultured tooth bud cells, prepared with the 0.4 mg/mL collagenase and 0.2 mg/mL dispase enzyme concentrations, after 2, 4, and 10 days in culture. Five independently isolated cell preparations were examined for each type of analysis (Table ). Wilcoxon’s test revealed highly significant values of P = 0.0003. Scale bar is 2 mm.
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Figure 2. Radiographic and histological analysis of harvested and sectioned 12-week experimental and control implants. (A) Negative control implant site. (B) Localized radiopaque areas (arrows) indicate mineralized tissue formation at the cell-seeded implant site. (C,E) H&E-stained positive control implanted 4-dpn tooth buds exhibited well-formed tooth structures, with organized crown and root structures. (D,F) Goldner’s stained 4-dpn tooth bud implants revealed characteristic blue-stained dentin, and brown-stained immature enamel. (G,I) H&E-stained bioengineered dental tissues present in jaw implants. (H,J) Goldner’s stained sections revealed blue-stained dentin, brown-stained immature enamel, and gray-stained mature enamel tissues. Unseeded, negative control scaffolds did not form dental tissues (data not shown). All 16 of the experimental cell-seeded implants and all of the 14 implanted tooth bud controls produced radiopaque dental tissues. None of the negative control unseeded scaffolds produced radiopaque mineralized tissue. Abbreviations: am, ameloblasts; b, bone; d, dentin; e, enamel; ime, immature enamel; me, mature enamel; od, odontoblasts; p, pulp; pd, predentin. Scale bars are 100 µm.
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Figure 3. Immunohistochemical (IHC) analysis of mandibular implant bioengineered tooth tissues. Bioengineered dental tissues were positive for amelogenin (AM) (A,B), dentin sialophosphoprotein (DSPP) (D,E), periostin (PER) (G,H), and vimentum (VM) (J,K). Corresponding immunostaining of natural tooth tissues is shown (C,F,I,L). All isotype control immunostaining was negative. Three experimental PGA and three experimental PLGA cell-seeded scaffold implants were analyzed by IHC. Two negative control ‘scaffold alone’ controls and 2 positive control tooth bud implants were also analyzed by IHC. Abbreviations: AM, amelogenin; DSPP, dentin sialophosphoprotein; PER, periostin; VM, vimentum. Scale bars = 200 µm.
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Journal of Dental Research, Vol. 87, No. 8,
745-750 (2008)
DOI: 10.1177/154405910808700811
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