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Hard Tissue Formation in Subcutaneously Transplanted Rat Dental Pulp

¹ Department of Oral Histology, Matsumoto Dental University, 1780 Gobara Hirooka, Shiojiri, Nagano 399-0781, Japan;
² Institute for Dental Science, Matsumoto Dental University, Nagano, Japan;
³ Department of Menicon Cartilage and Bone Regeneration, Graduate School of Medicine, The University of Tokyo, Tokyo, Japan;
⁴ Division of Cariology, Operative Dentistry and Endodontics, Department of Oral Health Science, Course for Oral Life Science, Niigata University Graduate School of Medical and Dental Sciences, Niigata, Japan;
⁵ Division of Cardiovascular Science, Department of Organ Regeneration, Shinshu University Graduate School of Medicine, Nagano, Japan;
⁶ Department of Orthopedic Surgery, Shinshu University Graduate School of Medicine, Nagano, Japan; and
⁷ Department of Endodontics and Operative Dentistry, Matsumoto Dental University, Nagano, Japan

View larger version (106K): [in this window] [in a new window]   Figure 1. BSE analysis (a–c) and light micrographs of sections stained by H-E (d–f) or immunostained for GFP (g,h) or BrdU (i–l). Transplanted pulp after 3 (d,i), 7 (a,e,j), and 14 (b,f,g,h,k) days. (a) Calcified hard tissue of the inner boundary of the pulp is evident. (b) Most of the pulp tissue is calcified. (c) The area of mineralized tissue was measured from images obtained by BSE analysis. (d) Typical odontoblasts have disappeared (asterisks) in the periphery of the pulp. (e) Extracellular matrix (stars in e–g,j,k) has formed within the peripheral pulp. (f) Matrix formation extends into the pulp. (g,h) GFP-positive cells are detected within the matrix and on its surface. "h" is an enlargement of the area denoted by the rectangle in "g", with the arrowheads pointing to GFP-positive cells on the surface of the matrix. (i) BrdU-positive cells are not seen in the pulp. (j) Numerous positive cells are apparent in the center of the pulp. (k) Positive cells have decreased in number. (l) Number of BrdU-positive cells counted in the immunostained sections. (c,l) Data are expressed as the means ± SE from 5 specimens per group. ANOVA and Student’s t test show a significant difference between the 2 groups. p values < 0.05 are considered statistically significant. The boundary between the transplant and the surrounding connective tissue (CT) is indicated by the dotted line (d–g, i–k). BV, blood vessel. Scale bar: 300 µm (a,b), 120 µm (g, i–k), 100 µm (d–f), 40 µm (h).

View larger version (96K): [in this window] [in a new window]   Figure 2. Immunohistochemical staining for OCN (a), OPN (b), BSP (c), and DSP (d) in the pulp 14 days after transplantation, positive controls (eh) for immunohistochemical staining for OCN (e), OPN (f), BSP (g), and DSP (h), and negative controls (i,j). (a–c) OCN, OPN, and BSP are localized within newly formed tissue, where the immunoreactivity is detected in globular aggregates. (d) DSP immunoreactivity is undetectable within the matrix. (e–h) Sections of a six-week-old normal molar tooth. OCN, OPN, and BSP immunoreactivities are detected in dentin matrix (D). Additionally, DSP is localized heavily in the dentin. (i,j) Control sections of pulp 14 days after transplantation. No immunoreaction is seen with non-immune goat or rabbit serum. Scale bar: 30 µm (a–j).

View larger version (138K): [in this window] [in a new window]   Figure 3. Electron micrographs of internal (a) and superficial (b) hard tissue in the 14-day transplant. (a) Numerous vesicle-like structures (arrows in the insert) are present in the extracellular matrix. An intact cell (asterisk) containing well-developed cellular organelles is also seen. (b) Matrix vesicles (arrows in the insert) are observed in the extracellular matrix. Go, Golgi apparatus; N, nucleus; rER, rough-surfaced endoplasmic reticulum. Scale bar: 2 µm (a,b), 0.2 µm (inserts of a,b).

View larger version (134K): [in this window] [in a new window]   Figure 4. Electron micrographs demonstrating various stages of calcification (a–d) and OPN localization (e,f) after 7 days. (a,b) At initial calcification sites, apatite crystals are observed within and outside the cells. (c,d) Calcification progresses to collagen fibrils (arrows in "c") from calcified nodules (CN). (e) Immunoreactivity for OPN is seen in the peripheral region of a calcified nodule. (f) The expanded collagen fibrils (asterisks) are immunopositive for OPN. Scale bar: 0.5 µm (d,e,f), 0.2 µm (b,c), 0.1 µm (a).