Immunolocalization of a Human Cementoblastoma-conditioned Medium-derived Protein
H. Arzate1,*,
L.F. Jiménez-García2,
M.A. Álvarez-Pérez1,
A. Landa3,
I. Bar-Kana4, and
S. Pitaru4
1 Laboratorio de Biología Celular y Molecular, División de Estudios de Posgrado e Investigación, Facultad de Odontología, UNAM, Cd. Universitaria, 04510, México DF, Mexico;
2 Departamento de Biología, Facultad de Ciencias, UNAM, Mexico;
3 Departamento de Microbiología y Parasitología, Facultad de Medicina, UNAM; and
4 The Maurice and Gabriela Goldschleger School of Dental Medicine, Tel Aviv University, Israel;
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Figure 2. Immunoblotting of immunoaffinity-purified fractions with anti-CP polyclonal antibody. Lane 1: Immunopurified conditioned medium from cementoblastoma-derived cells conditioned medium (0.9 µg/500 mL) showed an intense 56-kDa species cross-reacting with anti-CP antibody. Lane 2: Periodontal ligament cells immunopurified conditioning medium (0.4 µg/500 mL) expressed the 56-kDa protein. Lane 3: Alveolar bone-derived cells conditioning medium (0.1 µg/500 mL) did not cross-react with the 56-kDa species. Lane 4: Control for human serum was negative. Lane 5: Immunopurified conditioning medium from cementoblastoma-derived cells showed that a 70-kDa band cross-reacted with a monoclonal antibody (3G9) raised against bovine CAP. From top to bottom, arrows indicate the migration of protein standards of 68, 44, 32, and 22 kDa, respectively.
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Figure 3. Immunostaining of CP in human periodontal tissues. (A) Longitudinal section of a human tooth. Cementoid surface is strongly stained. Open vascular channels and endosteal spaces in alveolar bone are positive (arrowheads and arrow, respectively). (B) Transverse section of a human tooth shows both cementoid and bone endosteal spaces stained positive. (C) A few periodontal ligament cells are positive as well as cementoblasts lining the cellular cementum. Cells located adjacent to the periodontal ligament blood vessels are strongly positive (arrowhead). A cementocyte with cytoplasmic elongations localized within the cellular cementum is shown to be positive (arrow). (D,E) Colony-like cell formation (possibly pre-cementoblasts) in the vicinity of blood vessels (arrow). (F) Cells surrounding endosteal spaces in alveolar bone cross-reacted strongly to anti-CP antibody. (G) Cementocytes inside the cementum matrix are positive. (H) Higher magnification of strongly labeled cementocytes showing cytoplasmic processes interconnecting them. (I) Cementoblasts just becoming embedded in the cementum matrix cross-reacted more strongly than periodontal ligament cells and pre-cementoblasts. (J) Acellular cementum was strongly stained with anti-CP antibody. (K) Control using pre-immune rabbit serum was negative. Histological H & E-stained sections show longitudinal aspects of periodontal structures: open vascular channels (arrowheads), endosteal spaces in alveolar bone (arrow) (L), a cementocyte, and periodontal ligament blood vessel (M). Cells representing pre-cementoblasts located in the vicinity of periodontal ligament blood vessels (N), and a cementoblast becoming embedded in cementum matrix (arrowhead) (O).
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Figure 4. Photomicrographs showing localization of CP in cementoblastoma-derived cells. Positive immunostaining of CP in cementoblastoma-derived (A) and periodontal ligament cells (B). Alveolar bone-derived cells staining appears close to the background levels (C).. Controls using pre-immune rabbit serum were negative for the 3 cell types (D, E, and F for cementoblastoma, periodontal ligament cells, and alveolar bone-derived cells, respectively). Magnification 20X. Bar = 100 µm.
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, alveolar bone-derived cells •, and gingival fibroblasts 
. Asterisk (*) indicates significant differences in cell attachment between periodontal ligament and alveolar bone-derived cells vs. gingival fibroblasts at P < 0.05.