Figure 1. Isolation and characterization of periodontal ligament. (A1) Cartoon depiction of a parasagittal cross-section through the jaw and attached tooth. Tooth slices (rectangle) were made in the apical region of the extracted tooth to avoid any contamination with gingival epithelial cells. Tooth slices, termed ‘organotypic tooth explants’, had dimensions of a half-millimeter depth, a width of 1–4 mm, and a height of 2–5 mm; variation was due to topography of the tooth and differences in circumference. (A2) Paraffin section (12 µm) from an organotypic tooth explant stained with hematoxylin and eosin. Organotypic tooth explants always consisted of cementum (C) and periodontal ligament (PDL) and dentin (D). In approximately 15% of the sections, a layer of alveolar bone (B) was present. (B1) Proliferative periodontal cells were then cultured in vitro from organotypic tooth explants. Greater than 80% of the cells cultured have a spindle shape resembling the morphological properties noted for human periodontal ligament cells (black arrows). (B2) Cultured cells from explants stain positive for collagen III (green fluorescence). Thus, periodontal ligament fibroblasts were isolated from the cellular population based on morphology and high levels of collagen III expression. (C1-G3) Staining of periodontal tissues for collagen III, osteopontin, osteocalcin, BMB-2/4, and bone sialoprotein in vivo was completed on 12-µm paraffin or fresh-frozen sections. Collagen III staining (brown, panel C2), osteopontin staining (brown, panel D2), osteocalcin staining (brown, arrows, panel E2), BMP-2/4 staining (brown, panels F1 and F2), and bone sialoprotein staining (brown, arrows, panels G2 and G3) of acute organotypic tooth explants. Panels C1, D1, E1, and G1 are controls where non-immune serum was added, instead of primary antibody, during the immunohistochemical procedure. In panels C1-G2, the bone (b), periodontal ligament (pdl), cementum (c), and dentin (d) tissue layers are indicated. Bars = 50 µm (B2, C1, C2, D1, G1, D2), 100 µm (E1, E2, G2), and 200 µm (F1). Panels F2 and G3 are high-magnification images of regions in panels F1 and G2, respectively.