Proliferation and differentiation sequence of osteoblast histogenesis under physiological conditions in rat periodontal ligament

To define the mechanism of osteoblast histogenesis, nuclear morphometry was utilized as a marker for precursor cell differentiation. One hour after ³H-thymidine injection, groups of 7-week-old rats were killed at hourly intervals over one complete 24-hr photoperiod (LD 12:12). S-phase and mitosis were assessed in autoradiographs of 3-μm sections of molar periodontal ligament (PDL) adjacent to a physiological bone-forming surface. Labeled nuclei were divided into four categories according to morphometry of nuclear size: A (40–79 μm³), B (80–119 μm³), C (120–169 μm³), and D (≥ 170 μm³) cells. C and D cells synthesize DNA during the light and divide in the following dark phase; the rhythm for A cells is the opposite. B cells demonstrated no preference and were subsequently determined to be nonosteogenic. Compared to A cells the S-phase photoperiod of C and D cells (combined) is approximately a one-to-one reciprocal relationship, suggesting two proliferating progenitors in series. Based on arrest points in the histogenesis sequence, five compartments are defined: (1) A cells, less differentiated, self-perpetuating precursors; (2) A′ cells, committed osteoprogenitors; (3) C cells, G₁ stage preosteoblasts; (4) D cells, G₂ stage preosteoblasts; and (5) Ob cells, morphologically distinct osteoblasts. Minimal elapsed time for the A →A′ → C → D → Ob sequence is about 60 hr (five alternating dark/light cycles). A stress/strain-mediated increase in nuclear volume (A′ → C) is an important, rate-limiting step in osteoblast differentiation.