Cell Kinetics of Osteoblast Histogenesis in Evolving Rabbit Secondary Haversian Systems Using a Double Labeling Technique with [³H]-Thymidine and Bromodeoxyuridine

Abstract

The mechanism for internal cortical bone remodeling is orchestrated by the evolving secondary Haversian systems (SHSs), which originate on the surfaces of Volkmann's canals. During this coupled process, a cortical tunnel advances by the cutting cone of osteoclasts and closes by the bone-forming trail of osteoblasts. This study investigated the hypothesis that osteoblast histogenesis, within evolving SHSs of larger animals, is a vascular-related process, i.e., less differentiated osteogenic cells reside in close proximity to the advancing central blood vessel (CBV) while differentiating osteoblast precursors migrate toward the bone surface and become osteoblasts. Using a double-labeling method with [3 H]-thymidine and bromodeoxyuridine (BrdU), this study examined cell kinetics in 120 SHSs per rabbit at 12 hour intervals up to 72 hours after labeling. A total of 7 rabbits were injected with alizarin complexone (-1 O days: 20 mg/kg/day), tetracycline (-3 days: 10 mg/kg/day), [3H]-thymidine (time zero: 0.25 μCi/gm), and BrdU (1 hour before sacrifice: 25 mg/kg). The femoral midshaft was used for undecalcified fluorescent microscopic analysis of new bone vs. old bone and two adjacent diaphyseal tissues were demineralized for nuclear volume morphometric analysis of cells via light microscopy. Evolving SHSs demonstrating intense remodeling activity were selected for detailed cell kinetic analysis. The results showed that BrdU labeled cells were consistently located at the leading edge of the CBV (within 160 μm of its tip) and that the [3H]thymidine labeled cells were progressively left behind the advancing CBV (160 μm from the tip of CBV by 72 hours). The labeling indices (sampled 1 hour after labeling) between BrdU (10.6 ± 0.3 %) and [3H]-thymidine (14.4 ± 1.3 %) were comparable. Lightly labeled A+A' cells (identified as osteoprogenitor cells) remained in close approximation to the surface of the CBV (within 25 μm) and C+D cells (preosteoblasts) were located closer to bone-forming surfaces (~50 μm away from the CBV). The number of osteoblasts were increased up to 60 hours and about 22.5 ± 6.6 % of them survived to become osteocytes. The B cell compartment, characteristic of osteogenic tissues with a dense connective tissue component such as the periodontal ligament (POL), was essentially absent in the SHSs in this study. Although the direction of evolving SHSs was highly variable (caudally directed: 53.4 ± 11.2 % and rostrally directed: 41.6 ± 8.1 %), the osteogenic process along the advancing CBV was remarkably consistent. These results support the hypothesis that osteoblast histogenesis, associated with cortical bone remodeling, is a vascular-oriented differentiation process closely related to the internal angiogenesis within the evolving SHS. The primary proliferating region supporting osteogenesis was consistently located at the advancing tip of the CBV, suggesting the presence of a self-renewing, perivascular proliferative pool of cells accompanying the advancing vessel. In addition, a secondary proliferating region of cells trails the advancing CBV, providing for lateral migration of preosteoblasts to bone surfaces where they complete their development into functional osteoblasts. This study provides further insight into the similarities and differences in osteoblast histogenesis within evolving SHSs from adult rabbits and the more extensively studied rat POL model.