Sustained proliferation accompanies distraction osteogenesis in the rat

Abstract

These studies were conducted to compare the local cellular proliferation patterns in the rat tibia during distraction osteogenesis with those during nondistracted fracture healing. Bone specimens from distraction osteogenesis and nondistracted fracture groups were analyzed 2, 10, and 20 days after surgery. Proliferation was determined by metabolic labeling with [3H]thymidine and by immunocytochemistry with an antibody to proliferating cell nuclear antigen. Videomicroscopy was used to count the cells staining positively within specified regions. The number of cells incorporating [3H]thymidine was positively correlated (r2 = 0.78) with the number of proliferating cell nuclear antigen positive cells on alternating serial slides. At day 2, the latter cells were largely confined to the bone marrow and periosteum in both groups, and the cell numbers per mm2 were also equivalent. At days 10 and 20, the proliferating cell nuclear antigen positive cells predominated in both the proximal and distal primary matrix front zones in the distraction osteogenesis group. In contrast, the proliferating cell nuclear antigen positive cells in the nondistracted fracture group were scattered throughout the healing area. Significantly more of these cells were in the primary matrix front zones than in any location within the nondistracted fracture-healing area. The number of these cells in the bone marrow adjacent to the surgical area declined from day 2 to day 10 in both groups. The results suggest that (a) proliferating cell nuclear antigen immunostaining is a reliable indicator of cycling cells; (b) by day 10, distraction osteogenesis is characterized by a zone-specific pattern of proliferating cells; and (c) distraction osteogenesis prolongs the stimulation of proliferation within the gap after fracture.

A novel rat model for the study of deficits in bone formation in type-2 diabetes

“…As previously described, after removal of soft tissues except periosteum, the lengthened tibiae were fixed in 10% neutral buffered formalin with the external fixator intact (Aronson et al 1997a, b, 2001. After 48 h of fixation, the lengthened tibiae were removed from the fixators and positioned for highresolution single-beam radiography.…”

“…The percentage of new bone area in the distraction gap was calculated by dividing the new bone area by the total gap area. The relative density of "mineralized" new bone was calculated as a percentage of the adjacent host bone density as follows (Aronson et al 1997a, 2001. First, selected regions of interest (3 mm 2 ), one in both the adjacent proximal and distal original bone, were scanned by density slice.…”

“…A rat model for DO has been used to isolate and study osteoblastogenesis in vivo, and to study bone repair processes associated with osteoporotic fractures (Aronson et al 1997a, 2001. Using the rat tibia, DO has been shown to induce rapid bone formation by stretching a fracture callus at a prescribed rate and rhythm.…”

Immunohistochemical Study of Osteopontin Expression During Distraction Osteogenesis in the Rat

“…The following procedures were approved by the institutional animal care and use committee and performed as previously described (Aronson et al 1997a). Briefly, under sodium pentobarbital anesthesia, 4-month-old male Sprague-Dawley (Harlan; Indianapolis, IN) rats were fitted with external fixators on the left tibia.…”

“…To compare the relationship of OPN expression to proliferation, serial sections to those stained for OPN were immunohistochemically labeled for PCNA expression as previously described (Aronson et al 1997a). Briefly, PC-10, a mouse monoclonal antibody against PCNA, (Biogenex; ready to use) or normal mouse IgG for negative control (Dako) was applied for 60 min.…”