Determination of proliferative characteristics of growth plate chondrocytes by labeling with bromodeoxyuridine

Abstract

Postnatal bone growth occurs by the process of endochondral ossification in cartilaginous growth plates at the ends of long bones. The rate and extent of long bone growth is determined by a combination of chondrocytic proliferation, matrix production, and increase in chondrocytic height in the direction of growth during cellular enlargement. In this study, single pulse and/or repeated pulse labeling with the thymidine analog bromodeoxyuridine (BrdU) was used to study the role of cellular proliferation in controlling long bone growth. Variables studied included progression of the label over time following a pulse, and patterns and progression of the label over time following repeated pulse labeling for 24 and 48 hours. Examination was made of the proliferative characteristics of chondrocytes, the spatial pattern of cellular proliferation, and cell cycle kinetics. With respect to the spatial pattern of proliferative chondrocytes, results suggest that chondrocytes within a column are more synchronized with each other than are chondrocytes in different columns. This is consistent with the concept that each column represents a clonal expansion of a stem cell, which may proceed independently from adjacent columns. Despite this apparent heterogeneity, all chondrocytes in the proliferative zone complete at least one cell cycle in 24-28 hours. This estimate of the cell cycle time is significantly shorter than previous estimates of cell cycle times in similar growth plates. Our results also suggest that chondrocytes entering the cell cycle in the proximal part of the growth plate spend an average of 4 days in the proliferative cell zone, representing approximately four cellular divisions. After leaving the cell cycle, an additional 48 hours is required for the label to reach the terminal chondrocyte, which represents the time required to complete hypertrophy. These data are important when considering hypotheses concerning both the role of controls on proliferation in the determination of overall rate of long bone growth, as well as the interplay between proliferation and hypertrophy in regulating the overall amount of growth achieved by a given growth plate.

Ontogeny of Skeletal Maturation in the Juvenile Rat

“…In brief, we calculated the proportion of BrdU-immunoreactive cellular profiles per total cellular profiles in the selected sections. This proportion was then modified by application of a correction factor to account for the probability of observing a nucleated cellular profile in any randomly selected cellular profile observed in a tissue section less than one cell-diameter in thickness (Farnum and Wilsman, 1993).…”

Restoration of growth plate function following radiotherapy is driven by increased proliferative and synthetic activity of expansions of chondrocytic clones

“…16 On the seventh day following radiation therapy, all animals received a single intraperitoneal injection of the thymidine analog 5-bromo-2 0 -deoxyuridine (BrdU, 25 mg/kg, Sigma, St. Louis, MO) to label the population of cells in S-phase DNA synthesis, indicating proliferative activity at the time of injection. [17][18][19] The injection of BrdU was considered time 0 in this experiment.…”

“…17 BrdU labeling was used only to determine the initial cell-cycle duration and temporal progression of maturation; PCNA was used only to determine growth fraction. In both indices, the number of labeled nucleated cellular profiles per total cellular profiles was counted ( Figs.…”

“…1 and 2). As these labels are restricted to nuclear localization during S-phase, and because not all cellular profiles in a given thin section contain a nucleus, a correction factor was calculated for each morphologically defined zone 9,17 to adjust the labeling percentage of cells to account for the probability of observing a nucleated cellular profile. Thus, the labeling indices presented represent the proportion of labeled cellular profiles per total nucleated cellular profiles in a section.…”

“…Thus, the labeling indices presented represent the proportion of labeled cellular profiles per total nucleated cellular profiles in a section. 17,19,24 These correction factors were calculated separately for the reserve (0.598), proliferative (0.785), transitional (0.636), and hypertrophic zones (0.498).…”