Session III - Clinically Relevant Research

The Effect of Nicotine on the Proliferation of Isolated Periosteal Cells and Bone Marrow Cells in Culture

Maureen A. Finnegan, MD, Wan-Shou Guo, Herman May, Masaya Tsunoda, Seiji Kinoshita

Parkland Memorial Hospital, Dallas, Texas, USA

Hypothesis: Several clinical studies have documented that patients who smoke cigarettes take a significantly longer period of time to heal tibial shaft fractures than do non-smokers. Most theories to date have postulated that hypoxia secondary to vascular constriction caused by nicotine is the most likely etiology. The present study proposes that nicotine has a direct cellular effect on certain stem cells involved in fracture repair, namely periosteal cells and bone marrow cells. The two cells populations, harvested from juvenile rabbits and grown to confluence in culture, were exposed to various doses of nicotine and the corresponding effects on DNA synthesis, GAG synthesis and collagen synthesis were measured.

Methods: Following harvest under sterile conditions from juvenile rabbits, isolated cells were suspended in Ham's F-12 medium containing 10% fetal bovine serum and 0.1% penicillin/streptomycin. Periosteal cells were harvested from the anteromedial surface of the proximal tibial diaphysis and bone marrow cells were aspirated from the femurs of donor immature rabbits.

Cells were then seeded in culture flasks at a density of 10,000 cells/ sq cm and incubated at 30°C in 5% CO2. At confluence cells were passaged and seeded in 96-well plates at 4000 cells per well. Nicotine (1-methyl-2 [3-pyridyl] pyffolidine, Sigma), in varying concentrations, was added 24 - 48 hours prior to testing. DNA synthesis was measured using [3H] thymidine. Eighteen hours prior to harvest, 1µL Ci/ml was added to the medium and the sample then placed in the scintillation counter. DNA content was determined using spectrophotometry following the addition of bis-benzimidazole (33258 Hoechst) to the sample. The sulfated glycosaminoglycan (GAG) was measured using a modified dimethylmethal blue (DMB) technique. Following nicotine exposure, the medium was mixed 1:1 with 2x DMB, and absorbance measured at 540 nm in a culture plate reader. Collagen synthesis was measured after exposure to [3H] proline eighteen hours prior to harvest. Cells were then digested with collagenase and the protein portion precipitated using 50% trichloroacetic acid. The precipitate was then counted by liquid scintillation counter.

Results: The maximal inhibitory effects of nicotine occurred at concentrations exceeding 120 µg/ml. Under light microscopy cells showed a tendency to rounded shape and an increase in size. Intracellular vacuoles were observed when nicotine concentrations reached 480 µg/ml. Lower doses of nicotine did not inhibit cell proliferation. However, proliferation dropped to 34% of control at nicotine concentrations of 240 pg/mi. Both collagen and glycosaminoglycan synthesis demonstrated a dose dependent inhibition starting at 120 µg/ml of nicotine.

Discussion: Clinical and basic research have documented that both cigarette smoke and nicotine by itself decrease vascular flow, but the direct effects of nicotine on musculoskeletal cells is less well documented. Although a few studies have investigated the effects of nicotine on osteoblast cell lines and osteosarcoma cells, none have looked at the effects on periosteal or bone marrow cells. This study documents a direct cellular effect of nicotine on cells involved in fracture repair, suggesting that cigarette smoking may affect fracture healing in numerous ways.

Conclusion: Nicotine, at doses exceeding 120 µg/ml, has a direct effect on the cellular functions of periosteal cells and bone marrow cells.