Abstract
Introduction Efficient control of osteoblast metabolism is crucial for the development of methods for enhancement of bone fracture repair and in the treatment of osteoporosis. If extracellular matrix elaboration by osteoblast could be controlled on the cellular level, new theurapeutical means might be developed. The current methods for osteoblast metabolic manipulation include mechanical, electromagnetic, hormonal and biochemical, i.e. growth factors and cytokines, means. All this methods have different degrees of therapeutic success. Finding of additional pathways of metabolic stimulation of osteoblast will provide an important insight for the understanding of human bone mass maintenance. The recent report of the existence of peripheral ben-zodiazepine receptor(PBR) in mammalian fibroblast arises the possibility of the existence of an unknown cellular pathway for mesenchymal cells metabolic regulation through this receptor. The PBR is a part of the mitochondrial permeability transition complex with important role in cell proliferation, differentiation, steroidogenesis, immunity and apoptosis, i.e. this complex is involved in most of the cellular metabolic activities. The PBR was identified in various organs, especially with enhanced steroidogenetic activity, but never has been investigated in bone. Therefore PBR’s identification in the human osteoblast may reveal a new cellular pathway of its metabolism.
Methods Cultures of confluent layers of osteoblast-like cells originated from human cancellous bone from distal femur. The samples were taken during osteoarthritic knee replacements. Chips of cancellous bone, 2 – 3 grams in total, were incubated in DMEM with heat-inactivated fetal calf serum (10%), 20mM HEPES buffer, 2mM L-glutamine, 100 μ M ascorbate-2-phosphate, 10nM dexam-etasone, 50 U ml-ml penicillin, 150μml-ml streptomicin at 37°C in humidified atmospheric environment of 95% air with 5% CO2 ( v:v ) for 30 days. Human osteoblast-like cells grew out from the chips as adherent to the plastic culture plates until confluency. The human bone cell cultures obtained by this method have been shown previously to express osteoblast-like characteristics.
The PBR in the homogenized osteblast-like cells was identified by using its selective ligand PK11195. The affinity and density of the PBR was estimated by the scatchard analysis.
Results We found that binding of the ligand [3H]PK11195 to the human osteoblast PBR is saturable with a single population of binding sites (r=0.92 – 0.95). The equilibrium dissociation constant (Kd) equaled 9.15-9.34 nM and density of receptors (Bmax) was 7,672–7,691 fmol/mg protein.
Discussion The PBR receptor was identified in the human osteoblast with affinity to the PK11195 in the same magnitude as previously found in other tissues. The density of the PBR in the osteoblast appeared higher comparing to uterus, kidney, brain and placenta from different mammalian origin. PBR’s density in osteoblast is comparable only to the adrenal tissue, that is known to have its highest values. PBR density in the human osteoblast is also higher than in the rat’s skeletal fibroblast, and although this may suggest a higher differrention of the osteoblast, the interspecies comparison might be misleading. These data suggest that the human osteoblast is one of the important sites rich with PBR. The exact role of the PBR in the human osteoblast metabolism is not known yet and will be further investigated.
The abstracts were prepared by Orah Naor, IOA Co-ordinator and Secretary. Correspondence should be addressed to Israel Orthopaedic Association, PO Box 7845, Haifa 31074, Israel.
