Objectives

The need for bone tissue supplementation exists in a wide range of clinical conditions involving surgical reconstruction in limbs, the spine and skull. The bone supplementation materials currently used include autografts, allografts and inorganic matrix components; but these pose potentially serious side-effects. In particular the availability of the autografts is usually limited and their harvesting causes surgical morbidity. Therefore for the purpose of supplementation of autologous bone graft, we have developed a method for autologous extracorporeal bone generation.

Introduction: The mitochondrial Translocator Protein 18 kDa (TSPO, previously named as the peripheral benzodiazepine receptor - PBR) is involved in cellular respiration, steroidogenesis and apoptosis. In our recent study we reported on the role of the synthetic pharmacological ligands to the TSPO in enhancing human osteoblast catabolism. There is also a previous evidence of the existence of an endogenous ligands to the TSPO, but their role in the human osteoblast physiology hasn’t been verified yet. Porphyrine IX has been found having affinity to the TSPO. Therefore we hypothesize that human osteoblast metabolism might be mediated by the porphyrine IX and the mode of its action is similar the synthetic ligand to the TSPO.

Methods: Cell cycle of the cultured human derived osteoblast- like cells, following exposure to Porphyrine IX, endogenous ligand to TSPO, and N,N-di-n-hexyl 2-(4- fluorophenyl)indole-3-acetamide (FGIN-1–27), synthetic ligand to the TSPO, was determined by flow cytometry (FACS). These ligands’ affect on cell number, metabolic activity, i.e. cellular fluorodeoxyglucose ([¹⁸F]-FDG) incorporation and alkaline phosphatase activity, and cell death rate, i.e. LDH activity in the culture media, were assayed. The semi-quantitative response of TSPO to exposure to these ligands was estimated by Western blotting. Six samples of cultured cells for each condition were used. The t test was implemented for the statistical analyses. P values below.05 considered as statistically significant

Results: Cell count significantly decreased following exposure to FGIN-1–27 or porphyrine IX. Cellular [¹⁸F]-FDG incorporation and alkaline phosphatase activity were suppressed by both ligands. Cell cycle analysis showed a significant decrease in the fraction of cells in the G1 and G2/M phases when exposed to each ligand with a higher proportion of necrotic and apoptotic cells.

Western blotting showed a decrease in TSPO abundance following treatment by both ligands. LDH activity in culture media significantly increased following exposure to FGIN-1–27 or porphyrine IX.

Discussion: We show that FGIN-1–27 and porphyrine IX have a similar cell death inducing affect on human osteoblast-like cell in vitro. This affect is parallel to the inhibition of the cellular metabolism. Since both ligands similarly reduce the availability of TSPO we postulate that their mode of action is similar by affecting this mitochondrial structure with sub sequential induction of cell death, i.e. apoptosis and necrosis. Therefore we suggest that human osteoblast metabolism and cell cycle are mediated through TSPO and that porphyrine IX might be an active endogenous ligand to the TSPO having a regulatory affect on the human bone cell cycle.

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 (K_d) equaled 9.15-9.34 nM and density of receptors (B_max) 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.