engleski

The important role of Pentraxin 3 in bone formation and fracture healing

Introduction: Various proinflammatory mediators have been shown to affect bone metabolism by modulating the activity and functional interactions of principal bone cells, bone forming osteoblasts and bone resorbing osteoclasts. Pentraxin 3 (PTX3) is a highly conserved member of the long pentraxins subfamily produced by somatic and immune cells in response to proinflammatory stimuli. Its biological roles have been described in various conditions, such as infections, female fertility and angiogenesis. We aimed to elucidate the role of PTX3 in physiological bone formation as well as in the process of fracture healing. Methods: Wild type (WT) mice and mice deficient for the PTX3 gene (PTX3-KO) (both genders, 8-weeks of age) on C57BL/6 background were used, after obtaining the approval from the Ethical committee. Cells from bone marrow, homogenized bone shafts and spleen were harvested and cultured to stimulate differentiation into osteoblast (by the addition of ascorbic acid 50 μg/ml, 10−8 M dexamethasone, and 8 mM β-glycerophosphate) or osteoclast lineage (by the addition of RANKL (40 ng/mL) and M-CSF (15 ng/mL)). Same populations were analyzed by flow-cytometry to assess the proportion of putative osteoclast and osteoblast progenitor cells. Bone metabolism in vivo was determined by histomorphometry and micro- computerized tomography (μCT) using standard structural bone parameters (trabecular volume (BV/TV), trabecular thickness, trabecular number and trabecular separation) and dynamic parameters of bone formation rate. Fracture model was introduced to further investigate the importance of PTX3 in bone healing process. The method of Bonnarens and Einhorn was modified to produce standardized closed tibial fracture in WT and PTX3-KO female mice after insertion of stainless steel pin into the tibial medullary cavity for bone fragment fixation. Mice were sacrificed three weeks post-fracture, and the quality and composition of callus tissue were assessed by μCT. Results: PTX3-KO mice had lower bone mass (BV/TV 2.72 ± 1.23 for females and 5.39 ± 1.73 for males) than their WT littermates (BV/TV 5.03 ± 0.87 for females and 7.04 ± 0.87 for males, p<0.05), as measured by histomorphometry and confirmed by μCT analysis of both long bones and axial skeleton (lumbar vertebrae). Moreover, PTX3-KO mice had lower trabecular thickness and greater trabecular separation. Although we found that PTX3 expression is increasing with the maturation of osteoblast and osteoclast cultures from WT mice, no differences were observed in the number of osteoblast and osteoclast progenitor cells in bone marrow compartment or in the number of ex vivo differentiated bone cells between WT and PTX3-KO mice. Nevertheless, osteogenic activity in vivo, assessed by dynamic histomorphometry, showed that PTX3-KO mice had lower bone formation rate than WT mice. Moreover, PTX3-KO mice formed significantly less callus tissue (BV/TV 15.33 ± 2.32) following bone fracture compared with WT mice (BV/TV 19.66 ± 4.32, p<0.05), Conclusion: Our results confirmed that PTX3 has a positive impact on physiological bone metabolism and bone mass homeostasis. Particularly, we showed that the bone healing process requires PTX3 for the induction of periosteal reaction, supporting previous studies that indicate important role of various inflammatory molecules in the initial phase of fracture repair. Our further studies are aimed to define cellular source of PTX3 at the sites of bone formation and exact mechanism of its action on osteoprogenitor cells.

PTX3; inflammation; bone biology; fracture healing

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