Decreased osteogenesis, increased cell senescence and elevated Dickkopf-1 secretion in human fracture non union stromal cells

The delicately orchestrated process of bone fracture healing is not always successful and long term non union of fractured bone occurs in 5-20% of all cases. Atrophic fracture non unions have been described as the most difficult to treat and this is thought to arise through a cellular and local failure of osteogenesis. However, little is known about the presence and osteogenic proficiency of cells in the local area of non union tissue. We have examined the growth and differentiation potential of cells isolated from human non union tissues compared with normal human bone marrow mesenchymal stromal cells (BMSC). We report the isolation and culture expansion of a population of non union stromal cells (NUSC) which have a CD profile similar to that of BMSC, i.e. CD34-ve, CD45-ve and CD105+ve. The NUSC demonstrated multipotentiality and differentiated to some extent along chondrogenic, adipogenic and osteogenic lineages. However, and importantly, the NUSC showed significantly reduced osteogenic differentiation and mineralization in vitro compared to BMSC. We also found increased levels of cell senescence in NUSC compared to BMSC based on culture growth kinetics and cell positivity for senescence associated beta galactosidase (SA-beta-Gal) activity. The reduced capacity of NUSC to form osteoblasts was associated with significantly elevated secretion of Dickkopf-1 (Dkk-1) which is an important inhibitor of Wnt signalling during osteogenesis, compared to BMSC. Conversely, treating BMSC with levels of rhDkk-1 that were equivalent to those levels secreted by NUSC inhibited the capacity of BMSC to undergo osteogenesis. Treating BMSC with NUSC conditioned medium also inhibited the capacity of the BMSC to undergo osteogenic differentiation when compared to their treatment with BMSC conditioned medium. Our results suggest that the development of fracture non union is linked with a localised reduced capacity of cells to undergo osteogenesis, which in turn is associated with increased cell senescence and Dkk-1 secretion.

The presence of pleiotrophin in the human intervertebral disc is associated with increased vascularization:an immunohistologic study

Study Design. An immunohistological study of surgical specimens of human intervertebral disc.Objective.To examine the presence of pleiotrophin in diseased or damaged intervertebral disc tissue and the association between its presence and the extent of tissue vascularization and innervation.Summary of Background Data. Increased levels of pleiotrophin, a growth and differentiation factor that is active in various pathophysiologic processes, including angiogenesis, has been associated with osteoarthritic changes of human articular cartilage. The association between pleiotrophin expression and pathologic conditions of the human intervertebral disc is unknown.Methods. Specimens of human lumbar intervertebral discs, obtained following surgical discectomy, were divided into 3 groups: nondegenerated discs (n = 7), degenerated discs (n = 6), and prolapsed discs (n = 11). Serial tissue sections of each specimen were immunostained to determine the presence of pleiotrophin, blood vessels (CD34-positive endothelial cells), and nerves (neurofilament 200 kDa [NF200]-positive nerve fibers).Results. Pleiotrophin immunoreactivity was seen in disc cells, endothelial cells, and in the extracellular matrix in most specimens of intervertebral disc but was most prevalent in vascularized tissue in prolapsed discs. There was a significant correlation between the presence of pleiotrophin-positive disc cells and that of CD34-positive blood vessels. NF200-positive nerves were seen in vascularized areas of more degenerated discs, but nerves did not appear to codistribute with blood vessels or pleiotrophin positivity in prolapsed discs.Conclusions. Pleiotrophin is present in pathologic human intervertebral discs, and its prevalence and distribution suggest that it may play a role in neovascularization of diseased or damaged disc tissue.

Cystathionine γ-lyase regulates arteriogenesis through NO-dependent monocyte recruitment

AIMS: Hydrogen sulfide (H2S) is a vasoactive gasotransmitter that is endogenously produced in the vasculature by the enzyme cystathionine γ-lyase (CSE). However, the importance of CSE activity and local H2S generation for ischaemic vascular remodelling remains completely unknown. In this study, we examine the hypothesis that CSE critically regulates ischaemic vascular remodelling involving H2S-dependent mononuclear cell regulation of arteriogenesis. METHODS AND RESULTS: Arteriogenesis including mature vessel density, collateral formation, blood flow, and SPY angiographic blush rate were determined in wild-type (WT) and CSE knockout (KO) mice at different time points following femoral artery ligation (FAL). The role of endogenous H2S in regulation of IL-16 expression and subsequent recruitment of monocytes, and expression of VEGF and bFGF in ischaemic tissues, were determined along with endothelial progenitor cell (CD34/Flk1) formation and function. FAL of WT mice significantly increased CSE activity, expression and endogenous H2S generation in ischaemic tissues, and monocyte infiltration, which was absent in CSE-deficient mice. Treatment of CSE KO mice with the polysulfide donor diallyl trisulfide restored ischaemic vascular remodelling, monocyte infiltration, and cytokine expression. Importantly, exogenous H2S therapy restored nitric oxide (NO) bioavailability in CSE KO mice that was responsible for monocyte recruitment and arteriogenesis. CONCLUSION: Endogenous CSE/H2S regulates ischaemic vascular remodelling mediated during hind limb ischaemia through NO-dependent monocyte recruitment and cytokine induction revealing a previously unknown mechanism of arteriogenesis.