Mesenchymal Stem Cells Coated with Synthetic Bone-Targeting Polymers Enhance Osteoporotic Bone Fracture Regeneration

Osteoporosis is a progressive skeletal disease characterized by reduced bone density leading to bone fragility and an elevated risk of bone fractures. In osteoporotic conditions, decrease in bone density happens due to the augmented osteoclastic activity and the reduced number of osteoblast progenitor cells (mesenchymal stem cells, MSCs). We investigated a new method of cell therapy with membrane-engineered MSCs to restore the osteoblast progenitor pool and to inhibit osteoclastic activity in the fractured osteoporotic bones. The primary active sites of the polymer are the N-hydroxysuccinimide and bisphosphonate groups that allow the polymer to covalently bind to the MSCs’ plasma membrane, target hydroxyapatite molecules on the bone surface and inhibit osteolysis. The therapeutic utility of the membrane-engineered MSCs was investigated in female rats with induced estrogen-dependent osteoporosis and ulnar fractures. The analysis of the bone density dynamics showed a 27.4% and 21.5% increase in bone density at 4 and 24 weeks after the osteotomy of the ulna in animals that received four transplantations of polymer-modified MSCs. The results of the intravital observations were confirmed by the post-mortem analysis of histological slices of the fracture zones. Therefore, this combined approach that involves polymer and cell transplantation shows promise and warrants further bio-safety and clinical exploration.

BMP-2 hatása a humán embrionális szájpadlásból származó mesenchymalis preosteoblast sejtek morfológiájára és proliferációjára

Fogászati implantáció során a hiányzó fogat vagy fogakat „műgyökerek”, beültetésével pótoljuk. Az integráció folyamatánakfelgyorsítása érdekében az egyik legújabb törekvés bioaktív anyagokkal, növekedési faktorokkal segíteni azimplantátum körül a csontképződést. Ilyen molekula az amerikai gyógyszer- és élelmiszerügyi hatóság (FDA) által iselfogadott növekedési faktor a Bone Morfogenic Protein 2 (BMP-2) is. A BMP-2 ilyen jellegű alkalmazását in vitro tesztelésrealkalmas sejtvonalakon vizsgálják. Egyike ezeknek az osteoblast progenitor jellegű humán embrionális szájpadlásmesenchyma (HEPM) sejtvonal. Kísérleteink során vizsgáltuk a BMP-2 homodimer fehérjék hatását HEPM sejtek morfológiájáravalamint proliferációjára, rövid idejű, háromnapos kezelés után.Eredményeink azt mutatták, hogy háromnapos kezelés hatására a BMP-2 koncentrációjának függvényében a kezeltsejtek hamarabb tapadtak le, de végleges morfológiájukban, valamint proliferációjukban nem volt különbség.A kezelés idejét növelve azonban a sejtek osztódási képessége lecsökkent, ami utalhat a sejtek differenciációjára.

Carfilzomib Induces Differentiation of Mesenchymal Stromal Cells Toward Osteoblast Via Activation of β-Catenin/TCF Signaling

Abstract 4008 The first-in-class reversible proteasome inhibitor (PI) bortezomib (Bzb) is effective in the treatment of multiple myeloma (MM), both as a single agent and in combination with lenalidomide and dexamethasone. An irreversible next-generation PI, carfilzomib (CFZ) is a peptide epoxyketone that functions through primary inhibition of chymotrypsin-like (ChT-L) activity of the β5 subunits of the core 20S proteasome, similar to Bzb. CFZ has preclinical efficacy against hematologic and solid malignancies both in vitro and in vivo. Clinical data have shown an increase in the bone anabolic marker alkaline phosphatase in MM patients treated with CFZ. Recent studies reported that in addition to its anti-MM effect, CFZ inhibits osteoclast differentiation and promotes osteogenic differentiation (Hurchla et al, Leukemia 2012). However, the molecular basis of CFZ effects on bone anabolism in MM is unclear. We have previously demonstrated that Bzb induced osteoblast differentiation via regulation of β-catenin/T-cell factor (TCF) signaling (Qiang et al, Blood 2009), a pivotal regulator of mesenchymal stromal cell (MSC) differentiation into osteoblasts (Qiang et al, Bone 2008; Qiang et al, Blood 2008a and 2008b). In the present study we attempted to identify the molecular mechanism(s) by which CFZ regulates MSC differentiation toward osteoblasts. CFZ induced a significant increase in calcium deposition in matrix mineralization by osteoblasts as shown by van Kossa and Alizarin Red (AR) staining in multiple mouse and human MSC cell lines, human bone marrow (BM) stromal cells, and primary MSC from patients with MM. An E-cadherin pull-down assay and subsequent immunoblotting analysis demonstrated that CFZ induces increases in free and active forms of β-catenin in the cytoplasm and nuclei of human osteoblast progenitor cell lines and bone stromal cell lines in a dose- and time-dependent fashion. Similar results were observed in primary MSC from 6 patients with MM and 2 healthy donors. CFZ induced increases in the ubiquitinated β-catenin, as determined by its decreased electrophoretic mobility in SDS-PAGE analysis. Increases in cytoplasmic and nuclear β-catenin protein in response to CFZ treatment were further confirmed by immunofluorescent analysis in osteoblast cell lines and in 4 MSC samples from patients with MM. CFZ treatment also increased TCF transcriptional activity in a dose-dependent manner as determined by luciferase activity in cells transfected with TOPflash plasmid DNAs. CFZ-induced increases in β-catenin protein levels and TCF transcriptional activity were independent of modifications in the expression of 19 extracellular members of the Wnt family ligands, 10 members of the Frizzled receptor family, LRP5/6 co-receptors, antagonists of 4 members of the DKK and sFRP family, and β-catenin, respectively, as determined by qRT-PCR analysis. CFZ did not increase intracellular levels of Dvl-3 proteins, a downstream target of Wnt pathway. Lithium chloride, an inhibitor of GSK3β did not synergize with CFZ -induced increases in β-catenin protein or TCF transcriptional activity, indicating that CFZ activates β-catenin/TCF signaling independent of activity of GSK3β. Blocking the activation of β-catenin/TCF signaling by expression of dominant negative TCF attenuated CFZ-induced matrix mineralization indicating that CFZ-induced MSC differentiation into osteoblast occurred through activation of β-catenin/TCF signaling. Comparison of the biologic effect of CFZ with Bzb demonstrated that CFZ was a more potent inducer of MSC differentiation than Bzb. CFZ also induced increases in Runx2 protein in nuclear fractions, but this effect was less pronounced than CFZ's effect on β-catenin. These results provide evidence that CFZ induces MSC differentiation into osteoblasts mainly via Wnt-independent activation of the β-catenin/TCF signaling pathway. The data also indicate that transcriptional activation is a downstream effect of CFZ in osteoblast progenitor cell lines, BM stromal cells and MM derived MSC. Together these data suggest that PI therapy in MM may function mainly by bypassing tumor-induced suppression of canonical Wnt signaling in the bone microenvironment. Disclosures: No relevant conflicts of interest to declare.

Proteasome Inhibitor, Bortezomib Induces Mesenchymal Stem Cells toward Osteoblast Differentiation through Wnt-Independent Activation of Beta-catenin/TCF Signaling

In multiple myeloma (MM), the proteasome inhibitor bortezomib induces mesenchymal stem cells (MSC) toward osteoblast differentiation. However, it is full unclear about the mechanism(s) underlying bortezomib in this process. Wnt/beta-catenin pathway plays a pivotal role in osteoblast differenciation and bone development. We have demonstrated that inhibition of Wnt/beta-catenin signaling by MM-derived Dkk1 suppresses osteoblast progenitor cell differentiation into osteoblasts (Qiang et al, Bone 2008) and deregulate RANKL and OPG expression in osteoblast cells (Qiang et al Blood 2008a). Increase Wnt signaling by overexpression of Wnt3a in myeloma cells diminished MM-trigged bone lesion in mouse model (Qiang et al Blood 2008b). In the present study we revealed that bortezomib promotes MSC differentiation into osteoblast cells via Wnt-independent activation of beta-catenin/TCF signaling. E-cadherin pull-down assay and subsequently immunoblotting analysis demonstrated that bortezomib induced increases in both free and active forms of beta-catenin protein in cytoplasm and nuclear in bell-shaped dose- and time-dependent manner in mouse and human osteoblast progenitor cell lines including C2C12, C3H10T1/2, Saos-2 and MG63. Similar results were illustrated in primary human 2 cases of normal MSC and MSC from 8 cases of MM pateints. Bortezomib induced increase in ubiquitinated beta-catenin was evidenced by obvious seen slow migration bands of beta-catenin protein in SDS-PAGE gel analysis indicating that bortezomib increased beta-catenin protein by modification of proteasome-mediated degradation of beta-catenin. Increase in cytoplasm and nuclear beta-catenin protein response to bortezomib treatment in the osteoblast cell lines and 4 cases MM derived MSC was further confirmed by immunofluorescent analysis. RT-PCT analysis of TCF family revealed that abundant TCF1 and TCF4 mRNA were expressed in all tested cell lines and in a primary normal MSC, and MM-derived MSC. Bortezomib treatment also resulted in TCF transcriptional activity in bell-shaped, dose-dependent pattern as determined by luciferase activity in these cells transfected with TOPflash plasmid DNAs. Maximal responses to bortezomib were seen at 12.5 nM for both C2C12 (p<0.001) and MG63 (P<0.01), 25 nM for C3H10T1/2 (p<0.001) (p<0.00001) compared with non-stimulation control. These results suggest that transcriptional activation was a downstream effect of bortezomib in osteoblast progenitor cell lines and MM derived MSC. Bortezomib induced increases in beta-catenin protein and TCF transcriptional activity were independent of modification expression of extracellular 19 members of Wnt family ligands, 10 members of Frizzled receptor family, LRP5/6 co-receptors, and antagonists of 4 members of Dkk and sFRP family, respectively, as determined by RT-PCR analysis. Bortezomib did not increase intracellular Dvl-3 proteins, a downstream target of Wnt pathway. Lithium chloride, an inhibitor of GSK3beta did not synergized bortezomib induced increases in beta-catenin protein or TCF transcriptional activity indicating that bortezomib active beta-catenin/TCF signaling independent of activity of GSK3beta. Blocking the catenin/TCF signaling by expressing dominant–activation of beta- negative TFC attenuated bortezomib-induced matrix mineralization indicating that bortezomib induced MSC differentiation into osteoblast through activation of beta-catenin/TCF signaling. Data from experiments in comparison with biological effect of bortezomib with Wnt3a demonstrated that bortezomib did not have effect on OPG and RANKL in these cells, while Wnt3a induces OPG mRNA and protein, but inhibited RANKL expression indicating that bortezomib may have not effect on osteoclastogensis. These results provide insights into a clinically relevant mechanism of action of bortezomib and as such a rationale for its use in the treatment of diseases related to suppression of Wnt/beta-catenin/TCF signaling.