Osteoblast Progenitors Research Articles

Changes of Migration, Immunoregulation and Osteogenic Differentiation of Mesenchymal Stem Cells in Different Stages of Inflammation.

Bone infection has always been the focus of orthopedic research. Mesenchymal stem cells (MSCs) are the natural progenitors of osteoblasts, and the process of osteogenesis is triggered in response to different signals from the extracellular matrix. MSCs exert important functions including secretion and immune regulation and also play a key role in bone regeneration. The biological behavior of MSCs in acute and chronic inflammation, especially the transformation between acute inflammation and chronic inflammation, has aroused great interest among researchers. This paper reviews the recent literature and summarizes the behavior and biological characteristics of MSCs in acute and chronic inflammation to stimulate further research on MSCs and treatment of bone diseases.

Total body proton and heavy-ion irradiation causes cellular senescence and promotes pro-osteoclastogenic activity in mouse bone marrow

Low-LET photon radiation-induced persistent alterations in bone marrow (BM) cells are well documented in total-body irradiated (TBI) rodents and also among radiotherapy patients. However, the late effects of protons and high-LET heavy-ion radiation on BM cells and its implications in osteoclastogenesis are not fully understood. Therefore, C57BL6/J female mice (8 weeks; n = 10/group) were irradiated to sham, and 1 Gy of the proton (0.22 keV/μm), or high-LET 56Fe-ions (148 keV/μm) and at 60 d post-exposure, mice were sacrificed and femur sections were obtained for histological, cellular and molecular analysis. Cell proliferation (PCNA), cell death (active caspase-3), senescence (p16), osteoclast (RANK), osteoblast (OPG), osteoblast progenitor (c-Kit), and osteoclastogenesis-associated secretory factors (like RANKL) were assessed using immunostaining. While no change in cell proliferation and apoptosis between control and irradiated groups was noted, the number of BM megakaryocytes was significantly reduced in irradiated mice at 60 d post-exposure. A remarkable increase in p16 positive cells indicated a persistent increase in cell senescence, whereas increased RANKL/OPG ratio, reductions in the number of osteoblast progenitor cells, and osteocalcin provided clear evidence that exposure to both proton and 56Fe-ions promotes pro-osteoclastogenic activity in BM. Among irradiated groups, 56Fe-induced alterations in the BM cellularity and osteoclastogenesis were significantly greater than the protons that demonstrated a radiation quality-dependent effect. This study has implications in understanding the role of IR-induced late changes in the BM cells and its involvement in bone degeneration among deep-space astronauts, and also in patients undergoing proton or heavy-ion radiotherapy.

The phytochemical plumbagin reciprocally modulates osteoblasts and osteoclasts.

Bone metabolism is essential for maintaining bone mineral density and bone strength through a balance between bone formation and bone resorption. Bone formation is associated with osteoblast activity whereas bone resorption is linked to osteoclast differentiation. Osteoblast progenitors give rise to the formation of mature osteoblasts whereas monocytes are the precursors for multi-nucleated osteoclasts. Chronic inflammation, auto-inflammation, hormonal changes or adiposity have the potential to disturb the balance between bone formation and bone loss. Several plant-derived components are described to modulate bone metabolism and alleviate osteoporosis by enhancing bone formation and inhibiting bone resorption. The plant-derived naphthoquinone plumbagin is a bioactive compound that can be isolated from the roots of the Plumbago genus. It has been used as traditional medicine for treating infectious diseases, rheumatoid arthritis and dermatological diseases. Reportedly, plumbagin exerts its biological activities primarily through induction of reactive oxygen species and triggers osteoblast-mediated bone formation. It is plausible that plumbagin's reciprocal actions- inhibiting or inducing death in osteoclasts but promoting survival or growth of osteoblasts- are a function of the synergy with bone-metabolizing hormones calcitonin, Parathormone and vitamin D. Herein, we develop a framework for plausible molecular modus operandi of plumbagin in bone metabolism.

Diabetes impairs fracture healing through disruption of cilia formation in osteoblasts.

Diabetes-associated fracture risk and impaired fracture healing represents a serious health threat. It is well known that type 1 diabetes mellitus (T1DM) impairs fracture healing due to its effect on osteoblasts and their progenitor cells. Previous studies have showed that primary cilia and intraflagellar transport protein 80 (IFT80) are critical for bone formation. However, whether TIDM impairs fracture healing due to influencing ciliary gene expression and cilia formation is unknown. Here, we investigated the effect of T1DM on primary cilia in a streptozotocin induced diabetes mouse model and examined the impact of cilia on fracture healing in osteoblasts by deletion of IFT80 in osteoblast linage using osterix (OSX)-cre (OSXcretTAIFT80f/f). The results showed that diabetes inhibited ciliary gene expression and primary cilia formation to an extent that was similar to normoglycemic mice with IFT80 deletion. Moreover, diabetic mice and normoglycemic mice with cilia loss in osteoblasts (OSXcretTAIFT80f/f) both exhibited delayed fracture healing with significantly reduced bone density and mechanical strength as well as with reduced expression of osteoblast markers, decreased angiogenesis and proliferation of bone lining cells at the fracture sites. In vitro studies showed that advanced glycation end products (AGEs) downregulated IFT80 expression in osteoblast progenitors. Moreover, AGEs and IFT80 deletion significantly reduced cilia number and length which inhibited differentiation of primary osteoblast precursors. Thus, this study for the first time report that primary cilia are essential for bone regeneration during fracture healing and loss of cilia caused by diabetes in osteoblasts resulted in defective diabetic fracture healing.

Multiple Myeloma Derived Mitochondrial Damps Induce Inflammation in the Bone Marrow Adipose Tissue Which Promotes Tumour Development

Multiple myeloma (MM) is a malignancy of the antibody secreting plasma cells, characterized by the localisation and accumulation of tumour cells in the bone marrow (BM) microenvironment. The mitochondrial DNA (mtDNA) belongs to a group of mitochondrial damage associated molecular patterns (mtDAMPs) and contains islands of unmethylated CpG nucleotide motifs, which have been shown to activate and promote memory B cell antibody secretion and proliferation. Studies have indicated that mitochondrial DNA (mtDNA) is elevated in the circulation of trauma and cancer patients, highlighting a need to explore the functional purpose of mtDNA in the BM microenvironment. Moreover, myeloma relies on the BM microenvironment for survival, proliferation and drug resistance. BM adipocytes are highly secretory cells that support myeloma cells through the release of various factors. It has been shown that myeloma cells induce a shift from osteoblast progenitors towards adipogenesis. Here, we hypothesise that MM cells release mtDAMPs that leads to the promotion of a state of chronic inflammation that supports multiple myeloma disease progression and expansion by inducing adipocyte inflammation.First, we engrafted immunocompromised NSG mice with human myeloma cell line MM1S, blood serum samples were taken and analysed by RT-qPCR to detect the presence of human mtDNA. We found elevated levels of human mtDNA in the serum of engrafted mice correlating to disease progression. Next, we showed that mtDNA was higher in serum patients with MM compared to healthy controls, moreover, we discovered that BM serum from MM patients had significantly higher levels of mtDNA than blood serum. Furthermore, this observation was recapitulated in myeloma engrafted NSG mice. Next, we examined the effect of mtDNA on various cell populations of the BM. We found that in vitro both BM derived macrophages and BM derived adipocytes had increased inflammatory signatures, including upregulation of IL-6, when treated with mtDNA from myeloma cells. MtDNA induced IL-6 expression was attenuated upon blocking of the TLR9 receptor with antagonist ODN 2088. To understand the role of BM adipocyte induced inflammation in MM, we treated adipocytes with mtDNA before culturing them with MM. We found that MM cells had enhanced proliferation on mtDNA activated adipocytes. We further show that MM cells cultured with mtDNA activated adipocytes have increased uptake of free fatty acids and seahorse analysis confirmed an increase in b-oxidation derived metabolism.Here we establish that MM releases mtDNA into the microenvironment promoting the disease progression via a pro-inflammatory BM. We also show that both BM macrophages and BM adipocytes play a supportive role in multiple myeloma expansion and contribute to the pro-inflammatory bone marrow microenvironment. DisclosuresNo relevant conflicts of interest to declare.

The Effect of Genetically Modified Lactobacillus plantarum Carrying Bone Morphogenetic Protein 2 Gene on an Ovariectomized Rat.

ObjectiveOsteoporosis result from age-related decline in the number of osteoblast progenitors in the bone marrow. Probiotics have beneficial effects on the host, when administered in appropriate amounts. This study investigated the effects of probiotics expressing specific genes, especially the effects of genetically modified bone morphogenetic protein (BMP)-2-expressing Lactobacillus plantarum CJNU 3003 (LP) on ovariectomized rats. MethodsTwenty-eight female Wistar rats (250–300 g, 12 weeks old) were divided into four groups : the sham (control), the ovariectomy (OVX)-induced osteoporosis group (OVX), the OVX and LP (OVX/LP), OVX and genetically modified BMP-2-expressing LP (OVX/LP with BMP) groups. The three groups underwent bilateral OVX and two of these groups were administered two different types of LP via oral gavage daily. At 16 weeks post-OVX, blood was collected from the heart and the bilateral tibiae were extracted and were scanned by ex-vivo micro-computed tomography and stained with hematoxylin-and-eosin (H&E) and Masson’s trichrome stain for pathological assessment. The serum levels of osteocalcin (OC), rat C-telopeptide of type I collagen (CTX-I), BMP-2, and receptor activator of nuclear factor-ĸB ligand (RANKL) were measured. ResultsThe 3D-micro-computed tomography images showed that the trabecular structure in the OVX/LP with BMP group was maintained compared with OVX and OVX/LP groups. No significant differences were detected in trabecular thickness (Tb.Th) between control and OVX/LP with BMP groups (p>0.05). Furthermore, a tendency toward increased BMD, trabecular bone volume, Tb.Th, and trabecular number and decreased trabecular separation was found in rats in the OVX/LP with BMP groups when compared with the OVX and OVX/LP groups (p>0.05). The H&E and Masson’s trichrome stained sections showed a thicker trabecular bone in the OVX/LP with BMP group compared with the OVX and OVX/LP groups. There was no difference in serum levels of OC, CTX and RANKL control and OVX/LP with BMP groups (p>0.05). In contrast, significant differences were found in OC and CTX-1 levels between the OVX and OVX/LP with BMP groups (p<0.05). ConclusionOur results showed that the expression of genetically modified BMP-2 showed inhibition effect for bone loss in a rat model of osteoporosis.

Clinical Applications of Cell-Scaffold Constructs for Bone Regeneration Therapy.

Bone tissue engineering (BTE) is a process of combining live osteoblast progenitors with a biocompatible scaffold to produce a biological substitute that can integrate into host bone tissue and recover its function. Mesenchymal stem cells (MSCs) are the most researched post-natal stem cells because they have self-renewal properties and a multi-differentiation capacity that can give rise to various cell lineages, including osteoblasts. BTE technology utilizes a combination of MSCs and biodegradable scaffold material, which provides a suitable environment for functional bone recovery and has been developed as a therapeutic approach to bone regeneration. Although prior clinical trials of BTE approaches have shown promising results, the regeneration of large bone defects is still an unmet medical need in patients that have suffered a significant loss of bone function. In this present review, we discuss the osteogenic potential of MSCs in bone tissue engineering and propose the use of immature osteoblasts, which can differentiate into osteoblasts upon transplantation, as an alternative cell source for regeneration in large bone defects.

The Effects of a Short Self-Assembling Peptide on the Physical and Biological Properties of Biopolymer Hydrogels.

Hydrogel scaffolds have attracted much interest in the last few years for applications in the field of bone and cartilage tissue engineering. These scaffolds serve as a convenient three-dimensional structure on which cells can grow while sensing the native environment. Natural polymer-based hydrogels are an interesting choice for such purposes, but they lack the required mechanical properties. In contrast, composite hydrogels formed by biopolymers and short peptide hydrogelators possess mechanical characteristics suitable for osteogenesis. Here, we describe how combining the short peptide hydrogelator, Pyrene-Lysine-Cysteine (PyKC), with other biopolymers, can produce materials that are suitable for tissue engineering purposes. The presence of PyKC considerably enhances the strength and water content of the composite hydrogels, and confers thixotropic behavior. The hyaluronic acid-PyKC composite hydrogels were shown to be biocompatible, with the ability to support osteogenesis, since MC3 T3-E1 osteoblast progenitor cells grown on the materials displayed matrix calcification and osteogenic differentiation. The osteogenesis results and the injectability of these composite hydrogels hold promise for their future utilization in tissue engineering.

Runner's niche: multipurpose stromal cells maintained by exercise

Using newly developed reporter and lineage-tracing mice, Shen et al. found perivascular stromal cells coexpressing osteolectin and leptin receptor in the bone marrow that specifically supported lymphoid progenitors, served as osteoblast progenitors, and were maintained by mechanical stimulation. Exercise may thus have joint positive influences on lymphopoiesis and bone formation.

Gli1+ progenitors mediate bone anabolic function of teriparatide via Hh and Igf signaling.

SUMMARYTeriparatide is the most widely prescribed bone anabolic drug in the world, but its cellular targets remain incompletely defined. The Gli1+ metaphyseal mesenchymal progenitors (MMPs) are a main source for osteoblasts in postnatal growing mice, but their potential response to teriparatide is unknown. Here, by lineage tracing, we show that teriparatide stimulates both proliferation and osteoblast differentiation of MMPs. Single-cell RNA sequencing reveals heterogeneity among MMPs, including an unexpected chondrocyte-like osteoprogenitor (COP). COP expresses the highest level of Hedgehog (Hh) target genes and the insulin-like growth factor 1 receptor (Igf1r) among all cell clusters. COP also expresses Pth1r and further upregulates Igf1r upon teriparatide treatment. Inhibition of Hh signaling or deletion of Igf1r from MMPs diminishes the proliferative and osteogenic effects of teriparatide. The study therefore identifies COP as a teriparatide target wherein Hh and insulin-like growth factor (Igf) signaling are critical for the osteoanabolic response in growing mice.

Runx2 Deficiency in Committed Osteoblasts Promotes Myeloma Cell Homing to and Progression in New Bone Sites

Multiple myeloma (MM) is a plasma cell malignancy that arises and preferentially grows in the bone marrow (BM), with frequent progression to new local and distant bone sites. Our previous studies demonstrated that primary MM cells secrete soluble factors that orchestrate changes in distant bone sites prior to the arrival of metastatic tumor cells. One such alteration is the simultaneous suppression of osteoblastogenesis and bone formation via inhibition of the critical Runt-related transcription factor 2 (Runx2) in osteoblast (OB) progenitors and committed OBs. While the mechanisms regulating MM-induced Runx2 suppression in these cells have been described, how this change in distant sites provides feedback to regulate MM cell homing and survival has not been determined.To study the effect of OB-Runx2 suppression at distant bone sites on MM dissemination and progression to these sites, we generated a syngeneic mouse model of MM in which Runx2 is specifically deleted in the committed OBs of C57BL6/KaLwRij mice (OB-Runx2-/- mice). Bone-homing and growth of luciferase-expressing 5TGM1 (5TGM1-Luc) MM cells was tested by tail vein injection in 5-week-old OB-Runx2+/+ (controls) and OB-Runx2-/- mice (n=7 per group). Compared with controls, OB-Runx2-/- mice showed significantly enhanced bone homing of MM cells and increased tumor growth in bone, which was confirmed by weekly luciferin imaging and ELISA for serum levels of IgG2bκ (a soluble marker of 5TGM1 MM cells). These results demonstrate that Runx2 deficiency in committed OBs in new bone sites promotes MM metastasis to and progression in these areas.To investigate the molecular mechanism by which OB-Runx2 deficiency promotes MM bone-homing and tumor progression, we harvested BM from 5-week-old OB-Runx2+/+ controls and OB-Runx2-/- mice with 1 ml PBS (n=3-4 per group) and centrifuged it to collect BM cells and supernatants. Measurement of 48 cytokines related to tumor cell bone-homing and bone remodeling revealed substantially increased levels of pro-metastatic cytokines in the BM supernatants of OB-Runx2-/- mice, including EGF, GM-CSF, M-CSF, ICAM-1, IGF-1, MMP-3, OPN, SDF-1, TGF- β, RANKL, VCAM-1, and IL-10. Flow cytometric analysis of the immune cell profile showed significantly more immune inhibitory cells, including myeloid-derived suppressor cells (MDSCs) and regulatory T and B cells, in the BM of OB-Runx2-/- mice. These data indicate that Runx2 deficiency in committed OBs induces an inflammatory and immunosuppressive pre-condition in the BM microenvironment, which attracts MM cells and allows their invasion into the bone tissue.Next, we evaluated the survival and proliferation status of MM cells and the immune cell response to MM cells in the BM under the condition of OB-Runx2 suppression. We injected 2 x 106 5TGM1-Luc cells into 5-week-old OB-Runx2+/+ control and OB-Runx2-/- mice via tail vein (n=5 per group). After 4 weeks, MM and BM cells isolated from the BM of these mice were analyzed by immunostaining and flow cytometry with antibodies specific for CD138, a marker of MM cells; Ki-67, a marker of cell proliferation; and Bcl-2, an anti-apoptotic protein. Results showed significantly more proliferating and non-apoptotic MM cells in the BM of Runx2 OB-/- mice. Additional flow cytometry analysis of isolated BM cells revealed a striking increase in immune-suppressive cell populations, significantly higher expression of CD8+ T cell inhibitory molecules (Arginase 1, iNOS, IL-10) in MDSCs, and dramatically decreased production of tumor-killing factors (Perforin, Granzyme B, and IFN-γ) in T cells in OB-Runx2-/- mice. Together, these findings indicate that Runx2 deficiency in committed OBs impairs antitumor immunity, leading to tumor proliferation and progression in bone.In conclusion, this study demonstrated that Runx2 inhibition in committed OBs in distant bone sites, induced by primary MM, can feed back to invite MM cell metastasis to and progression in these new bone areas. The increase in pro-metastatic cytokines and immunosuppressive cells induced by OB-Runx2 deficiency in new bone sites play a critical role in this process. These novel discoveries provide insights into the mechanisms of MM dissemination and progression and may inform the prognosis of outcomes in patients with MM. DisclosuresNo relevant conflicts of interest to declare.

AML Differentially Alters Fate and Function of Mesenchymal Stem- and Osteolineage Cells in the Leukemic Niche

Acute Myeloid Leukemia (AML) alters the composition of the supportive bone marrow (BM) stroma, leading to a leukemia-supportive microenvironment that promotes extrinsic chemotherapy resistance. We previously reported functional and gene expression changes in patient derived mesenchymal stem cells (MSC) that included the increased secretion of chemoprotective cytokines, sustained in the absence of AML cells through serial passage ex vivo . Other groups demonstrated altered DNA methylation patterns in patient MSCs that correlated with increased extrinsic drug resistance in vitro . However, a more rigorous analysis of changes in stromal fate and function requires longitudinal- including pre diagnostic- observations, impossible to obtain from patients and further complicated by patient-patient heterogeneity as well as expansion culture induced in vitro artifacts. Here, we developed a protocol for flow-cytometric isolation and culture- free analysis of stringently purified endosteal MSCs (CD45-, CD31-, CD51+, SCA-1+) and osteoblastic progenitor cells (OPCs; CD45-, CD31-, CD51+, SCA-1-). This provides a robust platform in our established AML NSG xenograft model (using Molm14, HL60 or U937 cell lines) for a direct analysis of two principle mesenchymal populations known to be involved in leukemia drug resistance. At a leukemic burden of 60-90% in the BM, we initially observed a significant decline in the proportion of OPC in xenograft-bearing mice compared to healthy controls. We hypothesized that this could be caused by a block in osteogenic differentiation in the MSCs, or an increase in turnover of the OPCs. Surprisingly, we found an increase in osteoblastic differentiation in MSCs derived from xenograft-bearing mice, evidenced by strong induction of Osterix, Runx2, and other regulators of osteogenesis. We also found a concomitant increase in Annexin V and phosphorylated P53 within the OPC population, indicating significant apoptotic turnover, whereas no apoptosis in MSCs was observed. It was recently demonstrated that AML blasts can induce an unfolded protein response (UPR) in bystander cells. Here, we found that while both MSCs and OPCs demonstrate increased expression of Bip, Chop, and Xbp1, OPCs appear to be more sensitive to AML-induced UPR stress, an observation entirely consistent with their increased secretory function. These studies also provide a potential mechanism to explain experimental and clinical observations of osteoblastic cell loss as an aspect of disease progression in the AML BM. The induction of UPR stress in the expanding MSCs in the AML niche along with prior evidence of persistent functional changes led us to determine if an epigenetic mechanism was involved. First, in a candidate screen we discovered that AML significantly dysregulates the expression of key regulators of DNA methylation patterning in MSCs. Notably, the de novo methyltransferases Dnmt3a and 3b, as well as Tet2, were significantly upregulated in MSCs from all three AML xenografts. This highly reproducible in vivo observation prompted us to perform whole genome bisulfite sequencing on MSCs derived from our Molm-14 xenograft model, revealing substantial changes between AML xenografts and control MSCs in the DNA methylation signatures of CpG islands within annotated promoter regions. Gene ontology analyses of these promoter regions identified significant AML induces changes in apoptotic, UPR stress, and differentiation programs, consistent with both our transcriptional profiling and the change in MSC/ OPC composition in the niche. In sum, this work provides a link to explain prior studies in patients and demonstrates the power of xenograft models to experimentally dissect the complex and dynamic relationship between leukemia and discrete stromal populations in the AML niche. We provide direct evidence, free of tissue culture bias, that AML effects changes in the BM niche that differentially affect two key cellular components responsible for extrinsic drug resistance and relapse in AML. DisclosuresNo relevant conflicts of interest to declare.

Activation Levels of b-Catenin Signaling in Osteoblasts Define a Threshold for the Development of Myeloid Neoplasia of Variable Severity

A growing body of evidence indicates that osteoblasts have a direct pathogenetic role in the development of myelodysplastic syndromes (MDS), acute myeloid leukemia (AML) and transformation of MDS to AML. Models of osteoblast-induced MDS or AML (OIAML) include b-catenin activation in osteoblasts inducing AML in mice and seen in more than 30% of MDS and AML patients; deletion of Dicer1 in osteoblast progenitors leading to MDS in mice; Noonan syndrome mutation (PTPN11) in stromal cells driving MPN in mice; and Schwachman-Diamond syndrome mutation in stromal cells driving MDS in mice and predicting AML progression in patients. To further understand the mechanism of OIAML we examined whether a defined threshold of AML-inducing signal by osteoblasts is required for disease development. For this purpose we constitutively activated b-catenin in osteoblasts using three different lines of 2.3Col1 a 1-Cre transgenic mice characterized by different recombination efficiencies in bone. Line 1, in which recombination efficiency was 80%. Line 2 was the same original line in which after several inter-breedings we detected a reduction in recombination efficiency to 20%. Line3, which has 75% recombination efficiency. Activation of b-catenin in osteoblasts using Line 1, led to the development of MDS which rapidly transformed to full-blown AML characterized by multi-organ infiltration with myeloid blasts, hematopoietic deregulation and lethality by 6 weeks of age. In contrast, activation of b-catenin in osteoblasts using Line 2 did not lead to AML development. Mice remained healthy and survived until at least 10 months of age, the entire time they were observed. Activation of b-catenin in osteoblasts using Line 3 led to anemia, with the appearance of myeloid blasts in the blood, marrow and spleen and segmented neutrophils in blood. In addition to what was observed with Line 1, Pelger Huet neutrophils and Howell-Jolly bodies were detected in the blood. The latter are RBCs with inclusions of nuclear chromatin remnants (damaged chromosome fragments) indicating genomic instability and a dysfunctional spleen, unable to clear them from the circulation, due to dense infiltration with blasts and megakaryocytes. A spectrum of atypical immature monocytes with abundant pale blue agranular cytoplasm with vacuoles were observed in the blood. Taken together these features resemble characteristics of human MDS/CMML. Mice die between 4 and 14 weeks of age, when they undergo “blast crisis” indicating transformation to AML. To examine whether and how these observations relate to the human disease, we compared the levels of b-catenin activation in osteoblasts (calculated as a function % osteoblasts (Lin-/CD34-/RUNX2+ cells) expressing activated b-catenin) with the different MDS diseases subtypes as those are defined by the 2008 World Health Organization (WHO) classification system. We found that increased levels of b-catenin activation in osteoblasts correlate with the severity of the MDS type and with conversion to AML. Collectively, these observations suggest that a minimal activation level of a key leukemogenic signal from the stroma, is required to initiate cancer development, and provides mechanistic insight into the formation and progression of preleukemic stem cells in AML and the transformation of MDS to AML. DisclosuresRaza:Onconova Therapeutics: Research Funding, Speakers Bureau; Kura Oncology: Research Funding; Novartis: Speakers Bureau; Genoptix: Speakers Bureau; Janssen R&D: Research Funding; Celgene Inc.: Research Funding; Syros Pharmaceuticals: Research Funding.

Wnt/\xdf-catenin-mediated p53 suppression is indispensable for osteogenesis of mesenchymal progenitor cells

The developmental origins of mesenchymal progenitor cells (MPCs) and molecular machineries regulating their fate and differentiation are far from defined owing to their complexity. Osteoblasts and adipocytes are descended from common MPCs. Their fates are collectively determined by an orchestra of pathways in response to physiological and external cues. The canonical Wnt pathway signals MPCs to commit to osteogenic differentiation at the expense of adipogenic fate. In contrast to ß-catenin, p53’s anti-osteogenic function is much less understood. Both activities are thought to be achieved through targeting Runx2 and/or Osterix (Osx, Sp7) transcription. Precisely, how Osx activity is dictated by ß-catenin or p53 is not clarified and represents a knowledge gap that, until now, has largely been taken for granted. Using conditional lineage-tracing mice, we demonstrated that chondrocytes gave rise to a sizable fraction of MPCs, which served as progenitors of chondrocyte-derived osteoblasts (Chon-ob). Wnt/ß-catenin activity was only required at the stage of chondrocyte-derived mesenchymal progenitor (C-MPC) to Chon-ob differentiation. ß-catenin– C-MPCs lost osteogenic ability and favored adipogenesis. Mechanistically, we discovered that p53 activity was elevated in ß-catenin– MPCs including ß-catenin– C-MPCs and deleting p53 from the ß-catenin– MPCs fully restored osteogenesis. While high levels of p53 were present in the nuclei of ß-catenin– MPCs, Osx was confined to the cytoplasm, implying a mechanism that did not involve direct p53-Osx interaction. Furthermore, we found that p53’s anti-osteogenic activity was dependent on its DNA-binding ability. Our findings identify chondrocytes as an additional source for MPCs and indicate that Wnt/ß-catenin discretely regulates chondrocyte to C-MPC and the subsequent C-MPC to osteoblast developments. Most of all we unveil a previously unrecognized functional link between ß-catenin and p53, placing p53’s negative role in the context of Wnt/ß-catenin signaling-induced MPC osteogenic differentiation.

Mesoporous zirconia surfaces with anti-biofilm properties for dental implants

Cytocompatible bioactive surface treatments conferring antibacterial properties to osseointegrated dental implants are highly requested to prevent bacteria-related peri-implantitis. Here we focus on a newly designed family of mesoporous coatings based on zirconia (ZrO2) microstructure doped with gallium (Ga), exploiting its antibacterial and pro-osseo-integrative properties. The ZrO2 films were obtained via sol–gel synthesis route using Pluronic F127 as templating agent, while Ga doping was gained by introducing gallium nitrate hydrate. Chemical characterization by means of x-ray photoelectron spectroscopy and glow discharge optical emission spectroscopy confirmed the effective incorporation of Ga. Then, coatings morphological and structural analysis were carried out by transmission electron microscopy and selected area electron diffraction unveiling an effective stabilization of both the mesoporous structure and the tetragonal ZrO2 phase. Specimens’ cytocompatibility was confirmed towards gingival fibroblast and osteoblasts progenitors cultivated directly onto the coatings showing comparable metabolic activity and morphology in respect to controls cultivated on polystyrene. The presence of Ga significantly reduced the metabolic activity of the adhered oral pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans in comparison to untreated bulk zirconia (p < 0.05); on the opposite, Ga ions did not significantly reduce the metabolism of the oral commensal Streptococcus salivarius (p > 0.05) thus suggesting for a selective anti-pathogens activity. Finally, the coatings’ ability to preserve cells from bacterial infection was proved in a co-culture method where cells and bacteria were cultivated in the same environment: the presence of Ga determined a significant reduction of the bacteria viability while allowing at the same time for cells proliferation. In conclusion, the here developed coatings not only demonstrated to satisfy the requested antibacterial and cytocompatibility properties, but also being promising candidates for the improvement of implantable devices in the field of implant dentistry.

A decrease in NAD+ contributes to the loss of osteoprogenitors and bone mass with aging

Age-related osteoporosis is caused by a deficit in osteoblasts, the cells that secrete bone matrix. The number of osteoblast progenitors also declines with age associated with increased markers of cell senescence. The forkhead box O (FoxO) transcription factors attenuate Wnt/β-catenin signaling and the proliferation of osteoprogenitors, thereby decreasing bone formation. The NAD+-dependent Sirtuin1 (Sirt1) deacetylates FoxOs and β-catenin in osteoblast progenitors and, thereby, increases bone mass. However, it remains unknown whether the Sirt1/FoxO/β-catenin pathway is dysregulated with age in osteoblast progenitors. We found decreased levels of NAD+ in osteoblast progenitor cultures from old mice, associated with increased acetylation of FoxO1 and markers of cell senescence. The NAD+ precursor nicotinamide riboside (NR) abrogated FoxO1 and β-catenin acetylation and several marker of cellular senescence, and increased the osteoblastogenic capacity of cells from old mice. Consistent with these effects, NR administration to C57BL/6 mice counteracted the loss of bone mass with aging. Attenuation of NAD+ levels in osteoprogenitor cultures from young mice inhibited osteoblastogenesis in a FoxO-dependent manner. In addition, mice with decreased NAD+ in cells of the osteoblast lineage lost bone mass at a young age. Together, these findings suggest that the decrease in bone formation with old age is due, at least in part, to a decrease in NAD+ and dysregulated Sirt1/FoxO/β-catenin pathway in osteoblast progenitors. NAD+ repletion, therefore, represents a rational therapeutic approach to skeletal involution.

Fibronectin derived from osteoblastic progenitors suppresses tumour growth

Fibronectin derived from osteoblastic progenitors suppresses tumour growth

Osteonecrosis of jaw secondary to stem cell transplant

<h3>Background</h3> Osteonecrosis of the jaw (ONJ) is necrosis of mandibular or maxillary bone, which may lead to bone exposure, regardless of the etiology. It is thought to be attributed to insufficient blood supply and altered bone turnover, resulting in a disrupted repair process and eventually leading to the collapse of bone in the setting of microfractures. We report what is, to the best of our knowledge, the first case of ONJ secondary to stem cell transplant (SCT). <h3>Case Summary</h3> A 69-year-old woman with a medical history significant for hypertension, osteopenia, and myelodysplastic syndrome, status post-haploidentical SCT in March 2018, presented for evaluation of asymptomatic exposed bone of the maxilla and mandible. She had no history of radiotherapy to her head and neck; however, she had undergone total body irradiation of 2 Gy in February 2018 as part of her conditioning regimen. She had no history of exposure to a bisphosphonate, receptor activator of nuclear factor-κB ligand inhibitor, antiangiogenic medicine, or corticosteroids. Her current medications included amlodipine, metoprolol, omeprazole, tacrolimus, sulfamethoxazole-trimethoprim, acyclovir, cholecalciferol, and folic acid. She did not have gingivitis or active periodontal disease. The patient had developed exposed necrotic bone sequestra in several areas of the buccal aspect of her maxillary and mandibular gingiva 2 months after her transplant in May 2018. The pathology report indicated fragments of necrotic bone with acute inflammation and bacterial overgrowth consistent with osteonecrosis. <h3>Conclusions</h3> There is evidence that the microenvironment of the marrow stromal system is severely and irreversibly damaged after SCT. The deficit in the quantity and quality of osteoblastic progenitors may compromise the ability to regenerate a normal osteogenic cell population, leading to an abnormality in bone remodeling/turnover. Although osteonecrosis of the appendicular skeleton is a common complication after SCT, there have been no reports of association with ONJ. Particularly interesting is the pattern of ONJ presentation in all the 4 quadrants in this patient.

Rothmund-Thomson Syndrome-Like RECQL4 Truncating Mutations Cause a Haploinsufficient Low-Bone-Mass Phenotype in Mice.

Rothmund-Thomson syndrome (RTS) is an autosomal recessive disorder characterized by defects in the skeletal system, such as bone hypoplasia, short stature, low bone mass, and an increased incidence of osteosarcoma. RTS type 2 patients have germ line compound biallelic protein-truncating mutations of RECQL4. As existing murine models employ Recql4 null alleles, we have attempted to more accurately model RTS by generating mice with patient-mimicking truncating Recql4 mutations. Truncating mutations impaired the stability and subcellular localization of RECQL4 and resulted in homozygous embryonic lethality and a haploinsufficient low-bone mass phenotype. Combination of a truncating mutation with a conditional Recql4 null allele demonstrated that the skeletal defects were intrinsic to the osteoblast lineage. However, the truncating mutations did not promote tumorigenesis. We utilized murine Recql4 null cells to assess the impact of human RECQL4 mutations using an in vitro complementation assay. While some mutations created unstable protein products, others altered subcellular localization of the protein. Interestingly, the severity of the phenotypes correlated with the extent of protein truncation. Collectively, our results reveal that truncating RECQL4 mutations in mice lead to an osteoporosis-like phenotype through defects in early osteoblast progenitors and identify RECQL4 gene dosage as a novel regulator of bone mass.

Translational cell biology of highly malignant osteosarcoma.

Highly malignant osteosarcoma (HMO) is the most frequent malignant bone tumor preferentially occurring in adolescents and children with a second more flat peak in patients over the age of 60. The younger patients benefit from combined neoadjuvant chemotherapy with 65-70% 5-year survival rate. In patients with metastatic HMO the 5-year survival rate is consistently poor with approximately 30%. In the last several years strategies for target therapies have been developed by using next generation sequencing (NGS) for defining targetable molecular factors. However, it has so far been challenging to establish an effective target therapy for so-called 'orphan tumors' without recognizable driver mutations, including HMO. The molecular genetic studies using NGS have shown that HMOs are genomically unstable tumors with highly complex chaotic karyotypes. Before the background of this genetic complexity more investigations should be performed in the future for defining targetable biological factors. As the prognosis could not be improved for 40 years one may expect improvements for patients only by gaining a deeper understanding of the cell and molecular biology of HMO. The cell of origin of HMO is being clarified now. The majority of studies indicate that an osteoblastic progenitor cell is probably the cell of origin of HMO and not an undifferentiated mesenchymal stem cell. This means that the established histopathological definition of HMO through verification of osteoid production by the osteoblastic cells is well justified and will probably be the cornerstone for a precise differential diagnosis of HMO also in the years to come.