Osteoclasts Derive Predominantly from Bone Marrow-Resident CX3CR1+ Precursor Cells in Homeostasis, whereas Circulating CX3CR1+ Cells Contribute to Osteoclast Development during Fracture Repair.

Osteoclasts (OC) originate from either bone marrow (BM)-resident or circulating myeloid OC progenitors (OCP) expressing the receptor CX3CR1. Multiple lines of evidence argue that OCP in homeostasis and inflammation differ. We investigated the relative contributions of BM-resident and circulating OCP to osteoclastogenesis during homeostasis and fracture repair. Using CX3CR1-EGFP/TRAP tdTomato mice, we found CX3CR1 expression in mononuclear cells, but not in multinucleated TRAP+ OC. However, CX3CR1-expressing cells generated TRAP+ OC on bone within 5 d in CX3CR1CreERT2/Ai14 tdTomato reporter mice. To define the role that circulating cells play in osteoclastogenesis during homeostasis, we parabiosed TRAP tdTomato mice (CD45.2) on a C57BL/6 background with wild-type (WT) mice (CD45.1). Flow cytometry (CD45.1/45.2) demonstrated abundant blood cell mixing between parabionts after 2 wk. At 4 wk, there were numerous tdTomato+ OC in the femurs of TRAP tdTomato mice but almost none in WT mice. Similarly, cultured BM stimulated to form OC demonstrated multiple fluorescent OC in cell cultures from TRAP tdTomato mice, but not from WT mice. Finally, flow cytometry confirmed low-level engraftment of BM cells between parabionts but significant engraftment in the spleens. In contrast, during fracture repair, we found that circulating CX3CR1+ cells migrated to bone, lost expression of CX3CR1, and became OC. These data demonstrate that OCP, but not mature OC, express CX3CR1 during both homeostasis and fracture repair. We conclude that, in homeostasis mature OC derive predominantly from BM-resident OCP, whereas during fracture repair, circulating CX3CR1+ cells can become OC.

Fully Automated Macro- and Microfluidic Production of [68Ga]Ga-Citrate on mAIO® and iMiDEVTM Modules.

68Ga-radionuclide has gained importance due to its availability via 68Ge/68Ga generator or cyclotron production, therefore increasing the number of 68Ga-based PET radiopharmaceuticals available in clinical practice. [68Ga]Ga-citrate PET has been shown to be prominent for detection of inflammation/infection of the musculoskeletal, gastrointestinal, respiratory, and cardiovascular systems. Automation and comparison between conventional and microfluidic production of [68Ga]Ga-citrate was performed using miniAllInOne® (Trasis) and iMiDEV™ (PMB-Alcen) synthetic modules. Fully automated procedures were elaborated for cGMP production of tracer. In order to facilitate the tracer approval as a radiopharmaceutical for clinical use, a new method for radiochemical identity determination by HPLC analysis to complement standard TLC radiochemical purity measurement was developed. The results showed higher radiochemical yields when using MCX cartridge on the conventional module mAIO®, while a PS-H+ cation exchanger was shown to be preferred for integration into the microfluidic cassette of iMiDEV™ module. In this study, the fully automated radiosynthesis of [68Ga]Ga-citrate using different synthesizers demonstrated reliable and reproducible radiochemical yields. In order to demonstrate the applicability of [68Ga]Ga-citrate, in vitro and in vivo studies were performed showing similar characteristics of the tracer obtained using macro- and microfluidic ways of production.

Assessment of a one-week ketogenic diet on brain glycolytic metabolism and on the status epilepticus stage of a lithium-pilocarpine rat model.

The ketogenic diet (KD) has been shown to be effective in refractory epilepsy after long-term administration. However, its interference with short-term brain metabolism and its involvement in the early process leading to epilepsy remain poorly understood. This study aimed to assess the effect of a short-term ketogenic diet on cerebral glucose metabolic changes, before and after status epilepticus (SE) in rats, by using [18F]-FDG PET. Thirty-nine rats were subjected to a one-week KD (KD-rats, n = 24) or to a standard diet (SD-rats, n = 15) before the induction of a status epilepticus (SE) by lithium-pilocarpine administrations. Brain [18F]-FDG PET scans were performed before and 4 h after this induction. Morphological MRIs were acquired and used to spatially normalize the PET images which were then analyzed voxel-wisely using a statistical parametric-based method. Twenty-six rats were analyzed (KD-rats, n = 15; SD-rats, n = 11). The 7 days of the KD were associated with significant increases in the plasma ß-hydroxybutyrate level, but with an unchanged glycemia. The PET images, recorded after the KD and before SE induction, showed an increased metabolism within sites involved in the appetitive behaviors: hypothalamic areas and periaqueductal gray, whereas no area of decreased metabolism was observed. At the 4th hour following the SE induction, large metabolism increases were observed in the KD- and SD-rats in areas known to be involved in the epileptogenesis process late-i.e., the hippocampus, parahippocampic, thalamic and hypothalamic areas, the periaqueductal gray, and the limbic structures (and in the motor cortex for the KD-rats only). However, no statistically significant difference was observed when comparing SD and KD groups at the 4th hour following the SE induction. A one-week ketogenic diet does not prevent the status epilepticus (SE) and associated metabolic brain abnormalities in the lithium-pilocarpine rat model. Further explorations are needed to determine whether a significant prevention could be achieved by more prolonged ketogenic diets and by testing this diet in less severe experimental models, and moreover, to analyze the diet effects on the later and chronic stages leading to epileptogenesis.

Multi-tracer and multiparametric PET imaging to detect the IDH mutation in glioma: a preclinical translational in vitro, in vivo, and ex vivo study.

BACKGROUND: This translational study explores multi-tracer PET imaging for the non-invasive detection of the IDH1 mutation which is a positive prognostic factor in glioma. METHODS: U87 human high-grade glioma (HGG) isogenic cell lines with or without the IDH1 mutation (CRISP/Cas9 method) were stereotactically grafted into rat brains, and examined, in vitro, in vivo and ex vivo. PET imaging sessions, with radiotracers specific for glycolytic metabolism ([18F]FDG), amino acid metabolism ([18F]FDopa), and inflammation ([18F]DPA-714), were performed sequentially during 3-4 days. The in vitro radiotracer uptake was expressed as percent per million cells. For each radiotracer examined in vivo, static analyses included the maximal and mean tumor-to-background ratio (TBRmax and TBRmean) and metabolic tumor volume (MTV). Dynamic analyses included the distribution volume ratio (DVR) and the relative residence time (RRT) extracted from a reference Logan model. Ex vivo analyses consisted of immunological analyses. RESULTS: In vitro, IDH1+ cells (i.e. cells expressing the IDH1 mutation) showed lower levels of [18F]DPA-714 uptake compared to IDH1- cells (p < 0.01). These results were confirmed in vivo with lower [18F]DPA-714 uptake in IDH+ tumors (3.90 versus 5.52 for TBRmax, p = 0.03). Different values of [18F]DPA-714 and [18F] FDopa RRT (respectively 11.07 versus 22.33 and 2.69 versus - 1.81 for IDH+ and IDH- tumors, p < 0.02) were also observed between the two types of tumors. RRT [18F]DPA-714 provided the best diagnostic performance to discriminate between the two cell lines (AUC of 100%, p < 0.01). Immuno-histological analyses revealed lower expression of Iba-1 and TSPO antibodies in IDH1+ tumors. CONCLUSIONS: [18F]DPA-714 and [18F] FDopa both correlate with the presence of the IDH1 mutation in HGG. These radiotracers are therefore good candidates for translational studies investigating their clinical applications in patients.

Synthesis of a DOTA-C-glyco bifunctional chelating agent and preliminary in vitro and in vivo study of [68Ga]Ga-DOTA-C-glyco-RGD.

The design of bifunctional chelating agents (BFCA) allowing straightforward radiometal labelling of biomolecules is a current challenge. We report herein the development of a bifunctional chelating agent based on a DOTA chelator linked to a C-glycosyl compound, taking advantage of the robustness and hydrophilicity of this type of carbohydrate derivative. This new BFCA was coupled with success by CuAAC with c(RGDfK) for αvß3 integrin targeting. As attested by in vitro evaluation, the conjugate DOTA-C-glyco-c(RGDfC) demonstrated high affinity for αvß3 integrins (IC50 of 42 nM). [68Ga]Ga-DOTA-C-glyco-c(RGDfK) was radiosynthesized straightforwardly and showed high hydrophilic property (log D 7.4 = -3.71) and in vitro stability (>120 min). Preliminary in vivo PET study of U87MG engrafted mice gave evidence of an interesting tumor-to-non-target area ratio. All these data indicate that [68Ga]Ga-DOTA-C-glyco-c(RGDfK) allows monitoring of αvß3 expression and could thus be used for cancer diagnosis. The DOTA-C-glycoside BFCA reported here could also be used with various ligands and chelating other (radio)metals opening a broad scope of applications in imaging modalities and therapy.

Modulation of Notch1 signaling regulates bone fracture healing.

Fracture healing involves interactions of different cell types, driven by various growth factors, and signaling cascades. Periosteal mesenchymal progenitor cells give rise to the majority of osteoblasts and chondrocytes in a fracture callus. Notch signaling has emerged as an important regulator of skeletal cell proliferation and differentiation. We investigated the effects of Notch signaling during the fracture healing process. Increased Notch signaling in osteochondroprogenitor cells driven by overexpression of Notch1 intracellular domain (NICD1) (αSMACreERT2 mice crossed with Rosa-NICD1) during fracture resulted in less cartilage, more mineralized callus tissue, and stronger and stiffer bones after 3 weeks. Periosteal cells overexpressing NICD1 showed increased proliferation and migration in vitro. In vivo data confirmed that increased Notch1 signaling caused expansion of alpha-smooth muscle actin (αSMA)-positive cells and their progeny including αSMA-derived osteoblasts in the callus without affecting osteoclast numbers. In contrast, anti-NRR1 antibody treatment to inhibit Notch1 signaling resulted in increased callus cartilage area, reduced callus bone mass, and reduced biomechanical strength. Our study shows a positive effect of induced Notch1 signaling on the fracture healing process, suggesting that stimulating the Notch pathway could be beneficial for fracture repair.

Splenomegaly, myeloid lineage expansion and increased osteoclastogenesis in osteogenesis imperfecta murine.

Osteogenesis imperfecta (OI) is a disease caused by defects in type I collagen production that results in brittle bones. While the pathology is mainly caused by defects in the osteoblast lineage, there is also elevated bone resorption by osteoclasts resulting in high bone turnover in severe forms of the disease. Osteoclasts originate from hematopoietic myeloid cells, however changes in hematopoiesis have not been previously documented in OI. In this study, we evaluated hematopoietic lineage distribution and osteoclast progenitor cell frequency in bone marrow, spleen and peripheral blood of osteogenesis imperfecta murine (OIM) mice, a model of severe OI. We found splenomegaly in all ages examined, and expansion of myeloid lineage cells (CD11b+) in bone marrow and spleen of 7-9week old male OIM animals. OIM spleens also showed an increased frequency of purified osteoclast progenitors. This phenotype is suggestive of chronic inflammation. Isolated osteoclast precursors from both spleen and bone marrow formed osteoclasts more rapidly than wild-type controls. We found that serum TNFα levels were increased in OIM, as was IL1α in OIM females. We targeted inflammation therapeutically by treating growing animals with murine TNFR2:Fc, a compound that blocks TNFα activity. Anti-TNFα treatment marginally decreased spleen mass in OIM females, but failed to reduce bone resorption, or improve bone parameters or fracture rate in OIM animals. We have demonstrated that OIM mice have changes in their hematopoietic system, and form osteoclasts more rapidly even in the absence of OI osteoblast signals, however therapy targeting TNFα did not improve disease parameters.

* Hypoxia for Mesenchymal Stem Cell Expansion and Differentiation: The Best Way for Enhancing TGFß-Induced Chondrogenesis and Preventing Calcifications in Alginate Beads.

We examined the respective influence of a sequential or a continuous hypoxia during expansion and transforming growth factor beta 1-driven chondrogenic differentiation of human bone marrow mesenchymal stem cells (MSCs). The differentiation was performed within alginate beads, a classical tool for the implantation of MSCs within the joint. The standard normoxic 2D (expansion) and 3D (differentiation) MSCs cultures served as reference. To determine the quality of chondrogenesis, we analyzed typical markers such as type II and X collagens, SOX9, COMP, versican, and aggrecan mRNAs using polymerase chain reaction and we assessed the production of type II collagen and hypoxia-inducible factor (HIF)-1α by histological stainings. We simultaneously assessed the expression of osteogenic mRNAs (Alkaline Phosphatase, RUNX2, and Osteocalcin) and the presence of micro-calcifications by Alizarin red and Raman spectroscopy. Chondrogenic differentiation is clearly improved by hypoxia in 3D. Best results were obtained when the entire process, that is, 2D expansion and 3D differentiation, was performed under continuous 5% hypoxic condition. In addition, no calcification (hydroxyapatite, proved by RAMAN) was observed after 2D hypoxic expansion even in the case of a normoxic differentiation, in contrast with controls. Finally, a better chondrogenic differentiation of human MSCs is achieved when a reduced oxygen tension is applied during both expansion and differentiation times, avoiding in vitro osteogenic commitment of cells and subsequently the calcification deposition.

Visual reporters for study of the osteoblast lineage.

Advancing our understanding of osteoblast biology and differentiation is critical to elucidate the pathological mechanisms responsible for skeletal diseases such as osteoporosis. Histology and histomorphometry, the classical methods to study osteoblast biology, identify osteoblasts based on their location and morphology and ability to mineralize matrix, but do not clearly define their stage of differentiation. Introduction of visual transgenes into the cells of osteoblast lineage has revolutionized the field and resulted in a paradigm shift that allowed for specific identification and isolation of subpopulations within the osteoblast lineage. Knowledge acquired from the studies based on GFP transgenes has allowed for more precise interpretation of studies analyzing targeted overexpression or deletion of genes in the osteoblast lineage. Here, we provide a condensed overview of the currently available promoter-fluorescent reporter transgenic mice that have been generated and evaluated to varying extents. We cover different stages of the lineage as transgenes have been utilized to identify osteoprogenitors, pre-osteoblasts, osteoblasts, or osteocytes. We show that each of these promoters present with advantages and disadvantages. The studies based on the use of these reporter mice have improved our understanding of bone biology. They constitute attractive models to target osteoblasts and help to understand their cell biology.

Osteogenic potential of alpha smooth muscle actin expressing muscle resident progenitor cells.

Heterotopic ossification (HO) is a pathological process where bone forms in connective tissues such as skeletal muscle. Previous studies have suggested that muscle-resident non-myogenic mesenchymal progenitors are the likely source of osteoblasts and chondrocytes in HO. However, the previously identified markers of muscle-resident osteoprogenitors label up to half the osteoblasts within heterotopic lesions, suggesting other cell populations are involved. We have identified alpha smooth muscle actin (αSMA) as a marker of osteoprogenitor cells in bone and periodontium, and of osteo-chondro progenitors in the periosteum during fracture healing. We therefore utilized a lineage tracing approach to evaluate whether αSMACreERT2 identifies osteoprogenitors in the muscle. We show that in the muscle, αSMACreERT2 labels both perivascular cells, and satellite cells. αSMACre-labeled cells undergo osteogenic differentiation in vitro and form osteoblasts and chondrocytes in BMP2-induced HO in vivo. In contrast, Pax7CreERT2-labeled muscle satellite cells were restricted to myogenic differentiation in vitro, and rarely contributed to HO in vivo. Our data indicate that αSMACreERT2 labels a large proportion of osteoprogenitors in skeletal muscle, and therefore represents another marker of muscle-resident cells with osteogenic potential under HO-inducing stimulus. In contrast, muscle satellite cells make minimal contribution to bone formation in vivo.

Dose-response of superparamagnetic iron oxide labeling on mesenchymal stem cells chondrogenic differentiation: a multi-scale in vitro study.

AIM: The aim of this work was the development of successful cell therapy techniques for cartilage engineering. This will depend on the ability to monitor non-invasively transplanted cells, especially mesenchymal stem cells (MSCs) that are promising candidates to regenerate damaged tissues. METHODS: MSCs were labeled with superparamagnetic iron oxide particles (SPIO). We examined the effects of long-term labeling, possible toxicological consequences and the possible influence of progressive concentrations of SPIO on chondrogenic differentiation capacity. RESULTS: No influence of various SPIO concentrations was noted on human bone marrow MSC viability or proliferation. We demonstrated long-term (4 weeks) in vitro retention of SPIO by human bone marrow MSCs seeded in collagenic sponges under TGF-ß1 chondrogenic conditions, detectable by Magnetic Resonance Imaging (MRI) and histology. Chondrogenic differentiation was demonstrated by molecular and histological analysis of labeled and unlabeled cells. Chondrogenic gene expression (COL2A2, ACAN, SOX9, COL10, COMP) was significantly altered in a dose-dependent manner in labeled cells, as were GAG and type II collagen staining. As expected, SPIO induced a dramatic decrease of MRI T2 values of sponges at 7T and 3T, even at low concentrations. CONCLUSIONS: This study clearly demonstrates (1) long-term in vitro MSC traceability using SPIO and MRI and (2) a deleterious dose-dependence of SPIO on TGF-ß1 driven chondrogenesis in collagen sponges. Low concentrations (12.5-25 µg Fe/mL) seem the best compromise to optimize both chondrogenesis and MRI labeling.

Respective interest of T2 mapping and diffusion tensor imaging in assessing porcine knee cartilage with MR at 3 Teslas.

Non-invasive quantitative assessment of articular cartilage integrity is essential for early detection and evaluation of osteoarthritis (OA) and for the follow-up of stem-cell-driven cartilage engineering. In this study, we investigated the feasibility of exploiting diffusion tensor imaging (DTI) on porcine knee joints with a clinical magnetic resonance (MR) scanner to extract micro-structural information in order to complement biochemical information quantified by T2 maps. We propose an MR protocol for quantifying T2 and cartilage microstructure with diffusion MR on a clinical scanner. Preliminary results were obtained on four pig knee joints using a 3 T GE clinical MRI scanner and an 8-channel knee coil array. The measured cartilage volume, T2 values, apparent diffusion coefficient and fractional anisotropy (FA) of femoral and tibial cartilage were respectively 9.8/2.3 mm2, 67.0/56.1 ms, 1.3/1.3×10-3 mm2/s and 0.4/0.3. This new protocol has the potential to be combined in vivo with quantitative assessment of both cartilage degradation and restoration in osteoarthritis.

Effect of dynamic loading on MSCs chondrogenic differentiation in 3-D alginate culture.

Mesenchymal stem cells (MSCs) are regarded as a potential autologous source for cartilage repair, because they can differentiate into chondrocytes by transforming growth factor-beta (TGF-ß) treatment under the 3-dimensional (3-D) culture condition. In addition to these molecular and biochemical methods, the mechanical regulation of differentiation and matrix formation by MSCs is only starting to be considered. Recently, mechanical loading has been shown to induce chondrogenesis of MSCs in vitro. In this study, we investigated the effects of a calibrated agitation on the chondrogenesis of human bone MSCs (MSCs) in a 3-D alginate culture (day 28) and on the maintenance of chondrogenic phenotypes. Biomechanical stimulation of MSCs increased: (i) types 1 and 2 collagen formation; (ii) the expression of chondrogenic markers such as COMP and SOX9; and (iii) the capacity to maintain the chondrogenic phenotypes. Notably, these effects were shown without TGF-ß treatment. These results suggest that a mechanical stimulation could be an efficient method to induce chondrogenic differentiation of MSCs in vitro for cartilage tissue engineering in a 3-D environment. Additionally, it appears that MSCs and chondrocyte responses to mechanical stimulation are not identical.

Expression of chondrogenic genes by undifferentiated vs. differentiated human mesenchymal stem cells using array technology.

This study investigated the gene expression profile of human mesenchymal stem cells seeded in collagen sponge for 28 days in three different mediums: (1) basal medium as control containing ITS alone, (2) ITS+TGF-ß1 alone or (3) ITS 1% supplemented sequentially by TGF-ß1 (D3-D14) followed by BMP-2 (D15-D28). Differential expression of 84 genes implicated in chondrogenic and osteogenic differentiation of MSCs was analyzed at D28 by real-time RT-PCR array technology. TGF-ß1 alone down-regulated two genes, CD36 and cathepsin K. Sixteen genes were significantly up-regulated, notably type 2 and type 10 collagens, COMP and Sox9. The sequential combination of TGF-ß1 and BMP-2 produced a similar profile with prominent expression of type 2 collagen and the alkaline phosphatase gene. Interestingly, in this in vitro condition, RUNX2 was not up-regulated, suggesting that the sequential combination of TGF-ß1/BMP2 enhances the hypertrophic chondrogenic profile without turning towards the osteoblastic pathway.