NADPH oxidase 4 deficiency attenuates experimental osteoarthritis in mice.

OBJECTIVE: Low-grade inflammation plays a pivotal role in osteoarthritis (OA) through exposure to reactive oxygen species (ROS). In chondrocytes, NADPH oxidase 4 (NOX4) is one of the major ROS producers. In this study, we evaluated the role of NOX4 on joint homoeostasis after destabilisation of the medial meniscus (DMM) in mice. METHODS: Experimental OA was simulated on cartilage explants using interleukin-1ß (IL-1ß) and induced by DMM in wild-type (WT) and NOX4 knockout (NOX4-/-) mice. We evaluated NOX4 expression, inflammation, cartilage metabolism and oxidative stress by immunohistochemistry. Bone phenotype was also determined by micro-CT and histomorphometry. RESULTS: Whole body NOX4 deletion attenuated experimental OA in mice, with a significant reduction of the OARSI score at 8 weeks. DMM increased total subchondral bone plate (SB.Th), epiphysial trabecular thicknesses (Tb.Th) and bone volume fraction (BV/TV) in both NOX4-/- and wild-type (WT) mice. Interestingly, DDM decreased total connectivity density (Conn.Dens) and increased medial BV/TV and Tb.Th only in WT mice. Ex vivo, NOX4 deficiency increased aggrecan (AGG) expression and decreased matrix metalloproteinase 13 (MMP13) and collagen type I (COL1) expression. IL-1ß increased NOX4 and 8-hydroxy-2'-deoxyguanosine (8-OHdG) expression in WT cartilage explants but not in NOX4-/-. In vivo, absence of NOX4 increased anabolism and decreased catabolism after DMM. Finally, NOX4 deletion decreased synovitis score, 8-OHdG and F4/80 staining following DMM. CONCLUSION: NOX4 deficiency restores cartilage homoeostasis, inhibits oxidative stress, inflammation and delays OA progression after DMM in mice. These findings suggest that NOX4 represent a potential target to counteract for OA treatment.

Correction to: Induced membrane maintains its osteogenic properties even when the second stage of Masquelet's technique is performed later.

The original version of this article unfortunately contained a mistake. The presentation and legends of Figs. 4 and 5 were incorrect. The corrected versions are given below. The original article has been corrected.

Induced membrane maintains its osteogenic properties even when the second stage of Masquelet's technique is performed later.

PURPOSE: Previous clinical studies have shown the effectiveness of bone repair using two-stage surgery called the induced membrane (IM) technique. The optimal wait before the second surgery is said to be 1 month. We have been successfully performing the IM technique while waiting an average of 6 months to carry out the second stage. We hypothesised that the IM maintains its beneficial capabilities, even at a later second stage, and that there is no relation between the speed of bone union and the wait between the first and second stage. We sought to explore the biological properties of 'older' IMs sampled to substantiate our clinical observations. METHODS: Thirty-four patients with a critical size defect were treated with the IM technique. In seven of these patients, pieces of the IM were collected 4.2-14.7 months after the first surgery. IM-derived cell phenotype and osteogenic potential were investigated using in vitro studies (n = 4) while IM nature and function were investigated by histology and immunohistochemistry (n = 3). RESULTS: The median wait before the second surgery was 5.8 months [range 1.2-14.7] and bone healing occurred at 7.6 months [range 2.5-49.9] for 26 patients. IMs aged 4.2-14.7 months contained mesenchymal stromal cells with in vitro osteogenic potential and corresponded to a multipotent tissue with osteogenic and chondrogenic capabilities contributing to osteogenesis over time. CONCLUSION: This preliminary study suggests the IM retains its powerful osteogenic properties over time and that waiting longer between the two surgeries does not delay bone union.

The paracrine effects of human induced pluripotent stem cells promote bone-like structures via the upregulation of BMP expression in a mouse ectopic model.

Use of human induced pluripotent stem cells (h-iPSCs) for bone tissue engineering is most appealing, because h-iPSCs are an inexhaustible source of osteocompetent cells. The present study investigated the contribution of undifferentiated h-iPSCs and elucidated aspects of the underlying mechanism(s) of the involvement of these cells to new bone formation. Implantation of undifferentiated h-iPSCs seeded on coral particles in ectopic sites of mice resulted in expression of osteocalcin and DMP-1, and in mineral content similar to that of the murine bone. The number of the implanted h-iPSCs decreased with time and disappeared by 30 days post-implantation. In contrast, expression of the murine osteogenic genes at day 15 and 30 post-implantation provided, for the first time, evidence that the implanted h-iPSCs affected the observed outcomes via paracrine mechanisms. Supporting evidence was provided because supernatant conditioned media from h-iPSCs (h-iPSC CM), promoted the osteogenic differentiation of human mesenchymal stem cells (h-MSCs) in vitro. Specifically, h-iPSC CM induced upregulation of the BMP-2, BMP-4 and BMP-6 genes, and promoted mineralization of the extracellular matrix. Given the current interest in the use of h-iPSCs for regenerative medicine applications, our study contributes new insights into aspects of the mechanism underlying the bone promoting capability of h-iPSCs.

Human Mesenchymal Stem Cell Failure to Adapt to Glucose Shortage and Rapidly Use Intracellular Energy Reserves Through Glycolysis Explains Poor Cell Survival After Implantation.

Mesenchymal stem cells (MSCs) hold considerable promise in tissue engineering (TE). However, their poor survival when exogenously administered limits their therapeutic potential. Previous studies from our group demonstrated that lack of glucose (glc) (but not of oxygen) is fatal to human MSCs because it serves as a pro-survival and pro-angiogenic molecule for human MSCs (hMSCs) upon transplantation. However, which energy-providing pathways MSCs use to metabolize glc upon transplantation? Are there alternative energetic nutrients to replace glc? And most importantly, do hMSCs possess significant intracellular glc reserves for ensuring their survival upon transplantation? These remain open questions at the forefront of TE based-therapies. In this study, we established for the first time that the in vivo environment experienced by hMSCs is best reflected by near-anoxia (0.1% O2 ) rather than hypoxia (1%-5% O2 ) in vitro. Under these near-anoxia conditions, hMSCs rely almost exclusively on glc through anerobic glycolysis for ATP production and are unable to use either exogenous glutamine, serine, or pyruvate as energy substrates. Most importantly, hMSCs are unable to adapt their metabolism to the lack of exogenous glc, possess a very limited internal stock of glc and virtually no ATP reserves. This lack of downregulation of energy turnover as a function of exogenous glc level results in a rapid depletion of hMSC energy reserves that explains their poor survival rate. These new insights prompt for the development of glc-releasing scaffolds to overcome this roadblock plaguing the field of TE based-therapies. Stem Cells 2018;36:363-376.

Bone regeneration in sheep using acropora coral, a natural resorbable scaffold, and autologous mesenchymal stem cells.

Tissue constructs containing mesenchymal stem cells (MSC) are an appealing strategy for repairing massive segmental bone defects. However, their therapeutic effectiveness does not match that of autologous bone grafts; among the complicating reasons, the scaffold resorbability has been identified as a critical feature for achieving bone regeneration. In the present study, the osteogenic potential of constructs obtained by expanding autologous MSC onto granules of Acropora coral, a natural fully-resorbable scaffold, was investigated. MSC adhered and proliferated well in vitro after 1 week. When implanted in vivo into long-bone, critical-size defects in sheep (n=5), these constructs exhibited a two-fold increase in bone formation 6 months postimplantation compared to Acropora scaffolds alone (n=5). Interestingly, osteogenesis, mediated by MSC, within these constructs was found continuous not only with the bony stumps, but also at the core of the implants. Scaffold resorption was almost complete at 6 months, leading to full bone regeneration in one animal. Acropora coral appear to be an appealing scaffold for bone tissue engineering because it supported in vitro MSC adhesion and proliferation. Moreover, these results provided evidence that MSC could promote bone regeneration in sheep when loaded one a natural fully resorbable scaffold.

Human embryonic stem cell-derived mesodermal progenitors display substantially increased tissue formation compared to human mesenchymal stem cells under dynamic culture conditions in a packed bed/column bioreactor.

Bone tissue engineering represents a promising strategy to obviate bone deficiencies, allowing the ex vivo construction of bone substitutes with unprecedented potential in the clinical practice. Considering that in the human body cells are constantly stimulated by chemical and mechanical stimuli, the use of bioreactor is emerging as an essential factor for providing the proper environment for the reproducible and large-scale production of the engineered substitutes. Human mesenchymal stem cells (hMSCs) are experimentally relevant cells but, regardless the encouraging results reported after culture under dynamic conditions in bioreactors, show important limitations for tissue engineering applications, especially considering their limited proliferative potential, loss of functionality following protracted expansion, and decline in cellular fitness associated with aging. On the other hand, we previously demonstrated that human embryonic stem cell-derived mesodermal progenitors (hES-MPs) hold great potential to provide a homogenous and unlimited source of cells for bone engineering applications. Based on prior scientific evidence using different types of stem cells, in the present study we hypothesized that dynamic culture of hES-MPs in a packed bed/column bioreactor had the potential to affect proliferation, expression of genes involved in osteogenic differentiation, and matrix mineralization, therefore resulting in increased bone-like tissue formation. The reported findings suggest that hES-MPs constitute a suitable alternative cell source to hMSCs and hold great potential for the construction of bone substitutes for tissue engineering applications in clinical settings.

Proangiogenic and prosurvival functions of glucose in human mesenchymal stem cells upon transplantation.

A major limitation in the development of cellular therapies using human mesenchymal stem cells (hMSCs) is cell survival post-transplantation. In this study, we challenged the current paradigm of hMSC survival, which assigned a pivotal role to oxygen, by testing the hypothesis that exogenous glucose may be key to hMSC survival. We demonstrated that hMSCs could endure sustained near-anoxia conditions only in the presence of glucose. In this in vitro cell model, the protein expressions of Hif-1α and angiogenic factors were upregulated by the presence of glucose. Ectopically implanted tissue constructs supplemented with glucose exhibited four- to fivefold higher viability and were more vascularized compared to those without glucose at day 14. These findings provided the first direct in vitro and in vivo demonstration of the proangiogenic and prosurvival functions of glucose in hMSC upon transplantation and identified glucose as an essential component of the ideal scaffold for transplanting stem cells.

Noninvasive bioluminescent quantification of viable stem cells in engineered constructs.

Bioluminescence from murine stem cells tagged with the luciferase gene reporter and distributed within three-dimensional scaffolds of two different materials is quantified in vitro and in vivo. The luminescence emitted from cells adhering to the scaffolds tested is monitored noninvasively using a bioluminescence imaging system. Monitoring the kinetics of luciferase expression via bioluminescence enables real-time assessment of cell survival and proliferation on scaffolds both in vitro and in vivo over prolonged (8 weeks) periods of time.

A perfusion bioreactor for engineering bone constructs: an in vitro and in vivo study.

A perfusion bioreactor, which was designed based on fluidized bed concepts, was validated for the culture of bone constructs of clinically relevant size. For this study, natural coral has been used as three-dimensional scaffolds. This biomaterial is a microporous, biocompatible, osteoconductive, and absorbable scaffold. This perfusion bioreactor provided a stable environment in terms of osmolarity, pH, and, most importantly, oxidative stress. Bone constructs engineered in this system resulted in significantly higher cell proliferation and homogenous cell distribution than those cultured under static conditions. Particularly relevant to the production of bioengineered bone in a clinical setting, custom-made bone constructs (each one with volume up to 30 cm(3)) could be produced using a such perfusion bioreactor. Last, but not least, the bone constructs of clinically relevant volume thus produced were shown to be osteogenic when transplanted subcutaneously in sheep.

In vitro and in vivo bioluminescent quantification of viable stem cells in engineered constructs.

Bioluminescent quantification of viable cells inside three-dimensional porous scaffolds was performed in vitro and in vivo. The assay quantified the bioluminescence of murine stem (C3H10T1/2) cells tagged with the luciferase gene reporter and distributed inside scaffolds of either soft, translucent, AN69 polymeric hydrogel or hard, opaque, coral ceramic materials. Quantitative evaluation of bioluminescence emitted from tagged cells adhering to these scaffolds was performed in situ using either cell lysates and a luminometer or intact cells and a bioluminescence imaging system. Despite attenuation of the signal when compared to cells alone, the bioluminescence correlated with the number of cells (up to 1.5 x 10(5)) present on each material scaffold tested, both in vitro and noninvasively in vivo (subcutaneous implants in the mouse model). The noninvasive bioluminescence measurement technique proved to be comparable to the cell-destructive bioluminescence measurement technique. Monitoring the kinetics of luciferase expression via bioluminescence enabled real-time assessment of cell survival and proliferation on the scaffolds tested over prolonged (up to 59 days) periods of time. This novel, sensitive, easy, fast-to-implement, quantitative bioluminescence assay has great, though untapped, potential for screening and determining noninvasively the presence of viable cells on biomaterial constructs in the tissue engineering and tissue regeneration fields.

Fibroblast growth factor receptor 2 promotes osteogenic differentiation in mesenchymal cells via ERK1/2 and protein kinase C signaling.

Mesenchymal stem cells (MSCs) are able to differentiate into several lineages including osteoblasts. The signaling mechanisms involved in the osteogenic differentiation of MSCs are however not fully understood. We investigated the role of fibroblast growth factor receptor 2 (FGFR2) in osteoblast committment and differentiation of murine mesenchymal C3H10T1/2 cells stably transfected with wild type (WT) or activated FGFR2 due to Apert S252W genetic mutation (MT). WT FGFR2 slightly increased, whereas MT FGFR2 strongly increased, FGFR2 tyrosine phosphorylation, indicating activation of the receptor. WT and MT FGFR2 increased C3H10T1/2 cell proliferation but not survival. Both WT and MT FGFR2 increased early and late osteoblast gene expression and matrix mineralization. Forced expression of WT and MT FGFR2 also increased osteoblast gene expression in MC3T3-E1 calvaria osteoblasts. In both cell types, MT FGFR2 was more effective than WT FGFR2. In contrast, WT and MT FGFR2 decreased adipocyte differentiation of C3H10T1/2 cells. WT and MT FGFR2 induced ERK1/2 but not JNK or PI3K/AKT phosphorylation. MT, but not WT, also increased protein kinase C (PKC) activity. Pharmacological inhibition of ERK1/2 prevented cell proliferation induced by WT and MT FGFR2. Using dominant-negative ERK and PKCalpha vectors, we demonstrated that WT and MT FGFR2 promoted osteoblast gene expression through ERK1/2 and PKCalpha signaling, respectively. This study identifies FGFR2 as a novel regulatory molecule that promotes osteogenic differentiation in murine MSCs. The promoting effect of WT and MT FGFR2 is mediated by ERK1/2 and PKCalpha pathways that play essential and distinct roles in FGFR2-induced osteogenic differentiation of mesenchymal cells.

Desferrioxamine-driven upregulation of angiogenic factor expression by human bone marrow stromal cells.

Bone marrow stromal cells (BMSCs) are the subject of intense research because of their biological properties and potential use for the repair of damaged tissues. Success of BMSC-based therapies, however, relies on a number of methodological improvements, including the establishment of a vascular network providing nutrients and oxygen to the transplanted cells and ensuring their immediate survival and long-term functionality. We described a method to enhance the autocrine expression of angiogenic factors by BMSCs. For this purpose, human BMSCs were treated with desferrioxamine (DFX). No PDGF-BB, VEGF-R1 or -R2 mRNA expression was detected under any of the conditions tested. mRNA and protein expression levels of TGFbeta1 were similar in BMSCs, whether they were exposed to DFX (50 microM) or to control conditions under normoxia for 48 h. In comparison with the results obtained with control conditions under normoxia, exposure of BMSCs to DFX for 48 h resulted in upregulation of bFGF at the protein (26-fold) but not at the mRNA levels and VEGF at both the mRNA (1.5-fold) and protein levels (4.5-fold). In comparison with the results obtained with control conditions under hypoxia, DFX induced a 50% increase in VEGF secretion but led to the same level of hypoxia inducible factor-1alpha protein expression (a transduction factor involved in angiogenic factor expression and known to be activated by DFX). Exposure of BMSCs to DFX resulted in oversecretion of angiogenic factors, suggesting that DFX-treated BMSCs could be used to supply angiogenic factors.

Long-bone critical-size defects treated with tissue-engineered grafts: a study on sheep.

Standardized particulate bone constructs, obtained by expanding autologous mesenchymal stem cells (MSCs) onto coral granules in vitro, were transplanted into long-bone, critical-size defects in sheep. Control experiments were also performed in which autologous bone grafts were implanted. Defect cavities were lined with a preformed vascularized membrane (induced by temporarily inserting a cement spacer for 6 weeks prior to bone construct implantation), which served as a mold keeping the engineered bone granules in place. Radiographic, histological, and computed tomographic tests performed 6 months later showed that the osteogenic abilities of the engineered construct and autograft were significantly greater than those of coral scaffold alone. No significant differences were found between the amount of newly formed bone in defects filled with coral/MSCs and those filled with autograft, yet radiological scores differed significantly between the two groups (21% and 100% healed cortices, respectively). The present study on a clinically relevant animal model provides the first evidence that standardized particulate bone constructs can be used to repair large bone defects and that their osteogenic ability approaches that of bone autograft, the bone repair benchmark. By proving feasibility, the present study makes possible the treatment of segmental bone losses with bone constructs engineered from granules, a process which is much simpler than preparing customized massive constructs using computer-assisted techniques. Important parameters, such as the rate of scaffold resorption and the number of MSCs to be seeded on the scaffolds, need to be optimized before reaching pertinent definitive conclusions.

Hypoxia affects mesenchymal stromal cell osteogenic differentiation and angiogenic factor expression.

Mesenchymal stromal cells (MSCs) seeded onto biocompatible scaffolds have been proposed for repairing bone defects. When transplanted in vivo, MSCs (expanded in vitro in 21% O(2)) undergo temporary oxygen deprivation due to the lack of pre-existing blood vessels within these scaffolds. In the present study, the effects of temporary (48 h) exposure to hypoxia (

Induction of a barrier membrane to facilitate reconstruction of massive segmental diaphyseal bone defects: an ovine model.

OBJECTIVES: To report an ovine model that can be used to evaluate the efficacy of bone substitutes for repair of segmental diaphyseal bone defects. STUDY DESIGN: Experimental study. ANIMALS: Eleven 2-year-old Pré-Alpes Sheep. METHODS: Mid-diaphyseal metatarsal bone defects (25 mm long) were stabilized by a dynamic compression plate over a polymethylmethacrylate (PMMA) cement spacer, and by external coaptation. The PMMA spacer was removed at 6 weeks by incising the encapsulating membrane. The defect remained unfilled (Group 1; n=5) or was filled with morselized autologous corticocancellous graft (Group 2; n=6), the membrane sutured closed, and external coaptation applied for 6 months, when healing was evaluated. RESULTS: Radiographic, computed tomographic, and histologic examinations at 6 months after the 2nd surgery revealed non-union in ungrafted defects whereas grafted defects showed bone healing. The induced membrane had blood vessels, CBFA1+ cells, and very few macrophages entrapped in a collagenous tissue positive for type I collagen. CONCLUSION: This ovine metatarsal defect model resulted in a critical-size defect (non-union) that healed when grafted. The PMMA-induced membrane constrained the graft, was well vascularized, and may have osteogenic properties. CLINICAL RELEVANCE: This model may be useful to evaluate new strategies in bone tissue engineering because the PMMA-induced membrane may help confine bone morphogenetic proteins, skeletal stem cells, or other agents to the defect cavity where they could be useful to enhance bone formation.

In vivo imaging with cellular resolution of bone marrow cells transplanted into the ischemic brain of a mouse.

The aim of the study was to monitor in vivo and noninvasively the fate of single bone marrow cells (BMCs) transplanted into the ischemic brain of unirradiated mice. In vivo imaging was performed through a closed cranial window, throughout the 2 weeks following cell transplantation, using laser-scanning confocal fluorescence microscopy. The window was chronically implanted above the left parieto-occipital cortex in C57BL/6J adult mice. BMC (3 x 10(5) nucleated cells in 0.5 microL medium) from 5-week-old transgenic mice, ubiquitously expressing green fluorescent protein (GFP), was transplanted into the ipsilateral cortex 24 h after the induction of focal ischemia by coagulation of the left middle cerebral artery (n = 15). Three nonischemic mice served as controls. Repeated in vivo imaging, up to a depth of 200 microm, revealed that BMCs survived within the ischemic and peri-ischemic cortex, migrated significantly towards the lesion, proliferated and adopted a microglia-like morphology over 2 weeks. These results were confirmed using ex vivo imaging after appropriate immunocytochemical treatments. This study indicates that confocal fluorescence microscopy is a reliable and unique tool to repeatedly assess with cellular resolution the in vivo dynamic fate of fluorescent cells transplanted into a mouse brain. These results also provide the first in vivo findings on the fate of single BMCs transplanted into the ischemic brain of unirradiated mice.

A technique for creating critical-size defects in the metatarsus of sheep for use in investigation of healing of long-bone defects.

OBJECTIVE: To develop a technique for use in investigation of healing of long-bone defects by creation of a critical-size defect in the left metarsal III and IV bone (metatarsus) of sheep. ANIMALS: 18 healthy adult sheep. PROCEDURE: Sheep were allocated to 4 groups (3, 3, 5, and 7 sheep in groups 1 to 4, respectively). An ostectomy with various segmental length-to-diaphyseal diameter ratios (0.5, 1.0, 2.0, and 2.0 for groups 1 to 4, respectively) was performed on the left metatarsus of each sheep. The defect was left empty in sheep of groups 1, 2, and 3, whereas the defect was filled with a massive corticocancellous bone autograft in sheep of group 4. RESULTS: All sheep tolerated the surgical procedure well and were able to use the affected limb the day after surgery. Radiographic and histologic examinations conducted 16 weeks after surgery revealed nonunion in all sheep of groups 1, 2, and 3, whereas consistent bone healing with abundant bone formation was observed in all sheep of group 4. CONCLUSIONS AND CLINICAL RELEVANCE: Analysis of these findings suggests that the sheep metatarsal model is a critical-size defect model with low morbidity. It should allow the assessment of new technologies for bone regeneration in conditions closely mimicking the clinical setting. IMPACT FOR HUMAN MEDICINE: Use of this technique in sheep should be of benefit for the preclinical study of osteoconductive, osteoinductive, or osteogenic biomaterials for use in humans.