Gli1 labels progenitors during chondrogenesis in postnatal mice.

Skeletal growth promoted by endochondral ossification is tightly coordinated by self-renewal and differentiation of chondrogenic progenitors. Emerging evidence has shown that multiple skeletal stem cells (SSCs) participate in cartilage formation. However, as yet, no study has reported the existence of common long-lasting chondrogenic progenitors in various types of cartilage. Here, we identify Gli1+ chondrogenic progenitors (Gli1+ CPs), which are distinct from PTHrP+ or FoxA2+ SSCs, are responsible for the lifelong generation of chondrocytes in the growth plate, vertebrae, ribs, and other cartilage. The absence of Gli1+ CPs leads to cartilage defects and dwarfishness phenotype in mice. Furthermore, we show that the BMP signal plays an important role in self-renewal and maintenance of Gli1+ CPs. Deletion of Bmpr1α triggers Gli1+ CPs quiescence exit and causes the exhaustion of Gli1+ CPs, consequently disrupting columnar cartilage. Collectively, our data demonstrate that Gli1+ CPs are common long-term chondrogenic progenitors in multiple types of cartilage and are essential to maintain cartilage homeostasis.

Attenuates of NAD+ impair BMSC osteogenesis and fracture repair through OXPHOS.

BACKGROUND: Controlling the adipo-osteogenic lineage commitment of bone marrow mesenchymal stem cell (BMSC) in favor of osteogenesis is considered a promising approach for bone regeneration and repair. Accumulating evidence indicates that oxidative phosphorylation (OXPHOS) is involved in regulating cell fate decisions. As an essential cofactor for OXPHOS, nicotinamide adenine dinucleotide (NAD) has been shown to correlate with the differentiation of stem cells. However, whether NAD manipulates BMSC lineage commitment through OXPHOS remains elusive. Therefore, it is critical to investigate the potential role of NAD on energy metabolism in mediating BMSC lineage commitment. METHODS: In this study, the mitochondrial respiration and intracellular NAD+ level were firstly compared between osteogenic and adipogenic cells. For validating the role of NAD in mitochondrial OXPHOS, the inhibitor of NAD+ salvage pathway FK866 and activator P7C3 were used to manipulate the NAD+ level during osteogenesis. Furthermore, a murine femur fracture model was established to evaluate the effect of FK866 on bone fracture repair. RESULTS: We elucidated that osteogenic committed BMSCs exhibited increased OXPHOS activity and a decreased glycolysis accompanied by an elevated intracellular NAD+ level. In contrast, adipogenic committed BMSCs showed little change in OXPHOS but an upregulated activity in glycolysis and a decline in intracellular NAD+ level in vitro. Moreover, attenuates of NAD+ via salvage pathway in BMSCs diminished osteogenic commitment due to mitochondria dysfunction and reduced activity of OXPHOS. The cells were rescued by supplementing with nicotinamide mononucleotide. In addition, treatment with NAD+ inhibitor FK866 impaired bone fracture healing in vivo. CONCLUSION: Our data reveals NAD+-mediated mitochondrial OXPHOS is indispensable for osteogenic commitment in BMSCs and bone repair, which might provide a potential therapeutic target for bone repair and regeneration.

Wnt7b Inhibits Osteoclastogenesis via AKT Activation and Glucose Metabolic Rewiring.

The imbalance between bone formation and bone resorption causes osteoporosis, which leads to severe bone fractures. It is known that increases in osteoclast numbers and activities are the main reasons for increasing bone resorption. Although extensive studies have investigated the regulation of osteoclastogenesis of bone marrow macrophages (BMMs), new pharmacological avenues still need to be unveiled for clinical purpose. Wnt ligands have been widely demonstrated as stimulators of bone formation; however, the inhibitory effect of the Wnt pathway in osteoclastogenesis is largely unknown. Here, we demonstrate that Wnt7b, a potent Wnt ligand that enhances bone formation and increases bone mass, also abolishes osteoclastogenesis in vitro. Importantly, enforced expression of Wnt in bone marrow macrophage lineage cells significantly disrupts osteoclast formation and activity, which leads to a dramatic increase in bone mass. Mechanistically, Wnt7b impacts the glucose metabolic process and AKT activation during osteoclastogenesis. Thus, we demonstrate that Wnt7b diminishes osteoclast formation, which will be beneficial for osteoporosis therapy in the future.

GPX7 Facilitates BMSCs Osteoblastogenesis via ER Stress and mTOR Pathway.

Emerging evidence indicates extensive oxidative stress is a consequence of obesity which impairs bone formation. Glutathione peroxidase 7 (GPX7) is a conserved endoplasmic reticulum (ER) retention protein, lacking of which causes accumulation of reactive oxygen species (ROS) and promotes adipogenesis. Since the imbalance between osteogenic and adipogenic differentiation of bone marrow mesenchymal stem cell (BMSC) leads to severe bone diseases such as osteoporosis, it is critical to investigate the potential protective role of Gpx7 in osteogenesis. Here, we provide evidence that deficiency of Gpx7 reduces osteogenesis, but increases adipogenesis in both human BMSCs (hBMSCs) and mouse mesenchymal stem cell line. Interestingly, further studies indicate this defect can be alleviated by the ER stress antagonist, but not the ROS inhibitor, unveiling an unexpected finding that, unlike adipogenesis, lacking of Gpx7 inhibits osteogenesis mediating by induced ER stress instead of enhanced ROS. Furthermore, the mTOR signalling pathway is found down-regulation during osteogenic differentiation in Gpx7-deficient condition, which can be rescued by relief of ER stress. Taken together, for the first time we identify a novel function of Gpx7 in BMSCs' osteogenic differentiation and indicate that Gpx7 may protect against osteoporotic deficits in humans through ER stress and mTOR pathway interplay.

Targeting reactive oxygen species in stem cells for bone therapy.

Reactive oxygen species (ROS) have emerged as key players in regulating the fate and function of stem cells from both non-hematopoietic and hematopoietic lineages in bone marrow, and thus affect the osteoblastogenesis-osteoclastogenesis balance and bone homeostasis. Accumulating evidence has linked ROS and associated oxidative stress with the progression of bone disorders, and ROS-based therapeutic strategies have appeared to achieve favorable outcomes in bone. We review current knowledge of the multifactorial roles and mechanisms of ROS as a target in bone pathology. In addition, we discuss emerging ROS-based therapeutic strategies that show potential for bone therapy. Finally, we highlight the opportunities and challenges facing ROS-targeted stem cell therapeutics for improving bone health.

Both aerobic glycolysis and mitochondrial respiration are required for osteoclast differentiation.

Excessive bone resorption over bone formation is the root cause for bone loss leading to osteoporotic fractures. Development of new antiresorptive therapies calls for a holistic understanding of osteoclast differentiation and function. Although much has been learned about the molecular regulation of osteoclast biology, little is known about the metabolic requirement and bioenergetics during osteoclastogenesis. Here, we report that glucose metabolism through oxidative phosphorylation (OXPHOS) is the predominant bioenergetic pathway to support osteoclast differentiation. Meanwhile, increased lactate production from glucose, known as aerobic glycolysis when oxygen is abundant, is also critical for osteoclastogenesis. Genetic deletion of Glut1 in osteoclast progenitors reduces aerobic glycolysis without compromising OXPHOS, but nonetheless diminishes osteoclast differentiation in vitro. Glut1 deficiency in the progenitors leads to osteopetrosis due to fewer osteoclasts specifically in the female mice. Thus, Glut1-mediated glucose metabolism through both lactate production and OXPHOS is necessary for normal osteoclastogenesis.

Efficacy of Recombinant Human BMP2 and PDGF-BB in Orofacial Bone Regeneration: A Systematic Review and Meta-analysis.

With the rapid development of tissue engineering therapies, there is a growing interest in the application of recombinant human growth factors (rhGFs) to regenerate human orofacial bones. However, despite reports of their ability to promote orofacial bone regeneration in animal experiments, their benefits in human clinical treatments remain unclear. Furthermore, the appropriate concentrations or indications of a specific rhGF remain ambiguous. Therefore it is essential to collect data from diverse clinical trials to evaluate their effects more precisely. Here we reviewed randomized clinical trials (RCT) that focused on the utilization of rhGFs in orofacial bone regeneration. Data from included studies were extracted, pooled and then quantitatively analyzed according to a pre-established protocol. Our results indicate that all current concentrations of rhBMP-2 produces insufficient effect on promoting either tooth extraction socket healing, sinus augmentation or reconstruction of alveolar clefts. However, 0.3 mg/ml rhPDGF-BB promotes the healing of tooth extraction sockets, though the effect does not reach a level of statistical significance. Summarily, we recommend concentrations of 0.3 mg/ml rhPDGF-BB only for the healing of tooth extraction sockets.

Increased glycolysis mediates Wnt7b-induced bone formation.

Wingless/integrated (Wnt) signaling has emerged as a major mechanism for promoting bone formation and a target pathway for developing bone anabolic agents against osteoporosis. However, the downstream events mediating the potential therapeutic effect of Wnt proteins are not fully understood. Previous studies have indicated that increased glycolysis is associated with osteoblast differentiation in response to Wnt signaling, but direct genetic evidence for the importance of glucose metabolism in Wnt-induced bone formation is lacking. Here, we have generated compound transgenic mice to overexpress Wnt family member 7B (Wnt7b) transiently in the osteoblast lineage of postnatal mice, with or without concurrent deletion of the glucose transporter 1 (Glut1), also known as solute carrier family 2, facilitated glucose transporter member 1. Overexpression of Wnt7b in 1-mo-old mice for 1 wk markedly stimulated bone formation, but the effect was essentially abolished without Glut1, even though transient deletion of Glut1 itself did not affect normal bone accrual. Consistent with the in vivo results, Wnt7b increased Glut1 expression and glucose consumption in the primary culture of osteoblast lineage cells, and deletion of Glut1 diminished osteoblast differentiation in vitro. Thus, Wnt7b promotes bone formation in part through stimulating glucose metabolism in osteoblast lineage cells.-Chen, H., Ji, X., Lee, W.-C., Shi, Y., Li, B., Abel, E. D., Jiang, D., Huang, W., Long, F. Increased glycolysis mediates Wnt7b-induced bone formation.

Epigenetic Control of Mesenchymal Stem Cell Fate Decision via Histone Methyltransferase Ash1l.

Previous research indicates that knocking out absent, small, or homeotic-like (Ash1l) in mice, a histone 3 lysine 4 (H3K4) trimethyltransferase, can result in arthritis with more severe cartilage and bone destruction. Research has documented the essential role of Ash1l in stem cell fate decision such as hematopoietic stem cells and the progenitors of keratinocytes. Following up on those insights, our research seeks to document the function of Ash1l in skeletal formation, specifically whether it controls the fate decision of mesenchymal progenitor cells. Our findings indicate that in osteoporotic bones, Ash1l was significantly decreased, indicating a positive correlation between bone mass and the expression of Ash1l. Silencing of Ash1l that had been markedly upregulated in differentiated C3H10T1/2 (C3) cells hampered osteogenesis and chondrogenesis but promoted adipogenesis. Consistently, overexpression of an Ash1l SET domain-containing fragment 3 rather than Ash1lΔN promoted osteogenic and chondrogenic differentiation of C3 cells and simultaneously inhibited adipogenic differentiation. This indicates that the role of Ash1l in regulating the differentiation of C3 cells is linked to its histone methyltransferase activity. Subcutaneous ex vivo transplantation experiments confirmed the role of Ash1l in the promotion of osteogenesis. Further experiments proved that Ash1l can epigenetically affect the expression of essential osteogenic and chondrogenic transcription factors. It exerts this impact via modifications in the enrichment of H3K4me3 on their promoter regions. Considering the promotional action of Ash1l on bone, it could potentially prompt new therapeutic strategy to promote osteogenesis. Stem Cells 2019;37:115-127.

Visual Osteoclast Fusion via A Fluorescence Method.

Osteoclasts are multinucleated giant cells. Fusion is an essential element in the formation of osteoclasts. However, the exact cellular events and mechanisms remain largely unknown because of limited and insufficient methods for observing fusion process. In this work, a fluorescence reporter strategy was established to monitor osteoclast fusion. After fusing with cells expressing Cre recombinase, those cells with double fluorescence switch its expression from red to green fluorescent protein. The effect of RANKL and PTH on osteoclast fusion were both quantitatively and visually detected utilizing this strategy. Furthermore, a combination of this strategy with a technique of fluorescence-activated cell sorting revealed two different populations of fused osteoclasts, tdTomato+ GFP+ cells (TG cells) and GFP+ cells (G cells). The results argue for the potential of combining this technique with other bio-technologies to gain more information about osteoclast fusion. Overall, these data demonstrated that this visual fluorescence switch strategy is useful for further analysis of osteoclast fusion mechanisms.