Osteoblast Progenitor Cells Research Articles

Single‑cell RNA sequencing analysis of human embryos from the late Carnegie to fetal development

BackgroundThe cell development atlas of transition stage from late Carnegie to fetal development (7–9 weeks) remain unclear. It can be seen that the early period of human embryos (7–9 weeks) is a critical research gap. Therefore, we employed single‑cell RNA sequencing to identify cell types and elucidate differentiation relationships.ResultsThe single‑cell RNA sequencing analysis determines eighteen cell clusters in human embryos during the 7–9 weeks period. We uncover two distinct pathways of cellular development and differentiation. Initially, mesenchymal progenitor cells differentiated into osteoblast progenitor cells and neural stem cells, respectively. Neural stem cells further differentiated into neurons. Alternatively, multipotential stem cells differentiated into adipocyte, hematopoietic stem cells and neutrophil, respectively. Additionally, COL1A2-(ITGA1 + ITGB1) mediated the cell communication between mesenchymal progenitor cells and osteoblast progenitor cells. NCAM1-FGFR1 facilitated the cell communication between mesenchymal progenitor cells and neural stem cells. Notably, NCAM1-NCAM1 as a major contributor mediated the cell communication between neural stem cells and neurons. Moreover, CGA-FSHR simultaneously mediated the communication between multipotential stem cells, adipocyte, hematopoietic stem cells and neutrophil. Distinct cell clusters activated specific transcription factors such as HIC1, LMX1B, TWIST1, and et al., which were responsible for their specific functions. These coregulators, such as HOXB13, VSX2, PAX5, and et al., may mediate cell development and differentiation in human embryos.ConclusionsWe provide the cell development atlas for human embryos (7–9 weeks). Two distinct cell development and differentiation pathways are revealed.

FOXC1 and FOXC2 regulate growth plate chondrocyte maturation towards hypertrophy in the embryonic mouse limb skeleton.

The Forkhead box transcription factors FOXC1 and FOXC2 are expressed in condensing mesenchyme cells at the onset of endochondral ossification. We used the Prx1-cre mouse to ablate Foxc1 and Foxc2 in limb skeletal progenitor cells. Prx1-cre;Foxc1Δ/Δ;Foxc2Δ/Δ limbs were shorter than controls, with worsening phenotypes in distal structures. Cartilage formation and mineralization was severely disrupted in the paws. The radius and tibia were malformed, whereas the fibula and ulna remained unmineralized. Chondrocyte maturation was delayed, with fewer Indian hedgehog-expressing, prehypertrophic chondrocytes forming and a smaller hypertrophic chondrocyte zone. Later, progression out of chondrocyte hypertrophy was slowed, leading to an accumulation of COLX-expressing hypertrophic chondrocytes and formation of a smaller primary ossification center with fewer osteoblast progenitor cells populating this region. Targeting Foxc1 and Foxc2 in hypertrophic chondrocytes with Col10a1-cre also resulted in an expanded hypertrophic chondrocyte zone and smaller primary ossification center. Our findings suggest that FOXC1 and FOXC2 direct chondrocyte maturation towards hypertrophic chondrocyte formation. At later stages, FOXC1 and FOXC2 regulate function in hypertrophic chondrocyte remodeling to allow primary ossification center formation and osteoblast recruitment.

Bone remodeling in survivors of pediatric hematopoietic stem cell transplantation: Impact of heavy resistance training.

Early-onset osteoporosis is a frequent late effect after pediatric hematopoietic stem cell transplantation (HSCT). It remains unknown if physical training can improve bone formation in these patients, as the transplantation procedure may cause sustained dysregulation of the bone-forming osteoblast progenitor cells. We aimed to explore the effect of resistance training on bone remodeling in long-term survivors of pediatric HSCT. In this prospective, controlled intervention study, we included seven HSCT survivors and 15 age- and sex-matched healthy controls. The participants completed a 12-week heavy load, lower extremity resistance training intervention with three weekly sessions. We measured fasting serum levels of the bone formation marker "N-terminal propeptide of type I procollagen" (P1NP), and the bone resorption marker "C-terminal telopeptide of type I collagen" (CTX). The hypothesis was planned before data collection began. The trial was registered at Clinicaltrials.gov before including the first participant, with trial registration no. NCT04922970. Resistance training led to significantly increased levels of fasting P1NP in both patients (from 57.62 to 114.99ng/mL, p=.03) and controls (from 66.02 to 104.62ng/mL, p<.001). No significant changes in fasting CTX levels were observed. Despite previous high-dose cytotoxic therapy, long-term survivors of pediatric HSCT respond to resistance training with improvement of bone formation, comparable to that of healthy controls. This suggests that resistance training might be a promising non-pharmacological approach to prevent the early decline in bone mass, and should be considered as part of a follow-up program to counteract long-term sequela after pediatric HSCT.

Lithium-Doped Titanium Dioxide-Based Multilayer Hierarchical Structure for Accelerating Nerve-Induced Bone Regeneration.

Despite considerable advances in artificial bone tissues, the absence of neural network reconstruction in their design often leads to delayed or ineffective bone healing. Hence, we propose a multilayer hierarchical lithium (Li)-doped titanium dioxide structure, constructed through microarc oxidation combined with alkaline heat treatment. This structure can induce the sustained release of Li ions, mimicking the environment of neurogenic osteogenesis characterized by high brain-derived neurotrophic factor (BDNF) expression. During in vitro experiments, the structure enhanced the differentiation of Schwann cells (SCs) and the growth of human umbilical vein endothelial cells (HUVECs) and mouse embryo osteoblast progenitor cells (MC3T3-E1). Additionally, in a coculture system, the SC-conditioned media markedly increased alkaline phosphatase expression and the formation of calcium nodules, demonstrating the excellent potential of the material for nerve-induced bone regeneration. In an in vivo experiment based on a rat distal femoral lesion model, the structure substantially enhanced bone healing by increasing the density of the neural network in the tissue around the implant. In conclusion, this study elucidates the neuromodulatory pathways involved in bone regeneration, providing a promising method for addressing bone deformities.

Cyclosporine a inhibits bone regeneration and induces bone loss in a rat model

Cyclosporine A (CSA) is an immunosuppressant that has been extensively studied for its side effects on inhibiting osseointegration of titanium implants. However, the impact of CSA on bone healing in postmenopausal osteoporosis remains unknown. Therefore, this study aimed to investigate the effect of CSA on bone repair in an ovariectomized (OVX) rat model through both in vitro and in vivo experiments. We examined the interventions of CSA on osteoblast progenitor cells MC3T3-E1 and assessed their effects on biological function using RT-qPCR, CCK-8 assay, alizarin red staining, and alkaline phosphatase staining. Furthermore, we evaluated the effects of CSA on bone regeneration and bone mass in both OVX rat models and femoral diaphysis bone defect models. The results from the CCK-8 experiment indicated a positive influence of experimental doses of CSA on osteogenic differentiation of MC3T3-E1 cells. ALP expression levels and calcified nodules were also evaluated, suggesting that CSA intervention promoted osteogenic differentiation in MC3T3-E1 cells. Additionally, specific gene expressions including OPN, Runx-2, OC, and Col1a1 were up-regulated after CSA intervention. Biomechanical parameters aligned with histological analysis as well as micro-CT scans confirmed worse bone microstructure and strength following CSA intervention. Our findings preliminarily suggest that whether it is normal or osteoporotic bones, CSA has adverse effects on bone health which are associated with elevated-bone turnover.

Abstract 2920: In vitro assays for investigating the effects of cancer therapeutics on bone

Abstract Bone microenvironment is an important factor in regulating bone metastasis. When cancer cells home to bone they secrete factors that stimulate osteoclast activity leading to increased bone resorption. Stimulatory factors released from the resorbed bone matrix in turn promote proliferation of cancer cells and this process, referred as the vicious cycle, often leads to osteolytic lesions in bone when osteoclastic bone resorption exceeds osteoblastic bone formation. The vicious cycle observed in osteolytic disease occurs also in osteoblastic metastases, and in addition cancer cells produce osteoblast-stimulating factors. Cancers with bone metastases often include both osteolytic and osteoblastic lesions. Our aim was to establish in vitro cell culture models to study the effects of cancer therapeutics on osteoblast and osteoclast differentiation and activity. In the osteoclast differentiation assay, human osteoclast precursor cells (Lonza) were cultured on bovine bone slices for 7 days. Denosumab was added in the cultures at day 0, and tartrate-resistant acid phosphatase isoform 5b (TRACP 5b) activity was measured from the culture medium collected at day 7. Osteoclast activity was studied by allowing osteoclasts resorb bone after 7 days’ maturation period for additional 3 days in the presence of odanacatib. The amount of C-terminal cross-linked telopeptides of type I collagen (CTX-I) was measured in the culture medium collected at day 10 to quantitate osteoclast activity. Denosumab and odanacatib showed strong concentration dependent inhibition of osteoclast differentiation and activity, respectively. A mouse osteoblast progenitor cell line KS483 (Pharmatest Services) was used to study differentiation and activity of osteoblasts with test compounds bone morphogenetic protein 2 (BMP-2) and estradiol (E2). In the osteoblast differentiation assay, the activity of cellular alkaline phosphatase (ALP), a marker of osteoblast differentiation, was measured at day 8. In the osteoblast activity assay, the cells were cultured for 13 days. N-terminal propeptide of type I procollagen (PINP) secreted into the culture medium was determined at day 11 to indicate the effects on organic bone formation and calcium deposition was determined at the end of the study as a marker of inorganic bone formation. BMP-2 and E2 stimulated osteoblast differentiation and activity indicated by the increase in ALP activity, PINP and calcium levels. We conclude that these culture systems can be used for studying the effects of cancer therapeutics and identifying new potential compounds affecting the disease process of bone metastases with different mechanisms of action. Citation Format: Jenni H. Mäki-Jouppila, Mervi Ristola, Jukka Rissanen, Katja Fagerlund. In vitro assays for investigating the effects of cancer therapeutics on bone [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 2920.

Porcine Mandibular Bone Marrow-Derived Mesenchymal Stem Cell (BMSC)-Derived Extracellular Vesicles Can Promote the Osteogenic Differentiation Capacity of Porcine Tibial-Derived BMSCs.

Existing research suggests that bone marrow-derived mesenchymal stem cells (BMSCs) may promote endogenous bone repair. This may be through the secretion of factors that stimulate repair processes or directly through differentiation into osteoblast-progenitor cells. However, the osteogenic potential of BMSCs varies among different tissue sources (e.g., mandibular versus long BMSCs). The main aim of this study was to investigate the difference in osteogenic differentiation capacity between mandibular BMSCs (mBMSCs) and tibial BMSCs (tBMSCs). Bioinformatics analysis of the GSE81430 dataset taken from the Gene Expression Omnibus (GEO) database was performed using GEO2R. BMSCs were isolated from mandibular and tibial bone marrow tissue samples. Healthy pigs (n = 3) (registered at the State Office for Nature, Environment, and Consumer Protection, North Rhine-Westphalia (LANUV) 81-02.04.2020.A215) were used for this purpose. Cell morphology and osteogenic differentiation were evaluated in mBMSCs and tBMSCs. The expression levels of toll-like receptor 4 (TLR4) and nuclear transcription factor κB (NF-κB) were analyzed using quantitative polymerase chain reaction (qPCR) and Western blot (WB), respectively. In addition, mBMSC-derived extracellular vesicles (mBMSC-EVs) were gained and used as osteogenic stimuli for tBMSCs. Cell morphology and osteogenic differentiation capacity were assessed after mBMSC-EV stimulation. Bioinformatic analysis indicated that the difference in the activation of the TLR4/NF-κB pathway was more pronounced compared to all other examined genes. Specifically, this demonstrated significant downregulation, whereas only 5-7 upregulated genes displayed significant variances. The mBMSC group showed stronger osteogenic differentiation capacity compared to the tBMSC group, confirmed via ALP, ARS, and von Kossa staining. Furthermore, qPCR and WB analysis revealed a significant decrease in the expression of the TLR4/NF-κB pathway in the mBMSC group compared to the tBMSC group (TLR4 fold changes: mBMSCs vs. tBMSCs p < 0.05; NF-κB fold changes: mBMSCs vs. tBMSCs p < 0.05). The osteogenic differentiation capacity was enhanced, and qPCR and WB analysis revealed a significant decrease in the expression of TLR4 and NF-κB in the tBMSC group with mBMSC-EVs added compared to tBMSCs alone (TLR4 fold changes: p < 0.05; NF-κB fold changes: p < 0.05). Our results indicate that mBMSC-EVs can promote the osteogenic differentiation of tBMSCs in vitro. The results also provide insights into the osteogenic mechanism of mBMSCs via TLR4/NF-κB signaling pathway activation. This discovery promises a fresh perspective on the treatment of bone fractures or malunions, potentially offering a novel therapeutic method.

Printable oxygen-generating biodegradable scaffold for thicker tissue-engineered medical products.

Due to the increasing demand to generate thick and vascularized tissue-engineered constructs, novel strategies are currently being developed. An effective example is the fabrication of a 3D scaffold containing oxygen-releasing biomaterials to solve the limitations of gasdiffusion and transport within transplanted tissues or devices. In this study, we developed a biodegradable scaffold made of polycaprolactone (PCL) mixed with oxygen-generating calcium peroxide (CPO) to design new structures for regenerative tissue using a 3D printer capable of forming arbitrarily shapes. When osteoblast progenitor cells (MC3T3-E1 cells) were cultured under hypoxic conditions on scaffolds fabricated with this technique, it was shown that cell death was reduced by the new scaffolds. Therefore, the results suggest that 3D-printed scaffolds made from biodegradable oxygen-releasing materials may be useful for tissue engineering and regeneration.

Independent mesenchymal progenitor pools respectively produce and maintain osteogenic and chondrogenic cells in zebrafish.

Skeletal tissues including cartilage and bones are characteristic features of vertebrates that are crucial for supporting body morphology and locomotion. Studies mainly in mice have shown that osteoblasts and chondroblasts are supplied from several progenitors like the sclerotome cells in the embryonic stage, osteo-chondroprogenitors in growing long bones, and skeletal stem cells of bone marrow in the postnatal period. However, the exact origins of progenitor cells, their lineage relationships, and their potential to differentiate into osteoblasts and chondroblasts from embryos to adult tissues are not well understood. In this study, we conducted clonal cell tracking in zebrafish and showed that sox9a+ cells are already committed to either chondrogenic or osteogenic fates during embryonic stages and that respective progenies are independently maintained as mesenchymal progenitor pools. Once committed, they never change their lineage identities throughout animal life, even through regeneration. In addition, we further revealed that only osteogenic mesenchymal cells replenish the osteoblast progenitor cells (OPCs), a population of reserved tissue stem cells found to be involved in the de novo production of osteoblasts during regeneration and homeostasis in zebrafish. Thus, our clonal cell tracking study in zebrafish firstly revealed that the mesenchymal progenitor cells that are fated to develop into either chondroblasts or osteoblasts serve as respective tissue stem cells to maintain skeletal tissue homeostasis. Such mesenchymal progenitors dedicated to producing either chondroblasts or osteoblasts would be important targets for skeletal tissue regeneration.

Lipolysis supports bone formation by providing osteoblasts with endogenous fatty acid substrates to maintain bioenergetic status

Bone formation is a highly energy-demanding process that can be impacted by metabolic disorders. Glucose has been considered the principal substrate for osteoblasts, although fatty acids are also important for osteoblast function. Here, we report that osteoblasts can derive energy from endogenous fatty acids stored in lipid droplets via lipolysis and that this process is critical for bone formation. As such, we demonstrate that osteoblasts accumulate lipid droplets that are highly dynamic and provide the molecular mechanism by which they serve as a fuel source for energy generation during osteoblast maturation. Inhibiting cytoplasmic lipolysis leads to both an increase in lipid droplet size in osteoblasts and an impairment in osteoblast function. The fatty acids released by lipolysis from these lipid droplets become critical for cellular energy production as cellular energetics shifts towards oxidative phosphorylation during nutrient-depleted conditions. In vivo, conditional deletion of the ATGL-encoding gene Pnpla2 in osteoblast progenitor cells reduces cortical and trabecular bone parameters and alters skeletal lipid metabolism. Collectively, our data demonstrate that osteoblasts store fatty acids in the form of lipid droplets, which are released via lipolysis to support cellular bioenergetic status when nutrients are limited. Perturbations in this process result in impairment of bone formation, specifically reducing ATP production and overall osteoblast function.

Angiotensin receptor blockers, but not angiotensin-converting enzyme inhibitors, inhibit abnormal bone changes in spondyloarthritis

Spondyloarthritis (SpA) is a chronic inflammatory disease that results in bone ankylosis. The tissue renin-angiotensin system (RAS) is an emerging pathway potentially implicated in SpA-associated bone changes. The aim of the present study was to determine the mechanisms underlying this relationship. Sakaguchi (SKG) mice injected with curdlan (SKGc), animal models for SpA, were treated with RAS modulators, angiotensin II receptor blockers (ARBs) or angiotensin-converting enzyme inhibitors (ACEis). Disease activity was assessed using clinical scores and computed tomography scans. Mouse primary bone marrow monocytes (BMMs), osteoblast (OB) progenitor cells, peripheral blood monocytes (PBMCs), and bone-derived cells (BdCs) from patients with radiographic axial SpA (r-axSpA) were used to investigate the role of RAS in SpA pathogenesis. The expression of RAS components was significantly increased in SKGc mouse joints, and ARBs significantly reduced erosion and systemic bone loss, whereas ACEis did not. Osteoclast (OC) differentiation from primary BMMs, mediated by TRAF6, was inhibited by ARBs but promoted by ACEis; the modulators also exerted opposite effects on OB differentiation. Expression of RAS molecules was higher in PBMCs and BdCs of patients with r-axSpA than in control participants. ARBs inhibited OB differentiation in the BdCs of patients with r-axSpA, whereas ACEis did not. Neither ARBs nor ACEis affected OB differentiation in the control participants. In SpA, a condition characterized by RAS overexpression, ARBs, but not ACEis, inhibited OC and OB differentiation and bone progression. The findings should be taken into account when treating patients with SpA using RAS modulators.

MiR-196b-5p Regulates Osteoblast and Osteoclast Differentiation and Bone Homeostasis by Targeting SEMA3A.

miR-196b-5p plays a role in various malignancies. We have recently reported its function in regulating adipogenesis. However, it remains to be clarified whether and how miR-196b-5p affects bone cells and bone homeostasis. In this study, in vitro functional experiments showed an inhibitory effect of miR-196b-5p on osteoblast differentiation. Mechanistic explorations revealed that miR-196b-5p directly targeted semaphorin 3a (Sema3a) and inhibited Wnt/β-catenin signaling. SEMA3A attenuated the impaired osteogenesis induced by miR-196b-5p. Osteoblast-specific miR-196b transgenic mice showed significant reduction of bone mass. Trabecular osteoblasts were reduced and bone formation was suppressed, whereas osteoclasts, marrow adipocytes, and serum levels of bone resorption markers were increased in the transgenic mice. The osteoblastic progenitor cells from the transgenic mice had decreased SEMA3A levels and exhibited retarded osteogenic differentiation, whereas those marrow osteoclastic progenitors exhibited enhanced osteoclastogenic differentiation. miR-196b-5p and SEMA3A oppositely regulated the expression of receptor activator of nuclear factor-κB ligand and osteoprotegerin. The calvarial osteoblastic cells expressing the transgene promoted osteoclastogenesis, whereas the osteoblasts overexpressing Sema3a inhibited it. Finally, in vivo transfection of miR-196b-5p inhibitor to the marrow reduced ovariectomy-induced bone loss in mice. Our study has identified that miR-196b-5p plays a key role in osteoblast and osteoclast differentiation and regulates bone homeostasis. Inhibition of miR-196b-5p may be beneficial for amelioration of osteoporosis. © 2023 American Society for Bone and Mineral Research (ASBMR).

Pulsed Electromagnetic Field Promotes Bone Anabolism in Postmenopausal Osteoporosis through the miR-6976/BMP/Smad4 Axis

Background. Insufficient bone formation is the key reason for the imbalance of bone metabolism and one of the main mechanisms for the occurrence and deterioration of postmenopausal osteoporosis (PMOP). Accumulating evidence has demonstrated that pulsed electromagnetic field (PEMF), as a physiotherapy, can treat osteoporosis by promoting osteogenic differentiation in osteoblasts. However, little is known about its mechanisms. Methods. In vivo, ovariectomized mice were administered PEMF for 4 weeks, and skeletal analysis was conducted. In vitro, hydrogen peroxide-treated mouse osteoblast precursor cells with or without PEMF intervention were subjected to osteogenic differentiation testing and miRNA microarrays. The potential target miRNAs were validated, followed by gene expression assays to further clarify their regulatory relationships with target pathways. Results. We found that PEMF reduced bone loss in ovariectomized mice and promoted osteogenic differentiation of hydrogen peroxide-treated osteoblast precursor cells via downregulation of miR-6976-5p. Mechanistically, reduced miR-6976-5p enhanced the nuclear transport of phosphorylated Smad1/5/9 by upregulating Smad4, thereby activating the BMP/Smad pathway. Additionally, the administration of miR-6976-5p inhibitors successfully promoted osteogenic differentiation in vitro, and its antagomirs protected bone mass in vivo. miR-6976-5p mimics and agomirs acted in the opposite way. Conclusion. These results provide evidence that PEMF alleviates estrogen deficiency-induced bone loss by activating osteoblastic progenitor cells and maintaining their osteogenic differentiation and shed light on the mechanisms involved, which may provide a potential option for the clinical application of PEMF in PMOP.

Effects of extract solution from magnesium alloys supplemented with different compositions of rare earth elements on in vitro epithelial and osteoblast progenitor cells.

Background: Magnesium alloys (Mg-alloys) have gained significant attention in recent years as a potential bioactive material for clinical applications. The incorporation of rare earth elements (REEs) into Mg-alloys has been of particular interest due to their potential to improve both mechanical and biological properties. Although there are diverse results in terms of cytotoxicity and biological effects of REEs, investigating the physiological benefits of Mg-alloys supplemented with REEs will help in the transition from theoretical to practical applications. Methods: In this study, two culture systems were used to evaluate the effects of Mg-alloys containing gadolinium (Gd), dysprosium (Dy), and yttrium (Y): human umbilical vein endothelial cells (HUVEC) and mouse osteoblastic progenitor cells (MC3T3-E1). Different compositions of Mg-alloys were assessed, and the effects of the extract solution on cell proliferation, viability, and specific cell functions were analyzed. Results: Within the range of weight percentages tested, the Mg-REE alloys did not exhibit any significant negative impacts on either cell line. Interestingly, moderate compositions (Mg-1.5Gd-1.5Dy-0.825Y-0.5Zr and Mg-2Gd-2Dy-1.1Y-0.5Zr) demonstrated a tendency to enhance osteoblastic activity and promote the vascularization process in both HUVEC and MC3T3-E1 cell lines. Discussion: The results of this study provide valuable insights into the potential benefits of REE-supplemented Mg-alloys for clinical applications. The observed enhancement in osteoblastic activity and promotion of vascularization processes suggest that optimizing the compositions of REEs in Mg-alloys could lead to the development of novel, more effective bioactive materials. Further investigations are required to understand the underlying mechanisms and to refine the alloy compositions for improved biocompatibility and performance in clinical settings.

Development of serum substitute medium for bone tissue engineering.

In tissue engineering, cells are grown often on scaffolds and subjected to chemical/mechanical stimuli. Most such cultures still use fetal bovine serum (FBS) despite its known disadvantages including ethical concerns, safety issues, and variability in composition, which greatly influences the experimental outcomes. To overcome the disadvantages of using FBS, chemically defined serum substitute medium needs to be developed. Development of such medium depends on cell type and application-which makes it impossible to define one universal serum substitute medium for all cells in any application. Here, we developed a serum substitute medium for bone tissue engineering (BTE) in a step-by-step process. Essential components were added to the medium while human bone marrow mesenchymal stromal cells (hBMSCs, osteoblast progenitor cells) were cultured in two-dimensional and three-dimensional substrates. In a 3-week culture, the developed serum substitute medium worked equally well as FBS containing medium in term of cell attachment to the substrate, cell survival, osteoblast differentiation, and deposition of extracellular matrix. In the next step, the use of serum substitute medium was evaluated when culturing cells under mechanical loading in the form of shear stress. The outcomes showed that the application of shear stress is essential to improve extracellular matrix formation while using serum substitute medium. The developed serum substitute medium could pave the way in replacing FBS for BTE studies eliminating the use of controversial FBS and providing a better-defined chemical environment for BTE studies.

The trajectory of osteoblast progenitor cells in patients with type 2 diabetes and the predictive model for their osteogenic differentiation ability

The fate of osteoprogenitor cells along with the progression of type 2 diabetes (T2DM) and factors determining the fate of those cells remains to be elucidated. This cross-sectional study included 18 normoglycemic, 27 prediabetic, and 73 T2DM to determine osteogenic differentiation across the continuum of dysglycemia and to construct a model to predict the fate of osteoprogenitor cells. This study demonstrated a preserved osteogenic differentiation ability of peripheral blood-derived mononuclear cells (PBMC) isolated from normoglycemic and prediabetic but a progressive decline in their osteogenic differentiation during the progression of T2DM. The rate of osteogenic differentiation rapidly declined by 4–7% annually during the first 10 years of diabetes and then slowed down. A predictive model composed of three independent risk factors, including age, duration of diabetes, and glomerular filtration rate, demonstrated an AuROC of 0.834. With a proposed cut-off of 21.25, this model had 72.0% sensitivity, 87.5% specificity, and 78.9% accuracy in predicting the fate of osteoprogenitor cells. In conclusion, this study provided a perspective on the osteogenic differentiation ability of the osteoprogenitor cells across a continuum of dysglycemia and a predictive model with good diagnostic performance for the prediction of the fate of osteoprogenitor cells in patients with T2DM.

Differentiation ability of Gli1+ cells during orthodontic tooth movement.

Orthodontic tooth movement (OTM) induces bone formation on the alveolar bone of the tension side; however, the mechanism of osteoblast differentiation is not fully understood. Gli1 is an essential transcription factor for hedgehog signaling and functions in undifferentiated cells during embryogenesis. In this study, we examined the differentiation of Gli1+ cells in the periodontal ligament (PDL) during OTM using a lineage-tracing analysis. After the final administration of tamoxifen for 2days to 8-week-old Gli1-CreERT2/ROSA26-loxP-stop-loxP-tdTomato (iGli1/Tomato) mice, Gli1/Tomato+ cells were rarely observed near endomucin+ blood vessels in the PDL. Osteoblasts lining the alveolar bone did not exhibit Gli1/Tomato fluorescence. To move the first molar of iGli1/Tomato mice medially, nickel-titanium closed-coil springs were attached between the upper anterior alveolar bone and the first molar. Two days after OTM initiation, the number of Gli1/Tomato+ cells increased along with numerous PCNA+ cells in the PDL of the tension side. As some Gli1/Tomato+ cells exhibited positive expression of osterix, an osteoblast differentiation marker, Gli1+ cells probably differentiated into osteoblast progenitor cells. On day 10, the newly formed bone labeled by calcein administration during OTM was detected on the surface of the original alveolar bone of the tension side. Gli1/Tomato+ cells expressing osterix localized to the surface of the newly formed bone. In contrast, in the PDL of the compression side, Gli1/Tomato+ cells proliferated before day 10 and expressed type I collagen, suggesting that the Gli1+ cells also differentiated into fibroblasts. Collectively, these results demonstrate that Gli1+ cells in the PDL can differentiate into osteoblasts at the tension side and may function in bone remodeling as well as fibril formation in the PDL during OTM.

Mesenchymal cell TRPM7 expression is required for bone formation via the regulation of chondrogenesis.

Transient receptor potential melastatin-subfamily member 7 (TRPM7) is a bifunctional protein containing a kinase fused to an ion channel permeated with cations, including Ca2+ and Mg2+. Trpm7-null mice show embryonic lethality. Paired related homeobox 1 (Prx1) is expressed in undifferentiated mesenchymal cells such as the progenitor cells of both chondrocytes and osteoblasts involved in limb skeleton formation. Prx1-Cre-dependent Trpm7 mesenchymal-deleted mice were generated to examine the role of TRPM7 in bone development. We found that Prx1-Cre;Trpm7fl/fl mice had shortened bones and impaired trabecular bone formation. Trabecular bone parameters, such as the bone volume (BV/TV), and trabecular number (Tb.N), were decreased in Prx1-Cre;Trpm7fl/fl mice. The cortical bone parameters of cortical bone area (Ct.Ar) and cortical bone thickness (Ct.Th) were also down-regulated in these mice. The bone formation rate in Prx1-Cre;Trpm7fl/fl mice was unchanged, but the hypertrophic area and cell size of the zone were smaller, and the expression of Col2a1, Col10a1 and Mmp13 was downregulated compared with control mice. These findings suggest impaired chondrogenesis in Prx1-Cre;Trpm7fl/fl mice compared to control mice. The receptor activator of nuclear factor-kappa B ligand (RANKL) expression was increased, and RANKL-positive cells and osteoclasts were markedly accumulated in the boundary region between the growth plate and trabecular bone. In contrast, TRPM7 KR mice, which are kinase-dead mutants in which the TRPM7 ion channel function has not been altered, showed no marked differences in trabecular or cortical bone parameters compared to wild-type mice. These findings suggest that TRPM7 is critical as a cation channel rather than as a kinase in bone development via the regulation of chondrogenesis.

Osteogenic Differentiation &lt;i&gt;in vitro&lt;/i&gt; off Human Osteoblasts is Associated with Only Slight Shift in Their Proteomics Profile

Fracture healing is a complex process in which the periosteum and endosteum become the main sources of osteoblast progenitor cells. However, cellular mechanisms and signaling cascades underlying the early stages of osteoblast progenitors differentiation in adult bone are still not well understood. Therefore, we performed shotgun proteomics analysis of primary culture of isolated human osteoblasts from femur of adult donors in undifferentiated conditions and on the sixth day of osteogenic differentiation in vitro. This is an early timepoint in which we have observed no extracellular matrix mineralization yet. 1612 proteins identified with at least two unique peptides were included in proteomics analysis. Data are available via ProteomeXchange with identifier PXD033697. Despite the fact, that matrix mineralization starts only after induction of osteogenic differentiation, we revealed unexpectedly weak physiological shift associated with a decrease of cells proliferative activity and changes in proteins inVved in extracellular matrix secretion and organization. We demonstrated that osteoblasts were positive for markers of later osteogenic differentiation stages during standard cultivation: osteopontin, osteocalcin, BMP-2/4 and RUNX2. Therefore, further differentiation required for matrix mineralization needs minimal physiological changes.

Circ-Iqsec1 induces bone marrow-derived mesenchymal stem cell (BMSC) osteogenic differentiation through the miR-187-3p/Satb2 signaling pathway

BackgroundBone marrow-derived mesenchymal stem cells (BMSCs) are general progenitor cells of osteoblasts and adipocytes and they are characterized as a fundamental mediator for bone formation. The current research studied the molecular mechanisms underlying circRNA-regulated BMSC osteogenic differentiation.MethodsNext-generation sequencing (NGS) was employed to study abnormal circRNA and mRNA expression in BMSCs before and after osteogenic differentiation induction. Bioinformatics analysis and luciferase reporting analysis were employed to confirm correlations among miRNA, circRNA, and mRNA. RT-qPCR, ALP staining, and alizarin red staining illustrated the osteogenic differentiation ability of BMSCs.ResultsData showed that circ-Iqsec1 expression increased during BMSC osteogenic differentiation. circ-Iqsec1 downregulation reduced BMSC osteogenic differentiation ability. The present investigation discovered that Satb2 played a role during BMSC osteogenic differentiation. Satb2 downregulation decreased BMSC osteogenic differentiation ability. Bioinformatics and luciferase data showed that miR-187-3p linked circ-Iqsec1 and Satb2. miR-187-3p downregulation or Satb2 overexpression restored the osteogenic differentiation capability of BMSCs post silencing circ-Iqsec1 in in vivo and in vitro experiments. Satb2 upregulation restored osteogenic differentiation capability of BMSCs post miR-187-3p overexpression.ConclusionTaken together, our study found that circ-Iqsec1 induced BMSC osteogenic differentiation through the miR-187-3p/Satb2 signaling pathway.