Apigenin and Rutaecarpine reduce the burden of cellular senescence in bone marrow stromal stem cells.

Introduction: Osteoporosis is a systemic age-related disease characterized by reduced bone mass and microstructure deterioration, leading to increased risk of bone fragility fractures. Osteoporosis is a worldwide major health care problem and there is a need for preventive approaches. Methods and results: Apigenin and Rutaecarpine are plant-derived antioxidants identified through functional screen of a natural product library (143 compounds) as enhancers of osteoblastic differentiation of human bone marrow stromal stem cells (hBMSCs). Global gene expression profiling and Western blot analysis revealed activation of several intra-cellular signaling pathways including focal adhesion kinase (FAK) and TGFß. Pharmacological inhibition of FAK using PF-573228 (5 µM) and TGFß using SB505124 (1µM), diminished Apigenin- and Rutaecarpine-induced osteoblast differentiation. In vitro treatment with Apigenin and Rutaecarpine, of primary hBMSCs obtained from elderly female patients enhanced osteoblast differentiation compared with primary hBMSCs obtained from young female donors. Ex-vivo treatment with Apigenin and Rutaecarpine of organotypic embryonic chick-femur culture significantly increased bone volume and cortical thickness compared to control as estimated by µCT-scanning. Discussion: Our data revealed that Apigenin and Rutaecarpine enhance osteoblastic differentiation, bone formation, and reduce the age-related effects of hBMSCs. Therefore, Apigenin and Rutaecarpine cellular treatment represent a potential strategy for maintaining hBMSCs health during aging and osteoporosis.

TNIK is a conserved regulator of glucose and lipid metabolism in obesity.

Obesity and type 2 diabetes (T2D) are growing health challenges with unmet treatment needs. Traf2- and NCK-interacting protein kinase (TNIK) is a recently identified obesity- and T2D-associated gene with unknown functions. We show that TNIK governs lipid and glucose homeostasis in Drosophila and mice. Loss of the Drosophila ortholog of TNIK, misshapen, altered the metabolite profiles and impaired de novo lipogenesis in high sugar-fed larvae. Tnik knockout mice exhibited hyperlocomotor activity and were protected against diet-induced fat expansion, insulin resistance, and hepatic steatosis. The improved lipid profile of Tnik knockout mice was accompanied by enhanced skeletal muscle and adipose tissue insulin-stimulated glucose uptake and glucose and lipid handling. Using the T2D Knowledge Portal and the UK Biobank, we observed associations of TNIK variants with blood glucose, HbA1c, body mass index, body fat percentage, and feeding behavior. These results define an untapped paradigm of TNIK-controlled glucose and lipid metabolism.

KIAA1199 deficiency enhances skeletal stem cell differentiation to osteoblasts and promotes bone regeneration.

Upon transplantation, skeletal stem cells (also known as bone marrow stromal or mesenchymal stem cells) can regulate bone regeneration by producing secreted factors. Here, we identify KIAA1199 as a bone marrow stromal cell-secreted factor in vitro and in vivo. KIAA1199 plasma levels of patients positively correlate with osteoporotic fracture risk and expression levels of KIAA1199 in patient bone marrow stromal cells negatively correlates with their osteogenic differentiation potential. KIAA1199-deficient bone marrow stromal cells exhibit enhanced osteoblast differentiation in vitro and ectopic bone formation in vivo. Consistently, KIAA1199 knockout mice display increased bone mass and biomechanical strength, as well as an increased bone formation rate. They also exhibit accelerated healing of surgically generated bone defects and are protected from ovariectomy-induced bone loss. Mechanistically, KIAA1199 regulates osteogenesis by inhibiting the production of osteopontin by osteoblasts, via integrin-mediated AKT and ERK-MAPK intracellular signaling. Thus, KIAA1199 is a regulator of osteoblast differentiation and bone regeneration and could be targeted for the treatment or management of low bone mass conditions.

In Vivo Longitudinal Monitoring of Disease Progression in Inflammatory Arthritis Animal Models Using Raman Spectroscopy.

Current techniques for monitoring disease progression and testing drug efficacy in animal models of inflammatory arthritis are either destructive, time-consuming, subjective, or require ionizing radiation. To accommodate this, we have developed a non-invasive and label-free optical system based on Raman spectroscopy for monitoring tissue alterations in rodent models of arthritis at the biomolecular level. To test different sampling geometries, the system was designed to collect both transmission and reflection mode spectra. Mice with collagen antibody-induced arthritis and controls were subject to in vivo Raman spectroscopy at the tibiotarsal joint every 3 days for 14 days. Raman-derived measures of bone content correlated well with micro-computed tomography bone mineral densities. This allowed for time-resolved quantitation of bone densities, which indicated gradual bone erosion in mice with arthritis. Inflammatory pannus formation, bone erosion, and bone marrow inflammation were confirmed by histological analysis. In addition, using library-based spectral decomposition, we quantified the progression of bone and soft tissue components. In general, the tissue components followed significantly different tendencies in mice developing arthritis compared to the control group in line with the histological analysis. In total, this demonstrates Raman spectroscopy as a versatile technique for monitoring alterations to both mineralized and soft tissues simultaneously in rodent models of musculoskeletal disorders. Furthermore, the technique presented herein allows for objective repeated within-animal measurements potentially refining and reducing the use of animals in research while improving the development of novel antiarthritic therapeutics.

High-fat diet-induced obesity augments the deleterious effects of estrogen deficiency on bone: Evidence from ovariectomized mice.

Several epidemiological studies have suggested that obesity complicated with insulin resistance and type 2 diabetes exerts deleterious effects on the skeleton. While obesity coexists with estrogen deficiency in postmenopausal women, their combined effects on the skeleton are poorly studied. Thus, we investigated the impact of high-fat diet (HFD) on bone and metabolism of ovariectomized (OVX) female mice (C57BL/6J). OVX or sham operated mice were fed either HFD (60%fat) or normal diet (10%fat) for 12 weeks. HFD-OVX group exhibited pronounced increase in body weight (~86% in HFD and ~122% in HFD-OVX, p < 0.0005) and impaired glucose tolerance. Bone microCT-scanning revealed a pronounced decrease in trabecular bone volume/total volume (BV/TV) (-15.6 ± 0.48% in HFD and -37.5 ± 0.235% in HFD-OVX, p < 0.005) and expansion of bone marrow adipose tissue (BMAT; +60.7 ± 9.9% in HFD vs. +79.5 ± 5.86% in HFD-OVX, p < 0.005). Mechanistically, HFD-OVX treatment led to upregulation of genes markers of senescence, bone resorption, adipogenesis, inflammation, downregulation of gene markers of bone formation and bone development. Similarly, HFD-OVX treatment resulted in significant changes in bone tissue levels of purine/pyrimidine and Glutamate metabolisms, known to play a regulatory role in bone metabolism. Obesity and estrogen deficiency exert combined deleterious effects on bone resulting in accelerated cellular senescence, expansion of BMAT and impaired bone formation leading to decreased bone mass. Our results suggest that obesity may increase bone fragility in postmenopausal women.

Administration of whey protein complexed vitamin D3 to vitamin D3-deficient growing Sprague-Dawley rats.

Vitamin D deficiency is a global health issue with consequences for bone health. Complexation of vitamin D3 with specific whey proteins might increase the bioavailability and enhance the effect of dietary supplementation on health outcomes. The current rat study was set up to investigate if complexation of vitamin D3 with whey protein isolate (WPI) or ß-lactoglobulin (B-LG) increases bioavailability of the vitamin and how it impacts markers of bone turnover and bone structure. For 8 weeks, growing male Sprague Dawley rats (n = 48) were fed a vitamin D-deficient diet and during the final 4 weeks gavage dosing of vitamin D3 either alone (VitD) or complexed with WPI (VitD + WPI) or ß-LG (VitD + B-LG) was administered. A placebo treatment (placebo) was also included. After sacrifice, samples of bone were collected and analyzed using biomechanical testing and µCT scanning. The concentrations of vitamin D3, vitamin D3 metabolites and bone markers (P1NP and CTX) were measured in serum. The results showed that VitD + B-LG appeared to induce lower levels of 25-hydroxy vitamin D3 in serum compared to VitD alone. Markers of bone turnover were generally higher in the VitD group compared to placebo and the VitD + WPI and VitD + B-LG treatments. No effects of treatments on bone strength or bone microstructure were detected. In conclusion, whey protein complexation of vitamin D3 supplements appeared to have no beneficial effects on circulating vitamin D3 metabolites but this did not impose changes in bone strength or trabecular bone microstructure.

Impaired Bone Fracture Healing in Type 2 Diabetes Is Caused by Defective Functions of Skeletal Progenitor Cells.

The mechanisms of obesity and type 2 diabetes (T2D)-associated impaired fracture healing are poorly studied. In a murine model of T2D reflecting both hyperinsulinemia induced by high-fat diet and insulinopenia induced by treatment with streptozotocin, we examined bone healing in a tibia cortical bone defect. A delayed bone healing was observed during hyperinsulinemia as newly formed bone was reduced by -28.4 ± 7.7% and was associated with accumulation of marrow adipocytes at the defect site +124.06 ± 38.71%, and increased density of SCA1+ (+74.99 ± 29.19%) but not Runx2+ osteoprogenitor cells. We also observed increased in reactive oxygen species production (+101.82 ± 33.05%), senescence gene signature (≈106.66 ± 34.03%), and LAMIN B1- senescent cell density (+225.18 ± 43.15%), suggesting accelerated senescence phenotype. During insulinopenia, a more pronounced delayed bone healing was observed with decreased newly formed bone to -34.9 ± 6.2% which was inversely correlated with glucose levels (R2 = 0.48, P < .004) and callus adipose tissue area (R2 = .3711, P < .01). Finally, to investigate the relevance to human physiology, we observed that sera from obese and T2D subjects had disease state-specific inhibitory effects on osteoblast-related gene signatures in human bone marrow stromal cells which resulted in inhibition of osteoblast and enhanced adipocyte differentiation. Our data demonstrate that T2D exerts negative effects on bone healing through inhibition of osteoblast differentiation of skeletal stem cells and induction of accelerated bone senescence and that the hyperglycemia per se and not just insulin levels is detrimental for bone healing.

In vivo non-invasive monitoring of tissue development in 3D printed subcutaneous bone scaffolds using fibre-optic Raman spectroscopy.

The development of novel biomaterials for regenerative therapy relies on the ability to assess tissue development, quality, and similarity with native tissue types in in vivo experiments. Non-invasive imaging modalities such as X-ray computed tomography offer high spatial resolution but limited biochemical information while histology and biochemical assays are destructive. Raman spectroscopy is a non-invasive, label-free and non-destructive technique widely applied for biochemical characterization. Here we demonstrate the use of fibre-optic Raman spectroscopy for in vivo quantitative monitoring of tissue development in subcutaneous calcium phosphate scaffolds in mice over 16 weeks. Raman spectroscopy was able to quantify the time dependency of different tissue components related to the presence, absence, and quantity of mesenchymal stem cells. Scaffolds seeded with stem cells produced 3-5 times higher amount of collagen-rich extracellular matrix after 16 weeks implantation compared to scaffolds without. These however, showed a 2.5 times higher amount of lipid-rich tissue compared to implants with stem cells. Ex vivo micro-computed tomography and histology showed stem cell mediated collagen and bone development. Histological measures of collagen correlated well with Raman derived quantifications (correlation coefficient in vivo 0.74, ex vivo 0.93). In the absence of stem cells, the scaffolds were largely occupied by adipocytes. The technique developed here could potentially be adapted for a range of small animal experiments for assessing tissue engineering strategies at the biochemical level.

Treating mouse skull defects with 3D-printed fatty acid and tricalcium phosphate implants.

Skull surgery, also known as craniectomy, is done to treat trauma or brain diseases and may require the use of an implant to reestablish skull integrity. This study investigates the performance of 3D printed bone implants in a mouse model of craniectomy with the aim of making biodegradable porous implants that can ultimately be fitted to a patient's anatomy. A nonpolymeric thermoplastic bioink composed of fatty acids and ß-tricalcium phosphate was used to 3D print the skull implants. Some of these were sintered to yield pure ß-tricalcium phosphate implants. The performance of nonsintered and sintered implants was then compared in two semi-quantitative murine calvarial defect models using computed tomography, histology, and luciferase activity. Both types of implants were biocompatible, but only sintered implants promoted defect healing, with osseointegration to adjacent bone and the formation of new bone and bone marrow tissue in the implant pores. Luciferase scanning and histology showed that mesenchymal stem cells seeded onto the implants engraft and proliferate on the implants after implantation and contribute to forming bone. The experiments indicate that fatty acid-based 3D printing enables the creation of biocompatible and bone-forming ß-tricalcium phosphate implants.

C57BL/6J substrain differences in response to high-fat diet intervention.

C57BL/6J-related mouse strains are widely used animal models for diet-induced obesity (DIO). Multiple vendors breed C57BL/6J-related substrains which may introduce genetic drift and environmental confounders such as microbiome differences. To address potential vendor/substrain specific effects, we compared DIO of C57BL/6J-related substrains from three different vendors: C57BL/6J (Charles Rivers), C57BL/6JBomTac (Taconic Bioscience) and C57BL/6JRj (Janvier). After local acclimatization, DIO was induced by either a high-fat diet (HFD, 60% energy from fat) or western diet (WD, 42% energy from fat supplemented with fructose in the drinking water). All three groups on HFD gained a similar amount of total body weight, yet the relative amount of fat percentage and mass of inguinal- and epididymal white adipose tissue (iWAT and eWAT) was lower in C57BL/6JBomTac compared to the two other C57BL/6J-releated substrains. In contrast to HFD, the three groups on WD responded differently in terms of body weight gain, where C57BL/6J was particularly prone to WD. This was associated with a relative higher amount of eWAT, iWAT, and liver triglycerides. Although the HFD and WD had significant impact on the microbiota, we did not observe any major differences between the three groups of mice. Together, these data demonstrate significant differences in HFD- and WD-induced adiposity in C57BL/6J-related substrains, which should be considered in the design of animal DIO studies.

Neoplastic Transformation of Human Mesenchymal Stromal Cells Mediated via LIN28B.

Bone marrow stromal (Mesenchymal) stem cells (MSCs) are multipotent bone cells capable of differentiating into mesoderm-type cells, such as osteoblasts and adipocytes. Existing evidence suggests that transformation of MSCs gives rise to sarcoma. In order to identify the molecular mechanism leading to spontaneous transformation of human bone marrow MSCs (hBMSCs), we performed comprehensive microRNA (miRNA) and mRNA profiling in the transformed hBMSC-Tum line compared to the parental clone. As a result, we identified multiple dysregulated molecular networks associated with the hBMSC transformed phenotype. LIN28B was upregulated 177.0-fold in hBMSC-Tum, which was associated with marked reduction in LET-7 expression and upregulated expression of its target HMGA2. Targeted depletion of LIN28B or exogenous expression of LET-7b suppressed hBMSC-Tum proliferation, colony formation, and migration. On the other hand, forced expression of LIN28B promoted malignant transformation of parental hBMSC cells as shown by enhanced in vitro colony formation, doxorubicin resistance, and in vivo tumor formation in immunocompromised mice. Analysis of LIN28B and HMGA2 expression levels in cohorts from The Cancer Genome Atlas sarcoma dataset revealed a strong inverse-relationship between elevated expression and overall survival (OS) in 260 patients (p = 0.005) and disease-free survival (DFS) in 231 patients (p = 0.02), suggesting LIN28B and HMGA2 are important regulators of sarcoma biology. Our results highlight an important role for the LIN28B/LET-7 axis in human sarcoma pathogenesis and suggest that the therapeutic targeting of LIN28B may be relevant for patients with sarcoma.

A simple and reliable protocol for long-term culture of murine bone marrow stromal (mesenchymal) stem cells that retained their in vitro and in vivo stemness in long-term culture.

BACKGROUND: Bone marrow derived stromal stem cells (BMSCs) are a clonogenic cell population that is characterized by self-renewal capacity and differentiation potential into osteoblasts, and other mesenchymal cell types. Mouse BMSCs (mBMSCs) are difficult to be cultured and propagated in vitro due to their replicative senescent phenotype, heterogeneity and high contamination with plastic adherent hematopoietic progenitors (HPCs). In this study, we described long-term culture of homogenous population of mBMSCs using simple and highly reproducible approach based on frequent subculturing (FS) at fixed split ratio in the presence of basic fibroblast growth factor (bFGF). RESULTS: Cultured mBMSCs using this protocol (mBMSCs-FS) showed long-term survival in culture > 70 population doubling (PD) and retained their characteristic surface markers and differentiation capacity into osteoblast and adipocyte lineages. When compared to the clonal bone marrow-derived cell line ST2, mBMSCs-FS displayed more enhanced osteoblast differentiation potential and responsiveness to osteogenic factors including BMPs, IGF-1, PDGF, TGFß1,3, FGF, cAMP, Wnt3a and VEGF. In addition, unlike ST2 cells, mBMSCs-FS maintained capacity to form ectopic bone and bone marrow stroma upon in vivo transplantation in immune-compromising mice, even at high PD levels. Interestingly, by applying the same FS + bFGF protocol, we succeeded to obtain long-term cultures of primary neonatal calvarial osteoprogenitor cells (OBs) that were cultured for more than 70 PD and maintained in vitro and in vivo osteoblast differentiation capacities. CONCLUSIONS: Our data provide a simple and reliable protocol for generating long-term cultures of mBMSCs and OBs with retained high in vitro and in vivo osteoblast differentiation capacities for use in pre-clinical and molecular mechanism studies.

Strontium functionalized scaffold for bone tissue engineering.

Drug functionalized scaffolds are currently being employed to improve local delivery of osteoprotective drugs with the aim of reducing their loading dose as well as unwanted systemic complications. In this study we tested a poly-(ε) caprolactone (PCL)-laponite-strontium ranelate (SRA) composite scaffold (PLS3) for its abilities to support growth and osteogenic differentiation of human marrow derived stromal stem cells (hMSC). The in vitro experiments showed the PLS3 scaffold supported cell growth and osteogenic differentiation. The in vivo implantation of hMSC seeded PLS3 scaffold in immunocompromised mice revealed vascularized ectopic bone formation. PLS3 scaffolds can be useful in bone regenerative applications in the fields of orthopaedics and dentistry.

TAFA2 Induces Skeletal (Stromal) Stem Cell Migration Through Activation of Rac1-p38 Signaling.

Understanding the mechanisms regulating recruitment of human skeletal (stromal or mesenchymal) stem cells (hMSC) to sites of tissue injury is a prerequisite for their successful use in cell replacement therapy. Chemokine-like protein TAFA2 is a recently discovered neurokine involved in neuronal cell migration and neurite outgrowth. Here, we demonstrate a possible role for TAFA2 in regulating recruitment of hMSC to bone fracture sites. TAFA2 increased the in vitro trans-well migration and motility of hMSC in a dose-dependent fashion and induced significant morphological changes including formation of lamellipodia as revealed by high-content-image analysis at single-cell level. Mechanistic studies revealed that TAFA2 enhanced hMSC migration through activation of the Rac1-p38 pathway. In addition, TAFA2 enhanced hMSC proliferation, whereas differentiation of hMSC toward osteoblast and adipocyte lineages was not altered. in vivo studies demonstrated transient upregulation of TAFA2 gene expression during the inflammatory phase of fracture healing in a closed femoral fracture model in mice, and a similar pattern was observed in serum levels of TAFA2 in patients after hip fracture. Finally, interleukin-1ß was found as an upstream regulator of TAFA2 expression. Our findings demonstrate that TAFA2 enhances hMSC migration and recruitment and thus is relevant for regenerative medicine applications. Stem Cells 2019;37:407-416.

Antibody-based inhibition of circulating DLK1 protects from estrogen deficiency-induced bone loss in mice.

Soluble delta-like 1 homolog (DLK1) is a circulating protein that belongs to the Notch/Serrate/delta family, which regulates many differentiation processes including osteogenesis and adipogenesis. We have previously demonstrated an inhibitory effect of DLK1 on bone mass via stimulation of bone resorption and inhibition of bone formation. Further, serum DLK1 levels are elevated and positively correlated to bone turnover markers in estrogen (E)-deficient rodents and women. In this report, we examined whether inhibition of serum DLK1 activity using a neutralizing monoclonal antibody protects from E deficiency-associated bone loss in mice. Thus, we generated mouse monoclonal anti-mouse DLK1 antibodies (MAb DLK1) that enabled us to reduce and also quantitate the levels of bioavailable serum DLK1 in vivo. Ovariectomized (ovx) mice were injected intraperitoneally twice weekly with MAb DLK1 over a period of one month. DEXA-, microCT scanning, and bone histomorphometric analyses were performed. Compared to controls, MAb DLK1 treated ovx mice were protected against ovx-induced bone loss, as revealed by significantly increased total bone mass (BMD) due to increased trabecular bone volume fraction (BV/TV) and inhibition of bone resorption. No significant changes were observed in total fat mass or in the number of bone marrow adipocytes. These results support the potential use of anti-DLK1 antibody therapy as a novel intervention to protect from E deficiency associated bone loss.

High-Fat Diet-Induced Obesity Promotes Expansion of Bone Marrow Adipose Tissue and Impairs Skeletal Stem Cell Functions in Mice.

Obesity represents a risk factor for development of insulin resistance and type 2 diabetes. In addition, it has been associated with increased adipocyte formation in the bone marrow (BM) along with increased risk for bone fragility fractures. However, little is known on the cellular mechanisms that link obesity, BM adiposity, and bone fragility. Thus, in an obesity intervention study in C57BL/6J mice fed with a high-fat diet (HFD) for 12 weeks, we investigated the molecular and cellular phenotype of bone marrow adipose tissue (BMAT), BM progenitor cells, and BM microenvironment in comparison to peripheral adipose tissue (AT). HFD decreased trabecular bone mass by 29%, cortical thickness by 5%, and increased BM adiposity by 184%. In contrast to peripheral AT, BMAT did not exhibit pro-inflammatory phenotype. BM progenitor cells isolated from HFD mice exhibited decreased mRNA levels of inflammatory genes (Tnfα, IL1ß, Lcn2) and did not manifest an insulin resistant phenotype evidenced by normal levels of pAKT after insulin stimulation as well as normal levels of insulin signaling genes. In addition, BM progenitor cells manifested enhanced adipocyte differentiation in HFD condition. Thus, our data demonstrate that BMAT expansion in response to HFD exerts a deleterious effect on the skeleton. Continuous recruitment of progenitor cells to adipogenesis leads to progenitor cell exhaustion, decreased recruitment to osteoblastic cells, and decreased bone formation. In addition, the absence of insulin resistance and inflammation in the BM suggest that BMAT buffers extra energy in the form of triglycerides and thus plays a role in whole-body energy homeostasis. © 2018 The Authors. Journal of Bone and Mineral Research Published by Wiley Periodicals, Inc.

Global MicroRNA Profiling in Human Bone Marrow Skeletal-Stromal or Mesenchymal-Stem Cells Identified Candidates for Bone Regeneration.

Bone remodeling and regeneration are highly regulated multistep processes involving posttranscriptional regulation by microRNAs (miRNAs). Here, we performed a global profiling of differentially expressed miRNAs in bone-marrow-derived skeletal cells (BMSCs; also known as stromal or mesenchymal stem cells) during in vitro osteoblast differentiation. We functionally validated the regulatory effects of several miRNAs on osteoblast differentiation and identified 15 miRNAs, most significantly miR-222 and miR-423, as regulators of osteoblastogenesis. In addition, we tested the possible targeting of miRNAs for enhancing bone tissue regeneration. Scaffolds functionalized with miRNA nano-carriers enhanced osteoblastogenesis in 3D culture and retained this ability at least 2 weeks after storage. Additionally, anti-miR-222 enhanced in vivo ectopic bone formation through targeting the cell-cycle inhibitor CDKN1B (cyclin-dependent kinase inhibitor 1B). A number of additional miRNAs exerted additive osteoinductive effects on BMSC differentiation, suggesting that pools of miRNAs delivered locally from an implanted scaffold can provide a promising approach for enhanced bone regeneration.

Optimizing combination of vascular endothelial growth factor and mesenchymal stem cells on ectopic bone formation in SCID mice.

INTRODUCTION: Insufficient blood supply may limit bone regeneration in bone defects. Vascular endothelial growth factor (VEGF) promotes angiogenesis by increasing endothelial migration. This outcome, however, could depend on time of application. Sheep mesenchymal stem cells (MSCs) in severe combined immunodeficient (SCID) mice were used in this study to evaluate optimal time points for VEGF stimulation to increase bone formation. METHODS: Twenty-eight SCID (NOD.CB17-Prkdcscid /J) mice had hydroxyapatite granules seeded with 5 × 105 MSCs inserted subcutaneous. Pellets released VEGF on days 1-7, days 1-14, days 1-21, days 1-42, days 7-14, and days 21-42. After 8 weeks, the implant-bone-blocks were harvested, paraffin embedded, sectioned, and stained with both hematoxylin and eosin (HE) and immunohistochemistry for human vimentin (hVim) staining. Blood samples were collected for determination of bone-related biomarkers in serum. RESULTS: The groups with 5 × 105 MSCs and VEGF stimulation on days 1-14 and days 1-21 showed more bone formation when compared to the control group of 5 × 105 MSCs alone (p < 0.01). Serum biomarkers had no significant values. The hVim staining confirmed the ovine origin of the observed ectopic bone formation. CONCLUSION: Optimal bone formation of MSCs was reached when stimulating with VEGF during the first 14 or 21 days after surgery. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 105A: 3326-3332, 2017.

CHD1 regulates cell fate determination by activation of differentiation-induced genes.

The coordinated temporal and spatial activation of gene expression is essential for proper stem cell differentiation. The Chromodomain Helicase DNA-binding protein 1 (CHD1) is a chromatin remodeler closely associated with transcription and nucleosome turnover downstream of the transcriptional start site (TSS). In this study, we show that CHD1 is required for the induction of osteoblast-specific gene expression, extracellular-matrix mineralization and ectopic bone formation in vivo. Genome-wide occupancy analyses revealed increased CHD1 occupancy around the TSS of differentiation-activated genes. Furthermore, we observed that CHD1-dependent genes are mainly induced during osteoblast differentiation and are characterized by higher levels of CHD1 occupancy around the TSS. Interestingly, CHD1 depletion resulted in increased pausing of RNA Polymerase II (RNAPII) and decreased H2A.Z occupancy close to the TSS, but not at enhancer regions. These findings reveal a novel role for CHD1 during osteoblast differentiation and provide further insights into the intricacies of epigenetic regulatory mechanisms controlling cell fate determination.

Legumain Regulates Differentiation Fate of Human Bone Marrow Stromal Cells and Is Altered in Postmenopausal Osteoporosis.

Secreted factors are a key component of stem cell niche and their dysregulation compromises stem cell function. Legumain is a secreted cysteine protease involved in diverse biological processes. Here, we demonstrate that legumain regulates lineage commitment of human bone marrow stromal cells and that its expression level and cellular localization are altered in postmenopausal osteoporotic patients. As shown by genetic and pharmacological manipulation, legumain inhibited osteoblast (OB) differentiation and in vivo bone formation through degradation of the bone matrix protein fibronectin. In addition, genetic ablation or pharmacological inhibition of legumain activity led to precocious OB differentiation and increased vertebral mineralization in zebrafish. Finally, we show that localized increased expression of legumain in bone marrow adipocytes was inversely correlated with adjacent trabecular bone mass in a cohort of patients with postmenopausal osteoporosis. Our data suggest that altered proteolytic activity of legumain in the bone microenvironment contributes to decreased bone mass in postmenopausal osteoporosis.