Bone Marrow-derived Stromal Cells Research Articles

Targeting Bone Tumours with 45S5 Bioactive Glass.

Despite advances in treatment modalities, bone tumour therapies still face significant challenges. Severe side effects of conventional approaches, such as chemo- and radiation therapy, result in poor survival rates and high tumour recurrence rates, which are the most common issues that need to be improved upon. The aim of this study was to evaluate the therapeutic properties of 45S5 bioactive glass (BG) for targeting bone tumours. The viability of the cells derived from osteosarcoma, chondrosarcoma, and giant cell tumours was significantly reduced in the presence of 45S5-BG. In contrast, the viability of non-malignant osteoblast-like cells, chondrocytes, and bone marrow-derived stromal cells was not or only slightly affected. While alterations to the particle surface induced by heat treatment, acid etching, or incubation in a simulated body fluid had only minor effects on cytotoxicity, reducing the particle size or sintering the material significantly improved the cytotoxic effect of 45S5-BG. Further, using a chicken chorioallantoic membrane assay, the co-transplantation of 45S5-BG resulted in a significant reduction in tumour formation in vivo. Given the known positive effects of BGs on bone regeneration, our findings suggest that 45S5-BG holds great potential for the development of new and effective bone tumour therapies, with minimal side effects on non-malignant cells and simultaneous contribution to bone healing.

Modulation of osteoblastogenesis by NRF2: NRF2 activation suppresses osteogenic differentiation and enhances mineralization in human bone marrow-derived mesenchymal stromal cells.

Mesenchymal stromal stem cells (MSCs) or skeletal stem cells (SSCs) play a major role in tissue repair due to their robust ability to differentiate into osteoblasts, chondrocytes, and adipocytes. Complex cell signaling cascades tightly regulate this differentiation. In osteogenic differentiation, Runt-related transcription factor 2 (RUNX2) and ALP activity are essential. Furthermore, during the latter stages of osteogenic differentiation, mineral formation mediated by the osteoblast occurs with the secretion of a collagenous extracellular matrix and calcium deposition. Activation of nuclear factor erythroid 2-related factor 2 (NRF2), an important transcription factor against oxidative stress, inhibits osteogenic differentiation and mineralization via modulation of RUNX2 function; however, the exact role of NRF2 in osteoblastogenesis remains unclear. Here, we demonstrate that NRF2 activation in human bone marrow-derived stromal cells (HBMSCs) suppressed osteogenic differentiation. NRF2 activation increased the expression of STRO-1 and KITLG (stem cell markers), indicating NRF2 protects HBMSCs stemness against osteogenic differentiation. In contrast, NRF2 activation enhanced mineralization, which is typically linked to osteogenic differentiation. We determined that these divergent results were due in part to the modulation of cellular calcium flux genes by NRF2 activation. The current findings demonstrate a dual role for NRF2 as a HBMSC maintenance factor as well as a central factor in mineralization, with implications therein for elucidation of bone formation and cellular Ca2+ kinetics, dystrophic calcification and, potentially, application in the modulation of bone formation.

Rankl genetic deficiency and functional blockade undermine skeletal stem and progenitor cell differentiation

BackgroundSkeletal Stem Cells (SSCs) are required for skeletal development, homeostasis, and repair. The perspective of their wide application in regenerative medicine approaches has supported research in this field, even though so far results in the clinic have not reached expectations, possibly due also to partial knowledge of intrinsic, potentially actionable SSC regulatory factors. Among them, the pleiotropic cytokine RANKL, with essential roles also in bone biology, is a candidate deserving deep investigation.MethodsTo dissect the role of the RANKL cytokine in SSC biology, we performed ex vivo characterization of SSCs and downstream progenitors (SSPCs) in mice lacking Rankl (Rankl−/−) by means of cytofluorimetric sorting and analysis of SSC populations from different skeletal compartments, gene expression analysis, and in vitro osteogenic differentiation. In addition, we assessed the effect of the pharmacological treatment with the anti-RANKL blocking antibody Denosumab (approved for therapy in patients with pathological bone loss) on the osteogenic potential of bone marrow-derived stromal cells from human healthy subjects (hBMSCs).ResultsWe found that, regardless of the ossification type of bone, osteochondral SSCs had a higher frequency and impaired differentiation along the osteochondrogenic lineage in Rankl−/− mice as compared to wild-type. Rankl−/− mice also had increased frequency of committed osteochondrogenic and adipogenic progenitor cells deriving from perivascular SSCs. These changes were not due to the peculiar bone phenotype of increased density caused by lack of osteoclast resorption (defined osteopetrosis); indeed, they were not found in another osteopetrotic mouse model, i.e., the oc/oc mouse, and were therefore not due to osteopetrosis per se. In addition, Rankl−/− SSCs and primary osteoblasts showed reduced mineralization capacity. Of note, hBMSCs treated in vitro with Denosumab had reduced osteogenic capacity compared to control cultures.ConclusionsWe provide for the first time the characterization of SSPCs from mouse models of severe recessive osteopetrosis. We demonstrate that Rankl genetic deficiency in murine SSCs and functional blockade in hBMSCs reduce their osteogenic potential. Therefore, we propose that RANKL is an important regulatory factor of SSC features with translational relevance.

Vanadium and strontium co-doped hydroxyapatite enriched polycaprolactone matrices for effective bone tissue engineering: A synergistic approach

Scientific research targeted at enhancing scaffold qualities has increased significantly during the last few decades. This emphasis frequently centres on adding different functions to scaffolds in order to increase their usefulness as instruments in the field of regenerative medicine. This study aims to investigate the efficacy of a multifunctional sustainable polymer scaffold, specifically Polycaprolactone (PCL) embedded with hydroxyapatite co-doped with vanadium and strontium (HVS), for bone tissue engineering applications. Polycaprolactone was used to fabricate the scaffold, while hydroxyapatite co-doped with vanadium and strontium (HVS) served as the nanofiller. A thorough investigation of the physicochemical and biological characteristics of the HVS nanofiller was carried out using cutting-edge techniques including Dynamic Light Scattering (DLS), and X-ray Photoelectron Spectroscopy (XPS) and in vitro cell studies. A cell viability rate of more than 70 % demonstrated that the synthesised nanofiller was cytotoxic, but in an acceptable range. The mechanical, biological, and physicochemical properties of the scaffold were extensively evaluated after the nanofiller was integrated. The water absorption characteristics of scaffold were enhanced by the addition of HVS nanofillers, leading to increased swelling, porosity, and hydrophilicity. These improvements speed up the flow of nutrients and the infiltration of cells into the scaffold. The scaffold has been shown to have important properties that stimulate bone cell activity, including better biodegradability and improved mechanical strength, which increased from 5.30 ± 0.37 to 10.58 ± 0.42 MPa. Further, its considerable antimicrobial qualities, blood-compatible nature, and capacity to promote biomineralization strengthen its appropriateness for usage in biomedical applications. Mainly, enhanced Alkaline phosphatase (ALP) activity, Alizarin Red Staining (ARS) activity, and excellent cell adhesive properties, indicating the outstanding osteogenic potential observed in rat bone marrow-derived stromal cells (rBMSC). These combined attributes highlight the pivotal role of these nanocomposite scaffolds in the field of bone tissue engineering.

Cell type-specific network analysis in Diversity Outbred mice identifies genes potentially responsible for human bone mineral density GWAS associations.

Genome-wide association studies (GWASs) have identified many sources of genetic variation associated with bone mineral density (BMD), a clinical predictor of fracture risk and osteoporosis. Aside from the identification of causal genes, other difficult challenges to informing GWAS include characterizing the roles of predicted causal genes in disease and providing additional functional context, such as the cell type predictions or biological pathways in which causal genes operate. Leveraging single-cell transcriptomics (scRNA-seq) can assist in informing BMD GWAS by linking disease-associated variants to genes and providing a cell type context for which these causal genes drive disease. Here, we use large-scale scRNA-seq data from bone marrow-derived stromal cells cultured under osteogenic conditions (BMSC-OBs) from Diversity Outbred (DO) mice to generate cell type-specific networks and contextualize BMD GWAS-implicated genes. Using trajectories inferred from the scRNA-seq data, we identify networks enriched with genes that exhibit the most dynamic changes in expression across trajectories. We discover 21 network driver genes, which are likely to be causal for human BMD GWAS associations that colocalize with expression/splicing quantitative trait loci (eQTL/sQTL). These driver genes, including Fgfrl1 and Tpx2, along with their associated networks, are predicted to be novel regulators of BMD via their roles in the differentiation of mesenchymal lineage cells. In this work, we showcase the use of single-cell transcriptomics from mouse bone-relevant cells to inform human BMD GWAS and prioritize genetic targets with potential causal roles in the development of osteoporosis.

Abstract 1501: Engineering tissue models to elucidate prostate cancer bone metastasis dynamics

Abstract Prostate cancer (PCa) ranks as the second most frequently diagnosed cancer in men and the fifth leading cause of male cancer-related deaths globally Advanced PCa exhibits a propensity for bone metastasis which significantly contributes to morbidity. It is challenging to understand its mechanisms and develop effective treatments due to complicated tumor microenvironments. The current study addresses this gap by focusing on the construction of in vitro and in vivo models to mimic tissue-specific microenvironments resembling primary prostate tumor sites and metastatic bone tissue, shedding light on the complex processes of cancer cell dissemination, osteomimicry, and bone metastasis. To recapitulate the complex interplay between primary tumor sites and bone microenvironments, the study employs a combination of tunable extracellular matrix mechanics, bone-derived growth factors, and bone marrow-derived stromal cells. These engineered niches emulate the contexts of both primary tumor and bone tissue. LNCAP-C4-2B prostate cancer cells are introduced into these niches to analyze various aspects, including cell proliferation, osteoblastic differentiation, dissemination, and in vivo tumor formation. The study introduces a 3D organoid culture that mimics the pre-metastatic niche, providing a platform for studying PCa cell proliferation and dissemination. A negative relationship is observed between 3D matrix stiffness and cell proliferation, while a positive link is identified with cell dissemination. Bone matrix-derived soluble factors (BMSF) are identified as key modulators of LNCAP-C4-2B cell behavior, suppressing LNCAP-C4-2B proliferation but enhancing osteomimicry in vitro, Notably, bone marrow-derived stromal cells, primed with bone matrix-derived growth factors, amplify tumor cell proliferation and chemotaxis in vitro, contributing to increased tumor formation in vivo. In vivo experiments demonstrate BMSFs eliciting local inflammatory responses and inhibiting in vivo tumor formation. Moreover, osteogenic bone marrow stromal cells are shown to play a promotive role in LNCAP-C4-2B proliferation and migration, emphasizing the influence of the bone microenvironment on the prostate-to-bone progression of PCa cells. In conclusion, this study highlights the potential of the engineered tissue models for dissecting tumor progression and screening therapeutic interventions. By leveraging tissue-engineered soft and hard tissues, our approach offers a versatile toolbox for comprehensively investigating PCa progression and bone metastasis. These insights extend to potential therapeutic screening applications, providing a valuable resource for advancing our understanding of cancer pathogenesis and treatment strategies. Citation Format: Bo Han, Zhi Yang, Shuqing Zhao, Gangning Liang, Ba Xuan Hoang, Josephine Fang. Engineering tissue models to elucidate prostate cancer bone metastasis dynamics [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 1501.

Brain-Derived Neurotrophic Factor (BDNF) Enhances Osteogenesis and May Improve Bone Microarchitecture in an Ovariectomized Rat Model.

Brain-derived neurotrophic factor (BDNF) has gained attention as a therapeutic agent due to its potential biological activities, including osteogenesis. However, the molecular mechanisms involved in the osteogenic activity of BDNF have not been fully understood. This study aimed to investigate the action of BDNF on the osteoblast differentiation in bone marrow stromal cells, and its influence on signaling pathways. In addition, to evaluate the clinical efficacy, an in vivo animal study was performed. Preosteoblast cells (MC3T3-E1), bone marrow-derived stromal cells (ST2), and a direct 2D co-culture system were treated with BDNF. The effect of BDNF on cell proliferation was determined using the CCK-8 assay. Osteoblast differentiation was assessed based on alkaline phosphatase (ALP) activity and staining and the protein expression of multiple osteoblast markers. Calcium accumulation was examined by Alizarin red S staining. For the animal study, we used ovariectomized Sprague-Dawley rats and divided them into BDNF and normal saline injection groups. MicroCT, hematoxylin and eosin (H&E), and tartrate-resistant acid phosphatase (TRAP) stain were performed for analysis. BDNF significantly increased ALP activity, calcium deposition, and the expression of osteoblast differentiation-related proteins, such as ALP, osteopontin, etc., in both ST-2 and the MC3T3-E1 and ST-2 co-culture systems. Moreover, the effect of BDNF on osteogenic differentiation was diminished by blocking tropomyosin receptor kinase B, as well as inhibiting c-Jun N-terminal kinase and p38 MAPK signals. Although the animal study results including bone density and histology showed increased osteoblastic and decreased osteoclastic activity, only a portion of parameters reached statistical significance. Our study results showed that BDNF affects osteoblast differentiation through TrkB receptor, and JNK and p38 MAPK signal pathways. Although not statistically significant, the trend of such effects was observed in the animal experiment.

Improved Efficacy of Delayed Treatment with Human Bone Marrow-Derived Stromal Cells Evaluated in Rats with Spinal Cord Injury.

The treatment of spinal cord injury (SCI) with uncultivated human bone marrow-derived stromal cells (bmSCs) prepared by negative selection has been proposed to be therapeutically superior to treatment with stem cells that were expanded in vitro. To explore their use in clinical trials, we studied the functional effects of delayed application at 7 days after SCI by testing different doses of bmSCs. Spinal cord contusion injury was induced in adult male Wistar rats at the thoracic level T9. Human bmSCs were prepared by negative selection without expansion in vitro (NeuroCellsTM). Treatment consisted of one 150 µL injection into the cisterna magna containing 0.5 or 2.5 million fresh bmSCs or 2.5 million bmSCs. The recovery of motor functions was evaluated during a surveillance period of six weeks (6 W), during which spinal cords were assessed histologically. Treatment resulted in a significant, dose-dependent therapeutic effect on the recovery of motor performance. The histological analysis revealed a lower degree of axonal degeneration and better survival of neurons and oligodendrocytes in bmSCs treated rats. Our results support delayed intrathecal application of bmSCs prepared by negative selection without expansion in vitro as a treatment of SCI.

Biological effects of a zinc-substituted borosilicate bioactive glass on human bone marrow derived stromal cells in vitro and in a critical-size femoral defect model in rats in vivo.

The borosilicate 0106-B1-bioactive glass (BG) composition (in wt%: 37.5 SiO2, 22.6 CaO, 5.9 Na2O, 4.0P2O5, 12.0 K2O, 5.5 MgO, 12.5 B2O3) has shown favorable processing characteristics and bone regeneration ability. This study investigated the addition of zinc (Zn) to 0106-B1-BG as an approach to improve this BG's biological properties. Different proportions of ZnO were substituted for CaO in 0106-B1-BG, resulting in three new BG-compositions: 1-Zn-BG, 2-Zn-BG, 3-Zn-BG (in wt%: 37.5 SiO2, 21.6/20.1/17.6 CaO, 4.0 P2O5, 5.9 Na2O, 12.0 K2O, 5.5 MgO, 12.5 B2O3 and 1.0/2.5/5.0 ZnO). Effects of the BG compositions on cytocompatibility, osteogenic differentiation, extracellular matrix deposition, and angiogenic response of human bone marrow-derived mesenchymal stromal cells (BMSCs) were evaluated in vitro. Angiogenic effects were assessed using a tube formation assay containing human umbilical vein endothelial cells. The in vivo osteogenic and angiogenic potentials of 3-Zn-BG were investigated in comparison to the Zn-free 0106-B1-BG in a rodent critical-size femoral defect model. The osteogenic differentiation of BMSCs improved in the presence of Zn. 3-Zn-BG showed enhanced angiogenic potential, as confirmed by the tube formation assay. While Zn-doped BGs showed clearly superior biological properties in vitro, 3-Zn-BG and 0106-B1-BG equally promoted the formation of new bone in vivo; however, 3-Zn-BG reduced osteoclastic cells and vascular structures in vivo. The acquired data suggests that the differences regarding the in vivo and in vitro results may be due to modulation of inflammatory responses by Zn, as described in the literature. The inflammatory effect should be investigated further to promote clinical applications of Zn-doped BGs.

Microtissue Culture Provides Clarity on the Relative Chondrogenic and Hypertrophic Response of Bone-Marrow-Derived Stromal Cells to TGF-β1, BMP-2, and GDF-5.

Chondrogenic induction of bone-marrow-derived stromal cells (BMSCs) is typically accomplished with medium supplemented with growth factors (GF) from the transforming growth factor-beta (TGF-β)/bone morphogenetic factor (BMP) superfamily. In a previous study, we demonstrated that brief (1-3 days) stimulation with TGF-β1 was sufficient to drive chondrogenesis and hypertrophy using small-diameter microtissues generated from 5000 BMSC each. This biology is obfuscated in typical large-diameter pellet cultures, which suffer radial heterogeneity. Here, we investigated if brief stimulation (2 days) of BMSC microtissues with BMP-2 (100 ng/mL) or growth/differentiation factor (GDF-5, 100 ng/mL) was also sufficient to induce chondrogenic differentiation, in a manner comparable to TGF-β1 (10 ng/mL). Like TGF-β1, BMP-2 and GDF-5 are reported to stimulate chondrogenic differentiation of BMSCs, but the effects of transient or brief use in culture have not been explored. Hypertrophy is an unwanted outcome in BMSC chondrogenic differentiation that renders engineered tissues unsuitable for use in clinical cartilage repair. Using three BMSC donors, we observed that all GFs facilitated chondrogenesis, although the efficiency and the necessary duration of stimulation differed. Microtissues treated with 2 days or 14 days of TGF-β1 were both superior at producing extracellular matrix and expression of chondrogenic gene markers compared to BMP-2 and GDF-5 with the same exposure times. Hypertrophic markers increased proportionally with chondrogenic differentiation, suggesting that these processes are intertwined for all three GFs. The rapid action, or "temporal potency", of these GFs to induce BMSC chondrogenesis was found to be as follows: TGF-β1 > BMP-2 > GDF-5. Whether briefly or continuously supplied in culture, TGF-β1 was the most potent GF for inducing chondrogenesis in BMSCs.

Polyimide biocomposites coated with tantalum pentoxide for stimulation of cell compatibility and enhancement of biointegration for orthopedic implant

Orthopedic implants are an important tool in the treatment of musculoskeletal conditions and helped many patients to improve their quality of life. Various inorganic-organic biocomposites have been broadly investigated particularly in the area of load-bearing orthopedic/dental applications. Polyimide (PI) is a promising organic material and shows excellent mechanical properties, biocompatibility, bio-stability, and its elastic modulus is similar to human bone but it lacks bioactivity, which is very important for cell adhesion and ultimately for bone regeneration. In this research, tantalum pentoxide (Ta2O5) coating was prepared on the surface of PI by polydopamine (PDA) bonding. The results showed that Ta2O5 was evenly coated on the surface of PI, and with the concentration of Ta2O5 in the PDA suspension increased, the content of Ta2O5 particles on the surface of PI increased significantly. In addition, the Ta2O5 coating significantly increased the roughness and hydrophilicity of the PI matrix. Cell experiments showed that PI surface coating Ta2O5 could promote the proliferation, adhesion, and osteogenic differentiation of bone marrow-derived stromal cells (BMSCs). The results demonstrated that fabricating Ta2O5 coating on the surface of PI through PDA bonding could improve the biocompatibility as well as bioactivity of PI, and increase the application potential of PI in the field of bone repair materials.

Tensin-3 is involved in osteogenic versus adipogenic fate of human bone marrow stromal cells

BackgroundThe tightly controlled balance between osteogenic and adipogenic differentiation of human bone marrow-derived stromal cells (BMSCs) is critical to maintain bone homeostasis. Age-related osteoporosis is characterized by low bone mass with excessive infiltration of adipose tissue in the bone marrow compartment. The shift of BMSC differentiation from osteoblasts to adipocytes could result in bone loss and adiposity.MethodsTNS3 gene expression during osteogenic and adipogenic differentiation of BMSCs was evaluated by qPCR and Western blot analyses. Lentiviral-mediated knockdown or overexpression of TNS3 was used to assess its function. The organization of cytoskeleton was examined by immunofluorescent staining at multiple time points. The role of TNS3 and its domain function in osteogenic differentiation were evaluated by ALP activity, calcium assay, and Alizarin Red S staining. The expression of Rho-GTP was determined using the RhoA pull-down activation assay.ResultsLoss of TNS3 impaired osteogenic differentiation of BMSCs but promoted adipogenic differentiation. Conversely, TNS3 overexpression hampered adipogenesis while enhancing osteogenesis. The expression level of TNS3 determined cell shape and cytoskeletal reorganization during osteogenic differentiation. TNS3 truncation experiments revealed that for optimal osteogenesis to occur, all domains proved essential. Pull-down and immunocytochemical experiments suggested that TNS3 mediates osteogenic differentiation through RhoA.ConclusionsHere, we identify TNS3 to be involved in BMSC fate decision. Our study links the domain structure in TNS3 to RhoA activity via actin dynamics and implicates an important role for TNS3 in regulating osteogenesis and adipogenesis from BMSCs. Furthermore, it supports the critical involvement of cytoskeletal reorganization in BMSC differentiation.

Single-Cell Transcriptomics of Bone Marrow Stromal Cells in Diversity Outbred Mice: A Model for Population-Level scRNA-Seq Studies.

Genome-wide association studies (GWASs) have advanced our understanding of the genetics of osteoporosis; however, the challenge has been converting associations to causal genes. Studies have utilized transcriptomics data to link disease-associated variants to genes, but few population transcriptomics data sets have been generated on bone at the single-cell level. To address this challenge, we profiled the transcriptomes of bone marrow-derived stromal cells (BMSCs) cultured under osteogenic conditions from five diversity outbred (DO) mice using single-cell RNA-seq (scRNA-seq). The goal of the study was to determine if BMSCs could serve as a model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells from large populations of mice to inform genetic studies. By enriching for mesenchymal lineage cells in vitro, coupled with pooling of multiple samples and downstream genotype deconvolution, we demonstrate the scalability of this model for population-level studies. We demonstrate that dissociation of BMSCs from a heavily mineralized matrix had little effect on viability or their transcriptomic signatures. Furthermore, we show that BMSCs cultured under osteogenic conditions are diverse and consist of cells with characteristics of mesenchymal progenitors, marrow adipogenic lineage precursors (MALPs), osteoblasts, osteocyte-like cells, and immune cells. Importantly, all cells were similar from a transcriptomic perspective to cells isolated in vivo. We employed scRNA-seq analytical tools to confirm the biological identity of profiled cell types. SCENIC was used to reconstruct gene regulatory networks (GRNs), and we observed that cell types show GRNs expected of osteogenic and pre-adipogenic lineage cells. Further, CELLECT analysis showed that osteoblasts, osteocyte-like cells, and MALPs captured a significant component of bone mineral density (BMD) heritability. Together, these data suggest that BMSCs cultured under osteogenic conditions coupled with scRNA-seq can be used as a scalable and biologically informative model to generate cell type-specific transcriptomic profiles of mesenchymal lineage cells in large populations. © 2023 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

A comparative in vitro and in vivo analysis of the biological properties of the 45S5-, 1393-, and 0106-B1-bioactive glass compositions using human bone marrow-derived stromal cells and a rodent critical size femoral defect model

Since the introduction of the 45S5-bioactive glass (BG), numerous new BG compositions have been developed. Compared to the 45S5-BG, 1393-BG shows favorable processing properties due to its low crystallization tendency and the 1393-BG-based borosilicate 0106-B1-BG exhibits improved angiogenic properties due to its boron content. Despite their close (chemical) relationship, the biological properties of the mentioned BG composition have not yet been comparatively examined. In this study, the effects of the BGs on proliferation, viability, osteogenic differentiation, and angiogenic factor production of human bone marrow-derived mesenchymal stromal cells were assessed. Scaffolds made of the BGs were introduced in a critical-sized femur defect model in rats in order to analyze their impact on bone defect regeneration. In vitro, 1393-BG and 0106-B1-BG outperformed 45S5-BG with regard to cell proliferation and viability. 1393-BG enhanced osteogenic differentiation; 0106-B1-BG promoted angiogenic factor production. In vivo, 0106-B1-BG and 45S5-BG outperformed 1393-BG in terms of angiogenic and osteoclastic response resulting in improved bone regeneration. In conclusion, the biological properties of BGs can be significantly modified by tuning their composition. Demonstrating favorable processing properties and an equally strong in vivo bone regeneration potential as 45S5-BG, 0106-B1-BG qualifies as a basis to incorporate other bioactive ions to improve its biological properties.

G protein-coupled receptor 183 mediates the sensitization of Burkitt lymphoma tumors to CD47 immune checkpoint blockade by anti-CD20/PI3Kδi dual therapy.

Immunotherapy-based regimens have considerably improved the survival rate of B-cell non-Hodgkin lymphoma (B-NHL) patients in the last decades; however, most disease subtypes remain almost incurable. TG-1801, a bispecific antibody that targets CD47 selectively on CD19+ B-cells, is under clinical evaluation in relapsed/refractory (R/R) B-NHL patients either as a single-agent or in combination with ublituximab, a new generation CD20 antibody. A set of eight B-NHL cell lines and primary samples were cultured in vitro in the presence of bone marrow-derived stromal cells, M2-polarized primary macrophages, and primary circulating PBMCs as a source of effector cells. Cell response to TG-1801 alone or combined with the U2 regimen associating ublituximab to the PI3Kδ inhibitor umbralisib, was analyzed by proliferation assay, western blot, transcriptomic analysis (qPCR array and RNA sequencing followed by gene set enrichment analysis) and/or quantification of antibody-dependent cell death (ADCC) and antibody-dependent cell phagocytosis (ADCP). CRISPR-Cas9 gene edition was used to selectively abrogate GPR183 gene expression in B-NHL cells. In vivo, drug efficacy was determined in immunodeficient (NSG mice) or immune-competent (chicken embryo chorioallantoic membrane (CAM)) B-NHL xenograft models. Using a panel of B-NHL co-cultures, we show that TG-1801, by disrupting the CD47-SIRPα axis, potentiates anti-CD20-mediated ADCC and ADCP. This led to a remarkable and durable antitumor effect of the triplet therapy composed by TG-1801 and U2 regimen, in vitro, as well as in mice and CAM xenograft models of B-NHL. Transcriptomic analysis also uncovered the upregulation of the G protein-coupled and inflammatory receptor, GPR183, as a crucial event associated with the efficacy of the triplet combination. Genetic depletion and pharmacological inhibition of GPR183 impaired ADCP initiation, cytoskeleton remodeling and cell migration in 2D and 3D spheroid B-NHL co-cultures, and disrupted macrophage-mediated control of tumor growth in B-NHL CAM xenografts. Altogether, our results support a crucial role for GPR183 in the recognition and elimination of malignant B cells upon concomitant targeting of CD20, CD47 and PI3Kδ, and warrant further clinical evaluation of this triplet regimen in B-NHL.

Relationships between Circulating Sclerostin, Bone Marrow Adiposity, Other Adipose Deposits and Lean Mass in Post-Menopausal Women.

Sclerostin is a Wnt signaling pathway inhibitor that negatively regulates bone formation. Bone-marrow-derived stromal cell (BMSC) differentiation is influenced by the Wnt pathway, leading to the hypothesis that higher levels of sclerostin might be associated with an increase in bone marrow adiposity (BMA). The main purpose of this study was to determine whether a relationship exists between circulating sclerostin and BMA in post-menopausal women with and without fragility fractures. The relationships between circulating sclerostin and body composition parameters were then examined. The outcomes measures included vertebral and hip proton density fat fraction (PDFF) using the water fat imaging (WFI) MRI method; DXA scans; and laboratory measurements, including serum sclerostin. In 199 participants, no significant correlations were found between serum sclerostin and PDFF. In both groups, serum sclerostin was correlated positively with bone mineral density (R = 0.27 to 0.56) and negatively with renal function (R = -0.22 to -0.29). Serum sclerostin correlated negatively with visceral adiposity in both groups (R = -0.24 to -0.32). Serum sclerostin correlated negatively with total body fat (R = -0.47) and appendicular lean mass (R = -0.26) in the fracture group, but not in the controls. No evidence of a relationship between serum sclerostin and BMA was found. However, serum sclerostin was negatively correlated with body composition components, such as visceral adiposity, total body fat and appendicular lean mass.

Connexin43 overexpression promotes bone regeneration by osteogenesis and angiogenesis in rat glucocorticoid-induced osteonecrosis of the femoral head

Glucocorticoids induced osteonecrosis of the femoral head (GIONFH) is a devastating orthopedic disease. Previous studies suggested that connexin43 is involved in the process of osteogenesis and angiogenesis. However, the role of Cx43 potentiates in the osteogenesis and angiogenesis of bone marrow-derived stromal stem cells (BMSCs) in GIONFH is still not investigated. In this study, BMSCs were isolated and transfected with green fluorescent protein or the fusion gene encoding GFP and Cx43. The osteogenic differentiation of BMSCs were detected after transfected with Cx43. In addition, the migration abilities and angiogenesis of human umbilical vein endothelial cells (HUVECs) were been detected after induced by transfected BMSCs supernatants in vitro. Finally, we established GC-ONFH rat model, then, a certain amount of transfected or controlled BMSCs were injected into the tibia of the rats. Immunohistological staining and micro-CT scanning results showed that the transplanted experiment group had significantly promoted more bone regeneration and vessel volume when compared with the effects of the negative or control groups. This study demonstrated for the first time that the Cx43 overexpression in BMSCs could promote bone regeneration as seen in the osteogenesis and angiogenesis process, suggesting that Cx43 may serve as a therapeutic gene target for GIONFH treatment.

Ultrasound-derived mechanical stimulation of cell-laden collagen hydrogels for bone repair.

Cell therapy is emerging as an effective treatment strategy for many diseases. Here we describe a novel approach to bone tissue repair that combines hydrogel-based cell therapy with low intensity pulsed ultrasound (LIPUS), an FDA approved treatment for fracture repair. Bone marrow-derived stromal cells (BMSCs) have been encapsulated in type I collagen hydrogels and mechanically stimulated using LIPUS-derived acoustic radiation force (ARF). We observed the expression and upward trend of load-sensitive, osteoblast-specific markers and determined that the extent of cell response is dependent on an optimal combination of both hydrogel stiffness and ARF intensity. Specifically, cells encapsulated in hydrogels of optimal stiffness respond at the onset of ultrasound by upregulating early bone-sensitive markers such as calcium, cyclooxygenase-2, and prostaglandin E2 , and later by supporting mineralized tissue formation after 21 days of culture. In vivo evaluation of a critical size calvarial defect in NOD scid gamma (NSG) mice indicated that the implantation of BMSC-laden hydrogels of optimal stiffness improved healing of calvarial defects after daily administration of ARF over 4 weeks. Collectively, these findings validate the efficacy of our system of localized cell delivery for treating bone defects where undifferentiated BMSCs are induced to the osteoblastic lineage. Further, in vivo healing may be enhanced via non-invasive transdermal mechanical stimulation of implanted cells using ARF.

Synergistic therapeutic effects of intracerebral transplantation of human modified bone marrow-derived stromal cells (SB623) and voluntary exercise with running wheel in a rat model of ischemic stroke

BackgroundMesenchymal stromal cell (MSC) transplantation therapy is a promising therapy for stroke patients. In parallel, rehabilitation with physical exercise could ameliorate stroke-induced neurological impairment. In this study, we aimed to clarify whether combination therapy of intracerebral transplantation of human modified bone marrow-derived MSCs, SB623 cells, and voluntary exercise with running wheel (RW) could exert synergistic therapeutic effects on a rat model of ischemic stroke.MethodsWistar rats received right transient middle cerebral artery occlusion (MCAO). Voluntary exercise (Ex) groups were trained in a cage with RW from day 7 before MCAO. SB623 cells (4.0 × 105 cells/5 μl) were stereotactically injected into the right striatum at day 1 after MCAO. Behavioral tests were performed at day 1, 7, and 14 after MCAO using the modified Neurological Severity Score (mNSS) and cylinder test. Rats were euthanized at day 15 after MCAO for mRNA level evaluation of ischemic infarct area, endogenous neurogenesis, angiogenesis, and expression of brain-derived neurotrophic factor (BDNF) and vascular endothelial growth factor (VEGF). The rats were randomly assigned to one of the four groups: vehicle, Ex, SB623, and SB623 + Ex groups.ResultsSB623 + Ex group achieved significant neurological recovery in mNSS compared to the vehicle group (p < 0.05). The cerebral infarct area of SB623 + Ex group was significantly decreased compared to those in all other groups (p < 0.05). The number of BrdU/Doublecortin (Dcx) double-positive cells in the subventricular zone (SVZ) and the dentate gyrus (DG), the laminin-positive area in the ischemic boundary zone (IBZ), and the mRNA level of BDNF and VEGF in SB623 + Ex group were significantly increased compared to those in all other groups (p < 0.05).ConclusionsThis study suggests that combination therapy of intracerebral transplantation SB623 cells and voluntary exercise with RW achieves robust neurological recovery and synergistically promotes endogenous neurogenesis and angiogenesis after cerebral ischemia, possibly through a mechanism involving the up-regulation of BDNF and VEGF.

A 3D Perfusable Platform for In Vitro Culture of Patient Derived Xenografts.

Many advanced cancer models, such as patient-derived xenografts (PDXs), offer significant benefits in their preservation of the native tumor's heterogeneity and susceptibility to treatments, but face significant barriers to use in their reliance on a rodent host for propagation and screening. PDXs remain difficult to implement in vitro, particularly in configurations that enable both detailed cellular analysis and high-throughput screening (HTS). Complex multilineage co-cultures with stromal fibroblasts, endothelium, and other cellular and structural components of the tumor microenvironment (TME) further complicate ex vivo implementation. Herein,the culture of multiple prostate cancer (PCa)-derived PDX models as 3D clusters within engineered biomimetic hydrogel matrices, in a HTS-compatible multiwell microfluidic format, alongside bone marrow-derived stromal cells and a perfused endothelial channel. Polymeric hydrogel matrices are customized for each cell type, enabling cell survival in vitro and facile imaging across all conditions. PCa PDXs demonstrate unique morphologies and reliance on TME partners, retention of known phenotype, and expected sensitivity or resistance to standard PCa therapeutics. This novel integration of technologies provides a fully human model, and expands the information to be gathered from each specimen, while avoiding the time and labor involved with animal-based testing.