Functional Osteoblasts Research Articles

Smurf1-targeting microRNA-136-5p-modified bone marrow mesenchymal stem cells combined with 3D-printed β-tricalcium phosphate scaffolds strengthen osteogenic activity and alleviate bone defects.

Suitable biomaterials with seed cells have promising potential to repair bone defects. However, bone marrow mesenchymal stem cells (BMSCs), one of the most common seed cells used in tissue engineering, cannot differentiate efficiently and accurately into functional osteoblasts. In view of this, a new tissue engineering technique combined with BMSCs and scaffolds is a major task for bone defect repair. Lentiviruses interfering with miR-136-5p or Smurf1 expression were transfected into BMSCs. The effects of miR-136-5p or Smurf1 on the osteogenic differentiation (OD) of BMSCs were evaluated by measuring alkaline phosphatase activity and calcium deposition. Then, the targeting relationship between miR-136-5p and Smurf1 was verified by bioinformatics website analysis and dual luciferase reporter assay. Then, a rabbit femoral condyle bone defect model was established. miR-136-5p/BMSCs/β-TCP scaffold was implanted into the defect, and the repair of the bone defect was detected by Micro-CT and HE staining. Elevating miR-136-5p-3p or suppressing Smurf1 could stimulate OD of BMSCs. miR-136-5p negatively regulated Smurf1 expression. Overexpressing Smurf1 reduced the promoting effect of miR-136-5p on the OD of BMSCs. miR-136-5p/BMSCs/β-TCP could strengthen bone density in the defected area and accelerate bone repair. SmurF1-targeting miR-136-5p-modified BMSCs combined with 3D-printed β-TCP scaffolds can strengthen osteogenic activity and alleviate bone defects.

Methodology and Characterization of a 3D Bone Organoid Model Derived from Murine Cells.

Here, we report on the development of a cost-effective, well-characterized three-dimensional (3D) model of bone homeostasis derived from commonly available stocks of immortalized murine cell lines and laboratory reagents. This 3D murine-cell-derived bone organoid model (3D-mcBOM) is adaptable to a range of contexts and can be used in conjunction with surrogates of osteoblast and osteoclast function to study cellular and molecular mechanisms that affect bone homeostasis in vitro or to augment in vivo models of physiology or disease. The 3D-mcBOM was established using a pre-osteoblast murine cell line, which was seeded into a hydrogel extracellular matrix (ECM) and differentiated into functional osteoblasts (OBs). The OBs mineralized the hydrogel ECM, leading to the deposition and consolidation of hydroxyapatite into bone-like organoids. Fourier-transform infrared (FTIR) spectroscopy confirmed that the mineralized matrix formed in the 3D-mcBOM was bone. The histological staining of 3D-mcBOM samples indicated a consistent rate of ECM mineralization. Type I collagen C-telopeptide (CTX1) analysis was used to evaluate the dynamics of OC differentiation and activity. Reliable 3D models of bone formation and homeostasis align with current ethical trends to reduce the use of animal models. This functional model of bone homeostasis provides a cost-effective model system using immortalized cell lines and easily procured supplemental compounds, which can be assessed by measuring surrogates of OB and OC function to study the effects of various stimuli in future experimental evaluations of bone homeostasis.

Mild photothermal effect of titania nanotubes array as a promising solution for peri-implantitis

Peri-implantitis is the main cause of implant failure with limited clinical management. Recently, the photothermal therapy (PTT) has attracted more and more attention, due to its convenient and efficient erasure of bacteria. Unfortunately, the high temperature may also cause healthy tissues damage, so that there is a call for the balance between “kill and heal” during PTT. Here, the disregarded photothermal effect of titania nanotubes array (TiNTs) is investigated, to realize the mild PTT (mPTT) idea. It is found that the delivered heat is inversely proportional to TiNTs diameters (30–80 nm) and in proportional to NIR light power (300–2000 mW/cm2). The maximum temperature elevation is reached within 4–6 min (13.4℃ under 300 mW/cm2), which belongs to the temperature range of mPTT. In addition, the peri-implantitis related cells including macrophages, endothelial progenitor cells and osteoblasts all exhibit favorable phenotype alterations after mPTT. Specifically, the macrophages are undergoing M2 polarization (anti-inflammation, pro-healing), endothelial progenitor cells show functional suppression (abnormal vascularization inhibition) and osteoblasts exhibit enhanced osteogenic differentiation (beneficial for new bone repair). In conclusion, these findings suggest that mPTT conveyed by TiNTs may control inflammation and promote bone regeneration, which may be a promising choice for peri-implantitis management.

MTORC1 induces plasma membrane depolarization and promotes preosteoblast senescence by regulating the sodium channel Scn1a

Senescence impairs preosteoblast expansion and differentiation into functional osteoblasts, blunts their responses to bone formation-stimulating factors and stimulates their secretion of osteoclast-activating factors. Due to these adverse effects, preosteoblast senescence is a crucial target for the treatment of age-related bone loss; however, the underlying mechanism remains unclear. We found that mTORC1 accelerated preosteoblast senescence in vitro and in a mouse model. Mechanistically, mTORC1 induced a change in the membrane potential from polarization to depolarization, thus promoting cell senescence by increasing Ca2+ influx and activating downstream NFAT/ATF3/p53 signaling. We further identified the sodium channel Scn1a as a mediator of membrane depolarization in senescent preosteoblasts. Scn1a expression was found to be positively regulated by mTORC1 upstream of C/EBPα, whereas its permeability to Na+ was found to be gated by protein kinase A (PKA)-induced phosphorylation. Prosenescent stresses increased the permeability of Scn1a to Na+ by suppressing PKA activity and induced depolarization in preosteoblasts. Together, our findings identify a novel pathway involving mTORC1, Scn1a expression and gating, plasma membrane depolarization, increased Ca2+ influx and NFAT/ATF3/p53 signaling in the regulation of preosteoblast senescence. Pharmaceutical studies of the related pathways and agents might lead to novel potential treatments for age-related bone loss.

Human oral mucosa-derived neural crest-like stem cells differentiate into functional osteoprogenitors that contribute to regeneration of critical size calvaria defects.

Regeneration of large bony defects is an unmet medical need. The therapeutic effect of fully developed bony constructs engineered in vitro from mineralized scaffold and adult stem cells is hampered by deficient long-term graft integration. The purpose of the present study was to investigate the regenerative capacity of a bony primordial construct consisting of human oral mucosa stem cells (hOMSC)-derived osteoprogenitors and absorbable Gelfoam® sponges. Gingiva and alveolar mucosa-derived hOMSC were differentiated into osteoprogenitors (Runx2 and osterix positive) and loaded into Gelfoam® sponges to generate primordial hOMSC constructs. These were implanted into critical size calvaria defects in the rat. Defects treated with human dermal fibroblasts (HDF) constructs; Gelfoam® sponges and untreated defects served as controls. After 120-day post-implantation defects treated with hOMSC constructs, HDF constructs and gelatin and untreated defects exhibited 86%, 30%, 21%, and 9% of new bone formation, respectively. Immunofluorescence analysis for human nuclear antigen (HNA), bone sialoprotein (BSP), and osteocalcin (OCN) revealed viable hOMSC-derived osteoblasts and osteocytes that formed most of the cell population of the newly formed bone at 30 and 120days post surgery. Few HNA-positive HDF that were negative for BSP and OCN were identified together with inflammatory cells in the soft tissue adjacent to new bone formation only at 30days post implantation. Collectively, the results demonstrate that primordial in vitro engineered constructs consisting of hOMSC-derived osteoprogenitors and absorbable gelatin almost completely regenerate critical size defects in an immunocompetent xenogeneic animal by differentiating into functional osteoblasts that retain the immunomodulatory ability of naïve hOMSC.

Circulating Cytokines Present in Multiple Myeloma Patients Inhibit the Osteoblastic Differentiation of Adipose and Bone Marrow Stem Cells

Introduction: Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We therefore examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Materials and Methods: We studied 11 MM-ASCs, 5 HD-ASCs and 3 HD-BM-MSCs. The plasmas were from myeloma patients with bone lesions at diagnosis (n=12), in complete remission (n=8) and from 5 pools of 100 healthy donors (HD-plasmas). HD-ASCs were differentiated into osteoblasts and adipocytes and HD-BM-MSCs into osteoblasts with the three types of plasmas as well as newly discovered cytokines. Results: Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Alizarin red coloration and alkaline phosphatase activity were strongly decreased along with a decreased RUNX2 and osteocalcin expression. However, adipocyte differentiation was unaltered. The osteoblastic differentiation deficiency was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas which was confirmed by ELISA (Figure). They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, myeloma patients in remission had a cytokine plasma level almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomittantly, we observed that MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. When myeloma patients were in remission, the cytokines levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition by the cytokines were also observed on HD-BM-MSCs suggesting that this could also be the case on myeloma-BM-MSCs.Legend to figure: Cytokine expression in MM, CR and HD-plasmas(A) Representative images of cytokine array blots probed with the plasma samples. The red boxes identify the cytokines significantly dysregulated in MM- as compared to HD- or CR-plasmas and further analyzed by ELISA. The blue boxes identified the cytokines similarly expressed in MM-/CR-/HD-plasmas. (B) Cytokine concentrations in the HD-plasmas (n=5), MM-plasmas (n=11) and CR-plasmas (n=8) were measured by ELISA. * p < 0.05, ** p < 0.01, *** p < 0.001, ns (not significant). [Display omitted] DisclosuresDelhommeau: Novartis: Consultancy; BMS: Consultancy; Celgene: Consultancy. Garderet: Celgene: Consultancy; Janssen: Consultancy; Amgen: Consultancy; Sanofi: Consultancy; Takeda: Consultancy.

P-028: Circulating cytokines present in multiple myeloma patients inhibit the osteoblastic differentiation of adipose and bone marrow stem cells

<h3>Background</h3> Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We aimed to identify the cause of this defect. <h3>Materials and Methods</h3> We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas), in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of MM-ASCs, healthy donor-derived ASCs (HD-ASCs) and healthy donor-bone marrow derived MSCs (HD-BM-MSCs). Alizarin red coloration, alkaline phosphatase activity, RUNX2 and osteocalcin expression determined osteoblastic differentiation while oil-red O staining, PPARγ, C/EBPα and CD36 expression reflected the adipogenic capacity. Cytokine arrays were used along with Elisa assays for identification and quantification of relevant cytokines. RNA sequencing was performed on MM and HD-ASCs. <h3>Results</h3> Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas but adipocyte differentiation was unaltered. This defect was reversible once the plasma-derived factors were removed. Using cytokine array and comparing MM-plasmas with HD-plasmas, we identified seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB and TARC), besides DKK1, highly increased in MM-plasmas. They separately inhibited the osteoblastic differentiation of HD-ASCs. In contrast, in CR-plasmas, the cytokine plasma levels were almost normal with barely no osteoblastic differentiation inhibition. In addition, the mixture of the 7 cytokines with and without DKK1 inhibited not only the HD-ASCs but also the HD-BM-MSCs. Concomitantly, MM-plasmas enhanced adipogenesis-related gene expression. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Moreover, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. Finally, we tried to identify the origin of these cytokines. In CR-plasmas, the cytokine levels were strongly decreased suggesting a malignant plasmocyte secretion. This was reinforced by the detection of the 7 cytokines in three different myeloma cell lines with an especially high secretion of PDGF-AA. <h3>Conclusion</h3> We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation. Of note, this inhibition, by these new circulating cytokines, was also observed on HD-BM-MSCs, suggesting that this could also be the case on myeloma-BM-MSCs.

Circulating cytokines present in multiple myeloma patients inhibit the osteoblastic differentiation of adipose stem cells.

Myeloma is characterized by bone lesions, which are related to both an increased osteoclast activity and a defect in the differentiation of medullary mesenchymal stem cells (MSCs) into osteoblasts. Outside the medullary environment, adipocyte-derived MSCs (ASCs) could represent a source of functional osteoblasts. However, we recently found a defect in the osteoblastic differentiation of ASCs from myeloma patients (MM-ASCs). We examined the effects of plasma from myeloma patients at diagnosis (MM-plasmas) and in complete remission (CR-plasmas) and from healthy donors on the osteoblastic differentiation of healthy donor-derived ASCs (HD-ASCs). Osteoblastogenesis in HD-ASCs was suppressed by MM-plasmas. Seven cytokines (ANG1, ENA-78, EGF, PDGF-AA/AB/BB, and TARC) were increased in MM-plasmas and separately inhibited the osteoblastic differentiation of HD-ASCs. Comparison of MM-ASCs and HD-ASCs by RNA sequencing showed that two master genes characterizing adipocyte differentiation, CD36 and PPARγ, were upregulated in MM-ASCs as compared to HD-ASCs. Finally, we demonstrated a significant increase in CD36 and PPARγ expression in HD-ASCs in the presence of MM-plasmas or the seven cytokines individually, similarly as in MM-ASCs. We conclude that specific cytokines in MM-plasmas, besides the well-known DKK1, inhibit the osteoblastic differentiation of MM- and HD-ASCs with a skewing towards adipocyte differentiation.

Osteogenic Response to Polysaccharide Nanogel Sheets of Human Fibroblasts After Conversion Into Functional Osteoblasts by Direct Phenotypic Cell Reprogramming.

Human dermal fibroblasts (HDFs) were converted into osteoblasts using a ALK inhibitor II (inhibitor of transforming growth factor-β signal) on freeze-dried nanogel-cross-linked porous (FD-NanoClip) polysaccharide sheets or fibers. Then, the ability of these directly converted osteoblasts (dOBs) to produce calcified substrates and the expression of osteoblast genes were analyzed in comparison with osteoblasts converted by exactly the same procedure but seeded onto a conventional atelocollagen scaffold. dOBs exposed to FD-NanoClip in both sheet and fiber morphologies produced a significantly higher concentration of calcium deposits as compared to a control cell sample (i.e., unconverted fibroblasts), while there was no statistically significant difference in calcification level between dOBs exposed to atelocollagen sheets and the control group. The observed differences in osteogenic behaviors were interpreted according to Raman spectroscopic analyses comparing different polysaccharide scaffolds and Fourier transform infrared spectroscopy analyses of dOB cultures. This study substantiates a possible new path to repair large bone defects through a simplified transplantation procedure using FD-NanoClip sheets with better osteogenic outputs as compared to the existing atelocollagen scaffolding material.

Hematopoietic stem cell-derived functional osteoblasts exhibit therapeutic efficacy in a murine model of osteogenesis imperfecta.

Currently, there is no cure for osteogenesis imperfecta (OI)-a debilitating pediatric skeletal dysplasia. Herein we show that hematopoietic stem cell (HSC) therapy holds promise in treating OI. Using single-cell HSC transplantation in lethally irradiated oim/oim mice, we demonstrate significant improvements in bone morphometric, mechanics, and turnover parameters. Importantly, we highlight that HSCs cause these improvements due to their unique property of differentiating into osteoblasts/osteocytes, depositing normal collagen-an attribute thus far assigned only to mesenchymal stem/stromal cells. To confirm HSC plasticity, lineage tracing was done by transplanting oim/oim with HSCs from two specific transgenic mice-VavR, in which all hematopoietic cells are GFP+ and pOBCol2.3GFP, where GFP is expressed only in osteoblasts/osteocytes. In both models, transplanted oim/oim mice demonstrated GFP+ HSC-derived osteoblasts/osteocytes in bones. These studies unequivocally establish that HSCs differentiate into osteoblasts/osteocytes, and HSC transplantation can provide a new translational approach for OI.

Transcriptomic alterations underline aging of osteogenic bone marrow stromal cells.

BACKGROUNDMultipotent bone marrow stromal cells (BMSCs) are adult stem cells that form functional osteoblasts and play a critical role in bone remodeling. During aging, an increase in bone loss and reduction in structural integrity lead to osteoporosis and result in an increased risk of fracture. We examined age-dependent histological changes in murine vertebrae and uncovered that bone loss begins as early as the age of 1 mo. AIMTo identify the functional alterations and transcriptomic dynamics of BMSCs during early bone loss. METHODSWe collected BMSCs from mice at early to middle ages and compared their self-renewal and differentiation potential. Subsequently, we obtained the transcriptomic profiles of BMSCs at 1 mo, 3 mo, and 7 mo. RESULTSThe colony-forming and osteogenic commitment capacity showed a comparable finding that decreased at the age of 1 mo. The transcriptomic analysis showed the enrichment of osteoblastic regulation genes at 1 mo and loss of osteogenic features at 3 mo. The BMSCs at 7 mo showed enrichment of adipogenic and DNA repair features. Moreover, we demonstrated that the WNT and MAPK signaling pathways were upregulated at 1 mo, followed by increased pro-inflammatory and apoptotic features. CONCLUSIONOur study uncovered the cellular and molecular dynamics of bone aging in mice and demonstrated the contribution of BMSCs to the early stage of age-related bone loss.

The Potential of a Tailored Biomimetic Hydrogel for In Vitro Cell Culture Applications: Characterization and Biocompatibility

In this study, the Pluronic F127 with modified tripeptide Gly-Arg-Gly-Asp copolymer (hereafter defined as 3BE) hydrogel was evaluated in terms of its biocompatibility potentials. The fibroblasts (Swiss 3T3 cell line) and human hair follicles-derived mesenchymal stem cells (HFMSCs) were cultured in different concentrations of the 3BE hydrogel (0%, 0.05%, 0.1%, 0.25%, and 0.5%, respectively). The cell morphology and differentiation potential of HFMSCs were observed through optical microscopy, and the cell viability was investigated via Live/Dead Kit and Cell Counting Kit-8 assay. Analytical results showed that HFMSC can differentiate into adipogenic, chondrogenic, and osteogenic lineages. The HFMSC and Swiss 3T3 cells would properly assemble into a spherical shape as cultured with the 3BE hydrogel. Most importantly, cell viability could be maintained above 70%. The formation of spheroid structures of cells within this hydrogel is predicted to promote cell differentiation potentials of HFMSC that benefit in generating functional adipocytes, chondrocytes, and osteoblasts. Therefore, these findings demonstrate that the 3BE hydrogel has great potential as a three-dimensional cell culture scaffold for tissue engineering applications.

Abstract PO-035: A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment

Abstract Traditional in-vitro models of solid bone tumors used in basic and preclinical research are unable to faithfully recapitulate human physiology. Specifically, monolayer cultures of osteosarcoma fail to recapitulate various features of 3D tumors, and they lack the native bone milieu, where tumor growth, metastasis, and response to therapy are critically dependent on cancer cell interactions with the bone matrix, supporting cells, and secreted regulatory factors. Based on our previous success in developing a biomimetic tissue model of Ewing sarcoma, we extended our approach towards bioengineering a novel human 3D tumor model of osteosarcoma within a bone microenvironment that can overcome some of these limitations. Briefly, osteosarcoma cell lines were used to generate tumor-like aggregates which were subsequently introduced into, and cultured within, engineered bone tissue scaffolds. Our bioengineered osteosarcoma model was able to recapitulate several aspects of native tumors and their microenvironment missing in monolayer cultures. Significantly higher expression of cancer-related genes (as seen in patients), re-activation of the hypoxic and glycolytic tumor phenotype, re-activation of hypoxia-mediated expression of vascular endothelial growth factor VEGF-α (a critical component of tumor angiogenesis), induction of vasculogenic mimicry, more physiologically relevant growth rates, and co-culture with adjacent functional osteoblasts within a demineralized native bone scaffold were all achieved. Additionally, we were able to capture a degree of both intra-tumor heterogeneity, with the presence of a necrotic core and variability in proliferation across the tumor aggregates, as well as inter-tumor heterogeneity, with notable differences observed between our models generated from metastatic and non-metastatic osteosarcoma cell lines. Finally, the osteosarcoma cell lines were transduced with a GFP-luciferase reporter, allowing us to visualize the engineered tumors in situ within the bone scaffolds, and to evaluate tumor cell responses to various canonical and experimental drug treatments. Notably, unlike monolayers, our bioengineered osteosarcoma model could be cultured stably for several weeks. This allowed us to apply a clinical treatment regimen cycle of linsitinib, an experimental drug in an ongoing phase II clinical trial for Ewing sarcoma, in also being effective at preventing the growth of non-metastatic osteosarcoma bioengineered tumors. Overall, we were able to successfully generate a new, more native-like 3D bioengineered model of osteosarcoma with significant advantages over traditional in-vitro culture that has the potential to explore novel questions regarding tumor biology, the tumor microenvironment, and translational applications. Citation Format: Alan Chramiec, Alessandro Marturano-Kruik, Keith Yeager, Luke Hao, Roberta Lock, Gordana Vunjak-Novakovic. A novel bioengineered model of osteosarcoma recapitulates critical tumor phenotypes within a native bone microenvironment [abstract]. In: Proceedings of the AACR Virtual Special Conference on Tumor Heterogeneity: From Single Cells to Clinical Impact; 2020 Sep 17-18. Philadelphia (PA): AACR; Cancer Res 2020;80(21 Suppl):Abstract nr PO-035.

Direct Conversion of Human Fibroblasts into Osteoblasts Triggered by Histone Deacetylase Inhibitor Valproic Acid

The generation of functional osteoblasts from human somatic cells could provide an alternative means of regenerative therapy for bone disorders such as osteoporosis. In this study, we demonstrated the direct phenotypic conversion of human dermal fibroblasts (HDFs) into osteoblasts by culturing them in osteogenic medium supplemented with valproic acid (VPA), a histone deacetylase (HDAC) inhibitor. HDFs cultured with the VPA in osteogenic medium exhibited expression of alkaline phosphatase and deposition of mineralized calcium matrices, which are phenotypical characteristics of functional osteoblasts. They also expressed osteoblast-specific genes such as alkaline phosphatase, osteopontin, and bone sialoprotein, which demonstrated their direct conversion into osteoblasts. In addition, co-treatment with VPA and a specific inhibitor for activin-like kinase 5 (ALK5i II) had a synergistic effect on direct conversion. It is considered that the inductive effect of VPA on the conversion into osteoblast-lineage is due to the opening of the nucleosome structure by HDAC inhibitor, which facilitates chromatin remodeling and cellular reprogramming. Our findings provide a novel insight into the direct conversion of human somatic cells into transgene-free osteoblasts with small chemical compounds, thus making bone regeneration using cellular reprogramming strategy more clinically feasible.

Not just for patterning anymore: Hox genes function in skeletal stem cells throughout life

Hox genes play fundamental and highly conserved roles in vertebrates patterning of the axial and limb skeleton during embryonic development. We have shown that Hox genes continue to be regionally‐expressed through postnatal development and adult life. Hox‐expressing cells are observed only within a subset of the stromal cells defined as progenitor‐enriched mesenchymal stem/stromal cells. Using a Hoxa11‐CreERT2 allele, we show that Hox‐expressing cells lineage label into chondrocytes, osteoblasts, osteocytes and adipose cells at all stages of life, during growth, homeostasis and in response to injury. Further, lineage labeling from any stage, including during embryogenesis, results in the permanent labeling of a self‐renewing skeletal stem cell population, confirming that Hox‐expressing cells are bona fide skeletal stem cells throughout the life of the animal. However, whether Hox genes continue to function after embryonic stages has never been demonstrated. Embryonic patterning roles and perinatal lethality has prevented this question from being addressed. Using our newly generated Hoxd11 conditional allele and temporally restricted deletion only at adult stages, we show that Hox genes continue to play critical roles in skeletal maintenance and repair throughout life. Loss of Hox function leads to dramatic and progressive perturbations in skeletal integrity with loss of collagen organization, and defective osteocyte and osteoblast formation. Loss of Hox function does not lead to loss of the skeletal stem cell population, but rather an inability of early‐stage progenitors to properly differentiate into functional chondrocytes and osteoblasts. How Hox transcription factors direct these differentiation processes throughout the life of the animal is the focus of current investigations.Support or Funding InformationNIH NIAMS R01 AR 061402 and R61 AR073523Hox Skeletal LineageFigure 1

Reprogramming of human fibroblasts into osteoblasts by insulin-like growth factor-binding protein 7.

The induced pluripotent stem cell (iPSC) is a promising cell source for tissue regeneration. However, the therapeutic value of iPSC technology is limited due to the complexity of induction protocols and potential risks of teratoma formation. A trans‐differentiation approach employing natural factors may allow better control over reprogramming and improved safety. We report here a novel approach to drive trans‐differentiation of human fibroblasts into functional osteoblasts using insulin‐like growth factor binding protein 7 (IGFBP7). We initially determined that media conditioned by human osteoblasts can induce reprogramming of human fibroblasts to functional osteoblasts. Proteomic analysis identified IGFBP7 as being significantly elevated in media conditioned with osteoblasts compared with those with fibroblasts. Recombinant IGFBP7 induced a phenotypic switch from fibroblasts to osteoblasts. The switch was associated with senescence and dependent on autocrine IL‐6 signaling. Our study supports a novel strategy for regenerating bone by using IGFBP7 to trans‐differentiate fibroblasts to osteoblasts.

Direct Reprogramming of Mouse Fibroblasts into Functional Osteoblasts.

Although induced pluripotent stem cells hold promise as a potential source of osteoblasts for skeletal regeneration, the induction of pluripotency followed by directed differentiation into osteoblasts is time consuming and low yield. In contrast, direct lineage reprogramming without an intervening stem/progenitor cell stage would be a more efficient approach to generate osteoblasts. We screened combinations of osteogenic transcription factors and identified four factors, Runx2, Osx, Dlx5, and ATF4, that rapidly and efficiently reprogram mouse fibroblasts derived from 2.3 kb type I collagen promoter-driven green fluorescent protein (Col2.3GFP) transgenic mice into induced osteoblast cells (iOBs). iOBs exhibit osteoblast morphology, form mineralized nodules, and express Col2.3GFP and gene markers of osteoblast differentiation. The global transcriptome profiles validated that iOBs resemble primary osteoblasts. Genomewide DNA methylation analysis demonstrates that within differentially methylated loci, the methylation status of iOBs more closely resembles primary osteoblasts than mouse fibroblasts. We further demonstrate that Col2.3GFP+ iOBs have transcriptome profiles similar to GFP+ cells harvested from Col2.3GFP mouse bone chips. Functionally, Col2.3GFP+ iOBs form mineralized bone structures after subcutaneous implantation in immunodeficient mice and contribute to bone healing in a tibia bone fracture model. These findings provide an approach to derive and study osteoblasts for skeletal regeneration. © 2019 American Society for Bone and Mineral Research.

The effect of platelet lysate in culture of PDLSCs: an in vitro comparative study.

BackgroundCellular therapy clinical applications require large-scale production of stem cells. Therefore, abundance, ease of isolation, and proliferative potential are the most important factors in choosing the appropriate source of cells for transplantation studies. Multipotent stem cells obtained from periodontal ligament (PDL) can be used in periodontal tissue regeneration. In this study, we aimed to evaluate and compare the characteristics of periodontal ligament stem cells (PDLSCs), extracted by either enzymatic digestion or explant methods, and expanded using two different serum types: fetal bovine serum (FBS) and xeno-free platelet lysate (PL).MethodsExpanded PDLSCs were assessed for their proliferation capacity, surface markers expression, colony formation, differentiation potential and ability to self-renewal. Most importantly, PDLSCs were evaluated for their ability to produce osteoblasts in vitro.ResultsPDLSCs isolated by explant method and expanded in PL serve as a promising source of stem cells for osteoblasts regeneration. These cells showed higher proliferation capacity, they retained their stemness characteristics throughout the passages and they revealed an increase in the expression level of osteogenic markers, without showing any karyotypic abnormalities after cell expansion.ConclusionsPDLSCs produced using explant extraction method and expanded in cell culture media supplemented with PL provide an excellent source of xeno-free cells for the generation of functional osteoblasts.

Eldecalcitol effects on osteoblastic differentiation and function in the presence or absence of osteoclastic bone resorption.

Eldecalcitol (ELD) is an active vitamin D3 analog, possesses anti-resorption properties and is an approved therapeutic drug for the treatment of osteoporosis in Japan. However, the effect of ELD on osteoblasts in a distinct cell microenvironment, including in the presence or absence of osteoclastic bone resorption, is undetermined. In the current study, the effect of bone resorption supernatant on the ELD-mediated regulation of viability, differentiation and receptor activator of ΝF-κB ligand/osteoprotegerin (RANKL/OPG) expression was assessed in MC3T3-E1 pre-osteoblast cells. The murine macrophage-like cell line RAW 264.7 was induced to differentiate into functional osteoblasts. Bone resorption supernatant was prepared by culturing osteoclast with a bovine cortical bone specimen. Mouse MC3T3-E1 cells were subsequently treated with ELD combined with differentiated osteoclast cell culture (OCS) or osteoclast bone resorption model supernatants. Cell counting kit-8, alkaline phosphatase (ALP) activity, reverse transcription-quantitative (RT-q) PCR and western blot analysis were used to assess cell viability, osteogenic activity and RANKL and OPG expression in MC3T3-E1 cells. The OCS and OCS + ELD treatment exhibited significantly increased MC3T3-E1 cell viability when compared with the control group. However, ELD, bone resorption culture supernatant (BRS) and ELD + BRS treatments significantly decreased MC3T3-E1 cell viability. The results of ALP activity analysis, RT-qPCR and western blot analysis demonstrated that ELD treatment and OCS decreased the osteogenic markers (ALP and RUNX2), however, BRS increased them. All treatments enhanced the expression of RANKL and RANKL/OPG ratio. The results of the current study revealed that ELD inhibits osteoblastic differentiation in vitro. However, in the presence of BRS, which mimics the local bone microenvironment in vivo, the net effect on osteogenesis was positive. Furthermore, osteoclasts and bone matrix-derived factors increased the RANKL/OPG ratio, thereby potentiating osteoclastic activity.

A preliminary study on the mechanism of skeletal abnormalities in Turner syndrome using inducing pluripotent stem cells (iPS)- based disease models.

Osteoporosis represent one of main characteristics of Turner syndrome (TS), a rare diseases caused by aberrant deletion of X chromosomes, however, the underlying pathological mechanism remains unknown yet. In this study, we used pluripotent stem cells (iPSCs) derived from a Turner syndrome patient and a health control to induce functional osteoblasts and osteoclasts, in order to compare their difference in these two differentiation. We successfully produced functional osteoblasts and osteoclasts from iPSCs through embryoid bodies (EBs) and mesoderm stages, as demonstrated obvious mineralized nodules and multi-nuclear giant cells with positive tartrate-resistant acid phosphatase (TRAP) staining, and significant up-regulated differentiation marker genes. Interestingly, we found that there was no significant difference in phenotype and marker genes expression between osteoblasts from Turner syndrome and healthy control iPSCs. In contrast, Turner syndrome showed increased osteoclastogenesis compared to the healthy control indicating higher frequency of multi-nuclear TRAP staining cells and elevated osteoclast marker genes TRAP, MMP9, CA2, OSCAR. Therefore, our results suggest that the low bone density of Turner syndrome patients may be caused by aberrant osteoclast differentiation, and further investigation towards osteoclast function under Turner syndrome is deserved.