The Notch pathway regulates the bone gain induced by PTH anabolic signaling.

Parathyroid hormone (PTH) signaling downstream of the PTH 1 receptor (Pth1r) results in both bone anabolic and catabolic actions by mechanisms not yet fully understood. In this study, we show that Pth1r signaling upregulates the expression of several components of the Notch pathway and that Notch signals contribute to the catabolic actions of PTH in bone. We found that constitutive genetic activation of PTH receptor signaling in osteocytes (caPth1rOt ) or treatment with PTH daily increased the expression of several Notch ligands/receptors in bone. In contrast, sustained elevation of endogenous PTH did not change Notch components expression. Deletion of the PTH receptor or sclerostin overexpression in osteocytes abolished Notch increases by PTH. Further, deleting the canonical Notch transcription factor Rbpjk in osteocytes decreased bone mass and increased resorption and Rankl expression in caPth1rOt mice. Moreover, pharmacological bone-targeted Notch inhibition potentiated the bone mass gain induced by intermittent PTH by reducing bone resorption and preserving bone formation. Thus, Notch activation lies downstream of anabolic signaling driven by PTH actions in osteocytes, and Notch pharmacological inhibition maximizes the bone anabolic effects of PTH.

Rac1 activation controls nuclear localization of beta-catenin during canonical Wnt signaling.

Canonical Wnt signaling critically regulates cell fate and proliferation in development and disease. Nuclear localization of beta-catenin is indispensable for canonical Wnt signaling; however, the mechanisms governing beta-catenin nuclear localization are not well understood. Here we demonstrate that nuclear accumulation of beta-catenin in response to Wnt requires Rac1 activation. The role of Rac1 depends on phosphorylation of beta-catenin at Ser191 and Ser605, which is mediated by JNK2 kinase. Mutations of these residues significantly affect Wnt-induced beta-catenin nuclear accumulation. Genetic ablation of Rac1 in the mouse embryonic limb bud ectoderm disrupts canonical Wnt signaling and phenocopies deletion of beta-catenin in causing severe truncations of the limb. Finally, Rac1 interacts genetically with beta-catenin and Dkk1 in controlling limb outgrowth. Together these results uncover Rac1 activation and subsequent beta-catenin phosphorylation as a hitherto uncharacterized mechanism controlling canonical Wnt signaling and may provide additional targets for therapeutic intervention of this important pathway.

CHIR99021-Treated Osteocytes with Wnt Activation in 3D-Printed Module Form an Osteogenic Microenvironment for Enhanced Osteogenesis and Vasculogenesis.

Finding a bone implant that has high bioactivity that can safely drive stem cell differentiation and simulate a real in vivo microenvironment is a challenge for bone tissue engineering. Osteocytes significantly regulate bone cell fate, and Wnt-activated osteocytes can reversely regulate bone formation by regulating bone anabolism, which may improve the biological activity of bone implants. To achieve a safe application, we used the Wnt agonist CHIR99021 (C91) to treat MLO-Y4 for 24 h, in a co-culture with ST2 for 3 days after withdrawal. We found that the expression of Runx2 and Osx increased, promoted osteogenic differentiation, and inhibited adipogenic differentiation in the ST2 cells, and these effects were eliminated by the triptonide. Therefore, we hypothesized that C91-treated osteocytes form an osteogenic microenvironment (COOME). Subsequently, we constructed a bio-instructive 3D printing system to verify the function of COOME in 3D modules that mimic the in vivo environment. Within PCI3D, COOME increased the survival and proliferation rates to as high as 92% after 7 days and promoted ST2 cell differentiation and mineralization. Simultaneously, we found that the COOME-conditioned medium also had the same effects. Therefore, COOME promotes ST2 cell osteogenic differentiation both directly and indirectly. It also promotes HUVEC migration and tube formation, which can be explained by the high expression of Vegf. Altogether, these results indicate that COOME, combined with our independently developed 3D printing system, can overcome the poor cell survival and bioactivity of orthopedic implants and provide a new method for clinical bone defect repair.

PPP3R1 Promotes MSCs Senescence by Inducing Plasma Membrane Depolarization and Increasing Ca2+ Influx.

Aging of mesenchymal stem cells(MSCs) has been widely reported to be strongly associated with aging-related diseases, including osteoporosis (OP). In particular, the beneficial functions of mesenchymal stem cells decline with age, limiting their therapeutic efficacy in age-related bone loss diseases. Therefore, how to improve mesenchymal stem cell aging to treat age-related bone loss is the current research focus. However, the underlying mechanism remains unclear. In this study, protein phosphatase 3, regulatory subunit B, alpha isoform, calcineurin B, type I (PPP3R1) was found to accelerate the senescence of mesenchymal stem cells, resulting in reduced osteogenic differentiation and enhanced adipogenic differentiation in vitro. Mechanistically, PPP3R1 induces changes in membrane potential to promote cellular senescence by polarizing to depolarizing, increasing Ca2+ influx and activating downstream NFAT/ATF3/p53 signaling. In conclusion, the results identify a novel pathway of mesenchymal stem cell aging that may lead to novel therapeutic approaches for age-related bone loss.

Functionalized 3D-Printed ST2/Gelatin Methacryloyl/Polcaprolactone Scaffolds for Enhancing Bone Regeneration with Vascularization.

Growth factors were often used to improve the bioactivity of biomaterials in order to fabricate biofunctionalized bone grafts for bone defect repair. However, supraphysiological concentrations of growth factors for improving bioactivity could lead to serious side effects, such as ectopic bone formation, radiculitis, swelling of soft tissue in the neck, etc. Therefore, safely and effectively applying growth factors in bone repair biomaterials comes to be an urgent problem that needs to be addressed. In this study, an appropriate concentration (50 ng/mL) of Wnt3a was used to pretreat the 3D-bioprinting gelatin methacryloyl(GelMA)/polycaprolactone(PCL) scaffold loaded with bone marrow stromal cell line ST2 for 24 h. This pretreatment promoted the cell proliferation, osteogenic differentiation, and mineralization of ST2 in the scaffold in vitro, and enhanced angiogenesis and osteogenesis after being implanted in critical-sized mouse calvarial defects. On the contrary, the inhibition of Wnt/ß-catenin signaling in ST2 cells reduced the bone repair effect of this scaffold. These results suggested that ST2/GelMA/PCL scaffolds pretreated with an appropriate concentration of Wnt3a in culture medium could effectively enhance the osteogenic and angiogenic activity of bone repair biomaterials both in vitro and in vivo. Moreover, it would avoid the side effects caused by the supraphysiological concentrations of growth factors. This functionalized scaffold with osteogenic and angiogenic activity might be used as an outstanding bone substitute for bone regeneration and repair.

Activating Wnt/ß-Catenin Signaling in Osteocytes Promotes Osteogenic Differentiation of BMSCs through BMP-7.

Bone formation is critically needed in orthopedic clinical practice. We found that, bone morphogenetic protein-7 (BMP-7) gene expression was significantly increased in fractured mice, which activates canonical Wnt signaling exclusively in osteocytes. Wnt and BMP signaling appear to exhibit synergistic or antagonistic effects in different kinds of cells. However, the communication between Wnt/ß-catenin signaling and BMP signaling in osteocytes is almost unknown. Our study verified in vitro that BMP-7 expression was significantly increased when Wnt signaling was activated in osteocytes. Next, BMP-7 in osteocytes was overexpressed using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was enhanced, when cocultured with osteocytes. On the contrary, BMP-7 in osteocytes was silenced using an adenovirus, the osteogenesis of bone marrow stem cells (BMSCs) was weakened. In addition, the osteogenesis of BMSCs was no longer promoted by Wnt-activated osteocytes when BMP-7 was silenced. Therefore, the results showed that BMP-7 mediated the anabolic actions of Wnt/ß-catenin signaling in osteocytes. Our study provides new evidence for the clinical application of BMP-7-overexpressed osteocytes.

Osteocytes mediate the anabolic actions of canonical Wnt/ß-catenin signaling in bone.

Osteocytes, >90% of the cells in bone, lie embedded within the mineralized matrix and coordinate osteoclast and osteoblast activity on bone surfaces by mechanisms still unclear. Bone anabolic stimuli activate Wnt signaling, and human mutations of components along this pathway underscore its crucial role in bone accrual and maintenance. However, the cell responsible for orchestrating Wnt anabolic actions has remained elusive. We show herein that activation of canonical Wnt signaling exclusively in osteocytes [dominant active (da)ßcat(Ot) mice] induces bone anabolism and triggers Notch signaling without affecting survival. These features contrast with those of mice expressing the same daß-catenin in osteoblasts, which exhibit decreased resorption and perinatal death from leukemia. daßcat(Ot) mice exhibit increased bone mineral density in the axial and appendicular skeleton, and marked increase in bone volume in cancellous/trabecular and cortical compartments compared with littermate controls. daßcat(Ot) mice display increased resorption and formation markers, high number of osteoclasts and osteoblasts in cancellous and cortical bone, increased bone matrix production, and markedly elevated periosteal bone formation rate. Wnt and Notch signaling target genes, osteoblast and osteocyte markers, and proosteoclastogenic and antiosteoclastogenic cytokines are elevated in bones of daßcat(Ot) mice. Further, the increase in RANKL depends on Sost/sclerostin. Thus, activation of osteocytic ß-catenin signaling increases both osteoclasts and osteoblasts, leading to bone gain, and is sufficient to activate the Notch pathway. These findings demonstrate disparate outcomes of ß-catenin activation in osteocytes versus osteoblasts and identify osteocytes as central target cells of the anabolic actions of canonical Wnt/ß-catenin signaling in bone.

WNT7B promotes bone formation in part through mTORC1.

WNT signaling has been implicated in both embryonic and postnatal bone formation. However, the pertinent WNT ligands and their downstream signaling mechanisms are not well understood. To investigate the osteogenic capacity of WNT7B and WNT5A, both normally expressed in the developing bone, we engineered mouse strains to express either protein in a Cre-dependent manner. Targeted induction of WNT7B, but not WNT5A, in the osteoblast lineage dramatically enhanced bone mass due to increased osteoblast number and activity; this phenotype began in the late-stage embryo and intensified postnatally. Similarly, postnatal induction of WNT7B in Runx2-lineage cells greatly stimulated bone formation. WNT7B activated mTORC1 through PI3K-AKT signaling. Genetic disruption of mTORC1 signaling by deleting Raptor in the osteoblast lineage alleviated the WNT7B-induced high-bone-mass phenotype. Thus, WNT7B promotes bone formation in part through mTORC1 activation.

BMP-Smad4 signaling is required for precartilaginous mesenchymal condensation independent of Sox9 in the mouse.

Bone morphogenetic proteins (BMPs) regulate multiple aspects of skeletal development in vertebrates. Although exogenously applied BMPs can induce chondrogenesis de novo, the role and mechanism of physiologic BMP signaling during precartilaginous mesenchymal condensation is not well understood. By deleting the type I BMP receptors or the transcription factor Smad4 in the limb bud mesenchyme, we find that loss of BMP-Smad signaling abolishes skeletal development due to a failure in mesenchymal condensation. In the absence of Smad4, expression of Sox9, an essential transcription factor for chondrogenesis, initiates normally in the proximal mesenchyme of the limb bud, but fails to maintain its level or expand to the more distal territory at the later stages. However, forced-expression of Sox9 does not restore cartilage formation in the Smad4-deficeint embryo. In vitro micromass cultures show that the Smad4-deficient cells fail to condense in a cell-autonomous manner, even though they express several cell adhesion molecules either normally or even at a higher level. Thus, BMP-Smad signaling critically controls mesenchymal condensation to initiate skeletal development likely through a Sox9-independent mechanism.

Physiological notch signaling maintains bone homeostasis via RBPjk and Hey upstream of NFATc1.

Notch signaling between neighboring cells controls many cell fate decisions in metazoans both during embryogenesis and in postnatal life. Previously, we uncovered a critical role for physiological Notch signaling in suppressing osteoblast differentiation in vivo. However, the contribution of individual Notch receptors and the downstream signaling mechanism have not been elucidated. Here we report that removal of Notch2, but not Notch1, from the embryonic limb mesenchyme markedly increased trabecular bone mass in adolescent mice. Deletion of the transcription factor RBPjk, a mediator of all canonical Notch signaling, in the mesenchymal progenitors but not the more mature osteoblast-lineage cells, caused a dramatic high-bone-mass phenotype characterized by increased osteoblast numbers, diminished bone marrow mesenchymal progenitor pool, and rapid age-dependent bone loss. Moreover, mice deficient in Hey1 and HeyL, two target genes of Notch-RBPjk signaling, exhibited high bone mass. Interestingly, Hey1 bound to and suppressed the NFATc1 promoter, and RBPjk deletion increased NFATc1 expression in bone. Finally, pharmacological inhibition of NFAT alleviated the high-bone-mass phenotype caused by RBPjk deletion. Thus, Notch-RBPjk signaling functions in part through Hey1-mediated inhibition of NFATc1 to suppress osteoblastogenesis, contributing to bone homeostasis in vivo.

The Osteocyte with SB216763-Activated Canonical Wnt Signaling Constructs a Multifunctional 4D Intelligent Osteogenic Module.

The enhancement of bioactivity in materials has become an important focus within the field of bone tissue engineering. Four-dimensional intelligent osteogenic module, an innovative fusion of 3D printing with the time axis, shows immense potential in augmenting the bioactivity of these materials, thereby facilitating autologous bone regeneration efficiently. This study focuses on novel bone repair materials, particularly bioactive scaffolds with a developmental osteogenic microenvironment prepared through 3D bioprinting technology. This research mainly creates a developmental osteogenic microenvironment named "DOME". This is primed by the application of a small amount of the small molecule drug SB216763, which activates canonical Wnt signaling in osteocytes, promoting osteogenesis and mineralization nodule formation in bone marrow stromal cells and inhibiting the formation of adipocytes. Moreover, DOME enhances endothelial cell migration and angiogenesis, which is integral to bone repair. More importantly, the DOME-PCI3D system, a 4D intelligent osteogenic module constructed through 3D bioprinting, stably supports cell growth (91.2% survival rate after 7 days) and significantly increases the expression of osteogenic transcription factors in bone marrow stromal cells and induces osteogenic differentiation and mineralization for 28 days. This study presents a novel approach for bone repair, employing 3D bioprinting to create a multifunctional 4D intelligent osteogenic module. This innovative method not only resolves challenges related to shape-matching and biological activity but also demonstrates the vast potential for applications in bone repair.

Establishing stable and highly osteogenic hiPSC-derived MSCs for 3D-printed bone graft through microenvironment modulation by CHIR99021-treated osteocytes.

Human induced pluripotent stem cell (hiPSC)-derived mesenchymal stem cells (iMSCs) are ideal candidates for the production of standardised and scalable bioengineered bone grafts. However, stable induction and osteogenic differentiation of iMSCs pose challenges in the industry. We developed a precise differentiation method to produce homogeneous and fully differentiated iMSCs. In this study, we established a standardised system to prepare iMSCs with increased osteogenic potential and improved bioactivity by introducing a CHIR99021 (C91)-treated osteogenic microenvironment (COOME). COOME enhances the osteogenic differentiation and mineralisation of iMSCs via canonical Wnt signalling. Global transcriptome analysis and co-culturing experiments indicated that COOME increased the pro-angiogenesis/neurogenesis activity of iMSCs. The superior osteogenic differentiation and mineralisation abilities of COOME-treated iMSCs were also confirmed in a Bio3D module generated using a polycaprolactone (PCL) and cell-integrated 3D printing (PCI3D) system, which is the closest model to in vivo research. This COOME-treated iMSCs differentiation system offers a new perspective for generating highly osteogenic, bioactive, and anatomically matched grafts for clinical applications. Statement of significance: Although human induced pluripotent stem cell-derived MSCs (iMSCs) are ideal seed cells for synthetic bone implants, the challenges of stable induction and osteogenic differentiation hinder their clinical application. This study established a standardised system for the scalable preparation of iMSCs with improved osteogenic potential by combining our precise iMSC differentiation method with the CHIR99021 (C91)-treated osteocyte osteogenic microenvironment (COOME) through the activation of canonical Wnt signalling. Moreover, COOME upregulated the pro-angiogenic and pro-neurogenic capacities of iMSCs, which are crucial for the integration of implanted bone grafts. The superior osteogenic ability of COOME-treated iMSCs was confirmed in Bio3D modules generated using PCL and cell-integrated 3D printing systems, highlighting their functional potential in vivo. This study contributes to tissue engineering by providing insights into the functional differentiation of iMSCs for bone regeneration.

Indian hedgehog requires additional effectors besides Runx2 to induce osteoblast differentiation.

Indian hedgehog (Ihh) is indispensable for osteoblast differentiation during embryonic development of the endochondral skeleton. In the absence of Ihh, cells of the osteoblast lineage fail to activate the expression of Runx2, a transcription factor integral to osteoblast differentiation. However, it is hitherto unclear whether the lack of Runx2 expression is solely responsible for the failure of osteoblast formation in Ihh-null embryos. Here, by creating a mouse allele that expresses Runx2 in a Cre-dependent manner, we show that force-expression of Runx2 in the skeletogenic cells restores bone formation in the Runx2-null, but not in the Ihh-null embryo. Thus, the mechanism through which Ihh induces osteoblast differentiation requires other effectors in addition to Runx2.

Sustained release of a highly specific GSK3ß inhibitor SB216763 in the PCL scaffold creates an osteogenic niche for osteogenesis, anti-adipogenesis, and potential angiogenesis.

The safe and effective use of Wnt signaling is a hot topic in developing osteogenic drugs. SB216763 (S33) is a widely used highly specific GSK3ß inhibitor. Here, we show that S33 initiates canonical Wnt signaling by inhibiting GSK3ß activity in the bone marrow stromal cell line ST2 and increases osteoblast marker alkaline phosphatase activity, osteoblast marker gene expression including Alpl, Col1α1, and Runx2, promoting osteogenic differentiation and mineralization of ST2 cells. In addition, S33 suppressed the expression of adipogenic transcription factors Pparg and Cebpa in ST2 cells to suppress adipogenesis. ICRT-14, a specific transcriptional inhibitor of Wnt signaling, reversed the effects of S33 on the differentiation of ST2 cells. S33 also increased the expression of osteoclast cytokines RANKL and Opg but decreased the RANKL/Opg ratio and had the potential to inhibit osteoclast differentiation. In addition, we printed the PSCI3D (polycaprolactone, S33, cell-integrated 3D) scaffolds using a newly established integrated 3D printing system for hard materials and cells. S33 sustained release in the hydrogel of the scaffold with 25.4% release on day 1% and 81.7% release over 7 days. Cells in the scaffolds had good cell viability. The ratio of live/dead cells remained above 94% for 7 days, while the cells in the scaffolds proliferated linearly, and the proliferative activity of the PSCI3D scaffold group increased 1.4-fold and 1.7-fold on days 4 and 7, respectively. Similarly, in PSCI3D scaffolds, osteogenic differentiation of st2 cells was increased. The alkaline phosphatase activity increased 1.4- and 4.0-fold on days 7 and 14, respectively, and mineralization increased 1.7-fold at 21 days. In addition, PSCI3D conditioned medium promoted migration and tubulogenesis of HUVECs, and S33 upregulated the expression of Vegfa, a key factor in angiogenesis. In conclusion, our study suggests that S33 functions in osteogenesis, anti-adipogenesis, and potential inhibition of osteoclast differentiation. And the sustained release of S33 in PSCI3D scaffolds creates a safe osteogenic niche, which promotes cell proliferation, osteogenesis, and angiogenesis and has application prospects.

A novel decellularized matrix of Wnt signaling-activated osteocytes accelerates the repair of critical-sized parietal bone defects with osteoclastogenesis, angiogenesis, and neurogenesis.

Cell source is the key to decellularized matrix (DM) strategy. This study compared 3 cell types, osteocytes with/without dominant active Wnt/ß-catenin signaling (daCO and WTO) and bone marrow stromal cells (BMSCs) for their DMs in bone repair. Decellularization removes all organelles and >95% DNA, and retained >74% collagen and >71% GAG, maintains the integrity of cell basement membrane with dense boundaries showing oval and honeycomb structure in osteocytic DM and smooth but irregular shape in the BMSC-DM. DM produced higher cell survival rate (90%) and higher proliferative activity. In vitro, daCO-DM induces more and longer stress fibers in BMSCs, conducive to cell adhesion, spreading, and osteogenic differentiation. 8-wk after implantation of the critical-sized parietal bone defect model, daCO-DM formed tight structures, composed of a large number of densely-arranged type-I collagen under polarized light microscope, which is similar to and integrated with host bone. BV/TV (>54%) was 1.5, 2.9, and 3.5 times of WTO-DM, BMSC-DM, and none-DM groups, and N.Ob/T.Ar (3.2 × 102/mm2) was 1.7, 2.9, and 3.3 times. At 4-wk, daCO-DM induced osteoclastogenesis, 2.3 times higher than WTO-DM; but BMSC-DM or none-DM didn't. daCO-DM increased the expression of RANKL and MCSF, Vegfa and Angpt1, and Ngf in BMSCs, which contributes to osteoclastogenesis, angiogenesis, and neurogenesis, respectively. daCO-DM promoted H-type vessel formation and nerve markers ß3-tubulin and NeuN expression. Conclusion: daCO-DM produces metabolic and neurovascularized organoid bone to accelerate the repair of bone defects. These features are expected to achieve the effect of autologous bone transplantation, suitable for transformation application.

Noncanonical Wnt signaling through G protein-linked PKCdelta activation promotes bone formation.

Wnt signaling regulates a variety of developmental processes in animals. Although the beta-catenin-dependent (canonical) pathway is known to control cell fate, a similar role for noncanonical Wnt signaling has not been established in mammals. Moreover, the intracellular cascades for noncanonical Wnt signaling remain to be elucidated. Here, we delineate a pathway in which Wnt3a signals through the Galpha(q/11) subunits of G proteins to activate phosphatidylinositol signaling and PKCdelta in the murine ST2 cells. Galpha(q/11)-PKCdelta signaling is required for Wnt3a-induced osteoblastogenesis in these cells, and PKCdelta homozygous mutant mice exhibit a deficit in embryonic bone formation. Furthermore, Wnt7b, expressed by osteogenic cells in vivo, induces osteoblast differentiation in vitro via the PKCdelta-mediated pathway; ablation of Wnt7b in skeletal progenitors results in less bone in the mouse embryo. Together, these results reveal a Wnt-dependent osteogenic mechanism, and they provide a potential target pathway for designing therapeutics to promote bone formation.

[Activation of Notch signaling in bone marrow stromal cells promotes osteogenesis in mice in vitro].

Objective To investigate the osteogenic differentiation of bone marrow stromal cells (BMSC) with Notch signaling activation in vitro. Methods The BMSC derived from Notch1-NICDflox/flox mice were infected with recombinant adenovirus expressing Cre or GFP respectively, and designated as Ad-Cre group and Ad-GFP group. The expression of Notch1 was evaluated by Western blot analysis. Alkaline phosphatase (ALP) activity was determined by ALP staining and biochemical quantification, and the calcium deposition was analyzed with Alizarin red S staining. Real-time fluorescence quantitative PCR (qPCR) was used to quantify the expression of Notch target genes(Hes1, Hey1, Hey2, HeyL), osteogenic differentiation-related genes(ALP, RUNX2, osterix, osteocalcin), and angiogenesis factors(VEGF, HIF-1α). Results Notch signaling in BMSC of Notch1-NICDflox/flox mice was activated successfully by Ad-Cre, as was evidenced by the significantly elevated expression of Notch1 and Notch target genes. Compared with the Ad-GFP group, ALP activity and the late calcium deposition were significantly increased upon treatment with Ad-Cre. qPCR results demonstrated that the expression of ALP, RUNX2, osterix, osteocalcin, VEGF, HIF-1α in the Ad-Cre group were significantly upregulated. Conclusion Activation of Notch signaling in BMSC in vitro significantly promotes osteogenic differentiation.

The Osteocyte Stimulated by Wnt Agonist SKL2001 Is a Safe Osteogenic Niche Improving Bioactivities in a Polycaprolactone and Cell Integrated 3D Module.

Finding and constructing an osteogenic microenvironment similar to natural bone tissue has always been a frontier topic in orthopedics. We found that osteocytes are targeting cells controlling bone anabolism produced by PTH (JBMR 2017, PMID: 27704638), and osteocytes with activated Wnt signaling orchestrate bone formation and resorption (PNAS 2015, PMID: 25605937). However, methods for taking advantage of the leading role of osteocytes in bone regeneration remain unexplored. Herein, we found that the osteocytes with SKL2001-activated Wnt signaling could be an osteogenic microenvironment (SOOME) which upregulates the expression of bone transcription factor Runx2 and Bglap and promotes the differentiation of bone marrow stromal cell ST2 into osteoblasts. Interestingly, 60 µM SKL2001 treatment of osteocytic MLO-Y4 for 24 h maintained Wnt signaling activation for three days after removal, which was sufficient to induce osteoblast differentiation. Triptonide, a Wnt inhibitor, could eliminate this differentiation. Moreover, on day 5, the Wnt signaling naturally decreased to the level of the control group, indicating that this method of Wnt-signaling induction is safe to use. We quickly verified in vivo function of SOOME to a good proximation in 3D bioprinted modules composed of reciprocally printed polycaprolactone bundles (for support) and cell bundles (for bioactivity). In the cell bundles, SOOME stably supported the growth and development of ST2 cells, the 7-day survival rate was as high as 91.6%, and proliferation ability increased linearly. Similarly, SOOME greatly promoted ST2 differentiation and mineralization for 28 days. In addition, SOOME upregulated the expression of angiopoietin 1, promoted endothelial cell migration and angiogenesis, and increased node number and total length of tubes and branches. Finally, we found that the function of SOOME could be realized through the paracrine pathway. This study reveals that osteocytes with Wnt signaling activated by SKL2001 are a safe osteogenic microenvironment. Both SOOME itself and its cell-free culture supernatant can improve bioactivity for osteoblast differentiation, with composite scaffolds especially bearing application value.

A Highly Selective GSK-3ß Inhibitor CHIR99021 Promotes Osteogenesis by Activating Canonical and Autophagy-Mediated Wnt Signaling.

The discovery and application of small molecules is one of the practical strategies of safe osteogenic drugs. The small molecule CHIR99021 (C91) is a highly specific, safe, and most effective GSK-3ß Inhibitor. This study found that it efficiently activates the canonical Wnt signaling of bone marrow stromal cell ST2 and promotes osteoblast differentiation and mineralization. C91 increases the production and biochemical activity of osteoblast marker alkaline phosphatase, the expression of osteoblast marker genes Alpl, Bglap, Runx2, and Sp7, and the formation of bone nodules. Triptonide is a transcription inhibitor of Wnt target gene, which diminishes C91-induced osteoblast differentiation in a dose-dependent manner. Meanwhile, C91 also induces autophagy through autophagosome formation and conversion of autophagy biomarker LC-3I into LC-3II. Autophagy inhibitor 3MA partially reduces C91-induced osteoblast differentiation and mineralization; autophagy inducer Rapamycin increases the expression of ß-catenin to promote osteogenic differentiation, but cannot alleviate the inhibition of Triptonide on C91-induced osteogenic differentiation, indicating the crosstalk of canonical Wnt signaling and autophagy regulates C91-induced osteoblast differentiation. Furthermore, in order to simulate the in vivo detection of C91 in osteogenesis process, we made a C91 slow-release hydrogel with our newly established polycaprolactone and cell-integrated 3D printing system (PCCI3D module). The sustained release C91 promotes the differentiation and mineralization of ST2 cells. C91 can also enhance the proliferative activity of ST2 cells. The release rate of C91 from hydrogel gradually decreases within 7 days. During this period, the C91 is released by 83.0% and the cell viability maintained at 96.4%. Therefore, the small molecule Wnt agonist C91 promotes osteogenesis through caonical and autophagy-mediated Wnt signaling pathway with an option for translational application.

Canonical Wnt signaling works downstream of iron overload to prevent ferroptosis from damaging osteoblast differentiation.

Excessive iron has emerged in a large population of patients suffering from degenerative or hematological diseases with a common outcome, osteoporosis. However, its underlying mechanism remains to be clarified in order to formulate effective prevention and intervention against the loss of bone-forming osteoblasts. We show herein that increased intracellular iron by ferric ammonium citrate (FAC) mimicking the so-called non-transferrin bound iron concentrations leads to ferroptosis and impaired osteoblast differentiation. FAC upregulates the expression of Trfr and DMT1 genes to increase iron uptake, accumulating intracellular labile ferrous iron for iron overload status. Then, the excessive ferrous iron generates reactive oxygen species (ROS) and lipid peroxidation products (LPO), causing ferroptosis with its typical mitochondrial morphological changes, such as shrinkaged and condensed membrane with diminution and loss of crista and outer membrane rupture. We further examined that ferroptosis is the main cause responsible for FAC-disrupted osteoblast differentiation, although apoptosis and senescence are concurrently induced as well. Mechanistically, we revealed that iron dose-dependently down-regulates the expression of Wnt target genes and inhibits the transcription of Wnt reporter TopFlash construct, so as to inhibit the canonical Wnt signaling. Wnt agonist, ferroptosis inhibitor, or antioxidant melatonin reverses iron-inhibited canonical Wnt signaling to restore osteoblast differentiation by reducing ROS and LPO production to prevent ferroptosis notably without reducing iron overload. This study proposes a working model against excessive iron-induced osteoporosis: iron chelator deferoxamine or the above three drugs prevent ferroptosis, restore traditional Wnt signaling to maintain osteoblast differentiation no matter whether iron overload is removed or not. Additionally, iron chelator should be used to a suitable extent because iron itself is necessary for osteogenic differentiation.