Osteoblast Differentiation Markers Research Articles

Osteolineage depletion of mitofusin2 enhances cortical bone formation in female mice.

Mitochondria are essential organelles that form highly complex, interconnected dynamic networks inside cells. The GTPase mitofusin 2 (MFN2) is a highly conserved outer mitochondrial membrane protein involved in the regulation of mitochondrial morphology, which can affect various metabolic and signaling functions. The role of mitochondria in bone formation remains unclear. Since MFN2 levels increase during osteoblast (OB) differentiation, we investigated the role of MFN2 in the osteolineage by crossing mice bearing floxed Mfn2 alleles with those bearing Prx-cre to generate cohorts of conditional knock out (cKO) animals. By ex vivo microCT, cKO female mice, but not males, display an increase in cortical thickness at 8, 18, and 30weeks, compared to wild-type (WT) littermate controls. However, the cortical anabolic response to mechanical loading was not different between genotypes. To address how Mfn2 deficiency affects OB differentiation, bone marrow-derived mesenchymal stromal cells (MSCs) from both wild-type and cKO mice were cultured in osteogenic media with different levels of β-glycerophosphate. cKO MSCs show increased mineralization and expression of multiple markers of OB differentiation only at the lower dose. Interestingly, despite showing the expected mitochondrial rounding and fragmentation due to loss of MFN2, cKO MSCs have an increase in oxygen consumption during the first 7days of OB differentiation. Thus, in the early phases of osteogenesis, MFN2 restrains oxygen consumption thereby limiting differentiation and cortical bone accrual during homeostasis in vivo.

Palmul-Tang, a Korean Medicine, Promotes Bone Formation via BMP-2 Pathway in Osteoporosis.

Osteoporosis is a common skeletal disease in post-menopausal women. Palmul-tang, an herbal medicine, has been treated for gynecological disease such as anemia, anorexia, anti-fatigue, unspecified menstruation and female infertility in East Asia. In this study, ameliorative effects of Palmul-tang soft extracts (PMT), a Korean Medicine, on osteoporosis were investigated. Ovariectomized (OVX) osteoporotic ICR mice were intragastrically administrated PMT for 4 weeks. The level of bone mineral density (BMD) was analyzed in bone tissues by dual X-ray absorptiometry. The bone medullary cavity and deposition of collagen were investigated by histological analysis. In addition, the BMP-2 signaling-related molecules, osteoblastic differentiation and formation markers, were determined in femoral tissues. The levels of BMD and bone mineral content were significantly increased in tibia, femurs and LV by treatment of PMT. PMT replenished bone marrow cavity and increased collagen deposition in bone marrow cells of femur. In addition, administration of PMT recovered serum ALP, bALP, osteocalcin and calcium levels in osteoporotic mice. Moreover, PMT treatment up-regulated the expressions of BMP-2, RUNX2 and OSX with its downstream factors, ALP, OPN and BSP-1, in the femoral tissues. Taken together, PMT restored the bone minerals and improvement of bone integrity by bone-forming BMP-2 signaling pathway. These results demonstrate that PMT could be an ameliorative agent for osteoporosis.

The TrkA agonist gambogic amide augments skeletal adaptation to mechanical loading.

The periosteal and endosteal surfaces of mature bone are densely innervated by sensory nerves expressing TrkA, the high-affinity receptor for nerve growth factor (NGF). In previous work, we demonstrated that administration of exogenous NGF significantly increased load-induced bone formation through the activation of Wnt signaling. However, the translational potential of NGF is limited by the induction of substantial mechanical and thermal hyperalgesia in mice and humans. Here, we tested the effect of gambogic amide (GA), a recently identified robust small molecule agonist for TrkA, on hyperalgesia and load-induced bone formation. Behavioral analysis was used to assess pain up to one week after axial forelimb compression. Contrary to our expectations, GA treatment was not associated with diminished use of the loaded forelimb or sensitivity to thermal stimulus. Furthermore, dynamic histomorphometry revealed a significant increase in relative periosteal bone formation rate as compared to vehicle treatment. Additionally, we found that GA treatment was associated with an increase in the number of osteoblasts per bone surface in loaded limbs as well as a significant increase in the fold change of Ngf, Wnt7b, and Axin2 mRNA expression as compared to vehicle (control). To test the effect of GA on osteoblasts directly, we cultured MC3T3-E1 cells for up to 21days in osteogenic differentiation media containing NGF, GA, or vehicle (control). Media containing GA induced the significant upregulation of the osteoblastic differentiation markers Runx2, Bglap2, and Sp7 in a dose-dependent manner, whereas treatment with NGF was not associated with any significant increases in these markers. Furthermore, consistent with our in vivo findings, we observed that administration of 50nM of GA upregulated expression of Ngf at both Day 3 and Day 7. However, cells treated with the highest dose of GA (500nM) had significantly increased apoptosis and impaired cell proliferation. In conclusion, our study indicates GA may be useful for augmenting skeletal adaptation to mechanical forces without inducing hyperalgesia.

Effects of Black Jade on Osteogenic Differentiation of Adipose Derived Stem Cells under Benzopyrene

Jade, a popular gemstone symbolizing beauty, grace, and longevity, is known to improve blood circulation; however, scientific research evidence is still lacking. The effect of black jade extract on the expression levels of apoptotic and osteogenic genes was validated using qPCR and flow cytometry. In combination with the use of a fluorescence microscope, osteogenic differentiation and the stained osteocytes count were analyzed. Under the pressure of benzo(a)pyrene, dermal cell apoptosis was accelerated and the osteogenic differentiation of adipose-derived stem cells (ASCs) was suppressed; but black jade extract counteracted the effects. Through an anti-apoptotic mechanism, the extract suppressed the expression of apoptotic proteins Bax and cytochrome C to 9 and 4.8 times, respectively, compared to that in dermal cells exposed to benzo(a)pyrene. During osteogenic differentiation of ASCs, the extract enhanced their differentiation despite being exposed to benzo(a)pyrene, and the relative levels of the osteoblast differentiation markers osteoponin, osteocalcin, and sclerostin were 1.87, 2.54, and 3.9 times higher, respectively, than those in the conditioned medium by benzo(a)pyrene. These effects of the extract indicate that black jade extract is very useful when applied as a functional biomaterial.

Asarylaldehyde enhances osteogenic differentiation of human periodontal ligament stem cells through the ERK/p38 MAPK signaling pathway

Periodontitis is an inflammatory disease that affects tooth-supporting tissues. Chronic inflammation can progress to periodontitis, which results in loss of alveolar bone. Asarylaldehyde is a potential substance for bone metabolism present in natural compounds. Here, we propose the application of asarylaldehyde in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) to prevent bone loss. We investigated the effect of asarylaldehyde on hPDLSCs together with bone differentiation media in vitro. The osteogenic differentiation effect was observed after treatment of hPDLSCs with several concentrations of asarylaldehyde. After 21 days, osteogenic cells were identified by mineralization. We also observed that asarylaldehyde increased the mRNA expression of osteoblast-specific markers in hPDLSCs. Interestingly, asarylaldehyde regulated the levels of alkaline phosphatase (ALP) transcriptional activity through the p38/extracellular-signal-regulated kinase (ERK) signaling pathway. Notably, asarylaldehyde induced hPDLSCs to promote osteogenic differentiation. These results suggest that asarylaldehyde plays a key role in the osteogenic differentiation of hPDLSCs. Asarylaldehyde may be a good candidate for the application of natural compounds in future in periodontal regeneration.

Primary Cilia as a Biomarker in Mesenchymal Stem Cells Senescence: Influencing Osteoblastic Differentiation Potency Associated with Hedgehog Signaling Regulation.

Bone tissue engineering-based therapy for bone lesions requires the expansion of seeding cells, such as autologous mesenchymal stem cells (MSCs). A major obstacle to this process is the loss of the phenotype and differentiation capacity of MSCs subjected to passage. Recent studies have suggested that primary cilia, primordial organelles that transduce multiple signals, particularly hedgehog signals, play a role in senescence. Therefore, we explored the relationships among senescence, primary cilia, and hedgehog signaling in MSCs. Ageing of MSCs by expansion in vitro was accompanied by increased cell doubling time. The osteogenic capacity of aged MSCs at passage 4 was compromised compared to that of primary cells. P4 MSCs exhibited reductions in the frequency and length of primary cilia associated with decreased intensity of Arl13b staining on cilia. Senescence also resulted in downregulation of the expression of hedgehog components and CDKN2A. Suppression of ciliogenesis reduced the gene expression of both Gli1, a key molecule in the hedgehog signaling pathway and ALP, a marker of osteoblastic differentiation. This study demonstrated that the senescence of MSCs induced the loss of osteoblastic differentiation potency and inactivated hedgehog signaling associated with attenuated ciliogenesis, indicating that primary cilia play a mediating role in and are biomarkers of MSC senescence; thus, future antisenescence strategies involving manipulation of primary cilia could be developed.

Thrombin inhibitor argatroban modulates bone marrow stromal cells behaviors and promotes osteogenesis through canonical Wnt signaling

AimsCoagulation is a common event that play a double-edged role in physiological and pathological process. Anti-coagulation methods were applied in joint surgery or scaffolds implantation to encourage new vascular formation and avoid coagulation block. However, whether anti-coagulation drug perform regulatory roles in bone structure is unknown. This study aims to explore a direct thrombin inhibitor, argatroban, effects on bone marrow stromal cells (BMSCs) and decipher the underlying mechanisms. Materials and methodsArgatroban effects on BMSCs were investigated in vivo and in vitro. The drug was applied in periodontal disease model mice and bone loss was evaluated by μCT and histology. BMSCs were treated with different doses argatroban or vehicle. Cellular reactions were analyzed using wound healing assay, qRT-PCR, Alizarin Red S staining and western blotting. Key findingsWe demonstrated that local injection of argatroban can rescue bone loss in periodontal disease in vivo. To explore the underlying mechanism, we examined that cell proliferation and differentiation capability. Proliferation and migration of BMSCs were both inhibited by applying lower dose of argatroban. Interestingly, without affecting osteoclastogenesis, osteogenic differentiation was significantly induced by argatroban, which were shown by extracellular mineralization and upregulation of early osteoblastic differentiation markers, alkaline phosphatase, Osteocalcin, transcription factors RUNX2 and Osterix. In addition, molecular analysis revealed that argatroban promoted β-catenin nuclear translocation and led to an increase of osteogenesis through activating canonical Wnt signaling. SignificanceTaken together, our results show the novel application of the anti-coagulation compound argatroban in the commitment of BMSCs-based alveolar bone regeneration and remodeling.

M2-like macrophage infiltration and transforming growth factor-β secretion during socket healing process in mice

ObjectiveMacrophages are involved in tissue inflammation and repair through cytokine secretion. However, the contribution of macrophages to healing and osteogenesis after tooth extraction remains unclear. Therefore, we investigated the distribution of osteoblastic cells and macrophages in the early healing process after tooth extraction. MethodsThe maxillary first molars of 6-week-old male mice were extracted. The maxilla was collected 1, 3, and 7 days after extraction. The states of socket healing, localization of osteoblastic markers, and macrophage infiltration were sequentially observed by micro-CT imaging and immunohistochemistry. ResultsOn day 3 after tooth extraction, α-smooth muscle actin (SMA)-positive cells, osteoprogenitor cells at fracture healing, were observed in the socket. Several α-SMA-positive cells also expressed Runx2, the early osteoblast differentiation marker. The infiltration of F4/80-positive, mature macrophages and CD206-positive, M2-like macrophages was noted in the socket. However, CD169-positive macrophages (Osteomac), which are involved in fracture healing, were not detected in the socket. F4/80-positive and CD206-positive macrophages also showed the localization of transforming growth factor-β (TGF-β), which promotes osteoprogenitor cell proliferation and early differentiation. Phosphorylated Smad3, a downstream mediator of the signal activity of TGF-β, was detected in α-SMA-positive cells. On day 7, the extracted socket contained a large amount of new bone. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclasts were detected on bone surfaces. ConclusionOur data indicate that M2-like macrophages regulate the proliferation and differentiation of α-SMA-positive cells by secreting TGF-β at the early stage of socket healing, and also suggest the importance of macrophages in healing and bone formation after tooth extraction.

Strontium-Modified Scaffolds Based on Mesoporous Bioactive Glasses/Polyvinyl Alcohol Composites for Bone Regeneration.

In the search of a new biomaterial for the treatment of bone defects resulting from traumatic events, an osteoporosis scenario with bone fractures, tumor removal, congenital pathologies or implant revisions for infection, we developed 3D scaffolds based on mesoporous bioactive glasses (MBGs) (85 − x)SiO2–5P2O5–10CaO–xSrO (x = 0, 2.5 and 5 mol.%). The scaffolds with meso-macroporosity were fabricated by pouring a suspension of MBG powders in polyvinyl alcohol (PVA) into a negative template of polylactic acid (PLA), followed by removal of the template by extraction at low temperature. SrO-containing MBGs exhibited excellent properties for bone substitution including ordered mesoporous structure, high textural properties, quick in vitro bioactive response in simulated body fluid (SBF) and the ability of releasing concentrations of strontium ions able to stimulate expression of early markers of osteoblastic differentiation. Moreover, the direct contact of MC3T3-E1 pre-osteoblastic cells with the scaffolds confirmed the cytocompatibility of the three compositions investigated. Nevertheless, the scaffold containing 2.5% of SrO induced the best cellular proliferation showing the potential of this scaffold as a candidate to be further investigated in vitro and in vivo, aiming to be clinically used for bone regeneration applications in non-load bearing sites.

Macrophage-derived small extracellular vesicles promote biomimetic mineralized collagen-mediated endogenous bone regeneration

Macrophages play an important role in material-related immune responses and bone formation, but the functionality of macrophage-derived extracellular vesicles (EVs) in material-mediated bone regeneration is still unclear. Here, we evaluated intracellular communication through small extracellular vesicles (sEVs) and its effects on endogenous bone regeneration mediated by biomimetic intrafibrillarly mineralized collagen (IMC). After implantation in the bone defect area, IMC generated more neobone and recruited more mesenchymal stem cells (MSCs) than did extrafibrillarly mineralized collagen (EMC). More CD63+CD90+ and CD63+CD163+ cells were detected in the defect area in the IMC group than in the EMC group. To determine the functional roles of sEVs, extracellular vesicles from macrophages cultured on different mineralized collagen were isolated, and they showed no morphological differences. However, macrophage-derived sEVs in the IMC group showed an enhanced Young’s modulus and exerted beneficial effects on the osteogenic differentiation of bone marrow MSCs by increasing the expression of the osteoblastic differentiation markers BMP2, BGLAP, COL1, and OSX and calcium nodule formation. Mechanistically, sEVs from IMC-treated macrophages facilitated MSC osteogenesis through the BMP2/Smad5 pathway, and blocking sEV secretion with GW4869 significantly impaired MSC proliferative, immunomodulative and osteogenic potential. Taken together, these findings show that macrophage-derived sEVs may serve as an emerging functional tool in biomaterial-mediated endogenous bone regeneration.

Differentiation-inducing factor-1 potentiates adipogenic differentiation and attenuates the osteogenic differentiation of bone marrow-derived mesenchymal stem cells

Mesenchymal stem cells (MSCs) are an attractive cell source for tissue regeneration and repair. However, their low differentiation efficacy currently impedes the development of MSC therapy. Therefore, in this study, we investigated the effects of differentiation-inducing factor-1 (DIF-1) on the differentiation efficacy of bone marrow-derived MSCs (BM-MSCs) into adipogenic or osteogenic lineages. BM-MSCs, which were obtained from Sprague-Dawley rats, were positive for the MSC markers (CD29, CD73, and CD90). DIF-1 alone neither affected cell surface antigen expression nor induced adipogenic or osteogenic differentiation. However, DIF-1 significantly enhanced the effects of adipogenic differentiation stimuli, which were evaluated as the number of oil red-O positive cells and the expression of adipocyte differentiation markers (peroxisome proliferator-activated receptor gamma, adipocyte fatty acid-binding protein, and adiponectin). In contrast, DIF-1 significantly attenuated the effects of osteogenic differentiation stimuli, which were evaluated as alizarin red-S positive calcium deposition, and the expression of osteoblast differentiation markers alkaline phosphatase, runt-related transcription factor 2, and osteopontin. We further investigated the mechanism by which DIF-1 affects MSC differentiation efficacy and found that glycogen synthase kinase-3 was the main factor mediating the action of DIF-1 on the adipogenic differentiation of BM-MSCs, whereas it was only partially involved in osteogenic differentiation. These results suggest that DIF-1 supports MSC differentiation toward the desired cell fate by enhancing the differentiation efficacy.

MiR-129-5p Inhibits Bone Formation Through TCF4.

Osteoporosis is a frequently occurring bone disease in middle-aged and aged men and women. However, current therapies on this disease are still not ideal. MicroRNAs (miRNAs) are a class of endogenous non-protein-coding RNA with a length of 18–25 nucleotides. miRNAs have been identified as important regulators for development, metabolism, carcinogenesis, and bone formation. miR-129-5p has been reported as a regulator of cancer and neuroscience, whereas studies about its function on bone formation is still limited. In this study, we investigated the function and mechanism of miR-129-5p on osteoblast differentiation and bone formation. We have assessed the expression of miRNAs in bone mesenchymal stem cells from aging and menopause osteoporosis C57BL6 mice. The expression of miR-129-5p was altered in all osteoporosis models. Besides, the expression of miR-129-5p was negatively correlated with osteoblastic differentiation markers in the femur tissues of C57BL/6 mice of different ages. We further demonstrated that overexpression of miR-129-5p inhibited osteoblast differentiation in MC3T3-E1 cell line, as well as bone formation of C57BL/6 mice. On the other hand, down-regulation of miR-129-5p enhanced osteoblast differentiation and bone formation. We also found that miR-129-5p inhibited Wnt/β-catenin pathway in osteoblast. The target gene of miR-129-5p has been forecasted and proved as Tcf4. We further found that plasmid containing Tcf4–3′ UTR sequence enhanced osteoblast differentiation, as well as Wnt/β-catenin pathway in MC3T3-E1 cells. To further investigate the rescue effect of miR-129-5p inhibitor, we manufactured bioengineered novel recombinant miR-129-5p inhibitor through Escherichia coli system and then tested its function. The results showed that the novel recombinant miR-129-5p inhibitor promoted osteoblast differentiation and greatly ameliorated menopause osteoporosis in C57BL6 mice. In conclusion, we have discovered miR-129-5p as an inhibitor of bone formation. miR-129-5p inhibited downstream transcription factors of Wnt/β-catenin pathway through targeting Tcf4. Moreover, novel recombinant miR-129-5p inhibitor showed rescue effect on osteoporosis. This study has revealed a new mechanism of osteogenic differentiation and provided novel therapeutic strategies for treatment of skeletal disorders.

Muscle-Derived Lumican Stimulates Bone Formation via Integrin α2β1 and the Downstream ERK Signal.

Skeletal muscle and bone are highly interrelated, and previous proteomic analyses suggest that lumican is one of muscle-derived factors. To further understand the role of lumican as a myokine affecting adjacent bone metabolism, we investigated the effects of lumican on osteoblast biology. Lumican expression was significantly higher in the cell lysates and conditioned media (CM) of myotubes than those of undifferentiated myoblasts, and the known anabolic effects of myotube CM on osteoblasts were reduced by excluding lumican from the CM. Lumican stimulated preosteoblast viability and differentiation, resulting in increased calvaria bone formation. The expression of osteoblast differentiation markers was consistently increased by lumican. Lumican increased the phosphorylation of ERK, whereas ERK inhibitors completely reversed lumican-mediated stimulation of Runx2 and ALP activities in osteoblasts. Results of a binding ELISA experiment in osteoblasts show that transmembrane integrin α2β1 directly interacted with lumican, and an integrin α2β1 inhibitor attenuated the stimulation of ERK and ALP activities by lumican. Taken together, the results indicate that muscle-derived lumican stimulates bone formation via integrin α2β1 and the downstream ERK signal, indicating that this is a potential therapeutic target for metabolic bone diseases.

SATB2 is not a reliable diagnostic marker for distinguishing between oral osteosarcoma and fibro-osseous lesions of the jaws

Special AT-rich binding protein 2 (SATB2) is an immunohistochemical marker for osteoblast differentiation. Our aim was to investigate SATB2 expression in oral osteosarcoma and other bone-producing oral tumors/reactive lesions to evaluate its usefulness as a diagnostic marker. A total of 74 intraosseous and soft tissue bone-producing surgical samples and 10 samples of reactive bone tissue were stained with SATB2, including osteosarcoma/chondrosarcoma (n=16), fibro-osseous lesions (n=42), central giant cell granuloma (n=6), osteoblastoma (n=1), and gingival lesions (n=9). Nuclear labeling of the stromal spindle cells and intensity of staining was scored and analyzed. The intraosseous (n=65/65) and soft tissue samples (n=9/9) diffusely expressed SATB2. The strongest expression was observed in juvenile aggressive ossifying fibroma (n=2/2). Weak SATB2 expression was observed in the stromal spindle cells adjacent to reactive bone tissue (periosteal bone reaction). Our results indicate that SATB2 is not a reliable diagnostic marker for oral osteosarcoma but has practical use in detecting cells with osteoblast differentiation in histologic samples with scant bone production or in differentiating between a periosteal bone reaction and neoplastic bone induced by the tumor mesenchymal cells. Targeting SATB2 as an alternative therapy in oral osteosarcoma, fibro-osseous lesions, and central giant cell granuloma should be further investigated.

Secretoneurin, a Neuropeptide, Enhances Bone Regeneration in a Mouse Calvarial Bone Defect Model.

This study investigates the effects of a neuropeptide, secretoneurin (SN), on bone regeneration in an experimental mouse model. The effects of SN on cell proliferation, osteoblast marker genes expression, and mineralization were evaluated using the CCK-8 assay, quantitative reverse transcriptase polymerase chain reaction (RT-PCR), and alizarin red S staining, respectively. To examine the effects of SN on bone regeneration in vivo, bone defects were created in the calvaria of ICR mice, and 0.5 or 1µg/ml SN was applied. New bone formation was analyzed by micro-computed tomography (micro-CT) and histology. New blood vessel formation was assessed by CD34 immunohistochemistry. SN had no significant effect on proliferation and mineralization of MC3T3-E1 cells. However, SN partially induced the gene expression of osteoblast differentiation markers such as runt-related transcription factor 2, alkaline phosphatase, collagen type I alpha 1, and osteopontin. A significant increase of bone regeneration was observed in SN treated calvarial defects. The bone volume (BV), BV/tissue volume, trabecular thickness and trabecular number values were significantly increased in the collagen sponge plus 0.5 or 1µg/ml SN group (p < 0.01) compared with the control group. Histologic analysis also revealed increased new bone formation in the SN-treated groups. Immunohistochemical staining of CD34 showed that the SN-treated groups contained more blood vessels compared with control in the calvarial defect area. SN increases new bone and blood vessel formation in a calvarial defect site. This study suggests that SN may enhance new bone formation through its potent angiogenic activity.

CRISPR/Cas9 mediated GFP-human dentin matrix protein 1 (DMP1) promoter knock-in at the ROSA26 locus in mesenchymal stem cell for monitoring osteoblast differentiation.

Dentin matrix protein 1 (DMP1) is highly expressed in mineralized tooth and bone, playing a critical role in mineralization and phosphate metabolism. One important role for the expression of DMP1 in the nucleus of preosteoblasts is the up-regulation of osteoblast-specific genes such as osteocalcin and alkaline phosphatase1 . The present study aimed to investigate the potential application of human DMP1 promoter as an indicator marker of osteoblastic differentiation. In the present study, we developed DMP1 promoter-DsRed-GFP knock-in mesenchymal stem cell (MSCs) via the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system that enabled automatic detection of osteoblast differentiation. With the application of a homology-directed knock-in strategy, a 2-kb fragment of DMP1 promoter, which was inserted upstream of the GFP and DsRed reporter cassette, was integrated into the human ROSA locus to generate double fluorescent cells. We further differentiated MSCs under osteogenic media to monitor the fate of MSCs. First, cells were transfected using CRISPR/Cas9 plasmids, which culminated in MSCs with a green fluorescence intensity, then GFP-positive cells were selected using puromycin. Second, the GFP-positive MSCs were differentiated toward osteoblasts, which demonstrated an increased red fluorescence intensity. The osteoblast differentiation of MSCs was also verified by performing alkaline phosphatase and Alizarin Red assays. We have exploited the DMP1 promoter as a predictive marker of MSC differentiation toward osteoblasts. Using the CRISPR/Cas9 technology, we have identified a distinctive change in the fluorescence intensities of GFP knock-in (green) and osteoblast differentiated MSCs 2 . The data show that DMP1-DsRed-GFP knock-in MSCs through CRISPR/Cas9 technology provide a valuable indicator for osteoblast differentiation. Moreover, The DMP1 promoter might be used as a predictive marker of MSCs differentiated toward osteoblasts.

Tankyrase inhibitor XAV-939 enhances osteoblastogenesis and mineralization of human skeletal (mesenchymal) stem cells

Tankyrase is part of poly (ADP-ribose) polymerase superfamily required for numerous cellular and molecular processes. Tankyrase inhibition negatively regulates Wnt pathway. Thus, Tankyrase inhibitors have been extensively investigated for the treatment of clinical conditions associated with activated Wnt signaling such as cancer and fibrotic diseases. Moreover, Tankyrase inhibition has been recently reported to upregulate osteogenesis through the accumulation of SH3 domain-binding protein 2, an adaptor protein required for bone metabolism. In this study, we investigated the effect of Tankyrase inhibition in osteoblast differentiation of human skeletal (mesenchymal) stem cells (hMSCs). A Tankyrase inhibitor, XAV-939, identified during a functional library screening of small molecules. Alkaline phosphatase activity and Alizarin red staining were employed as markers for osteoblastic differentiation and in vitro mineralized matrix formation, respectively. Global gene expression profiling was performed using the Agilent microarray platform. XAV-939, a Tankyrase inhibitor, enhanced osteoblast differentiation of hBMSCs as evidenced by increased ALP activity, in vitro mineralized matrix formation, and upregulation of osteoblast-related gene expression. Global gene expression profiling of XAV-939-treated cells identified 847 upregulated and 614 downregulated mRNA transcripts, compared to vehicle-treated control cells. It also points towards possible changes in multiple signaling pathways, including TGFβ, insulin signaling, focal adhesion, estrogen metabolism, oxidative stress, RANK-RANKL (receptor activator of nuclear factor κB ligand) signaling, Vitamin D synthesis, IL6, and cytokines and inflammatory responses. Further bioinformatic analysis, employing Ingenuity Pathway Analysis identified significant enrichment in XAV-939-treated cells of functional categories and networks involved in TNF, NFκB, and STAT signaling. We identified a Tankyrase inhibitor (XAV-939) as a powerful enhancer of osteoblastic differentiation of hBMSC that may be useful as a therapeutic option for treating conditions associated with low bone formation.

Surface modification of titanium with collagen/hyaluronic acid and bone morphogenetic protein 2/7 heterodimer promotes osteoblastic differentiation.

The aim of this study was to evaluate the effects of a collagen/hyaluronic acid coating without or with incorporated heterodimeric bone morphogenetic protein 2/7 (BMP2/7) on in-vitro osteoblastic differentiation on titanium discs. The multilayer collagen/hyaluronic acid coatings without or without incorporated BMP2/7 were deposited on titanium discs via a layer-by-layer technique. The effects of the coatings were evaluated by assessing the alkaline phosphatase (ALP) activity (an early osteoblastic differentiation marker) and the osteocalcin expression (a late osteoblastic differentiation marker). The expression levels of the osteoblastic genes, such as alkaline phosphatase 2 (AKP2) and osteocalcin (OC) were detected using real-time RT-PCR. ALP activity and OC expression were significantly increased when cells were cultured with collagen/hyaluronic acid+BMP2/7 heterodimer (p<0.05). The same result was found in cells with the expression of a BMP2/7 fusion gene, OC and AKP2. These results indicated that collagen/hyaluronic acid+BMP2/7 heterodimer-coated discs might have the potential to greatly enhance osseointegration than a either BMP2 or BMP7 solution or a mixture of BMP2 and BMP7 BMP2/7.

Discoidin Domain Receptor 1 Regulates Runx2 during Osteogenesis of Osteoblasts and Promotes Bone Ossification via Phosphorylation of p38.

Discoidin domain receptor 1 (Drd1) is a collagen-binding membrane protein, but its role in osteoblasts during osteogenesis remains undefined. We generated inducible osteoblast-specific Ddr1 knockout (OKOΔDdr1) mice; their stature at birth, body weight and body length were significantly decreased compared with those of control Ddr1f/f-4OHT mice. We hypothesize that Ddr1 regulates osteogenesis of osteoblasts. Micro-CT showed that compared to 4-week-old Ddr1f/f-4OHT mice, OKOΔDdr1 mice presented significant decreases in cancellous bone volume and trabecular number and significant increases in trabecular separation. The cortical bone volume was decreased in OKOΔDdr1 mice, resulting in decreased mechanical properties of femurs compared with those of Ddr1f/f-4OHT mice. In femurs of 4-week-old OKOΔDdr1 mice, H&E staining showed fewer osteocytes and decreased cortical bone thickness than Ddr1f/f-4OHT. Osteoblast differentiation markers, including BMP2, Runx2, alkaline phosphatase (ALP), Col-I and OC, were decreased compared with those of control mice. Ddr1 knockdown in osteoblasts resulted in decreased mineralization, ALP activity, phosphorylated p38 and protein levels of BMP2, Runx2, ALP, Col-I and OC during osteogenesis. Overexpression and knockdown of Ddr1 in osteoblasts demonstrated that DDR1 mediates the expression and activity of Runx2 and the downstream osteogenesis markers during osteogenesis through regulation of p38 phosphorylation.

P6. A roughened 3D-printed surface enhances stem cell proliferation and osteoblast differentiation

BACKGROUND CONTEXT The advancement of manufacturing techniques, such as 3D-printing, has facilitated the design of complex and previously unattainable implant surface topographies. Research has shown surface properties, especially surface roughness, of an implant directly influence cellular response at the cell-material interface. Alkaline phosphatase (ALP), an early cellular marker of osteoblast differentiation, has been studied extensively to understand osteogenic cellular responses to a substrate. The identification of implant surfaces that promote a osteogenic environment may help identify those that are optimal for healing. PURPOSE The objective of this study was to investigate the effect of a roughened 3D-printed surface on the cellular proliferation and ALP expression of human bone-marrow derived mesenchymal stem cells (BM-MSCs). STUDY DESIGN/SETTING In vitro cell culture study. PATIENT SAMPLE N/A OUTCOME MEASURES Cellular proliferation was measured using a colorimetric, viability assay (alamarBlue™) to estimate the cell count on each substrate at days 1, 3 and 7. ALP activity was measured from cell lysate using a standard ALP assay on day 7. ALP activity was normalized to the total protein in each lysate sample measured using a bicinchoninic acid (BCA) assay also performed on day 7. METHODS Roughness (Ra) was characterized for each substrate in the study using a profilometer. BM-MSCs were seeded at a density of 10,000 cells/cm2 onto smooth polyetheretherkeytone (PEEK), smooth titanium-alloy (TAV), and a roughened, 3DP titanium-alloy (r3DP) substrates (n=6 per group) and cultured for 7 days. Cells were given 4 hours to attach, after which cell medium was changed and the substrates were moved to a separate well plate to remove any unattached cells. Assays were performed to quantify specific ALP cellular signaling, indicative of osteoblast differentiation. RESULTS The r3DP substrates had 15-30 times greater Ra than PEEK and TAV (15.2µm, 1.0µm, and 0.4µm, respectively). Cell count on day 7 for r3DP was significantly greater than PEEK (p 0.05). ALP activity was significantly greater in the r3DP group (8.8±1.1nmol/µg) compared to both PEEK (5.7±1.4nmol/µg) and TAV (4.4±0.9 nmol/µg) (p 0.05). CONCLUSIONS Optimal cellular activity and osteogenic cell signaling are important for osteoblast differentiation, a process needed to foster a cellular environment favorable for healing. Cells on a roughened 3D-printed TAV substrate exhibited significantly higher proliferation compared to PEEK and significantly higher ALP activity than both TAV and PEEK. Therefore, the r3DP substrate facilitated a cellular environment more favorable for osteoblast differentiation than TAV and PEEK substrates. Future research in an animal model is necessary to evaluate the downstream effects of an implant with a roughened 3D-printed TAV surface on the healing process. FDA DEVICE/DRUG STATUS SABLE (Globus Medical) (Approved for this indication)