Sp7 Expression Research Articles

Bone development by Hedgehog and Wnt signaling, Runx2, and Sp7.

Hedgehog and canonical Wnt signaling pathways and the transcription factors Runx2 and Sp7 are essential for osteoblast differentiation. Ihh is necessary for the commitment of perichondrial mesenchymal cells to Runx2+ osteoprogenitors and for the formation of the bone collar and primary spongiosa. Runx2 is needed for osteoblast differentiation during both endochondral and intramembranous ossification. It regulates the commitment of mesenchymal cells to osteoblast-lineage cells and their proliferation by inducing the expression of Hedgehog, Fgf, Wnt, Pthlh signaling pathway genes, and Dlx5. The Runx2-induced expression of Fgfr2 and Fgfr3 is important for the proliferation of osteoblast-lineage cells. Runx2 induces Sp7 expression and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Runx2, Sp7, and canonical Wnt signaling induce the differentiation of preosteoblasts into osteoblasts. Canonical Wnt signaling, but not Sp7, enhances the proliferation of osteoblast-lineage cells. In mature osteoblasts, Runx2 plays an important role in the expression of major bone matrix protein genes, including Col1a1, Col1a2, Spp1, Ibsp, and Bglap/Bglap2. The canonical Wnt signaling pathway is also crucial for bone formation by mature osteoblasts. Sp7 is needed for osteocytes to acquire a sufficient number of processes and a reduction in these processes results in osteocyte apoptosis and cortical porosity.

Regulation of Skeletal Development and Maintenance by Runx2 and Sp7.

Runx2 (runt related transcription factor 2) and Sp7 (Sp7 transcription factor 7) are crucial transcription factors for bone development. The cotranscription factor Cbfb (core binding factor beta), which enhances the DNA-binding capacity of Runx2 and stabilizes the Runx2 protein, is necessary for bone development. Runx2 is essential for chondrocyte maturation, and Sp7 is partly involved. Runx2 induces the commitment of multipotent mesenchymal cells to osteoblast lineage cells and enhances the proliferation of osteoprogenitors. Reciprocal regulation between Runx2 and the Hedgehog, fibroblast growth factor (Fgf), Wnt, and parathyroid hormone-like hormone (Pthlh) signaling pathways and Dlx5 (distal-less homeobox 5) plays an important role in these processes. The induction of Fgfr2 (Fgf receptor 2) and Fgfr3 expression by Runx2 is important for the proliferation of osteoblast lineage cells. Runx2 induces Sp7 expression, and Runx2+ osteoprogenitors become Runx2+Sp7+ preosteoblasts. Sp7 induces the differentiation of preosteoblasts into osteoblasts without enhancing their proliferation. In osteoblasts, Runx2 is required for bone formation by inducing the expression of major bone matrix protein genes, including Col1a1 (collagen type I alpha 1), Col1a2, Spp1 (secreted phosphoprotein 1), Ibsp (integrin binding sialoprotein), and Bglap (bone gamma carboxyglutamate protein)/Bglap2. Bglap/Bglap2 (osteocalcin) regulates the alignment of apatite crystals parallel to collagen fibrils but does not function as a hormone that regulates glucose metabolism, testosterone synthesis, and muscle mass. Sp7 is also involved in Co1a1 expression and regulates osteoblast/osteocyte process formation, which is necessary for the survival of osteocytes and the prevention of cortical porosity. SP7 mutations cause osteogenesis imperfecta in rare cases. Runx2 is an important pathogenic factor, while Runx1, Runx3, and Cbfb are protective factors in osteoarthritis development.

Plasma Rich in Growth Factors in Bone Regeneration: The Proximity to the Clot as a Differential Factor in Osteoblast Cell Behaviour.

The osteogenic differentiation process, by which bone marrow mesenchymal stem cells and osteoprogenitors transform into osteoblasts, is regulated by several growth factors, cytokines, and hormones. Plasma Rich in Growth Factors (PRGF) is a blood-derived preparation consisting of a plethora of bioactive molecules, also susceptible to containing epigenetic factors such as ncRNAs and EVs, that stimulates tissue regeneration. The aim of this study was to investigate the effect of the PRGF clot formulation on osteogenic differentiation. Firstly, osteoblast cells were isolated and characterised. The proliferation of bone cells cultured onto PRGF clots or treated with PRGF supernatant was determined. Moreover, the gene expression of Runx2 (ID: 860), SP7 (ID: 121340), and ALPL (ID: 249) was analysed by one-step real-time quantitative polymerase chain reaction (RT-qPCR). Additionally, alkaline phosphatase (ALPL) activity determination was performed. The highest proliferative effect was achieved by the PRGF supernatant in all the study periods analysed. Concerning gene expression, the logRGE of Runx2 increased significantly in osteoblasts cultured with PRGF formulations compared with the control group, while that of SP7 increased significantly in osteoblasts grown on the PRGF clots. On the other hand, despite the fact that the PRGF supernatant induced ALPL up-regulation, significantly higher enzyme activity was detected for the PRGF clots in comparison with the supernatant formulation. According to our results, contact with the PRGF clot could promote a more advanced phase in the osteogenic process, associated to higher levels of ALPL activity. Furthermore, the PRGF clot releasate stimulated a higher proliferation rate in addition to reduced SP7 expression in the cells located at a distant ubication, leading to a less mature osteoblast stage. Thus, the spatial relationship between the PRGF clot and the osteoprogenitors cells could be a factor that influences regenerative outcomes.

Mechanical induction of osteoanabolic Wnt1 promotes osteoblast differentiation via Plat.

Physical activity-induced mechanical stimuli play a crucial role in preserving bone mass and structure by promoting bone formation. While the Wnt pathway is pivotal for mediating the osteoblast response to loading, the exact mechanisms are not fully understood. Here, we found that mechanical stimulation induces osteoblastic Wnt1 expression, resulting in an upregulation of key osteogenic marker genes, including Runx2 and Sp7, while Wnt1 knockdown using siRNA prevented these effects. RNAseq analysis identified Plat as a major target through which Wnt1 exerts its osteogenic influence. This was corroborated by Plat depletion using siRNA, confirming its positive role in osteogenic differentiation. Moreover, we demonstrated that mechanical stimulation enhances Plat expression, which, in turn leads to increased expression of osteogenic markers like Runx2 and Sp7. Notably, Plat depletion by siRNA prevented this effect. We have established that Wnt1 regulates Plat expression by activating β-Catenin. Silencing Wnt1 impairs mechanically induced β-Catenin activation, subsequently reducing Plat expression. Furthermore, our findings showed that Wnt1 is essential for osteoblasts to respond to mechanical stimulation and induce Runx2 and Sp7 expression, in part through the Wnt1/β-Catenin/Plat signaling pathway. Additionally, we observed significantly reduced Wnt1 and Plat expression in bones from ovariectomy (OVX)-induced and age-related osteoporotic mouse models compared with non-OVX and young mice, respectively. Overall, our data suggested that Wnt1 and Plat play significant roles in mechanically induced osteogenesis. Their decreased expression in bones from OVX and aged mice highlights their potential involvement in post-menopausal and age-related osteoporosis, respectively.

Fibroblast growth factor signalling regulates the development of tooth root.

Fibroblast growth factor (FGF) signalling plays a crucial role in the morphogenesis of multiple tissues including teeth. While the role of the signal has been studied in tooth crown development, little is known about root development. Of several FGF ligands involved in hard tissue formation, we suggest that FGF18 regulates the development of murine tooth roots. We implanted FGF18-soaked heparin beads into the lower first molar tooth buds at postnatal day 6 (P6), followed by transplantation under the kidney capsule. After 3 weeks, FGF18 significantly facilitated root elongation and periodontal tissue formation compared to the control. Insitu hybridisation showed that Fgf18 transcripts were initially localised in the dental pulp along Hertwig's epithelial root sheath at P6 and P10 and subsequently in the dental follicle cells at P14. Fgf receptors were expressed in various dental tissues during these stages. Invitro analysis using the dental pulp stem cells revealed that FGF18 inhibited cell proliferation and decreased expression levels of osteogenic markers, Runx2, Alpl and Sp7. Consistently, after 1 week of kidney capsule transplantation, FGF18 application did not induce the expression of Sp7 and Bsp, but upregulated Periostin in the apical region of dental mesenchyme in the grafted molar. These findings suggest that FGF18 facilitates molar root development by regulating the calcification of periodontal tissues.

Effect of runx2b deficiency in intermuscular bones on the regulatory network of lncRNA-miRNA-mRNA

Intermuscular bones (IBs) are mineralized spicules that negatively impact the quality and value of fish products. Runx2b is a crucial modulator in promoting bone formation through regulating osteoblast differentiation. Previous studies suggested that loss of runx2b gene completely inhibited IBs formation in zebrafish. However, how the whole transcriptome, including mRNA and non-coding RNA (ncRNA), affects the IBs development in runx2b−/− zebrafish are not known. The aim of this study was to identify the regulatory networks of differentially expressed (DE) lncRNAs, miRNAs, and mRNAs in zebrafish with and without IBs (runx2b+/+ fish and runx2b−/− fish) utilizing high-throughput sequencing techniques. All together there are 1051 mRNAs, 456 lncRNAs, and 18 miRNAs differentially expressed were found between these two strains. The analysis of Kyoto Encyclopedia of Genes and Genomes (KEGG) has highlighted significant pathways linked to the development of IBs, specifically the TGF-beta and Wnt signaling pathways, and a number of genes concentrated on these two signaling pathways related to the formation of IBs. Further, 1989 competing endogenous RNA (ceRNA) networks were created according to the correlation among mRNAs, miRNAs and lncRNAs. The ceRNA networks results revealed 52 ceRNA pairs related to the IBs formation, consisting of 52 mRNAs, 37 lncRNAs, and 6 miRNAs. Of these, we found that dre-miR-2189 was the key element of ceRNA pairs, interacting with 19 mRNAs and 11 lncRNAs, and MSTRG.13175.1 could regulate sp7 expression by interacting with dre-miR-2189 to function in osteogenic differentiation. Subsequent experiments at the cellular level also revealed the interaction mechanism. The outcomes indicated a crucial role of miRNAs and lncRNAs in the development of fish IBs, which offer new views into the functions of ncRNAs involved in IBs formation.

Anti-osteoporosis effects of mammalian lignans and their precursors from flaxseed and safflower seed using zebrafish model.

Secoisolariciresinol diglucoside (SDG) and tracheloside (TCL) are the main lignan components of flaxseed cake and safflower seed cake, which are by-products of oil extraction. Both SDG and TCL are metabolized into mammalian lignan enterolactone (EL) with the involvement of intestinal bacteria. In this research, we evaluated the anti-osteoporosis effects of SDG and the in vivo metabolites EL and enterodiol (ED) prepared in our previous work, as well as the newly isolated chemical constituents from safflower seed, including TCL, the lactone ring opening product of TCL (OTCL) and two alkaloids on the alloxan-induced zebrafish model. All the compounds showed significant anti-osteoporosis effects at 80µM, with p<0.05 for EL and p<0.001 for other compounds compared with the model. SDG and TCL showed the most significant and concentration-dependent effects, with p<0.001 compared with model at 20µM. The alkaloids, N-coumaroylserotonin glucoside and N-feruloylserotonin glucoside, also showed anti-osteoporosis at 20µM with p<0.01, whereas EL, ED, and OTCL showed no significant effects. Quantitative real-time polymerase chain reaction revealed that SDG and TCL upregulated the expression of osteogenic genes Runx2, SP7, OPG, Col1a1a, Alp, ON, OPN, and OCN in alloxan-treated zebrafish. The in vivo metabolite of lignans, EL, showed significant anti-inflammatory effect (p<0.01) at 20µM, which might also help to combat osteoporosis and other complications caused by excessive immune response in the body. The results provided scientific data for using the oil extraction by-products as sources of anti-osteoporosis compounds. PRACTICAL APPLICATION: This study found that lignans in flaxseed cake and safflower seed cake exhibited anti-osteoporosis effects by upregulating the expression of osteogenic genes, making the oil extraction by-products sources of anti-osteoporosis compounds.

Age-associated spinal stenosis in the turquoise killifish

Aging triggers spinal degeneration, including common spinal stenosis, which causes back and leg pain in older individuals, significantly impacting their quality of life. Here, we explored aging traits in turquoise killifish spines, potentially offering a model for age-linked spinal stenosis in humans. Aged turquoise killifish exhibited body shape deformation and increased vertebral collapse, which was further accelerated by spawning. High-resolution CT scans revealed suppressed cortical bone thickness and hemal arch area in vertebrae due to spawning, and osteophyte formation was observed in both aged and breeding fish populations. Scale mineralization mirrored these changes, increasing with age but being suppressed by spawning. The expression of sp7, sox9b, axin1, and wnt4a/b genes can be utilized to monitor age- and reproduction-dependent spine deformation. This study demonstrates that turquoise killifish and humans share certain phenotypes of age-related vertebral abnormalities, suggesting that turquoise killifish could serve as a potential model for studying human spinal stenosis.

Autologous Platelet Lysate Is an Alternative to Fetal Bovine Serum for Canine Adipose-Derived Mesenchymal Stem Cell Culture and Differentiation.

The use of fetal bovine serum (FBS) in regenerative medicine raises serious ethical and scientific concerns. We have cultured and differentiated the canine mesenchymal stem cells (cMSCs) in five different media combinations of autologous platelet lysate (A-PL) and FBS; consisting of 0% A-PL and 10% FBS (M-1), 2.5% A-PL and 7.5% FBS (M-2), 5% A-PL and 5% FBS (M-3), 7.5% A-PL and 2.5% FBS (M-4), and 10% A-PL and 0% FBS (M-5). The cMSCs were evaluated for their doubling time, differentiation efficiency, and expression of CD73, CD90, CD105, and PDGFRα. The mRNA expression of NT5E, THY1, ENG, PPARγ, FABP4, FAS, SP7, BGLAP, and SPP1 was also assessed. The results indicated non-significant differences in cellular proliferation/viability; positive expression of surface markers, and PDGFRα with substantial adipo/osteogenic differentiation. The expression of adipogenic (PPARγ, FABP4, FAS), and osteogenic (SP7, BGLAP, SPP1) genes were higher (p < 0.05) in the M5 group. In conclusion, A-PL in cMSCs culture did not negatively affect cellular proliferation and viability but also enhanced their genetic potential for multilineage differentiation. Our results indicate that A-PL can be used as an alternative for FBS to develop potent cMSCs under good manufacturing practice protocol for regenerative medicine.

Bone remodelling marker expression in grafted and ungrafted sheep tooth extraction sockets: A comparative randomised study

ObjectiveTo compare key markers of bone remodelling in a sheep tooth extraction model for sockets left to heal naturally or grafted with the bovine-derived xenograft Bio-Oss® covered with a collagen Bio-Gide® membrane. DesignRight side premolar teeth were removed from thirty Romney-cross ewes. Standardised sockets in each sheep were randomly allocated treatments, a grafted test and an empty control. At 4-, 8- and 16-weeks sheep were euthanized and tissue collected (N = 10/group). RANK, RANKL and OPG immunohistochemical analysis was performed (n = 3). RANK, RANKL, OPG, COL1A1, TIMP3, SP7 and MSX2 mRNA expression levels were determined using RT2-qPCR assays (n = 3). ResultsHistologically, more new woven bone was observed in the test group at all time points. Strong RANK and RANKL expression was found in both groups; at all time points with stronger RANK staining in the test group at 8 and 16 weeks. Strong OPG staining was localized to both osteoblasts and connective tissues. RANK receptor mRNA was expressed at a lower level in the test group (−4.26-fold; p = 0.02) at 4 weeks and SP7 at 16 weeks (−2.89-fold; p = 0.04). COL1A1 and TIMP3 mRNA expression increased significantly over time in the control group (p = 0.045, F = 5.4 and p = 0.003, F = 42.2 respectively). ConclusionSocket healing over time was comparable. The sheep tooth extraction model was found to be suitable for the evaluation of changes in the alveolar bone at the molecular level.

Low Magnesium Concentration Enforces Bone Calcium Deposition Irrespective of 1,25-Dihydroxyvitamin D3 Concentration.

Efficient coordination between Mg2+ and vitamin D maintains adequate Ca2+ levels during lactation. This study explored the possible interaction between Mg2+ (0.3, 0.8, and 3 mM) and 1,25-dihydroxyvitamin D3 (1,25D; 0.05 and 5 nM) during osteogenesis using bovine mesenchymal stem cells. After 21 days, differentiated osteocytes were subjected to OsteoImage analysis, alkaline phosphatase (ALP) activity measurements, and immunocytochemistry of NT5E, ENG (endoglin), SP7 (osterix), SPP1 (osteopontin), and the BGLAP gene product osteocalcin. The mRNA expression of NT5E, THY1, ENG, SP7, BGLAP, CYP24A1, VDR, SLC41A1, SLC41A2, SLC41A3, TRPM6, TRPM7, and NIPA1 was also assessed. Reducing the Mg2+ concentration in the medium increased the accumulation of mineral hydroxyapatite and ALP activity. There was no change in the immunocytochemical localization of stem cell markers. Expression of CYP24A1 was higher in all groups receiving 5 nM 1,25D. There were tendencies for higher mRNA abundance of THY1, BGLAP, and NIPA1 in cells receiving 0.3 mM Mg2+ and 5 nM 1,25D. In conclusion, low levels of Mg2+ greatly enhanced the deposition of bone hydroxyapatite matrix. The effect of Mg2+ was not modulated by 1,25D, although the expression of certain genes (including BGLAP) tended to be increased by the combination of low Mg2+ and high 1,25D concentrations.

Laser-Modified Ti Surface Improves Paracrine Osteogenesis by Modulating the Expression of DKK1 in Osteoblasts

Titanium surface modifications are widely used to modulate cellular behavior by recognition of topographical cues. However, how those modifications affect the expression of mediators that will influence neighboring cells is still elusive. This study aimed to evaluate the effects of conditioned media from osteoblasts cultured on laser-modified titanium surfaces on the differentiation of bone marrow cells in a paracrine manner and to analyze the expression of Wnt pathway inhibitors. Mice calvarial osteoblasts were seeded on polished (P) and Yb:YAG laser-irradiated (L) Ti surfaces. Osteoblast culture media were collected and filtered on alternate days to stimulate mice BMCs. Resazurin assay was performed every other day for 20 days to check BMC viability and proliferation. After 7 and 14 days of BMCs maintained with osteoblasts P and L-conditioned media, alkaline phosphatase activity, Alizarin Red staining, and RT-qPCR were performed. ELISA of conditioned media was conducted to investigate the expression of Wnt inhibitors Dickkopf-1 (DKK1) and Sclerostin (SOST). BMCs showed increased mineralized nodule formation and alkaline phosphatase activity. The L-conditioned media enhanced the BMC mRNA expression of bone-related markers Bglap, Alpl, and Sp7. L-conditioned media decreased the expression of DKK1 compared with P-conditioned media. The contact of osteoblasts with Yb:YAG laser-modified Ti surfaces induces the regulation of the expression of mediators that affect the osteoblastic differentiation of neighboring cells. DKK1 is among these regulated mediators.

Atf7ip Inhibits Osteoblast Differentiation via Negative Regulation of the Sp7 Transcription Factor

Epigenetic modifications are critical for cell differentiation and growth. As a regulator of H3K9 methylation, Setdb1 is implicated in osteoblast proliferation and differentiation. The activity and nucleus localization of Setdb1 are regulated by its binding partner, Atf7ip. However, whether Atf7ip is involved in the regulation of osteoblast differentiation remains largely unclear. In the present study, we found that Atf7ip expression was upregulated during the osteogenesis of primary bone marrow stromal cells and MC3T3-E1 cells, and was induced in PTH-treated cells. The overexpression of Atf7ip impaired osteoblast differentiation in MC3T3-E1 cells regardless of PTH treatment, as measured by the expression of osteoblast differentiation markers, Alp-positive cells, Alp activity, and calcium deposition. Conversely, the depletion of Atf7ip in MC3T3-E1 cells promoted osteoblast differentiation. Compared with the control mice, animals with Atf7ip deletion in the osteoblasts (Oc-Cre;Atf7ipf/f) showed more bone formation and a significant increase in the bone trabeculae microarchitecture, as reflected by μ-CT and bone histomorphometry. Mechanistically, Atf7ip contributed to the nucleus localization of Setdb1 in MC3T3-E1, but did not affect Setdb1 expression. Atf7ip negatively regulated Sp7 expression, and through specific siRNA, Sp7 knockdown attenuated the enhancing role of Atf7ip deletion in osteoblast differentiation. Through these data, we identified Atf7ip as a novel negative regulator of osteogenesis, possibly via its epigenetic regulation of Sp7 expression, and demonstrated that Atf7ip inhibition is a potential therapeutic measure for enhancing bone formation.

The Establishment of a Murine Mandibular Molar Extraction Socket Healing Model.

This study introduces the development of a molar extraction model in the murine mandible to provide a practicable model for studying alveolar bone regeneration and intramembranous ossification. C57/J6 mice were used to extract the mandibular first molar to establish this model. They were executed, and the bilateral mandibles harvested, at 1 week and 4 weeks post-surgery, respectively. Subsequent serial stereoscopic harvest, histological assessment, and immunofluorescence staining were performed to demonstrate successful surgery. Immediately after surgery, the stereoscopic images displayed an empty extraction socket. The hematoxylin and eosin (H&E) at 1 week and Masson staining at 4 weeks post-surgery showed that the area of the original root was partially and fully filled with bone trabeculae, respectively. The immunofluorescence staining showed that, compared with the homeostasis side, the Sp7 expression increased at 1 week post-surgery, suggesting vigorous osteogenesis in the alveolar fossa. All these results demonstrated a practicable murine tooth extraction socket healing model. Upcoming studies revealing the mechanisms of jawbone defect healing or socket healing could adopt this method.

PMON54 Exosomes Isolated from Conditioned Media of Immortalized Kisspeptin Neurons Exert Diverse Effects on Central and Peripheral in Vitro Cell Models.

Abstract Previous work in our laboratory explored the proteomic contents of exosome-like extracellular vesicles (EVs) released into the media in vitro from immortalized Kisspeptin (KP) neuronal cell lines KTaV-3 (derived from female mouse AVPV) and KTaR-1 (derived from KNDy neurons in the ARC). EVs were isolated from conditioned media via ultracentrifugation or filtration kit and validated using a NanoCyte, and LCMS-MS analysis revealed that relative abundance of exosomal cargo varied dependent upon estrogen (E2) exposure in vitro, with ∼150-170 proteins up-regulated and ∼200-220 proteins downregulated by E2 in EVs of KTaR-1 and KTaV-3 KP neurons. Since E2-regulated KP exosomal proteins included candidates implicated in the regulation of synaptic plasticity and signaling (i.e. annexins, semaphorins, connexins), we investigated the effects of exposure to purified EVs on gene expression in immortalized GnRH neurons (GT1-7 cells). Notably, EVs from 24h E2-treated KTaV-3 neurons induced increased expression of kiss1r in GT1-7 cells, in contrast to KTaV-3 neurons not exposed to E2, suggesting that AVPV KP neurons may signal an increase in KP receptivity in GnRH neurons in vivo via non-neuronal communication. Additionally, increases in expression of the synaptic scaffolding protein PSD-95 (dlg4) were seen in GT1-7 cells treated with E2-treated KTaV-3 EVs, suggesting AVPV KP neurons may use extracellular vesicles to modulate GnRH neuronal synaptic plasticity over the estrous cycle. While EVs isolated from KTaR-1 conditioned media did not alter GT1-7 kiss1r or dlg4 levels, treatment (24h) resulted in induction of selective gap junction hemichannel expression in GT1-7 cells. KTaR-1 (but not KTaV-3) EVs increased expression of Cx26 (gjb2) irrespective of E2 exposure, while induction of Cx43 (gja1) expression in GT1-7 cells was only observed following treatment with E2-deprived KTaR-1 EVs. Further, we found that KTaR-1 media and EVs can affect osteoblast function in vitro, including increases in sp7 and runx2 expression, E2-dependent modulation of wnt10b, and formation of bone matrix (evaluated by Alizarin Red assay) in cultured osteoblast lines, supporting recent studies implicating ARC KP neurons in bone remodeling. Lastly, we found significant levels of immunomodulatory pentraxins (PTX3) in KP EVs, with abundance dependent upon prior E2 exposure in KTaR-1 KP neurons, as revealed by ELISA. Together, results from these studies suggest that EVs may represent additional intercellular communication pathways utilized by Kiss-1 neurons to elicit changes in nearby neuronal populations and potentially even in the periphery, affecting inflammation and bone remodeling. Future studies will address mechanisms involved in the E2 regulation of exosomal cargo in these critical neuronal populations. Presentation: Monday, June 13, 2022 12:30 p.m. - 2:30 p.m.

Proteomic evaluation of human osteoblast responses to titanium implants over time.

Titanium is widely used in bone prostheses due to its excellent biocompatibility and osseointegration capacity. To understand the effect of sandblasted acid-etched (SAE) Ti implants on the biological responses of human osteoblast (HOb), their proteomic profiles were analyzed using nLC-MS/MS. The cells were cultured with the implant materials, and 2544 distinct proteins were detected in samples taken after 1, 3, and 7 days. Comparative analyses of proteomic data were performed using Perseus software. The expression of proteins related to EIF2, mTOR, insulin-secretion and IGF pathways showed marked differences in cells grown with SAE-Ti in comparison with cells cultured without Ti. Moreover, the proteomic profiles obtained with SAE-Ti were compared over time. The affected proteins were related to adhesion, immunity, oxidative stress, coagulation, angiogenesis, osteogenesis, and extracellular matrix formation functions. The proliferation, mineralization and osteogenic gene expression in HObs cultured with SAE-Ti were characterized in vitro. The results showed that the osteoblasts exposed to this material increase their mineralization rate and expression of COLI, RUNX2, SP7, CTNNB1, CAD13, IGF2, MAPK2, and mTOR. Overall, the observed proteomic profiles can explain the SAE-Ti osteogenic properties, widening our knowledge of key signaling pathways taking part in the early stages of the osseointegration process in this type of implantations.

Bioinspired Protein/Peptide Loaded 3D Printed PLGA Scaffold Promotes Bone Regeneration.

Background: This study was aimed to investigate the effect of three dimensional (3D)printed poly lactide-co-glycolide (PLGA) scaffolds combined with Gly-Phe-Hyp-Gly-Arg (GFOGER) and bone morphogenetic protein 9 (BMP-9) on the repair of large bone defects. Methods: 3D printing method was used to produce PLGA scaffolds, and the sample was viewed by both optical microscopy and SEM, XRD analysis, water absorption and compressive strength analysis, etc. The rabbits were divided into six groups randomly and bone defect models were constructed (6 mm in diameter and 9 mm in depth): control group (n = 2), sham group (n = 4), model group (n = 4) and model + scaffold group (n = 4 rabbits for each group, 0%,2% and 4%). The rabbits were sacrificed at the 4th and 12th weeks after surgery, and the samples were collected for quantitative analysis of new bone mineral density by micro-CT, histopathological observation, immunohistochemistry and Western blot to detect the protein expression of osteoblast-related genes. Results: This scaffold presented acceptable mechanical properties and slower degradation rates. After surface modification with GFOGER peptide and BMP-9, the scaffold demonstrated enhanced new bone mineral deposition and density over the course of a 12 week in vivo study. Histological analysis and WB confirmed that this scaffold up-regulated the expression of Runx7, OCN, COL-1 and SP7, contributing to the noted uniform trabeculae formation and new bone regeneration. Conclusions: The application of this strategy in the manufacture of composite scaffolds provided extensive guidance for the application of bone tissue engineering.

Complex effects of Mg-biomaterials on the osteoblast cell machinery: A proteomic study.

The cell-biomaterial interface is highly complex; thousands of molecules and many processes participate in its formation. Growing demand for improved biomaterials has highlighted the need to understand the structure and functions of this interface. Proteomic methods offer a viable alternative to the traditional in vitro techniques for analyzing such systems. Magnesium is a promoter of cell adhesion and osteogenesis. Here, we used the LC-MS/MS to compare the protein expression profiles of human osteoblasts (HOb) exposed to sol-gel coatings without (MT) and with Mg (MT1.5Mg) for 1, 3, and 7days. PANTHER, DAVID, and IPA databases were employed for protein identification and data analysis. Confocal microscopy and gene expression analysis were used for further characterization. Exposure to MT1.5Mg increased the HOb cell area and the expression of SP7, RUNX2, IBP3, COL3A1, MXRA8, and FBN1 genes. Proteomic analysis showed that MT1.5Mg affected the early osteoblast maturation (PI3/AKT, mTOR, ERK/MAPK), insulin metabolism, cell adhesion (integrin, FAK, actin cytoskeleton regulation) and oxidative stress pathways. Thus, the effects of Mg on cell adhesion and osteogenesis are rather complex, affecting several pathways rather than single processes. Our analysis also confirms the potential of proteomics in biomaterial characterization, showing a good correlation with in vitro results.

Fisetin: An Integrated Approach to Identify a Strategy Promoting Osteogenesis.

Flavonoids may modulate the bone formation process. Among flavonoids, fisetin is known to counteract tumor growth, osteoarthritis, and rheumatoid arthritis. In addition, fisetin prevents inflammation-induced bone loss. In order to evaluate its favorable use in osteogenesis, we assayed fisetin supplementation in both in vitro and in vivo models and gathered information on nanoparticle-mediated delivery of fisetin in vitro and in a microfluidic system. Real-time RT-PCR, Western blotting, and nanoparticle synthesis were performed to evaluate the effects of fisetin in vitro, in the zebrafish model, and in ex vivo samples. Our results demonstrated that fisetin at 2.5 µM concentration promotes bone formation in vitro and mineralization in the zebrafish model. In addition, we found that fisetin stimulates osteoblast maturation in cell cultures obtained from cleidocranial dysplasia patients. Remarkably, PLGA nanoparticles increased fisetin stability and, consequently, its stimulating effects on RUNX2 and its downstream gene SP7 expression. Therefore, our findings demonstrated the positive effects of fisetin on osteogenesis and suggest that patients affected by skeletal diseases, both of genetic and metabolic origins, may actually benefit from fisetin supplementation.

Pathological differences in the bone healing processes between tooth extraction socket and femoral fracture

Despite various reports on the bone healing processes of tooth extraction socket and long bone fracture, the differences of pathological changes during these healing processes remain elusive. This study aims to elucidate the underlying mechanisms behind the pathophysiology of bone regeneration between the tooth extraction socket and femoral fractures through a comparative study. Eight-week-old male mice were used in the experiments. The maxillary first molar was extracted, and intramedullary nailing femoral fracture (semistabilized fracture repair) was performed in the femur. Pathological changes in these bone injuries were investigated by micro-CT, histology, immunohistochemistry, and RT-PCR until day 7 post operation. Pathological changes in drill hole injury created in cortical bone of femur were also examined. Micro-CT analyses revealed increases in mineralized tissues in both the tooth extraction socket and femoral fracture. Histological examinations revealed that tooth socket was repaired by intramembranous ossification, and intramedullary nailing femoral fracture was healed by endochondral ossification. Immunohistochemical investigation revealed that tooth socket healing associated with Sp7-positive cells but not Sox9, aggrecan, and type II collagen, while femoral fracture models exhibited positive signals for all antibodies. RT-PCR analyses revealed the expression of Sp7, Col1a1, and Col2a1 in tooth socket healing, and the expression of Sp7, Col1a1, Runx2, Sox9, Acan, Col2a1, and Col10a1 in intramedullary nailing femoral fracture. Drill hole injury was repaired primarily by intramembranous ossification when the periosteum was removed before making the hole. The present study demonstrated that the absence of cartilage appearance during tooth extraction socket healing indicates it as distinctly different pathological features from the healing processes of semistabilized femoral fracture. This study contributes to the understanding of the molecular and cellular characteristics of bone healing among the different sites of bone injury.