Peptide-based drug development is a promising direction due to its excellent biological activity, minimal immunogenicity, high in vivo stability, and efficient tissue penetrability. GV1001, an amphiphilic peptide, has proven effective as an anti-cancer vaccine, but its effect on osteoblast differentiation is unknown. To identify proteins interacting with GV1001, biotin-conjugated GV1001 was constructed and confirmed by mass spectrometry. Proteomic analyses were performed to determine GV1001\'s interaction with osteogenic proteins. GV1001 was highly associated with peptidyl-prolyl isomerase A and co-immunoprecipitation assays revealed that GV1001 bound to peptidyl-prolyl cis-trans isomerase 1 (Pin1). GV1001 significantly increased alkaline phosphatase (ALP) activity, bone nodule formation, and the expression of osteogenic gene markers. GV1001-induced osteogenic activity was enhanced by Pin1 overexpression and abolished by Pin1 knockdown. GV1001 increased the protein stability and transcriptional activity of Runx2 and Osterix. Importantly, GV1001 administration enhanced bone mass density in the OVX mouse model, as verified by µCT analysis. GV1001 demonstrated protective effects against bone loss in OVX mice by upregulating osteogenic differentiation via the Pin1-mediated protein stabilization of Runx2 and Osterix. GV1001 could be a potential candidate with anabolic effects for the prevention and treatment of osteoporosis.

OBJECTIVE: To investigate the effect of TNF-α on osteogenic differentiation of stem cells from human exfoliated deciduous teeth (SHED), and to analyze the changes of ERK1/2-Runx2 signaling pathway in the regulation process.
METHODS: SHED cells were isolated and cultured from normal deciduous permanent teeth of healthy children aged 6-8 years old, and the third passage of SHED cells were taken and divided into control group (osteogenic inducer culture), observation group (osteogenic inducer and TNF-α co-culture) and agonist group (osteogenic inducer, TNF-α and ERK pathway agonist co-culture). The osteogenic differentiation was determined by alizarin red staining. The protein expression levels of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 in SHED cells were determined by Western blot. The expressions of Osterix, OPN, ERK1/2, pERK1/2 and Runx2 mRNA were detected by qRT-PCR. Statistical analysis was performed with SPSS 26.0 software package.
RESULTS: Comparison of osteogenic differentiation ability of the three groups of cells showed that red-brown mineralized nodules were observed in the three groups of cells. Compared among the three groups, the control group had the most mineralized nodules, followed by the activation group, and the observation group had the least mineralized nodules. Compared with the control group, the expression levels of Osterix and OPN protein and mRNA in the observation group and the agonist group were significantly decreased, while the expression levels of Osterix and OPN protein and mRNA in the agonist group were significantly higher than those in the observation group. There was no significant difference in the expression levels of ERK1/2 protein and mRNA among the three groups, while the expression levels of pERK1/2 and Runx2 protein and mRNA in the observation group and the agonist group were significantly higher than those in the control group, and the expression levels of pERK1/2 and Runx2 protein and mRNA in the agonist group were significantly higher than those in the observation group.
CONCLUSIONS: TNF-α can inhibit osteogenic differentiation of SHED cells, which may be related to the inhibition of ERK1/2-Runx2 signaling pathway.

BACKGROUND: Craniofacial skeletal deformities can be addressed by applying tensile force to sutures to prompt sutural bone formation. The intricate process of mechanical modulation in craniofacial sutures involves complex biomechanical signal transduction. The small GTPase Ras homolog gene family member A (RhoA) functions as a key mechanotransduction protein, orchestrating the dynamic assembly of the cytoskeleton by activating the Rho-associated coiled-coil containing protein kinase (ROCK). Transcriptional coactivator with PDZ-binding motif (TAZ) serves as a crucial mediator in the regulation of genes and the orchestration of biological functions within the mechanotransduction signaling pathway. However, the role of RhoA/ROCK-TAZ in trans-sutural distraction osteogenesis has not been reported.
METHODS: We utilized pre-osteoblast-specific RhoA deletion mice to establish an in vivo calvarial trans-sutural distraction model and an in vitro mechanical stretch model for pre-osteoblasts isolated from neonatal mice. Micro-CT and histological staining were utilized to detect the formation of new bone in the sagittal suture of the skull as well as the activation of RhoA, Osterix and TAZ. The activation of ROCK-limk-cofilin and the nuclear translocation of TAZ in pre-osteoblasts under mechanical tension were detected through Western blot, qRT-PCR, and immunofluorescence.
RESULTS: The osteogenic differentiation of pre-osteoblasts was facilitated by mechanical tension through the activation of RhoA and Rho-associated kinase (ROCK), while ablation of RhoA impaired osteogenesis by inhibiting pre-osteoblast differentiation after suture expansion. Furthermore, inhibiting RhoA expression could block tensile-stimulated nuclear translocation of TAZ by preventing F-actin assembly through ROCK-LIM-domain kinase (LIMK)-cofilin pathway. In addition, the TAZ agonist TM-25659 could attenuate impaired osteogenesis caused by ablation of RhoA in pre-osteoblasts by increasing TAZ nuclear accumulation.
CONCLUSIONS: This study demonstrates that mechanical stretching promotes the osteogenic differentiation of pre-osteoblasts in trans-sutural distraction osteogenesis, and this process is mediated by the RhoA/ROCK-TAZ signaling axis. Overall, our results may provide an insight for potential treatment strategies for craniosynostosis patients through trans-sutural distraction osteogenesis.

OBJECTIVE: To assess the short-term efficacy of multiple sessions of antimicrobial photodynamic therapy (aPDT), light-emitting-diode (LED) photobiomodulation, and topical ozone therapy applications following surgical regenerative treatments on clinical parameters, patient-centered outcomes, and mRNA expression levels of VEGF, IL-6, RunX2, Nell-1, and osterix in gingival crevicular fluid samples in patients with stage III/IV, grade C periodontitis.
METHODS: Forty-eight systemically healthy patients were assigned into four groups to receive adjunctive modalities with regenerative periodontal surgical treatment. A 970 ± 15 nm diode laser plus indocyanine-green for aPDT group, a 626 nm LED for photobiomodulation group, and topical gaseous ozone were applied at 0, 1, 3, and 7 postoperative days and compared to control group. The clinical periodontal parameters, early wound healing index (EHI), and postoperative patients\' morbidity were evaluated. The mRNA levels of biomarkers were assessed by real-time polymerase chain reaction.
RESULTS: No significant difference in the clinical parameters except gingival recession (GR) was identified among the groups. For group-by-time interactions, plaque index (PI) and probing pocket depths (PD) showed significant differences (p = 0.034; p = 0.022). In sites with initial PD > 7 mm, significant differences were observed between control and photobiomodulation groups in PD (p = 0.011), between control and aPDT, and control and photobiomodulation groups in CAL at 6-month follow-up (p = 0.007; p = 0.022). The relative osterix mRNA levels showed a statistically significant difference among the treatment groups (p = 0.014).
CONCLUSIONS: The additional applications of aPDT and LED after regenerative treatment of stage III/IV grade C periodontitis exhibited a more pronounced beneficial effect on clinical outcomes in deep periodontal pockets.

BACKGROUND: Mechanical stimulation (MS) significantly increases the release of adenine and uracil nucleotides from bone marrow-derived mesenchymal stem cells (BM-MSCs) undergoing osteogenic differentiation. Released nucleotides acting via ionotropic P2X7 and metabotropic P2Y6 purinoceptors sensitive to ATP and UDP, respectively, control the osteogenic commitment of BM-MSCs and, thus, bone growth and remodelling. Yet, this mechanism is impaired in post-menopausal (Pm)-derived BM-MSCs, mostly because NTPDase3 overexpression decreases the extracellular accumulation of nucleotides below the levels required to activate plasma membrane-bound P2 purinoceptors. This prompted us to investigate whether in vitro MS of BM-MSCs from Pm women could rehabilitate their osteogenic commitment and whether xenotransplantation of MS purinome-primed Pm cells promote repair of critical bone defects in an in vivo animal model.
METHODS: BM-MSCs were harvested from the neck of femora of Pm women (70 ± 3 years old) undergoing total hip replacement. The cells grew, for 35 days, in an osteogenic-inducing medium either submitted (SS) or not (CTR) to MS (90 r.p.m. for 30 min) twice a week. Increases in alkaline phosphatase activity and in the amount of osteogenic transcription factors, osterix and osteopontin, denoted osteogenic cells differentiation, while bone nodules formation was ascertain by the alizarin red-staining assay. The luciferin-luciferase bioluminescence assay was used to quantify extracellular ATP. The kinetics of the extracellular ATP (100 µM) and UDP (100 µM) catabolism was assessed by HPLC. The density of P2Y6 and P2X7 purinoceptors in the cells was assessed by immunofluorescence confocal microscopy. MS-stimulated BM-MSCs from Pm women were xenotransplanted into critical bone defects drilled in the great trochanter of femora of one-year female Wistar rats; bone repair was assessed by histological analysis 10 days after xenotransplantation.
RESULTS: MS-stimulated Pm BM-MSCs in culture (i) release 1.6-fold higher ATP amounts, (ii) overexpress P2X7 and P2Y6 purinoceptors, (iii) exhibit higher alkaline phosphatase activity and overexpress the osteogenic transcription factors, osterix and osteopontin, and (iv) form larger bone nodules, than CTR cells. Selective blockage of P2X7 and P2Y6 purinoceptors with A438079 (3 µM) and MRS 2578 (0.1 µM), respectively, prevented the osteogenic commitment of cultured Pm BM-MSCs. Xenotransplanted MS purinome-primed Pm BM-MSCs accelerated the repair of critical bone defects in the in vivo rat model.
CONCLUSIONS: Data suggest that in vitro MS restores the purinergic cell-to-cell communication fostering the osteogenic differentiation and osteointegration of BM-MSCs from Pm women, a strategy that may be used in bone regeneration and repair tactics.

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed \"Cre-only\" Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

The Linker of Nucleoskeleton and Cytoskeleton (LINC) complex is a crucial connective component between the nuclear envelope and the cytoskeleton involving various cellular processes including nuclear positioning, nuclear architecture, and mechanotransduction. How LINC complexes regulate bone formation in vivo, however, is not well understood. To start bridging this gap, here we created a LINC disruption murine model using transgenic mice expressing Cre recombinase enzyme under the control of the Osterix (Osx-Cre) which is primarily active in pre-osteoblasts and floxed Tg(CAG-LacZ/EGFP-KASH2) mice. Tg(CAG-LacZ/EGFP-KASH2) mice contain a lox-STOP-lox flanked LacZ gene which is deleted upon cre recombination allowing for the overexpression of an EGFP-KASH2 fusion protein. This overexpressed protein disrupts endogenous Nesprin-Sun binding leading to disruption of LINC complexes. Thus, crossing these two lines results in an  Osx- driven  LINC  disruption (ODLD) specific to pre-osteoblasts. In this study, we investigated how this LINC disruption affects exercise-induced bone accrual. ODLD cells had decreased osteogenic and adipogenic potential in vitro compared to non-disrupted controls and sedentary ODLD mice showed decreased bone quality at 8 weeks. Upon access to a voluntary running wheel, ODLD animals showed increased running time and distance; however, our 6-week exercise intervention did not significantly affect bone microarchitecture and bone mechanical properties.

OBJECTIVE: To explore the effect of protein arginine methyltransferase 5 (PRMT5) on tooth extraction sockets healing, we established an extraction sockets model in osteoblast-conditional Prmt5 knockout mice. The results provided clues for promoting extraction sockets healing in clinical settings.
METHODS: Maxillary first molars were extracted from 6 to 8-week-old mice to establish an extraction fossa model. Microcomputed tomography (Micro-CT), histology, and immunostaining assays were performed on samples harvested at 3-, 7-, and 14-day post-extraction. Prmt5-silenced cell lines  were employed to explore the regulatory mechanisms underlying the osteigenic differentiation.
RESULTS: PRMT5 expression was higher in the early stage of socket healing. Micro-CT analysis showed that the percentage of new bone in the extraction sockets was lower in OC-Cre; Prmt5fl/fl mice than in the control group, consistent with Masson staining. We found that, Prmt5 deficiency delayed the osteogenesis during extraction socket healing, which might be achieved through the decrease of H4R3me2s in the Sp7 promoter region.
CONCLUSIONS: PRMT5 in osteoblasts may promote the differentiation of osteoblasts by regulating the Sp7 promoter H4R3me2s and participate in the healing of tooth extraction sockets.

OBJECTIVE: After tooth extraction, preservation of the alveolar ridge by socket grafting attenuates bone resorption. Runt-related transcription factor 2 (RUNX2) and SP7/Osterix (OSX) are transcription factors playing an important role in osteoblast differentiation. The purpose of this study was to evaluate the effects of carbonate apatite (CO3Ap) on osteoblast-related gene and protein expression after socket grafting.
METHODS: Alveolar bone and new bone after CO3Ap grafting were collected at the time of implant placement. Levels of mRNA for RUNX2, SP7/OSX, bone morphogenetic protein 2 (BMP2), BMP7 and platelet derived growth factor B were determined by real-time PCR. Immunostaining was performed using antibodies against RUNX2, SP7/OSX, vimentin and cytokeratin. To evaluate bone resorption rates, cone-beam CT (CBCT) imaging was performed after socket grafting and before implant placement.
RESULTS: CBCT imaging showed that the average degree of bone resorption at the CO3Ap graft site was 7.15 ± 3.79%. At the graft sites, levels of SP7/OSX and BMP2 mRNA were significantly increased. Replacement of CO3Ap with osteoid was evident histologically, and in the osteoid osteoblast-like cells were stained for SP7/OSX and vimentin.
CONCLUSIONS: These results show that gene expression of both SP7/OSX and BMP2 can be induced by CO3Ap, suggesting that increased expression of SP7/OSX and vimentin may be involved in the BMP pathway.

Deubiquitinases (DUBs) are essential for bone remodeling by regulating the differentiation of osteoblast and osteoclast. USP17 encodes for a deubiquitinating enzyme, specifically known as ubiquitin-specific protease 17, which plays a critical role in regulating protein stability and cellular signaling pathways. However, the role of USP17 during osteoblast differentiation has not been elusive. In this study, we initially investigated whether USP17 could regulate the differentiation of osteoblasts. Moreover, USP17 overexpression experiments were conducted to assess the impact on osteoblast differentiation induced by bone morphogenetic protein 4 (BMP4). The positive effect was confirmed through alkaline phosphatase (ALP) expression and activity studies since ALP is a representative marker of osteoblast differentiation. To confirm this effect, Usp17 knockdown was performed, and its impact on BMP4-induced osteoblast differentiation was examined. As expected, knockdown of Usp17 led to the suppression of both ALP expression and activity. Mechanistically, it was observed that USP17 interacted with Osterix (Osx), which is a key transcription factor involved in osteoblast differentiation. Furthermore, overexpression of USP17 led to an increase in Osx protein levels. Thus, to investigate whether this effect was due to the intrinsic function of USP17 in deubiquitination, protein stabilization experiments and ubiquitination analysis were conducted. An increase in Osx protein levels was attributed to an enhancement in protein stabilization via USP17-mediated deubiquitination. In conclusion, USP17 participates in the deubiquitination of Osx, contributing to its protein stabilization, and ultimately promoting the differentiation of osteoblasts.