Osteogenic Differentiation Genes Research Articles

Secretome Derived From Mesenchymal Stem Cells Cultured as Monolayer Show Enhanced Bone Regeneration Compared to Secretome From 3D Spheroid - Clues From the Proteome.

Repair and reconstruction of critical-sized bone defects present a significant challenge due to poor clinical outcomes of conventional bone repair strategies, such as autologous and allogenic bone grafts. The present study underscores the potential of human dental pulp stem cell-derived trophic factors to promote bone repair and regeneration, thus evading the risks associated with cell-based therapy. This study utilizes pre-osteoblast cells to evaluate the osteogenic potential of 2 Dimensional (2D) and 3 Dimensional (3D) secretome from monolayer and spheroid cultures of dental pulp stem cells (DPSCs), respectively. In-vitro results on pre-osteoblast cells (MC3T3-EI) treated with 2D and 3D secretome reveal lower mineralization and mRNA expression of osteogenic specific genes in 3D secretome in comparison to 2D secretome. Furthermore, 2D secretome shows better bone regeneration ability in rat models of calvarial bone defect compared to the 3D secretome. The proteomic profiles of 2D and 3D secretomes are also in concordance with these results and reveal key molecules governing bone regeneration potential. This data highlights the influence of culture conditions on the secretory pattern of mesenchymal stem cells and provides valuable insights for the development of a more effective secretome-based cell-free alternative for novel bone repair and regeneration.

3D-printed Ti3C2/polycaprolactone composite scaffold with a DOPA-SDF1 surface modified for bone repair.

3D-printed Ti3C2/polycaprolactone composite scaffold with a DOPA-SDF1 surface modified for bone repair.

Piceatannol promotes the osteogenic differentiation of human periodontal ligament cells through MAPKs activation.

We investigated the effects of piceatannol on the osteogenic differentiation of human periodontal ligament fibroblasts (HPLFs). Treatment with piceatannol increased alkaline phosphatase activity and osteogenic differentiation gene molecules (Osteocalcin and Runx2), but this was attenuated by mitogen-activated protein kinase (MAPK) inhibitors (PD098059 or SB203580). Our data suggest that piceatannol promotes osteogenic differentiation of HPLFs by activating MAPKs.

Osteogenesis Differentiation and Molecular Mechanism Study of a Si and Mg Dual-Ion System Based on mRNA Transcriptomic Sequencing Analysis.

Both silicon (Si) and magnesium (Mg) ions play essential roles in bone health. However, the precise mechanisms by which these two ions enhance osteogenic differentiation remain to be fully elucidated. Herein, a Si-Mg dual-ion system was designed to investigate the effects of Si and Mg ions on the cytological behavior of mouse bone marrow mesenchymal stem cells (mBMSCs). The molecular mechanism of the Si-Mg dual-ion system regulating osteogenic differentiation of mBMSCs was investigated by transcriptome sequencing technology. In the single-ion system, the Si group with concentrations of 1.5 and 0.75 mM exhibited good combined effects (cell proliferation, alkaline phosphatase (ALP) activity, and osteogenic differentiation gene expression (Runx2, OPN, and Col-I)) of mBMSCs. The Mg group with concentrations of 5 and 2.5 mM showed better combined effects (cell proliferation, ALP activity, and osteogenic differentiation gene expression) of mBMSCs. In the dual-ion system, the silicon (0.75 mM)-magnesium (2.5 mM) experimental group significantly enhanced the proliferation, ALP activity, and osteogenesis-related gene expression (Runx2, OPN, and Col-I) of mBMSCs. The analysis of transcriptome sequencing results showed that Mg ions had a certain pro-stem cell osteogenic differentiation regulatory effect. Si ions had a stronger regulation on osteogenic differentiation than the Mg ions. The regulation of osteogenic differentiation by Si-Mg dual ions was synergistically enhanced compared to that of a single ion. In addition, the transforming growth factor beta (TGF-β) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway were involved in mediating the pro-stem cell osteogenic differentiation by Si-Mg dual ions. This study sheds light on investigating the molecular mechanism of dual-ion regulation of the osteogenic differentiation of mBMSCs and enriches the theory of ion-regulating osteogenic differentiation.

A novel PTH1R mutation causes primary failure of eruption via the cAMP-PI3K/AKT pathway

BackgroundPrimary failure of eruption (PFE) is a rare disorder characterized by a posterior open bite. While mutations in the parathyroid hormone 1 receptor (PTH1R) gene have been demonstrated to cause PFE, the underlying mechanisms remain largely unknown.MethodsWhole exome sequencing was conducted to identify PTH1R variants in a PFE family. MG63 cells that stably expressed the corresponding mutant PTH1R were established using lentiviruses. Next, osteogenesis was assessed by measuring cell alkaline phosphatase activity, conducting alizarin red staining, and evaluating osteoblast-specific gene expression. Then, computational analysis of binding affinity and RNA sequencing were carried out. Lastly, rescue experiments were performed to validate the mechanism underlying the pathogenesis of PFE.ResultsA novel PTH1R missense mutation (c.904G > A, p.E302K) was identified in a Chinese family affected by PFE. Moreover, the E302K mutation inhibited the expression of osteogenic-specific genes and proteins in MG63 cells. Computational analysis revealed the E302K mutation decreased the binding affinity of Gαs to the PTH1R protein. Consistently, cAMP accumulation assays demonstrated that the E302K mutation impaired the intracellular PTH1-34 -induced accumulation of cAMP. Further RNA sequencing analysis and validation experiments revealed that the PI3K-AKT signaling pathway was predominantly down-regulated in response to the E302K mutation. Finally, forskolin partially restored the effects of the E302K mutation on osteogenesis.ConclusionsThis study indicated that the E302K mutation in PTH1R decreased the binding affinity of PTH1R protein for Gαs, down-regulated the cAMP-PI3K/AKT signaling pathway, and inhibited osteogenesis, eventually leading to PFE. This study not only expands the genotypic spectrum of PTH1R mutations but also elucidates the underlying pathogenic mechanism of PTH1R-associated PFE.

Artificial light at night effects glucose metabolism in the developing jawbone by inhibiting melatonin secretion

Objective: To investigate the effects of artificial light at night on the growth of mandibles in mice and its regulatory mechanisms. Methods: A mouse model of artificial light at night (night light pollution group) and normal lighting (normal light group) was established by controlling light exposure time, with 4 mice in each group. Micro-CT was employed to analyze the differences in bone quantities of the mandibles between the two groups. Real-time fluorescence quantitative PCR (RT-qPCR) was used to examine the expression levels of osteogenic differentiation and metabolism-related genes in the cortical bone and condylar ossification center of the mandibles. Enzyme-linked immunosorbent assay was utilized to assess the diurnal variation of serum melatonin concentrations between the two groups. The artificial light at night experimental group received daily timed injections of a defined dose of melatonin to restore the diurnal variation of serum melatonin concentration in the mice, while the normal light group and the artificial light at night control group received the same volume of saline. Bone quantities, mandibular tissue morphologies, ossification differentiation in the condylar region and cortical bone, as well as glucose metabolism expression differences were assessed across the three groups. Results: The cortical bone thickness of the mandibles in the artificial light at night group [(0.196±0.017) mm] was significantly less than that in the control group [ (0.228±0.007) mm] (P=0.029). The bone volume fraction of the condylar ossification center in the artificial light at night group [(36.90±1.09) %] was significantly lower than that in the normal light group [(54.24±1.49) %] (P<0.001). The length of the mandible in the artificial light at night group [(10.86±0.17) mm] was significantly shorter than that in the normal light group [(11.41±0.32) mm] (P=0.032). RT-qPCR results indicated that the expressions of osteogenic-related genes alkaline phosphatase, liver/bone/kidney (Alpl), osteocalcin (Ocn), Runt-related transcription factor 2 (Runx2), and osterix (Osx) in the cortical bone and condylar ossification center of mice in the artificial light at night group were significantly lower than those in the normal light group (all P<0.05). The expression of metabolism-related genes protein kinase, AMP activated alpha 1 (Prkaa1), V-type proton ATPase subunit d1 (Atp6v0d1), and cytochrome C oxidase subunit Ⅳ isoform 1 (Cox4i1) in the cortical bone and condylar ossification center of mice in the artificial light at night group were also significantly reduced compared to normal mice (all P<0.01). The serum melatonin concentration peaked 8 hours after lights off in the normal light group, whereas the artificial light at night group exhibited a significantly reduced nocturnal serum melatonin concentration with no apparent peak compared to the normal light group. Micro-CT results demonstrated that after artificial light at night group mice received timed melatonin supplementation daily, the thickness of cortical bone, the bone volume fraction of the condylar ossification center, and the length of the mandible were all significantly higher than those in the artificial light at night group (all P<0.05). Histological staining results indicated that the cortical bone structure of the mandibles in the melatonin supplementation group was more organized than that of the artificial light at night group, with higher brain and muscle ARNT-like 1 (BMAL1) expression (P=0.003). RT-qPCR results further showed that the expression levels of Prkaa1, Atp6v0d1, and Cox4i1 in the cortical bone and condylar ossification center significantly increased in the melatonin supplementation group compared to the artificial light at night group (all P<0.05), but still significantly lower than those in the normal light group (all P<0.05). Additionally, the RT-qPCR results further revealed that the expression levels of osteogenic differentiation-related genes Alpl, Ocn, and Runx2 in the cortical bone and condylar ossification center of the melatonin supplementation group were significantly higher than those in the artificial light at night group (all P<0.01). Western blotting analysis indicated that the expression levels of glucose metabolism and osteogenic-related proteins RUNX2, OSX, ATP6V0D1, and COX Ⅳ, along with the phosphorylation levels of AMPKα1/α2, were significantly higher in the melatonin supplementation group compared to the artificial light at night group (all P<0.01). Conclusions: Artificial light at night can inhibit melatonin secretion in mice, reduce glucose metabolism in mandibular tissues, and affect both intramembranous and chondrogenic ossification activities, ultimately leading to inadequate mandibular development.

A glucocorticoid spike derails muscle repair to heterotopic ossification after spinal cord injury.

A glucocorticoid spike derails muscle repair to heterotopic ossification after spinal cord injury.

Loss of ZC4H2, an Arthrogryposis Multiplex Congenita Associated Gene, Promotes Osteoclastogenesis in Mice.

ZC4H2 encodes a C4H2-type zinc finger protein, mutations of which lead to a spectrum of diseases known as ZC4H2 associated rare disorders (ZARD). In addition to neurological phenotypes, the most typical symptoms of ZARD are multiple joint contractures of varying degrees, accompanied by abnormal development of muscles and bones, and osteoporosis in some cases. The pathogenic mechanisms of such bone related phenotypes, however, remain unclear. Here, we showed that ZC4H2 is expressed in the developing bones in mice. ZC4H2 knockout mice were neonatal-lethal and smaller in size, with reduced calcification of long bones. Upon induced loss of ZC4H2 postnatally, the femoral bones developed an osteoporosis-like phenotype, with reduced bone mineral density, bone-volume fraction, and trabecular bone number. Knockdown of ZC4H2 showed no clear effect on the expression of osteogenic differentiation genes in in vitro models using mesenchymal stem cells. Interestingly, ZC4H2 knockdown significantly enhanced osteoclast differentiation and bone resorption in induced bone marrow-derived macrophages. We further confirmed that the number of osteoclasts in the long bone of ZC4H2 knockout mice was increased, as well as the expression of the serum bone resorption/osteoporosis marker CTX-1. Our study unveils a new role of ZC4H2 in osteoclast differentiation and bone development, providing new clues on the pathology of ZARD.

A functional analysis of a resorbable citrate-based composite tendon anchor

A functional analysis of a resorbable citrate-based composite tendon anchor

Fibroblast growth factor 23-mediated regulation of osteoporosis: Assessed via Mendelian randomization and invitro study.

Despite numerous investigations on the influence of fibroblast growth factor 23 (FGF23), α-Klotho and FGF receptor-1 (FGFR1) on osteoporosis (OP), there is no clear consensus. Mendelian randomization (MR) analysis was conducted on genome-wide association studies (GWASs)-based datasets to evaluate the causal relationship between FGF23, α-Klotho, FGFR1 and OP. The primary endpoint was the odds ratio (OR) of the inverse-variance weighted (IVW) approach. Furthermore, we stably transfected FGF23-mimic or siRNA-FGF23 into human bone marrow mesenchymal stem cells (hBMSCs) in culture and determined its cell proliferation and the effects on osteogenic differentiation. Using MR analysis, we demonstrated a strong correlation between serum FGF23 levels and Heel- and femoral neck-BMDs, with subsequent ORs of 0.919 (95% CI: 0.860-0.983, p = 0.014) and 0.751 (95% CI: 0.587-0.962; p = 0.023), respectively. The expression levels of FGF23 were significantly increased in femoral neck of patients with OP than in the control cohort (p < 0.0001). Based on our invitro investigation, after overexpression of FGF23, compared to the control group, the BMSC's proliferation ability decreased, the expression level of key osteogenic differentiation genes (RUNX2, OCN and OSX) significantly reduced, mineralized nodules and ALP activity significantly decreased. After silencing FGF23, it showed a completely opposite trend. Augmented FGF23 levels are causally associated with increased risk of OP. Similarly, FGF23 overexpression strongly inhibits the osteogenic differentiation of hBMSCs, thereby potentially aggravating the pathological process of OP.

The effects of SDF-1 combined application with VEGF on femoral distraction osteogenesis in rats.

Bone regeneration and mineralization can be achieved by means of distraction osteogenesis (DO). In the present study, we investigated the effect of stromal cell-derived factor 1 (SDF-1) and vascular endothelial growth factor (VEGF) on the new bone formation during DO in rats. Forty-eight Sprague-Dawley rats were randomized into four groups of 12 rats each. We established the left femoral DO model in rats and performed a mid-femoral osteotomy, which was fixed with an external fixator. DO was performed at 0.25 mm/12 h after an incubation period of 5 days. Distraction was continued for 10 days, resulting in a total of 5 mm of lengthening. After distraction, the solution was locally injected into the osteotomy site, once a day 1 ml for 1 week. One group received the solvent alone and served as the control, and the other three groups were treated with SDF-1, VEGF, and SDF-1with VEGF in an aqueous. Sequential X-ray radiographs were taken two weekly. The regeneration was monitored with the use of micro-CT analysis, mechanical testing, and histology. Radiographs showed accelerated regenerate ossification in the SDF-1, VEGF, and SDF-1 with the VEGF group, with a larger amount of new bone compared with the control group, especially SDF-1 with the VEGF group. Micro-CT analysis and biomechanical tests showed Continuous injection of the SDF-1, VEGF, and SDF-1 with VEGF during the consolidation period significantly increased bone mineral density bone volume, mechanical maximum loading, and bone mineralization of the regenerate. Similarly, the expression of osteogenic-specific genes, as determined by real-time polymerase chain reaction , was significantly higher in SDF-1 with the VEGF group than in the other groups. Histological examination revealed more new trabeculae in the distraction gap and more mature bone tissue for the SDF-1 with the VEGF group. SDF-1 and VEGF promote bone regeneration and mineralization during DO, and there is a synergistic effect between the SDF-1 and VEGF. It is possible to provide a new and feasible method to shorten the period of treatment of DO.

Mir-150-5p inhibits the osteogenic differentiation of bone marrow-derived mesenchymal stem cells by targeting irisin to regulate the p38/MAPK signaling pathway

PurposeTo study the effect of miR-150-5p on the osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs), and further explore the relationship between its regulatory mechanism and irisin.MethodsWe isolated mouse BMSCs, and induced osteogenic differentiation by osteogenic induction medium. Using qPCR to detect the expression of osteogenic differentiation-related genes, western blot to detect the expression of osteogenic differentiation-related proteins, and luciferase reporter system to verify that FNDC5 is the target of miR-150-5p. Irisin intraperitoneal injection to treat osteoporosis in mice constructed by subcutaneous injection of dexamethasone.ResultsUp-regulation of miR-150-5p inhibited the proliferation of BMSCs, and decreased the content of osteocalcin, ALP activity, calcium deposition, the expression of osteogenic differentiation genes (Runx2, OSX, OCN, OPN, ALP and BMP2) and protein (BMP2, OCN, and Runx2). And down-regulation of miR-150-5p plays the opposite role of up-regulation of miR-150-5p on osteogenic differentiation of BMSCs. Results of luciferase reporter gene assay showed that FNDC5 gene was the target gene of miR-150-5p, and miR-150-5p inhibited the expression of FNDC5 in mouse BMSCs. The expression of osteogenic differentiation genes and protein, the content of osteocalcin, ALP activity and calcium deposition in BMSCs co-overexpressed by miR-150-5p and FNDC5 was significantly higher than that of miR-150-5p overexpressed alone. In addition, the overexpression of FNDC5 reversed the blocked of p38/MAPK pathway by the overexpression of miR-150-5p in BMSCs. Irisin, a protein encoded by FNDC5 gene, improved symptoms in osteoporosis mice through intraperitoneal injection, while the inhibitor of p38/MAPK pathway weakened this function of irisin.ConclusionmiR-150-5p inhibits the osteogenic differentiation of BMSCs by targeting irisin to regulate the/p38/MAPK signaling pathway, and miR-150-5p/irisin/p38 pathway is a potential target for treating osteoporosis.

In-depth investigation of FAM20A insufficiency effects on deciduous dental pulp cells: Altered behaviours, osteogenic differentiation, and inflammatory gene expression.

Loss-of-function mutations in FAM20A result in amelogenesis imperfecta IG (AI1G) or enamel-renal syndrome, characterized by hypoplastic enamel, ectopic calcification, and gingival hyperplasia, with some cases reporting spontaneous tooth infection. Despite previous reports on the consequence of FAM20A reduction in gingival fibroblasts and transcriptome analyses of AI1G pulp tissues, suggesting its involvement in mineralization and infection, its role in deciduous dental pulp cells (DDP) remains unreported. The aim of this study was to evaluate the properties of DDP obtained from an AI1G patient, providing additional insights into the effects of FAM20A on the mineralization of DDP. DDP were obtained from a FAM20A-AI1G patient (mutant cells) and three healthy individuals. Cellular behaviours were examined using flow cytometry, MTT, attachment and spreading, colony formation, and wound healing assays. Osteogenic induction was applied to DDP, followed by alizarin red S staining to assess their osteogenic differentiation. The expression of FAM20A-related genes, osteogenic genes, and inflammatory genes was analysed using real-time PCR, Western blot, and/or immunolocalization. Additionally, STRING analysis was performed to predict potential protein-protein interaction networks. The mutant cells exhibited a significant reduction in FAM20A mRNA and protein levels, as well as proliferation, migration, attachment, and colony formation. However, normal FAM20A subcellular localization was maintained. Additionally, osteogenic/odontogenic genes, OSX, OPN, RUNX2, BSP, and DSPP, were downregulated, along with upregulated ALP. STRING analysis suggested a potential correlation between FAM20A and these osteogenic genes. After osteogenic induction, the mutant cells demonstrated reduced mineral deposition and dysregulated expression of osteogenic genes. Remarkably, FAM20A, FAM20C, RUNX2, OPN, and OSX were significantly upregulated in the mutant cells, whilst ALP, and OCN was downregulated. Furthermore, the mutant cells exhibited a significant increase in inflammatory gene expression, that is, IL-1β and TGF-β1, whereas IL-6 and NFκB1 expression was significantly reduced. The reduction of FAM20A in mutant DDP is associated with various cellular deficiencies, including delayed proliferation, attachment, spreading, and migration as well as altered osteogenic and inflammatory responses. These findings provide novel insights into the biology of FAM20A in dental pulp cells and shed light on the molecular mechanisms underlying AI1G pathology.

Nanoclay Hydrogel Microspheres with a Sandwich-Like Structure for Complex Tissue Infection Treatment.

Addressing complex tissue infections remains a challenging task because of the lack of effective means, and the limitations of traditional bioantimicrobial materials in single-application scenarios hinder their utility for complex infection sites. Hence, the development of a bioantimicrobial material with broad applicability and potent bactericidal activity is necessary to treat such infections. In this study, a layered lithium magnesium silicate nanoclay (LMS) is used to construct a nanobactericidal platform. This platform exhibits a sandwich-like structure, which is achieved through copper ion modification using a dopamine-mediated metallophenolic network. Moreover, the nanoclay is encapsulated within gelatin methacryloyl (GelMA) hydrogel microspheres for the treatment of complex tissue infections. The results demonstrate that the sandwich-like micro- and nanobactericidal hydrogel microspheres effectively eradicated Staphylococcus aureus (S. aureus) while exhibiting excellent biocompatibility with bone marrow-derived mesenchymal stem cells (BMSCs) and human umbilical vein endothelial cells (HUVECs). Furthermore, the hydrogel microspheres upregulated the expression levels of osteogenic differentiation and angiogenesis-related genes in these cells. In vivo experiments validated the efficacy of sandwich-like micro- and nanobactericidal hydrogel microspheres when injected into deep infected tissues, effectively eliminating bacteria and promoting robust vascular regeneration and tissue repair. Therefore, these innovative sandwich-like micro- and nanobacteriostatic hydrogel microspheres show great potential for treating complex tissue infections.

Effects of sitagliptin activation of the stromal cell-derived factor-1/CXC chemokine receptor 4 signaling pathway on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells induced by lipopolysaccharide.

This study aimed to investigate the effects of sitagliptin on the proliferation, apoptosis, inflammation, and osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) in lipopolysaccharide (LPS)-induced inflammatory microenvironment and its molecular mechanism. hPDLSCs were cultured in vitro and treated with different concentrations of sitagliptin to detect cell viability and subsequently determine the experimental concentration of sitagliptin. An hPDLSCs inflammation model was established after 24 h of stimulation with 1 µg/mL LPS and divided into blank, control, low-concentration sitagliptin (0.5 µmol/L), medium-concentration sitagliptin (1 µmol/L), and high-concentration sitagliptin (2 µmol/L), high-concentrationsitagliptin+stromal cell derived factor-1 (SDF-1)/CXC chemokine receptor 4 (CXCR4) pathway inhibitor (AMD3100) (2 µmol/L+10 µg/mL) groups. A cell-counting kit-8 was used to detect the proliferation activity of hPDLSCs after 24, 48, and 72 h culture. The apoptosis of hPDLSCs cultured for 72 h was detected by flow cytometry. After inducing osteogenic differentiation for 21 days, alizarin red staining was used to detect the osteogenic differentiation ability of hPDLSCs. The alkaline phosphatase (ALP) activity in hPDLSCs was determined using a kit. The levels of inflammatory factors [tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6] in the supernatant of hPDLSCs culture were detected by enzyme-linked immunosorbent assay. The mRNA expressions of osteogenic differentiation genes [Runt-associated transcription factor 2 (RUNX2), osteocalcin (OCN), osteopontin (OPN)], SDF-1 and CXCR4 in hPDLSCs were detected by real-time fluorescence quantitative polymerase chain reaction (RT-qPCR). Western blot analysis was used to determine SDF-1 and CXCR4 protein expression in hPDLSCs. Compared with the blank group, the proliferative activity, number of mineralized nodules, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in the control group significantly decreased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 significantly increased (P<0.05). Compared with the control group, the proliferative activity, number of mineralized nodule, staining intensity, ALP activity, and RUNX2, OCN, OPN mRNA, SDF-1, and CXCR4 mRNA and protein expression levels of hPDLSCs in low-, medium-, and high-concentration sitagliptin groups increased. The apoptosis rate and levels of TNF-α, IL-1β, and IL-6 decreased (P<0.05). AMD3100 partially reversed the effect of high-concentration sitagliptin on LPS-induced hPDLSCs (P<0.05). Sitagliptin may promote the proliferation and osteogenic differentiation of hPDLSCs in LPS-induced inflammatory microenvironment by activating the SDF-1/CXCR4 signaling pathway. Furthermore, it inhibited the apoptosis and inflammatory response of hPDLSCs.

Platelet-Rich Plasma Lysate Enhances the Osteogenic Differentiation of Adipose-Derived Stem Cells.

Adipose-derived stem cells (ADSCs) have become an accepted source of cells in bone tissue engineering. This study aimed to investigate whether platelet-rich plasma (PRP) lysate can replace traditional fetal bovine serum as a culture medium with the enhanced proliferation and osteogenic potential of ADSCs. We divided the experiment into 5 groups where the ADSCs were cultured in an osteogenic medium containing 2.5%, 5%, 7.5%, and 10% PRP lysate with 10% fetal bovine serum as the control group. The cell proliferation, alkaline phosphatase (ALP) activity, ALP stain, alizarin red stain, osteocalcin (OCN) protein expression, and osteogenic-specific gene expression were analyzed and compared among these groups. The outcome showed that all PRP lysate-treated groups had good ALP stain and ALP activity performance. Better alizarin red stains were found in the 2.5%, 5%, and 7.5% PRP lysate groups. The 2.5% and 5% PRP lysate groups showed superior results in OCN quantitative polymerase chain reaction, whereas the 5% and 7.5% PRP lysate groups showed higher OCN protein expressions. Early RUNX2 (Runt-related transcription factor 2 () genes were the most expressed in the 5% PRP lysate group, followed by the 2.5% PRP lysate group, and then the 7.5% PRP lysate group. Thus, we concluded that 5% PRP lysate seemed to provide the optimal effect on enhancing the osteogenic potential of ADSCs. Platelet-rich plasma lysate-treated ADSCs were considered to be a good cell source for application in treating nonunion or bone defects in the future.

Exploring the Role of Wnt Ligands in Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

This study aimed to investigate the functions of 19types of Wnt ligands during the process of osteogenic differentiation inhuman periodontal ligament stem cells (hPDLSCs), with particular attention to WNT3A and WNT4. The expression levels of 19 types ofWnt ligands were examined using real-time quantitative polymerase chain reaction (real-time qPCR) during hPDLSCs osteogenic differentiation at 7, 10, and 14days. Knockdown of WNT3A and WNT4 expression was achieved using adenovirus vectors, and conditioned medium derived from WNT3A and WNT4 overexpression plasmids was employed to investigate their roles in hPDLSCs osteogenesis. Osteogenic-specific genes were analyzed using real-time qPCR. Alkaline phosphatase (ALP) and alizarin red S activities and staining were employed to assess hPDLSCs' osteogenic differentiation ability. During hPDLSCs osteogenic differentiation, the expression of 19types of Wnt ligands varied, with WNT3A and WNT4 showing significant upregulation. Inhibiting WNT3A and WNT4 expression hindered hPDLSCs' osteogenic capacity. Conditioned medium of WNT3A promoted early osteogenic differentiation, while WNT4 facilitated late osteogenesis slightly. Wnt ligands, particularly WNT3A and WNT4, play an important role in hPDLSCs' osteogenic differentiation, highlighting their potential as promoters of osteogenesis. Given the challenging nature of alveolar bone regeneration, therapeutic strategies that target WNT3A and WNT4 signaling pathways offer promising opportunities. Additionally, innovative gene therapy approaches aimed at regulating of WNT3A and WNT4 expression hold potential for improving alveolar bone regeneration outcomes.

Bone-derived PDGF-BB drives brain vascular calcification in male mice.

Brain vascular calcification is a prevalent age-related condition often accompanying neurodegenerative and neuroinflammatory diseases. The pathogenesis of large-vessel calcifications in peripheral tissue is well studied, but microvascular calcification in the brain remains poorly understood. Here, we report that elevated platelet-derived growth factor BB (PDGF-BB) from bone preosteoclasts contributed to cerebrovascular calcification in male mice. Aged male mice had higher serum PDGF-BB levels and a higher incidence of brain calcification compared with young mice, mainly in the thalamus. Transgenic mice with preosteoclast-specific Pdgfb overexpression exhibited elevated serum PDGF-BB levels and recapitulated age-associated thalamic calcification. Conversely, mice with preosteoclast-specific Pdgfb deletion displayed diminished age-associated thalamic calcification. In an ex vivo cerebral microvascular culture system, PDGF-BB dose-dependently promoted vascular calcification. Analysis of osteogenic gene array and single-cell RNA-Seq (scRNA-Seq) revealed that PDGF-BB upregulated multiple osteogenic differentiation genes and the phosphate transporter Slc20a1 in cerebral microvessels. Mechanistically, PDGF-BB stimulated the phosphorylation of its receptor PDGFRβ (p-PDGFRβ) and ERK (p-ERK), leading to the activation of RUNX2. This activation, in turn, induced the transcription of osteoblast differentiation genes in PCs and upregulated Slc20a1 in astrocytes. Thus, bone-derived PDGF-BB induced brain vascular calcification by activating the p-PDGFRβ/p-ERK/RUNX2 signaling cascade in cerebrovascular cells.

Effects of Systemic Peroxisome Proliferator-Activated Receptor Gamma Inhibition on Bone and Immune Cells in Aged Female Mice.

This study investigates the effects of peroxisome proliferator-activated receptor gamma (PPARγ) inhibition on bone and immune cell profiles in aged female mice, as well as in vitro stromal stem cell osteogenic differentiation and inflammation gene expression. The hypothesis was that inhibition of PPARγ would increase bone mass and alter immune and other cellular functions. Our results showed that treatment with PPARγ antagonist GW9662 for 6 weeks reduced bone volume and trabecular number and increased trabecular spacing. However, inhibition of PPARγ had no significant effect on marrow and spleen immune cell composition in aged female mice. In vitro experiments indicated that GW9662 treatment increased the expression of osteogenic genes but did not affect adipogenic genes. Additionally, GW9662 treatment decreased the expression of several inflammation-related genes. Overall, these findings suggest that PPARγ inhibition may have adverse effects on bone in aged female mice.

Ataluren prevented bone loss induced by ovariectomy and aging in mice through the BMP-SMAD signaling pathway

Ataluren prevented bone loss induced by ovariectomy and aging in mice through the BMP-SMAD signaling pathway