Effects of Selective Alveolar Decortication on the Periodontal Complex

Background Modification of bone turnover has been reported following selective alveolar decortication but the molecular signals in the periodontal ligament space (PDL) remain unanswered. The objective of this study was to understand how selective alveolar decortication affects the biological reactions in the periodontal ligament. Methods Selective alveolar decortication in wild-type mice (n=25) was performed on mandibular right buccal cortical plate adjacent to the mandibular right third molar and euthanized at 3, 7, 14 and 28 days. We also performed selective alveolar decortication in Lrp5ACT (n=5) mice and Ad-Dkk1 treated mice (n=5), and euthanized at 7 days. The periodontium around the mandibular third molars were examined using histology, immunohistochemical analyses for osteogenic markers, TGF-β, RANKL, TRAP and alkaline phosphatase activity. Results The expression of osteogenic markers in the wild-type PDL was maintained during healing time period after selective alveolar decortication. Increased osteoclast activity in the wild-type mice was observed at 3 and 7 days after selective alveolar decortication. The PDL in Lrp5G171V (Lrp5ACT) mice and adenovirus Dkk1 (Ad-Dkk1) treated mice also showed insignificant changes in the expression of osteogenic markers following selective alveolar decortication. In Lrp5ACT mice where there was a reduction of bone resorption, selective alveolar decortication caused a dramatic increase in osteoclast activity. Conclusions Selective alveolar decortication affects only bone turnover, but not the expression of osteogenic markers in the PDL.

Nanotopography in directing osteogenic differentiation of mesenchymal stem cells: potency and future perspective

Severe bone injuries can result in disabilities and thus affect a person's quality of life. Mesenchymal stem cells (MSCs) can be an alternative for bone healing by growing them on nanopatterned substrates that provide mechanical signals for differentiation. This review aims to highlight the role of nanopatterns in directing or inducing MSC osteogenic differentiation, especially in bone tissue engineering. Nanopatterns can upregulate the expression of osteogenic markers, which indicates a faster differentiation process. Combined with growth factors, nanopatterns can further upregulate osteogenic markers, but with fewer growth factors needed, thereby reducing the risks and costs involved. Nanopatterns can be applied in scaffolds for tissue engineering for their lasting effects, even in vivo, thus having great potential for future bone treatment.

The role of autophagy in the process of osseointegration around titanium implants with micro-nano topography promoted by osteoimmunity

Osteoimmunity plays an important role in the process of implant osseointegration. Autophagy is a conservative metabolic pathway of eukaryotic cells, but whether the interaction between autophagy and osteoimmunity plays a key role in osseointegration remains unclear. In this study, we prepared smooth titanium disks and micro-nano topography titanium disks, to study the immune microenvironment of RAW264.7 cells, and prepared the conditioned medium to study the effect of immune microenvironment on the osteogenesis and autophagy of MC3T3-E1 cells. Autophagy inhibitor 3-MA was used to inhibit autophagy to observe the change of expression of osteogenic markers. The results showed that the micro-nano topography titanium disks could stimulate RAW264.7 cells to differentiate into M2 type, forming an anti-inflammatory immune microenvironment; compared with the control group, the anti-inflammatory immune microenvironment promoted the proliferation and differentiation of osteoblasts better. The anti-inflammatory immune environment activated the autophagy level of osteoblasts, while the expression of osteogenic markers was down-regulated after inhibition of autophagy. These results indicate that anti-inflammatory immune microenvironment can promote cell proliferation and osteogenic differentiation, autophagy plays an important role in this process. This study further explains the mechanism of implant osseointegration in osteoimmune microenvironment, and provides reference for improving implant osseointegration.

Ferulic acid attenuates osteoporosis induced by glucocorticoid through regulating the GSK-3β/Lrp-5/ERK signalling pathways

Objective To evaluate in-vivo and in-vitro effects of ferulic acid (FA) on glucocorticoid-induced osteoarthritis (GIO) to establish its possible underlying mechanisms. Methods The effects of FA on cell proliferation, cell viability (MTT assay), ALP activity, and mineralization assay, and oxidative stress markers (ROS, SOD, GSH LDH and MDA levels) were investigated by MC3T3-E1 cell line. Wistar rats received standard saline (control group) or dexamethasone (GC, 2 mg−1 kg) or DEX+FA (50 and 100 mg−1 kg) orally for 8 weeks. Bone density, micro-architecture, bio-mechanics, bone turnover markers and histo-morphology were determined. The expression of OPG, RANKL, osteogenic markers, and other signalling proteins was assessed employing quantitative RT-PCR and Western blotting. Results The findings indicated the elevation of ALP mRNA expressions, osteogenic markers (Runx-2, OSX, Col-I, and OSN), and the β-Catenin, Lrp-5 and GSK-3β protein expressions. FA showed the potential to increase MC3T3-E1 cell differentiation, proliferation, and mineralization. FA increased oxidative stress markers (SOD, MDA, and GSH) while decreasing ROS levels and lactate dehydrogenase release in GIO rats. The OPG/RANKL mRNA expression ratio was increased by FA, followed by improved GSK-3β and ERK phosphorylation with enhanced mRNA expressions of Lrp-5 and β-catenin. Conclusion These findings showed that FA improved osteoblasts proliferation with oxidative stress suppression by controlling the Lrp-5/GSK-3β/ERK pathway in GIO, demonstrating the potential pathways involved in the mechanism of actions of FA in GIO therapy.

Loss of signal transducer and activator of transcription 3 impaired the osteogenesis of mesenchymal progenitor cells in vivo and in vitro

Background Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors. Methods Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers. Results Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.

Effects of iRoot SP on osteogenic differentiation of human stem cells from apical papilla

Background Research shows that nano-bioceramics can modulate the differentiation of dental stem cells. The novel ready-to-use calcium-silicate-based root-canal sealer iRoot SP is widely used in root filling. Accordingly, the aim of this study was to evaluate the effects of iRoot SP on proliferation and osteogenic differentiation in human stem cells from the apical papilla (hSCAPs). Methods hSCAPs were isolated and characterized in vitro, then cultured with various concentrations of iRoot SP extract. Cell proliferation was assessed by CCK-8 assay, and scratch-wound-healing assays were performed to evaluate cell-migration capacity. hSCAPs were then cultured in osteogenic medium supplemented with iRoot SP extracts. Alkaline phosphatase (ALP) activity assay was used to evaluate ALP enzyme levels. Alizarin red staining and cetylpyridinium chloride (CPC) assays were performed to assess calcified-nodule formation and matrix-calcium accumulation of hSCAPs. The mRNA and protein expression levels of the osteogenic markers OCN, OSX, Runx2, and DSPP were determined by qRT-PCR and Western blotting. The data were analyzed using one-way ANOVA and LSD-t tests. Results iRoot SP at low concentrations (2, 0.2, and 0.02 mg/mL) is nontoxic to hSCAPs. iRoot SP at concentrations of 0.02 and 0.2 mg/mL significantly increases cell-migration capacity. In terms of osteogenic differentiation, 0.2 mg/mL iRoot SP promotes intracellular ALP activity and the formation of mineralized nodules. Moreover, the expression of osteogenic markers at the mRNA and protein levels are upregulated by iRoot SP. Conclusion iRoot SP is an effective filling material for periapical bone regeneration.

miR-20a-5p contributes to osteogenic differentiation of human dental pulp stem cells by regulating BAMBI and activating the phosphorylation of Smad5 and p38

Background Human dental pulp stem cells (hDPSCs) are the preferable choice of seed cells for craniomaxillofacial bone tissue regeneration. As a member of the miR-17-92 cluster, miR-20a-5p functions as an important regulator during bone remodeling. This study aimed to investigate the roles and mechanisms of miR-20a-5p during osteogenesis of hDPSCs. Methods Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was conducted to determine the expression of miR-20a-5p during osteogenesis of hDPSCs. We interfered with the expression of miR-20a-5p in hDPSCs to clarify the function of miR-20a-5p on osteogenesis both in vitro and vivo. Direct bind sites between miR-20a-5p and BAMBI were confirmed by dual-luciferase reporter assay, and the underlying mechanisms were investigated with cell co-transfections. Results The expression of miR-20a-5p was showed to be upregulated during osteogenesis of hDPSCs. Inhibition of miR-20a-5p could weaken the intensity of ALP/ARS staining and downregulate the expression of mRNAs and proteins of osteogenic markers, while overexpression of miR-20a-5p could enhance the intensity of ALP/ARS staining and the expression of osteogenic markers. Both micro-CT reconstruction images and histological results showed that miR-20a-5p could promote the regeneration of calvarial defects. miR-20a-5p directly targeted bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI), and the latter one was an inhibitor of hDPSC osteogenesis. Silencing BAMBI partially reversed the suppression effect of miR-20a-5p knockdown on osteogenesis. Phosphorylation of Smad5 and p38 was decreased when miR-20a-5p was silenced, whereas p-Smad5 and p-p38 were upregulated when miR-20a-5p was overexpressed or BAMBI was silenced. Conclusions It is demonstrated that miR-20a-5p functioned as a regulator of BAMBI to activate the phosphorylation of Smad5 and p38 during osteogenic differentiation of hDPSCs.

Loss of Signal Transducer and Activator of Transcription 3 Impaired the Osteogenesis of Mesenchymal Progenitor Cells in Vivo and in Vitro

Background: Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors.Methods: Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and Micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers.Results: Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions: Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.

The role of Serpina3n in the reversal effect of ATRA on dexamethasone-inhibited osteogenic differentiation in mesenchymal stem cells

Background Glucocorticoid-induced osteoporosis (GIOP) is the most common secondary osteoporosis. Patients with GIOP are susceptible to fractures and the subsequent delayed bone union or nonunion. Thus, effective drugs and targets need to be explored. In this regard, the present study aims to reveal the possible mechanism of the anti-GIOP effect of all-trans retinoic acid (ATRA). Methods Bone morphogenetic protein 9 (BMP9)-transfected mesenchymal stem cells (MSCs) were used as an in vitro osteogenic model to deduce the relationship between ATRA and dexamethasone (DEX). The osteogenic markers runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin were detected using real-time quantitative polymerase chain reaction, Western blot, and immunofluorescent staining assay. ALP activities and matrix mineralization were evaluated using ALP staining and Alizarin Red S staining assay, respectively. The novel genes associated with ATRA and DEX were detected using RNA sequencing (RNA-seq). The binding of the protein–DNA complex was validated using chromatin immunoprecipitation (ChIP) assay. Rat GIOP models were constructed using intraperitoneal injection of dexamethasone at a dose of 1 mg/kg, while ATRA intragastric administration was applied to prevent and treat GIOP. These effects were evaluated based on the serum detection of the osteogenic markers osteocalcin and tartrate-resistant acid phosphatase 5b, histological staining, and micro-computed tomography analysis. Results ATRA enhanced BMP9-induced ALP, RUNX2 expressions, ALP activities, and matrix mineralization in mouse embryonic fibroblasts as well as C3H10T1/2 and C2C12 cells, while a high concentration of DEX attenuated these markers. When DEX was combined with ATRA, the latter reversed DEX-inhibited ALP activities and osteogenic markers. In vivo analysis showed that ATRA reversed DEX-inhibited bone volume, bone trabecular number, and thickness. During the reversal process of ATRA, the expression of retinoic acid receptor beta (RARβ) was elevated. RARβ inhibitor Le135 partly blocked the reversal effect of ATRA. Meanwhile, RNA-seq demonstrated that serine protease inhibitor, clade A, member 3N (Serpina3n) was remarkably upregulated by DEX but downregulated when combined with ATRA. Overexpression of Serpina3n attenuated ATRA-promoted osteogenic differentiation, whereas knockdown of Serpina3n blocked DEX-inhibited osteogenic differentiation. Furthermore, ChIP assay revealed that RARβ can regulate the expression of Serpina3n. Conclusion ATRA can reverse DEX-inhibited osteogenic differentiation both in vitro and in vivo, which may be closely related to the downregulation of DEX-promoted Serpina3n. Hence, ATRA may be viewed as a novel therapeutic agent, and Serpina3n may act as a new target for GIOP.

Loss of signal transducer and activator of transcription 3 impaired the osteogenesis of mesenchymal progenitor cells in vivo and in vitro

Background Signal transducer and activator of transcription 3 (Stat3) is a cytoplasmic transcription factor that participates in various biologic processes. Loss of Stat3 causes hyperimmunoglobulin E syndrome, presenting with skeletal disorders including osteoporosis, recurrent fractures, scoliosis, and craniosynostosis. The objective of this study is to explore the effect and mechanism of Stat3 on osteogenesis of mesenchymal progenitors. Methods Stat3 was conditionally knockout (CKO) in mesenchymal progenitors by crossing the pair-related homeobox gene 1-cre (Prx1-Cre) with Stat3-floxed strain mice. Whole-mount-skeletal staining, histology, and Micro-CT were used to assess the differences between Stat3 CKO and control mice. Further, in vitro experiments were conducted to evaluate the osteogenesis potential of primary isolated bone marrow mesenchymal stem cells (BMSCs) from both control and Stat3 CKO mice. After osteogenic induction for 14d, alizarin red staining was used to show the calcium deposit, while the western blotting was applied to detect the expression of osteogenic markers. Results Compared with the control, Stat3 CKO mice were present with shortened limbs, multiple fractures of long bone, and open calvarial fontanels. The abnormal growth plate structure and reduced collagen fiber were found in Stat3 CKO limbs. According to micro-CT analysis, the reduced cortical bone thickness and bone volume were found on Stat3 CKO mice. The in vitro osteogenic differentiation of BMSCs was inhibited in Stat3 CKO samples. After osteogenic induction for 14d, the significantly diminished calcium deposits were found in Stat3 CKO BMSCs. The decreased expression of osteogenic markers (OPN and COL1A1) was observed in Stat3 CKO BMSCs, compared with the control. Conclusions Stat3 played a critical role in bone development and osteogenesis. Loss of Stat3 impaired the osteogenesis of mesenchymal progenitors in vivo and in vitro.