Osteogenic Function Research Articles

A single-cell transcriptomic atlas of human stem cells from apical papilla during the committed differentiation.

Human stem cells derived from the apical papilla (SCAPs) are recognized for their multilineage differentiation potential and their capacity for functional tooth root regeneration. However, the molecular mechanisms underlying odonto/osteogenic differentiation remain largely unexplored. In this study, we utilized single-cell RNA sequencing (scRNA-seq) to conduct an in-depth analysis of the transcriptional changes associated with chemically induced osteogenesis in SCAPs. scRNA-seq identified SCAPs as distinct subpopulations. Differentially expressed genes (DEGs) and Gene Ontology (GO) analyses were conducted to evaluate the potential function of each cluster. Pseudotime trajectory analysis was employed to elucidate the potential differentiation processes of the identified SCAP populations. To investigate the osteo/odontogenic potential of Deiodinase Iodothyronine Type 2 (DIO2) on SCAPs, we performed alkaline phosphatase staining, western blot analysis, Alizarin Red S staining and immunofluorescence staining. Additionally, SCAP components were transplanted into mouse calvarial defects to evaluate osteogenesis invivo. The analysis of cell clusters derived from our scRNA-seq data revealed a significant shift in cellular composition when cells were cultured in a mineralization induction medium compared to those cultured in a complete medium. Both groups exhibited heterogeneity, with some cells intrinsically predisposed to osteogenesis and others appearing to be primed for proliferative functions. Notably, we identified a subpopulation characterized by high expression of DIO2, which exhibited pronounced osteogenic activity during differentiation. Our study is the first to reveal a shift in the cellular composition of SCAPs when cultured in a mineralization induction medium compared to a complete medium. Following both invitro and invivo validation, the DIO2+ subpopulation exhibited the highest transcriptional similarity to osteogenic function, suggesting its potential utility in tissue regeneration applications.

One-Step Gas Foaming Strategy for Constructing Strontium Nanoparticle Decorated 3D Scaffolds: a New Platform for Repairing Critical Bone Defects.

The management of critical-sized bone defects poses significant clinical challenges, particularly in the battlefield and trauma-related injuries. However, bone tissue engineering scaffolds that satisfy high porosity and good angiogenic and osteogenic functions are scarce. In this study, 3D nanofiber scaffolds decorated with strontium nanoparticles (3DS-Sr) were fabricated by combining electrospinning and gas foaming. Sodium borohydride (NaBH4) served a dual role as both a reducing and gas-foaming agent, enabling a one-step process for expansion and modification. In vitro experimental results demonstrated that 3DS-Sr possessed an integrated multilayered porous structure. It promoted angiogenesis by upregulating the expression of hypoxia-inducible factor-1α (HIF-1α) protein and phosphorylation of ERK through the sustained release of Sr2+ and created a favorable microenvironment for osteogenesis by activating the Wnt/β-catenin pathway. In vivo experiments indicated that 3DS-Sr promoted cranial bone regeneration by synergistically promoting the effects of vascularization and osteogenesis. In summary, this study proposed a bioactive bone scaffold in a "one stone, two birds" manner, providing a promising strategy for bone defect repair.

Ultraviolet laser induced periodic surface structures positively influence osteogenic activity on titanium alloys.

Endoprostheses might fail due to complications such as implant loosening or periprosthetic infections. The surface topography of implant materials is known to influence osseointegration and attachment of pathogenic bacteria. Laser-Induced Periodic Surface Structures (LIPSS) can improve the surface topography of orthopedic implant materials. In this preclinical in vitro study, laser pulses with a wavelength in the ultraviolet (UV) spectrum were applied for the generation of LIPSS to positively influence formation of extracellular matrix by primary human Osteoblasts (hOBs) and to reduce microbial biofilm formation in vitro. Laser machining was employed for generating UV-LIPSS on sample disks made of Ti6Al4V and Ti6Al7Nb alloys. Sample disks with polished surfaces were used as controls. Scanning electron microscopy was used for visualization of surface topography and adherent cells. Metal ion release and cellular metal levels were investigated by inductively coupled plasma mass spectrometry. Cell culture of hOBs on sample disks with and without UV-LIPSS surface treatments was performed. Cells were investigated for their viability, proliferation, osteogenic function and cytokine release. Biofilm formation was facilitated by seeding Staphylococcus aureus on sample disks and quantified by wheat germ agglutinin (WGA) staining. UV-LIPSS modification results in topographies with a periodicity of 223nm ≤ λ ≤ 278nm. The release of metal ions was found increased for UV-LIPSS on Ti6Al4V and decreased for UV-LIPSS on Ti6Al7Nb, while cellular metal levels remain unaffected. Cellular adherence was decreased for hOBs on UV-LIPSS Ti6Al4V when compared to controls while proliferation rate was unaffected. Metabolic activity was lower on UV-LIPSS Ti6Al7Nb when compared to the control. Alkaline phosphatase activity was upregulated for hOBs grown on UV-LIPSS on both alloys. Less pro-inflammatory cytokines were released for cells grown on UV-LIPSS Ti6Al7Nb when compared to polished surfaces. WGA signals were significantly lower on UV-LIPSS Ti6Al7Nb indicating reduced formation of a S. aureus biofilm. Our results suggest that UV-LIPSS texturing of Ti6Al7Nb positively influence bone forming function and cytokine secretion profile of hOBs in vitro. In addition, our results indicate diminished biofilm formation on UV-LIPSS treated Ti6Al7Nb surfaces. These effects might prove beneficial in the context of long-term arthroplasty outcomes.

Quercetin ameliorates senescence and promotes osteogenesis of BMSCs by suppressing the repetitive element‑triggered RNA sensing pathway.

Cell senescence impedes the self‑renewal and osteogenic capacity of bone marrow mesenchymal stem cells (BMSCs), thus limiting their application in tissue regeneration. The present study aimed to elucidate the role and mechanism of repetitive element (RE) activation in BMSC senescence and osteogenesis, as well as the intervention effect of quercetin. In an H2O2‑induced BMSC senescence model, quercetin treatment alleviated senescence as shown by a decrease in senescence‑associated β‑galactosidase (SA‑β‑gal)‑positive cell ratio, increased colony formation ability and decreased mRNA expression of p21 and senescence‑associated secretory phenotype genes. DNA damage response marker γ‑H2AX increased in senescent BMSCs, while expression of epigenetic markers methylation histone H3 Lys9, heterochromatin protein 1α and heterochromatin‑related nuclear membrane protein lamina‑associated polypeptide 2 decreased. Quercetin rescued these alterations, indicating its ability to ameliorate senescence by stabilizing heterochromatin structure where REs are primarily suppressed. Transcriptional activation of REs accompanied by accumulation of cytoplasmic double‑stranded (ds)RNA, as well as triggering of the RNA sensor retinoic acid‑inducible gene I (RIG‑I) receptor pathway in H2O2‑induced senescent BMSCs were shown. Similarly, quercetin treatment inhibited these responses. Additionally, RIG‑I knockdown led to a decreased number of SA‑β‑gal‑positive cells, confirming its functional impact on senescence. Induction of senescence or administration of dsRNA analogue significantly hindered the osteogenic capacity of BMSCs, while quercetin treatment or RIG‑I knockdown reversed the decline in osteogenic function. The findings of the current study demonstrated that quercetin inhibited the activation of REs and the RIG‑I RNA sensing pathway via epigenetic regulation, thereby alleviating the senescence of BMSCs and promoting osteogenesis.

Role and mechanism of histone demethylase PHF8 in weightlessness osteoporosis

Weightlessness osteoporosis, which progresses continuously and has limited protective effects, has become one of the major problems that need to be solved in manned spaceflight. Our study aims to investigate the regulatory role of PHF8 in disuse osteoporosis by observing the expression of PHF8 in bone marrow mesenchymal stem cells (BMSCs) under simulated weightlessness conditions. Therefore, we used the model of ground-based microgravity simulated by disuse osteoporosis patients and tail suspension in mice to simulate microgravity in vivo, and measured the expression of PHF8 in bone tissue. Subsequently, we used the 2D gyroscope to simulate the weightless effect on bone marrow mesenchymal stem cells. In the weightless condition, we detected the proliferation, apoptosis, osteogenesis, and osteogenic differentiation functions of BMSCs. We also detected the expression of osteogenic-related transcription factors after knocking down and overexpressing PHF8. Our results show that the weightless effect can inhibit the proliferation, osteogenesis, and osteogenic differentiation functions of BMSCs, while enhancing their apoptosis; and overexpression of PHF8 can partially alleviate the osteoporosis caused by simulated weightlessness, providing new ideas and clues for potential drug targets to prevent weightlessness and disuse osteoporosis.

SCUBE3 promotes osteogenic differentiation and mitophagy in human bone marrow mesenchymal stem cells through the BMP2/TGF-β signaling pathway.

In clinical settings, addressing large bone defects remains a significant challenge for orthopedic surgeons. The use of genetically modified bone marrow mesenchymal stem cells (BMSCs) has emerged as a highly promising approach for these treatments. Signal peptide-CUB-EGF domain-containing protein 3 (SCUBE3) is a multifunctional secreted glycoprotein, the role of which remains unclear in human hBMSCs. This study used various experimental methods to elucidate the potential mechanism by which SCUBE3 influences osteogenic differentiation of hBMSCs invitro. Additionally, the therapeutic efficacy of SCUBE3, in conjunction with porous GeLMA microspheres, was evaluated invivo using a mouse bone defect model. Our findings indicate that SCUBE3 levels increase significantly during early osteogenic differentiation of hBMSCs, and that reducing SCUBE3 levels can hinder this differentiation. Overexpressing SCUBE3 elevated osteogenesis gene and protein levels and enhanced calcium deposition. Furthermore, treatment with recombinant human SCUBE3 (rhSCUBE3) protein boosted BMP2 and TGF-β expression, activated mitophagy in hBMSCs, ameliorated oxidative stress, and restored osteogenic function through SMAD phosphorylation. Invivo, GELMA/OE treatment effectively accelerated bone healing in mice. In conclusion, SCUBE3 fosters osteogenic differentiation and mitophagy in hBMSCs by activating the BMP2/TGF-β signaling pathway. When combined with engineered hydrogel cell therapy, it could offer valuable guidance for the clinical management of extensive bone defects.

The Increased Apoptosis of Mesenchymal Stem Cells Mediated Osteopenia Due to Prenatal Nicotine Exposure in Female Offspring Rats via IGF1 Pathway.

Nicotine exposure is a common adverse environment during pregnancy and causes developmental toxicity of long bones in offspring. However, the effect of prenatal nicotine exposure (PNE) on bone mass accumulation in female offspring and its mechanism remained to be further investigated. In this study, we constructed a PNE rat model and collected the long bone and the bone marrow mesenchymal stem cells (BMSCs) from female offspring rats for the detection of bone mass, cell apoptosis, and the expressions of osteogenesis- and apoptosis-related genes. The results revealed that PNE induced low bone mass in female offspring rats and was associated with the suppression of osteogenic function. Moreover, the apoptosis of BMSCs derived from the PNE female offspring rats was raised, and the expression ratio of apoptosis marker genes BAX/BCL-2 was significantly increased. Further, PNE inhibited the expression and function of insulin-like growth factor l (IGF1) signaling pathway in BMSCs. However, the exogenous IGF1 treatment partially ameliorated the increased apoptosis of BMSCs derived from the PNE female offspring rats. In conclusion, PNE induced low bone mass in female offspring rats, which was attributed to the increased apoptosis of BMSCs due to functional inhibition of IGF1 signaling pathway.

Fibroblast growth factor 2 promotes osteo/odontogenic differentiation in stem cells from the apical papilla by inhibiting PI3K/AKT pathway

Fibroblast growth factor 2 (FGF2) is a crucial factor in odontoblast differentiation and dentin matrix deposition, which facilitates pulpodentin repair and regeneration. Nevertheless, the specific biological function of FGF2 in odontoblastic differentiation remains unclear because it is controlled by complex signalling pathways. This study aimed to investigate the mechanism underlying the effect of FGF2 on osteo/odontogenic differentiation of stem cells from the apical papilla (SCAP). SCAP were pretreated with conditioned media containing FGF2 for 1 week, followed by culturing in induced differentiation medium for another week. RNA sequencing (RNA-seq) combined with quantitative reverse transcription polymerase chain reaction (RT-qPCR) was used to evaluate the pathways affected by FGF2 in SCAP. Osteo/odontogenic differentiation of SCAP was determined using Alizarin red S staining, alkaline phosphatase staining, RT-qPCR, and western blotting. Pretreatment with FGF2 for 1 week increased the osteo/odontogenic differentiation ability of SCAP. RNA-seq and Kyoto Encyclopedia of Genes and Genomes pathway analyses revealed that phosphatidylinositol 3-kinase (PI3K)/AKT signalling is involved in the osteogenic function of FGF2. RT-qPCR results indicated that SCAP expressed FGF receptors, and western blotting showed that p-AKT was reduced in FGF2-pretreated SCAP. The activation of the PI3K/AKT pathway partially reversed the stimulatory effect of FGF2 on osteo/odontogenic differentiation of SCAP. Our findings suggest that pretreatment with FGF2 enhances the osteo/odontogenic differentiation ability of SCAP by inhibiting the PI3K/AKT pathway.

Coaxial 3D Printing Scaffolds with Sequential Antibacterial and Osteogenic Functions to Effectively Repair Infected Mandibular Defects

AbstractInfected mandibular defects pose a formidable challenge without an entirely satisfactory repair strategy. Here, a coaxial 3D printing scaffold comprising a shell loaded is dveloped with the antibiotic minocycline hydrochloride and a core wrapped with traditional Chinese medicine antler powers. This study is conducted to evaluate the efficacy of the scaffold in facilitating the repair of infected mandibular defects. In vitro investigations exhibit that this construct possesses a porous microstructure, exceptional mechanical properties, appropriate degradability, acceptable water absorption capacity, and satisfactory biocompatibility. Transcriptome analyses further reveal a notable upregulation of relevant genes and signaling pathways associated with cell activity, osteogenesis, and angiogenesis. In vivo rat (Φ = 5 mm) and rabbit (5 mm × 4 mm × 10 mm) mandibular defect models confirm the scaffold's ability to sequentially regulate bone healing by suppressing infection and inflammation, followed by promoting osteogenesis and angiogenesis. This advanced graft holds significant translational potential for infectious bone regeneration.

Application of Concentrated Growth Factors in Combination with Bio-Oss for Extraction of Mandibular Proximal Impacted Wisdom Teeth

In this study, we investigate the combined application of concentrated growth factor (CGF) and Bio-Oss (BO) for the treatment of periodontal bone defect (BD) after extraction of impacted wisdom teeth. Firstly, we assess the osteogenic properties of CGF in periodontal ligament stem cells (PDLSCs). The concentration-dependent effect of CGF on enhancing the activity of PDLSCs has been demonstrated. Furthermore, CGF effectively promotes differentiation and enhances the osteogenic function of PDLSCs, leading to improved expression of related osteogenic proteins. Subsequently, a total of 64 patients with loss of alveolar bone in the second molar, who received treatment at our hospital between July 2020 and July 2023, are included. They are randomly divided into a BO group (treated with BO) and a CGF/BO group (treated with CGF+BO). We observe that the combined application of CGF and BO demonstrated superior efficacy in alleviating pain, reducing swelling, and preventing dry socket incidence. In addition, it exhibited enhanced effectiveness in restoring periodontal tissue, including reducing probing depth, gingival depth, and clinical attachment loss. It also displayed better inhibition of gingival inflammatory response and gingival bleeding. Furthermore, it could enhance the restoration of periodontal BD, such as increasing the width of the alveolar bone and root depth, reducing the vertical distance from the apex of the alveolar ridge, as well as improving dental function. Therefore, the combined application of CGF and BO holds great potential in periodontal BD therapy for promoting the regeneration of periodontal bone, thus restoring dental function.

Dendrobine Ameliorates Glucocorticoid-Induced Osteoporosis by Promoting Osteogenesis through JNK/p38 MAPK Pathway Activation and GR Nuclear Translocation Inhibition.

Glucocorticoid-induced osteoporosis (GIOP) is the common reason for secondary osteoporosis. Dendrobine (DEN) is the major biologically active component of Dendrobium officinale with anti-inflammatory and antiaging properties. Whether DEN could alleviate osteogenic inhibition in GIOP rats is still unknown. The influence on osteogenic function caused by DEN on dexamethasone-treated bone marrow mesenchymal stem cells and rats was observed. The in vitro results showed that DEN reversed the inhibition of osteogenic differentiation by dexamethasone. Moreover, DEN supplementation attenuated dexamethasone-induced bone loss in vivo. DEN activated JNK and p38 MAPK pathways and restrained GR nuclear translocation, which could be prevented by the JNK (SP600125) or p38 (SB203580) pathway inhibitor. This study verified that DEN alleviated dexamethasone-induced nuclear translocation of GR, and inhibition of osteogenesis via JNK and p38 pathways, laying the foundation for DEN as a therapeutic agent for GIOP.

Phosvitin phosphopeptides alleviate the inhibitory effect of oxidative stress on osteoblast function through FoxO/Wnt signaling

Oxidative stress inhibits osteoblast differentiation and function, leading to the development of osteoporosis. The effects of Phosvitin phosphopeptides (PPPs) on the osteogenic function of osteoblast were determined based on oxidative stress MC3T3-E1 cell model induced by hydrogen peroxide. The results showed that 50 μg/mL of PPPs significantly increased cell viability from 56.00% (H2O2) to 106.80% for 72 h. DCFH-DA fluorescence assay demonstrated that PPPs could remarkably reduce reactive oxygen species levels induced by H2O2. The alkaline phosphatase activity of oxidative stress-treated osteoblast increased from 2.36 Kim'unit/100 mL (H2O2 group) to 8.16 Kim'unit/100 mL, along with an increase in the activity of total superoxide dismutase, glutathione peroxidase, catalase, and the number of mineralized nodules in response to PPPs treatment. Overall, the results of this work indicated that PPPs had a stimulating impact on the Wnt/β-catenin signaling pathway while simultaneously inhibiting the forkhead box class O (FoxO) signaling pathway in MC3T3-E1 osteoblast induced by H2O2. Consequently, these findings demonstrate that PPPs have the ability to protect the osteogenic function of MC3T3-E1 osteoblasts against oxidative damage through the activation of the FoxO/Wnt signaling pathway.

Inflammatory microenvironment regulation and osteogenesis promotion by bone-targeting calcium and magnesium repletion nanoplatform for osteoporosis therapy

Osteoporosis is the most common bone metabolic disease that affects the health of middle-aged and elderly people, which is hallmarked by imbalanced bone remodeling and a deteriorating immune microenvironment. Magnesium and calcium are pivotal matrix components that participate in the bone formation process, especially in the immune microenvironment regulation and bone remodeling stages. Nevertheless, how to potently deliver magnesium and calcium to bone tissue remains a challenge. Here, we have constructed a multifunctional nanoplatform composed of calcium-based upconversion nanoparticles and magnesium organic frameworks (CM-NH2-PAA-Ald, denoted as CMPA), which features bone-targeting and pH-responsive properties, effectively regulating the inflammatory microenvironment and promoting the coordination of osteogenic functions for treating osteoporosis. The nanoplatform can efficaciously target bone tissue and gradually degrade in response to the acidic microenvironment of osteoporosis to release magnesium and calcium ions. This study validates that CMPA possessing favorable biocompatibility can suppress inflammation and facilitate osteogenesis to treat osteoporosis. Importantly, high-throughput sequencing results demonstrate that the nanoplatform exerts a good inflammatory regulation effect through inhibition of the nuclear factor kappa-B signaling pathway, thereby normalizing the osteoporotic microenvironment. This collaborative therapeutic strategy that focuses on improving bone microenvironment and promoting osteogenesis provides new insight for the treatment of metabolic diseases such as osteoporosis.

Investigation of the impact of planar microelectrodes on macrophage-mediated mesenchymal stem cell osteogenesis.

Osseointegration commences with foreign body inflammation upon implant placement, where macrophages play a crucial role in the immune response. Subsequently, during the intermediate and late stages of osseointegration, mesenchymal stem cells (MSCs) migrate and initiate their osteogenic functions, while macrophages support MSCs in osteogenesis. The utilization of ferroelectric P(VDF-TrFE) covered ITO planar microelectrodes facilitated the simulation of various surface charge to investigate their effects on MSCs' osteogenic differentiation and macrophage polarization and the results indicated a parabolic increase in the promotional effect of both with the rise in piezoelectric coefficient. Furthermore, the surface charge with a piezoelectric coefficient of -18 exhibited the strongest influence on the promotion of M1 polarization of macrophages and the promotion of MSCs' osteogenic differentiation. The impact of macrophage polarization and MSC osteogenesis following the interaction of macrophages affected by surface charge and MSC was ultimately investigated. It was observed that macrophages affected by the surface charge of -18 piezoelectric coefficient still exerted the most profound induced osteogenic effect, validating the essential role of M1-type macrophages in the osteogenic differentiation of MSCs.

Astaxanthin-mediated Nrf2 activation ameliorates glucocorticoid-induced oxidative stress and mitochondrial dysfunction and impaired bone formation of glucocorticoid-induced osteonecrosis of the femoral head in rats

BackgroundOsteonecrosis of the femoral head caused by glucocorticoids (GIONFH) is a significant issue resulting from prolonged or excessive clinical glucocorticoid use. Astaxanthin, an orange-red carotenoid present in marine organisms, has been the focus of this study to explore its impact and mechanism on osteoblast apoptosis induced by dexamethasone (Dex) and GIONFH.MethodsIn this experiment, bioinformatic prediction, molecular docking and dynamics simulation, cytotoxicity assay, osteogenic differentiation, qRT-PCR analysis, terminal uridine nickend labeling (TUNEL) assay, determination of intracellular ROS, mitochondrial function assay, immunofluorescence, GIONFH rat model construction, micro-computed tomography (micro-CT) scans were performed.ResultsOur research demonstrated that a low dose of astaxanthin was non-toxic to healthy osteoblasts and restored the osteogenic function of Dex-treated osteoblasts by reducing oxidative stress, mitochondrial dysfunction, and apoptosis. Furthermore, astaxanthin rescued the dysfunction in poor bone quality, bone metabolism and angiogenesis of GIONFH rats. The mechanism behind this involves astaxanthin counteracting Dex-induced osteogenic damage by activating the Nrf2 pathway.ConclusionAstaxanthin shields osteoblasts from glucocorticoid-induced oxidative stress and mitochondrial dysfunction via Nrf2 pathway activation, making it a potential therapeutic agent for GIONFH treatment.

YTHDF2-Mediated m6A methylation inhibition by miR27a as a protective mechanism against hormonal osteonecrosis in BMSCs

BackgroundWith the increasing incidence of steroid-induced necrosis of the femoral head (SNFH), numerous scholars have investigated its pathogenesis. Current evidence suggests that the imbalance between lipogenesis and osteoblast differentiation in bone marrow mesenchymal stem cells (BMSCs) is a key pathological feature of SNFH. MicroRNAs (miRNAs) have strong gene regulatory effects and can influence the direction of cell differentiation. N6-methyladenosine (m6A) is a prevalent epigenetic modification involved in diverse pathophysiological processes. However, knowledge of how miRNAs regulate m6A-related factors that affect BMSC differentiation is limited.ObjectiveWe aimed to investigate the role of miR27a in regulating the expression of YTHDF2 in BMSCs.MethodsWe compared miR27a, YTHDF2, and total m6A mRNA levels in SNFH-affected and control BMSCs. CCK-8 and TUNEL assays were used to assess BMSC proliferation and apoptosis. Western blotting and qRT‒PCR were used to measure the expression of osteogenic (ALP, RUNX2, and OCN) and lipogenic (PPARγ and C/EBPα) markers. Alizarin Red and Oil Red O staining were used to quantify osteogenic and lipogenic differentiation, respectively. miR27a was knocked down or overexpressed to evaluate its impact on BMSC differentiation and its relationship with YTHDF2. Bioinformatics analyses identified YTHDF2 as a differentially expressed gene in SNFH (ROC analysis) and revealed potential signaling pathways through GSEA. The effects of YTHDF2 silencing on the lipogenic and osteogenic functions of BMSCs were assessed.ResultsmiR27a downregulation and YTHDF2 upregulation were observed in the SNFH BMSCs. miR27a knockdown/overexpression modulated YTHDF2 expression, impacting BMSC differentiation. miR27a silencing decreased m6A methylation and promoted osteogenic differentiation, while YTHDF2 silencing exerted similar effects. GSEA suggested potential signaling pathways associated with YTHDF2 in SNFH.ConclusionmiR27a regulates BMSC differentiation through YTHDF2, affecting m6A methylation and promoting osteogenesis. This finding suggests a potential therapeutic target for SNFH.

Construction of a Lentiviral Vector for Fgfr2 Overexpression and its Impact on the Biological Behavior of Cranial Suture Mesenchymal Stem Cells.

Suture mesenchymal stem cells (SuSCs), possessing self-renewal and multilineage differentiation abilities, play a crucial role in cranial bone growth. However, the impact of the disease-causing fibroblast growth factor receptor 2 (FGFR2) mutation on SuSCs in Crouzon syndrome has not been explored. This study aims to employ a lentivirus to overexpress Fgfr2 and investigate its role in the pathogenesis of Crouzon syndrome. Starting with the prevalent FGFR2 mutation site in patients with Crouzon syndrome, a lentiviral vector carrying the Fgfr2.C361Y mutation was developed and transfected into SuSCs, with a determined multiplicity of infection values. The experimental group, SuSCs+Fgfr2.C361Y, was compared with the empty vector and normal SuSC groups. Cell proliferation, cycle, apoptosis, and osteogenic functionality were assessed using CCK-8 assays, flow cytometry, ALP activity assays, and real-time quantitative polymerase chain reaction. The lentiviral vector effectively infected SuSCs, leading to heightened Fgfr2 expression, with optimal multiplicity of infection values of 80. The experimental group demonstrated decreased proliferation activity and a higher apoptosis rate compared with controls (P < 0.05). After osteogenic induction, the experimental group showed significantly higher ALP activity than controls (P < 0.05). Real-time quantitative polymerase chain reaction indicated lower mRNA expression levels of Gli1, Axin2, Pcna, Cdk2, and Bcl-2 in the experimental group than controls, whereas Bax, Runx2, and Bmp-2 showed higher expression (P < 0.05). This study constructed a lentivirus vector to upregulate Fgfr2 expression in SuSCs, suppressing stem cell stemness by inhibiting proliferation, promoting apoptosis, and accelerating premature osteogenic differentiation, resulting in premature suture closure. These findings establish the groundwork for further understanding the pathogenesis of Crouzon syndrome.

Biomimetic dually cross-linked injectable poly(l-glutamic acid) based nanofiber composite hydrogels with self-healing, osteogenic and angiogenic properties for bone regeneration

Biomimetic hydrogels with satisfactory mechanical properties and osteogenic/angiogenic abilities have attracted extensive attention in recent years. Injectable hydrogels with the advantages of minimally invasive implantation and adaptability to irregular defects exhibit superior application potential in the field of bone regeneration. However, poor mechanical properties and limited self-healing ability hinder their potential as supporting materials. Furthermore, the absence of osteogenic and angiogenic properties significantly affect their application in bone regeneration. For injectable hydrogels, simulating natural bone tissue ECM presents an opportunity for developing innovative materials for bone repair. In this work, a strategy to prepare biomimetic nanofiber-reinforced dually cross-linked (DC) injectable hydrogels with self-healing, osteogenic and angiogenic properties was proposed. The PLGA/MDex/CaP@PBLG nanofiber hydrogels were crosslinked through two reversible dynamic interactions including Schiff base reaction and physical chelation, thereby demonstrating good self-healing property. Meanwhile, the incorporation of CaP@PBLG mineralized nanofibers and alendronate sodium (BP) grafted on PLGA could endow the hydrogels with favorable osteogenic function. Moreover, the introduction of antioxidant ferulic acid (FA) could significantly stimulate angiogenic activity of the hydrogels. The successful regeneration of cranial defects in rats demonstrated a potential application of PLGA/MDex/CaP@PBLG nanofiber hydrogels in the field of bone regeneration.

CircPRKD3/miR-6783-3p responds to mechanical force to facilitate the osteogenesis of stretched periodontal ligament stem cells

BackgroundThe mechanotransduction mechanisms by which cells regulate tissue remodeling are not fully deciphered. Circular RNAs (circRNAs) are crucial to various physiological processes, including cell cycle, differentiation, and polarization. However, the effects of mechanical force on circRNAs and the role of circRNAs in the mechanobiology of differentiation and remodeling in stretched periodontal ligament stem cells (PDLSCs) remain unclear. This article aims to explore the osteogenic function of mechanically sensitive circular RNA protein kinase D3 (circPRKD3) and elucidate its underlying mechanotransduction mechanism.Materials and methodsPDLSCs were elongated with 8% stretch at 0.5 Hz for 24 h using the Flexcell® FX-6000™ Tension System. CircPRKD3 was knockdown or overexpressed with lentiviral constructs or plasmids. The downstream molecules of circPRKD3 were predicted by bioinformatics analysis. The osteogenic effect of related molecules was evaluated by quantitative real-time PCR (qRT‐PCR) and western blot.ResultsMechanical force enhanced the osteogenesis of PDLSCs and increased the expression of circPRKD3. Knockdown of circPRKD3 hindered PDLSCs from osteogenesis under mechanical force, while overexpression of circPRKD3 promoted the early osteogenesis process of PDLSCs. With bioinformatics analysis and multiple software predictions, we identified hsa-miR-6783-3p could act as the sponge of circPRKD3 to indirectly regulate osteogenic differentiation of mechanically stimulated PDLSCs.ConclusionsOur results first suggested that both circPRKD3 and hsa-miR-6783-3p could enhance osteogenesis of stretched PDLSCs. Furthermore, hsa-miR-6783-3p could sponge circPRKD3 to indirectly regulate RUNX2 during the periodontal tissue remodeling process in orthodontic treatment.

Chitosan modified with PAP as a promising delivery system for melatonin in the treatment of osteoporosis: targeting the divalent metal transporter 1.

The demands for novel and efficient therapies have gradually increased with the rising concerns of osteoporosis (OP). The most popular method in promoting bone regeneration during osteoporotic conditions consists of loading bioactive materials with different drugs to treat osteoporotic bones by either promoting the process of osteogenesis, or by inhibiting the activity of osteoclasts. By analyzing single cell sequencing results, we found that divalent metal transporter 1 (DMT1) played a role in OP. Based on our previous results, we found that melatonin (MT) suppressed expression of DMT1 induced by high glucose during OP, so we determined the efficacy of MT for the treatment of OP. However, the clinical effects of MT on OP were unsatisfactory. To enhance its biological efficacy, we combined MT with porous gelatin chitosan (chitosan) and the conductive material, PLA-b-AP-b-PLA (PAP), then determined how MT incorporation in chitosan@PAP nanoparticles affected the ability to promote MC3T3-E1 osteogenesis and mineralization, both in vitro and in vivo. The results confirmed the effect of MT on DMT1. We then prepared and characterized composites prepared as nanofibers, and determined the efficacy of MT combined with chitosan-PAP modified hydrogels as a slow-release system in a femur model of osteoporosis mice, with associated properties suitable for bone tissue engineering. The results indicated that MT-loaded chitosan@PAP nanospheres showed favorable osteogenic functions, both in vivo and in vitro, providing a practical solution for bone regeneration for OP patients.