Early Osteogenic Differentiation Research Articles

Additive manufacturing of bioactive and biodegradable poly (lactic acid)-tricalcium phosphate scaffolds modified with zinc oxide for guided bone tissue repair

Bioactive and biodegradable scaffolds that mimic the natural extracellular matrix of bone serve as temporary structures to guide new bone tissue growth. In this study, 3D-printed scaffolds composed of poly (lactic acid) (PLA)-tricalcium phosphate (TCP) (90-10 wt.%) were modified with 1%, 5%, and 10 wt.% of ZnO to enhance bone tissue regeneration. A commercial chain extender named Joncryl was incorporated alongside ZnO to ensure the printability of the composites. Filaments were manufactured using a twin-screw extruder and subsequently used to print 3D scaffolds via fused filament fabrication (FFF). The scaffolds exhibited a homogeneous distribution of ZnO and TCP particles, a reproducible structure with 300 μm pores, and mechanical properties suitable for bone tissue engineering, with an elastic modulus around 100 MPa. The addition of ZnO resulted in enhanced surface roughness on the scaffolds, particularly for ZnO microparticles, achieving values up to 241 nm. This rougher topography was responsible for enhancing protein adsorption on the scaffolds, with an increase of up to 85% compared to the PLA-TCP matrix. Biological analyses demonstrated that the presence of ZnO promotes mesenchymal stem cell (MSC) proliferation and differentiation into osteoblasts. Alkaline phosphatase (ALP) activity, an important indicator of early osteogenic differentiation, increased up to 29%. The PLA-TCP composite containing 5% ZnO microparticles exhibited an optimized degradation rate and enhanced bioactivity, indicating its promising potential for bone repair applications.

Oncostatin M promotes osteogenic differentiation of tendon-derived stem cells through the JAK2/STAT3 signalling pathway

PurposeOncostatin M (OSM) is involved in the regulation of osteogenic differentiation and has a major role in the development of heterotopic ossification. The role of OSM in osteogenic differentiation of tendon-derived stem cells (TDSCs) and its mechanism have not been reported. This study aim to investigate the role of OSM in osteogenic differentiation of TDSCs and study the mechanism.MethodsTDSCs were differentiated in osteogenic differentiation medium for 7 days. Recombinant OSM was added to the osteogenic differentiation medium for 7 and 14 days. The effect of Janus kinase 2 (JAK2) inhibitor AZD1480 and signal transducer and activator of transcription 3 (STAT3) inhibitor stattic in the presence of recombinant OSM on osteogenic differentiation of TDSCs was examined after differentiation for 7 and 14 days. Alkaline phosphatase and alizarin red staining were used to assess the effects on early and mid-stage osteogenic differentiation, respectively. Western blotting and qPCR were used to assess the expression of receptor and signalling pathway-related proteins and osteogenic marker genes, respectively.ResultsTDSCs were successfully induced to differentiate into osteoblasts. Recombinant OSM promoted osteogenic differentiation of TDSCs to early and mid-stages. After addition of AZD1480 or stattic, decreased alkaline phosphatase and alizarin red staining were observed in the early and mid-stages of osteogenic differentiation. Additionally, decreased expression of receptor and pathway-related proteins, and osteogenic genes was found by western blotting and qPCR, respectively.ConclusionOSM promotes osteogenic differentiation of TDSCs and the JAK2/STAT3 signalling pathway plays an important role.

Fabrication of a coral-like barium titanate nano-piezoelectric coating and its effect on promoting osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro

Objective: To investigate the biocompatibility of coral-like barium titanate nano-piezoelectric coatings and the influence of ultrasound-excited piezoelectric effect on the early osteogenic differentiation. Methods: The barium titanate nano-piezoelectric coating (the coating group) was prepared on the surface of titanium metal by anodic oxidation, hydrothermal reaction and high-temperature annealing, and polished titanium specimens were used as control group. The surface morphology, composition, and crystal phase and hydrophilicity of the two groups of titanium specimens were characterized using scanning electron microscopy, X-ray photoelectron spectroscopy, Raman spectroscopy and contact angle meter. The piezoelectric properties of the materials were characterized by piezoresponse force microscopy. Rat bone marrow mesenchymal stem cells (BMSC) were cultured and identified and seeded the surface of titanium specimens in two groups. The cells seeded on blank culture plates were used as blank group. After low intensity pulsed ultrasound intervention, cell proliferation and live/dead staining were detected to evaluate cytocompatibility of the coatings. Alkaline phosphatase (ALP) activity of each group was detected by ALP staining kit, and the expression of osteogenesis-related genes [integrin, bone morphogenetic protein 2 (BMP-2), Runt-related transcription factor 2 (RUNX2)] was detected by real-time fluorescent quantitative PCR (RT-qPCR) to evaluate the effect of the coating on promoting the early osteogenic differentiation of BMSC. Results: The surface of titanium specimens in the coating group showed a uniform coral-like morphology, and the diameter of the coral tentacles was 70-100 nm. The main component was tetragonal barium titanate. The surface hydrophilicity of the coating group (water contact angle 10.12°± 0.93°) was significantly better than that of the control group (water contact angle 78.32°±0.71°) (F= 10 165.91, P<0.001). The coating has a stable piezoelectric property with a piezoelectric constant of about 5 pC/N. Cell experiments showed that, with or without ultrasound, the cell proliferation activity of the coating group was significantly lower than that of the blank group and the control group on the third day (P<0.05). On the fifth day, with or without ultrasound, there was no significant difference in cell proliferation activity between the three groups (P>0.05). After 7 days of culture, the ALP activity of the coating group was significantly higher than that of the blank group and the control group (P<0.05). The results of RT-qPCR showed that the mRNA expression of integrin and BMP-2 in the coating group with ultrasound was significantly higher than that in the other groups with ultrasound, and was higher than that of the coating group without ultrasound (P<0.05). The expression of integrin mRNA in the control group with ultrasound was significantly higher than that in the control group without ultrasound (P<0.05). The expression of RUNX2 mRNA in the coating group with ultrasound was significantly higher than that in the coating group without ultrasound (P<0.05). Conclusions: The coral-like barium titanate nano-piezoelectric coating exhibits favorable biocompatibility and stable piezoelectric property, and facilitates the early osteogenic differentiation of BMSC under the excitation of low-intensity pulsed ultrasound.

Presenting dual-functional peptides on implant surface to direct in vitro osteogenesis and in vivo osteointegration

The complex biological process of osseointegration and the bio-inertness of bone implants are the major reasons for the high failure rate of long-term implants, and have also promoted the rapid development of multifunctional implant coatings in recent years. Herein, through the special design of peptides, we use layer-by-layer assembly technology to simultaneously display two peptides with different biological functions on the implant surface to address this issue. A variety of surface characterization techniques (ellipsometry, atomic force microscopy, photoelectron spectroscopy, dissipation-quartz crystal microbalance) were used to study in detail the preparation process of the dual peptide functional coating and the physical and chemical properties, such as the composition, mechanical modulus, stability, and roughness of the coating. Compared with single peptide functional coatings, dual-peptide functionalized coatings had much better performances on antioxidant, cellular adhesion in early stage, proliferation and osteogenic differentiation in long term, as well as in vivo osteogenesis and osseointegration capabilities. These findings will promote the development of multifunctional designs in bone implant coatings, as a coping strategy for the complexity of biological process during osteointegration.

Effect of Curcumin-containing Nanofibrous Gelatin-hydroxyapatite Scaffold on Proliferation and Early Osteogenic Differentiation of Dental Pulp Stem Cells.

In recent years, the electrospinning method has received attention because of its usage in producing a mimetic nanocomposite scaffold for tissue regeneration. Hydroxyapatite and gelatin are suitable materials for producing scaffolds, and curcumin has the osteogenesis induction effect. This study aimed to evaluate the toxicity and early osteogenic differentiation stimulation of nanofibrous gelatin-hydroxyapatite scaffold containing curcumin on dental pulp stem cells (DPSCs). The objective of the present investigation was the evaluation of the proliferative effect and primary osteogenic stimulation of DPSCs with a nanofibrous gelatin-hydroxyapatite scaffold containing curcumin. Hydroxyapatite and gelatin were used as suitable and biocompatible materials to make a scaffold suitable for stimulating osteogenesis. Curcumin was added to the scaffold as an osteogenic differentiation- enhancing agent. The effect of nano-scaffold on the proliferation of DPSCs was evaluated. The activity of alkaline phosphatase (ALP) as the early osteogenic marker was considered to assess primary osteogenesis stimulation in DPSCs. The nanofibrous gelatin-hydroxyapatite scaffold containing curcumin significantly increased the proliferation and the ALP activity of DPSCs (P<0.05). The proliferative effect was insignificant in the first 2 days, but the scaffold increased cell proliferation by more than 40% in the fourth and sixth days. The prepared scaffold increased the activity of the ALP of DPSCs by 60% compared with the control after 14 days (p<0.05). The produced nanofibrous gelatin-hydroxyapatite scaffold containing curcumin can be utilized as a potential candidate in tissue engineering and regeneration of bone and tooth. The prepared scaffold in the present study could be a beneficial biomaterial for tissue engineering and the regeneration of bone and tooth soon.

Hydrogel loaded with bone marrow stromal cell-derived exosomes promotes bone regeneration by inhibiting inflammatory responses and angiogenesis.

Bone healing is a complex process involving early inflammatory immune regulation, angiogenesis, osteogenic differentiation, and biomineralization. Fracture repair poses challenges for orthopedic surgeons, necessitating the search for efficient healing methods. To investigate the underlying mechanism by which hydrogel-loaded exosomes derived from bone marrow mesenchymal stem cells (BMSCs) facilitate the process of fracture healing. Hydrogels and loaded BMSC-derived exosome (BMSC-exo) gels were characterized to validate their properties. In vitro evaluations were conducted to assess the impact of hydrogels on various stages of the healing process. Hydrogels could recruit macrophages and inhibit inflammatory responses, enhance of human umbilical vein endothelial cell angiogenesis, and promote the osteogenic differentiation of primary cranial osteoblasts. Furthermore, the effect of hydrogel on fracture healing was confirmed using a mouse fracture model. The hydrogel effectively attenuated the inflammatory response during the initial repair stage and subsequently facilitated vascular migration, promoted the formation of large vessels, and enabled functional vascularization during bone repair. These effects were further validated in fracture models. We successfully fabricated a hydrogel loaded with BMSC-exo that modulates macrophage polarization and angiogenesis to influence bone regeneration.

Remnant polarization and structural arrangement in P(VDF-TrFE) electrospun fiber meshes affect osteogenic differentiation of human mesenchymal stromal cells

Many tissues and cells are influenced by mechano-electric stimulation, thus the application of piezoelectric materials has recently received considerable attention in tissue engineering. This report investigated electrospun fiber meshes based on poly(vinylidenefluoride-co-trifluoroethylene) [P(VDF-TrFE)] as instructive biomaterials for osteogenic differentiation of human mesenchymal stromal cells (hMSCs). The influence played by methyl ethyl ketone (MEK), used as a solvent in place of dimethylformamide (DMF)/acetone mixture, and the effect of rotating velocity of the electrospinning collector on fiber morphology, mechanical and piezoelectric properties were studied. The solvent had noticeable effects on morphology and piezoelectric properties of electrospun fibers, with MEK outperforming DMF/acetone. By increasing the collector velocity up to 4000 rpm, the fiber diameter reduced and the mutual alignment of the fibers increased, corresponding to enhanced mechanical properties and piezo-active β-phase content. However, as a consequence of the diverse mechanical properties of random and aligned fibrous architectures, which ultimately affected the piezoelectric properties, randomly-oriented fibers exhibited higher remnant piezoelectric properties (Vout and d31 piezoelectric coefficient) than aligned ones. On these scaffolds, hMSCs showed an excellent capability of early osteogenic differentiation, leading to high calcium production. Fiber surface topology, fiber mesh morphology and remnant piezoelectric properties played a determinant role on hMSC osteogenic commitment.

Sonic Hedgehog potentiates BMP9-induced osteogenic differentiation of mesenchymal stem cells

Bone morphogenetic protein 9 (BMP9) has remarkable potential to induce the differentiation of mesenchymal stem cells (MSCs) towards the osteoblastic lineage. Additionally, research suggests that certain growth factors have the ability to potentiate BMP9-induced osteogenic differentiation of MSCs. Sonic Hedgehog (Shh) plays an indispensable role in the regulation of skeletal development. The objective of this research was to assess the potential influence of Shh on BMP9-induced osteogenic differentiation of MSCs. Our findings indicated that Shh effectively enhanced BMP9-induced early and late osteogenic differentiation of MSCs, and increased BMP9-induced expression/transcriptional activity of osteogenesis-related transcription factors. Besides, it was observed that Shh promoted BMP9-induced ectopic bone formation of MSCs in vivo. Moreover, BMP9 was able to facilitate the repair of bone defects in rats, while Shh further accelerated this reparative process. Mechanistically, Shh enhanced the activation of the Smad1/5/8 signaling pathway which was induced by BMP9. Furthermore, GANT-61, an inhibitor of Gli1 and Gli2, attenuated the enhancing effect of Shh on BMP9-induced osteogenic differentiation of MSCs. Collectively, the co-administration of BMP9 and Shh may present a promising therapeutic approach for the treatment of fracture nonunion, delayed fracture healing, and bone defects.

Flower-like Ti(HPO4)2 bioceramic-laden 3D printed platform for enhanced bone regeneration via BMP signaling pathway

Regenerative bone implants have been designed to promote new bone formation, however, the search for a suitable implant remains challenging despite the use of various biomaterials (e.g., bone cement, hydroxyapatite, allograft). In this study, we developed a highly bioactive flower-like titanium phosphate (Ti(HPO4)2) bioceramic and poly(ε-caprolactone) 3D composite with a hierarchical structure and interconnected pores. The newly synthesized Ti(HPO4)2 bioceramic, exhibiting an amorphous surface resembling native bone, high porosity, and enhanced ion release properties, was incorporated into 3D scaffolds using an optimized pneumatic 3D printing process. These scaffolds demonstrated their potential for bone regeneration by promoting early osteogenic differentiation and rapid mineralization with human bone marrow mesenchymal stem cells (hBM-MSCs). Moreover, it was revealed their structural stability and tissue regeneration effects after implantation in mouse calvaria. The developed 3D-printed PCL/Ti(HPO4)2 scaffold exhibited enhanced performance in bone regeneration and angiogenesis effects, activated by the bone morphogenetic protein (BMP) signaling pathway. Therefore, this study demonstrates an advanced platform with excellent physicochemical and biological properties for bone regeneration.

The mechanism of lncRNA MALAT1 targeting the miR-124-3p/IGF2BP1 axis to regulate osteogenic differentiation of periodontal ligament stem cells.

This study aimed to investigate the regulatory roles of lncRNA MALAT1, miR-124-3p, and IGF2BP1 in osteogenic differentiation of periodontal ligament stem cells (PDLSCs). We characterized PDLSCs by employing quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot analyses to evaluate the expression of key osteogenic markers including ALPL, SPP1, and RUNX2. Manipulation of lncRNA MALAT1 and miR-124-3p expression levels was achieved through transfection techniques. In addition, early osteogenic differentiation was assessed via Alkaline phosphatase (ALP) staining, and mineral deposition was quantified using Alizarin Red S (ARS) staining. Cellular localization of lncRNA MALAT1 was determined through Fluorescence In Situ Hybridization (FISH). To elucidate the intricate regulatory network, we conducted dual-luciferase reporter assays to decipher the binding interactions between lncRNA MALAT1 and miR-124-3P as well as between miR-124-3P and IGF2BP1. Overexpression of lncRNA MALAT1 robustly promoted osteogenesis in PDLSCs, while its knockdown significantly inhibited the process. We confirmed the direct interaction between miR-124-3p and lncRNA MALAT1, underscoring its role in impeding osteogenic differentiation. Notably, IGF2BP1 was identified as a direct binding partner of lncRNA MALAT1, highlighting its pivotal role within this intricate network. Moreover, we determined the optimal IGF2BP1 concentration (50ng/ml) as a potent enhancer of osteogenesis, effectively countering the inhibition induced by si-MALAT1. Furthermore, in vivo experiments utilizing rat calvarial defects provided compelling evidence, solidifying lncRNA MALAT1's crucial role in bone formation. Our study reveals the regulatory network involving lncRNA MALAT1, miR-124-3p, and IGF2BP1 in PDLSCs' osteogenic differentiation. These findings enhance our understanding of lncRNA-mediated osteogenesis, offering potential therapeutic implications for periodontal tissue regeneration and the treatment of bone defects.

Early Predicting Osteogenic Differentiation of Mesenchymal Stem Cells Based on Deep Learning Within One Day.

Osteogenic differentiation of mesenchymal stem cells (MSCs) is proposed to be critical for bone tissue engineering and regenerative medicine. However, the current approach for evaluating osteogenic differentiation mainly involves immunohistochemical staining of specific markers which often can be detected at day 5-7 of osteogenic inducing. Deep learning (DL) is a significant technology for realizing artificial intelligence (AI). Computer vision, a branch of AI, has been proved to achieve high-precision image recognition using convolutional neural networks (CNNs). Our goal was to train CNNs to quantitatively measure the osteogenic differentiation of MSCs. To this end, bright-field images of MSCs during early osteogenic differentiation (day 0, 1, 3, 5, and 7) were captured using a simple optical phase contrast microscope to train CNNs. The results showed that the CNNs could be trained to recognize undifferentiated cells and differentiating cells with an accuracy of 0.961 on the independent test set. In addition, we found that CNNs successfully distinguished differentiated cells at a very early stage (only 1day). Further analysis showed that overall morphological features of MSCs were the main basis for the CNN classification. In conclusion, MSCs differentiation detection can be achieved early and accurately through simple bright-field images and DL networks, which may also provide a potential and novel method for the field of cell detection in the near future.

Comparison of the osteogenic potential of fibroblasts from different sources

ObjectiveWith the growing interest in the role of fibroblasts in osteogenesis, this study presents a comparative evaluation of the osteogenic potential of fibroblasts derived from three distinct sources: human gingival fibroblasts (HGFs), mouse embryonic fibroblasts (NIH3T3 cells), and mouse subcutaneous fibroblasts (L929 cells). MC3T3-E1 pre-osteoblast cells were employed as a positive control for osteogenic behavior. DesignOur assessment involved multiple approaches, including vimentin staining for cell origin verification, as well as ALP and ARS staining in conjunction with RT-PCR for osteogenic characterization. ResultsOur findings revealed the superior osteogenic differentiation capacity of HGFs compared to MC3T3-E1 and NIH3T3 cells. Analysis of ALP staining confirmed that early osteogenic differentiation was most prominent in MC3T3-E1 cells at 7 days, followed by NIH3T3 and HGFs. However, ARS staining at 21 days demonstrated that HGFs produced the highest number of calcified nodules, indicating their robust potential for late-stage mineralization. This late-stage osteogenic potential of HGFs was further validated through RT-PCR analysis. In contrast, L929 cells displayed no significant osteogenic differentiation potential. ConclusionsIn light of these findings, HGFs emerge as the preferred choice for seed cells in bone tissue engineering applications. This study provides valuable insights into the potential utility of HGFs in the fields of bone tissue engineering and regenerative medicine.

Self‐Activating Phosphorus‐Rich Substrate Import Enzyme Catalytic Domains for Bone Regeneration

AbstractEnzymes play a key role in natural mineralization, but their function is disabling in bone metabolism when patients suffer severe degenerations like hypophosphatasia. To regulate bone homeostasis, a self‐activating phosphorus‐rich system is proposed on calcium glycerophosphate (CaGP) modified polyurethane (PU) fibrous substrate, wherein ample enzyme catalytic domains are imported with intestinal alkaline phosphatase (IALP) chelating and activating by calcium in situ that is testified by chemical potential evaluation. Therefore, Ca‐bonding IALP catalyzes organophosphate to phosphate locally to achieve a calcified symbiotic state, regulating mineralization and accelerating osteoinduction in vitro and in vivo. The self‐activating enzyme system promotes early osteogenic differentiation and angiogenesis, resulting in rapid healing of cranial defect with a “long‐tail” effect compared to an enzyme‐assisted system. Transcriptomic and metabolomic analyses demonstrate positive associations with neovascularization and calcium/phosphorus metabolism, specifically upregulated glycerophospholipids and calcium ions in favor of osteogenic differentiation under cytokine‐free conditions. These results provide a potential enzyme‐activating therapy in bone homeostasis and regeneration.

Preparation of PDA-GO/CS composite scaffold and its effects on the biological properties of human dental pulp stem cells

Reduced graphene oxide (rGO) is an graphene oxide (GO) derivative of graphene, which has a large specific surface area and exhibited satisfactory physicochemical characteristics. In this experiment, GO was reduced by PDA to generate PDA-GO complex, and then PDA-GO was combined with Chitosan (CS) to synthesize PDA-GO/CS composite scaffold. PDA-GO was added to CS to improve the degradation rate of CS, and it was hoped that PDA-GO/CS composite scaffolds could be used in bone tissue engineering. Physicochemical and antimicrobial properties of the different composite scaffolds were examined to find the optimal mass fraction. Besides, we examined the scaffold’s biocompatibility by Phalloidin staining and Live and Dead fluorescent staining.Finally, we applied ALP staining, RT-qPCR, and Alizarin red S staining to detect the effect of PDA-GO/CS on the osteogenic differentiation of human dental pulp stem cells (hDPSCs). The results showed that PDA-GO composite was successfully prepared and PDA-GO/CS composite scaffold was synthesized by combining PDA-GO with CS. Among them, 0.3%PDA-GO/CS scaffolds improves the antibacterial activity and hydrophilicity of CS, while reducing the degradation rate. In vitro, PDA-GO/CS has superior biocompatibility and enhances the early proliferation, migration and osteogenic differentiation of hDPSCs. In conclusion, PDA-GO/CS is a new scaffold materialsuitable for cell culture and has promising application prospect as scaffold for bone tissue engineering.

The response of human osteoblasts on bovine xenografts with and without hyaluronate used in bone augmentation

The aim of the in vitro study was to asses the effect of hyaluronate in conjunction with bovine derived xenografts on the viability, proliferation on day 4, 7 and 10, expression of early osteogenic differentiation marker Alkaline phosphatase on day 14 and 21, collagen, calcium deposition on day 14, 21 and 28 and cellular characteristics, as assessed through live cell image analysis, confocal laser scanning microscopy and scanning electron microscopy, in primary human osteoblasts compared to three bovine xenografts without hyaluronate. All experiments were performed in triplicates. Data were compared between groups and timepoints using one-way analysis of variance (ANOVA). Bonferroni post hoc test were further used for multiple comparison between groups (p < .05) An increase in cell viability (p < .05) and enhanced ALP activity was observed in all xenografts. Specimens containing hyaluronate showed a highest significant difference (23755 ± 29953, p < .0001). The highest levels of calcium (1.60 ± 0.30) and collagen (1.92 ± 0.09, p < .0001) deposition were also observed with hyaluronate loaded groups. The osteoblasts were well attached and spread on all xenograft groups. However, a higher number of cells were observed with hyaluronate functionalized xenograft (76.27 ± 15.11, (p < .0001) in live cell image analysis and they migrated towards the graft boundaries. The biofunctionalization of xenografts with hyaluronate improves their in vitro performance on human osteoblasts. This suggests that hyaluronate might be able to improve the bone regeneration when using such xenografts.

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.

Titanium-based substrate modified with nanoenzyme for accelerating the repair of bone defect

Titanium (Ti) and titanium alloy are the most common metal materials in clinical orthopedic surgery. However, in the initial stage of surgery and implantation, the production of excessive reactive oxygen species (ROS) can induce oxidative stress (OS) microenvironment. OS will further inhibit the growth of new bone, resulting in surgical failure. In this study, based on the fact that nanoscale manganese dioxide (MnO2) can show H2O2-like enzyme activity, a MnO2 nanocoating was prepared on mciro-nano structured surface of Ti substrate via a two-step method of alkaline thermal and hydrothermal treatment. The results of scanning electron microscopy (SEM), X-ray diffractometer (XRD) and X-ray photoelectron spectroscopy (XPS) showed that the nano-MnO2 coating was successfully fabricated on the surface of Ti substrate. The results of measurement of H2O2, dissolved O2 and intracellular ROS in vitro showed that the treated Ti substrate could efficiently eliminate H2O2 and reduce ROS. Furthermore, the modified Ti substrate could promote the early adhesion, proliferation and osteogenic differentiation of MSCs, which was demonstrated by experimental results of cell morphology, cell viability, alkaline phosphatase, collagen, and mineralization deposition. The results of quantitative real-time polymerase chain reaction (qRT-PCR) of MSCs adhered the modified Ti substrate showed that the expression of genes related to osteogenic differentiation significantly increased. More importantly, the modified Ti implant could eliminate ROS at the injury site, reduce OS and promote the regeneration of bone tissue, which was demonstrated via hematoxylin/eosin, Masson's trichrome and immunohistochemical staining. In conclusion, the modified Ti implant presented here had the effect of reducing OS and promoting osseointegration. Relevant research ideas and results provide new methods for the research and development of functional implants, which have potential application value in the field of orthopedics.

Decoupling the roles of mechanical stretching and piezoelectrical stimulation in calcium signal mediated osteogenic differentiation of MSCs on biomimetic nanofibers

Piezoelectric materials implanted in vivo will be affected by physiological stress, leading to the generation of dynamic piezoelectrical signals. The cells identify both piezoelectrical and mechanical stimuli, and the way they behave is regulated through an electromechanical coupling process. Understanding the specific functions of individual stimulation on the osteogenic differentiation of mesenchymal stem cells (MSCs) would aid the development of ideal biomaterials for bone tissue engineering. In this work, by utilizing the controllable piezoelectric properties of polylactic acid materials, we prepared biomimetic piezoelectric poly (L-lactic acid) nanofibers and its non-piezoelectric isomer poly (D, l-lactide) nanofibers using the electrospinning technology. The dynamic piezoelectric and mechanical stimuli on the nanofibers were theoretically and experimentally investigated. A home-made in-situ mechanical loading platform was used to apply predetermined tensile loading on the nanofibers under cell culture conditions. The results indicated that piezo-potential ranging from 11 to 103 μV could be produced by cell adhesion forces varied between 1 and 15 nN, while mechanical loading ranging from 1 % to 15 % strain resulted in piezoelectrical signals in the magnitude of several volts. These piezoelectric signals could influence cell adhesion through enhancing cell aspect ratio and cell area. During the process of cell contraction and re-spreading caused by the mechanical loading, the piezoelectric signals were capable of accelerating the recovery of spreading and adhesion. Furthermore, individual piezoelectric and mechanical signals could modulate intracellular calcium signals, and significantly promote osteogenic differentiation of MSCs. Specifically, the mechanical stimulation could activate PIEZO signaling pathway, resulting in a greater upregulation of early osteogenic differentiation markers of Collagen type I and Bone morphogenetic protein-2 compared to the piezoelectric signal. However, the combination of the two stimuli could obviously and effectively enhance osteogenic differentiation of MSCs in a synergistic manner. These findings offer fresh understandings regarding the electromechanical influences in the process of bone tissue regeneration, which afford additional possibilities for optimizing piezoelectric biomaterials for biomedical applications in a more reasonable manner.

Enhancing osteogenic differentiation in adipose-derived mesenchymal stem cells with Near Infra-Red and Green Photobiomodulation

Worldwide, osteoporosis is the utmost predominant degenerative bone condition. Stem cell regenerative therapy using adipose-derived mesenchymal stem cells (ADMSCs) is a promising therapeutic route for osteoporosis. Photobiomodulation (PBM) has sparked considerable international appeal due to its’ ability to augment stem cell proliferation and differentiation properties. Furthermore, the differentiation of ADMSCs into osteoblast cells and cellular proliferation effects have been established using a combination of osteogenic differentiation inducers and PBM. This in vitro study applied dexamethasone, β-glycerophosphate disodium, and ascorbic acid as differentiation inducers for osteogenic induction differentiation media. In addition, PBM at a near-infrared (NIR) wavelength of 825 nm, a green (G) wavelength of 525 nm, and the novel combination of both these wavelengths using a single fluence of 5 J/cm2 had been applied to stimulate proliferation and differentiation effectivity of immortalised ADMSCs into early osteoblasts. Flow cytometry and ELISA were used to identify osteoblast antigens using early and late osteoblast protein markers. Alizarin red Stain was employed to identify calcium-rich deposits by cells within culture. The morphology of the cells was examined, and biochemical assays such as an EdU proliferation assay, MTT proliferation and viability assay, Mitochondrial Membrane Potential assay, and Reactive Oxygen Species assay were performed. The Central Scratch Test determined the cells' motility potential. The investigative outcomes revealed that a combination of PBM treatment and osteogenic differentiation inducers stimulated promising early osteogenic differentiation of immortalised ADMSCs. The NIR-Green PBM combination did appear to offer great potential for immortalised ADMSC differentiation into early osteoblasts amongst selected assays, however, further investigations will be required to establish the effectivity of this novel wavelength combination. This research contributes to the body of knowledge and assists in the establishment of a standard for osteogenic differentiation in vitro utilising PBM.

Regulation of the integrin αVβ3- actin filaments axis in early osteogenic differentiation of human mesenchymal stem cells under cyclic tensile stress

BackgroundIntegrins are closely related to mechanical conduction and play a crucial role in the osteogenesis of human mesenchymal stem cells. Here we wondered whether tensile stress could influence cell differentiation through integrin αVβ3.MethodsWe inhibited the function of integrin αVβ3 of human mesenchymal stem cells by treating with c(RGDyk). Using cytochalasin D and verteporfin to inhibit polymerization of microfilament and function of nuclear Yes-associated protein (YAP), respectively. For each application, mesenchymal stem cells were loaded by cyclic tensile stress of 10% at 0.5 Hz for 2 h daily. Mesenchymal stem cells were harvested on day 7 post-treatment. Western blotting and quantitative RT-PCR were used to detect the expression of alkaline phosphatase (ALP), RUNX2, β-actin, integrin αVβ3, talin-1, vinculin, FAK, and nuclear YAP. Immunofluorescence staining detected vinculin, actin filaments, and YAP nuclear localization.ResultsCyclic tensile stress could increase the expression of ALP and RUNX2. Inhibition of integrin αVβ3 activation led to rearrangement of actin filaments and downregulated the expression of ALP, RUNX2 and promoted YAP nuclear localization. When microfilament polymerization was inhibited, ALP, RUNX2, and nuclear YAP nuclear localization decreased. Inhibition of YAP nuclear localization could reduce the expression of ALP and RUNX2.ConclusionsCyclic tensile stress promotes early osteogenesis of human mesenchymal stem cells via the integrin αVβ3-actin filaments axis. YAP nuclear localization participates in this process of human mesenchymal stem cells.7pqm7m_g_KeHaQ8nD7WfNiVideo