Osteogenic Differentiation Potential Research Articles

Exploring SSEA3 as an emerging biomarker for assessing the regenerative potential of dental pulp-derived stem cells

BackgroundHuman dental pulp-derived stem cells (hDPSCs) have emerged as a promising source for adult stem cell-based regenerative medicine. Stage-specific embryonic antigen 3 (SSEA3) is a cell surface marker associated with Multilineage-differentiating stress-enduring (Muse) cells, a subpopulation of human bone marrow-derived stem cells (hBMSCs), known for their potent regenerative potential and safety profile. In this study, we investigated the influence of the prolonged culture period and the number of culture passages on the regenerative capacity of hDPSCs and explored the association between SSEA3 expression and their regenerative abilities. MethodshDPSCs were isolated and cultured for up to 20 passages. Cell proliferation, migration, and osteogenic, adipogenic and neurogenic differentiation potential were assessed at passages 5, 10, and 20. Flow cytometry and immunofluorescence were employed to analyze SSEA3 expression. RNA sequencing (RNA-seq) was performed on SSEA3-positive and SSEA3-negative hDPSCs to identify differentially expressed genes and associated pathways. ResultsOur findings demonstrated a progressive decline in hDPSCs proliferation and migration capacity with increasing passage number. Conversely, cell size exhibited a positive correlation with passage number. Early passage hDPSCs displayed superior osteogenic and adipogenic differentiation potential. Notably, SSEA3 expression exhibited a significant negative correlation with passage numbers, reflecting the observed decline in differentiation capacity. RNA-seq analysis revealed distinct transcriptional profiles between SSEA3-positive and SSEA3-negative hDPSCs. SSEA3-positive cells displayed upregulation of genes associated with ectodermal differentiation and downregulation of genes involved in cell adhesion. ConclusionsThis study elucidates the impact of passaging on hDPSC behavior and suggests SSEA3 as a valuable biomarker for evaluating stemness and regenerative potential. SSEA3-positive hDPSCs, functionally analogous to Muse cells, represent a promising cell population for developing targeted regenerative therapies with potentially improved clinical outcomes.

Controlled cell proliferation and immortalization of human dental pulp stem cells with a doxycycline-inducible expression system.

Human dental pulp stem cells are a potentially useful resource for cell-based therapies and tissue repair in dental and medical applications. However, the primary culture of isolated dental pulp stem cells has notably been limited. A major requirement of an ideal human dental pulp stem cell culture system is the preservation of efficient proliferation and innate stemness over prolonged passaging, while also ensuring ease of handling through standard, user-friendly culture methods. In this study, we have engineered a novel human dental pulp stem cell line, distinguished by the constitutive expression of telomerase reverse transcriptase (TERT), and the conditional expression of the R24C mutant cyclin-dependent kinase 4 (CDK4R24C) and Cyclin D1. We have named this cell line Tet-off K4DT hDPSCs. Furthermore, we have conducted a comprehensive comparative analysis of their biological attributes in relation to a previously immortalized human dental pulp stem cells, hDPSC-K4DT, which were immortalized by the constitutive expression of CDK4R24C, Cyclin D1 and TERT. In Tet-off K4DT cells, the expression of the K4D genes can be precisely suppressed by the inclusion of doxycycline. Remarkably, Tet-off K4DT cells demonstrated an extended cellular lifespan, increased proliferative capacity, and enhanced osteogenic differentiation potential when compared to K4DT cells. Moreover, Tet-off K4DT cells had no observable genomic aberrations and also displayed a sustained expression of stem cell markers even at relatively advanced passages. Taken together, the establishment of this new cell line holds immense promise as powerful experimental tool for both fundamental and applied research involving dental pulp stem cells.

Murine iPSC-Loaded Scaffold Grafts Improve Bone Regeneration in Critical-Size Bone Defects.

In certain situations, bones do not heal completely after fracturing. One of these situations is a critical-size bone defect where the bone cannot heal spontaneously. In such a case, complex fracture treatment over a long period of time is required, which carries a relevant risk of complications. The common methods used, such as autologous and allogeneic grafts, do not always lead to successful treatment results. Current approaches to increasing bone formation to bridge the gap include the application of stem cells on the fracture side. While most studies investigated the use of mesenchymal stromal cells, less evidence exists about induced pluripotent stem cells (iPSC). In this study, we investigated the potential of mouse iPSC-loaded scaffolds and decellularized scaffolds containing extracellular matrix from iPSCs for treating critical-size bone defects in a mouse model. In vitro differentiation followed by Alizarin Red staining and quantitative reverse transcription polymerase chain reaction confirmed the osteogenic differentiation potential of the iPSCs lines. Subsequently, an in vivo trial using a mouse model (n = 12) for critical-size bone defect was conducted, in which a PLGA/aCaP osteoconductive scaffold was transplanted into the bone defect for 9 weeks. Three groups (each n = 4) were defined as (1) osteoconductive scaffold only (control), (2) iPSC-derived extracellular matrix seeded on a scaffold and (3) iPSC seeded on a scaffold. Micro-CT and histological analysis show that iPSCs grafted onto an osteoconductive scaffold followed by induction of osteogenic differentiation resulted in significantly higher bone volume 9 weeks after implantation than an osteoconductive scaffold alone. Transplantation of iPSC-seeded PLGA/aCaP scaffolds may improve bone regeneration in critical-size bone defects in mice.

Improved protective properties of Mg alloy AZ31 using bis-[triethoxysilylpropyl] tetrasulfide silane films modified with nano TiO2 by electrochemically assisted deposition

Magnesium (Mg) and its alloys were favored by biomedical practitioners thanks to availability of bioactivity and degradability. However, the mismatch between the degradation properties of Mg alloys and the rate of osteogenesis often led to implant failure and bacterial infections within the desired period. The goal of this study was to improve the corrosion resistance of Mg alloys, providing theoretical guidance for solving the problems of implantable Mg-based materials. In this experiment, we prepared a dense and uniform BTESPT/TiO2 film layer on the surface of Mg substrate by electrochemically assisted deposition. The BTESPT/TiO2 film layer provided a physical barrier to avoid direct contact between AZ31 and the corrosive medium. When the addition amount was 2 g/L TiO2, the coating had the best corrosion resistance behavior, its corrosion current density could be up to 9.973×10−8 A/cm2. The BTESPT/TiO2 revealed good cell viability as well as osteogenic differentiation potential on MC3T3-E1 cells.

Influence of Hypoxic Condition on Cytotoxicity, Cellular Migration, and Osteogenic Differentiation Potential of Aged Periodontal Ligament Cells.

This study aimed to investigate and compare the influence of hypoxic conditions on cytotoxicity, cellular migration, and osteogenic differentiation of aged periodontal ligament (PDL) cells. Isolated human PDL cells from aged and young subjects were cultured under hypoxic conditions, which were treated with hydrogen peroxide (H2O2) (0, 25, 50, 100, 200, and 500 µM). To assess cytotoxicity, lactate dehydrogenase release was determined by the optical density at 490 nm, and the percentage of cell death was calculated. An in vitro wound healing assay was performed over 24 to 48 hours for cellular migration determination. Osteogenic differentiation was determined by alizarin red staining and osteogenic gene expression, including the expression of runt-related transcription factor 2 (RUNX2), alkaline phosphatase (ALP), and osteopontin (OPN). There was a significant difference in the percentage of cell death with high hypoxic condition (200 and 500 µM) compared to low hypoxic conditions on both day 1 and 2. The highest cellular migration was depicted at 50 µM in both young and aged groups of the in vitro wound healing assay. Osteogenic gene expression of RUNX2 in the aged group was increased at 25 and 50 µM hypoxic condition at day 7, but the expression was gradually decreased after 14 days. On the contrary, the expression of ALP and OPN in the aged group was increased at day 14. Only OPN had been found to be statistically significantly different when compared with gene expression at day 7 and 14 (p < 0.05). The results showed no statistically significant differences when compared with the young and aged groups in all genes and all concentrations. The concentration of low hypoxic condition (25-50 µM) was proposed to promote cell viability, cellular migration, and osteogenic differentiation in aged PDL cells. We suggested that the potential of aged PDL cells for use in cell therapy for periodontal regeneration might possibly be similar to that of young PDL cells.

Biomimetic bone-periosteum scaffold for spatiotemporal regulated innervated bone regeneration and therapy of osteosarcoma

The complexity of repairing large segment defects and eradicating residual tumor cell puts the osteosarcoma clinical management challenging. Current biomaterial design often overlooks the crucial role of precisely regulating innervation in bone regeneration. Here, we develop a Germanium Selenium (GeSe) co-doped polylactic acid (PLA) nanofiber membrane-coated tricalcium phosphate bioceramic scaffold (TCP-PLA/GeSe) that mimics the bone-periosteum structure. This biomimetic scaffold offers a dual functionality, combining piezoelectric and photothermal conversion capabilities while remaining biodegradable. When subjected to ultrasound irradiation, the US-electric stimulation of TCP-PLA/GeSe enables spatiotemporal control of neurogenic differentiation. This feature supports early innervation during bone formation, promoting early neurogenic differentiation of Schwann cells (SCs) by increasing intracellular Ca2+ and subsequently activating the PI3K-Akt and Ras signaling pathways. The biomimetic scaffold also demonstrates exceptional osteogenic differentiation potential under ultrasound irradiation. In rabbit model of large segment bone defects, the TCP-PLA/GeSe demonstrates promoted osteogenesis and nerve fibre ingrowth. The combined attributes of high photothermal conversion capacity and the sustained release of anti-tumor selenium from the TCP-PLA/GeSe enable the synergistic eradication of osteosarcoma both in vitro and in vivo. This strategy provides new insights on designing advanced biomaterials of repairing large segment bone defect and osteosarcoma.

Cytotoxicity effects and differentiation potential of ormocer-based and nanohybrid composite resins on human dental pulp stem cells

Objectiveto compare conventional nanohybrid (Ceram.x Spectra) and ormocer-based (Admira fusion) dental composite resins effects on human dental pulp stem cells (hDPSCs) in terms of cytotoxicity, self-renewal, migration and osteogenic differentiation. MethodshDPSCs were cultured in presence of different dilutions (undiluted, form 1:2 to 1:100) of CeramX (CX) and Admira fusion (AD) eluates and viability assay in standard or osteogenic conditions were performed. Samples and eluates were prepared according to ISO 10993–12. In addition, apoptosis, self-renewal and migration activity evaluations were carried out. Osteogenic differentiation potential was tested by Alkaline Phosphatase Activity, alizarin red staining and gene expression of specific markers (ALP, RUNX2, OCN, OPN and COL1α1). Statistical analysis was performed by means of a One-way analysis of variance (One-way ANOVA) followed by a Tukey’s test for multiple comparison; results were presented as mean ± standard error of mean (SEM). ResultsAdmira Fusion demonstrated to be highly biocompatible and showed positive effects on hDPSCs proliferation and differentiation; on the contrary, conventional nanohybrid composite showed to be more cytotoxic and without any notable effect on stem cells differentiation. Moreover, the obtained results were further corroborated by a significant upregulation of osteogenic differentiation markers obtained in presence of ormocer-based composite resin eluate. Specifically, in AD 1:50 group expression levels of ALP, Runx2, Col1α1 were double than control (ALP, p = 0.045; Runx2, p = 0.003; Col1α1, p = 0.001) and CX 1:50 (ALP, p = 0.006; RUNX2, p = 0.029; Col1α1, p = 0.005). Moreover, in the same group, OPN and OCN resulted about 5 times more expressed as compared to control (OPN, p = 0.009; OCN, p = 0.0005) and CX 1:50 (OPN, p = 0.012; OCN, p = 0.0006). SignificanceThe less cytotoxicity obtained by AD than conventional nanohybrid composite may be attributed to a reduced monomers release in the oral environment, supporting the hypothesis of limited adverse effect and enhanced healing potential, mainly when the material is positioned in close contact with pulp tissue.

Enhancement of Bone Tissue Regeneration with Multi-Functional Nanoparticles by Coordination of Immune, Osteogenic, and Angiogenic Responses.

Inorganic nanoparticles are promising materials for bone tissue engineering due to their chemical resemblance to the native bone structure. However, most studies are unable to capture the entirety of the defective environment, providing limited bone regenerative abilities. Hence, this study aims to develop a multifunctional nanoparticle to collectively control the defective bone niche, including immune, angiogenic, and osteogenic systems. The nanoparticles, self-assembled by biomimetic mineralization and tannic acid (TA)-mediated metal-polyphenol network (MPN), are released sustainably after the incorporation within a gelatin cryogel. The released nanoparticles display a reduction in M1 macrophages by means of reactive oxygen species (ROS) elimination. Consequently, osteoclast maturation is also reduced, which is observed by the minimal formation of multinucleated cells (0.4%). Furthermore, the proportion of M2 macrophages, osteogenic differentiation, and angiogenic potential are consistently increased by the effects of magnesium from the nanoparticles. This orchestrated control of multiple systems influences the in vivo vascularized bone regeneration in which 80% of the critical-sized bone defect is regenerated with new bones with mature lamellar structure and arteriole-scale micro-vessels. Altogether, this study emphasizes the importance of the coordinated modulation of immune, osteogenic, and angiogenic systems at the bone defect site for robust bone regeneration.

Targeting of C-ROS-1 Activity Using a Controlled Release Carrier to Treat Craniosynostosis in a Preclinical Model of Saethre-Chotzen Syndrome

Saethre-Chotzen syndrome (SCS) is one of the most prevalent craniosynostosis, caused by a loss-of-function mutation in the TWIST-1 gene, with current treatment options relying on major invasive transcranial surgery. TWIST-1 haploinsufficient osteogenic progenitor cells exhibit increased osteogenic differentiation potential due to an upregulation of the transmembrane tyrosine kinase receptor, C-ROS-1, a TWIST-1 target gene known to promote bone formation. The present study assessed the efficacy of suppressing C-ROS-1 activity using a known chemical inhibitor to C-ROS-1, crizotinib, to halt premature coronal suture fusion in a preclinical mouse model of SCS. Crizotinib (1 μM, 2 μM, or 4 μM) was administered locally over the calvaria of Twist‐1del/+ heterozygous mice prior to coronal suture fusion using either a nonresorbable collagen sponge (quick drug release) or a resorbable sodium carboxymethylcellulose microdisk (slow sustained release). Coronal suture fusion rates and bone parameters were determined by μCT imaging and histomorphometric analysis of calvaria postcoronal suture fusion. Results demonstrated a dose-dependent increase in the efficacy of crizotinib to maintain coronal suture patency, with no adverse effects to brain, kidney, liver, and spleen tissue, or blood cell parameters. Moreover, crizotinib delivered on microdisks resulted in a greater efficacy at a lower concentration to reduce bone formation at the coronal suture sites compared to sponges. However, the bone inhibitory effects were found to be diminished by over time following cessation of treatment. Our findings lay the foundation for the development of a pharmacological nonsurgical, targeted approach to temporarily maintain open coronal sutures in SCS patients. This study could potentially be used to develop similar therapeutic strategies to treat different syndromic craniosynostosis conditions caused by known genetic mutations.

Neferine alleviates ovariectomy-induced osteoporosis by enhancing osteogenic differentiation of bone marrow mesenchymal stem cells via regulation of the p38MAPK pathway

ABSTRACT Objective Osteoporosis, a skeletal ailment marked by bone metabolism imbalance and disruption of bone microarchitecture, Neferine, a bisbenzylisoquinoline alkaloid with diverse pharmacological activities, has received limited attention in the context of osteoporosis treatment. Methods We employed a bilateral ovariectomy (OVX) rat model to induce osteoporosis and subsequently administered Neferine treatment for four weeks following successful model establishment. Throughout the modeling and treatment phases, we closely monitored rat body weights. We assessed alterations in bone tissue microstructure through micro-CT, HE staining, and safranin O-fast green staining. Levels of bone formation and resorption markers in serum were evaluated using ELISA assay. Western blot analysis was employed to determine the expression levels of p38MAPK, p-p38MAPK, and bone formation-related genes in bone tissue. We isolated and cultured OVX rat BMSCs (OVX-BMSCs) and induced osteogenic differentiation while simultaneously introducing Neferine and the p38MAPK inhibitor SB203580 for intervention. Results Neferine treatment effectively curbed the rapid weight gain in OVX rats, ameliorated bone loss, and decreased serum levels of TRAP, CTX-I, PINP, and BALP. Most notably, Neferine promoted the expression of bone formation-related factors in bone tissue of OVX rats, while concurrently activating the p38MAPK signaling pathway. In in vitro experiments, Neferine facilitated the expression of bone formation-related factors in OVX-BMSCs, increased the osteogenic differentiation potential of OVX-BMSCs, and activated the p38MAPK signaling pathway. Nevertheless, SB203580 partially reversed Neferine’s promotive effect. Conclusion Neferine can boost the osteoblastic differentiation of BMSCs and alleviate OVX-induced osteoporosis in rats by activating the p38MAPK signaling pathway.

Enhanced Electroactive Phases of Poly(vinylidene Fluoride) Fibers for Tissue Engineering Applications.

Nanofibrous materials generated through electrospinning have gained significant attention in tissue regeneration, particularly in the domain of bone reconstruction. There is high interest in designing a material resembling bone tissue, and many scientists are trying to create materials applicable to bone tissue engineering with piezoelectricity similar to bone. One of the prospective candidates is highly piezoelectric poly(vinylidene fluoride) (PVDF), which was used for fibrous scaffold formation by electrospinning. In this study, we focused on the effect of PVDF molecular weight (180,000 g/mol and 530,000 g/mol) and process parameters, such as the rotational speed of the collector, applied voltage, and solution flow rate on the properties of the final scaffold. Fourier Transform Infrared Spectroscopy allows for determining the effect of molecular weight and processing parameters on the content of the electroactive phases. It can be concluded that the higher molecular weight of the PVDF and higher collector rotational speed increase nanofibers' diameter, electroactive phase content, and piezoelectric coefficient. Various electrospinning parameters showed changes in electroactive phase content with the maximum at the applied voltage of 22 kV and flow rate of 0.8 mL/h. Moreover, the cytocompatibility of the scaffolds was confirmed in the culture of human adipose-derived stromal cells with known potential for osteogenic differentiation. Based on the results obtained, it can be concluded that PVDF scaffolds may be taken into account as a tool in bone tissue engineering and are worth further investigation.

Enhanced osteogenic differentiation potential of Arnica montana and Bellis perennis in C3H10T1/2 multipotent mesenchymal stem cells.

Arnica montanaandBellis perennisare two medicinal plants that are thought to accelerate bone repair in homoeopathic literature. Mesenchymal stem cells (MSCs) are multipotent stem cells with the ability to differentiate and regenerate bone or osteogenesis.Hence, we aimed to determine the role ofArnica montanaandBellis perennison the osteogenic differentiation of the C3H10T1/2 stem cell line. The cell proliferation ofArnica montanaandBellis perenniswas evaluated by MTT assay. Osteogenic differentiation of C3H10T1/2 was induced by the addition of β-glycerophosphate, ascorbic acid and dexamethasone in the differentiation medium over 3 weeks. Cells were treated with Arnica montana andBellis perennis individually as well as in combination. The osteogenic differentiation potential ofArnica montana andBellis perennis to differentiate C3H10T1/2 into osteoblasts was measured by alkaline phosphatase activity, alizarin red staining and the expression of Osteocalcin using immunostaining and qRT-PCR. Arnica montana and Bellis perennis could enhance C3H10T1/2 cell proliferation at 1600µg. Further, the compound showed the ability to augment osteogenesis as confirmed by increased expression of alkaline phosphatase and enhanced calcium accumulation as seen by the Alizarin Red staining and quantification. Enhanced osteogenesis was further supported by the increased expression of osteocalcin in the treated cells with individual and combined doses of Arnica montana and Bellis perennis. Therefore, the findings provide additional support for the positive impact of Arnica montana and Bellis perennis on bone formation. Our findings suggest that homoeopathic compoundsArnica montanaandBellis perenniscan augment osteogenesis individually as well as in combination.

Identification of Periostin Positive Cell Population during the Long-Term Culture of Mouse Tendon-Derived Cells in Late Passage.

Tendon-derived cells exhibit phenotypic changes and gradually lose their proliferative capacity during serial passages in vitro. This study aimed to characterize the changes in the growth and stem cell characteristics of tendon-derived cells over a long-term culture. Mouse flexor digitorum profundus tendon-derived cells were obtained by enzymatic digestion and seeded at an initial density of 5,000/cm2. Cells were characterized by morphology, growth, senescence staining, trilineage differentiation assays, real-time polymerase chain reaction, immunocytochemistry, flow cytometry, and RNA sequencing analysis. Tendon-derived cells underwent a proliferative stage in the first three passages, followed by a gradual senescence. However, a novel cell population expressing periostin (Postn+) emerged during the long-term culture from passages 5-8, which possessed a high rate of proliferation without significant senescence over successive passages. Compared to early passage cells, Postn+ cells exhibited enhanced osteogenic differentiation potential and attenuated chondrogenic differentiation potential, decreased expression of SSEA-1, Oct3/4, tenomodulin, scleraxis, CD90.2, CD73, CD105, Sca-1, and CD44, and increased expression of collagen III and CD34. RNA-sequencing analysis of Postn+ and early passage cells identified 908 differentially expressed genes, with extracellular matrix-receptor interaction and focal adhesion as the top pathways, and integrins as hub genes. This study highlights the dynamics of tendon-derived cells during serial passages. We identify a Postn+ cell population during long-term culture in late passages, with high proliferative ability and prominent osteogenic differentiation potential. Further investigations are needed to elucidate the origin and potential applications of Postn+ tendon-derived cells.

Unraveling Key m6A Modification Regulators Signatures in Postmenopausal Osteoporosis through Bioinformatics and Experimental Verification.

Bone marrow mesenchymal stem cells (BMSCs) show significant potential for osteogenic differentiation. However, the underlying mechanisms of osteogenic capability in osteoporosis-derived BMSCs (OP-BMSCs) remain unclear. This study aims to explore the impact of YTHDF3 (YTH N6-methyladenosine RNA binding protein 3) on the osteogenic traits of OP-BMSCs and identify potential therapeutic targets to boost their bone formation ability. We examined microarray datasets (GSE35956 and GSE35958) from the Gene Expression Omnibus (GEO) to identify potential m6A regulators in osteoporosis (OP). Employing differential, protein interaction, and machine learning analyses, we pinpointed critical hub genes linked to OP. We further probed the relationship between these genes and OP using single-cell analysis, immune infiltration assessment, and Mendelian randomization. Our in vivo and in vitro experiments validated the expression and functionality of the key hub gene. Differential analysis revealed seven key hub genes related to OP, with YTHDF3 as a central player, supported by protein interaction analysis and machine learning methodologies. Subsequent single-cell, immune infiltration, and Mendelian randomization studies consistently validated YTHDF3's significant link to osteoporosis. YTHDF3 levels are significantly reduced in femoral head tissue from postmenopausal osteoporosis (PMOP) patients and femoral bone tissue from PMOP mice. Additionally, silencing YTHDF3 in OP-BMSCs substantially impedes their proliferation and differentiation. YTHDF3 may be implicated in the pathogenesis of OP by regulating the proliferation and osteogenic differentiation of OP-BMSCs.

LncRNA in periodontal tissue-derived cells on osteogenic differentiation in the periodontitis field.

Periodontitis can lead to the destruction of periodontal tissues and potentially tooth loss. Numerous periodontal tissue-derived cells display osteogenic differentiation potential. The presence of differentially expressed long non-coding RNAs (lncRNAs) in these cells indicate their ability to regulate the process of osteogenic differentiation. We aim to elucidate the various lncRNA-mediated regulatory mechanisms in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis at epigenetic modification, transcriptional, and post-transcriptional levels. We systematically searched the PubMed, Web of Science, and ScienceDirect databases to identify relevant literature in the field of periodontitis discussing the role of lncRNAs in regulating osteogenic differentiation of periodontal tissue-derived cells. The identified literature was subsequently summarized for comprehensive review. In this review, we have comprehensively summarized the regulatory mechanisms of lncRNAs in the osteogenic differentiation of periodontal tissue-derived cells in the field of periodontitis and discussed how these lncRNAs provide novel perspectives for understanding the pathogenesis and progression of periodontitis. These results indicate the pivotal role of lncRNAs as regulators in the osteogenic differentiation of periodontal tissue-derived cells, providing a solid basis for future investigations on the role of lncRNAs in the periodontitis field.

Comparing the bone regeneration potential between a trabecular bone and a porous scaffold through osteoblast migration and differentiation: A multiscale approach.

Both cell migration and osteogenic differentiation are critical for successful bone regeneration. Therefore, understanding the mechanobiological aspects that govern these two processes is essential in designing effective scaffolds that promote faster bone regeneration. Studying these two factors at different locations is necessary to manage bone regeneration in various sections of a scaffold. Hence, a multiscale computational model was used to observe the mechanical responses of osteoblasts placed in different positions of the trabecular bone and gyroid scaffold. Fluid shear stresses in scaffolds at cell seeded locations (representing osteogenic differentiation) and strain energy densities in cells at cell substrate interface (representing cell migration) were observed as mechanical response parameters in this study. Comparison of these responses, as two critical factors for bone regeneration, between the trabecular bone and gyroid scaffold at different locations, is the overall goal of the study. This study reveals that the gyroid scaffold exhibits higher osteogenic differentiation and cell migration potential compared to the trabecular bone. However, the responses in the gyroid only mimic the trabecular bone in two out of nine positions. These findings can guide us in predicting the ideal cell seeded sites within a scaffold for better bone regeneration and in replicating a replaced bone condition by altering the physical parameters of a scaffold.

CD34+ synovial fibroblasts exhibit high osteogenic potential in synovial chondromatosis.

Synovial chondromatosis (SC) is a disorder of the synovium characterized by the formation of osteochondral nodules within the synovium. This study aimed to identify the abnormally differentiated progenitor cells and possible pathogenic signaling pathways. Loose bodies and synovium were obtained from patients with SC during knee arthroplasty. Single-cell RNA sequencing was used to identify cell subsets and their gene signatures in SC synovium. Cells derived from osteoarthritis (OA) synovium were used as controls. Multi-differentiation and colony-forming assays were used to identify progenitor cells. The roles of transcription factors and signaling pathways were investigated through computational analysis and experimental verification. We identified an increased proportion of CD34+ sublining fibroblasts in SC synovium. CD34+CD31- cells and CD34-CD31- cells were sorted from SC synovium. Compared with CD34- cells, CD34+ cells had larger alkaline phosphatase (ALP)-stained area and calcified area after osteogenic induction. In addition, CD34+ cells exhibited a stronger tube formation ability than CD34- cells. Our bioinformatic analysis suggested the expression of TWIST1, a negative regulator of osteogenesis, in CD34- sublining fibroblasts and was regulated by the TGF-β signaling pathway. The experiment showed that CD34+ cells acquired the TWIST1 expression during culture and the combination of TGF-β1 and harmine, an inhibitor of Twist1, could further stimulate the osteogenesis of CD34+ cells. Overall, CD34+ synovial fibroblasts in SC synovium have multiple differentiation potentials, especially osteogenic differentiation potential, and might be responsible for the pathogenesis of SC.

Mgp High-Expressing MSCs Orchestrate the Osteoimmune Microenvironment of Collagen/Nanohydroxyapatite-Mediated Bone Regeneration.

Activating autologous stem cells after the implantation of biomaterials is an important process to initiate bone regeneration. Although several studies have demonstrated the mechanism of biomaterial-mediated bone regeneration, a comprehensive single-cell level transcriptomic map revealing the influence of biomaterials on regulating the temporal and spatial expression patterns of mesenchymal stem cells (MSCs) is still lacking. Herein, the osteoimmune microenvironment is depicted around the classical collagen/nanohydroxyapatite-based bone repair materials via combining analysis of single-cell RNA sequencing and spatial transcriptomics. A group of functional MSCs with high expression of matrix Gla protein (Mgp) is identified, which may serve as a pioneer subpopulation involved in bone repair. Remarkably, these Mgp high-expressing MSCs (MgphiMSCs) exhibit efficient osteogenic differentiation potential and orchestrate the osteoimmune microenvironment around implanted biomaterials, rewiring the polarization and osteoclastic differentiation of macrophages through the Mdk/Lrp1 ligand-receptor pair. The inhibition of Mdk/Lrp1 activates the pro-inflammatory programs of macrophages and osteoclastogenesis. Meanwhile, multiple immune-cell subsets also exhibit close crosstalk between MgphiMSCs via the secreted phosphoprotein 1 (SPP1) signaling pathway. These cellular profiles and interactions characterized in this study can broaden the understanding of the functional MSC subpopulations at the early stage of biomaterial-mediated bone regeneration and provide the basis for materials-designed strategies that target osteoimmune modulation.

FBLN5 was Regulated by PRDM9, and Promoted Senescence and Osteogenic Differentiation of Human Periodontal Ligament Stem Cells.

Periodontal ligament stem cells (PDLSCs) are ideal seed cells for periodontal tissue regeneration. Our previous studies have indicated that the histone methyltransferase PRDM9 plays an important role in human periodontal ligament stem cells (hPDLSCs). Whether FBLN5, which is a downstream gene of PRDM9, also has a potential impact on hPDLSCs is still unclear. Senescence was assessed using β-galactosidase and Enzyme-linked immunosorbent assay (ELISA). Osteogenic differentiation potential of hPDLSCs was measured through Alkaline phosphatase (ALP) activity assay and Alizarin red detection, while gene expression levels were evaluated using western blot and RT-qPCR analysis. FBLN5 overexpression promoted the osteogenic differentiation and senescence of hPDLSCs. FBLN5 knockdown inhibited the osteogenic differentiation and senescence of hPDLSCs. Knockdown of PRDM9 decreased the expression of FBLN5 in hPDLSCs and inhibited senescence of hPDLSCs. Additionally, both FBLN5 and PRDM9 promoted the expression of phosphorylated p38 MAPK, Erk1/2 and JNK. The p38 MAPK pathway inhibitor SB203580 and the Erk1/2 pathway inhibitor PD98059 have the same effects on inhibiting the osteogenic differentiation and senescence of hPDLSCs. The JNK pathway inhibitor SP600125 reduced the senescence of hPDLSCs. FBLN5 promoted senescence and osteogenic differentiation of hPDLSCs via activation of the MAPK signaling pathway. FBLN5 was positively targeted by PRDM9, which also activated the MAPK signaling pathway.

Characterization of the biological and transcriptomic landscapes of bone marrow-derived mesenchymal stem cells in patients with multiple myeloma

BackgroundMesenchymal stem/stromal cells (MSCs) have been acknowledged as the most important stromal cells in the bone marrow (BM) microenvironment for physiologic hematopoiesis and the concomitant hematologic malignancies. However, the systematic and detailed dissection of the biological and transcriptomic signatures of BM-MSCs in multiple myeloma (MM) are largely unknown.MethodsIn this study, we isolated and identified BM-MSCs from 10 primary MM patients and 10 healthy donors (HD). On the one hand, we compared the multifaceted biological characteristics of the indicated two BM-MSCs, including biomarker expression pattern, multilineage differentiation potential, stemness and karyotyping, together with the cellular vitality and immunosuppressive property. On the other hand, we took advantage of RNA-SEQ and bioinformatics analysis to verify the similarities and differences at the transcriptomic level between MM-MSCs and HD-MSCs.ResultsAs to biological phenotypes and biofunctions, MM-MSCs revealed conservation in immunophenotype, stemness and differentiation towards adipocytes and chondrocytes with HD-MSCs, whereas with impaired osteogenic differentiation potential, cellular vitality and immunosuppressive property. As to transcriptomic properties, MM-MSCs revealed multidimensional alterations in gene expression profiling and genetic variations.ConclusionsOverall, our date systematic and detailed reflected the multifaceted similarities and variations between MM-MSCs and HD-MSCs both at the cellular and molecular levels, and in particular, the alterations of immunomodulation and cellular viability of MM-MSCs, which wound benefit the further exploration of the pathogenesis and new drug application (NDA) of multiple myeloma from the view of BM-MSCs.