Early Osteoblast Research Articles

Secreted protein TNA: a promising biomarker for understanding the adipose-bone axis and its impact on bone metabolism

BackgroundOsteoporosis (OP) is a systemic bone disease characterized by reduced bone mass and deterioration of bone microstructure, leading to increased bone fragility. Platelets can take up and release cytokines, and a high platelet count has been associated with low bone density. Obesity is strongly associated with OP, and adipose tissue can influence platelet function by secreting adipokines. However, the biological relationship between these factors remains unclear.MethodsWe conducted differential analysis to identify OP platelet-related plasma proteins. And, making comprehensive analysis, including functional enrichment, protein-protein interaction network analysis, and Friends analysis. The key protein, Tetranectin (TNA/CLEC3B), was identified through screening. Then, we analyzed TNA’s potential roles in osteogenic and adipogenic differentiation using multiple RNA-seq data sets and validated its effect on osteoclast differentiation and bone resorption function through in vitro experiments.ResultsSix OP-platelet-related proteins were identified via differential analysis. Then, we screened the key protein TNA, which was found to be highly expressed in adipose tissue. RNA-seq data suggested that TNA may promote early osteoblast differentiation. In vitro experiments showed that knockdown of TNA expression significantly increased the expression of osteoclast markers, thereby promoting osteoclast differentiation and bone resorption.ConclusionsWe identified TNA as a secreted protein that inhibits osteoclast differentiation and bone resorption. While, it potentially promoted early osteoblast differentiation from bioinformatic results. TNA may play a role in bone metabolism through the adipose-bone axis.

Integrating Mechanics and Bioactivity: A Detailed Assessment of Elasticity and Viscoelasticity at Different Scales in 2D Biofunctionalized PEGDA Hydrogels for Targeted Bone Regeneration.

Methods for promoting and controlling the differentiation of human mesenchymal stem cells (hMSCs) in vitro before in vivo transplantation are crucial for the advancement of tissue engineering and regenerative medicine. In this study, we developed poly(ethylene glycol) diacrylate (PEGDA) hydrogels with tunable mechanical properties, including elasticity and viscoelasticity, coupled with bioactivity achieved through the immobilization of a mixture of RGD and a mimetic peptide of the BMP-2 protein. Despite the key relevance of hydrogel mechanical properties for cell culture, a standard for its characterization has not been proposed, and comparisons between studies are challenging due to the different techniques employed. Here, a comprehensive approach was employed to characterize the elasticity and viscoelasticity of these hydrogels, integrating compression testing, rheology, and atomic force microscopy (AFM) microindentation. Distinct mechanical behaviors were observed across different PEGDA compositions, and some consistent trends across multiple techniques were identified. Using a photoactivated cross-linker, we controlled the functionalization density independently of the mechanical properties. X-ray photoelectrin spectroscopy and fluorescence microscopy were employed to evaluate the functionalization density of the materials before the culturing of hMSCs on them. The cells cultured on all functionalized hydrogels expressed an early osteoblast marker (Runx2) after 2 weeks, even in the absence of a differentiation-inducing medium compared to our controls. Additionally, after only 1 week of culture with osteogenic differentiation medium, cells showed accelerated differentiation, with clear morphological differences observed among cells in the different conditions. Notably, cells on stiff but stress-relaxing hydrogels exhibited an overexpression of the osteocyte marker E11. This suggests that the combination of the functionalization procedure with the mechanical properties of the hydrogel provides a potent approach to promoting the osteogenic differentiation of hMSCs.

Exploration of the miR-187-3p/CNR2 pathway in modulating osteoblast differentiation and treating postmenopausal osteoporosis through mechanical stress.

This study aimed to explore how mechanical stress affects osteogenic differentiation via the miR-187-3p/CNR2 pathway. To conduct this study, 24 female C57BL/6 mice, aged 8 weeks, were used and divided into four groups. The Sham and OVX groups did not undergo treadmill exercise, while the Sham + EX and OVX + EX groups received a 8-week treadmill exercise. Post-training, bone marrow and fresh femur samples were collected for further analysis. Molecular biology analysis, histomorphology analysis, and micro-CT analysis were conducted on these samples. Moreover, primary osteoblasts were cultured under osteogenic conditions and divided into GM group and CTS group. The cells in the CTS group underwent a sinusoidal stretching regimen for either 3 or 7 days. The expression of early osteoblast markers (Runx2, OPN, and ALP) was measured to assess differentiation. The study findings revealed that mechanical stress has a regulatory impact on osteoblast differentiation. The expression of miR-187-3p was observed to decrease, facilitating osteogenic differentiation, while the expression of CNR2 increased significantly. These observations suggest that mechanical stress, miR-187-3p, and CNR2 play crucial roles in regulating osteogenic differentiation. Both invivo and invitro experiments have confirmed that mechanical stress downregulates miR-187-3p and upregulates CNR2, which leads to the restoration of distal femoral bone mass and enhancement of osteoblast differentiation. Therefore, mechanical stress promotes osteoblasts, resulting in improved osteoporosis through the miR-187-3p/CNR2 signaling pathway. These findings have broad prospect and provide molecular biology guidance for the basic research and clinical application of exercise in the prevention and treatment of PMOP.

Vasoactive intestinal peptide exerts an osteoinductive effect in human mesenchymal stem cells.

Several neuropeptides present in bone tissues, produced by nerve fibers and bone cells, have been reported to play a role in regulating the fine-tuning of osteoblast and osteoclast functions to maintain bone homeostasis. This study aims to characterize the influence of the neuropeptide vasoactive intestinal peptide (VIP) on the differentiation process of human mesenchymal stem cells (MSCs) into osteoblasts and on their anabolic function. We describe the mRNA and protein expression profile of VIP and its receptors in MSCs as they differentiate into osteoblasts, suggesting the presence of an autocrine signaling pathway in these cells. Our findings reveal that VIP enhances the expression of early osteoblast markers in MSCs under osteogenic differentiation and favors both bone matrix formation and proper cytoskeletal reorganization. Finally, our data suggest that VIP could be exerting a direct modulatory role on the osteoblast to osteoclast signaling by downregulating the receptor activator of nuclear factor-κB ligand/osteoprotegerin ratio. These results highlight the potential of VIP as an osteoinductive differentiation factor, emerging as a key molecule in the maintenance of human bone homeostasis.

Conditional loss of CaMKK2 in Osterix-positive osteoprogenitors enhances osteoblast function in a sex-divergent manner

Ca2+/calmodulin-dependent protein kinase kinase 2 (CaMKK2) is a multi-functional, serine/threonine protein kinase with predominant roles in inflammation, systemic energy metabolism, and bone remodeling. We previously reported that global ablation of CaMKK2 or its systemic pharmacological inhibition led to bone mass accrual in mice by stimulating osteoblasts and inhibiting osteoclasts. However, a direct, cell-intrinsic role for the kinase in the osteoblast lineage has not been established. Here we report that conditional deletion of CaMKK2 from osteoprogenitors, using the Osterix 1 (Osx1) - GFP::Cre (tetracycline-off) mouse line, resulted in increased trabecular bone mass due to an acute stimulation of osteoblast function in male and female mice. The acute simulation of osteoblasts and bone formation following conditional ablation of osteoprogenitor-derived CaMKK2 was sustained only in female mice. Periosteal bone formation at the cortical bone was enhanced only in male conditional knockout mice without altering cortical bone mass or strength. Prolonged deletion of CaMKK2 in early osteoblasts was accompanied by a stimulation of osteoclasts in both sexes, indicating a coupling effect. Notably, alterations in trabecular and cortical bone mass were absent in the doxycycline-removed “Cre-only” Osx1-GFP::Cre mice. Thus, the increase in osteoblast function at the trabecular and cortical bone surfaces following the conditional deletion of CaMKK2 in osteoprogenitors is indicative of a direct but sex-divergent role for the kinase in osteoblasts.

Linoleic acid blunts early osteoblast differentiation and impairs oxidative phosphorylation in vitro

BackgroundLinoleic acid (LNA), an essential polyunsaturated fatty acid (PUFA), plays a crucial role in cellular functions. However, excessive intake of LNA, characteristic of Western diets, can have detrimental effects on cells and organs. Human observational studies have shown an inverse relationship between plasma LNA concentrations and bone mineral density. The mechanism by which LNA impairs the skeleton is unclear, and there is a paucity of research on the effects of LNA on bone-forming osteoblasts. MethodsThe effect of LNA on osteoblast differentiation, cellular bioenergetics, and production of oxidized PUFA metabolites in vitro, was studied using primary mouse bone marrow stromal cells (BMSC) and MC3T3-E1 osteoblast precursors. ResultsLNA treatment decreased alkaline phosphatase activity, an early marker of osteoblast differentiation, but had no effect on committed osteoblasts or on mineralization by differentiated osteoblasts. LNA suppressed osteoblast commitment by blunting the expression of Runx2 and Osterix, key transcription factors involved in osteoblast differentiation, and other key osteoblast-related factors involved in bone formation. LNA treatment was associated with increased production of oxidized LNA- and arachidonic acid-derived metabolites and blunted oxidative phosphorylation, resulting in decreased ATP production. ConclusionOur results show that LNA inhibited early differentiation of osteoblasts and this inhibitory effect was associated with increased production of oxidized PUFA metabolites that likely impaired energy production via oxidative phosphorylation.

The ecto-nucleotide pyrophosphatase/phosphodiesterase 2 promotes early osteoblast differentiation and mineralization in stromal stem cells.

The phosphodiesterase enzymes mediate calcium-phosphate deposition in various tissues, although which enzymes are active in bone mineralization is unclear. Using gene array analysis, we found that a member of ecto-nucleotide pyrophosphatase/phosphodiesterase family, ENPP2, was strongly down-regulated with age in stromal stem cells that produce osteoblasts and make bone. This is in keeping with reduced bone formation in older animals. Thus, we hypothesized that ENPP2 is, at least in part, an early mediator of bone formation and thus may reflect reduced bone formation with age. Since ENPP2 has not previously been shown to have a role in osteoblast differentiation, we studied its effect on bone differentiation from stromal stem cells, verified by flow cytometry for stem cell antigens. In these remarkably uniform osteoblast precursors, we did transfection with ENPP2 DsiRNA, scrambled DsiRNA, or no transfection to make cells with normal or greatly reduced ENPP2 and analyzed osteoblast differentiation and mineralization. Osteoblast differentiation down-regulation was shown by alizarin red binding, silver staining, and alkaline phosphatase activity. Differences were confirmed by real-time PCR for alkaline phosphatase (ALPL), osteocalcin (BGLAP), and ENPP2 and by Western Blot for Enpp2. These were decreased, ∼50%, in osteoblasts transfected with ENPP2 DsiRNA compared with cells transfected with a scrambled DsiRNA or not transfected (control) cells. This finding is the first evidence for the role of ENPP2 in osteoblast differentiation and mineralization.NEW & NOTEWORTHY We report the discovery that the ecto-nucleotide pyrophosphatase/phosphodiesterase, ENPP2, is an important regulator of early differentiation of bone-forming osteoblasts.

Altered Osteoblast Metabolism with Aging Results in Lipid Accumulation and Oxidative Stress Mediated Bone Loss.

Cellular aging is associated with dysfunction of numerous tissues affecting multiple organ systems. A striking example of this is related to age-related bone loss, or osteoporosis, increasing fracture incidence. Interestingly, the two compartments of bone, cortical and cancellous or trabecular, rely on different mechanisms for development and maintenance during 'normal' aging. At a cellular level, the aging process disturbs a multitude of intracellular pathways. In particular, alterations in cellular metabolic functions thereby impacting cellular bioenergetics have been implicated in multiple tissues. Therefore, this study aimed to characterize how metabolic processes were altered in bone forming osteoblasts in aged mice compared to young mice. Metabolic flux analyses demonstrated both stromal cells and mature, matrix secreting osteoblasts from aged mice exhibited mitochondrial dysfunction. This was also accompanied by a lack of adaptability or metabolic flexibility to utilize exogenous substrates compared to osteoblasts cultured from young mice. Additionally, lipid droplets accumulated in both early stromal cells and mature osteoblasts from aged mice, which was further depicted as increased lipid content within the bone cortex of aged mice. Global transcriptomic analysis of the bone further supported these metabolic data as enhanced oxidative stress genes were up-regulated in aged mice, while osteoblast-related genes were down-regulated when compared to the young mice. Collectively, these data suggest that aging results in altered osteoblast metabolic handling of both exogenous and endogenous substrates which could contribute to age-related osteoporosis.

A convergent fabrication of graphene oxide/silk fibroin/hydroxyapatite nanocomposites delivery improved early osteoblast cell adhesion and bone regeneration

For controlled bone regeneration, two-dimensional materials are essential because they provide an isolated area for bone regeneration and protect the surrounding tissues. Excellent mechanical and hydrophilic qualities have made graphene oxide (GO) a common choice for bone regeneration. A biomimetic mineralization process combined with one-step vacuum filtering created mechanically strong and biocompatible two-dimensional (2D) membranes. Silk fibroin (SF) and hydroxyapatite (HA) increased the membranes' mechanical strength and hydrophilicity on the surface. Osteoblast adhesion, differentiation, and mineralization improved with little HA administered. A calvarial defect model was used to test the impact of the produced composite membranes on bone formation in vivo after they were implanted. Histological examination demonstrates that bone regeneration may be accelerated by GO/SF/HA composite membranes without needing endogenous cytokines. GO/SF/HA nanocomposite membranes with distinctive porous architectures, exceptional mechanical characteristics, and excellent bone regeneration capability are viable nanocomposites for bone regeneration.

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.

Single‐ and Multiscale Laser Patterning of 3D Printed Biomedical Titanium Alloy: Toward an Enhanced Adhesion and Early Differentiation of Human Bone Marrow Stromal Cells

AbstractThis study explores the enhancement of biocompatible titanium‐based implants through surface functionalization for improved bone healing. Specifically, a near‐beta type Ti‐13Nb‐13Zr alloy is 3D printed using laser powder bed fusion and subsequently textured using nanosecond (ns) and picosecond (ps) direct laser interference patterning (DLIP) to create single‐scale and multi‐scale surface textures. On these textures, the cell behavior, morphology, metabolic activity and osteogenic differentiation potential of human bone marrow stromal cells are assessed using fluorescence microscopy and MTS assays. Moreover, tissue non‐specific alkaline phosphatase activity served as an early osteoblast production marker. Compared to untextured specimens, both types of textures exhibited higher metabolic activity and cell proliferation. Single‐scale ns‐DLIP textures encouraged cell extensions anchored in groove regions, while ps‐DLIP textures with hierarchical structures promoted cell extensions attaching to nanostructures on sidewalls. The groove width and nanotopographies in groove areas facilitated cell spreading. Surface topography, roughness, and surface chemistry (surface energy, wettability) influenced cell adhesion, proliferation, and differentiation. A comprehensive evaluation of DLIP‐generated surface textures, including their topography and chemical states, complements the factors affecting in vitro cell behavior. Overall, this research demonstrates the potential of surface‐functionalized 3Dprinted titanium for a novel generation of biocompatible implants.

Evaluation of human amnion denuded derived mesenchymal stem cell on 3D porous hydroxyapatite composite scaffolds for osteogenic differentiation: Prolonged in vitro study

Recent studies using composite porous scaffolds have globally demonstrated the biocompatibility of mesenchymal stem cell sources. It is especially important in hard tissue bioengineering due to its desirable features and stemness effectivity. This study has emphasized primarily the evaluation of the morphological aspects of human amnion-denuded derived mesenchymal stem cells (hAMMSC) in combination with the fabrication of three-dimensional (3D) porous hydroxyapatite (Hap) composite scaffolds for bone tissue engineering based on the principal component of bone mineralization. We presented a novel combination of hAMMSC and nano-crystalline powder hydroxyapatite/bioactive glass (Hap/BG) composite scaffolds fabricated through the hydrothermal method via a novel formulation method from a previous study of stem cell in vitro prolonged culture. The 3D porous scaffold, 6 mm in size, was fabricated from 70 nm nanocrystalline powder and interacted with hAMMSC cultured in osteo-inductive conditions for 30 days. The characterization of 3D porous scaffolds was analyzed via Fourier transform infrared spectroscopy (FTIR) and transmission electron microscopy (TEM) to explore the particle structure, morphology, bioactivity, and porosity of the composite powder and scaffolds in contact with hAMMSCs culture. Furthermore, biocompatibility was assessed using the PrestoBlueTM viability assay and scanning electron microscopy observation (SEM), which revealed the attachment, morphology, and spreading of hAMMSCs on the 3D porous scaffolds of Hap/BG by showing spindle-like morphology and forming the spreading filopodia bridge-like structure within the scaffolds. Subsequently, the secondary objective is the characterization of human AMMSC differentiation capacity assessed by early osteoblast differentiation and mineralization behaviour investigated by early detected assay via alkaline phosphatase activity (ALP) activity marker and energy dispersive X-ray (EDX) analysis simultaneously of the mineralization within bone matrix elements. It was comparatively evaluated via ALP and EDX for calcium quantification elements. Immunomodulatory properties were analyzed via ELISA kits. In conclusion, our findings indicate that 3D porous scaffolds made of Hap/BG composites could be great candidates for future regenerative medicine applications due to their support towards stem cell growth and enhancement of bone cell capacity.

Expression of Stro-1, Runx-2, Osterix, and Alp in Alveolar Bone Regeneration Process Following the Administration of Hydroxyapatite Gypsum Puger (HAGP) Scaffold

Introduction: Tooth extraction before denture placement could result in trauma and damage to up to 50% of the alveolar bone, inducing bone resorption, and affecting the patient’s quality of life. Hydroxyapatite Gypsum Puger (HAGP) can be used as an alternative to bone graft material which degrades slowly, affecting the proliferation and activity of cells that are responsible for bone tissue engineering. This study aimed to analyze the regeneration mechanism of alveolar bone by administering the HAGP scaffold and observing the Stro-1, Runx-2, Osterix, and ALP expression. Methods: Laboratory experimental research was conducted and we used 150-355µm HAGP scaffold particles, applied in vivo inside alveolar sockets of the rats for 7, 14, and 28 days, followed by immunohistochemical examination of Stro-1, Runx-2, Osterix, and ALP expressions. Results: The HAGP scaffold group showed that the Stro-1 expression was significantly higher than the K(-) group, and the Runx-2 expression increased on day 7 and decreased on day 28 in the HAGP and K(-) groups. Osterix expression increased from day 7, 14, to day 28. The high expression of Osterix on day 28 means it took over the Runx-2 function. In ALP there was a significant increase on day 7. ALP expression was a sign of early osteoblast differentiation and production by cells, this extracellular matrix mineralization is an indicator of the osteogenic process. Conclusion: Alveolar bone regeneration mechanism in rats revealed that the expression of Stro-1, Runx-2, Osterix, and ALP was higher in the HAGP scaffold group compared to the control group on days 7,14, and 28.

The potential of RANKL reverse signaling as a novel pharmacological target

RANKL is a bidirectional signaling molecule; activation of the RANKL reverse signaling is triggered by the cross-linking of multiple RANKL trimers to form a molecular cluster. In adults, RANKL is expressed primarily in bone tissue and the immune system. In bone tissue, the functions of RANKL forward and reverse signaling have been separately analyzed; the forward signaling is responsible for bone resorption by inducing the maturation of osteoclasts, while the reverse signaling is activated by vesicular RANK, one of the osteoclast-derived coupling factors, leading to the promotion of early osteoblast differentiation. In the immune system, RANKL is expressed on lymphocytes and the interaction with antigen-presenting cells such as RANK-expressing dendritic cells should result in the activation of both the forward and the reverse signaling, however, the discrimination of the function of each pathway has not been achieved yet. To activate RANKL reverse signaling, a multivalent protein construct of anti-RANKL single-chain Fv multimerized with peptide linkers would be effective, since this type of construct can induce cluster formation by cross-linking RANKL trimers. It was also found that the divalent construct with minimal molecular size can cross-link the RANKL trimer without affecting the forward signaling. On the other hand, the design of constructs to inhibit the activation of RANKL reverse signaling needs to be tested experimentally. Particularly, it may be necessary to obtain small molecules that act on the RANKL intracellular domain to achieve selective inhibition of the reverse signaling without affecting the forward signaling.

A NOVEL SPHEROIDAL PROGENITOR CELL DIFFERENTIATION AND BONE-LIKE IN VITRO BIOMINERALIZATION MODEL

Many age-related diseases affect our skeletal system, but bone health-targeting drug development strategies still largely rely on 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings.MC3T3-E1 pre-osteoblast spheroids were cultured in V-shaped plates for 28 days in alpha-MEM (10% FCS, 1% L-Gln, 1X NEAA) with 1% pen/strep, changed every two days, and differentiation was induced by 10mM b-glycerophosphate and 50µg/ml ascorbic-acid. Osteogenic cell differentiation was assessed through profiling mRNA expression of selected osteogenic markers by efficiency corrected normalized 2^DDCq RT-qPCR. Biomineralization in spheroids was evaluated by histochemistry (Alizarin Red/von Kossa staining), Alkaline phosphatase (Alp) activity, Fourier transform infrared spectroscopy (FTIR) analyses, micro-CT analyses, and scanning electron microscopy on critical point-dried samples. GraphPad Prism 9 analyses comprised Shapiro-Wilk and Brown-Forsythe tests as well as 2-way ANOVA with Tukey post-hoc and non-parametric Kruskal-Wallis with Dunn post-hoc tests.During mineralization, as opposed to non-mineralizing conditions, characteristic mRNA expression profiles of selected early and late osteoblast differentiation markers (e.g., RunX, Alp, Col1a1, Bglap) were observed between day 0 and 28 of culture; Alp was strongly upregulated (p<0.001) from day 7 on, followed by its enzymatic activity (p<0.001). Bglap and Col1a1 expression peaked on (p<0.001) and from day 14 on (p<0.05), respectively. IHC revealed osteocalcin staining in the spheroid core regions at day 14, while type I collagen staining of the cores was most prominent from day 21 on. Alizarin Red and Von Kossa confirmed central and radially outwards expanding mineralization patterns between day 14 and day 28, which was accompanied by a steady increase in extracellular calcium deposition over time (p<0.001). Micro-CT analyses allowed quantitative appreciation of the overall increase in mineral density over time (day21, p<0.05; d28, p<0.001), while SEM-EDX and FTIR ultimately confirmed a bone-like hydroxyapatite mineral deposition in 3D.A novel and thoroughly characterized versatile bone-like 3D biomineralization in vitro model was established, which allows for studying effects of pharmacological interventions on bone mineralization ex vivo under physiomimetic conditions. Ongoing studies currently aim at elucidating in how far it specifically recapitulates intramembranous ossification.

Transcriptomic Changes toward Osteogenic Differentiation of Mesenchymal Stem Cells on 3D-Printed GelMA/CNC Hydrogel under Pulsatile Pressure Environment.

Biomimetic soft hydrogels used in bone tissue engineering frequently produce unsatisfactory outcomes. Here, it is investigated how human bone-marrow-derived mesenchymal stem cells (hBMSCs) differentiated into early osteoblasts on remarkably soft 3D hydrogel (70 ± 0.00049Pa). Specifically, hBMSCs seeded onto cellulose nanocrystals incorporated methacrylate gelatin hydrogels are subjected to pulsatile pressure stimulation (PPS) of 5-20kPa for 7 days. The PPS stimulates cellular processes such as mechanotransduction, cytoskeletal distribution, prohibition of oxidative stress, calcium homeostasis, osteogenic marker gene expression, and osteo-specific cytokine secretions in hBMSCs on soft substrates. The involvement of Piezo 1 is the main ion channel involved in mechanotransduction. Additionally, RNA-sequencing results reveal differential gene expression concerning osteogenic differentiation, bone mineralization, ion channel activity, and focal adhesion. These findings suggest a practical and highly scalable method for promoting stem cell commitment to osteogenesis on soft matrices for clinical reconstruction.

In vitro biological evaluation of epigallocatechin gallate (EGCG) release from three-dimensional printed (3DP) calcium phosphate bone scaffolds.

Three-dimensional printed (3DP) tricalcium phosphate (TCP) scaffolds can guide bone regeneration, especially for patient-specific defect repair applications in low-load bearing sites. Epigallocatechin gallate (EGCG), a green tea compound, has gained attention as a safer alternative treatment for bone disorders. The 3DP TCP scaffold is designed for localized EGCG delivery, which can enhance in vitro osteogenic ability, anti-osteoclastogenic activity, vascularization formation, and chemoprevention. In the cocultures of human bone marrow-derived mesenchymal stem cells (hMSCs) and monocytes (THP-1), EGCG release enhances osteogenic differentiation of hMSCs at day 16 compared to the control; this is indicated by a 2.8- and 4.0-fold upregulation of Runt-related transcription factor 2 (Runx2) and bone gamma-carboxyglutamic acid-containing protein (BGLAP), the early and late osteoblast differentiation marker expressions. However, EGCG significantly downregulates the receptor activator of nuclear factor-κB ligand (RANKL) expression by 7.0-fold, indicating that EGCG suppresses RANKL-induced osteoclast maturation. EGCG also stimulates endothelial tube formation at as early as 3 hours when human umbilical vein endothelial cells (HUVECs) grow on Matrigel. It reduces human osteosarcoma MG-63 cell viability by 66% compared to the control at day 11. An in vitro release kinetics study demonstrates that EGCG shows a ∼64% release within a day followed by a sustained release in the physiological environment (pH 7.4) because its phenolic hydroxyl groups are easily deprotonated at physiological pH. These findings contribute to developing a multifunctional scaffold for the treatment of low load-bearing patient-specific bone defects after trauma and tumor excision.

Trifloroside Induces Bioactive Effects on Differentiation, Adhesion, Migration, and Mineralization in Pre-Osteoblast MC3T3E-1 Cells

Gentianae Scabrae Radix is used in traditional medicine and is known to possess bioactive compounds, including secoiridoid glycosides, flavonoids, lignans, and triterpenes. Trifloroside (TriFs) is a secoiridoid glycoside known for its antioxidant activity; however, its other effects have not been studied. In the present study, we investigated the biological effects of TriFs isolated from the roots of Gentianae Scabrae Radix using pre-osteoblast MC3T3E-1 cells. No cellular toxicity was observed with 1 μM TriFs, whereas 5-100 μM TriFs showed a gradual increase in cell viability. Alkaline phosphatase staining and microscopic observations revealed that 1-10 μM TriFs stimulated osteogenic activity during early osteoblast differentiation. Trifloroside also increased mineral apposition during osteoblast maturation. Biochemical analyses revealed that TriFs promoted nuclear RUNX2 expression and localization by stimulating the major osteogenic BMP2-Smad1/5/8-RUNX2 pathway. Trifloroside also increased p-GSK3β, β-catenin, p-JNK, and p-p38, but not Wnt3a, p-AKT, and p-ERK. Moreover, TriFs increased the MMP13 levels and promoted cell migration and adhesion. In contrast, TriFs-induced osteoblast differentiation and maturation had negligible effects on autophagy and necrosis. Our findings suggest that TriFs induces osteogenic effects through differentiation, adhesion, migration, and mineral apposition. Therefore, TriFs is suggested as a potential drug target in osteoblast-mediated bone diseases.

Surface Free Energy Dominates the Biological Interactions of Postprocessed Additively Manufactured Ti-6Al-4V.

Additive manufacturing (AM) has emerged as a disruptive technique within healthcare because of its ability to provide personalized devices; however, printed metal parts still present surface and microstructural defects, which may compromise mechanical and biological interactions. This has made physical and/or chemical postprocessing techniques essential for metal AM devices, although limited fundamental knowledge is available on how alterations in physicochemical properties influence AM biological outcomes. For this purpose, herein, powder bed fusion Ti-6Al-4V samples were postprocessed with three industrially relevant techniques: polishing, passivation, and vibratory finishing. These surfaces were thoroughly characterized in terms of roughness, chemistry, wettability, surface free energy, and surface ζ-potential. A significant increase in Staphylococcus epidermidis colonization was observed on both polished and passivated samples, which was linked to high surface free energy donor γ– values in the acid–base, γAB component. Early osteoblast attachment and proliferation (24 h) were not influenced by these properties, although increased mineralization was observed for both these samples. In contrast, osteoblast differentiation on stainless steel was driven by a combination of roughness and chemistry. Collectively, this study highlights that surface free energy is a key driver between AM surfaces and cell interactions. In particular, while low acid–base components resulted in a desired reduction in S. epidermidis colonization, this was followed by reduced mineralization. Thus, while surface free energy can be used as a guide to AM device development, optimization of bacterial and mammalian cell interactions should be attained through a combination of different postprocessing techniques.

Effect of Menin Deletion in Early Osteoblast Lineage on the Mineralization of an In Vitro 3D Osteoid-like Dense Collagen Gel Matrix.

Bone has a complex microenvironment formed by an extracellular matrix (ECM) composed mainly of mineralized type I collagen fibres. Bone ECM regulates signaling pathways important in the differentiation of osteoblast-lineage cells, necessary for bone mineralization and in preserving tissue architecture. Compared to conventional 2D cell cultures, 3D in vitro models may better mimic bone ECM and provide an environment to support osteoblastic differentiation. In this study, a biomimetic 3D osteoid-like dense collagen gel model was used to investigate the role of the nuclear protein menin plays in osteoblastic differentiation and matrix mineralization. Previous in vitro and in vivo studies have shown that when expressed at later stages of osteoblastic differentiation, menin modulates osteoblastogenesis and regulates bone mass in adult mice. To investigate the role of menin when expressed at earlier stages of the osteoblastic lineage, conditional knockout mice in which the Men1 gene is specifically deleted early (i.e., at the level of the pluripotent mesenchymal stem cell lineage), where generated and primary calvarial osteoblasts were cultured in plastically compressed dense collagen gels for 21 days. The proliferation, morphology and differentiation of isolated seeded primary calvarial osteoblasts from knockout (Prx1-Cre; Men1f/f) mice were compared to those isolated from wild-type (Men1f/f) mice. Primary calvarial osteoblasts from knockout and wild-type mice did not show differences in terms of proliferation. However, in comparison to wild-type cells, primary osteoblast cells derived from knockout mice demonstrated deficient mineralization capabilities and an altered gene expression profile when cultured in 3D dense collagen gels. In summary, these findings indicate that when expressed at earlier stages of osteoblast differentiation, menin is important in maintaining matrix mineralization in 3D dense collagen gel matrices, in vitro.