Human Bone Mesenchymal Stem Cells Research Articles

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

MiR-26b-5p/TET3 regulates the osteogenic differentiation of human bone mesenchymal stem cells and bone reconstruction in female rats with calvarial defects

Study on liver tissue derived-extracellular vesicles regulating the osteogenic differentiation ability of mesenchymal stem cells and promoting the healing of jaw bone defects

Objective: To explore the biological process of liver tissue-derived extracellular vesicle (LT-EV) in promoting osteogenic differentiation of mesenchymal stem cells and healing of jaw defects to provide a feasible treatment method for the clinical treatment of jaw bone defects. Methods: Enzymatic hydrolysis and differential centrifugation were used to extract LT-EV, scanning electron microscopy, Western blotting, and nanoparticle tracking analyzers were used to identify and characterize LT-EV, and further to explore the biological functions of LT-EV through proteomics and Kyoto Encyclopedia of Genes and Genomes. Flow cytometry was used to detect LT-EV plasma concentration and to calculate the plasma half-life of LT-EV. Small animal in vivo imaging system was used to detect the biological distribution of LT-EV 24 hours after injection. Six C57BL/6 mice were divided into control group and LT-EV group (3 mice in each group) by simple random sampling method. All mice underwent jaw bone defect surgery and tail vein injection every 7 days (the control group was injected with phosphoric buffer saline, LT-EV group was injected with LT-EV), micro-CT was used to evaluate the degree of mouse jaw bone healing 28 days after surgery, HE staining was used to analyze the multi-organ biosafety of LT-EV, and immunofluorescence staining was used to detect the jaw bone expression of osteogenic marker proteins in the defect area. Human jaw bone mesenchymal stem cells (hJBMSC) induced by osteogenic differentiation were treated with LT-EV (obtained from orthognathic surgery patients provided by the Department of Traumatology and Orthognathic Surgery of School of Stomatology of The Fourth Military Medical University resected normal jaw bone fragments), and the difference in osteogenic differentiation ability between the hJBMSC group and the control group (phosphate buffer saline treatment) was compared, and the in vitro bone differentiation promoting effect of LT-EV was verified through alkaline phosphatase (ALP) staining and real-time fluorescence quantitative PCR. Results: The yield of LT-EV was high, and proteomics and Kyoto Encyclopedia of Genes and Genomes showed that LT-EV contained a series of proteins that regulated cell biological functions. LT-EV injected into the tail vein could reach the mouse jaw bone defect area and promote the regeneration and repair of the jaw bone defect [the bone volume fractions of the LT-EV group and the control group were (36.06±4.20)% and (18.58±5.61)%, respectively; t=4.32, P=0.013], and had good biosafety. LT-EV could promote osteogenic differentiation of hJBMSC in vitro. Compared to the control group, ALP staining and osteogenic gene expression levels were significantly enhanced after osteogenic differentiation of hJBMSC (P<0.05). Conclusions: LT-EV exhibits a high yield, ease of acquisition, high biological safety, and excellent bone-promoting effects. It holds promise as a novel cell-free therapy strategy for regenerating craniofacial bone defects.

Branched Channels in Porous β-Tricalcium Phosphate Scaffold Promote Vascularization.

Rapid and efficient vascularization is still considerably challenging for a porous β-tricalcium phosphate (β-TCP) scaffold to achieve. To overcome this challenge, branched channels were created in the porous β-TCP scaffold by using 3D printing and a template-casting method to facilitate the instant flow of blood supply. Human bone mesenchymal stem cells (hBMSCs) and human umbilical vein endothelial cells (HUVECs) were seeded in the channeled porous scaffolds and characterized through a double-stranded DNA (dsDNA) assay, alkaline phosphatase (ALP) assay, and cell migration. Channeled porous β-TCP scaffolds were then implanted in the subcutaneous pockets of mice. Histological staining and immunohistochemical staining on vascularization and bone-related markers were carried out on the embedded paraffin sections. Results from in vitro experiments showed that branched channels significantly promoted HUVECs' infiltration, migration, proliferation, and angiogenesis, and also promoted the proliferation and osteogenesis differentiation of hBMSCs. In vivo implantation results showed that, in the early stage after implantation, cells significantly migrated into branched channeled scaffolds. More matured blood vessels formed in the branched channeled scaffolds compared to that in nonchanneled and straight channeled scaffolds. Beside promoting vascularization, the branched channels also stimulated the infiltration of bone-related cells into the scaffolds. These results suggested that the geometric design of branched channels in the porous β-TCP scaffold promoted rapid vascularization and potentially stimulated bone cells recruitment.

Cryopreservation with DMSO affects the DNA integrity, apoptosis, cell cycle and function of human bone mesenchymal stem cells

Cryopreservation (CP) enables pooling and long-term banking of various types of cells, which is indispensable for the cell therapeutics. Dimethyl sulfoxide (DMSO) is universally used as a cryoprotectant in basic and clinical research. Although, the use of DMSO has been under serious debate due to significant clinical side effects correlated with infusions of cellular therapy products containing DMSO, the effect of CP with DMSO on the cell properties and functions remains unknown. Here, we experimentally found that the CP of human bone mesenchymal stem cells (hBMSCs) with 10 % DMSO results 10–15 % of cells apoptosis upon immediate freeze-thaw, ca. 3.8 times of DNA damage/repair relative to the fresh ones after post-thaw cultured in 48 h, and cell cycle arrests at G0/G1 after post-thaw cultured in 24 h. Moreover, CP with 10 % DMSO significantly increases the reactive oxygen species (ROS) level of the frozen-thawed MSCs which may be one of the causes impair cellular properties and functions. Indeed, we found that the differentiation and migration ability of post-thaw cultured hBMSCs decrease as the expression of adipogenic, osteogenic genes and F-actin reduces in the comparison with those of the fresh cells.

Study on the effect of dynamic chemical polishing treatment on porous titanium alloy scaffolds fabricated using laser powder bed fusion

This study addresses the issue of a significant amount of unmelted powder adhering to the surface of Laser Powder Bed Fusion (LPBF) porous titanium alloy scaffolds, and the current inability of existing technologies to effectively handle such surface defects. Porous titanium alloy scaffolds with a triple periodical minimal surface (TPMS) Gyroid structure were prepared using powder bed fusion technology for dynamic chemical polishing. The spatial and surface morphology, as well as the mechanical properties of the scaffolds, were evaluated before and after treatment. Concurrently, fluid finite element simulation was employed to analyze the flow state of the solution inside the porous scaffold during dynamic chemical polishing. Cell culture samples were prepared and subjected to dynamic chemical polishing to study the specific effects of scaffold surface morphology on cell activity. The results indicate that the surface treatment effect, internal and external homogeneity, and dynamic and static mechanical properties of the porous scaffolds with a Gyroid structure are significantly improved after dynamic chemical polishing. Additionally, it was found that dynamic chemical polishing could effectively reduce the micrometer roughness and increase the nanometer roughness of the sample surface, thereby enhancing the adhesion, proliferation, and differentiation activities of Human Bone Mesenchymal Stem cells.

Pueraria lobata-derived exosome-like nanovesicles alleviate osteoporosis by enhacning autophagy

Osteoporosis (OP) is the most common bone disorder worldwide, especially in postmenopausal women. However, many OP drugs are not suitable for long term use due to major adverse effects. Therefore, there is an urgent need to identify more effective and safe therapeutic drugs. Pueraria lobata has been reported to promote osteoblast growth in bone regeneration, but the exact mechanisms still need further exploration. The current study found that Pueraria lobata-derived exosome-like nanovesicles (PELNs) promoting primary human bone mesenchymal stem cells (hBMSCs) differentiation and mineralization both in vitro and in ovariectomized (OVX)-induced osteoporotic rats. Interestingly, the relative abundance of harmful strains significantly decreased in the intestine of the osteoporosis SD rat model administrated PELNs via the regulation of trimethylamine-N-oxide (TMAO), a metabolite of gut microbiota. Moreover, RNA sequencing revealed that the osteogenic activity of PELNs is revealed to autophagy signaling. In vitro and in vivo experiments also showed that the treatment with PELNs promoted the differentiation and function of hBMSCs by elevating autophagy via the degradation of TMAO. Collectively, PELNs demonstrate promise as a therapeutic approach for OP, with TMAO emerging as a potential target of OP treatment.

Ischemic myocardial inflammatory signaling in starvation versus hypoxia-derived extracellular vesicles: A comparative analysis

BackgroundCoronary artery disease remains a leading cause of death worldwide. Bone mesenchymal stem cell–derived extracellular vesicles (EVs) have shown promise in the setting of myocardial ischemia. Furthermore, the properties of the EVs can be modified via preconditioning of progenitor cells. Previous research from our lab demonstrated a significant decrease in proinflammatory signaling following treatment with EVs derived from starvation preconditioning of human bone mesenchymal stem cells (MVM EVs) in a porcine model of chronic myocardial ischemia. However, rodent models have demonstrated that the use of EVs derived from hypoxia preconditioning of bone mesenchymal stem cells (HYP EVs) may have extended benefits compared to MVM EVs. This study evaluated the effect of HYP EVs on inflammation in a swine model of chronic myocardial ischemia. We hypothesized that HYP EVs would have a greater anti-inflammatory effect than MVM EVs or saline (CON). MethodsYorkshire swine fed a standard diet underwent placement of an ameroid constrictor to the left circumflex artery. Two weeks later, the animals received intramyocardial injection of saline (CON; n = 6), starvation-derived EVs (MVM; n = 10), or hypoxia-derived EVs (HYP; n = 7). After 5 weeks, myocardial perfusion was assessed, and left ventricular myocardial tissue was harvested. Protein expression was measured using immunoblotting. Data were analyzed via the Kruskal-Wallis test or one-way analysis of variance based on the results of a Shapiro-Wilk test. Coronary perfusion was plotted against relative cytokine concentration and analyzed with the Spearman rank-sum test. ResultsHYP EV treatment was associated with decreased expression of proinflammatory markers interleukin (IL)-6 (P = .03), Pro-IL-1ß (P = .01), IL-17 (P < .01), and NOD-like receptor protein 3 (NLRP3; P < .01) compared to CON. Ischemic tissue from the MVM group showed significantly decreased expression of pro-inflammatory markers NLRP3 (P < .01), IL-17 (P < .01), and HLA class II histocompatibility antigen (P < .01) compared to CON. The MVM group also had decreased expression of anti-inflammatory IL-10 (P = .01) compared to CON counterparts. There were no significant differences in expression of tumor necrosis factor-α, interferon-γ, IL-12, Toll-like receptor-2, and nuclear factor kappa-light-chain-enhancer of activated B cells in either group . There was no correlation between coronary perfusion and cytokine concentration in the MVM or HYP groups, either at rest or with pacing. ConclusionsHYP EVs and MVM EVs appear to result in relative decreases in the degree of inflammation in chronically ischemic swine myocardium, independent of coronary perfusion. It is possible that this observed decrease may partially explain the myocardial benefits seen with both HYP and MVM EV treatment.

Development and optimisation of hydroxyapatite-polyethylene glycol diacrylate hydrogel inks for 3D printing of bone tissue engineered scaffolds

In the event of excessive damage to bone tissue, the self-healing process alone is not sufficient to restore bone integrity. Three-dimensional (3D) printing, as an advanced additive manufacturing technology, can create implantable bone scaffolds with accurate geometry and internal architecture, facilitating bone regeneration. This study aims to develop and optimise hydroxyapatite-polyethylene glycol diacrylate (HA-PEGDA) hydrogel inks for extrusion 3D printing of bone tissue scaffolds. Different concentrations of HA were mixed with PEGDA, and further incorporated with pluronic F127 (PF127) as a sacrificial carrier. PF127 provided good distribution of HA nanoparticle within the scaffolds and improved the rheological requirements of HA-PEGDA inks for extrusion 3D printing without significant reduction in the HA content after its removal. Higher printing pressures and printing rates were needed to generate the same strand diameter when using a higher HA content compared to a lower HA content. Scaffolds with excellent shape fidelity up to 75-layers and high resolution (∼200 µm) with uniform strands were fabricated. Increasing the HA content enhanced the compression strength and decreased the swelling degree and degradation rate of 3D printed HA-PEGDA scaffolds. In addition, the incorporation of HA improved the adhesion and proliferation of human bone mesenchymal stem cells (hBMSCs) onto the scaffolds. 3D printed scaffolds with 2 wt% HA promoted osteogenic differentiation of hBMSCs as confirmed by the expression of alkaline phosphatase activity and calcium deposition. Altogether, the developed HA-PEGDA hydrogel ink has promising potential as a scaffold material for bone tissue regeneration, with excellent shape fidelity and the ability to promote osteogenic differentiation of hBMSCs.

Apoptotic vesicles derived from human red blood cells promote bone regeneration via carbonic anhydrase 1.

Apoptotic vesicles (apoVs) are nanoscale vesicles derived from billions of apoptotic cells involved in the maintenance of the human body's homeostasis. Previous researches have shown that some apoVs, such as those derived from mesenchymal stem cells, contribute to bone formation. However, those apoVs cannot be extracted from patients in large quantities, and cell expansion is needed before apoV isolation, which limits their clinical translation. Mature RBCs, which have no nuclei or genetic material, are easy to obtain, showing high biological safety as a source of extracellular vesicles (EVs). Previous studies have demonstrated that RBC-derived EVs have multiple biological functions, but it is unknown whether RBCs produce apoVs and what effect these apoVs have on bone regeneration. In this study, we isolated and characterized RBC-derived apoVs (RBC-apoVs) from human venous blood and investigated their role in the osteogenesis of human bone mesenchymal stem cells (hBMSCs). We showed that RBCs could produce RBC-apoVs that expressed both general apoVs markers and RBC markers. RBC-apoVs significantly promoted osteogenesis of hBMSCs and enhanced bone regeneration in rat calvarial defects. Mechanistically, RBC-apoVs regulated osteogenesis by transferring carbonic anhydrase 1 (CA1) into hBMSCs and activating the P38 MAPK pathway. Our results indicated that RBC-apoVs could deliver functional molecules from RBCs to hBMSCs and promote bone regeneration, pointing to possible therapeutic use in bone tissue engineering.

Abstract P3153: Human Bone Mesenchymal Stem Cell Lentiviral Transduction For The Production Of Extracellular Vesicles Targeted To The Heart Limits Cell Line Expansion

Objectives: Human bone mesenchymal stem cell derived-extracellular vesicles (HBMSC-EV) have been found to have regenerative and anti-inflammatory effects in heart disease, but one of the main obstacles of this treatment modality is the lack of practical delivery to the heart. To make intravenous delivery possible, this study sought to create a human bone mesenchymal stem cell (HBMSC) that secretes HBMSC-EV which will avoid macrophage uptake and home to the heart through lentiviral genetic modification. Methods: The following HBMSC extracellular genetic modifications were selected: CD-47, an anti-phagocytic marker; cardiac targeting peptide (CTP, sequence APWHLSSQYSRT) and ischemic myocardium targeting peptide (IMTP, sequence CSTSMLKAC), peptides both found to have selective cardiac and ischemic myocardium uptake, respectively, through previous biopanning research. Through a 3 rd generation lentiviral system and subsequent antibiotic selection process, wild-type HBMSC were altered to carry these modifications. Results: Three HMBSC lines were created: 1) CD-47 only, 2) CD-47 and CTP anchored to the outer cell membrane by Lamp-2b, and 3) CD-47 and IMTP anchored by Lamp-2b. Preliminary findings show hindered cell proliferation and adherence - cell doubling time was significantly slowed by passage 6 (versus passage 10 in wild-type HBMSC). Also, the genetically modified HBMSC required a 1:2 subculture ratio (versus 1:3-4 in wild-type HBMSC). Conclusions: These genetically-modified HBMSC, engineered with a goal to be better suited for HBMSC-EV production in the treatment of cardiac disease, have significant limitations in cell proliferation and expansion, which will be a significant obstacle to the production of a meaningful quantity of HBMSC-EV. Ongoing studies are being carried out to characterize changes in HBMSC differentiation and in the cell-matrix.

Astragalus polysaccharide promotes osteogenic differentiation of human bone marrow derived mesenchymal stem cells by facilitating ANKFY1 expression through miR-760 inhibition.

Astragalus polysaccharide (APS) participates in various processes, such as the enhancement of immunity and inhibition of tumours. APS can affect osteoporosis (OP) by regulating the osteogenic differentiation of human bone mesenchymal stem cells (hBMSCs). This study was designed to elucidate the mechanism of APS in hBMSC proliferation and osteoblast differentiation. Reverse transcriptase polymerase chain reaction (RT-PCR) and Western blotting were performed to determine the expression of microRNA (miR)-760 and ankyrin repeat and FYVE domain containing 1 (ANKFY1) in OP tissues and hBMSCs. Cell viability was measured using the Cell Counting Kit-8 assay. The expression of cyclin D1 and osteogenic marker genes (osteocalcin (OCN), alkaline phosphatase (ALP), and runt-related transcription factor 2 (RUNX2)) was evaluated using quantitative reverse transcriptase polymerase chain reaction (qRT-PCR). Mineral deposits were detected through Alizarin Red S staining. In addition, Western blotting was performed to detect the ANKFY1 protein levels following the regulation of miR-760. The relationship between miR-760 and ANKFY1 was determined using a luciferase reporter assay. The expression of miR-760 was upregulated in OP tissues, whereas ANKFY1 expression was downregulated. APS stimulated the differentiation and proliferation of hBMSCs by: increasing their viability; upregulating the expression levels of cyclin D1, ALP, OCN, and RUNX2; and inducing osteoblast mineralization. Moreover, APS downregulated the expression of miR-760. Overexpression of miR-760 was found to inhibit the promotive effect of APS on hBMSC differentiation and proliferation, while knockdown of miR-760 had the opposite effect. ANKFY1 was found to be the direct target of miR-760. Additionally, ANKFY1 participated in the APS-mediated regulation of miR-760 function in hBMSCs. APS promotes the osteogenic differentiation and proliferation of hBMSCs. Moreover, APS alleviates the effects of OP by downregulating miR-760 and upregulating ANKFY1 expression.

Retraction Note: Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/β-catenin signaling pathway

Retraction Note: Polydatin improves osteogenic differentiation of human bone mesenchymal stem cells by stimulating TAZ expression via BMP2-Wnt/β-catenin signaling pathway

Inhibition of Mertk Signaling Enhances Bone Healing after Tooth Extraction

Regeneration of alveolar bone is an essential step in restoring healthy function following tooth extraction. Growth of new bone in the healing extraction socket can be variable and often unpredictable when systemic comorbidities are present, leading to the need for additional therapeutic targets to accelerate the regenerative process. One such target is the TAM family (Tyro3, Axl, Mertk) of receptor tyrosine kinases. These proteins have been shown to help resolve inflammation and maintain bone homeostasis and thus may have therapeutic benefits in bone regeneration following extraction. Treatment of mice with a pan-TAM inhibitor (RXDX-106) led to accelerated alveolar bone fill following first molar extraction in a mouse model without changing immune infiltrate. Treatment of human alveolar bone mesenchymal stem cells with RXDX-106 upregulated Wnt signaling and primed the cells for osteogenic differentiation. Differentiation of human alveolar bone mesenchymal stem cells with osteogenic media and TAM-targeted inhibitor RXDX-106 (pan-TAM), ASP-2215 (Axl specific), or MRX-2843 (Mertk specific) showed enhanced mineralization with pan-TAM or Mertk-specific inhibitors and no change with Axl-specific inhibitor. First molar extractions in Mertk–/– mice had increased alveolar bone regeneration in the extraction socket relative to wild type controls 7 d postextraction. Flow cytometry of 7-d extraction sockets showed no difference in immune cell numbers between Mertk –/– and wild type mice. RNAseq of day 7 extraction sockets showed increased innate immune-related pathways and genes associated with bone differentiation in Mertk –/– mice. Together, these results indicate that TAM receptor signaling, specifically through Mertk, can be targeted to enhance bone regeneration after injury.

A 3D Bioprinted Nanoengineered Hydrogel with Photoactivated Drug Delivery for Tumor Apoptosis and Simultaneous Bone Regeneration via Macrophage Immunomodulation.

One of the significant challenges in bone tissue engineering (BTE) is the healing of traumatic tissue defects owing to the recruitment of local infection and delayed angiogenesis. Herein, a 3D printable multi-functional hydrogel composing polyphenolic carbon quantum dots (CQDs, 100 µg mL-1 ) and gelatin methacryloyl (GelMA, 12 wt%) is reported for robust angiogenesis, bone regeneration and anti-tumor therapy. The CQDs are synthesized from a plant-inspired bioactive molecule, 1, 3, 5-trihydroxybenzene. The 3D printed GelMA-CQDs hydrogels display typical shear-thinning behavior with excellent printability. The fabricated hydrogel displayed M2 polarization of macrophage (Raw 264.7) cells via enhancing anti-inflammatory genes (e.g., IL-4 and IL10), and induced angiogenesis and osteogenesis of human bone mesenchymal stem cells (hBMSCs). The bioprinted hBMSCs are able to produce vessel-like structures after 14 d of incubation. Furthermore, the 3D printed hydrogel scaffolds also show remarkable near infra-red (NIR) responsive properties under 808 nm NIR light (1.0W cm-2 ) irradiation with controlled release of antitumor drugs (≈49%) at pH 6.5, and thereby killing the osteosarcoma cells. Therefore, it is anticipated that the tissue regeneration and healing ability with therapeutic potential of the GelMA-CQDs scaffolds may provide a promising alternative for traumatic tissue regeneration via augmenting angiogenesis and accelerated immunomodulation.

3D printing of chitooligosaccharide-polyethylene glycol diacrylate hydrogel inks for bone tissue regeneration.

To date, lack of functional hydrogel inks has limited 3D printing applications in tissue engineering. This study developed a series of photocurable hydrogel inks based on chitooligosaccharide (COS)-polyethylene glycol diacrylate (PEGDA) for extrusion-based 3D printing of bone tissue scaffolds. The scaffolds were prepared by aza-Michael addition of COS and PEGDA followed by photopolymerisation of unreacted PEGDA. The hydrogel inks showed sufficient shear thinning properties required for extrusion 3D printing. The printed scaffolds exhibited excellent shape fidelity and fine microstructure with a resolution of 250 μm. By increasing the COS content, the swelling ratio of the scaffolds decreased, while the compressive strength increased. 3D printed COS-PEGDA scaffolds showed high viability of human bone mesenchymal stem cells in vitro. In addition, scaffolds containing 2 wt% COS showed significantly higher alkaline phosphatase activity, calcium deposition, and bioactivity in simulated body fluid compared to the control (PEGDA). Altogether, 3D printed COS-PEGDA scaffolds represent promising candidates for bone tissue regeneration.

An efficient metal–organic framework-based drug delivery platform for synergistic antibacterial activity and osteogenesis

Bone implants for clinical application should be endowed with antibacterial activity, biocompatibility, and even osteogenesis-promoting properties. In this work, metal–organic framework (MOF) based drug delivery platform was used to modify titanium implants for improved clinical applicability. Methyl Vanillate@Zeolitic Imidazolate Framework-8 (MV@ZIF-8) was immobilized on the polydopamine (PDA) modified titanium. The sustainable release of the Zn2+ and MV causes substantial oxidative damage to Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus). The increased reactive oxygen species (ROS) significantly up-regulates the expression of oxidative stress and DNA damage response genes. Meanwhile, the structural disruption of lipid membranes caused by the ROS, the damage caused by Zinc active sites and the damage accelerated by the MV are both involved in inhibiting bacterial proliferation. The up-regulated expression of the osteogenic-related genes and proteins indicated that the MV@ZIF-8 could effectively promote the osteogenic differentiation of the human bone mesenchymal stem cells (hBMSCs). RNA sequencing and Western blotting analysis revealed that the MV@ZIF-8 coating activates the canonical Wnt/β-catenin signaling pathway through the regulation of tumor necrosis factor (TNF) pathway, thereby promoting the osteogenic differentiation of the hBMSCs. This work demonstrates a promising application of the MOF-based drug delivery platform in bone tissue engineering.

Integrated and Bifunctional Bilayer 3D Printing Scaffold for Osteochondral Defect Repair

AbstractBioinspired scaffolds with two distinct regions resembling stratified anatomical architecture provide potential strategies for osteochondral defect repair and are studied in preclinical animals. However, delamination of the two layers often causes tissue disjunction between the regenerated cartilage and subchondral bone, leading to few commercially available clinical applications. This study develops an integrated poly(ε‐caprolactone) (PCL)‐based scaffold for repairing osteochondral defects. An extracellular matrix (ECM)‐incorporated 3D printing composite scaffold (ECM/PCL) coated with ECM hydrogel (E‐co‐E/PCL) is fabricated as the upper layer, and magnesium oxide nanoparticles coated with polydopamine (MgO@PDA)‐incorporated composite scaffold (MD/PCL) is fabricated using 3D printing as the bottom layer. The physicochemical and mechanical properties of the bilayer scaffold meet the requirements in designing and fabricating the osteochondral scaffold, especially a strong interface possessed between the two layers. By in vitro study, the integrated scaffold stimulates proliferation, chondrogenic differentiation, and osteogenic differentiation of human bone mesenchymal stem cells. Moreover, the integrated bilayer scaffold exhibits well repair ability to facilitate simultaneous regeneration of cartilage and subchondral bone after implanting into the osteochondral defect in rats. In addition, cartilage “tidemarks” completely regenerated after 12 weeks of implantation of the bilayer scaffold, which indicates no tissue disjunctions formed between the regenerated cartilage and subchondral bone.

Electrically stimulated 3D bioprinting of gelatin-polypyrrole hydrogel with dynamic semi-IPN network induces osteogenesis via collective signaling and immunopolarization

In recent years, three-dimensional (3D) bioprinting of conductive hydrogels has made significant progress in the fabrication of high-resolution biomimetic structures with gradual complexity. However, the lack of an effective cross-linking strategy, ideal shear-thinning, appropriate yield strength, and higher print fidelity with excellent biofunctionality remains a challenge for developing cell-laden constructs, hindering the progress of extrusion-based 3D printing of conductive polymers. In this study, a highly stable and conductive bioink was developed based on polypyrrole-grafted gelatin methacryloyl (GelMA-PPy) with a triple cross-linking (thermo-photo-ionically) strategy for direct ink writing-based 3D printing applications. The triple-cross-linked hydrogel with dynamic semi-inner penetrating polymer network (semi-IPN) displayed excellent shear-thinning properties, with improved shape fidelity and structural stability during 3D printing. The as-fabricated hydrogel ink also exhibited “plug-like non-Newtonian” flow behavior with minimal disturbance. The bioprinted GelMA-PPy-Fe hydrogel showed higher cytocompatibility (93%) of human bone mesenchymal stem cells (hBMSCs) under microcurrent stimulation (250 mV/20 min/day). Moreover, the self-supporting and tunable mechanical properties of the GelMA-PPy bioink allowed 3D printing of high-resolution biological architectures. As a proof of concept, we printed a full-thickness rat bone model to demonstrate the structural stability. Transcriptomic analysis revealed that the 3D bioprinted hBMSCs highly expressed gene hallmarks for NOTCH/mitogen-activated protein kinase (MAPK)/SMAD signaling while down-regulating the Wnt/β-Catenin and epigenetic signaling pathways during osteogenic differentiation for up to 7 days. These results suggest that the developed GelMA-PPy bioink is highly stable and non-toxic to hBMSCs and can serve as a promising platform for bone tissue engineering applications.

Characteristic Evaluation of Recombinant MiSp/Poly(lactic-co-glycolic) Acid (PLGA) Nanofiber Scaffolds as Potential Scaffolds for Bone Tissue Engineering.

Biomaterial-based nanofibrous scaffolds are the most effective alternative to bone transplantation therapy. Here, two recombinant minor ampullate spidroins (spider silk proteins), R1SR2 and NR1SR2C, were blended with Poly(lactic-co-glycolic) Acid (PLGA), respectively, to generate nanofiber scaffolds by electrospinning. The N-terminal (N), C-terminal (C), repeating (R1 and R2) and spacer (S) modules were all derived from the minor ampullate spidroins (MiSp). The physical properties and structures of the blended scaffolds were measured by scanning electron microscopy (SEM), water contact angle measurement, Fourier transform infrared spectroscopy (FTIR), Differential scanning calorimetry (DSC), and Tensile mechanical testing. The results showed that blending of MiSp (R1SR2 and NR1SR2C) reduced the diameter of nanofibers, increased the porosity and glass transition temperatures of nanofibrous scaffolds, and effectively improved the hydrophilicity and ultimate strain of scaffolds. It is worth noting that the above changes were more significant in the presence of the N- and C-termini of MiSp. In cell culture assays, human bone mesenchymal stem cells (HBMSCs) grown on NR1SR2C/PLGA (20/80) scaffolds displayed markedly enhanced proliferative and adhesive abilities compared with counterparts grown on pure PLGA scaffolds. Jointly, these findings indicated recombinant MiSp/PLGA, particularly NR1SR2C/PLGA (20/80) blend nanofibrous scaffolds, is promising for bone tissue engineering.

EGFL7 Secreted By Human Bone Mesenchymal Stem Cells Promotes Osteoblast Differentiation Partly Via Downregulation Of Notch1-Hes1 Signaling Pathway.

Epidermal growth factor-like domain protein 7 (EGFL7) is a secreted protein that is differentially expressed in the bone microenvironment; however, the effect of EGFL7 on the osteogenesis of human bone marrow mesenchymal stem cells (hBMSCs) is largely unknown. EGFL7 expression in the fracture microenvironment was analyzed based on the Gene Expression Omnibus (GEO) database. Knockdown of EGFL7 by small interfering RNA (siRNA) and in vitro stimulation with recombinant human EGFL7 (rhEGFL7) protein were used to assess alterations in downstream signaling and changes in the osteogenic differentiation and proliferation of hBMSCs. A γ-secretase inhibitor was used to further explore whether inhibition of Notch signaling rescued the osteogenic-inhibitory effect of EGFL7 knockdown in hBMSCs. A femoral defect model was established to verify the effect of recombinant mouse EGFL7 on bone healing in vivo. EGFL7 expression increased during hBMSC osteogenesis. Knockdown of EGFL7 impaired hBMSC osteogenesis and activated Notch1/NICD/Hes1 signaling. rhEGFL7 promoted hBMSC osteogenesis and downregulated Notch1 signaling. The osteoblast-inhibitory effect of EGFL7 knockdown was rescued by Notch1 signaling inhibition. Recombinant EGFL7 led to enhanced bone healing in mice with femoral defects. EGFL7 promotes osteogenesis of hBMSCs partly via downregulation of Notch1 signaling.