Effective Osteogenic Priming of Mesenchymal Stem Cells through LNA-ASOs-Mediated Sfrp1 Gene Silencing

Mesenchymal stem cell (MSC) transplantation has emerged as a promising approach for bone regeneration. Importantly, the beneficial effects of MSCs can be improved by modulating the expression levels of specific genes to stimulate MSC osteogenic differentiation. We have previously shown that Smurf1 silencing by using Locked Nucleic Acid-Antisense Oligonucleotides, in combination with a scaffold that sustainably releases low doses of BMP-2, was able to increase the osteogenic potential of MSCs in the presence of BMP-2 doses significantly smaller than those currently used in the clinic. This would potentially allow an important reduction in this protein in MSs-based treatments, and thus of the side effects linked to its administration. We have further improved this system by specifically targeting the Wnt pathway modulator Sfrp1. This approach not only increases MSC bone regeneration efficiency, but is also able to induce osteogenic differentiation in osteoporotic human MSCs, bypassing the need for BMP-2 induction, underscoring the regenerative potential of this system. Achieving successful osteogenesis with the sole use of LNA-ASOs, without the need of administering pro-osteogenic factors such as BMP-2, would not only reduce the cost of treatments, but would also open the possibility of targeting these LNA-ASOs specifically to MSCs in the bone marrow, allowing us to treat systemic bone loss such as that associated with osteoporosis.

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TRIM16 Positively Regulates Osteogenic Differentiation of Human Periodontal Ligament Stem Cells by Modulating the Ubiquitination and Degradation of RUNX2

10.21203/rs.3.rs-38207/v1 ◽

2020 ◽

Author(s):

Yi Zhao ◽

Qiaoli Zhai ◽

Hong Liu ◽

Xun Xi ◽

Shuai Chen ◽

...

Keyword(s):

Stem Cells ◽

Alkaline Phosphatase ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Periodontal Ligament ◽

Osteogenic Potential ◽

Common Disease ◽

Periodontal Ligament Stem Cells ◽

Alizarin Red ◽

Periodontal Tissues

Abstract BackgroundPeriodontal disease is a common disease that compromises the integrity of tooth-supporting tissues. Bone regeneration is the ultimate goal of periodontal therapies, in which osteogenic differentiation of human periodontal ligament stem cells plays a critical role. The tripartite motif (TRIM)16 is downregulated in periodontal tissues of patients with periodontitis and involved in osteogenic differentiation of human bone marrow mesenchymal stem cells(hBMSCs).However, the role of TRIM16 in the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) is largely unknown.MethodshPDLSCs were isolated and identified by immunophenotype assays using flow cytometry. Overexpression plasmids and specific short-hairpin RNAs (shRNAs) were constructed to manipulate the expression of target molecules. Alkaline phosphatase (ALP) staining, alizarin red staining (ARS) and enzyme‐linked immunosorbent assays (ELISA) were used to evaluate osteogenic potential capacity. Reverse transcription quantitative PCR (RT-qPCR) and Western blot analysis were performed to determine the expression of osteogenic-related markers and activation of relevant signaling pathways. Co-immunoprecipitation assays were performed to confirm the interactions between proteins and the ubiquitination of RUNX2. A LC-MS/MS analysis was performed to explore the different expression proteins in present of TRIM16.ResultsTRIM16 significantly promoted alkaline phosphatase activity and mineralized nodule formation, and positively regulated the osteogenic differentiation of hPDLSCs by enhancing protein expression of RUNX2, COL1A1 and OCN. Mechanistically, TRIM16 serves as a pivotal factor that stabilizes RUNX2 protein levels by decreasing CHIP-mediated K48-linked ubiquitination degradation of the RUNX2 protein. Besides, TRIM16 significantly increased expression of COL1A1 via activation of p38MAPK/RUNX2.ConclusionThis study identified a novel mechanism of TRIM16 in regulating stability of the RUNX2 protein, which may promote the osteogenic differentiation of hPDLSCs. TRIM16 may be a potential target of stem cell based-bone regeneration for periodontal therapies.

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MiR-144-5p, an exosomal miRNA from bone marrow-derived macrophage in type 2 diabetes, impairs bone fracture healing via targeting Smad1

Journal of Nanobiotechnology ◽

10.1186/s12951-021-00964-8 ◽

2021 ◽

Vol 19 (1) ◽

Author(s):

Dong Zhang ◽

Yifan Wu ◽

Zonghuan Li ◽

Hairen Chen ◽

Siyuan Huang ◽

...

Keyword(s):

Bone Marrow ◽

Fracture Healing ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Fracture Repair ◽

Osteogenic Potential ◽

Patients With Diabetes ◽

The Impact

Abstract Background Patients with diabetes have an increased risk of nonunion and delayed union of fractures. Macrophages have been shown as a key player in diabetic complications. However, it remains obscure how diabetic milieu affects macrophage-derived exosomes and its implications on osteogenic differentiation of BMSCs. In this study, we aim to define the impact of diabetic milieu on macrophage-derived exosomes, role of extracellular vesicles in intercellular communication with BMSCs, and subsequent effects on osteogenic differentiation and fracture repair. Results The osteogenic potential and the ability of fracture repair of exosomes derived from diabetic bone marrow-derived macrophages (dBMDM-exos) were revealed to be lower, as compared with non-diabetic bone marrow-derived macrophages (nBMDM-exos) in vitro and in vivo. Interestingly, miR-144-5p levels were sharply elevated in dBMDM-exos and it could be transferred into BMSCs to regulate bone regeneration by targeting Smad1. In addition, the adverse effects of dBMDM-exos on the osteogenic potential and the ability of fracture repair were reversed through the suppression of miR-144-5p inhibition in vitro and vivo. Conclusions The results demonstrated an important role of exosomal miR-144-5p in bone regeneration, offering insight into developing new strategy for the improvement of fracture healing in patients with diabetes mellitus. Graphic Abstract

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Translational Research for Orthopedic Bone Graft Development

Materials ◽

10.3390/ma14154130 ◽

2021 ◽

Vol 14 (15) ◽

pp. 4130

Author(s):

Maria J. C. Vilela ◽

Bruno J. A. Colaço ◽

José Ventura ◽

Fernando J. M. Monteiro ◽

Christiane L. Salgado

Keyword(s):

Production Process ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Laser Scanning ◽

Complex Structure ◽

Laser Scanning Microscopy ◽

Confocal Laser ◽

Mechanical Resistance ◽

Human Mscs ◽

Biological Studies

Designing biomaterials for bone-substitute applications is still a challenge regarding the natural complex structure of hard tissues. Aiming at bone regeneration applications, scaffolds based on natural collagen and synthetic nanohydroxyapatite were developed, and they showed adequate mechanical and biological properties. The objective of this work was to perform and evaluate a scaled-up production process of this porous biocomposite scaffold, which promotes bone regeneration and works as a barrier for both fibrosis and the proliferation of scar tissue. The material was produced using a prototype bioreactor at an industrial scale, instead of laboratory production at the bench, in order to produce an appropriate medical device for the orthopedic market. Prototypes were produced in porous membranes that were e-beam irradiated (the sterilization process) and then analysed by scanning electron microscopy (SEM), confocal laser scanning microscopy (CLSM), dynamic mechanical analysis (DMA), cytotoxicity tests with mice fibroblasts (L929), human osteoblast-like cells (MG63) and human MSC osteogenic differentiation (HBMSC) with alkaline phosphatase (ALP) activity and qPCR for osteogenic gene expression. The prototypes were also implanted into critical-size bone defects (rabbits’ tibia) for 5 and 15 weeks, and after that were analysed by microCT and histology. The tests performed for the physical characterization of the materials showed the ability of the scaffolds to absorb and retain water-based solvents, as well as adequate mechanical resistance and viscoelastic properties. The cryogels had a heteroporous morphology with microporosity and macroporosity, which are essential conditions for the interaction between the cells and materials, and which consequently promote bone regeneration. Regarding the biological studies, all of the studied cryogels were non-cytotoxic by direct or indirect contact with cells. In fact, the scaffolds promoted the proliferation of the human MSCs, as well as the expression of the osteoblastic phenotype (osteogenic differentiation). The in vivo results showed bone tissue ingrowth and the materials’ degradation, filling the critical bone defect after 15 weeks. Before and after irradiation, the studied scaffolds showed similar properties when compared to the results published in the literature. In conclusion, the material production process upscaling was optimized and the obtained prototypes showed reproducible properties relative to the bench development, and should be able to be commercialized. Therefore, it was a successful effort to harness knowledge from the basic sciences to produce a new biomedical device and enhance human health and wellbeing.

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Association of Dental Pulp Stem Cells and Ricinus Bone Compound in a Model of Bone Defect

JOURNAL OF BIOENGINEERING AND TECHNOLOGY APPLIED TO HEALTH ◽

10.34178/jbth.v3i3.129 ◽

2020 ◽

Vol 3 (3) ◽

pp. 267-278

Author(s):

Alan Jesus ◽

Adriano Jesus ◽

Flávia Lima ◽

Luiz Freitas ◽

Cássio Meira ◽

...

Keyword(s):

Stem Cells ◽

Bone Regeneration ◽

Osteogenic Differentiation ◽

Bone Defect ◽

Dental Pulp ◽

Dental Pulp Stem Cells ◽

Osteogenic Potential ◽

Autogenous Bone ◽

Control Group

Autogenous bone grafting is needed in some bone tissue defects; however, it causes secondary surgical wounds and morbidity. Tissue bioengineering may be an alternative approach for bone regeneration. Here we investigated the osteogenic potential of dental pulp stem cells from deciduous teeth (DPSC) in association with a Ricinus bone compound (RBC) in a model of bone defect. The influence of the biomaterial RBC on the proliferation and osteogenic differentiation of DPSC was assessed in vitro by MTT metabolism and alizarin red staining, respectively. The morphologic analysis was performed using the optic and scanning electron (SEM) microscopies. For the in vivo study, 54 Wistar rats submitted to calvarial defects were filled with RBC or RBC+DPSC. A control group had the defects filled only with blood clots. Analyses were performed 15, 30 and 60 days after treatment using digital radiography, optical microscopy, SEM and chemical analysis by electron dispersive spectroscopy. The Ricinus bone compound (RBC) did not inhibit the osteogenic differentiation in vitro. No spontaneous regeneration was observed in the control group. The area of the calvarial defect of the RBC+DPSC group showed greater radiopacity on day 15. The RBC presented no reabsorption, was biocompatible and showed osteointegration, working as a mechanical filling. Only sparse ossification areas were found and those were larger and more developed on the RBC+DPSC group when compared to animals treated only with RBC. RBC in association with DPSC is a promising combination for applications in bone regeneration.

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Human Mesenchymal Stem Cell Response to Lactoferrin-based Composite Coatings

Materials ◽

10.3390/ma12203414 ◽

2019 ◽

Vol 12 (20) ◽

pp. 3414 ◽

Cited By ~ 3

Author(s):

Madalina Icriverzi ◽

Anca Bonciu ◽

Laurentiu Rusen ◽

Livia Elena Sima ◽

Simona Brajnicov ◽

...

Keyword(s):

Bone Regeneration ◽

Osteogenic Differentiation ◽

Bioactive Compounds ◽

Composite Coatings ◽

Laser Evaporation ◽

Human Mscs ◽

Research Subjects ◽

Ongoing Research ◽

Cell Based Therapy ◽

Composite Layers

The potential of mesenchymal stem cells (MSCs) for implantology and cell-based therapy represents one of the major ongoing research subjects within the last decades. In bone regeneration applications, the various environmental factors including bioactive compounds such as growth factors, chemicals and physical characteristics of biointerfaces are the key factors in controlling and regulating osteogenic differentiation from MSCs. In our study, we have investigated the influence of Lactoferrin (Lf) and Hydroxyapatite (HA) embedded within a biodegradable PEG-PCL copolymer on the osteogenic fate of MSCs, previous studies revealing an anti-inflammatory potential of the coating and osteogenic differentiation of murine pre-osteoblast cells. The copolymer matrix was obtained by the Matrix Assisted Pulsed Laser Evaporation technique (MAPLE) and the composite layers containing the bioactive compounds (Lf, HA, and Lf-HA) were characterised by Scanning Electron Microscopy and Atomic Force Microscopy. Energy-dispersive X-ray spectroscopy contact angle and surface energy of the analysed coatings were also measured. The characteristics of the composite surfaces were correlated with the viability, proliferation, and morphology of human MSCs (hMSCs) cultured on the developed coatings. All surfaces were found not to exhibit toxicity, as confirmed by the LIVE/DEAD assay. The Lf-HA composite exhibited an increase in osteogenic differentiation of hMSCs, results supported by alkaline phosphatase and mineralisation assays. This is the first report of the capacity of biodegradable composite layers containing Lf to induce osteogenic differentiation from hMSCs, a property revealing its potential for application in bone regeneration.

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K-Carrageenan Stimulates Pre-Osteoblast Proliferation and Osteogenic Differentiation: A Potential Factor for the Promotion of Bone Regeneration?

Molecules ◽

10.3390/molecules26206131 ◽

2021 ◽

Vol 26 (20) ◽

pp. 6131

Author(s):

Wei Cao ◽

Jianfeng Jin ◽

Gang Wu ◽

Nathalie Bravenboer ◽

Marco N. Helder ◽

...

Keyword(s):

Bone Regeneration ◽

Osteogenic Differentiation ◽

Metabolic Activity ◽

Fold Increase ◽

Bone Tissue Regeneration ◽

Osteogenic Potential ◽

Maximum Effect ◽

Osteoblast Proliferation ◽

Matrix Mineralization ◽

Osteoblast Adhesion

Current cell-based bone tissue regeneration strategies cannot cover large bone defects. K-carrageenan is a highly hydrophilic and biocompatible seaweed-derived sulfated polysaccharide, that has been proposed as a promising candidate for tissue engineering applications. Whether κ-carrageenan can be used to enhance bone regeneration is still unclear. In this study, we aimed to investigate whether κ-carrageenan has osteogenic potential by testing its effect on pre-osteoblast proliferation and osteogenic differentiation in vitro. Treatment with κ-carrageenan (0.5 and 2 mg/mL) increased both MC3T3-E1 pre-osteoblast adhesion and spreading at 1 h. K-carrageenan (0.125–2 mg/mL) dose-dependently increased pre-osteoblast proliferation and metabolic activity, with a maximum effect at 2 mg/mL at day three. K-carrageenan (0.5 and 2 mg/mL) increased osteogenic differentiation, as shown by enhanced alkaline phosphatase activity (1.8-fold increase at 2 mg/mL) at day four, and matrix mineralization (6.2-fold increase at 2 mg/mL) at day 21. K-carrageenan enhanced osteogenic gene expression (Opn, Dmp1, and Mepe) at day 14 and 21. In conclusion, κ-carrageenan promoted MC3T3-E1 pre-osteoblast adhesion and spreading, metabolic activity, proliferation, and osteogenic differentiation, suggesting that κ-carrageenan is a potential osteogenic inductive factor for clinical application to enhance bone regeneration.

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Panax notoginseng Saponin Promotes Bone Regeneration in Distraction Osteogenesis via the TGF-β1 Signaling Pathway

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2021/2895659 ◽

2021 ◽

Vol 2021 ◽

pp. 1-11

Author(s):

Di Liu ◽

Zhenchen Zhao ◽

Weidong Jiang ◽

Peiqi Zhu ◽

Xiaoning An ◽

...

Keyword(s):

Bone Regeneration ◽

Osteogenic Differentiation ◽

Distraction Osteogenesis ◽

Panax Notoginseng ◽

Osteogenic Potential ◽

Mrna Levels ◽

Alizarin Red ◽

Smad Pathway ◽

Tgf Β1

Distraction osteogenesis (DO) is an efficient strategy that is employed for the treatment of large bone defects in craniomaxillofacial surgery. Despite its utility, however, DO is associated with a prolonged consolidation phase and a high complication rate that hinder its more widespread utilization. Panax notoginseng saponin (PNS) is a traditional Chinese medicine that is frequently administered for the treatment of a range of conditions. Herein, we explored the ability of PNS treatment to influence osteogenic differentiation using both rabbit bone marrow mesenchymal cells (BMSCs) and a model of mandibular DO. BMSC proliferation was assessed via CCK-8 assay, while osteogenic differentiation was monitored through ALP and alizarin red S staining. A PCR approach was used to evaluate the expression of genes associated with osteogenesis (ALP, Runx2, and OCN) and genes linked to the TGF pathway (TβR-II, SMAD2, SMAD3, and PPM1A). For in vivo experiments, treated BMSCs were locally injected into the DO gap, with PNS being injected into treated rabbits every other day throughout the experimental period. The quality of the regenerative process was assessed via scanning electron microscopy (SEM), energy dispersive spectroscopy (EDS), X-ray imaging, and hematoxylin and eosin (H&E) staining. These analyses revealed that PNS was able to promote BMSC osteogenesis and mandibular generation, driving the upregulation of osteogenesis-related genes at the mRNA levels through the modulation of the TGF-β1/Smad pathway. Consistently, the overexpression or silencing of TβR-II in PNS-treated BMSCs was sufficient to modulate their osteogenic potential. Analyses of in vivo mandibular DO outcomes revealed significantly augmented new bone growth in the PNS-treated group relative to control animals, with maximal osteogenesis in the group overexpressing rabbit TβR-II. Together, these results highlight the PNS as a promising and cost-effective therapeutic tool with the potential to enhance bone regeneration in clinical contexts through the modulation of the TGF-β1/Smad pathway.

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Macrophage-derived small extracellular vesicles promote biomimetic mineralized collagen-mediated endogenous bone regeneration

International Journal of Oral Science ◽

10.1038/s41368-020-00100-6 ◽

2020 ◽

Vol 12 (1) ◽

Author(s):

Anqi Liu ◽

Shanshan Jin ◽

Cuicui Fu ◽

Shengji Cui ◽

Ting Zhang ◽

...

Keyword(s):

Bone Regeneration ◽

Extracellular Vesicles ◽

Osteoblastic Differentiation ◽

Osteogenic Potential ◽

Nodule Formation ◽

Differentiation Markers ◽

Functional Roles ◽

Beneficial Effects ◽

Defect Area ◽

Mineralized Collagen

AbstractMacrophages play an important role in material-related immune responses and bone formation, but the functionality of macrophage-derived extracellular vesicles (EVs) in material-mediated bone regeneration is still unclear. Here, we evaluated intracellular communication through small extracellular vesicles (sEVs) and its effects on endogenous bone regeneration mediated by biomimetic intrafibrillarly mineralized collagen (IMC). After implantation in the bone defect area, IMC generated more neobone and recruited more mesenchymal stem cells (MSCs) than did extrafibrillarly mineralized collagen (EMC). More CD63+CD90+ and CD63+CD163+ cells were detected in the defect area in the IMC group than in the EMC group. To determine the functional roles of sEVs, extracellular vesicles from macrophages cultured on different mineralized collagen were isolated, and they showed no morphological differences. However, macrophage-derived sEVs in the IMC group showed an enhanced Young’s modulus and exerted beneficial effects on the osteogenic differentiation of bone marrow MSCs by increasing the expression of the osteoblastic differentiation markers BMP2, BGLAP, COL1, and OSX and calcium nodule formation. Mechanistically, sEVs from IMC-treated macrophages facilitated MSC osteogenesis through the BMP2/Smad5 pathway, and blocking sEV secretion with GW4869 significantly impaired MSC proliferative, immunomodulative and osteogenic potential. Taken together, these findings show that macrophage-derived sEVs may serve as an emerging functional tool in biomaterial-mediated endogenous bone regeneration.

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