Osteogenic Differentiation Of Mesenchymal Stem Cells Research Articles

MTOR is involved in LRP5-induced osteogenic differentiation of normal and aged periodontal ligament stem cells in vitro.

Periodontal ligament stem cells (PDLSCs) plays an important role in tissue engineering. As the age increased, the cell viability and osteogenic differentiation of PDLSCs all decreased. Low density lipoprotein receptor related protein 5 (LRP5) was found to promote bone marrow mesenchymal stem cells osteogenic differentiation. Therefore, our study explored the effect of LRP5 on normal and aged PDLSCs and relative mechanism. Here, we found that the expression of LRP5 in PDLSCs of 24week-old mice was decreased compared with PDLSCs of 5week-old mice (n = 5). . LRP5 overexpression in PDLSCs increased the intensity of alkaline phosphatase and alizarin red staining, accompanied with upregulated the levels of RUNX family transcription factor 2, collagen type I, and β-Catenin. LRP5 knockdown displayed the opposite results in PDLSCs in vitro.LRP5 overexpression in aged PDLSCs restored part ability of osteogenic differentiation. Meantime, LRP5 increased the protein expression of phosphorylation of mammalian target of rapamycin (p-mTOR) in normal and aged PDLSCs. Immunofluorescence showed that LRP5 increased the accumulation of p-mTOR nucleus. The effect of LRP5 in promoting osteogenic differentiation of PDLSCs can be antagonized by mTOR inhibitor rapamycin. These findings suggest that LRP5 positively regulate osteogenic differentiation of normal and aged PDLSCs and may be a potential target for enlarging the application of PDLSCs in tissue regeneration.

Impairment of APPL1/Myoferlin facilitates adipogenic differentiation of mesenchymal stem cells by blocking autophagy flux in osteoporosis

An imbalance of human mesenchymal stem cells (hMSCs) adipogenic and osteogenic differentiation is crucial in the pathogenesis of osteoporosis, and elucidation of the underlying mechanism is urgently needed. APPL1, an adaptor protein of the adiponectin receptor, was recently shown to be closely related to bone mass. However, the role of APPL1 in the imbalance of hMSC differentiation in osteoporosis is unclear. Therefore, we aimed to explore the mechanisms by which APPL1 alters hMSCs adipogenic differentiation in osteoporosis. Here, we found that APPL1 expression was downregulated in elderly patients with osteoporosis and in mouse osteoporosis model. APPL1 negatively regulated hMSC adipogenic differentiation in vivo and in vitro. Mechanistically, by enhancing ubiquitination-mediated Myoferlin degradation, downregulated APPL1 expression increased the risk of lysosome dysfunction during hMSCs adipogenic differentiation. Lysosomal dysfunction inhibited autophagy flux by suppressing autophagosome degradation and promoted hMSC differentiation towards the adipocyte lineage. Our findings suggest that APPL1/Myoferlin downregulation promoted hMSCs adipogenic differentiation by inhibiting autophagy flux, further impairing the balance of hMSCs adipogenic and osteogenic differentiation in osteoporosis; the APPL1/ Myoferlin axis may be a promising diagnostic and therapeutic target for osteoporosis.

Fabrication and in vitro evaluation of 3D printed porous silicate substituted calcium phosphate scaffolds for bone tissue engineering.

Silicate-substituted calcium phosphate (Si-CaP) ceramics, alternative materials for autogenous bone grafting, exhibit excellent osteoinductivity, osteoconductivity, biocompatibility, and biodegradability; thus, they have been widely used for treating bone defects. However, the limited control over the spatial structure and weak mechanical properties of conventional Si-CaP ceramics hinder their wide application. Here, we used digital light processing (DLP) printing technology to fabricate a novel porous 3D printed Si-CaP scaffold to enhance the scaffold properties. Scanning electron microscopy, compression tests, and computational fluid dynamics simulations of the 3D printed Si-CaP scaffolds revealed a uniform spatial structure, appropriate mechanical properties, and effective interior permeability. Furthermore, compared to Si-CaP groups, 3D printed Si-CaP groups exhibited sustained release of silicon (Si), calcium (Ca), and phosphorus (P) ions. Furthermore, 3D printed Si-CaP groups had more comprehensive and persistent osteogenic effects due to increased osteogenic factor expression and calcium deposition. Our results show that the 3D printed Si-CaP scaffold successfully improved bone marrow mesenchymal stem cells(BMSCs) adhesion, proliferation, and osteogenic differentiation and possessed a distinct apatite mineralization ability. Overall, with the help of DLP printing technology, Si-CaP ceramic materials facilitate the fabrication of ideal bone tissue engineering scaffolds with essential elements, providing a promising approach for bone regeneration.

A cell-free difunctional demineralized bone matrix scaffold enhances the recruitment and osteogenesis of mesenchymal stem cells by promoting inflammation resolution.

The dialogue between host macrophages (Mφs) and endogenous mesenchymal stem cells (MSCs) promotes M2 Mφs polarization to resolve early-stage inflammation, thereby effectively guiding in situ bone regeneration. Once inflammation is unresolved/incontrollable, it will induce the impediment of MSCs homing at bone defect site, implying the seasonable resolution of inflammation in balancing bone homeostasis. Repeatedly, evidence elucidated that specialized pro-resolving mediators (SPMs) could conduce to proper resolve inflammation and promote the repairing of bone defect. A difunctional demineralized bone matrix (DBM) scaffold co-modified by maresin 1 (MaR1) and aptamer 19S (Apt19S) was fabricated to facilitate the osteogenesis of MSCs. To confirm the osteogenesis and immunomodulatory role of the difunctional DBM scaffold, the proliferation, recruitment, and osteogenic differentiation of MSCs and the Mφs M2 subtype polarization were evaluated in vitro. Then, the DBM scaffolds were implanted into mice model with critical size calvarial defect to evaluate bone repair efficiency. Finally, the specific resolution mechanism in Mφs cultured on the difunctional DBM scaffold was further in-depth investigated. This difunctional DBM scaffold exhibited an enhanced function on the recruitment, proliferation, migration, osteogenesis of MSCs and the resolution of inflammation, finally improved bone-scaffold integration. At the same time, MaR1 modified on the difunctional DBM scaffold increased the biosynthesis of 12-lipoxygenase (12-LOX) and 12S-hydroxy-eicosatetraenoic acid (12S-HETE), and also directly stimulated lipid droplets (LDs) biogenesis in Mφs, which suggested that MaR1 regulated Mφ lipid metabolism at bone repair site. Findings based on this synergy strategy demonstrated that Mφ lipid metabolism was essential in bone homeostasis, which might provide a theoretical direction for the treatment-associated application of MaR1 in inflammatory bone disease.

Development of a multi-scale model to simulate mesenchymal stem cell osteogenic differentiation within hydrogels in a rotating wall bioreactor

Demand for mesenchymal stem cell (MSC)-based therapies for various clinical applications has been steadily increasing. Suitable implants can be obtained by in-vitro osteogenic differentiation of stem cells to meet this demand, at least in part. This paper proposes a multiscale population balance model for the osteogenic differentiation of mesenchymal stem cells encapsulated within alginate-gelatin hydrogels in a rotating wall bioreactor. The mathematical model incorporates key genes and metabolic pathways linked to proliferation and osteogenesis and captures spatial as well as cell cycle heterogeneity within the hydrogels inside the bioreactor. The discretized formulation of the model consists of 12,563 simultaneous ordinary differential equations. Concentrations of key metabolites (e.g., glucose, pyruvate, glutamine, lactate) were compared with experimental data from static well plate cultures, rendering the model predictions as adequate. Gene activation (Runx2 and osteonectin) in alginate-gelatin-bead-encapsulated cells was slightly delayed compared with well-plate cultures. Global sensitivity analysis revealed that parameters related to gene expression (decay rates and gene transcription activation constants) carry the most significance and should be the focus of future parameter estimation. Hydrogel bead size does not meaningfully impact simulation outcomes for bead diameters in the 2–3 mm range. The mathematical model proposed in this work can function as a foundation for model-based bioreactor and bioprocess optimization when coupled with a high-performance computing system.

MicroRNA-29b/graphene oxide-polyethyleneglycol-polyethylenimine complex incorporated within chitosan hydrogel promotes osteogenesis.

MicroRNAs (miRNAs) play a pivotal role in regulating a number of physiologic and pathologic processes including bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation, making them a candidate used to promote osteogenesis. However, due to intrinsic structure and characteristics, “naked” miRNAs are unstable in serum and could not pass across the cellular membrane. Nano delivery systems seem to be a solution to these issues. Recently, graphene oxide (GO)-based nanomaterials are considered to be promising for gene delivery due to their unique physiochemical characteristics such as high surface area, biocompatibility, and easy modification. In this work, a GO-based nanocomplex functionalized by polyethyleneglycol (PEG) and polyethylenimine (PEI) was prepared for loading and delivering miR-29b, which participates in multiple steps of bone formation. The nanocomplex revealed good biocompatibility, miRNA loading capacity, and transfection efficiency. The miR-29b/GO-PEG-PEI nanocomplex was capsulated into chitosan (CS) hydrogel for osteogenesis. In vitro and in vivo evaluation indicated that miR-29b/GO-PEG-PEI@CS composite hydrogel was able to promote BMSC osteogenic differentiation and bone regeneration. All these results indicate that PEG/PEI functionalized GO could serve as a promising candidate for miRNA cellular delivery, and the miR-29b/GO-PEG-PEI@CS hydrogel has the potential for repairing bone defects in vivo.

The Effect of Cell Source on Mesenchymal Stem Cell Behavior and Osteogenic Differentiation of Buccal Fat Pad and Dental Pulp Stem Cells, an In Vitro Study

Objective: The buccal fat pad is a source of adipose stem cells accessible from the oral cavity and dental pulp is a common dental source of stem cells. This study aimed to assess the biological behaviors and osteogenic differentiation of human buccal fat pad adipose-derived stem cells (hBFP-ADSCs) and human dental pulp derived stem cells (hDPSCs).Material and Methods: Buccal fat pads (595.4±80 mg) and dental pulp from 2 wisdom teeth were obtained from 3 matched donors and hBFP-ADSCs and hDPSC cells, respectively, and isolated. The isolated cells were sequentially expanded through passages 5, 10, and 15 for analysis. Cell growth, osteogenic differentiation, and cell senescence were investigated.Results: HBFP-ADSCs and hDPSCs exhibited different cell morphologies and behaviors. Cell expansion was associated with the decrease of cell growth and in vitro mineralization of hDPSCs. The cell yields of hBFP-ADSCs and hDPSCs at the primary passage were 3.2x10^5 ±9.9x10^3 cells/100 mg and 1.13x10^6 ±2.4x10^5 cells/tooth, respectively. The expanded cells exhibited a limited life span and maintained normal karyotypes at the late cell expansion stage.Conclusion: HBFP-ADSCs showed steady cell growth, high osteogenic differentiation potential, tissue availability, and minimal in vitro cell expansion. The availability and accessibility of hBFP-ADSCs would enable clinical applicability as a stem cell source for bone regeneration.

Nacre-mimetic hydroxyapatite/chitosan/gelatin layered scaffolds modifying substance P for subchondral bone regeneration

Nacre, exhibiting excellent mechanical strengths due to its hierarchical structures, becomes a potential model to design bone implants. Herein, we firstly fabricated nacre-mimetic hydroxyapatite/chitosan/gelatin (HA/CS/Gel) layered scaffolds and incorporated substance P (SP) peptides. The CS scaffolds with a layered architecture could regulate the deposition of HA nanoplates on the interlamellar CS sheets by using CaCO3 as precursors. The HA/CS/Gel layered scaffolds exhibited a great flexure strength of 11.42 MPa due to a brick-and-mortar structure. The biocompatible components, layered macropores and SP peptides in the HA/CS/Gel therapeutic scaffolds facilitated the spreading and proliferation mesenchymal stem cells (MSCs). Notably, the incorporation of SP peptides induced MSC osteogenic differentiation and extracellular matrix mineralization. Rabbit knee subchondral defect models further proved that the HA/CS/Gel-SP layered scaffolds promoted in vivo subchondral bone regeneration. Hence, the combination of nacre-mimetic bone implants and therapeutic drugs may become an attractive strategy for subchondral bone regeneration.

Cells-Micropatterning Biomaterials for Immune Activation and Bone Regeneration.

Natural tissues are composed of ordered architectural organizations of multiple tissue cells. The spatial distribution of cells is crucial for directing cellular behavior and maintaining tissue homeostasis and function. Herein, an artificial bone bioceramic scaffold with star‐, Tai Chi‐, or interlacing‐shaped multicellular patterns is constructed. The “cross‐talk” between mesenchymal stem cells (MSCs) and macrophages can be effectively manipulated by altering the spatial distribution of two kinds of cells in the scaffolds, thus achieving controllable modulation of the scaffold‐mediated osteo‐immune responses. Compared with other multicellular patterns, the Tai Chi pattern with a 2:1 ratio of MSCs to macrophages is more effective in activating anti‐inflammatory M2 macrophages, improving MSCs osteogenic differentiation, and accelerating new bone formation in vivo. In brief, the Tai Chi pattern generates a more favorable osteo‐immune environment for bone regeneration, exhibiting enhanced immunomodulation and osteogenesis, which may be associated with the activation of BMP‐Smad, Oncostatin M (OSM), and Wnt/β‐catenin signaling pathways in MSCs mediated by macrophage‐derived paracrine signaling mediators. The study suggests that the manipulation of cell distribution to improve tissue formation is a feasible approach that can offer new insights for the design of tissue‐engineered bone substitutes with multicellular interactions.

Strontium-loaded titanium implant with rough surface modulates osseointegration by changing sfrp4 in canonical and noncanonical Wnt signaling pathways

A rough morphology and strontium (Sr) can activate the Wnt pathway to regulate bone mesenchymal stem cells (rBMSCs) osteogenic differentiation, but the mechanism remains unclear. We constructed smooth Ti (ST) surfaces, rough Ti (RT) surfaces subjected to hydrofluoric acid etching, strontium-loaded smooth Ti (ST-Sr) surfaces subjected to magnetron sputtering, and rough strontium-loaded Ti (RT-Sr) surfaces. We systematically studied the in vitro osteogenic differentiation of rBMSCs on these four surfaces by alkaline phosphatase measurement, Alizarin Red staining and polymerase chain reaction (PCR). We also investigated whether crosstalk of the canonical and noncanonical Wnt signaling pathways regulated by sfrp4, which is an inhibitor of canonical and noncanonical Wnt, is the underlying mechanism via PCR on rBMSCs in different stages of osteogenic differentiation. We confirmed the effect of sfrp4 through an in vivo sfrp4-siRNA test. The in vitro osteogenic differentiation of rBMSCs decreased in the order RT-Sr, RT, ST-Sr, and ST. Regarding the mechanism, rough morphology and Sr both enhanced the canonical Wnt pathway to promote osseointegration. Additionally, rough morphology can inhibit sfrp4 to activate the noncanonical Wnt pathway, and then, the activated noncanonical Wnt pathway can suppress the canonical Wnt pathway at the early stage of osteogenic differentiation. Sr continuously enhanced sfrp4 to inhibit the canonical Wnt pathway instead of activating the noncanonical Wnt pathway. Interestingly, the effect of rough morphology on sfrp4 changed from inhibition to enhancement, and the enhancing effect of Sr on sfrp4 was gradually attenuated. The results of the in vivo sfrp4-siRNA test showed that osseointegration decreased in the order RT-Sr, RT-Sr-siRNA, and ST. Our results suggest that the lack of sfrp4 could suppress osseointegration, indicating that sfrp4 acts as a crucial regulatory molecule for the canonical and noncanonical Wnt pathways during the response of rBMSCs to rough morphology and Sr.

The Role of Flavonoids in the Osteogenic Differentiation of Mesenchymal Stem Cells.

Mesenchymal stem cells (MSCs) play an important role in developing bone tissue engineered constructs due to their osteogenic and chondrogenic differentiation potential. MSC-based tissue engineered constructs are generally considered a safe procedure, however, the long-term results obtained up to now are far from satisfactory. The main causes of these therapeutic limitations are inefficient homing, engraftment, and directional differentiation. Flavonoids are a secondary metabolite, widely existed in nature and have many biological activities. For a long time, researchers have confirmed the anti-osteoporosis effect of flavonoids through in vitro cell experiments, animal studies. In recent years the regulatory effects of flavonoids on mesenchymal stem cells (MSCs) differentiation have been received increasingly attention. Recent studies revealed flavonoids possess the ability to modulate self-renewal and differentiation potential of MSCs. In order to facilitate further research on MSCs osteogenic differentiation of flavonoids, we surveyed the literature published on the use of flavonoids in osteogenic differentiation of MSCs, and summarized their pharmacological activities as well as the underlying mechanisms, aimed to explore their promising therapeutic application in bone disorders and bone tissue engineered constructs.

Microfluidic-preparation of PLGA microcarriers with collagen patches for MSCs expansion and osteogenic differentiation

This study was aimed to develop a capillary-based microfluidic methodology for preparing poly (lactic-co-glycolic acid) (PLGA) microcarriers patterned with collagen patchy structure (PLGA-Col), which are superior in enhancing proliferation and osteogenic differentiation of mesenchymal stem cells (MSCs) seeded on them. Primary W/O emulsions were first prepared using aqueous collagen and PLGA solutions. A two-phase microfluidic device was then designed and the emulsions were used as the inner liquid phase to obtain PLGA-Col microcarriers. The content of collagen was varied to yield PLGA-Col-L (lower collagen content) and PLGA-Col-H (higher collagen content). The proliferation behavior of the cells on the microcarriers was evaluated by live/dead cell staining and CCK-8 assays, while the osteogenic differentiation behavior was evaluated by ALP and qRT-PCR assays. Pristine PLGA microcarriers were also prepared to serve as a control. Three groups, PLGA, PLGA-Col-L, and PLGA-Col-H were then obtained. PLGA-Col was asymmetric with a rough/porous collagen patchy structure. All of these microcarriers could maintain morphology integrity for at least 3 weeks in the aqueous medium before undergoing obvious swelling (5w) and further erosion (7w). It was demonstrated that PLGA-Col could not only enhance the MSCs adhesion/proliferation but also mediate their subsequent osteogenic differentiation. PLGA-Col-L was suitable for supporting the MSCs proliferation, while PLGA-Col-H was more efficient in promoting the osteogenic differentiation of MSCs. Hence, the incorporation of collagen as a patchy structure into PLGA microspheres through the current microfluidic method could be an effective strategy to provide a superior type of microcarriers with well-defined functionality.

Osteogenesis-Inducing Chemical Cues Enhance the Mechanosensitivity of Human Mesenchymal Stem Cells for Osteogenic Differentiation on a Microtopographically Patterned Surface.

Mechanical cues are widely used for regulating cell behavior because of their overarching, extensive, and non‐invasive advantages. However, unlike chemical cues, mechanical cues are not efficient enough to determine cell fate independently and improving the mechanosensitivity of cells is rather challenging. In this study, the combined effect of chemical and mechanical cues on the osteogenic differentiation of human mesenchymal stem cells is examined. These results show that chemical cues such as the presence of an osteogenic medium, induce cells to secrete more collagen, and induce integrin for recruiting focal adhesion proteins that mature and cascade a series of events with the help of the mechanical force of the scaffold material. High‐resolution, highly ordered hollow‐micro‐frustum‐arrays using double‐layer lithography, combined with modified methacrylate gelatin loaded with pre‐defined soluble chemicals to provide both chemical and mechanical cues to cells. This approach ultimately facilitates the achievement of cellular osteodifferentiation and enhances bone repair efficiency in a model of femoral fracture in vivo in mice. Moreover, the results also reveal these pivotal roles of Integrin α2/Focal adhesion kinase/Ras homolog gene family member A/Large Tumor Suppressor 1/Yes‐associated protein in human mesenchymal stem cells osteogenic differentiation both in vitro and in vivo. Overall, these results show that chemical cues enhance the microtopographical sensitivity of cells.

Exendin‐4 enhances osteogenic differentiation of adipose tissue mesenchymal stem cells through the receptor activator of nuclear factor‐kappa B and osteoprotegerin signaling pathway

The capability of mesenchymal stem cells (MSCs) to repair bone damage and defects has long been investigated. The receptor activator of nuclear factor-kappa B (RANK), its ligand (RANKL) and the decoy receptor osteoprotegerin (OPG) axis is crucial to keep the equilibrium between osteoblastic and osteoclastic activity. Exendin-4 utilization increased bone formation and enhanced bone integrity. This study aimed to investigate the mentioned axis and determine the effect of exendin-4 upon adipose mesenchymal stem cells (Ad-MSCs) osteogenic differentiation. Ad-MSCs were isolated from rat epididymal fat, followed by characterization and then differentiation into osteocytes both in the presence or absence of exendin-4. Osteogenic differentiation was evaluated by alizarin red staining and the expression of osteogenic markers; using reverse transcriptase-quantitative polymerase chain reaction, western blotting and enzyme-linked immunoassay. MSCs derived from rat epididymal fat were isolated and characterized, along with their differentiation into osteocytes. The differentiated cells were alizarin red-stained, showing increased staining intensity upon addition of exendin-4. Moreover, the addition of exendin-4 elevated the messenger RNA expression levels of osteogenic markers; runt-related transcription factor-2 (RUNX-2), osteocalcin, and forkhead box protein O-1while reducing the expression of the adipogenic marker peroxisome-proliferator-activated receptor-gamma. Exendin-4 addition elevated OPG levels in the supernatant of osteogenic differentiated cells. Moreover, exendin-4 elevated the protein levels of glucagon-like peptide-1 receptor and RUNX-2, while decreasing both RANK and RANKL. In conclusion, osteogenic differentiation of Ad-MSCs is associated with increased osteoblastic rather than osteoclastic activity. The findings of this study suggest that exendin-4 can enhance Ad-MSCs osteogenic differentiation partially through the RANK/RANKL/OPG axis.

Osteogenesis-Related Long Noncoding RNA GAS5 as a Novel Biomarker for Osteonecrosis of Femoral Head.

Background: The lack of effective biomarkers makes it difficult to achieve early diagnosis and intervention for osteonecrosis of the femoral head (ONFH). Hence, we aimed to identify novel long noncoding RNA (lncRNA) biomarkers for ONFH. Methods: High-throughput RNA sequencing was performed to detect lncRNA and mRNA expression levels in subchondral bone samples from three patients with ONFH and three patients with femoral neck fractures. Integrated bioinformatics analyses were conducted to identify lncRNAs associated with ONFH development and their potential functions and signaling pathways. A co-expression network was constructed based on the gene time-series expression data in GSE113253. After selecting lncRNA GAS5 as a novel biomarker for ONFH, bone marrow mesenchymal stem cell (BMSC) osteogenic differentiation assays were performed to verify the association between lncRNA GAS5 and osteogenic differentiation. Alkaline phosphatase (ALP) staining and quantitative reverse transcription polymerase chain reaction (RT-qPCR) were used to measure the osteogenic phenotype and lncRNA GAS5 expression. Finally, for further validation, ONFH rat models were established, and lncRNA GAS5 expression in subchondral bone was detected by RT-qPCR. Results: We identified 126 and 959 differentially expressed lncRNAs and genes, respectively. lncRNA GAS5 expression level was significantly downregulated in patients with ONFH compared to the control group patients. The BMSC osteogenic differentiation assays showed that ALP activity increased gradually from days 3 to 7, while the lncRNA GAS5 expression level was significantly upregulated in the osteogenic differentiation induction groups. Furthermore, in vivo experiments suggested that the bone volume/tissue volume value and trabecular thickness significantly decreased in the ONFH rat model group compared to the control group, whereas the trabecular space significantly increased in the ONFH group compared to the control group. In addition, the lncRNA GAS5 expression level significantly decreased in the ONFH rat model group. Conclusion: The lncRNA GAS5 expression level was highly associated with BMSC osteogenic differentiation and was significantly downregulated in both the subchondral trabecular bone tissue of ONFH patients and ONFH rat models. Therefore, lncRNA GAS5 can serve as an ONFH osteogenic biomarker to provide an effective target for early diagnosis and molecular therapy of ONFH.

Promoting in-vivo bone regeneration using facile engineered load-bearing 3D bioactive scaffold

The worldwide incidence of bone disorders has trended steeply upward and is expected to get doubled by 2030. The biological mechanism of bone repair involves both osteoconductivity and osteoinductivity. Despite the self-healing functionality after injury, bone tissue faces a multitude of pathological challenges. Several innovative approaches have been developed to prepare biomaterial-based bone grafts. To design a suitable bone material, the freeze-drying technique has achieved significant importance among the other conventional methods. However, the functionality of the polymeric freeze-dried scaffold in in-vivo osteogenesis is in a nascent stage. In this study facile, freeze-dried, biomaterial-based load-bearing three-dimensional porous composite scaffolds have been prepared. The biocompatible scaffolds have been made by using chitosan (C), polycaprolactone (P), hydroxyapatite (H), glass ionomer (G), and graphene (gr). Scaffolds of eight different groups (C, P, CP, CPH, CPHG, CPHGgr1, CPHGgr2, CPHGgr3) have been designed and characterized to evaluate their applicability in orthopedics. To evaluate the efficacy of the scaffolds a series of physio-chemical, morphological, and in-vitro and in-vivo biological experiments have been performed. From the obtained results it was observed that the CPHGgr1 is the ideal compatible material for Wharton’s jelly-derived mesenchymal stem cells (MSCs) and the blood cells. The in-vitro bone-specific gene expression study revealed that the scaffold assists MSCs osteogenic differentiation. Additionally, the in-vivo study on the mice model was also performed for a period of four and eight weeks. The subcutaneous implantation of the designed scaffolds did not show any altered physiological condition in the animals, which indicated the in-vivo biocompatibility of the designed material. The histopathological study revealed that after eight weeks of implantation, the CPHGgr1 scaffold supported significantly better collagen deposition and calcification. The facile designing of the CPHGgr1 multicomponent nanocomposite provided an osteo-regenerative biomaterial with desired mechanical strength as an ideal regenerative material for cancellous bone tissue regeneration.

BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models.

Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.

Stratified-structural hydrogel incorporated with magnesium-ion-modified black phosphorus nanosheets for promoting neuro-vascularized bone regeneration

Angiogenesis and neurogenesis play irreplaceable roles in bone repair. Although biomaterial implantation that mimics native skeletal tissue is extensively studied, the nerve-vascular network reconstruction is neglected in the design of biomaterials. Our goal here is to establish a periosteum-simulating bilayer hydrogel and explore the efficiency of bone repair via enhancement of angiogenesis and neurogenesis. In this contribution, we designed a bilayer hydrogel platform incorporated with magnesium-ion-modified black phosphorus (BP) nanosheets for promoting neuro-vascularized bone regeneration. Specifically, we incorporated magnesium-ion-modified black phosphorus (BP@Mg) nanosheets into gelatin methacryloyl (GelMA) hydrogel to prepare the upper hydrogel, whereas the bottom hydrogel was designed as a double-network hydrogel system, consisting of two interpenetrating polymer networks composed of GelMA, PEGDA, and β-TCP nanocrystals. The magnesium ion modification process was developed to enhance BP nanosheet stability and provide a sustained release platform for bioactive ions. Our results demonstrated that the upper layer of hydrogel provided a bionic periosteal structure, which significantly facilitated angiogenesis via induction of endothelial cell migration and presented multiple advantages for the upregulation of nerve-related protein expression in neural stem cells (NSCs). Moreover, the bottom layer of the hydrogel significantly promoted bone marrow mesenchymal stem cells (BMSCs) activity and osteogenic differentiation. We next employed the bilayer hydrogel structure to correct rat skull defects. Based on our radiological and histological examinations, the bilayer hydrogel scaffolds markedly enhanced early vascularization and neurogenesis, which prompted eventual bone regeneration and remodeling. Our current strategy paves way for designing nerve-vascular network biomaterials for bone regeneration.

Heterotopic ossification vs. fracture healing: Extracellular vesicle cargo proteins shed new light on bone formation

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare disease in which bone tissue forms in extraskeletal sites, which is known as heterotopic ossification (HO). Extracellular vesicles (EVs) are small phospholipid-enclosed particles released by various cells which have an emerging, but not completely understood role in various (patho)physiological processes. In order to further study the pathophysiology of FOP we conducted a small observational study comparing the proteomic profiles of EV cargo, derived from pooled plasma of four patient groups: FOP patient (N = 1) during active disease phase (flare-up), FOP patients during remission (N = 2), patients after long bone fracture (N = 20) and healthy controls (N = 10). After isolation of EVs – their protein cargo was determined using liquid chromatography / mass spectrometry, after which a functional gene enrichment analysis was performed. Our results show a sizeable difference of the proteomics profiles in which EVs from the bone fracture group show significant activity of integrin interactions, Wnt, VEGF, IGF-1 and PDGF pathways; conversely, FOP patients' EVs indicate that HO occurs via processes of innate immunity and the Ephrin B signaling pathway. We hypothesize that the Ephrin B signaling (expressed in EVs) contributes to HO by aiding in mesenchymal stem cell recruitment and osteogenic differentiation, as well as by contributing to the inflammatory response, including macrophage chemotaxis and activation. This is, to our knowledge, the first published analysis of EV protein cargo in FOP.

Cannabidiol Promotes Osteogenic Differentiation of Bone Marrow Mesenchymal Stem Cells in the Inflammatory Microenvironment via the CB2-dependent p38 MAPK Signaling Pathway.

Chronic inflammation of bone tissue often results in bone defects and hazards to tissue repair and regeneration. Cannabidiol (CBD) is a natural cannabinoid with multiple biological activities, including anti-inflammatory and osteogenic potential. This study aimed to investigate the efficacy and mechanisms of CBD in the promotion of bone marrow mesenchymal stem cells (BMSCs) osteogenic differentiation in the inflammatory microenvironment. BMSCs isolated from C57BL/6 mice, expressed stem cell characteristic surface markers and presented multidirectional differentiation potential. The CCK-8 assay was applied to evaluate the effects of CBD on BMSCs' vitality, and demonstrating the safety of CBD on BMSCs. Then, BMSCs were stimulated with lipopolysaccharide (LPS) to induce inflammatory microenvironment. We found that CBD intervention down-regulated mRNA expression levels of inflammatory cytokines and promoted cells proliferation in LPS-treated BMSCs, also reversed the protein and mRNA levels downregulation of osteogenic markers caused by LPS treatment. Moreover, CBD intervention activated the cannabinoid receptor 2 (CB2) and the p38 mitogen-activated protein kinase (MAPK) signaling pathway. While AM630, a selective CB2 inhibitor, reduced phosphorylated (p)-p38 levels. In addition, AM630 and SB530689, a selective p38 MAPK inhibitor, attenuated the enhancement of osteogenic markers expression levels by CBD in inflammatory microenvironment, respectively. CBD promoted osteogenic differentiation of BMSCs via the CB2/p38 MAPK signaling pathway in the inflammatory microenvironment.