Non‐Invasive Monitoring of Osteogenic Differentiation on Microtissue Arrays under Physiological Conditions Using Scanning Electrochemical Microscopy

In this paper, we present a non‐invasive assay using scanning electrochemical microscopy (SECM) for detecting osteogenic differentiation at physiological conditions (pH 7.5) on arrays of C2C12 microtissues. Upon exposure to bone morphogenic protein 2 (BMP‐2), C2C12 microtissues differentiate and express alkaline phosphatase (ALP), which is indirectly detected through an enzymatic assay producing an electroactive species. The latter is detected using SECM by scanning at constant height over live microtissues at physiological pH (7.5) as well as more alkaline pH (8.5). As a control, expression of ALP is confirmed using a standard colorimetric assay. Detecting differentiation on live samples at physiological conditions represents a significant improvement for continuous monitoring of tissue differentiation or further use of the microtissues for, e.g., regenerative medicine.     

应用原子力显微镜研究淫羊霍苷诱导人脐带间充质干细胞成骨分化

利用碱性磷酸酶(ALP)染色和钙结节(Vonkossa)染色的方法对诱导21 d的淫羊霍苷诱导人脐带间充质干细胞进行鉴定;应用原子力显微镜(AFM)观察淫羊霍苷的形貌和人脐带间充质干细胞诱导0、5、10、15、21 d后的细胞形貌。结果表明,经成骨诱导分化21 d后,ALP染色呈强阳性,Vonkossa染色可见明显钙结节。AFM分析表明,淫羊霍苷在盖玻片上呈分散状分布,在细胞表面上聚集并呈微米域分布。实验发现,由于吸附在细胞表面时,被细胞膜分子包裹,更有利于在细胞表面的吸附,进入细胞内部,细胞表面的淫羊霍苷颗粒较在盖玻片上时增大,由淫羊霍苷颗粒进入细胞后在细胞表面留下一些小孔,可知其通过进入细胞内部诱导成骨分化。分化后,细胞表面有小突触,是由成骨分化后细胞内形成钙结节造成。     

Ultra-small nanodots coated with oligopeptides providing highly negative charges to enhance osteogenic differentiation of hBMSCs better than osteogenic induction medium

For bone regenerative engineering,it is a promising method to form skeletal tissues differentiating from human bone morrow mesenchyme stem cells(hBMSCs).However,it is still a critical challenge to efficiently control ostogenesis and clearly reveal the influence factor.To this end,the fluorescent gold nanodots(Au NDs) with highly negative charges as osteogenic induction reagent are successfully synthesized,which display better than commercial osteogenic induction medium through the investigations of ALP activity(2.5 folds) and cytoskeleton staining(1.5 folds).Two kinds of oligopeptides with different bio-structures(cysteine,Cys and glutathione,GSH) are selected for providing surficial charges on Au NDs.It is revealed that Au-Cys with more negative charges(-51 mV) play better role than Au-GSH(-19 mV) in osteogenic differentiation,when both of them have same size(~2 nm),sphere shape and show similar cell uptake amount.To explore deeply,osteogenesis related signaling pathways are monitored,revealing that the enhancement of osteogenic differentiation was through autophagy signaling pathway triggered by Au-Cys.And the promotion of highly negative charges in osteogenic diffe rentiation was further proved via sliver nanodots(Ag NDs,Ag-Cys and Ag-GSH) and carbon nanodots(CDs,Cys-CDs and GSH-CDs).This work indicates part of insights during hBMSCs differentiation and provides a novel strategy in osteogenic differentiation process.     

Design and Assessment of a Dynamic Perfusion Bioreactor for Large Bone Tissue Engineering Scaffolds

Bioreactors can be used to apply fluid flow in vitro to scaffolds to improve mass transport of media and apply mechanical forces to cells. In this study, we developed and tested an autoclavable, modular perfusion bioreactor suitable for large scaffolds. We investigated the effects of fluid flow induced shear stress (FFSS) on osteogenic differentiation of human embryonic stem cell-derived mesenchymal progenitors (hES-MP cells) cultured on large polyurethane (PU) scaffolds (30 mm diameter × 5 mm thickness) in osteogenesis induction media (OIM). After seeding, scaffolds were either maintained in static conditions or transferred to the bioreactor 3 days post-seeding and a continuous flow rate of 3.47 mL/min was applied. Alkaline phosphatase activity (ALP) was used to evaluate osteogenic differentiation and resazurin salt reduction (RR) to measure metabolic activity after 10 days. Cultures subjected to flow contained significantly more metabolically active cells and higher total DNA content, as well as significantly higher ALP activity compared to scaffolds grown in static culture. These results confirm the responsiveness of hES-MP cells to fluid flow stimuli, and present a cost-effective, user-friendly bioreactor capable of supporting the growth and differentiation of mesenchymal progenitor cells within scaffolds capable of filling large bone defects.     

Development of an osteoblast-based 3D continuous-perfusion microfluidic system for drug screening

In this work, we demonstrated that biological cells could be cultured in a continuous-perfusion glass microchip system for drug screening. We used mouse Col1a1GFP MC-3T3 E1 osteoblastic cells, which have a marker gene system expressing green fluorescent protein (GFP) under the control of osteoblast-specific promoters. With our microchip-based cell culture system, we realized automated long-term monitoring of cells and sampling of the culture supernatant system for osteoblast differentiation assay using a small number of cells. The system successfully monitored cells for 10 days. Under the 3D microchannel condition, shear stress (0.07 dyne/cm² at a flow rate of 0.2 μL/min) was applied to the cells and it enhanced the GFP expression and differentiation of the osteoblasts. Analysis of alkaline phosphatase (ALP), which is an enzyme marker of osteoblasts, supported the results of GFP expression. In the case of differentiation medium containing bone morphogenetic protein 2, we found that ALP activity in the culture supernatant was enhanced 10 times in the microchannel compared with the static condition in 48-well dishes. A combined system of a microchip and a cell-based sensor might allow us to monitor osteogenic differentiation easily, precisely, and noninvasively. Our system can be applied in high-throughput drug screening assay for discovering osteogenic compounds.     

Activated protein C differentially regulates both viability and differentiation of osteoblasts mediated by bisphosphonates

Activated protein C (APC) is a cytoprotective anticoagulant that can promote cutaneous healing. We examined the effect of APC on viability and differentiation of the osteoblastic line, MG63, in the presence and absence of bisphosphonates (BPs). Osteoblasts were cultured and treated for 24 or 48 h with Alendronate (Aln), Zoledronate (Zol) or Pamidronate (Pam) at concentrations ranging from 10⁻⁴ to 10⁻⁶ ℳ. Cell differentiation was measured using type 1 collagen production, Alizarin red staining and alkaline phosphatase activity, whereas cell viability was assessed using MTT and crystal violet assays. All three BPs induced MG63 cell death in a dose- and time-dependent manner. Pam- and Zol-related cell death was prevented by APC treatment; however, cell death induced by Aln was accelerated by APC. APC induced MG63 cell differentiation that was enhanced by Aln, but inhibited by Pam or Zol. Endothelial protein C receptor (EPCR) was expressed by MG63 cells and mediated the protective effect of APC on Zol-induced viability. In summary, we have demonstrated that (1) APC favorably regulates MG63 viability and differentiation toward bone growth, (2) APC differentially regulates the effects of specific BPs and (3) at least part of the effects of APC is mediated through EPCR. These findings highlight the potential importance of the PC pathway in bone physiology and provide strong evidence that APC may influence bone cells and has potential to be a therapeutic drug for bone regeneration, depending on concurrent BP treatment.     

25 Hz electromagnetic field exposure has no effect on cell cycle distribution and apoptosis in U-937 and HCA-2/1cch cells

It is reported that exposure to 50 Hz extremely low-frequency electromagnetic field (ELF-EMF) can produce apoptosis and small variations in cell cycle distribution on different cell lines. In order to study the effect of ELF-EMF on tumoral cells in vitro, two cell lines (U-937, from a histiocytic lymphoma, and HCA-2/1cch, from a human colon adenocarcinoma) were exposed to 25 Hz, 1.5 mT, for 2 h and 45 min. Cell cycle distribution, apoptosis (spontaneous and dexamethasone-induced) and cell growth were evaluated. Neither significant alteration in cell cycle phases nor induction of apoptosis was observed. Nevertheless, the relative cell number was found to decrease to 55.84+/-7.35% (p <0.05, Student's t-test) for HCA-2/1cch cells after exposure to EMF in the presence of dexamethasone. The presence of dexamethasone during the EMF exposure could probably produce a decrease in the cell growth of this cell line.     

Electrochemical Monitoring of Saos‐2 Cell Differentiation on Pyrolytic Carbon Electrodes

In this study we establish an electrochemical platform based on two dimensional (2D) pyrolytic carbon electrodes for in vitro analysis of osteoblast differentiation. Electrochemical impedance spectroscopy (EIS) was used to monitor cell adhesion and proliferation, while an electrochemical assay based on square wave voltammetry (SWV) was applied to measure the activity of the differentiation marker alkaline phosphatase (ALP). 2D pyrolytic carbon electrodes were fabricated and used to monitor Saos‐2 cell differentiation for a period of up to 21 days. With this method it was possible to detect a faster increase of ALP activity for cells cultured in medium supplemented with differentiation factors compared to cells cultured in growth medium. This was confirmed by the results obtained with Alizarin Red staining, showing that cells subjected to osteogenic medium went through the entire differentiation process, from proliferation to mineralization. Finally, for the first time, real‐time monitoring of ALP activity combined with continuous EIS monitoring of the same cell culture was achieved using the pyrolytic carbon electrodes.     

Laponite–Gelatin Nanofibrous Microsphere Promoting Human Dental Follicle Stem Cells Attachment and Osteogenic Differentiation for Noninvasive Stem Cell Transplantation

Nanofibrous microspheres (NFM) are emerging as prominent next-generation biomimetic injectable scaffold system for stem cell delivery and different tissue regeneration where nanofibrous topography facilitates ECM-like stem cells niches. Addition of osteogenic bioactive nanosilicate platelets within NFM can provide osteoconductive cues to facilitate matrix mediated osteogenic differentiation of stem cells and enhance the efficiency of bone tissue regeneration. In this study, gelatin nanofibrous microspheres are prepared containing fluoride-doped laponite XL21 (LP) using the emulsion mediated thermal induce phase separation (TIPS) technique. Systematic studies are performed to understand the effect of physicochemical properties of biomimicking NFM alone and with different concentrations of LP on human dental follicle stem cells (hDFSCs), their cellular attachment, proliferation, and osteogenic differentiation. The study highlights the effect of LP nanosilicate with biomimicking nanofibrous injectable scaffold system aiding in enhancing stem cell differentiation under normal physiological conditions compared to NFM without LP. The laponite–NFM shows suitability as excellent injectable biomaterials system for stem cell attachment, proliferation and osteogenic differentiation for stem cell transplantation and bone tissue regeneration.     

球形羟基磷灰石纳米颗粒的可控合成及其对间充质干细胞生长分化的影响

本文采用CTAB为添加剂进行球形纳米相羟基磷灰石(nHAP)的可控合成，并采用透射电子显微镜(TEM)、X-射线衍射仪(XRD)、傅立叶变换红外光谱仪(FTIR)和精密接触角测量仪对所制得的纳米颗粒的物性进行了表征。结果表明所制得的纳米颗粒为部分结晶的羟基磷灰石，颗粒为均匀球形，粒径约为20 nm，具有很好的亲水性。由该纳米颗粒构成的生长基质有利于骨髓间充质干细胞的贴壁、增殖以及成骨分化，是一种良好的骨组织工程支架材料。     

Comparison of the low-frequency magnetic field effects on bacteria Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus

This work studies biological effects of low-frequency electromagnetic fields. We have exposed three different bacterial strains-Escherichia coli, Leclercia adecarboxylata and Staphylococcus aureus to the magnetic field (t<30 min, B(m)=10 mT, f=50 Hz) in order to compare their viability (number of colony-forming units (CFU)). We have measured the dependence of CFU on time of exposure and on the value of the magnetic field induction B(m). Viability decreases with longer exposure time and/or higher induction B(m) for all strains, but the quantity of the effect is strain-dependent. The highest decrease of the viability and the biggest magnetic field effect was observed with E. coli. The smallest magnetic field effect appears for S. aureus. From the measurement of the growth dynamics we have concluded that the decrease of the CFU starts immediately after the magnetic field was switched on.     

MicroRNA‐2861 and nanofibrous scaffold synergistically promote human induced pluripotent stem cells osteogenic differentiation

Tissue engineering using new strategies has become a growing and promising method for treating large tissue lesions in the body. On the other hand, microRNAs (miRNAs), which are small non‐coding regulatory RNAs, are a new class of genetic materials that can have effective pharmacological roles. The combination of these two themes has created promising prospects for the treatment of diseases. Herein, human induced pluripotent stem cells (iPSCs) were transduced with miRNA‐2861 and then the osteogenic differentiation potential of transduced iPSCs and non‐transduced iPSCs was investigated while cultured on the electrospun poly lactic‐co‐glycolic acid (PLGA) nanofibrous scaffold and culture plate. MiR‐2861‐transduced iPSCs showed a significantly higher viability, mineralization, alkaline phosphatase (ALP) activity, calcium content, and bone‐related gene expression in comparison with those iPSCs that non‐transduced. The results also indicated that this increase is improved when miR‐2861 transduced iPSCs are cultured on the PLGA nanofibrous scaffold synergistically. This synergy was also confirmed by the results obtained from of Western blot analysis. It can be concluded that, miR‐2861, by negative regulation of those proteins that decrease/inhibit osteogenic differentiation and PLGA nanofibrous scaffold by preparation of a suitable artificial extracellular matrix, have a great positive impact in improving iPSCs osteogenic differentiation potential and this blend can be proposed to use in bone tissue engineering application.     

Bone regeneration on macroporous aqueous-derived silk 3-D scaffolds

Spinner flask culture under osteogenic conditions was used to study osteogenic outcomes from human bone marrow-derived mesenchymal stem cells (hMSCs) seeded on aqueous-derived porous silk scaffolds. Of particular novelty was the use of larger sized scaffolds (15 mm diameter, 5 mm thick) and large pore sizes ( approximately 900-1 000 micron diameter). Cultures were maintained for 84 d in the spinner flasks and compared to static controls under otherwise similar conditions. The spinner flask cultures demonstrated enhanced cell proliferation compared to static cultures and the improved fluid flow promoted significantly improved osteogenic related outcomes based on elevated alkaline phosphatase (ALP) activity and the deposition of mineralized matrix. The expression of osteogenic differentiation associated markers based on real time PCR also demonstrated increased responses under the dynamic spinner flask culture conditions. Histological analysis showed organized bone-like structures in the constructs cultured in the spinner flasks after 56 d of culture. These structures stained intensely with von Kossa. The combination of improved transport due to spinner flask culture and the use of macroporous 3D aqueous-derived silk scaffolds with large pore sizes resulted in enhanced outcomes related to bone tissue engineering, even with the use of large sized scaffolds in the study. These results suggest the importance of the structure of the silk biomaterial substrate (water vs. solvent based preparation) and large pore sizes in improved bone-like outcomes during dynamic cultivation.     

Influence of 40 Hz and 100 Hz Vibration on SH-SY5Y Cells Growth and Differentiation—A Preliminary Study

(1) Background: A novel bioreactor platform of neuronal cell cultures using low-magnitude, low-frequency (LMLF) vibrational stimulation was designed to discover vibration influence and mimic the dynamic environment of the in vivo state. To better understand the impact of 40 Hz and 100 Hz vibration on cell differentiation, we join biotechnology and advanced medical technology to design the nano-vibration system. The influence of vibration on the development of nervous tissue on the selected cell line SH-SY5Y (experimental research model in Alzheimer’s and Parkinson’s) was investigated. (2) Methods: The vibration stimulation of cell differentiation and elongation of their neuritis were monitored. We measured how vibrations affect the morphology and differentiation of nerve cells in vitro. (3) Results: The highest average length of neurites was observed in response to the 40 Hz vibration on the collagen surface in the differentiating medium, but cells response did not increase with vibration frequency. Also, vibrations at a frequency of 40 Hz or 100 Hz did not affect the average density of neurites. 100 Hz vibration increased the neurites density significantly with time for cultures on collagen and non-collagen surfaces. The exposure of neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation. The 40 Hz vibration has the best impact on neuronal-like cell growth and differentiation. (4) Conclusions: The data demonstrated that exposure to neuronal cells to 40 Hz and 100 Hz vibration enhanced cell differentiation and proliferation. This positive impact of vibration can be used in tissue engineering and regenerative medicine. It is planned to optimize the processes and study its molecular mechanisms concerning carrying out the research.     

Bmp 2 and Bmp 7 Induce Odonto- And Osteogenesis of Human Tooth Germ Stem Cells

Bone morphogenetic proteins (BMPs) initiate, promote, and maintain odontogenesis and osteogenesis. In this study, we studied the effect of bone morphogenic protein 2 (BMP 2) and bone morphogenic protein 7 (BMP 7) as differentiation inducers in tooth and bone regeneration. We compared the effect of BMP 2 and BMP 7 on odontogenic and osteogenic differentiation of human tooth germ stem cells (hTGSCs). Third molar-derived hTGSCs were characterized with mesenchymal stem cell surface markers by flow cytometry. BMP 2 and BMP 7 were transfected into hTGSCs and the cells were seeded onto six-well plates. One day after the transfection, hTGSCs were treated with odontogenic and osteogenic mediums for 14 days. For confirmation of odontogenic and osteogenic differentiation, mRNA levels of BMP2, BMP 7, collagen type 1 (COL1A), osteocalsin (OCN), and dentin sialophosphoprotein (DSPP) genes were measured by quantitative real-time PCR. In addition to this, immunocytochemistry was performed by odontogenic and osteogenic antibodies and mineralization obtained by von Kossa staining. Our results showed that the BMP 2 and BMP 7 both promoted odontogenic and osteogenic differentiation of hTGSCs. Data indicated that BMP 2 treatment and BMP 7 treatment induce odontogenic differentiation without affecting each other, whereas they induce osteogenic differentiation by triggering expression of each other. These findings provide a feasible tool for tooth and bone tissue engineering.     

DOPA-IGF-1 Coated HA/PLGA Microspheres Promoting Proliferation and Osteoclastic Differentiation of Rabbit Bone Mesenchymal Stem Cells

To explore the ability of dihydroxyphenylalanine-insulin-like growth factor-1 (DOPA-IGF-1) coated hydroxyapatite/poly(lactic-co-glycolic acid)(HA/PLGA) microspheres to promote the proliferation and osteoclastic differentiation of rabbit bone mesenchymal stem cells(rBMSCs), HA/PLGA microspheres with different HA content (10%, 30%, 50%, mass fraction) were prepared by electrospinning method and HA/PLGA microspheres with 50% HA were coated with IGF-1 and DOPA-IGF-1, respectively. They were co-cultured with rBMSCs, respctively. Cell counting kit-8(CCK-8) detection, confocal laser scanning microscopy(CLSM), alkaline phosphatase(ALP) detection and osteogenesis related genes COL IA1, Runx2 and bone morphogenetic protein-2(BMP-2) detection were conducted to detect the proliferation activity, cell morphology, differentiation ability and the expression level of osteogenesis-related genes of cells cultured on all microspheres groups. The results showed that rBMSCs proliferation increased in an HA content dependent manner, and cells proliferated more in the IGF-1 coated and DOPA-IGF-1 coated groups, in particular in DOPA-IGF-1 coated group, and the differences were more remarkable over time (P<0.05). HA/PLGA microspheres promoted the proliferation and osteogenic differentiation of rBMSCs, and DOPA-IGF-1 coating enhanced the proliferation and osteogenic differentiation of rBMSCs.