Immunomodulatory and Anti-inflammatory effect of Neural Stem/Progenitor Cells in the Central Nervous System.

Neuroinflammation is a critical event that responds to disturbed homeostasis and governs various neurological diseases in the central nervous system (CNS). The excessive inflammatory microenvironment in the CNS can adversely affect endogenous neural stem cells, thereby impeding neural self-repair. Therapies with neural stem/progenitor cells (NSPCs) have shown significant inhibitory effects on inflammation, which is mainly achieved through intercellular contact and paracrine signalings. The intercellular contact between NSPCs and immune cells, the activated CNS- resident microglia, and astrocyte plays a critical role in the therapeutic NSPCs homing and immunomodulatory effects. Moreover, the paracrine effect mainly regulates infiltrating innate and adaptive immune cells, activated microglia, and astrocyte through the secretion of bioactive molecules and extracellular vesicles. However, the molecular mechanism involved in the immunomodulatory effect of NSPCs is not well discussed. This article provides a systematic analysis of the immunomodulatory mechanism of NSPCs, discusses efficient ways to enhance its immunomodulatory ability, and gives suggestions on clinical therapy.

Investigation of low-level laser therapy potentiality on proliferation and differentiation of human osteoblast-like cells in the absence/presence of osteogenic factors.

Several studies have shown that low-level laser irradiation (LLLI) has beneficial effects on bone regeneration. The objective of this study was to examine the in vitro effects of LLLI on proliferation and differentiation of a human osteoblast-like cell line (Saos-2 cell line). Cultured cells were exposed to different doses of LLLI with a semiconductor diode laser (659 nm; 10 mW power output). The effects of laser on proliferation were assessed daily up to seven days of culture in cells irradiated once or for three consecutive days with laser doses of 1 or 3 J/cm(2). The obtained results showed that laser stimulation enhances the proliferation potential of Saos-2 cells without changing their telomerase pattern or morphological characteristics. The effects on cell differentiation were assessed after three consecutive laser irradiation treatments in the presence or absence of osteo-inductive factors on day 14. Enhanced secretion of proteins specific for differentiation toward bone as well as calcium deposition and alkaline phosphatase activity were observed in irradiated cells cultured in a medium not supplemented with osteogenic factors. Taken together these findings indicate that laser treatment enhances the in vitro proliferation of Saos-2 cells, and also influences their osteogenic maturation, which suggest it is a helpful application for bone tissue regeneration.

A comparative analysis of the in vitro effects of pulsed electromagnetic field treatment on osteogenic differentiation of two different mesenchymal cell lineages.

Human mesenchymal stem cells (MSCs) are a promising candidate cell type for regenerative medicine and tissue engineering applications. Exposure of MSCs to physical stimuli favors early and rapid activation of the tissue repair process. In this study we investigated the in vitro effects of pulsed electromagnetic field (PEMF) treatment on the proliferation and osteogenic differentiation of bone marrow MSCs (BM-MSCs) and adipose-tissue MSCs (ASCs), to assess if both types of MSCs could be indifferently used in combination with PEMF exposure for bone tissue healing. We compared the cell viability, cell matrix distribution, and calcified matrix production in unstimulated and PEMF-stimulated (magnetic field: 2 mT, amplitude: 5 mV) mesenchymal cell lineages. After PEMF exposure, in comparison with ASCs, BM-MSCs showed an increase in cell proliferation (p<0.05) and an enhanced deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p<0.05). Calcium deposition was 1.5-fold greater in BM-MSC-derived osteoblasts (p<0.05). The immunofluorescence related to the deposition of bone matrix proteins and calcium showed their colocalization to the cell-rich areas for both types of MSC-derived osteoblast. Alkaline phosphatase activity increased nearly 2-fold (p<0.001) and its protein content was 1.2-fold higher in osteoblasts derived from BM-MSCs. The quantitative reverse-transcription polymerase chain reaction (qRT-PCR) analysis revealed up-regulated transcription specific for bone sialoprotein, osteopontin, osteonectin, and Runx2, but at a higher level for cells differentiated from BM-MSCs. All together these results suggest that PEMF promotion of bone extracellular matrix deposition is more efficient in osteoblasts differentiated from BM-MSCs.

In vitro osteogenesis of human stem cells by using a three-dimensional perfusion bioreactor culture system: a review.

Tissue engineering (by culturing cells on appropriate scaffolds, and using bioreactors to drive the correct bone structure formation) is an attractive alternative to bone grafting or implantation of bone substitutes. Osteogenesis is a biological process that involves many molecular intracellular pathways organized to optimize bone modeling. The use of bioreactor systems and especially the perfusion bioreactor, provides both the technological means to reveal fundamental mechanisms of cell function in a 3D environment, and the potential to improve the quality of engineered tissues. In this mini-review all the characteristics for the production of an appropriate bone construct are analyzed: the stem cell source, scaffolds useful for the seeding of pre-osteoblastic cells and the effects of fluid flow on differentiation and proliferation of bone precursor cells. By automating and standardizing tissue manufacture in controlled closed systems, engineered tissues may reduce the gap between the process of bone formation in vitro and subsequent graft of bone substitutes in vivo.

Characterization of an inducible promoter in different DNA copy number conditions.

BACKGROUND: The bottom-up programming of living organisms to implement novel user-defined biological capabilities is one of the main goals of synthetic biology. Currently, a predominant problem connected with the construction of even simple synthetic biological systems is the unpredictability of the genetic circuitry when assembled and incorporated in living cells. Copy number, transcriptional/translational demand and toxicity of the DNA-encoded functions are some of the major factors which may lead to cell overburdening and thus to nonlinear effects on system output. It is important to disclose the linearity working boundaries of engineered biological systems when dealing with such phenomena. RESULTS: The output of an N-3-oxohexanoyl-L-homoserine lactone (HSL)-inducible RFP-expressing device was studied in Escherichia coli in different copy number contexts, ranging from 1 copy per cell (integrated in the genome) to hundreds (via multicopy plasmids). The system is composed by a luxR constitutive expression cassette and a RFP gene regulated by the luxI promoter, which is activated by the HSL-LuxR complex. System output, in terms of promoter activity as a function of HSL concentration, was assessed relative to the one of a reference promoter in identical conditions by using the Relative Promoter Units (RPU) approach. Nonlinear effects were observed in the maximum activity, which is identical in single and low copy conditions, while it decreases for higher copy number conditions. In order to properly compare the luxI promoter strength among all the conditions, a mathematical modeling approach was used to relate the promoter activity to the estimated HSL-LuxR complex concentration, which is the actual activator of transcription. During model fitting, a correlation between the copy number and the dissociation constant of HSL-LuxR complex and luxI promoter was observed. CONCLUSIONS: Even in a simple inducible system, nonlinear effects are observed and non-trivial data processing is necessary to fully characterize its operation. The in-depth analysis of model systems like this can contribute to the advances in the synthetic biology field, since increasing the knowledge about linearity and working boundaries of biological phenomena could lead to a more rational design of artificial systems, also through mathematical models, which, for example, have been used here to study hard-to-predict interactions.

Video evaluation of the kinematics and dynamics of the beating cardiac syncytium: an alternative to the Langendorff method.

Many important observations and discoveries in heart physiology have been made possible using the isolated heart method of Langendorff. Nevertheless, the Langendorff method has some limitations and disadvantages such as the vulnerability of the excised heart to contusions and injuries, the probability of preconditioning during instrumentation, the possibility of inducing tissue edema, and high oxidative stress, leading to the deterioration of the contractile function. To avoid these drawbacks associated with the use of a whole heart, we alternatively used beating mouse cardiac syncytia cultured in vitro in order to assess possible ergotropic, chronotropic, and inotropic effects of drugs. To achieve this aim, we developed a method based on image processing analysis to evaluate the kinematics and the dynamics of the drug-stimulated beating syncytia starting from the video recording of their contraction movement. In this manner, in comparison with the physiological no-drug condition, we observed progressive positive ergotropic, positive chronotropic, and positive inotropic effects of 10 µM isoproterenol (ß-adrenergic agonist) and early positive ergotropic, negative chronotropic, and positive inotropic effects of 10 µM phenylephrine (alpha-adrenergic agonist), followed by a late phase with negative ergotropic, positive chronotropic, and negative inotropic trends. Our method permitted a systematic study of in vitro beating syncytia, producing results consistent with previous works. Consequently, it could be used in in vitro studies of beating cardiac patches, as an alternative to Langendorff's heart in biochemical and pharmacological studies, and especially when the Langendorff technique is inapplicable (e.g., in studies about human cardiac syncytium in physiological and pathological conditions, patient-tailored therapeutics, and syncytium models derived from induced pluripotent/embryonic stem cells with genetic mutations). Furthermore, the method could be helpful in heart tissue engineering and bioartificial heart research to "engineer the heart piece by piece." In particular, the proposed method could be useful in the identification of a suitable cell source, in the development and testing of "smart" biomaterials, and in the design and use of novel bioreactors and microperfusion systems.

Multiplexing and demultiplexing logic functions for computing signal processing tasks in synthetic biology.

Building biological devices to perform computational and signal processing tasks is one of the main research issues in synthetic biology. Herein, two modular biological systems that could mimic multiplexing and demultiplexing logic functions are proposed and discussed. These devices, called multiplexer (mux) and demultiplexer (demux), respectively, have a remarkable importance in electronic, telecommunication, and signal processing systems and, similarly, they could play a crucial role if implemented in a living organism, such as Escherichia coli. BioBrick standard parts were used to design mux and demux and to construct two genetic circuits that could carry out the desired tasks. A modular approach, mimicking basic logic gates (AND, OR, and NOT) with protein/autoinducer or protein/DNA interactions and interconnecting them to create the final circuits, was adopted. A mathematical model of the designed gene networks was been defined and simulations performed to validate the expected behavior of the systems. In addition, circuit subparts were tested in vivo and the results used to determine some of the parameters of the mathematical model. According to both the experimental and simulated results, guidelines for future finalization of mux and demux are provided.

In vitro osteoblastic differentiation of human mesenchymal stem cells and human dental pulp stem cells on poly-L-lysine-treated titanium-6-aluminium-4-vanadium.

Three-dimensional (3D) titanium-6-aluminium-4-vanadium (Ti6Al4V) is a widely used biomaterial for orthopedic prosthesis and dental implants; thanks to its very high-mechanical strength and resistance to corrosion. Human mesenchymal stem cells (hMSCs) and dental pulp stem cells (hDPSCs) are responsible for bone regeneration following colonization of prosthesis or dental implants. Both hMSCs and hDPSCs have lower ability to colonize this biomaterial in comparison with tissue culture-treated plastic. Both hMSCs and hDPSCs show lack of focal adhesion kinase (FAK) activation when grown on Ti6Al4V. This signal is restored in the presence of poly-L-lysine (poly-L-lys). Poly-L-lys has been used as part of organoapatite or together with zinc and calcium ions. Our results suggest that poly-L-lys alone induces FAK activation through ß1-INTEGRIN, because the presence of ß1-INTEGRIN blocking antibody avoided FAK autophosphorylation. Presence of poly-L-lys also increases expression of osteoblastic differentiation marker genes in hMSCs and hDPSCs grown on Ti6Al4V.

In vitro calcified matrix deposition by human osteoblasts onto a zinc-containing bioactive glass.

Bioactive glasses synthesized by the sol-gel technique possess many of the qualities associated with an ideal scaffold material for a bone graft substitute. In view of the potential clinical applications, we performed a detailed in vitro study of the biological reactivity of synthesized 58S bioactive glass containing-zinc, in terms of osteoblast morphology, proliferation, and deposition of a mineralized extracellular matrix (ECM). Human Sarcoma Osteoblast (SAOS-2) cells were used to i) assess cytotoxicity by lactate dehydrogenase (LDH) release and ii) evaluate the deposition of a calcified extracellular matrix by ELISA assay and quantitative RT-PCR (qRT-PCR). In comparison with pure silica and 58S, the 58S-Zn0.4 bioglass showed a significant increase in cellular proliferation and deposition of ECM components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, type-I and -III collagens. Calcium deposition was significantly higher than on pure silica and 58S samples. Also Alkaline phosphatase (ALP) activity and its protein content was higher with respect to pure silica and 58S. qRT-PCR analysis revealed the up-regulation of type-I collagen, bone sialoprotein and osteopontin genes. All together these results demonstrate the cytocompatibility of 58S-Zn0.4 bioglass and its capability to promote osteoblast differentiation.

Low-power ultrasounds as a tool to culture human osteoblasts inside cancellous hydroxyapatite.

Bone graft substitutes and cancellous biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of cancellous hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering. In this study, we have followed a tissue-engineering strategy where ultrasonically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix inside a porous hydroxyapatite scaffold. The ultrasonic stimulus had the following parameters: average power equal to 149 mW and frequency of 1.5 MHz. In comparison with control conditions, the ultrasonic stimulus increased the cell proliferation and the surface coating with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The mechanical stimulus aimed at obtaining a better modification of the biomaterial internal surface in terms of cell colonization and coating with bone matrix. The modified biomaterial could be used, in clinical applications, as an implant for bone repair.

In vitro enhancement of SAOS-2 cell calcified matrix deposition onto radio frequency magnetron sputtered bioglass-coated titanium scaffolds.

In bone tissue engineering, bioglass coating of titanium (Ti) scaffolds has drawn attention as a method to improve osteointegration and implant fixation. In this in vitro study, bioactive glass layers with an approximate thickness of 1 microm were deposited at 200 degrees C onto a three-dimensional Ti-6Al-4V scaffold using a radio frequency (r.f.) magnetron sputtering system. After incubation with SAOS-2 human osteoblasts, in comparison with the uncoated scaffolds, the bioglass-coated scaffolds showed a twofold increase in cell proliferation (p < 0.05) up to 68.4 x 10(6), and enhanced the deposition of extracellular matrix components such as decorin, fibronectin, osteocalcin, osteonectin, osteopontin, and type-I and -III collagens (p < 0.05). Calcium deposition was twofold greater on the bioglass-coated scaffolds (p < 0.05). The immunofluorescence related to the preceding bone matrix proteins and calcium showed their colocalization to the cell-rich areas. Alkaline phosphatase activity increased twofold (p < 0.001) and its protein content was threefold higher with respect to the uncoated sample. Quantitative reverse transcriptase-polymerase chain reaction analysis revealed upregulated transcription specific for type-I collagen and osteopontin (p < 0.001). All together, these results demonstrate that the bioglass coating of the three-dimensional Ti scaffolds by the r.f. magnetron sputtering technique determines an in vitro increase of the bone matrix elaboration and may potentially have a clinical benefit.

In vitro electromagnetically stimulated SAOS-2 osteoblasts inside porous hydroxyapatite.

One of the key challenges in reconstructive bone surgery is to provide living constructs that possess the ability to integrate in the surrounding tissue. Bone graft substitutes, such as autografts, allografts, xenografts, and biomaterials have been widely used to heal critical-size long bone defects due to trauma, tumor resection, congenital deformity, and tissue degeneration. In particular, porous hydroxyapatite is widely used in reconstructive bone surgery owing to its biocompatibility. In addition, the in vitro modification of hydroxyapatite with osteogenic signals enhances the tissue regeneration in vivo, suggesting that the biomaterial modification could play an important role in tissue engineering. In this study we have followed a biomimetic strategy where electromagnetically stimulated SAOS-2 human osteoblasts proliferated and built their extracellular matrix inside a porous hydroxyapatite scaffold. The electromagnetic stimulus had the following parameters: intensity of the magnetic field equal to 2 mT, amplitude of the induced electric tension equal to 5 mV, frequency of 75 Hz, and pulse duration of 1.3 ms. In comparison with control conditions, the electromagnetic stimulus increased the cell proliferation and the surface coating with bone proteins (decorin, osteocalcin, osteopontin, type-I collagen, and type-III collagen). The physical stimulus aimed at obtaining a better modification of the biomaterial internal surface in terms of cell colonization and coating with bone matrix.

Effects of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 human osteogenic cell line.

The aim of the work was to understand the consequences of low-amplitude, high-frequency vibrations on proliferation and differentiation of SAOS-2 cells (sarcoma osteogenetic), an osteoblastic and tumorigenic cell line. We realized a bioreactor composed of an eccentric motor that produces a displacement of 11 mm at frequencies between 1 and 120 Hz on a plate connected to the motor. The cultures of SAOS-2 cells were fixed on the plate, and the linear acceleration provoked by the motor to the cultures was measured. We used 30 Hz as stimulating frequency after a preliminary test on the effect of different frequencies on differentiation of cells. Afterward, SAOS-2 cells were stimulated with 30 Hz for different durations, every day for 4 days. The expression of some genes involved in the differentiation process was analyzed first with a reverse transcriptase-polymerase chain reaction and afterward with a real-time polymerase chain reaction on the most expressed genes. Moreover, the proliferation of cells was evaluated. The results suggest a strong increase in the expression of the genes involved in tissue differentiation in the treated groups with respect to the controls. On the other hand, the proliferation seems to be slowed down, so probably the acceleration perceived by the mechanosensors of the cells changes the cellular cycle by blocking the duplication to early differentiate toward bone tissue.

Effects of electromagnetic stimulation on calcified matrix production by SAOS-2 cells over a polyurethane porous scaffold.

There is increasing interest in designing new biomaterials that could potentially be used in the form of scaffolds as bone substitutes. In this study we used a hydrophobic crosslinked polyurethane in a typical tissue-engineering approach, that is, the seeding and in vitro culturing of cells using a porous scaffold. Using an electromagnetic bioreactor (magnetic field intensity, 2 mT; frequency, 75 Hz), we investigated the effect of the electromagnetic stimulation on SAOS-2 human osteoblast proliferation and calcified matrix production. Cell proliferation was twice as high; expression of decorin, osteocalcin, osteopontin, type I collagen, and type III collagen was greater (1.3, 12.2, 12.1, 10.0, and 10.5 times as great, respectively); and calcium deposition was 5 times as great as under static conditions without electromagnetic stimulation. RT-PCR analysis revealed the electromagnetically upregulated transcription specific for decorin, fibronectin, osteocalcin, osteopontin, transforming growth factor-beta, type I collagen, and type III collagen. The immunolocalization of the extracellular matrix constituents showed their colocalization in the cell-rich areas. The bioreactor and the polyurethane foam were designed to obtain cell colonization and calcified matrix deposition. This cultured biomaterial could be used, in clinical applications, as an osteoinductive implant for bone repair.