TIMPs Expression as A Maternal Cell Free Plasma Biomarker of Severe Preeclampsia: A Case-Control Study.

OBJECTIVE: Preeclampsia (PE) is a pregnancy related disorder with prevalence of 6-7%. Insufficient trophoblastic invasion leads to incomplete remodeling of spiral arteries and consequent decrease in feto-placental perfusion. Altered placental expression of tissue inhibitors of matrix metalloproteinase (TIMPs) is considered to be involved in this process while the balance between matrix metalloproteinases (MMPs) and TIMPs contributes to remodeling of the placenta and uterine arteries by degradation and refurbishing of extracellular matrix (ECM). Therefore, TIMPs, fetal expression pattern was evaluated with the aim of its potential to be used as a determinant for the (early) detection of PE. MATERIALS AND METHODS: In this case-control study, cell free fetal RNA (cffRNA) released by placenta into the maternal blood was used to determine expression patterns of TIMP1, 2, 3 and 4 in the severe preeclamptic women in comparison with the normal pregnant women. Whole blood from 20 preeclamptic and 20 normal pregnant women in their 28-32 weeks of gestational age was collected. The second control group consisted of 20 normal pregnant women in either 14 or 28 weeks of gestation (each 10). cffRNA was extracted from plasma and real-time polymerase chain reaction (PCR) was done to determine the expression levels of TIMP1, 2, 3 and 4 genes. RESULTS: Statistical analysis of the results showed significant higher expression of TIMP1-4 in the preeclamptic women in comparison with the control group (P=0.029, 0.037, 0.037 and 0.049, respectively). Also, an increased level of TIMPs expression was observed by comparing 14 to 28 weeks of gestational age in the normal pregnant women in the second control group. CONCLUSION: An increased cffRNA expression level of TIMPs may be correlated with the intensity of placental vascular defect and may be used as a determinant of complicated pregnancies with severe preeclampsia.

Effect of porosity on mechanical and biological properties of bioprinted scaffolds.

Treatment of tissue defects commonly represents a major problem in clinics due to difficulties involving a shortage of donors, inappropriate sizes, abnormal shapes, and immunological rejection. While many scaffold parameters such as pore shape, porosity percentage, and pore connectivity could be adjusted to achieve desired mechanical and biological properties. These parameters are crucial scaffold parameters that can be accurately produced by 3D bioprinting technology based on the damaged tissue. In the present research, the effect of porosity percentage (40%, 50%, and 60%) and different pore shapes (square, star, and gyroid) on the mechanical (e.g., stiffness, compressive and tensile behavior) and biological (e.g., biodegradation, and cell viability) properties of porous polycaprolactone (PCL) scaffolds coated with gelatin have been investigated. Moreover, human foreskin fibroblast cells were cultured on the scaffolds in the in-vitro procedures. MTT assay (4, 7, and 14 days) was utilized to determine the cytotoxicity of the porous scaffolds. It is revealed that the porous scaffolds produced by the bioprinter did not produce a cytotoxic effect. Among all the porous scaffolds, scaffolds with a pore size of about 500 µm and porosity of 50% showed the best cell proliferation compared to the controls after 14 days. The results demonstrated that the pore shape, porosity percentage, and pore connectivity have an important role in improving the mechanical and biological properties of porous scaffolds. These 3D bioprinted biodegradable scaffolds exhibit potential for future application as polymeric scaffolds in hard tissue engineering applications.

A smart magnetic hydrogel containing exosome promotes osteogenic commitment of human adipose-derived mesenchymal stem cells.

Objectives: Exosomes, as nano-sized extracellular vehicles acting as cell-to-cell communicators, are novel promising therapeutics in the area of bone tissue engineering. Moreover, magnetic nanoparticles, whose integration with other appropriate components is viewed as an intriguing approach to strengthen bone tissue engineering efficacy. We investigated the effect of magnetic enriched with exosomes on osteogenic differentiation. Materials and Methods: Exosomes were isolated from human adipose-derived mesenchymal stem cells by Exo-spin™ kit (MSC-EX). Alginate (Alg) scaffold containing 1% (w/w) cobalt ferrite nanoparticles (CoFe2O4) was produced. MSC-EX were gently loaded onto Alg and Alg-cobalt ferrite (Alg-CF) scaffolds yielding Alg-EX and Alg-CF-EX scaffolds. The effects of MSC-Ex and magnetic hydrogel composite under an external static magnetic field (SMF) on proliferation and differentiation of MSCs were evaluated by alkaline phosphatase (ALP) activity measurement, alizarin red staining, and energy dispersive X-ray (EDX) analysis. Results: Our results showed that Alg and Alg-CF scaffolds were not only cytotoxic but also supported AdMSCs proliferation. MSC-EX loading of the scaffolds enhanced AdMSCs proliferation significantly. According to the results, Alg-CF-EX scaffolds under magnetic stimulation exhibited the most potent effect on osteogenic differentiation of cultured AdMSCs as evidenced by higher ALP activity and mineralization. Conclusion: We provided evidence that the combination of Alg hydrogel, CFNPs, and MSC-EX resulted in the construction of a bone tissue-engineering scaffold that highly supports the osteogenic commitment of MSCs.

Comparative study of mouse adipose- and bone marrow mesenchymal stem cells in diabetic model with critical limb ischemia.

The aim of this research is to compare the capabilities of Adipose tissue mesenchymal stem cells (AT-MSCs) and bone marrow mesenchymal stem cells (BM-MSCs) in the treatment of diabetic male mice with CLI model. Supernatants were collected from C57BL/6 mice isolated AT-MSCs and BM-MSCs, afterward their effects on human umbilical vein endothelial (HUVEC) migration potential were evaluated. Diabetes mellitus type 1 was induced by streptozotocin injection. Diabetic mice with CLI model were divided into three groups and injected with AT-MSCs, BM-MSCs, or PBS then the efficacy of them was assessed. Survival of MSCs was analysed by SRY-specific gene. The conditioned medium of AT-MSCs and BM-MSCs stimulated HUVECs migration and the donor cells were detected till 21 day in two groups. BM-MSCs and AT-MSCs improved significantly functional recovery and ischemia damage. Neovascularization in ischemic muscle was significantly higher in mice treated with AT-MSCs and BM-MSCs and they improved muscle regeneration. In vivo and in vitro findings show that AT-MSCs and BM-MSCs transplantation could be proposed as a promising therapy to promote angiogenesis and muscle regeneration through secretion of proangiogenic factors, cytokines and growth factors in diabetic mice with CLI model wherein blood supply is insufficient and disrupted.

The effect of electromagnetic field on decreasing and increasing of the growth and proliferation rate of dermal fibroblast cell.

Maintaining the health of dermal fibroblast cells and controlling their growth and proliferation would directly affect the health of skin tissues. The present study encompassed three control and three experimental specimens, which were different in terms of the duration of exposure to electromagnetic field (EMF) and intensity. With a decrease in intensity from 2 to 1 mT during 24, 48, and 72 h after exposing the cells to EMF, the frequency of the sample fibroblast cells increased by 60.3%, 144.9%, and 90.1%, respectively. With an increase in intensity from 3 to 4 mT during 48 and 72 h of exposure to EMF, the frequencies of the sample fibroblast cells decreased by 6.8% and 86.7%, respectively. It seems to be possible to achieve the most desirable condition to help the restoration of wounds and skin lesions through decreasing the exposure intensity from 2 to 0.5 mT and increasing EMF exposure time from 24 to 72 h simultaneously and non-invasively. The most desirable approach to improve the treatment of skin cancers non-invasively is to increase the intensity from 3 to 5 mT and to enhance EMF exposure time from 48 to 72 h.

Gene expression analysis of MMPs in women with preeclampsia using cell-free fetal RNA in maternal plasma.

OBJECTIVE: Nucleic acids released from the placenta into the mother's blood circulation system provide a valuable source of potential biomarkers for early detection of pregnancy complications such as preeclampsia (PE). PE affects nearly 5-10% of pregnancies worldwide and is a major contributor to the maternal and neonatal mortality and morbidity. It is known that altered placental expression of matrix metalloproteinases (MMPs) may cause shallow cytotrophoblastic invasion and ultimately lead to preeclampsia. The present study aimed to evaluate pattern of placental/fetal expression of the MMP family (MMP-2, MMP-9, MMP-14, MMP-15 and MMP-26) in preeclamptic women and compare it to normal pregnancies, using cell free fetal RNA (cff-RNA). METHODS: Blood samples were obtained from 20 pregnant women diagnosed with severe PE (28-32 weeks) and 40 control healthy pregnant women in two groups of either matched gestational age (N = 20) or 14 and 28 weeks pregnancies (each 10). cff-RNA was extracted from plasma, followed by reverse transcription of cff-RNA. Expression of MMP genes was measured using quantitative reverse transcription PCR (qRT-PCR). RESULTS: The expression levels of MMP-2, MMP-9 and MMP-15 were significantly increased, while MMP-14 expression level was significantly reduced and the expression of MMP-26 showed a relative increase in PE pregnancies compared to the control group. Additionally, increased level of MMPs expression was observed by comparing 14 and 28 weeks gestation age in normal pregnancy. CONCLUSION: Using cff-RNA, circulatory expression level of MMP-2, MMP-9, MMP-14 and MMP-15 were significantly altered in preeclampsia compared to normal pregnancies.

Development of An Artificial Male Germ Cell Niche Using Electrospun Poly Vinyl Alcohol/Human Serum Albumin/Gelatin Fibers.

OBJECTIVE: Recent achievements in stem cell biotechnology, nanotechnology and tissue engineering have led to development of novel approaches in regenerative medicine. Azoospermia is one of the challenging disorders of the reproductive system. Several efforts were made for isolation and culture of testis-derived stem cells to treat male infertility. However, tissue engineering is the best approach to mimic the three dimensional microenvironment of the testis in vitro. We investigated whether human testis-derived cells (hTCs) obtained by testicular sperm extraction (TESE) can be cultured on a homemade scaffold composed of electrospun nanofibers of homogeneous poly (vinyl alcohol)/human serum albumin/gelatin (PVA/HSA/gelatin). MATERIALS AND METHODS: In this experimental lab study, human TCs underwent two steps of enzymatic cell isolation and five culture passages. Nanofibrous scaffolds were characterized by scanning electron microscopy (SEM) and Fouriertransform infrared spectroscopy (FTIR). Attachment of cells onto the scaffold was shown by hematoxylin and eosin (H and E) staining and SEM. Cell viability study using MTT [3-(4, 5-dimethyl-2-thiazolyl) -2, 5-diphenyl -2H- tetrazolium bromide] assay was performed on days 7 and 14. RESULTS: Visualization by H and E staining and SEM indicated that hTCs were seeded on the scaffold. MTT test showed that the PVA/HSA/gelatin scaffold is not toxic for hTCs. CONCLUSION: It seems that this PVA/HSA/gelatin scaffold is supportive for growth of hTCs.

Specific targeting of a pore-forming toxin (listeriolysin O) to LHRH-positive cancer cells using LHRH targeting peptide.

Conventional drug delivery systems have many limitations including cytotoxicity and affecting non-specific cells. Cell-targeting peptides (CTPs) as a potential class of targeting moiety have some advantages over previous targeting moieties such as monoclonal antibodies, offer additional benefits to design systems using CTPs. Here we have engineered listeriolysin O (LLO) pore-forming toxin by adding a luteinizing hormone-releasing hormone (LHRH) targeting peptide to its N-terminus. Two versions of the toxin, with and without targeting peptide, were sub-cloned into a bacterial expression plasmid. BL21 DE3 cells were used for induction of expression and recombinant proteins were purified using nickel-immobilized metal affinity chromatography column. In order to treat MDA-MB-231 and SKOV3 cell lines as LHRH receptor positive and negative cells, two mentioned LLO toxins were used to evaluate their cytotoxicity and specificity. Our results reveal that the IC50 of LLO toxin on MDA-MB-231 and SKOV3 cells was 0.32 and 0.41 µg/ml respectively. Furthermore, IC50 of fusion LHRH-LLO toxin on the cells was 0.88 and 19.55 µg/ml. Cytotoxicity of engineered LHRH-LLO toxin on negative cells was significantly 48-fold lower than wild-type LLO toxin. But this difference has been lowered to only 2.7-fold less cytotoxicity in positive cells. To the best of our knowledge, the current work as the first study regarding engineered toxin revealed that CDC family members could be used to target the specific cell-type.

Dual-aptamer based electrochemical sandwich biosensor for MCF-7 human breast cancer cells using silver nanoparticle labels and a poly(glutamic acid)/MWNT nanocomposite.

This paper reports on a sensitive and selective method for the detection of Michigan Cancer Foundation-7 (MCF-7) human breast cancer cells and MUC1 biomarker by using an aptamer-based sandwich assay. A biocompatible nanocomposite consisting of multiwall carbon nanotubes (MWCNT) and poly(glutamic acid) is placed on a glassy carbon electrode (GCE). The sandwich assay relies on the use of a mucin 1 (MUC1)-binding aptamer that is first immobilized on the surface of modified GCE. Another aptamer (labeled with silver nanoparticles) is applied for secondary recognition of MCF-7 cells in order to increase selectivity and produce an amplified signal. Differential pulse anodic stripping voltammetry was used to follow the electrochemical signal of the AgNPs. Under the optimal condition, the sensor responds to MCF-7 cells in the concentration range from 1.0 × 102 to 1.0 × 107 cells·mL-1 with a detection limit of 25 cells. We also demonstrate that the MUC1 tumor marker can be detected by the present biosensor. The assay is highly selective and sensitive, acceptably stable and reproducible. This warrants the applicability of the method to early diagnosis of breast cancer. Graphical abstract Schematic of the fabrication of an aptamer-based sandwich biosensor for Michigan Cancer Foundation-7 cells (MCF-7). A MWCNT-poly(glutamic acid) nanocomposite was used as a biocompatible matrix for MUC1-aptamer immobilization. Stripping voltammetry analysis of AgNPs was performed using aptamer conjugated AgNPs as signalling probe.

Human embryonic stem cells and good manufacturing practice: Report of a 1- day workshop held at Stem Cell Biology Research Center, Yazd, 27th April 2017.

This report explains briefly the minutes of a 1-day workshop entitled; "human embryonic stem cells (hESCs) and good manufacturing practice (GMP)" held by Stem Cell Biology Research Center based in Yazd Reproductive Sciences Institute at Shahid Sadoughi University of Medical Sciences, Yazd, Iran on 27th April 2017. In this workshop, in addition to the practical sessions, Prof. Harry D. Moore from Centre for Stem Cell Biology, University of Sheffield, UK presented the challenges and the importance of the biotechnology of clinical-grade human embryonic stem cells from first derivation to robust defined culture for therapeutic applications.

Use of nanoscale mechanical stimulation for control and manipulation of cell behaviour.

The ability to control cell behaviour, cell fate and simulate reliable tissue models in vitro remains a significant challenge yet is crucial for various applications of high throughput screening e.g. drug discovery. Mechanotransduction (the ability of cells to convert mechanical forces in their environment to biochemical signalling) represents an alternative mechanism to attain this control with such studies developing techniques to reproducibly control the mechanical environment in techniques which have potential to be scaled. In this review, the use of techniques such as finite element modelling and precision interferometric measurement are examined to provide context for a novel technique based on nanoscale vibration, also known as "nanokicking". Studies have shown this stimulus to alter cellular responses in both endothelial and mesenchymal stem cells (MSCs), particularly in increased proliferation rate and induced osteogenesis respectively. Endothelial cell lines were exposed to nanoscale vibration amplitudes across a frequency range of 1-100 Hz, and MSCs primarily at 1 kHz. This technique provides significant potential benefits over existing technologies, as cellular responses can be initiated without the use of expensive engineering techniques and/or chemical induction factors. Due to the reproducible and scalable nature of the apparatus it is conceivable that nanokicking could be used for controlling cell behaviour within a wide array of high throughput procedures in the research environment, within drug discovery, and for clinical/therapeutic applications. STATEMENT OF SIGNIFICANCE: The results discussed within this article summarise the potential benefits of using nanoscale vibration protocols for controlling cell behaviour. There is a significant need for reliable tissue models within the clinical and pharma industries, and the control of cell behaviour and stem cell differentiation would be highly beneficial. The full potential of this method of controlling cell behaviour has not yet been realised.

Production of Nanoscale Vibration for Stimulation of Human Mesenchymal Stem Cells.

Mechanical stimulation is becoming a common technique for manipulating cell behaviour in bioengineering with applications in tissue engineering and possibly regenerative therapy. Living organisms show biological responses in vivo and in vitro to various types of mechanical stimulation including vibration. The development of apparatus to produce vertical motions of nanoscale amplitude is detailed and their effect on mouse endothelial (Le2) and human mesenchymal stem cells (hMSCs) is investigated. Piezo ceramic actuators and aluminium reinforcement were utilised along with laser interferometry to ensure amplitude consistency at the nanometre level across a cell culture substrate. Peak force applied to the cells was estimated to be of nN magnitude at frequencies of 500 and 1000 Hz. Morphological changes in the cytoskeleton were found for both cell types along with increased MSC proliferation after 1 week of stimulation at 500 Hz. Changes in the nuclear size of MSCs after stimulation were also found.

Nanoscale stimulation of osteoblastogenesis from mesenchymal stem cells: nanotopography and nanokicking.

AIM: Mesenchymal stem cells (MSCs) have large regenerative potential to replace damaged cells from several tissues along the mesodermal lineage. The potency of these cells promises to change the longer term prognosis for many degenerative conditions currently suffered by our aging population. We have endeavored to demonstrate our ability to induce osteoblatogenesis in MSCs using high-frequency (1000-5000 Hz) piezo-driven nanodisplacements (16-30 nm displacements) in a vertical direction. MATERIALS & METHODS: Osteoblastogenesis has been determined by the upregulation of osteoblasic genes such as osteonectin (ONN), RUNX2 and Osterix, assessed via quantitative real-time PCR; the increase of osteocalcin (OCN) and osteopontin (OPN) at the protein level and the deposition of calcium phosphate determined by histological staining. RESULTS: Intriguingly, we have observed a relationship between nanotopography and piezo-stimulated mechanotransduction and possibly see evidence of two differing osteogenic mechanisms at work. These data provide confidence in nanomechanotransduction for stem cell differentiation without dependence on soluble factors and complex chemistries. CONCLUSION: In the future it is envisaged that this technology may have beneficial therapeutic applications in the healthcare industry, for conditions whose overall phenotype maybe characterized by weak or damaged bones (e.g., osteoporosis and bone fractures), and which can benefit from having an increased number of osteoblastic cells in vivo.

Cell interactions at the nanoscale: piezoelectric stimulation.

Nanometric movements of the substrate on which endothelial cells are growing, driven by periodic sinusoidal vibration from 1 Hz to 50 Hz applied by piezo actuators, upregulate endothelin-1 and Kruppel-like factor 2 expression, and increase cell adhesion. These movements are in the z (vertical) axis and ranges from 5 to 50 nm and are similar in vertical extent to protrusions from the cells themselves already reported in the literature. White noise vibrations do not to produce these effects. Vibrational sweeps, if suitably confined within a narrow frequency range, produce similar stimulatory effects but not at wider sweeps. These effects suggest that coherent vibration is crucial for driving these cellular responses. In addition to this, the applied stimulations are observed to be close to or below the random seismic noise of the surroundings, which may suggest stochastic resonance is being employed. The stimulations also interact with the effects of nanometric patterning of the substrates on cell adhesion and Kruppel-like factor 2 and endothelin-1 expression thus linking cell reactions to nanotopographically patterned surfaces with those to mechanical stimulation.

Osteogenesis of mesenchymal stem cells by nanoscale mechanotransduction.

It is likely that mesenchymal stem cells will find use in many autologous regenerative therapies. However, our ability to control cell stem growth and differentiation is presently limited, and this is a major hurdle to the clinical use of these multipotent cells especially when considering the desire not to use soluble factors or complex media formulations in culture. Also, the large number of cells required to be clinically useful is currently a hurdle to using materials-based (stiffness, chemistry, nanotopography, etc.) culture substrates. Here we give a first demonstration of using nanoscale sinusoidal mechanotransductive protocols (10-14 nm displacements at 1 kHz frequency), "nanokicking", to promote osteoblastogenesis in human mesenchymal stem cell cultures. On the basis of application of the reverse piezo effect, we use interferometry to develop the optimal stem cell stimulation conditions, allowing delivery of nanoscale cues across the entire surface of the Petri dishes used. A combination of immunofluorescence, PCR, and microarray has then been used to demonstrate osteoblastogenesis, and the arrays implicate RhoA as central to osteoblastic differentiation in agreement with materials-based strategies. We validate this with pharmacological inhibition of RhoA kinase. It is easy to envisage such stimulation protocols being up-scaled to form large-scale osteoblast bioreactors as standard cell culture plates and incubators are used in the protocol.