Comparison of the efficiency of ultrafiltration, precipitation, and ultracentrifugation methods for exosome isolation.

Extracellular vesicles (EVs) are enclosed by a lipid-bilayer membrane and secreted by all types of cells. They are classified into three groups: apoptotic bodies, microvesicles, and exosomes. Exosomes play a number of important roles in the intercellular communication and crosstalk between tissues in the body. In this study, we use three common methods based on different principles for exosome isolation, namely ultrafiltration, precipitation, and ultracentrifugation. We use field emission scanning electron microscopy (FESEM) and dynamic light scattering (DLS) analyses for characterization of exosomes. The functionality and effect of isolated exosomes on the viability of hypoxic cells was investigated by alamarBlue and Flow-cytometry. The results of the FESEM study show that the ultrafiltration method isolates vesicles with higher variability of shapes and sizes when compared to the precipitation and ultracentrifugation methods. DLS results show that mean size of exosomes isolated by ultrafiltration, precipitation, and ultracentrifugation methods are 122, 89, and 60 nm respectively. AlamarBlue analysis show that isolated exosomes increase the viability of damaged cells by 11%, 15%, and 22%, respectively. Flow-cytometry analysis of damaged cells also show that these vesicles increase the content of live cells by 9%, 15%, and 20%, respectively. This study shows that exosomes isolated by the ultracentrifugation method are characterized by smaller size and narrow size distribution. Furthermore, more homogenous particles isolated by this method show increased efficiency of the protection of hypoxic cells in comparison with the exosomes isolated by the two other methods.

Advanced bioengineering of female germ cells to preserve fertility.

Oogenesis and folliculogenesis are considered as complex and species-specific cellular differentiation processes, which depend on the in vivo ovarian follicular environment and endocrine cues. Considerable efforts have been devoted to driving the differentiation of female primordial germ cells toward mature oocytes outside of the body. The recent experimental attempts have laid stress on offering a suitable microenvironment to assist the in vitro folliculogenesis and oogenesis. Despite developing a variety of bioengineering techniques and generating functional mature gametes through in vitro oogenesis in earlier studies, we still lack knowledge of appropriate microenvironment conditions for building biomimetic culture systems for female fertility preservation. Therefore, this review paper can provide a source for a large body of scientists developing cutting-edge in vitro culture systems for female germ cells or setting up the next generation of reproductive medicine as feasible options for female infertility treatment. The focal point of this review outlines advanced bioengineering technologies such as 3D biofabricated hydrogels/scaffolds and microfluidic systems utilized with female germlines for fertility preservation through in vitro folliculogenesis and oogenesis.

Advanced bioengineering of male germ stem cells to preserve fertility.

In modern life, several factors such as genetics, exposure to toxins, and aging have resulted in significant levels of male infertility, estimated to be approximately 18% worldwide. In response, substantial progress has been made to improve in vitro fertilization treatments (e.g. microsurgical testicular sperm extraction (m-TESE), intra-cytoplasmic sperm injection (ICSI), and round spermatid injection (ROSI)). Mimicking the structure of testicular natural extracellular matrices (ECM) outside of the body is one clear route toward complete in vitro spermatogenesis and male fertility preservation. Here, a new wave of technological innovations is underway applying regenerative medicine strategies to cell-tissue culture on natural or synthetic scaffolds supplemented with bioactive factors. The emergence of advanced bioengineered systems suggests new hope for male fertility preservation through development of functional male germ cells. To date, few studies aimed at in vitro spermatogenesis have resulted in relevant numbers of mature gametes. However, a substantial body of knowledge on conditions that are required to maintain and mature male germ cells in vitro is now in place. This review focuses on advanced bioengineering methods such as microfluidic systems, bio-fabricated scaffolds, and 3D organ culture applied to the germline for fertility preservation through in vitro spermatogenesis.

Effects of strontium ions with potential antibacterial activity on in vivo bone regeneration.

Bioactive glasses (BGs) have attracted added attention in the structure of the scaffolds for bone repair applications. Different metal ions could be doped in BGs to induce specific biological responses. Among these ions, strontium (Sr) is considered as an effective and safe doping element with promising effects on bone formation and regeneration. In this experiment, we evaluated the antibacterial activities of the gelatin-BG (Gel-BG) and Gel-BG/Sr scaffolds in vitro. The osteogenic properties of the prepared scaffolds were also assessed in rabbit calvarial bone defects for 12 weeks. Both scaffolds showed in vivo bone formation during 12 weeks with the newly formed bone area in Gel-BG/Sr scaffold was higher than that in Gel-BG scaffolds after the whole period. Based on the histological results, Gel-BG/Sr exhibited acceleration of early-stage bone formation in vivo. The results of antibacterial investigation for both scaffolds showed complete growth inhibition against Escherichia coli (E. coli). Although Gel-BG revealed no antibacterial effect on Staphylococcus aureus (S. aureus), the Gel-BG/Sr was able to partially inhibit the growth of S. aureus, as detected by threefold reduction in growth index. Our results confirmed that Sr doped BG is a favorable candidate for bone tissue engineering with superior antibacterial activity and bone regeneration capacity compared with similar counterparts having no Sr ion.

Microfluidic-Based Droplets for Advanced Regenerative Medicine: Current Challenges and Future Trends.

Microfluidics is a promising approach for the facile and large-scale fabrication of monodispersed droplets for various applications in biomedicine. This technology has demonstrated great potential to address the limitations of regenerative medicine. Microfluidics provides safe, accurate, reliable, and cost-effective methods for encapsulating different stem cells, gametes, biomaterials, biomolecules, reagents, genes, and nanoparticles inside picoliter-sized droplets or droplet-derived microgels for different applications. Moreover, microenvironments made using such droplets can mimic niches of stem cells for cell therapy purposes, simulate native extracellular matrix (ECM) for tissue engineering applications, and remove challenges in cell encapsulation and three-dimensional (3D) culture methods. The fabrication of droplets using microfluidics also provides controllable microenvironments for manipulating gametes, fertilization, and embryo cultures for reproductive medicine. This review focuses on the relevant studies, and the latest progress in applying droplets in stem cell therapy, tissue engineering, reproductive biology, and gene therapy are separately evaluated. In the end, we discuss the challenges ahead in the field of microfluidics-based droplets for advanced regenerative medicine.

Effects of collagen/ß-tricalcium phosphate bone graft to regenerate bone in critically sized rabbit calvarial defects.

Bone defects remain a significant health issue and a major cause of morbidity in elderly patients. Composites based on collagen/calcium phosphate have been widely used for bone repair in clinical applications, owing to their comparability to bone extracellular matrix. This study aimed to evaluate the effects of a scaffold of collagen/calcium phosphate (COL/ß-TCP) on bone formation to assess its potential use as a bone substitute to repair bone defects. Bilateral full-thickness critically sized calvarial defects (8 mm in diameter) were created in New Zealand white rabbits and treated with COL/ß-TCP or COL scaffolds. One defect was also left unfilled as a control. Bone regeneration was assessed through histological evaluation using hematoxylin and eosin and Masson's trichrome staining after 4 and 8 weeks. Alizarin Red staining was also utilized to observe the mineralization process. Our findings indicated that COL/ß-TCP implantation could better enhance bone regeneration than COL and exhibited both new bone growth and scaffold material degradation.

Panax ginseng Extract Improves Follicular Development after Mouse Preantral Follicle 3D Culture.

OBJECTIVE: Panax ginseng is a popular traditional herb that has been used in complementary and alternative medicine in eastern Asia, and it possesses pharmacologically active compounds like ginsenosides (GSs). This study aimed to investigate the impact of Panax ginseng extract (PGE) at different concentrations on in vitro follicular function and development in a three-dimensional (3D) culture system fabricated using sodium alginate after 12 days of culture. MATERIALS AND METHODS: In this experimental study, preantral follicles (n=661) were mechanically isolated from the ovaries of 14-day-old female NMRI mice using 29-gauge insulin syringes. Follicles were individually capsulated within sodium alginate, and divided into four groups including control and experimental groups 1, 2, and 3. Then, they were cultured for 12 days in the medium supplemented with different concentrations of PGE (0, 50, 100, and 500 µg/ mL, for control groups and groups 1, 2 and 3, respectively). At the end of the culture period, the mean diameter and maturation of follicles, follicular steroid production, mRNA expression level of proliferating cell nuclear antigen (PCNA) and follicle stimulating hormone receptor (FSH-R), and reactive oxygen species (ROS) levels in collected metaphase-II (MII) oocytes were determined. RESULTS: The mean diameter of follicles in group 2 was significantly increased as compared to other groups (P<0.001). The percentages of the survival and maturation rate and levels of secreted hormones were higher in group 2 than the other groups (P<0.05). Follicles cultured in the presence of PGE 100 µg/mL had higher levels of proliferation cell nuclear antigen (PCNA) and follicle stimulating hormone receptor (FSH-R) mRNA expression in comparison to other groups (P<0.05). Moreover, oocytes collected from groups 2 and 3 had lower levels of ROS as compared to other groups (P<0.05). CONCLUSION: Our results suggest that PGE at the concentration of 100 µg/mL induces higher follicular function and development in the 3D culture system.

Development of a bioactive porous collagen/ß-tricalcium phosphate bone graft assisting rapid vascularization for bone tissue engineering applications.

We developed collagen (COL) and collagen/beta tricalcium phosphate (COL/ß-TCP) scaffolds with a ß-TCP/collagen weight ratio of 4 by freeze-drying. Mouse bone marrow-derived mesenchymal stem cells (BMMSCs) were cultured on these scaffolds for 14 days. Samples were characterized by physicochemical analyses and their biological properties such as cell viability and alkaline phosphatase (ALP) activity was, also, examined. Additionally, the vascularization potential of the prepared scaffolds was tested subcutaneously in Wistar rats. We observed a microporous structure with large porosity (∼95-98%) and appropriate pore size (120-200 µm). The COL/ß-TCP scaffolds had a much higher compressive modulus (970 ± 1.20 KPa) than pure COL (0.8 ± 1.82 KPa). In vitro model of apatite formation was established by immersing the composite scaffold in simulated body fluid for 7 days. An ALP assay revealed that porous COL/ß-TCP can effectively activate the differentiation of BMMSCs into osteoblasts. The composite scaffolds also promoted vascularization with good integration with the surrounding tissue. Thus, introduction of ß-TCP powder into the porous collagen matrix effectively improved the mechanical and biological properties of the collagen scaffolds, making them potential bone substitutes for enhanced bone regeneration in orthopedic and dental applications. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 73-85, 2018.

Three-dimensional wet-electrospun poly(lactic acid)/multi-wall carbon nanotubes scaffold induces differentiation of human menstrual blood-derived stem cells into germ-like cells.

Infertility caused by the disruption or absence of germ cells is a major and largely incurable medical problem. Germ cells (i.e., sperm or egg) play a key role in the transmission of genetic and epigenetic information across generations. Generation of gametes derived in vitro from stem cells hold promising prospects which could potentially help infertile men and women. Menstrual blood-derived stem cells are a unique stem cell source. Evidence suggests that menstrual blood-derived stem cells exhibit a multi-lineage potential and have attracted extensive attention in regenerative medicine. To maintain the three-dimensional structure of natural extra cellular matrices in vitro, scaffolds can do this favor and mimic a microenvironment for cell proliferation and differentiation. According to previous studies, poly(lactic acid) and multi-wall carbon nanotubes have been introduced as novel and promising biomaterials for the proliferation and differentiation of stem cells. Some cell types have been successfully grown on a matrix containing carbon nanotubes in tissue engineering but there is no report for this material to support stem cells differentiation into germ cells lineage. This study designed a 3D wet-electrospun poly(lactic acid) and poly(lactic acid)/multi-wall carbon nanotubes composite scaffold to compare infiltration, proliferation, and differentiation potential of menstrual blood-derived stem cells toward germ cell lineage with 2D culture. Our primary data revealed that the fabricated scaffold has mechanical and biological suitable qualities for supporting and attachments of stem cells. The differentiated menstrual blood-derived stem cells tracking in scaffolds using scanning electron microscopy confirmed cell attachment, aggregation, and distribution on the porous scaffold. Based on the differentiation assay by RT-PCR analysis, stem cells and germ-like cells markers were expressed in 3D groups as well as 2D one. It seems that poly(lactic acid)/multi-wall carbon nanotubes scaffold-seeded menstrual blood-derived stem cells could be viewed as a novel, safe, and accessible construct for these cells, as they enhance germ-like generation from menstrual blood-derived stem cells.

Cryptosporidiosis in Iranian Farm Workers and Their Household Members: A Hypothesis about Possible Zoonotic Transmission.

Objectives. The prevalence of Cryptosporidium and the risk factors of zoonotic transmission in Najafabad, Isfahan, Iran dairy farms were examined. Methods. One fecal sample was collected from all calves less than 6 months old in eight dairy farms around Najafabad (Isfahan province, Central Iran) as well as individuals working in these farms and their household members. A two-step nested PCR protocol was used to amplify the 18S rRNA gene (830 bp). Results. Cryptosporidium was identified in the stool of 36 (prevalence 8.5%) of 96 farm workers and 326 household members. Furthermore, 31 (14.2%) of 218 calf samples were positive. Cryptosporidium parvum was identified in 15 (72%) of the positive farm workers and 10 (65%) of the positive household members. Of the positive calves, 20 (64.5%) were infected with C. parvum, indicating possible zoonotic transmission in these farms. Contact with calves (P < 0.0001) was the most significant risk factor of C. parvum infection. A considerable negative association was observed between C. parvum infection and cleaning of shoes/boots after daily work (P = 0.004), hand washing (P = 0.013), and use of piped water (P < 0.006). In the multivariate analysis with logistic regression, only contact with calves was significant. Conclusion. Zoonotic transmission of C. parvum due to contact with calves is predominant among farm workers and their household members of this region and appropriate health measures must be applied to control the infection and decrease of zoonotic transmission of this parasite.

Prevalence, molecular characteristics and risk factors for cryptosporidiosis among Iranian immunocompromised patients.

Cryptosporidium spp. is a major cause of diarrhea in developing countries, mainly affecting people with compromised immune systems in general and HIV-infected individuals with low CD4 + T-cell counts in particular. This infection is self-limiting in healthy persons; however, it can be severe, progressive and persistent in those who are immunocompromised. There are few published studies concerning cryptosporidiosis and Cryptosporidium genotypes in Iranian immunocompromised patients and none of them describe risk factors. This study was undertaken to identify prevalence, genotypes and risk factors for cryptosporidiosis in immunocompromised patients. Three fecal samples were obtained at two day intervals from each of the 183 patients and processed with modified Ziehl-Neelsen staining methods and 18S rRNA gene amplification and sequencing. The overall infection prevalence was 6%. Cryptosporidium parvum was identified in isolates from five HIV-infected patients, one patient who had undergone bone marrow transplantation and one with chronic lymphocytic leukemia. Cryptosporidium hominis was identified in isolates from two HIV-infected patients and two patients with acute lymphocytic leukemia. According to univariate analysis, the statistically significant factors were diarrhea (OR = 21.7, CI = 2.83-78.4, P= 0.003), CD4 + lymphocytes less than 100 cells/mm(3) (OR = 41.3, CI = 13.45-114.8, P < 0.0001), other microbial infections (OR = 7.1321.7, CI = 1.97-25.73, P = 0.006), weight loss (OR = 73.78, CI = 15.5-350, P < 0.0001), abdominal pain (OR = 10.29, CI = 2.81-37.74.4, P= 0.001), dehydration (OR = 72.1, CI = 17.6-341.5, P < 0.0001), vomiting (OR = 4.87, CI = 1.4-16.9, P= 0.015), nausea (OR = 9.4, CI = 2.38-37.2, P < 0.001), highly active antiretroviral therapy (OR = 0.089, CI = 0.01-0.8, P= 0.015) and diarrhea in household members (OR = 7.37, CI = 2.04-26.66, P= 0.001). After multivariate analysis and a backward deletion process, only < 100 CD4 + T-lymphocytes/mm(3) maintained a significant association with infection. The authors recommend that this infection should be suspected in patients with diarrhea, weight loss and dehydration in general and in diarrheal individuals with < 100 CD4 + T-lymphocytes/mm(3).