Cytocompatibility of electrospun poly-L-lactic acid membranes for Bruch's membrane regeneration using human embryonic stem cell-derived retinal pigment epithelial cells.

Cell replacement therapy is under development for dry age-related macular degeneration (AMD). A thin membrane resembling the Bruch's membrane is required to form a cell-on-membrane construct with retinal pigment epithelial (RPE) cells. These cells have been differentiated from human embryonic stem cells (hESCs) in vitro. A carrier membrane is required for cell implantation, which is biocompatible for cell growth and has dimensions and physical properties resembling the Bruch's membrane. Here a nanofiber electrospun poly-L-lactic acid (PLLA) membrane is tested for capacity to support cell growth and maturation. The requirements for laminin coating of the membrane are identified here. A porous electrospun nanofibrous PLLA membrane of ∼50 nm fiber diameter was developed as a prototype support for functional RPE cells grown as a monolayer. The need for laminin coating applied to the membrane following treatment with poly-L-ornithine (PLO), was identified in terms of cell growth and survival. Test membranes were compared in terms of hydrophilicity after laminin coating, mechanical properties of surface roughness and Young's modulus, porosity and ability to promote the attachment and proliferation of hESC-RPE cells in culture for up to 8 weeks. Over this time, RPE cell proliferation, morphology, and marker and gene expression, were monitored. The functional capacity of cell monolayers was identified in terms of transepithelial electrical resistance (TEER), phagocytosis of cells, as well as expression of the cytokines, vascular endothelial growth factor (VEGF) and pigment epithelium-derived factor (PEDF). PLLA polymer fibers are naturally hydrophobic, so their hydrophilicity was improved by pretreatment with PLO for subsequent coating with the bioactive protein laminin. They were then assessed for amount of laminin adsorbed, contact angle and uniformity of coating using scanning electron microscopy (SEM). Pretreatment with 100% PLO gave the best result over 10% PLO treatment or no treatment prior to laminin adsorption with significantly greater surface stiffness and modulus. By 6 weeks after cell plating, the coated membranes could support a mature RPE monolayer showing a dense apical microvillus structure and pigmented 3D polygonal cell morphology. After 8 weeks, PLO (100%)-Lam coated membranes exhibited the highest cell number, cell proliferation, and RPE barrier function measured as TEER. RPE cells showed the higher levels of specific surface marker and gene expression. Microphthalmia-associated transcription factor expression was highly upregulated indicating maturation of cells. Functionality of cells was indicated by expression of VEGF and PEDF genes as well as phagocytic capacity. In conclusion, electrospun PLLA membranes coated with PLO-Lam have the physical and biological properties to support the distribution and migration of hESC-RPE cells throughout the whole structure. They represent a good membrane candidate for preparation of hESC-RPE cells as a monolayer for implantation into the subretinal space of AMD patients.

The effect of body mass index on the morbidity and outcomes of COVID-19 in Iranian patients with multiple sclerosis.

Background: It seems that patients with multiple sclerosis (MS) are at a higher risk for coronavirus disease 2019 (COVID-19) implications due to being subjected to immunomodulatory or immunosuppressive treatments. Besides, obesity as a risk factor may lead to more adverse consequences. The relationship between obesity and COVID-19 morbidity and outcomes in Iranian patients with MS still remains unclear. Methods: A cross-sectional study was conducted in Sina Hospital, Tehran, Iran. Patients with MS were asked to complete an online questionnaire in the Google Form format. Demographic information, clinical information including MS disease-related factors, COVID-19-related factors, and anthropometric information were recorded. Totally, 492 patients filled the questionnaire during two weeks in November 2021, by the response rate of 21.6%. Body mass index (BMI) was categorized based on the standard classification of the World Health Organization (WHO). The logistic regression was used to examine the risk of morbidity and chi-square test/one-way analysis of variance (ANOVA) was employed to determine the difference regarding severity and symptoms among groups. Results: In the fully adjusted model, the odds ratio (OR) of COVID-19 morbidity in class II obese participants was significantly 5.41 times higher than that in the normal BMI group [OR: 5.41, 95% confidence interval (CI): 1.00-29.09]. COVID-19 severity was significantly different among BMI groups (P = 0.024). Respiratory symptoms (P = 0.05) as well as gastrointestinal (GI) symptoms (P < 0.01) of COVID-19 were more prevalent among class I and class II obese patients compared with overweight, normal weight, and underweight groups. Moreover, no one in the class I and class II obesity groups reported COVID-19 morbidity without any symptoms (P = 0.04). Conclusion: The results of the current study support the view that obesity could play a key role in susceptibility to COVID-19 morbidity and severity of the symptoms in patients with MS. The findings recommended that neurologists pay more attention to patients' BMI during this pandemic.

COVID-19 vaccine hesitancy in Iranian patients with multiple sclerosis.

BACKGROUND: World Health Organization (WHO) mentioned COVID-19 vaccination as the safest way to eradicate this pandemic. In the meantime, vaccine hesitancy (a delay in accepting or rejecting the vaccine despite the availability of vaccination services) is a barrier. Hence, we studied this obstacle in the Iranian multiple sclerosis (MS) population. OBJECTIVE: MS patients eligible for vaccination were asked to complete a google form survey. Demographic information, MS disease-related factors, flu vaccination history, COVID-19 vaccination history, cause of vaccination refusal, past history of COVID-19 infection, and their compliance with public health guidelines after vaccination were recorded. RESULTS: 1479 patients participated in this study. 6.9% of participants have not received the vaccination. Sinopharm was the most commonly used vaccine (92.9%). Vaccine hesitancy was associated with young age, lower education, unemployment, negative flu vaccination history, no previous episode of COVID-19 infection, less concern about COVID-19, and the expectation of not getting infected with the virus after vaccination. Participants mentioned concerns about the side effects of the vaccines as the most prevalent cause of avoiding vaccination (58.0%). Patients' concern of SARS-CoV-2 significantly decreased after vaccination (p-value < 0.001). CONCLUSION: Our findings in this study elucidate that a minor group of patients with MS has vaccine hesitancy, which may expose them to more severe COVID 19. The treating physicians should ask the history of vaccination and try to persuade such patients with scientific knowledge transformation. The long-term consequences of not being vaccinated should be clarified to such patients especially those who are receiving immunosuppressive agents.

Acupuncture for multiple sclerosis: A literature review.

BACKGROUND: Acupuncture as a complementary and alternative medicine (CAM) modality appears to be a helpful integrative therapy for multiple sclerosis (MS). Due to the chronicity of the disease and persistent symptoms, a large number of patients seek to use CAM for the MS treatment. Therefore, the present review aimed to determine the effectiveness of acupuncture in the treatment of multiple sclerosis. METHODS: PUBMED database was searched for English articles (at least English abstracts) in September 2021, including all articles published since the earliest literature until September 2021. Review articles were searched for relevant data. The searched keywords in titles and abstracts included ((acupuncture) OR (electroacupuncture)) AND (multiple sclerosis). RESULTS: Totally, out of 75 studied articles, 31 were included in this research. The advantages of acupuncture are mainly reflected in regulating neuro-immune system, improving the quality of life, reducing fatigue, improving the bladder function, reducing the spasm and pain of the limbs, delaying the progression of the disease, and reducing relapses. CONCLUSIONS: According to the review of the recent articles, traditional Chinese acupuncture and scalp acupuncture appear to help improve the symptoms of multiple sclerosis (including fatigue, neural functional deficits, pain, gait impairments, and bladder dysfunction) and reduce relapses. Therefore, acupuncture could be used as an integrative therapy in patients with MS.

In vivo bone regeneration assessment of offset and gradient melt electrowritten (MEW) PCL scaffolds.

BACKGROUND: Biomaterial-based bone tissue engineering represents a promising solution to overcome reduced residual bone volume. It has been previously demonstrated that gradient and offset architectures of three-dimensional melt electrowritten poly-caprolactone (PCL) scaffolds could successfully direct osteoblast cells differentiation toward an osteogenic lineage, resulting in mineralization. The aim of this study was therefore to evaluate the in vivo osteoconductive capacity of PCL scaffolds with these different architectures. METHODS: Five different calcium phosphate (CaP) coated melt electrowritten PCL pore sized scaffolds: 250 µm and 500 µm, 500 µm with 50% fibre offset (offset.50.50), tri layer gradient 250-500-750 µm (grad.250top) and 750-500-250 µm (grad.750top) were implanted into rodent critical-sized calvarial defects. Empty defects were used as a control. After 4 and 8 weeks of healing, the new bone was assessed by micro-computed tomography and immunohistochemistry. RESULTS: Significantly more newly formed bone was shown in the grad.250top scaffold 8 weeks post-implantation. Histological investigation also showed that soft tissue was replaced with newly formed bone and fully covered the grad.250top scaffold. While, the bone healing did not happen completely in the 250 µm, offset.50.50 scaffolds and blank calvaria defects following 8 weeks of implantation. Immunohistochemical analysis showed the expression of osteogenic markers was present in all scaffold groups at both time points. The mineralization marker Osteocalcin was detected with the highest intensity in the grad.250top and 500 µm scaffolds. Moreover, the expression of the endothelial markers showed that robust angiogenesis was involved in the repair process. CONCLUSIONS: These results suggest that the gradient pore size structure provides superior conditions for bone regeneration.

Role of offset and gradient architectures of 3-D melt electrowritten scaffold on differentiation and mineralization of osteoblasts.

BACKGROUND: Cell-scaffold based therapies have the potential to offer an efficient osseous regenerative treatment and PCL has been commonly used as a scaffold, however its effectiveness is limited by poor cellular retention properties. This may be improved through a porous scaffold structure with efficient pore arrangement to increase cell entrapment. To facilitate this, melt electrowriting (MEW) has been developed as a technique able to fabricate cell-supporting scaffolds with precise micro pore sizes via predictable fibre deposition. The effect of the scaffold's architecture on cellular gene expression however has not been fully elucidated. METHODS: The design and fabrication of three different uniform pore structures (250, 500 and 750 µm), as well as two offset scaffolds with different layout of fibres (30 and 50%) and one complex scaffold with three gradient pore sizes of 250-500 - 750 µm, was performed by using MEW. Calcium phosphate modification was applied to enhance the PCL scaffold hydrophilicity and bone inductivity prior to seeding with osteoblasts which were then maintained in culture for up to 30 days. Over this time, osteoblast cell morphology, matrix mineralisation, osteogenic gene expression and collagen production were assessed. RESULTS: The in vitro findings revealed that the gradient scaffold significantly increased alkaline phosphatase activity in the attached osteoblasts while matrix mineralization was higher in the 50% offset scaffolds. The expression of osteocalcin and osteopontin genes were also upregulated compared to other osteogenic genes following 30 days culture, particularly in offset and gradient scaffold structures. Immunostaining showed significant expression of osteocalcin in offset and gradient scaffold structures. CONCLUSIONS: This study demonstrated that the heterogenous pore sizes in gradient and fibre offset PCL scaffolds prepared using MEW significantly improved the osteogenic potential of osteoblasts and hence may provide superior outcomes in bone regeneration applications.

Global, Regional, and National Cancer Incidence, Mortality, Years of Life Lost, Years Lived With Disability, and Disability-Adjusted Life-Years for 29 Cancer Groups, 1990 to 2017: A Systematic Analysis for the Global Burden of Disease Study.

Importance: Cancer and other noncommunicable diseases (NCDs) are now widely recognized as a threat to global development. The latest United Nations high-level meeting on NCDs reaffirmed this observation and also highlighted the slow progress in meeting the 2011 Political Declaration on the Prevention and Control of Noncommunicable Diseases and the third Sustainable Development Goal. Lack of situational analyses, priority setting, and budgeting have been identified as major obstacles in achieving these goals. All of these have in common that they require information on the local cancer epidemiology. The Global Burden of Disease (GBD) study is uniquely poised to provide these crucial data. Objective: To describe cancer burden for 29 cancer groups in 195 countries from 1990 through 2017 to provide data needed for cancer control planning. Evidence Review: We used the GBD study estimation methods to describe cancer incidence, mortality, years lived with disability, years of life lost, and disability-adjusted life-years (DALYs). Results are presented at the national level as well as by Socio-demographic Index (SDI), a composite indicator of income, educational attainment, and total fertility rate. We also analyzed the influence of the epidemiological vs the demographic transition on cancer incidence. Findings: In 2017, there were 24.5 million incident cancer cases worldwide (16.8 million without nonmelanoma skin cancer [NMSC]) and 9.6 million cancer deaths. The majority of cancer DALYs came from years of life lost (97%), and only 3% came from years lived with disability. The odds of developing cancer were the lowest in the low SDI quintile (1 in 7) and the highest in the high SDI quintile (1 in 2) for both sexes. In 2017, the most common incident cancers in men were NMSC (4.3 million incident cases); tracheal, bronchus, and lung (TBL) cancer (1.5 million incident cases); and prostate cancer (1.3 million incident cases). The most common causes of cancer deaths and DALYs for men were TBL cancer (1.3 million deaths and 28.4 million DALYs), liver cancer (572 000 deaths and 15.2 million DALYs), and stomach cancer (542 000 deaths and 12.2 million DALYs). For women in 2017, the most common incident cancers were NMSC (3.3 million incident cases), breast cancer (1.9 million incident cases), and colorectal cancer (819 000 incident cases). The leading causes of cancer deaths and DALYs for women were breast cancer (601 000 deaths and 17.4 million DALYs), TBL cancer (596 000 deaths and 12.6 million DALYs), and colorectal cancer (414 000 deaths and 8.3 million DALYs). Conclusions and Relevance: The national epidemiological profiles of cancer burden in the GBD study show large heterogeneities, which are a reflection of different exposures to risk factors, economic settings, lifestyles, and access to care and screening. The GBD study can be used by policy makers and other stakeholders to develop and improve national and local cancer control in order to achieve the global targets and improve equity in cancer care.

Effects of Gradient and Offset Architectures on the Mechanical and Biological Properties of 3-D Melt Electrowritten (MEW) Scaffolds.

This study describes the fabrication and characterization of three-dimensional (3-D) poly(ε-caprolactone) (PCL) scaffolds with defined pore architectures prepared using the melt electrowriting (MEW) technique. Three homogeneous pore-sized (250, 500, and 750 µm) scaffolds, two fiber offset (30/70% and 50/50%), and a three-layered (250 µm bottom-500 µm middle-750 µm top) gradient pore-sized scaffolds were designed and printed with ∼10 µm fibers. The mechanical properties (tensile and compression tests), total surface area, porosity of these scaffolds, and their ability to promote the attachment and proliferation of human osteoblasts were then compared. All scaffolds induced good tensile properties; however, they reacted differently during compressive testing. The offset 30/70 scaffold had the highest surface area to volume ratio which enhanced osteoblast attachment after 3 days of cell culture. While the highest initial level of osteoblast attachment at day 1 was found on the 250 µm homogeneous scaffold, the highest degree of cell proliferation and infiltration at day 30 was observed in the three-layered graded porosity scaffold. In terms of physical and biological properties to support bone cell distribution and migration through the entire structure of the scaffold, our results suggest that melt electrowritten offset and gradient scaffolds are good candidate platforms for cell infiltration and growth compared to homogeneous scaffolds.

Influence of oriented nanofibrous PCL scaffolds on quantitative gene expression during neural differentiation of mouse embryonic stem cells.

Neural differentiation of mouse embryonic stem cells in combination with three-dimensional electrospun nanofibers as an artificial extracellular matrix can be utilized to reconstruct a spinal cord defect. In this study, random and parallel-aligned nanofibrous poly ɛ-caprolactone was fabricated using electrospinning. Its hydrophobicity was modified by O2 plasma treatment to facilitate enhanced cell attachment. Embryoid bodies (EBs), which contain all three embryonic germ layers, were cultured on poly ɛ-caprolactone scaffolds to study the effect of fiber orientation on cell morphology and differentiation. Cell morphology and neuron-specific gene and protein expressions were, respectively, evaluated by scanning electron microscopy, real-time polymerase chain reaction, and immunocytochemistry. Although two types of nanofibrous scaffolds showed neural marker expression at the protein level, cells on randomly oriented scaffolds showed short-range topographical guidance and stretched across multiple directions, whereas cells on the parallel scaffolds exhibited long extension with enhanced neuron outgrowth along the fiber, producing oriented extracellular matrix, leading to direct cell migration and nerve regeneration. Quantitative real-time polymerase chain reaction showed that both aligned and random electrospun nanofibers downregulated the precursor neural marker Nestin compared with that in the control group, a gelatin-coated tissue culture plate (T). Analysis also showed higher expression of dorso-ventral neural markers (Isl1/2 and Lim1/2) than motor neuron progenitor markers (Pax6, Nkx6.1, and olig2) in aligned nanofibers than in the T group. Moreover, aligned nanofibers showed higher expression of mature neural specific markers such as ß-tub and Map2 than those in the randomly oriented scaffolds. Therefore, we conclude that nanofibers with different orientations can support the neural lineage, but aligned nanofibrous scaffolds are superior candidates to promote the advancement of neural precursors to achieve maturity during the differentiation process.

Controlled surface morphology and hydrophilicity of polycaprolactone toward selective differentiation of mesenchymal stem cells to neural like cells.

Differentiation of mesenchymal stem cells (MSCs) into neuron cells has great potential in therapy of damaged nerve tissue. It has been shown that three-dimensional biomaterials have great ability to up regulate the expression of neuronal proteins. In this study, O2 plasma technology was used to enhance hydrophilicity of poly (ε-caprolactone) (PCL) toward selective differentiation of MSCs into neural cells. Random and aligned PCL nanofibers scaffolds were fabricated by electrospinning method and their physicochemical and mechanical properties were carried out by scanning electron microscope (SEM), contact angle, and tensile measurements. Contact angle studies of PCL and plasma treated PCL (p-PCL) nanofibers revealed significant change on the surface properties PCL nanofibers from the view point of hydrophilicity. Physiochemical studies revealed that p-PCL nanofibers were extremely hydrophilic compared with untreated PCL nanofibers which were highly hydrophobic and nonabsorbent to water. Differentiation of MSCs were carried out by inducing growth factors including basic fibroblast growth factor, nerve growth factor, and brain derived growth factor, NT3, 3-isobutyl-1-methylxanthine (IBMX) in Dulbecco's modified Eagle's medium/F12 media. Differentiated MSCs on nanofibrous scaffold were examined by immunofluorescence assay and was found to express the neuronal proteins; ß-tubulin III and Map2, on day 15 after cell culture. The real-time polymerase chain reaction (RT-PCR) analysis showed that p-PCL nanofibrous scaffold could upregulate expression of Map-2 and downregulate expression of Nestin genes in nerve cells differentiated from MSCs. This study indicates that mesenchymal stem cell cultured on nanofibrous scaffold have potential differentiation to neuronal cells on and could apply in nerve tissue repair.

The Effects of Plasma Treated Electrospun Nanofibrous Poly (ε-caprolactone) Scaffolds with Different Orientations on Mouse Embryonic Stem Cell Proliferation.

OBJECTIVE: Assessments of cell reactions such as motility, orientation and activation to the topography of the substratum will assist with the fabrication of a proper implantable scaffold for future tissue engineering applications.The current challenge is to analyze the orientation effect of elecrospun nanofibers of poly (ε-caprolactone) (PCL) on viability and proliferation of mouse embryonic stem cells (mESCs). MATERIALS AND METHODS: In this experimental study, we used the electrospinning method to fabricate nanofibrous PCL scaffolds. Chemical and mechanical characterizations were specified by the contact angle and tensile test. O2plasma treatment was used to improve surface hydrophilicity. We used the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to evaluate mESCs adhesion and proliferation before and after surface modification. The influence of the orientation of the nanofibers on mESCs growth was evaluated by scanning electron microscopy (SEM). Statistical analysis was performed using one-way analysis of variance (ANOVA) With differences considered statistically significant at p≤0.05. RESULTS: The results showed that plasma treatment improved the hydrophilic property of PCL scaffolds. MTT assay showed a significant increase in proliferation of mESCs on plasma treated PCL (p-PCL) scaffolds compared to non-treated PCL (p=0.05). However gelatin coated tissue culture plate (TCP) had a better effect in initial cell attachment after one day of cell seeding. There was more cell proliferation on day 3 in aligned plasma treated (AP) nanofibers compared to the TCP. SEM showed optical density of the cell colonies. Aligned nanofibrous scaffolds had larger colony sizes and spread more than random nanofibrous scaffolds. CONCLUSION: This study showed that plasma treating of scaffolds was a more suitable substrate for growth and cell attachment. In addition, aligned nanofibrous scaffolds highly supported the proliferation and spreading of mESCs when compared to random nanofibrous scaffolds and TCP.