MMP inhibition as a novel strategy for extracellular matrix preservation during whole liver decellularization.

As the only reliable treatment option for end-stage liver diseases, conventional liver transplantation confronts major supply limitations. Accordingly, the decellularization of discarded livers to produce bioscaffolds that support recellularization with progenitor/stem cells has emerged as a promising translational medicine approach. The success of this approach will substantially be determined by the extent of extracellular matrix (ECM) preservation during the decellularization process. Here, we assumed that the matrix metalloproteinase (MMP) inhibition could reduce the ECM damage during the whole liver decellularization of an animal model using a perfusion-based system. We demonstrated that the application of doxycycline as an MMP inhibitor led to significantly higher preservation of collagen, glycosaminoglycans, and hepatic growth factor (HGF) contents, as well as mechanical and structural features, including tensile strength, fiber integrity, and porosity. Notably, produced bioscaffolds were biocompatible and efficiently supported cell viability and proliferation in vitro. We also indicated that produced bioscaffolds efficiently supported HepG2 cell function upon seeding onto liver ECM discs using albumin and urea assay. Additionally, MMP inhibitor pretreated decellularized livers were more durable in contact with collagenase digestion compared to control bioscaffolds in vitro. Using zymography, we confirmed the underlying mechanism that results in these promising effects is through the inhibition of MMP2 and MMP9. Overall, we demonstrated a novel method based on MMP inhibition to ameliorate the ECM structure and composition preservation during liver decellularization as a critical step in fabricating transplantable bioengineered livers.

Design, implementation, and validation of an application for childbirth preparation classes to improve the performance of midwifery students: Study protocol.

BACKGROUND: Childbirth preparation classes are incredibly useful for midwifery students as future medical personnel. Nowadays, given the outbreak of Covid-19 pandemic and as mobile applications are extensively welcomed, virtual space can be used for education in the area of childbirth preparation classes. Given the lack of an application for childbirth preparation classes, this study will be conducted to design, implement and validate such an application to improve the performance of midwifery students in the areas of pregnancy and safe delivery. MATERIALS AND METHODS: The present study will be conducted in three phases. In the first phase, content will be provided to Information Technology experts based on the content of the national guidelines for physiological delivery in Iran, and the application will be designed and validated for the use of midwifery students, then develop app for other medical students, midwives and physicians. In the second phase, the assessment will be based on Kirkpatrick's model. In the third stage, develop app for other medical students, midwives and physicians based on the results of the first and second phase. SPSS version 17 will be used in this phase for analysis of data through descriptive and analytical tests. CONCLUSION: Owing to the expansion of virtual space and the outbreak of Covid-19 pandemic, design, validation, and evaluation of an application for childbirth preparation classes is an exceptionally significant necessity which contributes to the process of educating midwifery students.

Wet-spinnability and crosslinked Fiber properties of alginate/hydroxyethyl cellulose with varied proportion for potential use in tendon tissue engineering.

Researchers have examined different bio-inspired materials in tissue engineering and regenerative medicine to fabricate scaffolds to address tendon regeneration requirements. We developed fibers based on alginate (Alg) and hydroxyethyl cellulose (HEC) by wet-spinning technique to mimic the fibrous sheath of ECM. Various proportions (25:75, 50:50, 75:25) of 1 % Alg and 4 % HEC were blended to this aim. Two steps of crosslinking with different concentrations of CaCl2 (2.5 and 5 %) and glutaraldehyde (2.5 %) were used to improve physical and mechanical properties. The fibers were characterized by FTIR, SEM, swelling, degradation, and tensile tests. The in vitro proliferation, viability, and migration of tenocytes on the fibers were also evaluated. Moreover, the biocompatibility of implanted fibers was investigated in an animal model. The results showed ionic and covalent molecular interactions between the components. In addition, by properly maintaining surface morphology, fiber alignment, and swelling, lower concentrations of HEC in the blending provided good degradability and mechanical features. The mechanical strength of fibers was in the range of collagenous fibers. Increasing the crosslinking led to significantly different mechanical behaviors in terms of tensile strength and elongation at break. Because of good in vitro and in vivo biocompatibility, tenocyte proliferation, and migration, the biological macromolecular fibers could serve as desirable tendon substitutes. This study provides more practical insight into tendon tissue engineering in translational medicine.

Effects of Different Perfusing Routes through The Portal Vein, Hepatic Vein, and Biliary Duct on Whole Rat Liver Decellularization.

OBJECTIVE: Organ transplantation is the last therapeutic choice for end-stage liver failure, which is limited by the lack of sufficient donors. Decellularized liver can be used as a suitable matrix for liver tissue engineering with clinical application potential. Optimizing the decellularization procedure would obtain a biological matrix with completely removed cellular components and preserved 3-dimensional structure. This study aimed to evaluate the decellularization efficacy through three anatomical routes. MATERIALS AND METHODS: In this experimental study, rat liver decellularization was performed through biliary duct (BD), portal vein (PV), and hepatic vein (HV); using chemical detergents and enzymes. The decellularization efficacy was evaluated by measurement of DNA content, extracellular matrix (ECM) total proteins, and glycosaminoglycans (GAGs). ECM preservation was examined by histological and immunohistochemical (IHC) staining and scanning electron microscopy (SEM). Scaffold biocompatibility was tested by the MTT assay for HepG2 and HUVEC cell lines. RESULTS: Decellularization through HV and PV resulted in a transparent scaffold by complete cell removal, while the BD route produced an opaque scaffold with incomplete decellularization. H and E staining confirmed these results. Maximum DNA loss was obtained using 1% and 0.5% sodium dodecyl sulfate (SDS) in the PV and HV groups and the DNA content decreased faster in the HV group. At the final stages, the proteins excreted in the HV and PV groups were significantly less than the BD group. The GAGs level was diminished after decellularization, especially in the PV and HV groups. In the HV and PV groups the collagen amount was significantly more than the BD group. The IHC and SEM images showed that the ECM structure was preserved and cellular components were entirely removed. MTT assay showed the biocompatibility of the decellularized scaffold. CONCLUSION: The results revealed that the HV is a more suitable route for liver decellularization than the PV and BD.

Osteogenic Differentiation Effect of BMP-9 with Phenamil and Simvastatin on Intact Human Amniotic Epithelial Stem Cells

Background: Background: Bone tissue engineering has shown to be a promising strategy for repairing bone defects without causing harmful side effects to the patient. Three main building blocks of tissue engineering, including seeding cells, scaffold, and signaling molecules, are required for adequate bone regeneration. The human amniotic membrane (hAM) is the innermost of the placental membranes. In addition to providing a source of stem cells and growth factors, hAM has several features that make it an appropriate scaffold containing stem cells for use in tissue engineering purposes. The present investigation aimed to assess the effect of bone morphogenetic protein-9 (BMP-9) combined with phenamil and simvastatin on osteogenic induction of hAM with its human amniotic membrane epithelial cells (hAECs). Method: Methods: Using six different osteogenic medium (OMs), we cultured hAM for 14 days. The basic OMs were chosen as the first group and other media were made by adding BMP-9, phenamil, simvastatin, BMP-9 alongside phenamil, and BMP-9 alongside simvastatin to the basic OMs. Finally, viability assay, tissue mineralization, calcium and phosphate content determination, and measurement of lactic acid dehydrogenase (LDH), and alkaline phosphatase (ALP) activity were performed. Results: Results: Among all study groups, groups containing simvastatin showed a significantly lower level of viability. Although all media could induce osteogenic features, the hAECs cultured in media containing BMP-9 and phenamil demonstrated a wider area of mineralization and a significantly higher level of calcium and phosphate content, LDH, and ALP activity. Conclusion: Conclusion: Our findings indicated that the use of phenamil together with BMP-9 could synergistically show in situ osteogenic induction in hAECs, which could be a new insight into translational medicine.

Design, implementation and evaluation of a virtual clinical training protocol for midwifery internship in a gynecology course during COVID-19 pandemic: A semi-experimental study.

BACKGROUND: Over the past year, the occurrence of COVID-19 pandemic has challenged clinical education for health care students, due to the possibility of exposure to the virus and increased spread of the disease. Clinical training of midwifery students in gynecologic problems, based on the Iran midwifery education curriculum, was also disrupted during this pandemic. OBJECTIVES: This study was aimed at designing, implementing and evaluating a virtual clinical training protocol for midwifery internship in a Gynecology course. DESIGN: A semi-experimental study. SETTINGS: Faculty of Nursing and Midwifery, Mazandaran University of Medical Sciences, Sari, Iran. PARTICIPANTS: Forty-seven midwifery interns in Gynecology course were recruited during two semesters in 2020. METHODS: Five steps based on the Analysis, Design, Development, Implementation, and Evaluation (ADDIE) model were taken, which included 1) educational and skills needs assessment, 2) design, 3) development via focused group interviews and brainstorming with the presence of the midwifery department members in three sessions, 4) implementation including pretest and posttest, webinar, uploading the information of virtual patients, questions and correct answers, and 5) evaluation including knowledge assessment by a designed questionnaire and skills evaluation by the modified-Mini-CEX checklist. Data were analyzed using mean, standard deviation and paired t-test. RESULTS: After training, a significant increase (p < 0.001) was observed in scores of knowledge and interview skills, clinical judgment, consultation, efficiency, professionalism, clinical competence and total score of clinical skills. CONCLUSIONS: Training for gynecological diseases through virtual clinic promoted knowledge and clinical skills of midwifery interns. To enhance education, a virtual clinic may be used in crisis situations and in combination with teaching under normal circumstances by strengthening the infrastructure and removing barriers.

Developing a pro-angiogenic placenta derived amniochorionic scaffold with two exposed basement membranes as substrates for cultivating endothelial cells.

Decellularized and de-epithelialized placenta membranes have widely been used as scaffolds and grafts in tissue engineering and regenerative medicine. Exceptional pro-angiogenic and biomechanical properties and low immunogenicity have made the amniochorionic membrane a unique substrate which provides an enriched niche for cellular growth. Herein, an optimized combination of enzymatic solutions (based on streptokinase) with mechanical scrapping is used to remove the amniotic epithelium and chorion trophoblastic layer, which resulted in exposing the basement membranes of both sides without their separation and subsequent damages to the in-between spongy layer. Biomechanical and biodegradability properties, endothelial proliferation capacity, and in vivo pro-angiogenic capabilities of the substrate were also evaluated. Histological staining, immunohistochemistry (IHC) staining for collagen IV, and scanning electron microscope demonstrated that the underlying amniotic and chorionic basement membranes remained intact while the epithelial and trophoblastic layers were entirely removed without considerable damage to basement membranes. The biomechanical evaluation showed that the scaffold is suturable. Proliferation assay, real-time polymerase chain reaction for endothelial adhesion molecules, and IHC demonstrated that both side basement membranes could support the growth of endothelial cells without altering endothelial characteristics. The dorsal skinfold chamber animal model indicated that both side basement membranes could promote angiogenesis. This bi-sided substrate with two exposed surfaces for cultivating various cells would have potential applications in the skin, cardiac, vascularized composite allografts, and microvascular tissue engineering.

Biofabrication of chitosan/chitosan nanoparticles/polycaprolactone transparent membrane for corneal endothelial tissue engineering.

We aimed to construct a biodegradable transparent scaffold for culturing corneal endothelial cells by incorporating chitosan nanoparticles (CSNPs) into chitosan/polycaprolactone (PCL) membranes. Various ratios of CSNP/PCL were prepared in the presence of constant concentration of chitosan and the films were constructed by solvent casting method. Scaffold properties including transparency, surface wettability, FTIR, and biocompatibility were examined. SEM imaging, H&E staining, and cell count were performed to investigate the HCECs adhesion. The phenotypic maintenance of the cells during culture was investigated by flow cytometry. Transparency and surface wettability improved by increasing the CSNP/PCL ratio. The CSNP/PCL 50/25, which has the lowest WCA, showed comparable transparency with human acellular corneal stroma. The scaffold was not cytotoxic and promoted the HCECs proliferation as evaluated by MTT assay. Cell counting, flow cytometry, SEM, and H&E results showed appropriate attachment of HCECs to the scaffold which formed a compact monolayer. The developed scaffold seems to be suitable for use in corneal endothelial regeneration in terms of transparency and biocompatibility.

The Bottlenecks in Translating Placenta-Derived Amniotic Epithelial and Mesenchymal Stromal Cells Into the Clinic: Current Discrepancies in Marker Reports.

Placenta-derived amniotic cells have prominent features for application in regenerative medicine. However, there are still discrepancies in the characterization of human amniotic epithelial and mesenchymal stromal cells. It seems crucial that the characterization of human amniotic membrane cells be investigated to determine whether there are currently discrepancies in their characterization reports. In addition, possible causes for the witnessed discrepancies need to be addressed toward paving the way for further clinical application and safer practices. The objective of this review is to investigate the marker characterization as well as the potential causes of the discrepancies in the previous reports on placenta-derived amniotic epithelial and mesenchymal stromal cells. The current discrepancies could be potentially due to reasons including passage number and epithelial to mesenchymal transition (EMT), cell heterogeneity, isolation protocols and cross-contamination, the region of cell isolation on placental disk, measuring methods, and gestational age.

Amniotic membrane and its epithelial and mesenchymal stem cells as an appropriate source for skin tissue engineering and regenerative medicine.

One of the main goals of tissue engineering and regenerative medicine is to develop skin substitutes for treating deep dermal and full thickness wounds. In this regard, both scaffold and cell source have a fundamental role to achieve exactly the same histological and physiological analog of skin. Amnion epithelial and mesenchymal cells possess the characteristics of pluripotent stem cells which have the capability to differentiate into all three germ layers and can be obtained without any ethical concern. Amniotic cells also produce different growth factors, angio-modulatory cytokines, anti-bacterial peptides and a wide range of anti-inflammatory agents which eventually cause acceleration in wound healing. In addition, amniotic membrane matrix exhibits characteristics of an ideal scaffold and skin substitute through various types of extracellular proteins such as collagens, laminins and fibronectins which serve as an anchor for cell attachment and proliferation, a bed for cell delivery and a reservoir of drugs and growth factors involved in wound healing process. Recently, isolation of amniotic cells exosomes, surface modification and cross-linking approaches, construction of amnion based nanocomposites and impregnation of amnion with nanoparticles, construction of amnion hydrogel and micronizing process promoted its properties for tissue engineering. In this manuscript, the recent progress was reviewed which approve that amnion-derived cells and matrix have potential to be involved in skin substitutes; an enriched cell containing scaffold which has a great capability to be translated into the clinic.

Differentiation of Rat bone marrow Mesenchymal stem cells into Adipocytes and Cardiomyocytes after treatment with platelet lysate.

BACKGROUND: Mesenchymal stem cells (MSCs) are multipotential cells and their therapeutic potency is under intense investigation. Studying the effect of different induction factors on MSCs could increase our knowledge about the differentiation potency of these cells. One of the most important sources of these factors in mammalian body is platelet. Platelet lysate (PL) contains many growth factors and therefore, it can be used as a differentiation inducer. In the present study, the effect of PL on differentiation of rat bone marrow MSCs into cardiomyocytes was studied. MATERIALS AND METHODS: To study the differentiation-inducing effect of PL, MSCs were treated with 2.5, 5 and 10% PL. Early results of this study showed that PL in high concentrations (10%) induces adipogenic differentiation of MSCs. Therefore, to evaluate differentiation to cardiomyocytes, MSCs were cultured in media containing lower levels of PL (2.5% and 5%) and then cardiomyogenic differentiation was induced by treatment with 5-azacytidine. Differentiation of MSCs was evaluated using direct observation of beating cells, immunostaining and real-time PCR techniques. RESULTS: The results of qPCR showed that treatment with PL alone increased the expression of cardiac alpha actinin (CAA) being predictable by earlier observation of beating cells in PL-treated groups. The results of staining assays against cardiac alpha actinin also showed that there were stained cells in PL-treated groups. CONCLUSION: The results of the present study showed that PL is a powerful induction factor for differentiation of MSCs into different cell lines such as cardiomyocytes and adipocytes.

Relationship between Revised Graduated Index (R-GINDEX) of prenatal care utilization & preterm labor and low birth weight.

Prenatal care refers to accurate and consistent performance of the principles important to maintain healthy pregnancy outcomes and also for mother and child health. One of the new indices to assess the adequacy of care is Revised Graduated Index of Prenatal Care Utilization (R-GINDEX).The study aims to assess the relationship between quantitative prenatal care factors and preterm labor and low birth weight using R-GINDEX. This historical cohort study has been conducted on 420 mothers during the first two years after delivery in 2010. The adequacy of care was calculated by R-GINDEX. Based on this index, participants have been divided into three care groups including inadequate, adequate and intensive care groups. A significant relationship has been found between R-GINDEX and preterm birth and low birth weight (P<0.05). Thus the probability of premature labor in inadequate care group (RR=3.93) and low birth weight (RR= 2.53) was higher than that of the adequate and intensive care group. The results showed that the quantity of prenatal care is effective in reducing preterm birth and low birth weight.

Treatment with platelet lysate induces endothelial differentation of bone marrow mesenchymal stem cells under fluid shear stress.

By considering stem cell-based therapies as a new hope for the treatment of some tragic diseases, marrow stromal cells or marrow mesenchymal stem cells (MSCs) were considered as a suitable and safe multipotential cell source for this new therapeutic approach. For this purpose, many investigations have been performed on differentiation of MSCs toward specific cell lines to overcome the demand for providing the organ specific cells for cell therapy or preparation of engineered tissues. In the present study, differentiation of MSCs to endothelial cells (ECs) by mechanical and chemical stimulation was evaluated. Fluid shear stress (FSS) was used as mechanical inducer, while platelet lysate (PL) and estradiol (E) were used as chemical induction factors. MSCs were placed under FSS with different forces (2, 5 and 10dyn/cm(2)) for different periods (6, 12 and 24 hours). In some groups, PL and E were added to the culture media to evaluate their effect on expression of EC specific markers. This investigation revealed that FSS with low tension (2.5-5 dyn/cm(2)) for a long time (24 hours) or high tension (10 dyn/cm(2)) in short time (6 hours) in the presence of PL could differentiate MSCs toward ECs. The presence of PL was necessary for initiation of endothelial differentiation, and in the absence of PL, there was not any expression of CD34 and Cadherin5 (Cdh5) among cells. Adding E to the culture medium did not change the rate of endothelial differentiation under FSS. Generated endothelial progenitors could produce von Willebrand factor (vWF) after two weeks culture and also they formed tubular structures after culture on matrigel.

Differentiation of bone marrow mesenchymal stem cells into chondrocytes after short term culture in alkaline medium.

BACKGROUND: Bone marrow mesenchymal stem cells (MSCs) are one of the undifferentiated multipotential cell sources of human body. MSCs have the capacity to form a variety of cell types, especially chondrocytes and osteocytes. Learning about responses of MSCs to external milieu and chemical factors such as pH could recommend new approaches for preparation of suitable scaffolds for bone and cartilage tissue engineering. In present study, the effect of alkaline medium on chondrogenic and osteogenic differentiation of rat MSCs was evaluated. METHODS: MSCs were harvested from bone marrow of animals and then the response of passage1 and 2 of MSCs (P1 MSCs & P2 MSCs) to the culture in alkaline medium (pH: 8) was evaluated. Cytochemical and immunocytochemical staining were performed to distinguish chondrocytes and osteocytes. Real-time PCR was performed to evaluate the type II collagen and osteopontin mRNA levels. RESULTS: Staining for type II collagen, a chondrocytic specific marker, revealed that after one-week culture in alkaline medium, a considerable amount of P1 MSCs had shown chondrocytic morphology. By prolonging the culture period up to 4 weeks, osteogenic cells with expanded matrix and mineralized areas around them were appeared. Results of real-time PCR showed that P1 MSCs after one week culture in alkaline medium expressed highest rate of type II collagen and osteopontin mRNA among all groups. CONCLUSION: This study demonstrated that alkaline medium is a potent chondrogenic differentiation inducer for MSCs in their first passage.

Relationship between adequacy of prenatal care utilization index and pregnancy outcomes.

BACKGROUND: Prenatal care is a comprehensive antepartum care program involving a coordinated approach to medical care and psychosocial support that is optimally offered before conception. Inadequate care during pregnancy can lead to undesirable outcomes, including preterm labor and low birth weight. One of these new, accurate, and comprehensive indicator measurements is adequacy of prenatal care utilization index. This study aimed to assess the adequacy of care and its relationship with preterm labor and low birth weight. MATERIALS AND METHODS: This analytic historical cohort study was performed on 420 mothers who referred to health centers in Sari during 2010. Data were collected by interviews and questionnaires. Based on the adequacy of prenatal care utilization, this care was classified into four groups: intensive, adequate, intermediate, and inadequate. Data were analyzed using chi-square test, analysis of variance (ANOVA), Spearman correlation coefficient, and relative risk (RR). RESULTS: Of the 420 mothers who were studied, inadequate care was observed in 151 (36%) cases, which was the highest percentage of care. There was a significant relationship between the adequacy of prenatal care utilization and preterm labor and low birth weight (for both P < 0.05). Performing inadequate care, the rate of preterm labor was RR = 1.36 times and the rate of low birth weight was RR = 1.08 times more than in adequate and intensive care. CONCLUSION: According to the programs that reduced the number of referrals of pregnant mothers (standardization protocol for mothers in Iran), this study confirms the efficacy of adequacy of prenatal care on reducing preterm labor and low birth weight. This study emphasizes on minimum care performance and the importance of conducting further studies to assess the relationship between quantities of care and other outcomes.