Biofabrication of size-controlled liver microtissues incorporated with ECM-derived microparticles to prolong hepatocyte function

Multicellular microtissues of primary human hepatocytes(PHHs)co-cultured with other supporting cell types are a promis-ing model for drug screening and toxicological studies.However,these liver microtissues(LMs)rapidly lose their functions during ex vivo culture.Here,in order to mimic the cellular and structural hepatic microenvironment,we co-cultured PHHs with human mesenchymal stromal cells(MSCs)and human umbilical vein endothelial cells(HUVECs)in the presence of cell-sized microparticles(MPs)derived from liver extracellular matrix(LEMPs).The microwell culture platform enabled biofabrication of size-controlled multicellular microtissues(PHH:HUVEC:MSC=3:2:1)with efficient LEMP incorporation(about 70%at a 2:1 ratio of cells:MP).The biofabricated liver microtissues(BLMs)were cultured ex vivo for 14 days and compared to the cell-only LM in terms of gene and protein expression,functional activity,cytochrome P450(CYP450)enzyme inducibility,and drug sensitivity.The results supported superior hepatic-related gene expression,functional activity,and polarity for PHH in BLM compared to LM.CYP450 enzyme inducibility and dose-responsive sensitivity to toxic drugs were significantly higher in the BLM group.In conclusion,microtissue engineering by incorporation of tissue-specific microparticles within a multicellular microtissue can offer some advantages for drug discovery studies and cell transplantation applications.In the near future,this approach could generate a scalable platform of several functional biofabricated microtissues representing different organs.

Gelatin-based hydrogel functionalized with taurinemoieties for in vivo skin tissue regeneration

Functionalized hydrogels stimulate the migration and morphogenesis of endothelial cells(ECs)and are useful substrates for wound healing.The present study investigates the feasibility of covalent conjugation of taurine(Tau)on a gelatin-based hydrogel.This hydrogel is expected to maintain positive charged growth factors such as basic fibroblast growth factor(bFGF)and vascular endothelial growth factors(VEGFs)near ECs within the hydrogel microenvironment.The gelatin was conjugated with hydroxyl phenol(Ph)and Tau moieties,and in following that Ph residues were crosslinked through a horseradish peroxidase-catalyzed reaction.The migration characteristics of ECs were analyzed by scratch migration assay and microparticle-based cell migration assay.Cellular morphology and amounts of angiopoietin 1(Ang 1),bFGF,and VEGF proteins were evaluated for encapsulated cells.The potential of synthesized hydrogels in wound healing was assessed by the percentage of reduction from the original wound size and histopathological analyses of rat skin.The incorporated Tau molecules within the hydrogel remained stable through covalent bonds during incubation.During extended incubation,the gelatin-based hydrogel conjugated with Tau improved the migration distance and number of existing migrated ECs.Immobilized Tau within the gelatin-based hydrogel induced high motility of ECs,accompanied by robust cytoskeleton extension and a cell subpopulation that expressed CD44 and CD31 receptors as well as enhancement of Ang 1,bFGF,and VEGF.We found that injectable Gel-Ph-Tau effectively improves wound-healing parameters.

Sequential expression of miR-221-3p and miR-338-3p in Schwann cells as a therapeutic strategy to promote nerve regeneration and functional recovery

The functional properties of endogenous Schwann cells(SCs)during nerve repair are dynamic.Optimizing the functional properties of SCs at different stages of nerve repair may have therapeutic benefit in improving the repair of damaged nerves.Previous studies showed that miR-221-3p promotes the proliferation and migration of SCs,and miR-338-3p promotes the myelination of SCs.In this study,we established rat models of sciatic nerve injury by bridging the transected sciatic nerve with a silicone tube.We injected a miR-221 lentiviral vector system together with a doxycycline-inducible Tet-On miR-338 lentiviral vector system into the cavity of nerve conduits of nerve stumps to sequentially regulate the biological function of endogenous SCs at different stages of nerve regeneration.We found that the biological function of SCs was sequentially regulated,the diameter and density of myelinated axons were increased,the expression levels of NF200 and myelin basic protein were increased,and the function of injured peripheral nerve was improved using this system.miRNA Target Prediction Database prediction,Nanopore whole transcriptome sequencing,quantitative PCR,and dual luciferase reporter gene assay results predicted and verified Cdkn1b and Nrp1 as target genes of miR-221-3p and miR-338-3p,respectively,and their regulatory effects on SCs were confirmed in vitro.In conclusion,here we established a new method to enhance nerve regeneration through sequential regulation of biological functions of endogenous SCs,which establishes a new concept and model for the treatment of peripheral nerve injury.The findings from this study will provide direct guiding significance for clinical treatment of sciatic nerve injury.

Bioactive 3D-printed chitosan-based scaffolds for personalized craniofacial bone tissue engineering

Regeneration of craniofacial bone defects is a key issue in the bone regeneration field.Hence,novel treatment strategies,such as tissue engineering using porous scaffolds,have been developed.An ideal tissue-engineered scaffold for bone tissue regeneration should possess pores to facilitate nutrients transmission and support repar-ative tissue ingrowth,bioactivity for osteoconduction and osseointegration,and biocompatibility to improve cell attachment,proliferation,and extracellular matrix formation.In the present study,we manufactured chitosan-based hydrogels substituted with alginate with optimized properties by extrusion-based three-dimensional(3D)printing.3D printing of the scaffolds enables the designing and developing of complex architectures for cranio-facial reconstruction using computer-aided design(CAD).Different ratios(2.5,5,and 10%)of hydroxyapatite were added to the hydrogel,printed,and subsequently lyophilized to augment the physical and biological char-acteristics of the scaffolds.Hydroxyapatite incorporation into the chitosan-based scaffolds increased the porosity and pore size of the printed scaffolds.In addition,the presence of hydroxyapatite amplified apatite formation and decreased the size of formed apatite crystals.All the scaffold samples showed biocompatible properties and did not have toxicity toward rat bone marrow mesenchymal stem cells.Furthermore,the scaffolds containing 5%w/w hydroxyapatite exhibited significant growth in cell viability compared to the control.Overall,it is concluded that chitosan-based scaffolds adorned with hydroxyapatite are considerable for regenerating craniofacial bone defects.

Efficacy of nanofibrous conduits in repair of longsegment sciatic nerve defects

Our previous studies have histomorphologically confirmed that nanofibrous poly(3-hydroxybutyrateco-3-hydroxyvalerate) conduit can be used to repair 30-mm-long sciatic nerve defects.However,the repair effects on rat behaviors remain poorly understood.In this study,we used nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) conduit and autologous sciatic nerve to bridge 30-mm-long rat sciatic nerve gaps.Within 4 months after surgery,rat sciatic nerve functional recovery was evaluated per month by behavioral analyses,including toe out angle,toe spread analysis,walking track analysis,extensor postural thrust,swimming test,open-field analysis and nociceptive function.Results showed that rat sciatic nerve functional recovery was similar after nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) conduit and autologous nerve grafting.These findings suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) conduit is suitable in use for repair of long-segment sciatic nerve defects.

Combined use of Y-tube conduits with human umbilical cord stem cells for repairing nerve bifurcation defects

Given the anatomic complexity at the bifurcation point of a nerve trunk,enforced suturing between stumps can lead to misdirection of nerve axons,thereby resulting in adverse consequences.We assumed that Y-tube conduits injected with human umbilical cord stem cells could be an effective method to solve such problems,but studies focused on the best type of Y-tube conduit remain controversial.Therefore,the present study evaluated the applicability and efficacy of various types of Y-tube conduits containing human umbilical cord stem cells for treating rat femoral nerve defects on their bifurcation points.At 12 weeks after the bridging surgery that included treatment with different types of Y-tube conduits,there were no differences in quadriceps femoris muscle weight or femoral nerve ultrastructure.However,the Y-tube conduit group with longer branches and a short trunk resulted in a better outcome according to retrograde labeling and electrophysiological analysis.It can be concluded from the study that repairing a mixed nerve defect at its bifurcation point with Y-tube conduits,in particular those with long branches and a short trunk,is effective and results in good outcomes.

Citalopram increases the differentiation efficacy of bone marrow mesenchymal stem cells into neuronal-like cells

Several studies have demonstrated that selective serotonin reuptake inhibitor antidepressants can promote neuronal cell proliferation and enhance neuroplasticity both in vitro and in vivo. It is hypothesized that citalopram, a selective serotonin reuptake inhibitor, can promote the neuronal differentiation of adult bone marrow mesenchymal stem cells. Citalopram strongly enhanced neuronal characteristics of the cells derived from bone marrow mesenchymal stem cells. The rate of cell death was decreased in citalopram-treated bone marrow mesenchymal stem cells than in control cells in neurobasal medium. In addition, the cumulative population doubling level of the citalopram-treated cells was significantly increased compared to that of control cells. Also BrdU incorporation was elevated in citalopram-treated cells. These findings suggest that citalopram can improve the neuronal-like cell differentiation of bone marrow mesenchymal stem cells by increasing cell proliferation and survival while maintaining their neuronal characteristics.

Nanofibrous nerve conduits for repair of 30-mm-long sciatic nerve defects

It has been confirmed that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit can promote peripheral nerve regeneration in rats. However, its efficiency in repair of over 30-mm-long sciatic nerve defects needs to be assessed. In this study, we used a nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit to bridge a 30-mm-long gap in the rat sciatic nerve. At 4 months after nerve conduit implantation, regenerated nerves were macroscopically observed and histologically assessed. In the nanofibrous graft, the rat sciatic nerve trunk had been reconstructed by restoration of nerve continuity and formation of myelinated nerve fiber. There were Schwann cells and glial cells in the regenerated nerves. Masson's trichrome staining showed that there were no pathological changes in the size and structure of gastrocnemius muscle cells on the operated side of rats. These findings suggest that nanofibrous poly(3-hydroxybutyrate-co-3-hydroxyvalerate) nerve conduit is suitable for repair of long-segment sciatic nerve defects.

Optimization and comparison of two different 3D culture methods to prepare cell aggregates as a bioink for organ printing

The ultimate goal of tissue engineering is to design and fabricate functional human tissues that are similar to natural cells and are capable of regeneration.Preparation of cell aggregates is one of the important steps in 3D tissue engineering technology,particularly in organ printing.Two simple methods,hanging drop(HD)and conical tube(CT)were utilized to prepare cell aggregates.The size and viability of the aggregates obtained at different initial cell densities and pre-culture duration were compared.The proliferative ability of the cell aggregates and their ability to spread in culture plates were also investigated.In both methods,the optimum average size of the aggregates was less than 500μm.CT aggregates were smaller than HD aggregates.5,000 cells per drop HD aggregates showed a marked ability to attach and spread on the culture surface.The proliferative ability reduced when the initial cell density was increased.Comparing these methods,we found that the HD method having better size controlling ability as well as enhanced ability to maintain higher rates of viability,spreading,and proliferation.In conclusion,smaller HD aggregates might be a suitable choice as building blocks for making bioink particles in bioprinting technique.

Effect of hydro-alcoholic extract of Olea europaea on apoptosis-related genes and oxidative stress in a rat model of torsion/detorsion-induced ovarian damage

Objective: To evaluate the impact of Olea (O.) europaea extract on markers of oxidative stress and apoptosis of ovarian tissues in a rat model of torsion/detorsion-induced ovarian damage. Methods: A total of 28 Wistar female rats were randomly assigned into 4 groups, with 7 rats in each group. The sham group received a 2.5 cm longitudinal incision in the midline part of the abdomen which was then sutured with 5-0 nylon thread;the torsion/detorsion group underwent torsion induction for 3 h followed by reperfusion for 10 days;the torsion/detorsion+O. europaea group received 300 mg/kg hydro-alcoholic extract of O. europaea 30 min before detorsion, followed by reperfusion for 10 days;and the O. europaea group only received 300 mg/kg hydro-alcoholic extract of O. europaea for 10 days. After the treatment period, blood samples were taken;the levels of estrogen, glutathione peroxidase, superoxide dismutase, and malondialdehyde were assayed. The histological changes, as well as the rate of apoptosis in ovarian tissues, were also carried out by histomorphometric analysis at day 10 post-procedure. Results: Histological comparisons demonstrated a significant detrimental change in the torsion/detorsion group as compared with other groups. The number of pre-antral and antral follicles and corpus luteum was significantly decreased in the torsion/detorsion group compared with the sham group, while treatment with O. europaea could enhance their numbers (P<0.05). The index of apoptosis and the number of atretic body in the ovarian tissue were significantly higher in the torsion/detorsion group compared with the sham group (P<0.05). The concentrations of glutathione peroxidase, estrogen, and superoxide dismutase as well as the mRNA expression of Bcl-2 were considerably diminished in the torsion/detorsion group while they were elevated in the torsion/detorsion+O. europaea group (P<0.05) compared with the torsion/detorsion group. The serum malondialdehyde level and the mRNA expression of Bax were markedly increased during ischemia, while treatment with O. europaea significantly diminished the increased concentrations of malondialdehyde and Bax level in the torsion/detorsion+O. europaea group (P<0.05). Conclusions: O. europaea extract can reduce the degree of tissue damage induced by oxidative stress and apoptosis in the ovary following ovarian ischemia/reperfusion.

N-methyl-D-aspartate receptor subunit expression in memory-related brain areas of lead-exposed rats

Lead exposure induces decreased hippocampal N-methyl-D-aspartic acid(NMDA) receptor gene and protein expressions,which influences the molecular mechanisms of learning and memory.However,lead poisoning-induced differences in NMDA subunit expression,and the correlation of lead poisoning with learning and memory,remain poorly understood.The present study measured differences in expression of NMDA receptor subunits NR1,NR2A,and NR2B in memory-related brain regions of rats who underwent different doses of lead exposure.Results demonstrated decreased NR1,NR2A,and NR2B subunit expressions in some memory-related brain areas.The inhibitory effect of 4.8 mmol/L lead exposure on hippocampal NR2B was most significant,although NR2A expression also significantly decreased following 14.4 mmol/L lead exposure.There was no difference in NR1 expression following exposure to < 4.8 mmol/L lead,although the inhibitory effect of 19.6 mmol/L lead exposure was strongest for NR1 expression in the hippocampus.Inhibitory avoidance test results revealed that greater concentrations of lead exposure resulted in decreased learning and memory.Therefore,lead toxicity was dependent on NMDA receptor subunit composition,and NR1,NR2A,and NR2B expressions were associated with time and concentration of lead exposure.

Differentiation of Wharton's jelly mesenchymal stem cells into neurons in alginate scaffold

Alginate scaffold has been considered as an appropriate biomaterial for promoting the differentiation of embryonic stem cells toward neuronal cell lineage. We hypothesized that alginate scaffold is suitable for culturing Wharton's jelly mesenchymal stem cells(WJMSCs) and can promote the differentiation of WJMSCs into neuron-like cells. In this study, we cultured WJMSCs in a three-dimensional scaffold fabricated by 0.25% alginate and 50 m M Ca Cl2 in the presence of neurogenic medium containing 10 μM retinoic acid and 20 ng/m L basic fibroblast growth factor. These cells were also cultured in conventional two-dimensional culture condition in the presence of neurogenic medium as controls. After 10 days, immunofluorescence staining was performed for detecting β-tubulin(marker for WJMSCs-differentiated neuron) and CD271(motor neuron marker). β-Tubulin and CD271 expression levels were significantly greater in the WJMSCs cultured in the three-dimensional alginate scaffold than in the conventional two-dimensional culture condition. These findings suggest that three-dimensional alginate scaffold cell culture system can induce neuronal differentiation of WJMSCs effectively.

Protein biomarkers of neural system

The utilization of biomarkers for in vivo and in vitro research is growing rapidly. This is mainly due to the enormous potential of biomarkers in evaluating molecular and cellular abnormalities in cell models and in tissue, and evaluating drug responses and the effectiveness of therapeutic intervention strategies. An important way to analyze the development of the human body is to assess molecular markers in embryonic specialized cells, which include the ectoderm, mesoderm, and endoderm. Neuronal development is controlled through the gene networks in the neural crest and neural tube, both components of the ectoderm. The neural crest differentiates into several different tissues including, but not limited to, the peripheral nervous system, enteric nervous system, melanocyte, and the dental pulp. The neural tube eventually converts to the central nervous system. This review provides an overview of the differentiation of the ectoderm to a fully functioning nervous system, focusing on molecular biomarkers that emerge at each stage of the cellular specialization from multipotent stem cells to completely differentiated cells. Particularly, the otic placode is the origin of most of the inner ear cell types such as neurons, sensory hair cells, and supporting cells. During the development, different auditory cell types can be distinguished by the expression of the neurogenin differentiation factor1 (Neuro D1), Brn3a, and transcription factor GATA3. However, the mature auditory neurons express other markers including bIII tubulin, the vesicular glutamate transporter (VGLUT1), the tyrosine receptor kinase B and C (Trk B, C), BDNF, neurotrophin 3 (NT3), Calretinin, etc.

Mechanical and histological properties of an electrospun scaffold with a modified surface by plasma polymerization implanted in an in vivo model

This article presents the construction of scaffolds composed of polylactic acid(PLA)with different concentrations of hydroxyapatite(HA)by electrospinning,which were superficially modified with polypyrrole(PPy/I)by plasma polymerization.A preliminary study was conducted of the biological and mechanical behavior of the scaffolds when they were implanted in the back of rabbits for 30 days;bone cells differentiated from mesenchymal stem cells(MSCs)were used.The bone cell and scaffold structures were characterized by histological,immunohistochemical,and mechanical stress tests.Hematoxylin–eosin staining showed good tissue conformation.The immunohistochemical tests highlighted the presence of the main bone tissue proteins,such as collagen,osteocalcin,and osteopontin.The PLA/HA scaffolds were observed to exhibit cell adhesion and proliferation properties;however,the response was much better in the scaffolds that had a higher concentration of HA and that were coated with PPy/I.The results of the mechanical tests of the scaffolds indicated that the plasma treatment improved the adhesion and cell proliferation properties and contributed to the mechanical support,allowing the formation of neotissues with good viability of cell growth.

SKL promotes patency and endothelialization of small-caliber vascular grafts in rabbit carotid artery model

The Klotho.(KL)gene is related to aging.In this study,SKL(secreted KL)and heparin were cross:linked to the acellular small intestinal submucosa(SIS).Based on this,small-diameter tissue engineered blood vessels(TEVs)were constructed.The role of SKL on improving the patency of TEVs through promoting cell adherence was investigated.The SIS membrane was cross linked with heparin and SKL,respectively,to prepare heparin group and SKL group artificial blood vessels.The CCK8 assay showed that SKL treatment promoted endothelial cells proliferation.Western blot showed that SKL upregulated the levels of FAK and RHOA.

Effect of deforolimus and VEGF on angiogenesis in endometrial stromal cells following three-dimensional culture

The presence of endometrial tissue outside of the uterine cavity is named endometriosis and is the most common gynecologic disorder in women. Determining the inhibitory effect of a Deforolimus on angiogenesis in a three-dimensional (3-D) culture of human endometrial stromal cells (hEnCs) in vitro. The important mechanism in the pathogenesis of endometriosis is angiogenesis, and deforolimus has been shown to have anti-angiogenic activity. This was an in vitro study of human endometrial stromal cells in 3-D culture of fibrin matrix. Endometrial stromal cells isolated and placed in a 3-D fibrin matrix culture system for angiogenesis with VEGF and inhibit angiogenesis by deforolimus. Finally these cells analyzed by CD31 antibodies. After 3 weeks, in cells treated with VEGF, endothelial cell branching was observed and rudimentary capillary-like structures formed. In the presence of 5μM of deforolimus, angiogenesis was reduced. The deforolimus were shown to be effective in inhibiting the mechanisms of angiogenesis.

Macrolide-Clarithromycin Task-Force for the Treatment and Prophylaxis of Covid-19 as a Single Agent

SARS-CoV-2 is a novel RNA coronavirus responsible of a deadly pandemic: the clinical illness COVID-19. With only one authorized drug for emergency use in critically ill patients: Remdesivir, there is not any other approved drug or vaccine yet with proven potential to overcome this infection. We exposed here many scientific evidences to support our novel idea that a macrolide, basically Clarithromycin, could be effective as a single agent for treatment and prophylaxis of COVID-19. Clarithromycin could change the history of this pandemic. It could reduce the costs of treatment and the potential adverse effects when combining more than one drug such as with Hydroxychloroquine. Clarithromycin treatment and prophylaxis as a single agent could be much more simple, safe and cheaper as giving Chloroquine or Hydroxychloroquine alone or in combination with Azithromycin as well as other therapeutic options.

Repair of critical size rat calvarial defects using endometrial-derived stem cells embedded within gelatin/apatite nanocomposite scaffold

Human stromal stem cells derived from endometrium (hESCs) are a type of multipotent stromal cells of the proven ability to differentiate into osteogenic lineage. Thus, it was suggested that these cells may be used to repair skeletal defects. In this study, Human ESCs were extracted from female endometrium and harvested. Biomimetic gelatin/apatite (Gel/Ap) scaffolds with and without harvested cells were implanted in a Critical size calvarial defects in the cranial bone of adult male rat. To CT-Scan and Histological studies were performed to investigate the level of bone formation after 8 weeks of surgery. Results confirmed that the treated defects with the bare and hESCs grafted Gel/Ap scaffold showed significant bone formation and maturation in comparison with the control group.

Nanocomposite Electrospun Scaffold Based on Polyurethane/Polycaprolactone Incorporating Gold Nanoparticles and Soybean Oil for Tissue Engineering Applications

Electrospun nanofibers combined with a wide range of functional additives can be used for a various tissue engineering applications due to their desired biomimetic and physicochemical properties.Therefore,the present study was conducted to obtain a highly efficient nanocomposite electrospun scaffold with appropriate physicochemical performance and biological properties based on Polycaprolactone/Polyurethane(PCL/PU)mixed with gold nanoparticles(GNPs)and soybean oil(SO).In the present study,the desired nanofibers were fabricated by electrospinning PCL/PU mixed solution with GNPs and SO.The nanocomposite electrospun PU/PCL/SO/GNP nanofibers were characterized in terms of chemical composition by attenuated total reflectance-Fourier transform infrared spectroscopy(ATR-FTIR),morphological structure by field-emission scanning electron microscopy(FE-SEM),and mechanical and biological properties.The surface topography and wettability were determined by atomic force microscopy(AFM)and water contact angle measurements,respectively.It was found that the presence of GNPs along with SO in the structure of PCL/PU nanofiber created a smoother surface in terms of surface roughness and also a more homogeneous fibrous structure.In addition,it was observed that both SO and GNPs caused an increase in the electrical conductivity of the fibrous mats.In the biocompatibility evaluations by measuring cell viability and cell adherence to the scaffold's surfaces,it was found that adding of SO and GNPs supports fibroblasts.Taken together,the fabricated nanocomposite fibrous scaffolds can be a potential candidate for various tissue engineering purposes.

Surface modification of small intestine submucosa in tissue engineering

With the development of tissue engineering,the required biomaterials need to have the ability to promote cell adhesion and proliferation in vitro and in vivo.Especially,surface modification of the scaffold material has a great influence on biocompatibility and functionality of materials.The small intestine submucosa(SIS)is an extracellular matrix isolated from the submucosal layer of porcine jejunum,which has good tissue mechanical properties and regenerative activity,and is suitable for cell adhesion,proliferation and differentiation.In recent years,SIS is widely used in different areas of tissue reconstruction,such as blood vessels,bone,cartilage,bladder and ureter,etc.This paper discusses the main methods for surface modification of SIS to improve and optimize the performance of SIS bioscaffolds,including functional group bonding,protein adsorption,mineral coating,topography and formatting modification and drug combination.In addition,the reasonable combination of these methods also offers great improvement on SIS surface modification.This article makes a shallow review of the surface modification of SIS and its application in tissue engineering.