Comparison of the effects of preservation methods on structural, biological, and mechanical properties of the human amniotic membrane for medical applications.

Amniotic membrane (AM), the innermost layer of the placenta, is an exceptionally effective biomaterial with divers applications in clinical medicine. It possesses various biological functions, including scar reduction, anti-inflammatory properties, support for epithelialization, as well as anti-microbial, anti-fibrotic and angio-modulatory effects. Furthermore, its abundant availability, cost-effectiveness, and ethical acceptability make it a compelling biomaterial in the field of medicine. Given the potential unavailability of fresh tissue when needed, the preservation of AM is crucial to ensure a readily accessible and continuous supply for clinical use. However, preserving the properties of AM presents a significant challenge. Therefore, the establishment of standardized protocols for the collection and preservation of AM is vital to ensure optimal tissue quality and enhance patient safety. Various preservation methods, such as cryopreservation, lyophilization, and air-drying, have been employed over the years. However, identifying a preservation method that effectively safeguards AM properties remains an ongoing endeavor. This article aims to review and discuss different sterilization and preservation procedures for AM, as well as their impacts on its histological, physical, and biochemical characteristics.

Exosomes for angiogenesis induction in ischemic disorders.

Ischaemic disorders are leading causes of morbidity and mortality worldwide. While the current therapeutic approaches have improved life expectancy and quality of life, they are unable to "cure" ischemic diseases and instate regeneration of damaged tissues. Exosomes are a class of extracellular vesicles with an average size of 100-150 nm, secreted by many cell types and considered a potent factor of cells for paracrine effects. Since exosomes contain multiple bioactive components such as growth factors, molecular intermediates of different intracellular pathways, microRNAs and nucleic acids, they are considered as cell-free therapeutics. Besides, exosomes do not rise cell therapy concerns such as teratoma formation, alloreactivity and thrombotic events. In addition, exosomes are stored and utilized more convenient. Interestingly, exosomes could be an ideal complementary therapeutic tool for ischemic disorders. In this review, we discussed therapeutic functions of exosomes in ischemic disorders including angiogenesis induction through various mechanisms with specific attention to vascular endothelial growth factor pathway. Furthermore, different delivery routes of exosomes and different modification strategies including cell preconditioning, gene modification and bioconjugation, were highlighted. Finally, pre-clinical and clinical investigations in which exosomes were used were discussed.

The role of spinal cord tractography in detecting lesions following selective bladder afferent and efferent fibers injury: A novel method for induction of neurogenic lower urinary tract dysfunction in rabbit.

OBJECTIVE: Neurogenic lower urinary tract dysfunction (NLUTD), a challenging disorder, is defined by lack of bladder control due to the abnormalities in neural pathways and can be classified based on the location of lesions within the nervous system, thus investigating the neural pathways can help us to know the site of the lesion and specify the class of the NLUTD. Diffusion Tensor Imaging (DTI) tractography, a noninvasive advanced imaging method, is capable of detecting central nervous system pathologies, even if routine magnetic resonance imaging shows no abnormality. Accordingly, tractography is an ideal technique to evaluate patients with NLUTD and visualize the pathology site within the spine. This study aimed to introduce a novel method of spinal cord injury (SCI) to establish NLUTD in the rabbit and to investigate the potential of tractography in tracing neural tracts of the spinal cord in an induced NLUTD animal model. MATERIALS AND METHODS: An animal model of NLUTD was induced through cauterization of the spinal cord at the level T12-L1 in 12 rabbits. Then rabbits were assessed via DTI, urodynamic studies (UDS), voiding cystourethrogram (VCUG), and pathology assessments using antineurofilament 200 (NF200) antibody, anti-S100, anti-Smooth Muscle Actin, anti-Myogenin, and anti-MyoD1. RESULTS: The tractography visualized lesions within spinal cord fibers. DTI parameters including fractional anisotropy (FA) value and tract density were significantly decreased (FA: p-value = 0.01, Tract density: p-value = 0.05) after injury. The mean diffusivity (MD) was insignificantly increased compared to before the injury. Also, the results of UDS and pathology assessments corroborated that applying SCI and the establishment of the NLUTD model was completely successful. CONCLUSION: In the present study, we investigated the auxiliary role of tractography in detecting the spinal cord lesions in the novel established rabbit model of NLUTD. The introduced method of NLUTD induction was without the leg's neurological deficit, easily applicable, low-cost, and was accompanied by minimal surgical preparation and a satisfactory survival rate in comparison with other SCI animal models.

The differentiation effect of bone morphogenetic protein (BMP) on human amniotic epithelial stem cells to express ectodermal lineage markers.

Stem cells are a promising tool for treatment of a variety of degenerative diseases. Human amniotic epithelial stem cells (hAECs) have desirable and unique characteristics that make them a proper candidate for cell therapy. In this study, we have investigated the effects of BMP-4 (bone morphogenetic protein-4) and its inhibition on differentiation of AECs into ectodermal lineages. Analysis of AEC-derived ectodermal lineages (neurons and keratinocytes) was performed by using flow cytometry technique for Map2 and ß-tubulin (as neuron markers), Olig2 and MBP (as oligodendrocyte markers), and K14 and K10 (as keratinocyte markers). The results of this study illustrated that noggin (as BMP antagonist), BMP4, and both BMP4 and heparin (together or separately) increased neural and keratinocyte marker expression, respectively. The expression of markers MAP2, olig2, and K14 in hAECs has been significantly decreased 21 days after exposure to differentiation medium (without growth factors) compared with isolation day, which supports the hypothesis that AECs can be dedifferentiated into pluripotent cells. Moreover, activation and inhibition of BMP signaling have no effects on viability of hAECs. The results of this study showed that BMP signaling and its inhibition are the key factors for ectodermal lineage differentiation of amnion-derived stem cells.

Substrate stiffness affects the morphology and gene expression of epidermal neural crest stem cells in a short term culture.

According to the intrinsic plasticity of stem cells, controlling their fate is a critical issue in cell-based therapies. Recently, a growing body of evidence has suggested that substrate stiffness can affect the fate decisions of various stem cells. Epidermal neural crest stem cells as one of the main neural crest cell derivatives hold great promise for cell therapies due to presenting a high level of plasticity. This study was conducted to define the influence of substrate stiffness on the lineage commitment of these cells. Here, four different polyacrylamide hydrogels with elastic modulus in the range of 0.7-30 kPa were synthesized and coated with collagen and stem cells were seeded on them for 24 hr. The obtained data showed that cells can attach faster to hydrogels compared with culture plate and cells on <1 kPa stiffness show more neuronal-like morphology as they presented several branches and extended longer neurites over time. Moreover, the transcription of actin downregulated on all hydrogels, while the expression of Nestin, Tubulin, and PDGFR-α increased on all of them and SOX-10 and doublecortin gene expression were higher only on <1 kPa. Also, it was revealed that soft hydrogels can enhance the expression of glial cell line-derived neurotrophic factor, neurotrophin-3, and vascular endothelial growth factor in these stem cells. On the basis of the results, these cells can respond to the substrate stiffness in the short term culture and soft hydrogels can alter their morphology and gene expression. These findings suggested that employing proper substrate stiffness might result in cells with more natural profiles similar to the nervous system and superior usefulness in therapeutic applications.

14-3-3ζ protein protects against brain ischemia/reperfusion injury and induces BDNF transcription after MCAO in rat.

Brain ischemia is a leading cause of death and disability worldwide that occurs when blood supply of the brain is disrupted. Brain-derived neurotrophic factor (BDNF) is a protective factor in neurodegenerative conditions. Nevertheless, there are some problems when exogenous BDNF is to be used in the clinic. 14-3-3ζ is a pro-survival highly-expressed protein in the brain that protects neurons against death. This study evaluates 14-3-3ζ effects on BDNF transcription at early time point after ischemia and its possible protective effects against ischemia damage. Human 14-3-3ζ protein was purified after expression. Rats were assigned into four groups, including sham, ischemia, and two treatment groups. Stereotaxic cannula implantation was carried out in the right cerebral ventricle. After one week, rats underwent middle cerebral artery occlusion (MCAO) surgery and received 14-3-3ζ (produced in our laboratory or standard form as control) in the middle of ischemia time. At 6 h of reperfusion after ischemia, brain parts containing the hippocampus, the cortex, the piriform cortex-amygdala and the striatum were collected for real time PCR analysis. At 24 h of reperfusion after ischemia, neurological function evaluation and infarction volume measurement were performed. The present study showed that 14-3-3ζ could up-regulate BDNF mRNA at early time point after ischemia in the hippocampus, in the cortex and in the piriform cortex-amygdala and could also improve neurological outcome and reduce infarct volume. It seems that 14-3-3ζ could be a candidate factor for increasing endogenous BDNF in the brain and a potential therapeutic factor against brain ischemia.

The effect of cryopreservation on anti-cancer activity of human amniotic membrane.

Human amniotic membrane (AM) is an appropriate candidate for treatment of cancer due to special properties, such as inhibition of angiogenesis and secretion of pro-apoptotic factors. This research was designed to evaluate the impact of cryopreservation on cancer cell death induction and anti-angiogenic properties of the AM. Cancer cells were treated with fresh and cryopreserved amniotic condition medium during 24 h and cancer cell viability was determined by MTT assay. To evaluate angiogenesis, the rat aorta ring assay was performed for both fresh and cryopreserved AM within 7 days. In addition, four anti-angiogenic factors Tissue Inhibitor of Matrix Metalloproteinase-1 and 2 (TIMP-1 and TIMP-2), Thrombospondin, and Endostatin were measured by ELISA assay before and after cryopreservation. The results showed that the viability of cultured cancer cells dose-dependently decreased after treatment with condition medium of fresh and cryopreserved tissue and no significant difference was observed between the fresh and cryopreserved AM. The results revealed that the amniotic epithelial stem cells inhibit the penetration of fibroblast-like cells and angiogenesis. Moreover, the penetration of fibroblast-like cells in both epithelial and mesenchymal sides of fresh and cryopreserved AM was observed after removing of epithelial cells. The cryopreservation procedure significantly decreased anti-angiogenic factors TIMP-1, TIMP-2, Thrombospondin, and Endostatin which shows that angio-modulatory property is not fully dependent on proteomic and metabolomic profiles of the AM. These promising results demonstrate that cancer cell death induction and anti-angiogenic properties of the AM were maintained within cryopreservation; a procedure which can circumvent limitations of the fresh AM.

Induction of apoptosis, stimulation of cell-cycle arrest and inhibition of angiogenesis make human amnion-derived cells promising sources for cell therapy of cancer.

Amniotic membrane (AM), the nearest layer of fetal membranes to the fetus, contains two types of cells with unique characteristics that make them excellent candidates for clinical applications. Amniotic epithelial and mesenchymal cells have low immunogenicity, anti-inflammation, anti-fibrosis and anti-bacterial properties and no ethical issues. Although amniotic cells have stem cell properties and express transcription factors specific for pluripotent stem cells, they are not tumorigenic after transplantation. In the last decade, a new line of research has been initiated with a focus on the anti-proliferative effects of amniotic epithelial and mesenchymal cells on tumor growth. Amnion-derived epithelial and mesenchymal cells inhibit tumor growth and invasion through three pathways: the induction of apoptosis, the stimulation of cell-cycle arrest and the inhibition of angiogenesis. In this review, the various aspects of the anti-cancer properties of amnion-derived cells and the underlying mechanisms are discussed with emphasis on the translation of the cell therapy of cancer from experimental into clinical practice.

The effects of cryopreservation on angiogenesis modulation activity of human amniotic membrane.

Amniotic membrane (AM), as the innermost layer of placenta, has side dependent effects on the angiogenesis. Cryopreservation is a necessary process to avoid the challenging problems of fresh tissues; a procedure which makes the AM ready-to-use. Since the cryopreservation can influence the AM characteristics for experimental and clinical purposes, in this study the effects of cryopreservation were evaluated on angiogenesis modulation activity of the AM compared to fresh tissues in an animal model. The AM was implanted mesenchymal side up or epithelial side up in a rat dorsal skinfold chamber. The length and number of branches of formed capillaries were measured via intravital microscopy after 7 days. The amount of IL-8 (interleukin-8) and TIMP-2 (Tissue Inhibitor of Matrix Metalloproteinase-2) as two factors in amniotic cells which have great impacts on angiogenesis were evaluated using ELISA assay. The epithelial surface of cryopreserved AM had inhibitory effects on vessel formation. The cryopreserved amniotic mesenchymal side increased the vessel length and sprout. The result of cryopreserved AM on angiogenesis was similar to that of fresh tissues. The levels of IL-8 and TIMP-2 in cryopreserved samples were significantly less than fresh AMs which shows that angio-modulatory properties are not limited to the effects of amnion epithelial and mesenchymal stem cells and the other components such as extracellular matrix may contribute in angio-modulatory effects. These promising results show that inducing and inhibitory effects of the AM, which make it an appropriate candidate for different clinical situations, were maintained after cryopreservation.

Human amniotic epithelial cells induce apoptosis of cancer cells: a new anti-tumor therapeutic strategy.

BACKGROUND AIMS: Amniotic membrane (AM), the innermost layer of human placenta, is composed of a single layer of epithelial cells, a basement membrane and an avascular stroma. The AM has many functions and properties, among which angiogenic modulatory and immunoregulatory effects are applicable in cancer therapy. Because these functions belong to amniotic epithelial cells, in this study we compared the anti-cancer effect of amniotic epithelial cells and the whole AM. METHODS: The effect of the AM and the amniotic epithelial cells on cancer cell apoptosis was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay, terminal deoxynucleotidyl transferase dUTP nick end labeling assay and immunocytochemistry. The effect of the AM on angiogenesis in conditions both with and without epithelial cells was also evaluated using rat aortic ring assay. RESULTS: There was a decrease in cancer cell viability after adding either AM or amniotic epithelial cell supernatant to cancer cells. A significant increase in caspase-3 and caspase-8 expression in cancer cells treated with amniotic epithelial cell supernatant was observed. The recorded media also demonstrated the possible induction of apoptosis in cancer cells treated with the amniotic epithelial cell supernatant. In the aorta ring assay, the AM showed an anti-angiogenic effect in the presence of its epithelial cells; however, this effect was altered to initiate angiogenesis when amniotic epithelial cells were removed from the AM. CONCLUSIONS: These results suggest that amniotic epithelial cells, with their anti-angiogenic effect and induction of apoptosis, are candidates for cancer therapeutic agents in the near future.

Amnion-derived hydrogels as a versatile platform for regenerative therapy: from lab to market.

In recent years, the amnion (AM) has emerged as a versatile tool for stimulating tissue regeneration and has been of immense interest for clinical applications. AM is an abundant and cost-effective tissue source that does not face strict ethical issues for biomedical applications. The outstanding biological attributes of AM, including side-dependent angiogenesis, low immunogenicity, anti-inflammatory, anti-fibrotic, and antibacterial properties facilitate its usage for tissue engineering and regenerative medicine. However, the clinical usage of thin AM sheets is accompanied by some limitations, such as handling without folding or tearing and the necessity for sutures to keep the material over the wound, which requires additional considerations. Therefore, processing the decellularized AM (dAM) tissue into a temperature-sensitive hydrogel has expanded its processability and applicability as an injectable hydrogel for minimally invasive therapies and a source of bioink for the fabrication of biomimetic tissue constructs by recapitulating desired biochemical cues or pre-defined architectural design. This article reviews the multi-functionality of dAM hydrogels for various biomedical applications, including skin repair, heart treatment, cartilage regeneration, endometrium regeneration, vascular graft, dental pulp regeneration, and cell culture/carrier platform. Not only recent and cutting-edge research is reviewed but also available commercial products are introduced and their main features and shortcomings are elaborated. Besides the great potential of AM-derived hydrogels for regenerative therapy, intensive interdisciplinary studies are still required to modify their mechanical and biological properties in order to broaden their therapeutic benefits and biomedical applications. Employing additive manufacturing techniques (e.g., bioprinting), nanotechnology approaches (e.g., inclusion of various bioactive nanoparticles), and biochemical alterations (e.g., modification of dAM matrix with photo-sensitive molecules) are of particular interest. This review article aims to discuss the current function of dAM hydrogels for the repair of target tissues and identifies innovative methods for broadening their potential applications for nanomedicine and healthcare.

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.

Serum-free cryopreservation of human amniotic epithelial cells before and after isolation from their natural scaffold.

Amniotic epithelial cells are a promising source for stem cell-based therapy through their potential capacity to differentiate into the cell lineages of all three germ layers. Long-term preservation is necessary to have a ready-to-use source of stem cells, when required. Reduced differentiation capability, decrease of viability and use of fetal bovine serum (FBS) are three drawbacks of clinical application of cryopreserved stem cells. In this study, we used human amniotic fluid instead of animal serum, and evaluated viability and multipotency of amniotic epithelial cells after cryopreservation in suspension and compared with those cryopreserved on their natural scaffold (in situ cryopreservation). There was no significant difference in viability of the cells cryopreserved in amniotic fluid and FBS. Also, the same results were achieved for expression of pluripotency marker OCT-4 when FBS was replaced by amniotic fluid in the samples with the same cryoprotectant. The cells cryopreserved in presence of scaffold had a higher level of viability compared to the cells cryopreserved in suspension. Although, the number of the cells expressed OCT-4 significantly decreased within cryopreservation in suspension, no decrease in expression of OCT-4 was observed when the cells cryopreserved with their natural scaffold. Upon culturing of post-thawed cells in specific lineage differentiating mediums, the markers of neuronal, hepatic, cardiomyocytic and pancreatic were found in differentiated cells. These results show that replacement of FBS by amniotic fluid and in situ cryopreservation of amniotic epithelial cells is an effective approach to overcome limitations related to long-term preservation including differentiation during cryopreservation and decrease of viability.

The effects of preservation procedures on antibacterial property of amniotic membrane.

Amniotic membrane (AM), the innermost layer of the fetal membranes, has been widely employed in the surgical reconstruction and tissue engineering. Expression of the antimicrobial peptides such as defensins, elafin and SLPI which are essential elements of the innate immune system results in antibacterial properties of the AM. Preservation is necessary to reach a ready-to-use source of the AM. However, these methods might change the properties of the AM. The aim of this study was to evaluate antibacterial properties of the AM after preservation. Antibacterial property of the fresh AM was compared with cryopreserved and freeze-dried AM by modified disk diffusion method. Staphylococcus aureus ATCC 25923, Pseudomonas aeruginosa ATCC 27853, Escherichia coli ATCC 25922 and two clinical isolated strains of E. coli were cultured in Mueller Hinton agar and a piece of the AM was placed on agar surface. After 24h incubation, the inhibition zone was measured. In addition, one of the most important antibacterial peptides, elafin, was measured by ELISA assay before and after preservations procedures. Antibacterial properties of the AM were maintained after cryopreservation and freeze-drying. However, the inhibition zone was depending on the bacterial strains. The cryopreservation and freeze-drying procedures significantly decreased elafin which shows that antibacterial property is not limited to the effects of amniotic cells and the other components such as extracellular matrix may contribute in antibacterial effects. The promising results of this study show that the preserved AM is a proper substitute of the fresh AM to be employed in clinical situations.

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.

Angiogenesis and Re-endothelialization in decellularized scaffolds: Recent advances and current challenges in tissue engineering.

Decellularization of tissues and organs has recently become a promising approach in tissue engineering and regenerative medicine to circumvent the challenges of organ donation and complications of transplantations. However, one main obstacle to reaching this goal is acellular vasculature angiogenesis and endothelialization. Achieving an intact and functional vascular structure as a vital pathway for supplying oxygen and nutrients remains the decisive challenge in the decellularization/re-endothelialization procedure. In order to better understand and overcome this issue, complete and appropriate knowledge of endothelialization and its determining variables is required. Decellularization methods and their effectiveness, biological and mechanical characteristics of acellular scaffolds, artificial and biological bioreactors, and their possible applications, extracellular matrix surface modification, and different types of utilized cells are factors affecting endothelialization consequences. This review focuses on the characteristics of endothelialization and how to optimize them, as well as discussing recent developments in the process of re-endothelialization.

Antiproliferative and apoptotic effects of conditioned medium released from human amniotic epithelial stem cells on breast and cervical cancer cells.

INTRODUCTION: Human amniotic membrane (hAM) and its cells have been proposed for several clinical applications, including cancer therapy. However, reports on the anticancer effects of human amniotic epithelial stem cells-conditioned media (hAECs-CM) are limited. This work aims to evaluate the anticancer effects of hAECs-CM on cervical cancer and breast cancer cell lines in vitro. METHODS: Human term placentas were gained from uncomplicated Cesarean sections from healthy donor women. After amnion peeling from the chorion, its epithelial stem cells were isolated and cultured, and its conditioned medium (CM) was collected for experiments. MTT assay was performed to assess cancer cells viability. Migration rate of cancer cells was examined via wound healing assay. Cell-cycle distribution and apoptosis were determined using flow cytometry. RESULTS: Based on MTT assay hAECs-CM was cytotoxic against cancerous cell lines in a dose-time-dependent manner. After 48 h of treatment with hAECs-CM pure, the cell viability of breast cancer cells includes MCF-7 and MDA-MB-231 reached to 73.2% and 65.5%, respectively. In the same situation, HeLa cervical cancer cell line revealed the lowest viability by 47.3%. The wound-healing assay displayed an incomplete wound closure of scratched MDA-MB-231 cells and significant inhibition of cell migration after hAECs-CM treatment. The results also revealed that hAECs-CM exerted anti-proliferation activity by prompting cell cycle arrest and apoptosis of cancer cells.Conclusions: hAECs-CM is a potent candidate for inducing apoptosis and simultaneously inhibition of the proliferation and migration of cancer cells via inhibiting cell cycle blockade.

Immunogenicity of decellularized extracellular matrix scaffolds: a bottleneck in tissue engineering and regenerative medicine.

Tissue-engineered decellularized extracellular matrix (ECM) scaffolds hold great potential to address the donor shortage as well as immunologic rejection attributed to cells in conventional tissue/organ transplantation. Decellularization, as the key process in manufacturing ECM scaffolds, removes immunogen cell materials and significantly alleviates the immunogenicity and biocompatibility of derived scaffolds. However, the application of these bioscaffolds still confronts major immunologic challenges. This review discusses the interplay between damage-associated molecular patterns (DAMPs) and antigens as the main inducers of innate and adaptive immunity to aid in manufacturing biocompatible grafts with desirable immunogenicity. It also appraises the impact of various decellularization methodologies (i.e., apoptosis-assisted techniques) on provoking immune responses that participate in rejecting allogenic and xenogeneic decellularized scaffolds. In addition, the key research findings regarding the contribution of ECM alterations, cytotoxicity issues, graft sourcing, and implantation site to the immunogenicity of decellularized tissues/organs are comprehensively considered. Finally, it discusses practical solutions to overcome immunogenicity, including antigen masking by crosslinking, sterilization optimization, and antigen removal techniques such as selective antigen removal and sequential antigen solubilization.

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.

Retinoic acid loaded with chitosan nanoparticles improves spermatogenesis in scrotal hyperthermia in mice.

OBJECTIVE: High temperatures can trigger cellular oxidative stress and disrupt spermatogenesis, potentially leading to male infertility. We investigated the effects of retinoic acid (RA), chitosan nanoparticles (CHNPs), and retinoic acid loaded with chitosan nanoparticles (RACHNPs) on spermatogenesis in mice induced by scrotal hyperthermia (Hyp). METHODS: Thirty mice (weighing 25 to 30 g) were divided into five experimental groups of six mice each. The groups were as follows: control, Hyp induced by a water bath (43 °C for 30 minutes/day for 5 weeks), Hyp+RA (2 mg/kg/day), Hyp+CHNPs (2 mg/kg/72 hours), and Hyp+RACHNPs (4 mg/kg/72 hours). The mice were treated for 35 days. After the experimental treatments, the animals were euthanized. Sperm samples were collected for analysis of sperm parameters, and blood serum was isolated for testosterone measurement. Testis samples were also collected for histopathology assessment, reactive oxygen species (ROS) evaluation, and RNA extraction, which was done to compare the expression levels of the bax, bcl2, p53, Fas, and FasL genes among groups. Additionally, immunohistochemical staining was performed. RESULTS: Treatment with RACHNPs significantly increased stereological parameters such as testicular volume, seminiferous tubule length, and testicular cell count. Additionally, it increased testosterone concentration and improved sperm parameters. We observed significant decreases in ROS production and caspase-3 immunostaining in the RACHNP group. Moreover, the expression levels of bax, p53, Fas, and FasL significantly decreased in the groups treated with RACHNPs and RA. CONCLUSION: RACHNPs can be considered a potent antioxidative and antiapoptotic agent for therapeutic strategies in reproductive and regenerative medicine.