In-vitro and in-vivo evaluation of angiogenic potential of a novel lithium chloride loaded silk fibroin / alginate 3D porous scaffold with antibacterial activity, for promoting diabetic wound healing.

Healing diabetic ulcers with chronic inflammation is a major challenge for researchers and professionals, necessitating new strategies. To rapidly treat diabetic wounds in rat models, we have fabricated a composite scaffold composed of alginate (Alg) and silk fibroin (SF) as a wound dressing that is laden with molecules of lithium chloride (LC). The physicochemical, bioactivity, and biocompatibility properties of Alg-SF-LC scaffolds were investigated in contrast to those of Alg, SF, and Alg-SF ones. Afterward, full-thickness wounds were ulcerated in diabetic rats in order to evaluate the capacity of LC-laden scaffolds to regenerate skin. The characterization findings demonstrated that the composite scaffolds possessed favorable antibacterial properties, cell compatibility, high swelling, controlled degradability, and good uniformity in the interconnected pore microstructure. Additionally, in terms of wound contraction, re-epithelialization, and angiogenesis improvement, LC-laden scaffolds revealed better performance in diabetic wound healing than the other groups. This research indicates that utilizing lithium chloride molecules loaded in biological materials supports the best diabetic ulcer regeneration in vivo, and produces a skin replacement with a cellular structure comparable to native skin.

Advanced strategies for single embryo selection in assisted human reproduction: A review of clinical practice and research methods.

Among the primary objectives of contemporary assisted reproductive technology research are achieving the births of healthy singletons and improving overall fertility outcomes. Substantial advances have been made in refining the selection of single embryos for transfer, with the aim of maximizing the likelihood of successful implantation. The principal criterion for this selection is embryo morphology. Morphological evaluation systems are based on traditional parameters, including cell count and fragmentation, pronuclear morphology, cleavage rate, blastocyst formation, and various sequential embryonic assessments. To reduce the incidence of multiple pregnancies and to identify the single embryo with the highest potential for growth, invasive techniques such as preimplantation genetic screening are employed in in vitro fertilization clinics. However, new approaches have been suggested for clinical application that do not harm the embryo and that provide consistent, accurate results. Noninvasive technologies, such as time-lapse imaging and omics, leverage morphokinetic parameters and the byproducts of embryo metabolism, respectively, to identify noninvasive prognostic markers for competent single embryo selection. While these technologies have garnered considerable interest in the research community, they are not incorporated into routine clinical practice and still have substantial room for improvement. Currently, the most promising strategies involve integrating multiple methodologies, which together are anticipated to increase the likelihood of successful pregnancy.

In vitro proliferation and differentiation of mouse spermatogonial stem cells in decellularized human placenta matrix.

Utilizing natural scaffold production derived from extracellular matrix components presents a promising strategy for advancing in vitro spermatogenesis. In this study, we employed decellularized human placental tissue as a scaffold, upon which neonatal mouse spermatogonial cells (SCs) were cultured three-dimensional (3D) configuration. To assess cellular proliferation, we examined the expression of key markers (Id4 and Gfrα1) at both 1 and 14 days into the culture. Our quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis revealed a notable increase in Gfrα1 gene expression, with the 3D culture group exhibiting the highest levels. Furthermore, the relative frequency of Gfrα1-positive cells significantly rose from 38.1% in isolated SCs to 46.13% and 76.93% in the two-dimensional (2D) and 3D culture systems, respectively. Moving forward to days 14 and 35 of the culture period, we evaluated the expression of differentiating markers (Sycp3, acrosin, and Protamine 1). Sycp3 and Prm1 gene expression levels were upregulated in both 2D and 3D cultures, with the 3D group displaying the highest expression. Additionally, acrosin gene expression increased notably within the 3D culture. Notably, at the 35-day mark, the percentage of Prm1-positive cells in the 3D group (36.4%) significantly surpassed that in the 2D group (10.96%). This study suggests that the utilization of placental scaffolds holds significant promise as a bio-scaffold for enhancing mouse in vitro spermatogenesis.

Therapeutic potential of exosomes in spermatogenesis regulation and male infertility.

BACKGROUND: Spermatogenesis is a fundamental process crucial for male reproductive health and fertility. Exosomes, small membranous vesicles released by various cell types, have recently garnered attention for their role in intercellular communication. OBJECTIVE: This review aims to comprehensively explore the role of exosomes in regulating spermatogenesis, focusing on their involvement in testicular development and cell-to-cell communication. METHODS: A systematic examination of literature was conducted to gather relevant studies elucidating the biogenesis, composition, and functions of exosomes in the context of spermatogenesis. RESULTS: Exosomes play a pivotal role in orchestrating the complex signaling networks required for proper spermatogenesis. They facilitate the transfer of key regulatory molecules between different cell populations within the testes, including Sertoli cells, Leydig cells, and germ cells. CONCLUSION: The emerging understanding of exosome-mediated communication sheds light on novel mechanisms underlying spermatogenesis regulation. Further research in this area holds promise for insights into male reproductive health and potential therapeutic interventions.

In vivo and in vitro sperm production: an overview of the challenges and advances in male fertility restoration.

Male infertility can be caused by genetic anomalies, endocrine disorders, inflammation, and exposure to toxic chemicals or gonadotoxic treatments. Therefore, several recent studies have concentrated on the preservation and restoration of fertility to enhance the quality of life for affected individuals. It is currently recommended to biobank the tissue extracted from testicular biopsies to provide a later source of spermatogonial stem cells (SSCs). Another successful approach has been the in vitro production of haploid male germ cells. The capacity of SSCs to transform into sperm, as in testicular tissue transplantation, SSC therapy, and in vitro or ex vivo spermatogenesis, makes them ideal candidates for in vivo fertility restoration. The transplantation of SSCs or testicular tissue to regenerate spermatogenesis and create embryos has been achieved in nonhuman mammal species. Although the outcomes of human trials have yet to be released, this method may soon be approved for clinical use in humans. Furthermore, regenerative medicine techniques that develop tissue or cells on organic or synthetic scaffolds enriched with bioactive molecules have also gained traction. All of these methods are now in different stages of experimentation and clinical trials. However, thanks to rigorous studies on the safety and effectiveness of SSC-based reproductive treatments, some of these techniques may be clinically available in upcoming decades.

Testicular niche repair after gonadotoxic treatments: Current knowledge and future directions.

The testicular niche, which includes the germ cells, somatic cells, and extracellular matrix, plays a crucial role in maintaining the proper functions of the testis. Gonadotoxic treatments, such as chemotherapy and radiation therapy, have significantly improved the survival rates of cancer patients but have also been shown to have adverse effects on the testicular microenvironment. Therefore, repairing the testicular niche after gonadotoxic treatments is essential to restore its function. In recent years, several approaches, such as stem cell transplantation, gene therapy, growth factor therapy, and pharmacological interventions have been proposed as potential therapeutic strategies to repair the testicular niche. This comprehensive review aims to provide an overview of the current understanding of testis damage and repair mechanisms. We will cover a range of topics, including the mechanism of gonadotoxic action, repair mechanisms, and treatment approaches. Overall, this review highlights the importance of repairing the testicular niche after gonadotoxic treatments and identifies potential avenues for future research to improve the outcomes for cancer survivors.

Polycaprolactone/Testicular Extracellular Matrix/Graphene Oxide-Based Electrospun Tubular Scaffolds for Reproductive Medicine: Biomimetic Architecture of Seminiferous Tubules.

Numerous scaffolds are developed in the field of testicular bioengineering. However, effectively replicating the spatial characteristics of native tissue, poses a challenge in maintaining the requisite cellular arrangement essential for spermatogenesis. In order to mimic the structural properties of seminiferous tubules, the objective is to fabricate a biocompatible tubular scaffold. Following the decellularization process of the testicular tissue, validation of cellular remnants' elimination from the specimens is conducted using 4',6-diamidino-2-phenylindole staining, hematoxylin and eosin staining, and DNA content analysis. The presence of extracellular matrix (ECM) components is confirmed through Alcian blue, Orcein, and Masson's trichrome staining techniques. The electrospinning technique is employed to synthesize the scaffolds using polycaprolactone (PCL), extracted ECM, and varying concentrations of graphene oxide (GO) (0.5%, 1%, and 2%). Subsequently, comprehensive evaluations are performed to assess the properties of the synthetic scaffolds. These evaluations encompass Fourier-transform infrared spectroscopy, scanning electron microscopy imaging, scaffold degradation testing, mechanical behavior analysis, methylthiazolyldiphenyl-tetrazolium bromide assay, and in vivo biocompatibility assessment. The PCL/decellularized extracellular matrix with 0.5% GO formulation exhibits superior fiber morphology and enhanced mechanical properties, and outperforms other groups in terms of in vitro biocompatibility. Consequently, these scaffolds present a viable option for implementation in "in vitro spermatogenesis" procedures, holding promise for future sperm production from spermatogonial cells.

Ultrastructural study: in vitro and in vivo differentiation of mice spermatogonial stem cells.

Mouse testicular tissue is composed of seminiferous tubules and interstitial tissue. Mammalian spermatogenesis is divided into three stages: spermatocytogenesis (mitotic divisions) in which spermatogonial stem cells (SSCs) turn into spermatocytes, followed by two consecutive meiotic divisions in which spermatocytes form spermatids. Spermatids differentiate into spermatozoa during spermiogenesis. Various factors affect the process of spermatogenesis and the organization of cells in the testis. Any disorder in different stages of spermatogenesis will have negative effects on male fertility. The aim of the current study was to compare the in vitro and in vivo spermatogenesis processes before and after transplantation to azoospermic mice using ultrastructural techniques. In this study, mice were irradiated with single doses of 14 Gy 60Co radiation. SSCs isolated from neonatal mice were cultured in vitro for 1 week and were injected into the seminiferous tubule recipient's mice. Testicular cells of neonatal mice were cultured in the four groups on extracellular matrix-based 3D printing scaffolds. The transplanted testes (8 weeks after transplantation) and cultured testicular cells in vitro (after 3 weeks) were then processed for transmission electron microscopy studies. Our study's findings revealed that the morphology and ultrastructure of testicular cells after transplantation and in vitro culture are similar to those of in vivo spermatogenesis, indicating that spermatogenic cell nature is unaltered in vitro.

The Effect of Chitosan/Alginate/Graphene Oxide Nanocomposites on Proliferation of Mouse Spermatogonial Stem Cells.

Male survivors of childhood cancer have been known to be afflicted with azoospermia. To combat this, the isolation and purification of spermatogonial stem cells (SSCs) are crucial. Implementing scaffolds that emulate the extracellular matrix environment is vital for promoting the regeneration and proliferation of SSCs. This research aimed to evaluate the efficiency of nanocomposite scaffolds based on alginate, chitosan, and graphene oxide (GO) in facilitating SSCs proliferation. To analyze the cytotoxicity of the scaffolds, an MTT assay was conducted at 1, 3, and 7 days, and the sample containing 30 µg/mL of GO (ALGCS/GO30) exhibited the most favorable results, indicating its optimal performance. The identity of the cells was confirmed using flow cytometry with C-Kit and GFRα1 markers. The scaffolds were subjected to various analyses to characterize their properties. FTIR was employed to assess the chemical structure, XRD to examine crystallinity, and SEM to visualize the morphology of the scaffolds. To evaluate the proliferation of SSCs, qRT-PCR was used. The study's results demonstrated that the ALGCS/GO30 nanocomposite scaffold exhibited biocompatibility and facilitated the attachment and proliferation of SSCs. Notably, the scaffold displayed a significant increase in proliferation markers compared to the control group, indicating its ability to support SSC growth. The expression level of the PLZF protein was assessed using the Immunocytochemistry method. The observations confirmed the qRT-PCR results, which indicated that the nanocomposite scaffolds had higher levels of PLZF protein expression than scaffolds without GO. The biocompatible ALGCS/GO30 is a promising alternative for promoting SSC proliferation in in vitro applications.

Differentiation of human primary testicular cells in the presence of SCF using the organoid culture system.

PURPOSE: Development of organoids using human primary testicular cells has remained a challenge due to the complexity of the mammalian testicular cytoarchitecture and culture methods. In this study, we generated testicular organoids derived from human primary testicular cells. Then, we evaluated the effect of stem cell factor (SCF) on cell differentiation and apoptosis in the testicular organoid model. METHODS: The testicular cells were harvested from the three brain-dead donors. Human spermatogonial stem cells (SSCs) were characterized using immunocytochemistry (ICC), RT-PCR and flow cytometry. Testicular organoids were generated from primary testicular cells by hanging drop culture method and were cultured in three groups: control group, experimental group 1 (treated FSH and retinoic acid (RA)), and experimental group 2 (treated FSH, RA and SCF), for five weeks. We assessed the expression of SCP3 (Synaptonemal Complex Protein 3) as a meiotic gene, PRM2 (Protamine 2) as a post-meiotic marker and apoptotic genes of Bax (BCL2-Associated X Protein) and Bcl-2 (B-cell lymphoma 2), respectively by using RT-qPCR. In addition, we identified the expression of PRM2 by immunohistochemistry (IHC). RESULTS: Relative expression of SCP3, PRM2 and Bcl-2 were highest in group 2 after five weeks of culture. In contrast, BAX expression level was lower in experimental group 2 in comparison with other groups. IHC analyses indicated the highest expression of PRM2 as a postmeiotic marker in group 2 in comparison to 2D culture and control groups but not find significant differences between experimental group 1 and experimental group 2 groups. Morphological evaluations revealed that organoids are compact spherical structures and in the peripheral region composed of uncharacterized elongated fibroblast-like cells. CONCLUSION: Our findings revealed that the testicular organoid culture system promote the spermatogonial stem cell (SSC) differentiation, especially in presence of SCF. Developed organoids are capable of recapitulating many important properties of a stem cell niche.