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The Effect of Chitosan/Alginate/Graphene Oxide Nanocomposites on Proliferation of Mouse Spermatogonial Stem Cells
Alaa Moeinzadeh1,2, Behnaz Ashtari3,4, Heriberto Garcia5
1Department of Anatomy, School of Medicine, Iran University of Medical Sciences, Tehran, Iran.
Abstract:
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.
Insights
This study developed a biocompatible nanocomposite scaffold using alginate, chitosan, and graphene oxide (GO) to enhance spermatogonial stem cell (SSC) proliferation, offering a promising solution for male infertility after cancer treatment.
Area of Science:
- Biomaterials Science
- Stem Cell Biology
- Reproductive Medicine
Background:
- Male survivors of childhood cancer often face azoospermia, necessitating methods to preserve or restore fertility.
- Spermatogonial stem cells (SSCs) are critical for sperm production, and their isolation and proliferation are key challenges.
- Extracellular matrix-mimicking scaffolds are essential for supporting SSC regeneration and growth.
Purpose of the Study:
- To evaluate the efficacy of alginate-chitosan-graphene oxide (GO) nanocomposite scaffolds in promoting SSC proliferation.
- To assess the biocompatibility and cytotoxicity of these novel scaffolds for in vitro applications.
Main Methods:
- Nanocomposite scaffolds (alginate, chitosan, GO) were fabricated and characterized using FTIR, XRD, and SEM.
- Cytotoxicity was evaluated via MTT assay.
- SSC proliferation and identity were assessed using qRT-PCR, flow cytometry (C-Kit, GFRα1 markers), and immunocytochemistry (PLZF protein).
Main Results:
- The ALGCS/GO30 nanocomposite scaffold demonstrated optimal biocompatibility and minimal cytotoxicity.
- Flow cytometry confirmed SSC identity.
- qRT-PCR and immunocytochemistry revealed significantly enhanced SSC proliferation and PLZF protein expression on the ALGCS/GO30 scaffold compared to controls.
Conclusions:
- The ALGCS/GO30 nanocomposite scaffold is biocompatible and effectively supports SSC attachment and proliferation in vitro.
- This scaffold represents a promising alternative for enhancing SSC growth, potentially aiding fertility restoration in cancer survivors.
