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Purpose <p>This study evaluated the effects of decellularized ovarian bioscaffolds and resveratrol-loaded polymeric nanoparticles (RLPN) on the in vitro culture, viability, and ultrastructural preservation of bovine secondary follicles.</p> Methods <p>Decellularized extracellular matrix (dECM) from cortical fragments was obtained by freeze–thaw cycles and sequential incubation in Triton X-100 and sodium dodecyl sulfate. Decellularization efficiency and extracellular matrix integrity were assessed by hematoxylin–eosin, Hoechst staining, scanning electron microscopy, and quantification of collagen and glycosaminoglycans. Bovine secondary follicles were isolated and cultured for 12&#xa0;days in either a two-dimensional (2D) system or in dECM scaffolds in medium supplemented with 0.02, 0.2, or 2.0&#xa0;µM RLNP, blank nanoparticles, or unencapsulated resveratrol. Follicular viability and ultrastructure were evaluated by calcein-AM/ethidium homodimer-1 staining and transmission electron microscopy. Expression of mRNA for catalase, superoxide dismutase, glutathione peroxidase 1, peroxiredoxin 6, and nuclear factor erythroid 2-related factor 2 was assessed by qRT-PCR. Quantitative data were analyzed by unpaired t-tests or one-way ANOVA, followed by Tukey’s test (<i>P</i> &lt; 0.05).</p> Results <p>Hematoxylin–eosin and Hoechst staining confirmed effective cell removal, while collagen, glycosaminoglycans, and ECM ultrastructure were preserved. Follicles cultured in the three-dimensional (3D) system showed increased viability, further enhanced by 0.02 or 2.00&#xa0;µM RLPN. Follicles cultured with 0.02&#xa0;µM RLPN exhibited well-preserved morphology, including intact zona pellucida, oocyte membrane, and organelles. qRT-PCR analysis revealed reduced mRNA expression of antioxidant-related genes in follicles cultured with RLNP.</p> Conclusion <p>The decellularization protocol effectively removed cellular content and preserved ECM structure and ultrastructure. 3D culture system combined with supplementation of 0.02&#xa0;µM RLNP supported follicular viability and ultrastructural preservation and was associated with transcriptional changes in antioxidant-related genes.</p>

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Decellularized ovarian bioscaffolds and resveratrol-loaded polymeric nanoparticles support in vitro viability and ultrastructural preservation of bovine preantral follicles

  • 期刊论文

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  • 发表日期：

    2026年3月17日

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  • Francisco das Chagas Costa,
  • Ernando Igo Teixeira de Assis,
  • Danisvânia Ripardo Nascimento,
  • Miguel Fernandes de Lima Neto,
  • Andreza Aguiar Silva,
  • Venância Antônia Nunes Azevedo,
  • Regislane Pinto Ribeiro,
  • Bianca Régia Silva,
  • Mariana Aragão Matos Donato,
  • Alice Vitória Frota Reis,
  • Josimar Oliveira Eloy,
  • José Roberto Viana Silva

摘要

Purpose

This study evaluated the effects of decellularized ovarian bioscaffolds and resveratrol-loaded polymeric nanoparticles (RLPN) on the in vitro culture, viability, and ultrastructural preservation of bovine secondary follicles.

Methods

Decellularized extracellular matrix (dECM) from cortical fragments was obtained by freeze–thaw cycles and sequential incubation in Triton X-100 and sodium dodecyl sulfate. Decellularization efficiency and extracellular matrix integrity were assessed by hematoxylin–eosin, Hoechst staining, scanning electron microscopy, and quantification of collagen and glycosaminoglycans. Bovine secondary follicles were isolated and cultured for 12 days in either a two-dimensional (2D) system or in dECM scaffolds in medium supplemented with 0.02, 0.2, or 2.0 µM RLNP, blank nanoparticles, or unencapsulated resveratrol. Follicular viability and ultrastructure were evaluated by calcein-AM/ethidium homodimer-1 staining and transmission electron microscopy. Expression of mRNA for catalase, superoxide dismutase, glutathione peroxidase 1, peroxiredoxin 6, and nuclear factor erythroid 2-related factor 2 was assessed by qRT-PCR. Quantitative data were analyzed by unpaired t-tests or one-way ANOVA, followed by Tukey’s test (P < 0.05).

Results

Hematoxylin–eosin and Hoechst staining confirmed effective cell removal, while collagen, glycosaminoglycans, and ECM ultrastructure were preserved. Follicles cultured in the three-dimensional (3D) system showed increased viability, further enhanced by 0.02 or 2.00 µM RLPN. Follicles cultured with 0.02 µM RLPN exhibited well-preserved morphology, including intact zona pellucida, oocyte membrane, and organelles. qRT-PCR analysis revealed reduced mRNA expression of antioxidant-related genes in follicles cultured with RLNP.

Conclusion

The decellularization protocol effectively removed cellular content and preserved ECM structure and ultrastructure. 3D culture system combined with supplementation of 0.02 µM RLNP supported follicular viability and ultrastructural preservation and was associated with transcriptional changes in antioxidant-related genes.