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Scientific reports2025; 15(1); 19759; doi: 10.1038/s41598-025-04013-4

Development of a two-layer 3D equine endometrial tissue model using genipin-crosslinked collagen scaffolds and 3D printing.

Abstract: Advances in endometrial tissue engineering have enabled the combination of modified scaffolding materials with modern cell culture technologies. Genipin and three-dimensional (3D) printing have advanced cell-tissue engineering by enabling the precise layering of cell-containing matrices while ensuring low cytotoxicity. This study aimed to advance equine endometrial tissue engineering by designing customized collagen scaffolds using 3D printing technology, while optimizing the genipin concentration to avoid toxicity. Genipin was tested at concentrations of 4, 2, 1, 0.5, 0.25, 0.125, and 0 mM on equine endometrial epithelial cells (eECs) and mesenchymal stromal cells (eMSCs). Its effects on cell morphology and scaffold properties were evaluated in collagen-based conventional equine endometrial tissue (3D-ET) by assessing percentage of cells spreading within each genipin concentration. Additionally, genipin-collagen scaffolds at 2, 1, 0.5, 0.25, and 0 mM were analyzed for viscoelastic properties using rheological testing. Based on these assessments, 0.5 mM genipin was identified as the optimal concentration and was to develop in vitro 3D-ET. Key 3D printing parameters, including extrusion pressure, printing temperature, pre-printing time, and velocity, were optimized. The structural integrity of the advanced 3D-ET was assessed via phase contrast microscopy. Cellular characterization was performed using Pan-cytokeratin and Vimentin staining. For the characterization of printed 3D-ET, mucin production was assessed using Alcian blue staining, while estrogen receptor alpha (ERα) expression was evaluated by immunofluorescence. A study of oxytocin-stimulated prostaglandin synthesis capacity was performed in an advanced 3D-ET for 24 h, and expression of key genes was analyzed quantitatively using real-time PCR. Genipin exhibited dose-dependent toxicity, with 0.5 mM identified as the optimal concentration based on its support of proliferative activity, cell morphology, and viscoelastic properties. Only eMSCs were successfully 3D-printed in a collagen scaffold with 0.5 mM genipin. While the 3D-printed biomaterial failed to support eECs viability; eECs survived and formed glands only when a conventional seeding method was used. Consequently, a dual-layer 3D-ET model was developed in which eMSCs were printed with 0.5 mM genipin-collagen, and eECs were overlain using conventional methods. This model preserved the structural integrity necessary for glandular-like development and maintained the functional characteristics of equine endometrial tissue. Mucin production was observed, while ERα was detected in the cytoplasm and translocated into the nucleus.Notably, after OT challenge prostaglandin-endoperoxide synthase 2 (PTGS2) expression was significantly elevated in the treatment group compared to controls (p < 0.05). This advanced 3D-ET model offers a robust platform for studying tissue-specific functions and could be further developed by incorporating immune or endothelial cells or creating complex structures such as glands or vessels.
© 2025. The Author(s).
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Publication Date: 2025-06-05 PubMed ID: 40473768PubMed Central: PMC12141639DOI: 10.1038/s41598-025-04013-4Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The study focuses on improving the equine endometrial tissue engineering using three-dimensional (3D) printing technology and a chemical called genipin. The research identified the optimal genipin concentration that supports the growth of cells without toxicity and combined it with collagen scaffolds in 3D printing to replicate an equine endometrial tissue structure.

Understanding Genipin and its Role

  • The researchers experimented with various concentrations of genipin, a compound known for its low cytotoxicity, on equine endometrial epithelial cells (eECs) and mesenchymal stromal cells (eMSCs).
  • They observed the effects of genipin on cell morphologies (shapes) and scaffold properties in collagen-based endometrial tissue.
  • The genipin concentration was adjusted to find the concentration that offers the best balance between cellular support and toxicity. It was eventually discovered that 0.5mM was safe and effective.

3D Printing of Endometrial Tissue

  • 3D printing technology was used to create customized collagen scaffolds, physical structures in which the eMSCs could be ‘printed’.
  • Various 3D printing parameters — including the extrusion pressure, printing temperature, pre-printing time, and velocity — were optimized to print the cells accurately.

Model Assessment and Characteristics

  • The structural integrity and cellular characteristics of the subsequent 3D printed tissue were assessed. It was found that the combination of genipin and 3D-printing encourages gland-like development and maintains the function of endometrial tissue.
  • Further analysis was conducted on the expression of estrogen receptor alpha (ERα) and mucin production, essential aspects of endometrial health and function.

Limits of Current Models

  • It was discovered that while the genipin-collagen scaffold successfully supported eMSCs, the model could not support the viability of eECs. Therefore, a dual-layer model was developed where eMSCs were printed using the genipin-collagen scaffold, and eECs overlaid using conventional methods.
  • This model maintains the structural integrity necessary for gland-like development and the functional characteristics of equine endometrial tissue.

Potential for Further Research

  • The research acknowledges that further development could include incorporating immune or endothelial cells or creating complex structures such as glands or vessels, providing a more robust platform for studying tissue-specific functions.

Cite This Article

APA
Santiviparat S, Suthithanakom S, Bhanpattanakul S, Srisuwattanasagul S, Melde K, Stout TAE, Tharasanit T. (2025). Development of a two-layer 3D equine endometrial tissue model using genipin-crosslinked collagen scaffolds and 3D printing. Sci Rep, 15(1), 19759. https://doi.org/10.1038/s41598-025-04013-4

Publication

Scientific reports
ISSN: 2045-2322
NlmUniqueID: 101563288
Country: England
Language: English
Volume: 15
Issue: 1
Pages: 19759
PII: 19759

Researcher Affiliations

Santiviparat, Sawita
  • Center of Excellence for Veterinary Clinical Stem Cells and Bioengineering, Chulalongkorn University, Bangkok, Thailand.
  • Center of Excellence in Animal Fertility Chulalongkorn University (CU-AF), Chulalongkorn University, Bangkok, Thailand.
  • Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Suthithanakom, Setthibhak
  • Institute for Molecular Systems Engineering and Advanced Materials, Heidelberg University, Im Neuenheimer Feld 225, 69120, Heidelberg, Germany.
  • Micro/Nano Electromechanical Integrated Device Research Unit, Faculty of Engineering, Chulalongkorn University, Bangkok, Thailand.
  • Max Plank Institute for Medical Research, Jahnstr.29, 69120, Heidelberg, Germany.
Bhanpattanakul, Sudchaya
  • Center of Excellence for Veterinary Clinical Stem Cells and Bioengineering, Chulalongkorn University, Bangkok, Thailand.
  • Department of Pathology, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand.
Srisuwattanasagul, Sayamon
  • Department of Anatomy, Faculty of Veterinary, Science Chulalongkorn University, Bangkok, Thailand.
Melde, Kai
  • Institute for Molecular Systems Engineering and Advanced Materials, Heidelberg University, Im Neuenheimer Feld 225, 69120, Heidelberg, Germany.
  • Max Plank Institute for Medical Research, Jahnstr.29, 69120, Heidelberg, Germany.
Stout, Tom A E
  • Maxwell H. Gluck Equine Research Center, University of Kentucky, Lexington, KY, USA.
Tharasanit, Theerawat
  • Center of Excellence for Veterinary Clinical Stem Cells and Bioengineering, Chulalongkorn University, Bangkok, Thailand. Theerawat.t@chula.ac.th.
  • Center of Excellence in Animal Fertility Chulalongkorn University (CU-AF), Chulalongkorn University, Bangkok, Thailand. Theerawat.t@chula.ac.th.
  • Department of Obstetrics, Gynecology and Reproduction, Faculty of Veterinary Science, Chulalongkorn University, Bangkok, Thailand. Theerawat.t@chula.ac.th.

MeSH Terms

  • Animals
  • Printing, Three-Dimensional
  • Iridoids / chemistry
  • Iridoids / pharmacology
  • Female
  • Endometrium / cytology
  • Endometrium / metabolism
  • Tissue Scaffolds / chemistry
  • Horses
  • Tissue Engineering / methods
  • Collagen / chemistry
  • Mesenchymal Stem Cells / cytology
  • Mesenchymal Stem Cells / drug effects
  • Epithelial Cells / cytology
  • Epithelial Cells / drug effects
  • Cells, Cultured

Conflict of Interest Statement

Declarations. Competing interests: The authors declare no competing interests. Conflict of interest: The authors have no conflicts of interest to declare.

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Citations

This article has been cited 1 times.
  1. Grumezescu V, Duta L. Collagen Type I as a Biological Barrier Interface in Biomimetic Microfluidic Devices: Properties, Applications, and Challenges. Biomimetics (Basel) 2026 Jan 13;11(1).
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