3D-printed scaffolds with controlled-releasing compounds for ectopic activation of dormant ovarian follicles

Abstract

Throughout a woman’s reproductive life, hundreds of functioning follicles are activated for development, while thousands of dormant follicles remain in the ovaries. In vitro dormant follicle activation is an effective clinical strategy for women with fertility needs facing ovarian dysfunction. However, in vivo dormant follicle activation remains a challenge due to difficulties such as delivering stimulators to local sites. Here, we developed a compound-preloaded microporous scaffold by combining three-dimensional (3D) printing techniques with pharmacological activators to support and stimulate the activation and development of primordial follicles. The gelatin/alginate composite scaffolds exhibited exceptional mechanical properties and biological compatibility, effectively supporting the survival of ovarian granulosa cells for more than 7 days, which is essential for oocyte development. Furthermore, the ovarian tissue scaffold complex successfully survived after transplantation under the mouse kidney capsule, demonstrating its excellent biocompatibility. After pre-mixing widely used clinical activators into the bioink, the scaffold could gradually release the mixture of compounds, effectively activating primordial follicles. By transplanting the ovary–scaffold complex containing activators into the mouse abdominal subcutaneous pocket, dormant follicles could be activated subcutaneously and developed into growing follicles. The number of growing follicles is approximately three times higher compared to the group without activators. In conclusion, by integrating biomaterials, activators, and 3D printing technology, we have developed 3D-printed biological scaffolds that can ectopically support primordial follicle activation and development in vivo. This novel approach could provide a promising strategy for treating ovarian insufficiency and endocrine disorders in the clinic, without the need for in situ ovarian tissue grafting.

Keywords

3D printing / Scaffold / Dormant ovarian follicles / Activation / Transplantation

Funding

This study was supported by the National Natural Science Foundation of China (82301907), Guangdong Basic and Applied Basic Research Foundation (2024A1515010059 and 2024A1515030279), Shenzhen Medical Research Fund (B2404004), Shenzhen Science and Technology Program (JCYJ20220818101218040, JCYJ20220818103608017, JCYJ20220818103607015, JCYJ20220818102811025, JSGGKQTD20210831174330015), Shenzhen Key Laboratory of Metabolic Health (Grant No. ZDSYS20210427152400001), Sino-European Center of Biomedicine and Health.

Conflict of interest

The authors declare there is no conflict of interest.

References

[1] Liu CM, Ding LJ, Li JY, Dai JW, Sun HX. Advances in the study of ovarian dysfunction with aging. Hereditas. 2019; 41(9): 816-826. doi: 10.16288/j.yczz.19-134

[2] Harada M. Cellular senescence in the pathogenesis of ovarian dysfunction. J Obstet Gynaecol Res. 2024; 50(5): 800-808. doi: 10.1111/jog.15918

[3] Zhang T, He M, Zhang J, et al. Mechanisms of primordial follicle activation and new pregnancy opportunity for premature ovarian failure patients. Front Physiol. 2023; 14: 1113684. doi: 10.3389/fphys.2023.1113684

[4] Ford EA, Beckett EL, Roman SD, McLaughlin EA, Sutherland JM. Advances in human primordial follicle activation and premature ovarian insufficiency. Reproduction. 2020; 159(1): R15-R29. doi: 10.1530/rep-19-0201

[5] Luyckx V, Dolmans MM, Vanacker J, et al. A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. Fertil Steril. 2014; 101(4): 1149-1156. doi: 10.1016/j.fertnstert.2013.12.025

[6] Vanacker J, Luyckx V, Dolmans MM, et al. Transplantation of an alginate-matrigel matrix containing isolated ovarian cells: first step in developing a biodegradable scaffold to transplant isolated preantral follicles and ovarian cells. Biomaterials. 2012; 33(26): 6079-6085. doi: 10.1016/j.biomaterials.2012.05.015

[7] Shikanov A, Xu M, Woodruff TK, Shea LD. Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development. Biomaterials. 2009; 30(29): 5476-5485. doi: 10.1016/j.biomaterials.2009.06.054

[8] Xu M, Fazleabas AT, Shikanov A, et al. In vitro oocyte maturation and preantral follicle culture from the luteal-phase baboon ovary produce mature oocytes. Biol Reprod. 2011; 84(4): 689-697. doi: 10.1095/biolreprod.110.088674

[9] Kreeger PK, Deck JW, Woodruff TK, Shea LD. The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. Biomaterials. 2006; 27(5): 714-723. doi: 10.1016/j.biomaterials.2005.06.016

[10] Shikanov A, Zhang Z, Xu M, et al. Fibrin encapsulation and vascular endothelial growth factor delivery promotes ovarian graft survival in mice. Tissue Eng Part A. 2011; 17(23-24): 3095-3104. doi: 10.1089/ten.TEA.2011.0204

[11] Kniazeva E, Hardy AN, Boukaidi SA, Woodruff TK, Jeruss JS, Shea LD. Primordial follicle transplantation within designer biomaterial grafts produce live births in a mouse infertility model. Sci Rep. 2015; 5: 17709. doi: 10.1038/srep17709

[12] Henning NFC, Jakus AE, Laronda MM. Building organs using tissue-specific microenvironments: perspectives from a bioprosthetic ovary. Trends Biotechnol. 2021; 39(8): 824-837. doi: 10.1016/j.tibtech.2021.01.008

[13] Jin Z, Li Y, Yu K, et al. 3D printing of physical organ models: recent developments and challenges. Adv Sci. 2021; 8(17): e2101394. doi: 10.1002/advs.202101394

[14] Nair R, Kasturi M, Mathur V, Seetharam RN, S Vasanthan K. Strategies for developing 3D printed ovarian model for restoring fertility. Clin Transl Sci. 2024; 17(7): e13863. doi: 10.1111/cts.13863

[15] Pennarossa G, Arcuri S, De Iorio T, Gandolfi F, Brevini TAL. Current advances in 3D tissue and organ reconstruction. Int J Mol Sci. 2021; 22(2): 830. doi: 10.3390/ijms22020830

[16] Wu T, Gao YY, Su J, et al. Three-dimensional bioprinting of artificial ovaries by an extrusion-based method using gelatin-methacryloyl bioink. Climacteric. 2022;25(2): 170-178. doi: 10.1080/13697137.2021.1921726

[17] Laronda MM, Rutz AL, Xiao S, et al. A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice. Nat Commun. 2017; 8: 15261. doi: 10.1038/ncomms15261

[18] Aydin S, Yaşlı M, Yildiz Ş, Urman B. Advancements in three-dimensional bioprinting for reproductive medicine: a systematic review. Reprod Biomed Online. 2024; 49(4): 104273. doi: 10.1016/j.rbmo.2024.104273

[19] Gandolfi F, Ghiringhelli M, Brevini TAL. Bioengineering the ovary to preserve and reestablish female fertility. Anim Reprod. 2020; 16(1): 45-51. doi: 10.21451/1984-3143-ar2018-0099

[20] Zivari‐Ghader T, Dolati S, Mehdizadeh A, Davaran S, Rashidi MR, Yousefi M. Recent scaffold‐based tissue engineering approaches in premature ovarian failure treatment. J Tissue Eng Regen Med. 2022; 16(7): 605‐620. doi: 10.1002/term.3306

[21] Gao F, Xu Z, Liang Q, et al. Osteochondral regeneration with 3D-printed biodegradable high-strength supramolecular polymer reinforced-gelatin hydrogel scaffolds. Adv Sci. 2019; 6(15): 1900867. doi: 10.1002/advs.201900867

[22] Gao F, Xu ZY, Liang QF, et al. Direct 3D printing of high strength biohybrid gradient hydrogel scaffolds for efficient repair of osteochondral defect. Adv Funct Mater. 2018; 28(13): 1706644. doi: 10.1002/adfm.201706644

[23] Zhai X, Ruan C, Ma Y, et al. 3D-bioprinted osteoblast- Laden nanocomposite hydrogel constructs with induced microenvironments promote cell viability, differentiation, and osteogenesis both in vitro and in vivo. Adv Sci. 2018; 5(3): 1700550. doi: 10.1002/advs.201700550

[24] Sahoo J, Murthy PN, Biswal S, Sahoo SK, Mahapatra AK. Comparative study of propranolol hydrochloride release from matrix tablets with KollidonSR or hydroxy propyl methyl cellulose. AAPS PharmSciTech. 2008; 9(2): 577-582. doi: 10.1208/s12249-008-9092-2

[25] Yang YL, Sun LF, Yu Y, et al. Deficiency of Gpr1 improves steroid hormone abnormality in hyperandrogenized mice. Reprod Biol Endocrinol. 2018; 16(1): 50. doi: 10.1186/s12958-018-0363-9

[26] Sosnik A. Alginate particles as platform for drug delivery by the oral route: state-of-the-art. ISRN Pharm. 2014; 2014: 926157. doi: 10.1155/2014/926157

[27] Jaipan P, Nguyen A, Narayan RJ. Gelatin-based hydrogels for biomedical applications. MRS Commun. 2017; 7(3): 416-426. doi: 10.1557/mrc.2017.92

[28] Pepling ME. From primordial germ cell to primordial follicle: mammalian female germ cell development. Genesis. 2006; 44(12): 622-632. doi: 10.1002/dvg.20258

[29] O’Connell JM, Pepling ME. Primordial follicle formation – some assembly required. Curr Opin Endocr Metab Res. 2021;18;118-127. doi: 10.1016/j.coemr.2021.03.005

[30] Wesevich V, Kellen AN, Pal L. Recent advances in understanding primary ovarian insufficiency. F1000Res. 2020; 9: F1000 Faculty Rev-1101. doi: 10.12688/f1000research.26423.1

[31] Gill EL, Li X, Birch MA, Huang YYS. Multi-length scale bioprinting towards simulating microenvironmental cues. Biodes Manuf. 2018; 1(2): 77-88. doi: 10.1007/s42242-018-0014-1

[32] Xu C. Freeform vertical and horizontal fabrication of alginate-based vascular-like tubular constructs using inkjetting. J Manuf Sci Eng. 2014; 136(6): 061020.

[33] Sun X, Su Y, He Y, et al. New strategy for in vitro activation of primordial follicles with mTOR and PI3K stimulators. Cell Cycle. 2015; 14(5): 721-731. doi: 10.1080/15384101.2014.995496

[34] Castrillon DH, Miao L, Kollipara R, Horner JW, DePinho RA. Suppression of ovarian follicle activation in mice by the transcription factor Foxo3a. Science. 2003; 301(5630): 215-218. doi: 10.1126/science.1086336

[35] Liu L, Rajareddy S, Reddy P, et al. Infertility caused by retardation of follicular development in mice with oocyte-specific expression of Foxo3a. Development. 2007; 134(1): 199-209. doi: 10.1242/dev.02667

[36] Reddy P, Liu L, Adhikari D, et al. Oocyte-specific deletion of Pten causes premature activation of the primordial follicle pool. Science. 2008; 319(5863): 611-613. doi: 10.1126/science.1152257

[37] Adhikari D, Flohr G, Gorre N, et al. Disruption of Tsc2 in oocytes leads to overactivation of the entire pool of primordial follicles. Mol Hum Reprod. 2009; 15(12): 765-770. doi: 10.1093/molehr/gap092

[38] Adhikari D, Zheng W, Shen Y, et al. Tsc/mTORC1 signaling in oocytes governs the quiescence and activation of primordial follicles. Hum Mol Genet. 2010; 19(3): 397-410. doi: 10.1093/hmg/ddp483

[39] Cheng Y, Kim J, Li XX, Hsueh AJ. Promotion of ovarian follicle growth following mTOR activation: synergistic effects of AKT stimulators. PLoS One. 2015; 10(2): e0117769. doi: 10.1371/journal.pone.0117769