A bioprosthetic ovary created using 3D printed microporous scaffolds restores ovarian function in sterilized mice

Emerging additive manufacturing techniques enable investigation of the effects of pore geometry on cell behavior and function. Here, we 3D print microporous hydrogel scaffolds to test how varying pore geometry, accomplished by manipulating the advancing angle between printed layers, affects the survival of ovarian follicles. 30° and 60° scaffolds provide corners that surround follicles on multiple sides while 90° scaffolds have an open porosity that limits follicle–scaffold interaction. As the amount of scaffold interaction increases, follicle spreading is limited and survival increases. Follicle-seeded scaffolds become highly vascularized and ovarian function is fully restored when implanted in surgically sterilized mice. Moreover, pups are born through natural mating and thrive through maternal lactation. These findings present an in vivo functional ovarian implant designed with 3D printing, and indicate that scaffold pore architecture is a critical variable in additively manufactured scaffold design for functional tissue engineering.

[1]  J. Segars,et al.  Dynamic Reciprocity Between Cells and Their Microenvironment in Reproduction1 , 2015, Biology of reproduction.

[2]  L. Shea,et al.  A new hypothesis regarding ovarian follicle development: ovarian rigidity as a regulator of selection and health , 2010, Journal of Assisted Reproduction and Genetics.

[3]  Ali Khademhosseini,et al.  3D biofabrication strategies for tissue engineering and regenerative medicine. , 2014, Annual review of biomedical engineering.

[4]  A. Fazleabas,et al.  In Vitro Oocyte Maturation and Preantral Follicle Culture from the Luteal-Phase Baboon Ovary Produce Mature Oocytes1 , 2011, Biology of reproduction.

[5]  L. Kjeldsen,et al.  Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia. , 2010, Fertility and sterility.

[6]  Lisa E. Freed,et al.  Accordion-Like Honeycombs for Tissue Engineering of Cardiac Anisotropy , 2008, Nature materials.

[7]  L. Shea,et al.  Distribution of extracellular matrix proteins type I collagen, type IV collagen, fibronectin, and laminin in mouse folliculogenesis , 2006, Histochemistry and Cell Biology.

[8]  J. Smitz,et al.  Fertility preservation in women with cancer , 2014, The Lancet.

[9]  G. Prestwich,et al.  Photocrosslinkable hyaluronan-gelatin hydrogels for two-step bioprinting. , 2010, Tissue engineering. Part A.

[10]  L. Shea,et al.  Interpenetrating fibrin-alginate matrices for in vitro ovarian follicle development. , 2009, Biomaterials.

[11]  F. Lin,et al.  Generation of three-dimensional hepatocyte/gelatin structures with rapid prototyping system. , 2006, Tissue engineering.

[12]  Hai Yao,et al.  Regeneration of the articular surface of the rabbit synovial joint by cell homing: a proof of concept study , 2010, The Lancet.

[13]  D. Paul,et al.  Female infertility in mice lacking connexin 37 , 1997, Nature.

[14]  T. Strowitzki,et al.  Preservation of Fertility in Patients with Cancer , 2009 .

[15]  Jiwon Kim,et al.  Synthetic hydrogel supports the function and regeneration of artificial ovarian tissue in mice , 2016, npj Regenerative Medicine.

[16]  A. des Rieux,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. , 2012, Biomaterials.

[17]  S. Corathers,et al.  Late endocrine effects of childhood cancer , 2016, Nature Reviews Endocrinology.

[18]  J. Kim,et al.  Recreating the female reproductive tract in vitro using iPSC technology in a linked microfluidics environment , 2013, Stem Cell Research & Therapy.

[19]  E. Ernst,et al.  Outcomes of transplantations of cryopreserved ovarian tissue to 41 women in Denmark. , 2015, Human reproduction.

[20]  E. Rosseeva,et al.  An NMR Study of Biomimetic Fluorapatite – Gelatine Mesocrystals , 2015, Scientific Reports.

[21]  J. Plendl.  Angiogenesis and Vascular Regression in the Ovary * , 2000, Anatomia, histologia, embryologia.

[22]  R. Gosden,et al.  Transplantation of frozen-thawed mouse primordial follicles. , 1993, Human reproduction.

[23]  L. Shea,et al.  Primordial Follicle Transplantation within Designer Biomaterial Grafts Produce Live Births in a Mouse Infertility Model , 2015, Scientific Reports.

[24]  Min Xu,et al.  Tissue-engineered follicles produce live, fertile offspring. , 2006, Tissue engineering.

[25]  Adam E Jakus,et al.  Initiation of puberty in mice following decellularized ovary transplant. , 2015, Biomaterials.

[26]  Antonios G Mikos,et al.  Gelatin as a delivery vehicle for the controlled release of bioactive molecules. , 2005, Journal of controlled release : official journal of the Controlled Release Society.

[27]  Martin M. Matzuk,et al.  Intercellular Communication in the Mammalian Ovary: Oocytes Carry the Conversation , 2002, Science.

[28]  Nupura S. Bhise,et al.  Direct-write bioprinting of cell-laden methacrylated gelatin hydrogels , 2014, Biofabrication.

[29]  Fertility preservation in patients undergoing gonadotoxic therapy or gonadectomy: a committee opinion. , 2019, Fertility and sterility.

[30]  Mark A. Skylar-Scott,et al.  Three-dimensional bioprinting of thick vascularized tissues , 2016, Proceedings of the National Academy of Sciences.

[31]  N B Schwartz,et al.  Dynamic changes in inhibin messenger RNAs in rat ovarian follicles during the reproductive cycle. , 1988, Science.

[32]  Beatriz Peñalver Bernabé,et al.  Promoting extracellular matrix remodeling via ascorbic acid enhances the survival of primary ovarian follicles encapsulated in alginate hydrogels , 2014, Biotechnology and bioengineering.

[33]  L. Shea,et al.  Size-specific follicle selection improves mouse oocyte reproductive outcomes. , 2015, Reproduction.

[34]  J. Lewis,et al.  3D Bioprinting of Vascularized, Heterogeneous Cell‐Laden Tissue Constructs , 2014, Advanced materials.

[35]  L. Brinson,et al.  Multi-modal magnetic resonance elastography for noninvasive assessment of ovarian tissue rigidity in vivo. , 2015, Acta biomaterialia.

[36]  P. R. van Weeren,et al.  Gelatin-methacrylamide hydrogels as potential biomaterials for fabrication of tissue-engineered cartilage constructs. , 2013, Macromolecular bioscience.

[37]  Adam E Jakus,et al.  Advancing the field of 3D biomaterial printing , 2016, Biomedical materials.

[38]  C. V. van Blitterswijk,et al.  The effect of PEGT/PBT scaffold architecture on the composition of tissue engineered cartilage. , 2005, Biomaterials.

[39]  Anthony Atala,et al.  3D bioprinting of tissues and organs , 2014, Nature Biotechnology.

[40]  A. Hasegawa,et al.  Pup birth from mouse oocytes in preantral follicles derived from vitrified and warmed ovaries followed by in vitro growth, in vitro maturation, and in vitro fertilization. , 2006, Fertility and sterility.

[41]  H. Shibahara,et al.  Live births from isolated primary/early secondary follicles following a multistep culture without organ culture in mice. , 2013, Reproduction.

[42]  Amir A. Zadpoor,et al.  Additive Manufacturing of Biomaterials, Tissues, and Organs , 2016, Annals of Biomedical Engineering.

[43]  Subbu Venkatraman,et al.  Printing cell-laden gelatin constructs by free-form fabrication and enzymatic protein crosslinking , 2015, Biomedical microdevices.

[44]  A. Nagler,et al.  Searching for evidence of disease and malignant cell contamination in ovarian tissue stored from hematologic cancer patients. , 2008, Human reproduction.

[45]  L. Shea,et al.  Noninvasive Index of Cryorecovery and Growth Potential for Human Follicles In Vitro1 , 2010, Biology of reproduction.

[46]  J. Couchman,et al.  Dynamics of extracellular matrix in ovarian follicles and corpora lutea of mice , 2009, Cell and Tissue Research.

[47]  C. Plancha,et al.  Three‐dimensional environments preserve extracellular matrix compartments of ovarian follicles and increase FSH‐dependent growth , 1999, Molecular reproduction and development.

[48]  J. Donnez,et al.  A new step toward the artificial ovary: survival and proliferation of isolated murine follicles after autologous transplantation in a fibrin scaffold. , 2014, Fertility and sterility.

[49]  S. Hollister.  Porous scaffold design for tissue engineering , 2005, Nature materials.

[50]  L. Shea,et al.  Fibrin encapsulation and vascular endothelial growth factor delivery promotes ovarian graft survival in mice. , 2011, Tissue engineering. Part A.

[51]  P. Working,et al.  Localization of inhibin and activin binding sites in the testis during development by in situ ligand binding. , 1994, Biology of reproduction.

[52]  L. Shea,et al.  The in vitro regulation of ovarian follicle development using alginate-extracellular matrix gels. , 2006, Biomaterials.

[53]  D. Albertini,et al.  Gap junctions between the oocyte and companion follicle cells in the mammalian ovary , 1976, The Journal of cell biology.

[54]  Kui Liu,et al.  Two classes of ovarian primordial follicles exhibit distinct developmental dynamics and physiological functions , 2013, Human molecular genetics.

[55]  R. Gosden.  Restitution of fertility in sterilized mice by transferring primordial ovarian follicles. , 1990, Human reproduction.

[56]  Lonnie D Shea,et al.  Novel approach for the three-dimensional culture of granulosa cell-oocyte complexes. , 2003, Tissue engineering.

[57]  J. Malda,et al.  Biofabrication of reinforced 3D-scaffolds using two-component hydrogels. , 2015, Journal of materials chemistry. B.

[58]  Wouter J A Dhert,et al.  Porous bioprinted constructs in BMP-2 non-viral gene therapy for bone tissue engineering. , 2013, Journal of materials chemistry. B.

[59]  Alexandra L. Rutz,et al.  A Multimaterial Bioink Method for 3D Printing Tunable, Cell‐Compatible Hydrogels , 2015, Advanced materials.

[60]  Hod Lipson,et al.  Direct Freeform Fabrication of Seeded Hydrogels in Arbitrary Geometries , 2022 .

[61]  C. Highley,et al.  Direct 3D Printing of Shear‐Thinning Hydrogels into Self‐Healing Hydrogels , 2015, Advanced materials.

[62]  C. Bethea,et al.  RU 486 blocks and fluoxetine augments progesterone-induced prolactin secretion in monkeys. , 1997, Neuroendocrinology.

[63]  Bon-Kyoung Koo,et al.  Modeling mouse and human development using organoid cultures , 2015, Development.

[64]  Ali Khademhosseini,et al.  Hydrogel Templates for Rapid Manufacturing of Bioactive Fibers and 3D Constructs , 2015, Advanced healthcare materials.

[65]  Vivian K. Lee,et al.  Printing of Three-Dimensional Tissue Analogs for Regenerative Medicine , 2016, Annals of Biomedical Engineering.

[66]  P. Dubruel,et al.  The 3D printing of gelatin methacrylamide cell-laden tissue-engineered constructs with high cell viability. , 2014, Biomaterials.