Bioprosthetics: 3D printing as ovary restoration strategy LARONDA.pdf · preservation for male and female patients undergoing gonadotoxic treatments • Infertility is one of the

Bioprosthetics: 3D printing as ovary restoration strategy

Monica M. Laronda, Ph.D. Warren & Eloise Batts Scholar Director of Basic & Translational Research Fertility & Hormone Preservation & Restoration Program Assistant Professor, Department of Pediatrics Ovarian Club XII – December 2018 Hong Kong

Stanley Manne Children’s Research Institute

Why pursue fertility preservation?

• American Society of Clinical Oncology & American Society for Reproductive Medicine & American Academy of Pediatrics recommend a discussion about fertility preservation for male and female patients undergoing gonadotoxic treatments

• Infertility is one of the primary concerns of cancer survivors

75% of young adults who are childless at the time of diagnosis report a desire to parent in the future. Cancer survivors report PTSD symptoms related to infertility as long as 10

years post-treatment. Parents want fertility preservation options presented regardless of

infertility risk/prognosis*.

Schover, et al. (2009) Pediatric Blood Cancer; *J Vaught, KE Corkum, EE Rowell, et al. (2017) unpublished


Gonadotoxicity from Disease or Treatments

Childhood Cancer Survivors are significantly more likely to be infertile or have difficulty getting pregnant than their siblings. Genetic Causes for

gonadal dysfunction: • Turner Syndrome • Fragile X Syndrome • Germ cell tumor • Galactosemia

Cancer.gov (0-19 y.o., rev: 1/13/15); Woodruff (2013) Nat Rev Endo Barton, et al. (2013) The Lancet Oncology; W Gao, et al. (2015) JARG; Modified from: Wallace, et al. Int. J. Radiation Oncology Biol. Phys., (2005


Ovarian tissue cryopreservation (OTC)

Ann & Robert H. Lurie Children’s Hospital: IRB-approved research protocol since 2010

Study aims: To cryopreserve gonadal tissue for patient’s own future use Obtain research tissue biopsy (optional) and processing media For optimizing freeze/thaw techniques For developing technology for immature follicle maturation For establishing pediatric-specific tissue processing techniques

Long term patient follow-up for timing and type of tissue and hormone restoration


Protocol Inclusion Criteria Pre or post pubertal individual Will undergo imminent surgery, chemotherapy or radiation therapy that has implications

on future fertility and reproductive hormone potential: • any health condition or malignancy that requires removal of all or part of one or both ovaries,

• whole abdomen or pelvic irradiation ≥10Gy in post-pubertal girls or ≥15Gy in pre-pubertal girls

• total body irradiation, and

• alkylating-intensive chemotherapy

• cyclophosphamide cumulative dose ≥7.5 g/m2

• any treatment regimen containing procarbazine

• busulfan cumulative dose >600 mg/m2

• alkylating chemotherapy conditioning prior to stem cell transplantation

• combination of any alkylating agent with total body irradiation or whole abdomen or pelvic radiation

• cranial radiation ≥30 Gy

• summed alkylating agent dose score ≥3 (Green et al., 2009)

• cyclophosphamide equivalent dose (CED) ≥ 4,000 mg/m2 (Green et al., 2014)

Patient may have newly diagnosed or relapsed disease.

Modified from: Cordeiro, MH, SY Kim, TK Woodruff. Chaper 1 (2015) Anderson & Spears Cancer Treatment and the Ovary; FE Duncan, et al. (2015) Jrnl Adolescent & Young Adult Oncology National Physicians Cooperative Patient: 4 y.o. scale bar = 100 µm

Fertility Preservation: Child

Release of EGGS

at Puberty

oocyte granulosa theca


Oocytes are a Finite Resource

Modified from: Wallace & Kelsey (2010) PLoS ONE and Wallace, et al. Int. J. Radiation Oncology Biol. Phys., (2002)

Sex Hormones

Reviewed in: SL Eddie, JJ Kim, TK Woodruff, JE Burdette. Exp Bio and Med (2014) 239; RA Lobo. Nat Rev Endo (2016) 164; P Schmidt. Menopause (2012) 19 S Mostoufi-Moab, et al. J of Clinical Oncology (2016) 34; JP Langrish, et al. (UK) Hypertension (2009);

System / Organ

Disease / Disorder Developed in POI or

menopause Bone Decreased bone mass,

Osteoporosis Muscle Decreased mass, strength Vascular High blood pressure Skin Decreased laxity, wound healing Brain Decreased short and long-term

memory


Ovarian tissue preservation Unilateral Oophorectomy Remaining ovary compensates and maintains same level of hormones There is little difference in age at menopause Remaining ovary can release more eggs when stimulated for ART

Rowell, E. in Pediatric and Adolescent Oncofertility (eds. Woodruff, T. K. & Gosiengfiao, Y. C.) 30, 243–249 (Springer International Publishing, 2017).

Intra-operative photo of mesovarium and small ovary in a 2 yo girl.

ovary

Fallopian

Preservation Removal

ovary laparoscope

uterus

ovary

Fallopian


Re-implanting post-pubertal ovarian tissue restores fertility & hormones.

> 130 reported live births1 Hormone production up to 12 years,

average 2 – 5 years1,2

Possibility of reintroducing cancer cells3

Need to improve transplant and in vitro maturation to restore function long-term in all patients

Meirow, et al. (2005) NEJM

1. Donnez, J. & Dolmans, M.-M. New England Journal of Medicine 377, (2017). 2. Pacheco, F. & Oktay, K. Reprod Sci 24, (2017). 3. Laronda, M. M. et al. Biomaterials 50, 20–29 (2015) ; Scale bar = 50 µm

SALL

4 / D

API

acute lymphoblastic leukemia cells in ovarian cortical tissue


National Physicians Cooperative Patient: 4 y.o. scale bar = 100 µm

Engineering an Ovary

Isolate desired cells; Remove cancer

oocyte granulosa

Support structure


Matrices to graft ovarian follicles

Reviewed in: Amorim & Shikanov (2016) Future Oncology

Telfer & Gosden (1990) Repro. • collagen-encapsulated

pre-antral follicles • kidney capsule • embryos after stimulation

and IVF

Carroll & Gosden (1993) Human Repro. • plasma clot-encapsulated

primordial follicles • ovarian bursa (6-12 wks) • pups, no genetic

verification from transplant, few small follicles left

Laronda, et al. (2015) Biomaterials • ovarian ECM scaffold

primordial follicles & granulosa cells

• kidney capsule (21 days) • antral follicles, few

follicles left

Shikanov, et al. (2011) Tissue Eng. Part A & Kniazeva, et al. (2015) Sci. Rep. • fibrin(-HBP)-VEGF

encapsulated ovarian tissue pieces OR primordial follicles

• ovarian bursa (14-21 days)

• pups

kidney


Scale bar = 5 mm Laronda, et al. Biomaterials (2015) doi:10.1016/j.biomaterials.2015.01.051

Decellularized Ovary


Scale bars = 2 μm; 2 μm; 50 μm

Primary ovarian cells cultured on ovarian ECM develop secretory blebs and follicle-

like pores. Decellularized Ovary Scaffold Primary Ovary Cells

Laronda, et al. Biomaterials (2015) doi:10.1016/j.biomaterials.2015.01.051


Puberty was initiated in ovariectomized mice with ovary

grafts. Decellularized Ovary Scaffold Primary Ovary Cells

Implanted into Adolescent

Ovariectomized Mouse

• Initiated Puberty •Supported Oocyte Differentiation

• Increasing Serum Estradiol

Laronda, et al. Biomaterials (2015) doi:10.1016/j.biomaterials.2015.01.051


Scaffold Criteria: • Create a 3D feel • Mechanically rigid to

handle during implantation

• Open pores for nutrient flow, growth and ovulation

• Bioactive, bio-safe material

Gelatin scaffold with a microporous architecture

Development of Scaffold

Jakus, Laronda, et al. (2017) Advanced Functional Materials; Laronda, Rutz, et al. (2017) Nature Communications


Z D

epth

Ana

lysi

s Different 3D printed advancing angles

result in different pore design. Intersecting (30º) Intersecting (60º) Grid (90º)

Folli

cle

/ Str

ut

Laronda, Rutz, et al. (2017) Nature Communications


Follicles prefer more contact points.

Scale bar = 50 µm

30º; D8

oocyte

basement membrane



Follicles interact with scaffolds pockets

stroma / cytoskeleton / DNA vinculin / dapi



Mic

rotu

bule

s / D

NA

a b

d c

+hCG

Follicles release MII eggs in vitro.

Bioprosthetic Ovary Transplant

Both ovaries removed from adult mouse

Rest of reproductive tract remains in tact

3D printed scaffold with seeded with follicles


Bioprosthesis 3wks post-surgery

Primary follicle

Secondary follicles

vessel

Scale bar = 200 and 50 µm

Large follicles

Uterine horn

CL

Bioprosthesis 8wks post-surgery



Bioprosthetic ovaries develop functional vasculature.

PECAM / DNA Scale bar = 50 µm



Bioprostethic ovary recipients reared healthy pups.

Implanted into Non-GREEN

Ovariectomized Mouse

GREEN pup from Ovary Implant (- pup from different litter) Laronda, Rutz, et al. (2017) Nature Communications


Transplant recipient (EGFP-) with EGFP+ pup

Bioprostethic ovary recipients reared healthy pups.

Females with Bioprosthetic Ovary:

• Cycling (vaginal histology)

• 3 had pups from transplant

• Supported pups with milk until wean

• Pups produced Grand-pups



Scale bar = 1 μm

Next Steps: Compartmentalization

Bovine ovary

theca cells granulosa

cells

oocyte

surface epithelium

primordial follicles

vessels

Laronda, et al. (2015) Biomaterials doi:10.1016/j.biomaterials.2015.01.051

cort

ex

med

ulla



1. Define the differences between cortex and medulla 2. Observe folliculogenesis under altered environments 3. Modify ink / scaffold design to achieve desired outcome

slice ovaries

decellularize

create matrisome map



•Printing different support cells •Printing different structural proteins •Printing different rigidity properties

stroma / cytoskeleton

100 µm


Acknowledgements

Fertility & Hormone Preservation & Restoration Team including Erin Rowell, MD Kristine Corkum, MD Courtney Harris, MD Laronda Lab Members Kelly Even Nathaniel Henning Sofia Petukhova Kelly McKinnon, PhD

Thank you Pat Magoon and Tom Shanley, M.D. & Lurie

Children’s Vision 2025 Initiative Mary and Ralph Gesualdo Stanley Manne

Funding Sources Warren & Eloise Batts Endowment (MML) Burroughs Wellcome Fund Career at the

Scientific Interface Award (MML) This work was supported by the Center for Reproductive Health

After Disease (P50HD076188) from the National Institutes of Health National Center for Translational Research in Reproduction and Infertility (NCTRI).

Artificial Ovary: patent application #15/545,175

LarondaLab.org


In vitro Maturation of Small Follicles

Isolated Follicle Culture Growth of secondary follicles to

healthy MII eggs

Cortical Strip Culture Maintain health of primordial follicles

UK/US group obtained MII eggs from cortical strip follicles

Jeruss and Woodruff (2009) NEJM; Woodruff (2013) Nature Reviews Endocrinology; Donnez and Dolmans (2017) NEJM; Laronda et al. (2014) JARG; Jakus, Laronda, et al. (2017) Adv Funct Mat; Xiao, et al (2015) Science Reports; McLaughlin, Telfer, et al. (2018) Mol. Human Repro

Xiao, et al. (2015) Science Reports

Laronda, et al. (2015) JARG

McLaughlin, et al. (2018) Mol. Human Repro.

Bioprosthetics: 3D printing as ovary restoration strategy Why pursue fertility preservation?Gonadotoxicity from Disease �or TreatmentsOvarian tissue cryopreservation (OTC) Protocol Inclusion CriteriaFertility Preservation: ChildOocytes are a Finite ResourceSex HormonesOvarian tissue preservationRe-implanting post-pubertal ovarian tissue restores fertility & hormones.Engineering an OvaryMatrices to graft ovarian folliclesDecellularized OvaryPrimary ovarian cells cultured on ovarian ECM develop secretory blebs and follicle-like pores.Puberty was initiated in ovariectomized mice with ovary grafts.Development of ScaffoldDifferent 3D printed advancing angles result in different pore design.Follicles prefer more contact points.Follicles interact with scaffolds pocketsFollicles release MII eggs in vitro.投影片編號 21Bioprosthetic ovaries develop functional vasculature.Bioprostethic ovary recipients �reared healthy pups.Bioprostethic ovary recipients �reared healthy pups.Next Steps: CompartmentalizationNext Steps: CompartmentalizationNext Steps: CompartmentalizationAcknowledgementsIn vitro Maturation of Small Follicles

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Bioprosthetics: 3D printing as ovary restoration strategy LARONDA.pdf · preservation for male and female patients undergoing gonadotoxic treatments • Infertility is one of the