3D ink preparation and 3D printing

One gram of gelatin (porcine, type A; Sigma-Aldrich) was dissolved in 10 ml of phosphate-buffered saline solution (PBS; Gibco) (pH=7.4) at 37 °C. The solution was subsequently loaded in a stainless steel printing cartridge, and a lab-made agarose (Sigma-Aldrich) piston was placed on top of the solution. The cartridge was then maintained at 30 °C for at least 3 h to cool the solution into a gel. With an EnvisionTEC 3D-Bioplotter, gelatin was printed from a 100 μm stainless steel nozzle onto glass slides maintained at 10 °C. Extruding pressures ranged from 1.8 to 4.5 bar to control ink flow rates, and the ink was printed at a speed of 10 mm s−1 into 15 × 15 mm squares, 5 layers thick. The first layer was completely solid (no spacing between struts) whereas the distance between struts (from middle of one strut to middle of adjacent strut) on all subsequent layers was 600 μm. This resulted in scaffolds with struts ∼250 μm and pore size (edge of one strut to the other) ∼350 μm. Three types of microporous internal architectures were printed with advancing angles between layers of 30°, 60° and 90°.

Scaffold preparation

After printing, structures were kept in a closed container with water (to keep humidity) and on ice. Structures were crosslinked for 1 h with a 15 mM N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide (EDC; Sigma-Aldrich)/6 mM N-hydroxysuccinimide (NHS; Sigma-Aldrich) solution in deionized water to stabilize gelatin scaffolds for culture at physiological temperature. Scaffolds were then washed with deionized water, and sterilized by overnight incubation in 70% ethanol as well as 1 h of UV exposure. Scaffolds were then stored in sterile PBS at 4 °C.

Ink and scaffold characterization

Oscillatory shear rheology was performed with the gelatin ink on an Anton-Paar MCR 302 rheometer. Rheology was conducted with a cone (2°)-plate fixture and at 10 rad s−1. Temperature of the stage was controlled between 15 and 40 °C. Gelatin was loaded onto the stage while warm (37 °C) and after lowering the cone fixture into position, the edges were covered with mineral oil to prevent dehydration. Temperature sweep was conducted at 0.5 °C min−1 and at 1% strain. The architecture of scaffolds was analysed from photographs and images were taken with a Photojojo macrolens and cell phone camera along with a Leica M205 C stereoscope. For 3D imaging, scaffolds were fluorescently labelled with NHS-rhodamine (Thermo-Scientific) and were washed with PBS. NHS-rhodamine was dissolved in dimethyl sulfoxide (Sigma-Aldrich) at 10 mg ml−1. A diluted PBS solution of the 100 × concentrate was used for labelling for 1–2 h. Labelled scaffolds were then imaged on a Nikon A1R laser scanning confocal microscope. Compression testing was performed on an LF Plus mechanical tester at 0.5 mm s−1 (Lloyd instruments, 50N load cell). Gels (200 μl) were prepared between glass coverslips and crosslinked with 15 mM EDC/6 mM NHS solution in deionized water for 1 h to yield flat gel cylinders of 7 mm diameter and 1 mm height. Modulus was taken over 0–10% strain.

Follicle seeding and culture

Both in vitro and in vivo experiments designed with scaffolds seeded with follicles are outlined in Table 1. Scaffolds were prepared by using 2, 3 or 4 mm biopsy punches onto the printed design and using a scalpel to lift each piece off the glass slide. A thin microspatula or flat forceps were used to place the scaffold punches on 0.4 μm pore 12 mm transwells (Millipore; PICM01250) in a 24-well plate (Corning; 353047). Each well was filled with 400 μl of growth media (αMEM Glutamax, Life Technologies, 32561) supplemented with 3 mg ml−1 bovine serum albumin (BSA) (MP Biomedicals, 210370025), 10 mIU ml−1 recombinant follicle-stimulating hormone (from A.F. Parlow, National Hormone and Peptide Program, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD, USA), 1 mg ml−1 bovine fetuin (Millipore, 341506), 5 μg ml−1 insulin, 5 μg ml−1 transferrin and 5 μg ml−1 selenium (Sigma-Aldrich, I1884). Multilayer secondary follicles (150–180 μm) were isolated from 16-day-old CD-1 strain (Harlan) female mice. All mice were housed in a controlled barrier facility at Northwestern University’s Center of Comparative Medicine under constant temperature, humidity and light (12 h light/12 h dark). Food and water were provided ad libitum. All animal experiments were approved by the Institutional Animal Care and Use Committee and were performed in accordance with National Institutes of Health Guidelines. Ovaries were removed from the bursas and follicles were mechanically isolated with insulin needles in a glass dish containing dissociation media made of Leibovitz’s L-15 Medium (Life Technologies, 11415) with 0.5% penicillin–streptomycin (Cellgro, 30-002-CI) and 10% fetal bovine serum (Life Technologies, 10082139). Only follicles that displayed intact morphology were selected for seeding and culture. Follicles were seeded by micropipetting onto scaffolds and removing excess fluid from the bottom of the scaffold. This allowed the follicles to fall through the open porosity and populate the entire depth of the scaffold. Follicles on scaffolds were cultured at 37 °C in 5% CO 2 in air for up to 8 days. Half of the growth media (200 μl) was replaced every other day. Spent media was stored at −20 °C and analysed for estradiol (below). Follicles were imaged after seeding and at each media change using a dissecting scope (Lecia, MZ95). Follicles were scored as alive if the oocyte was visible, round and generally centralized within the follicle through light microscopy. Four separate experiments with a total of 130 follicles were included and divided into groups of seven or eight follicles. Follicles were excluded if they were not healthy (degenerated oocyte or punctured granulosa cell layer) by 24 h after seeding, as we assumed these follicles were unhealthy because of the isolation procedure. The number of strut contacts was identified by light microscopy on day 2 of culture and scoring was consistent in those that were also observed with confocal microscopy. There were four replicate groups of follicles with one contact, eight groups with two contacts and five groups with three contacts. There were three groups of follicles in 30° scaffolds, six groups in 60° and seven groups in 90° scaffolds. Data in Fig. 2 are represented as the mean±s.e.m. and also listed in the text. An ordinary one-way ANOVA with Holm–Sidak’s multiple comparisons test was performed with α=0.05. The biological replicates were spread equally among the groups compared and normal distribution was observed among the replicates. The Brown-Forsythe and Bartlett’s tests found no significant variation between groups.

Table 1 Description of three experimental designs used to investigate ovarian follicles in the 3D printed scaffolds. Full size table

Follicle–scaffold interaction 3D analysis

GFP-expressing follicles were seeded into NHS-rhodamine labelled scaffolds and cultured. Two scaffolds per geometry (30°/60°/90°) with four follicles each were analysed (24 follicles total). The experiment was conducted once. After 2 days, the follicles were analysed by confocal fluorescence microscopy (Nikon A1R laser scanning) at an image slice thickness of 5.3 μm. Images were analysed as image stacks as well as 3D reconstructions in NIS Elements software. Confocal images were compared to light microscopy images during culture. Eight follicles were eliminated from analysis because either the follicle moved during transfer for imaging or artifacts in the collected images obscured analysis (e.g., air bubbles between follicle and scaffold). To quantify the number of contacts between follicles and scaffold struts, the image was scrolled through to identify slices where the follicle was flush with the scaffold. Side contacts were measured at the image slice with the longest length of contact (the longest area of green fluorescent follicular cells along a strut). Bottom scaffold contacts were determined if red fluorescence was observed underneath of green fluorescence within 20 μm but were not quantified. Strut contacts underneath the follicle (bottom contacts) could not be quantified due to diminished follicle fluorescence. For individual lengths of contacts per number of contacts, there were four measured lengths for follicles making one strut contact, 11 lengths for follicles making two strut contacts, 12 lengths for follicles making three strut contacts for 17 total follicles. All follicles making three contacts had at least one contact that could not be measured and, therefore, were not included in the total length analysis. For the total length of contact for follicles making one and two total contacts and whose contacts were all side contacts (no bottom, only side contacts were measurable), there four measured total lengths per one and two total contacts for eight follicles total. An ordinary one-way ANOVA with Holm–Sidak’s multiple comparisons test was performed with α=0.05. The bars in Fig. 2 represent mean±s.e.m.

For scanning electron microscopy analysis, the samples were fixed in a solution containing 2.5% EM Grade glutaraldehyde, 2% paraformaldehyde and 0.1 M PBS for 1 h at room temperature and overnight at 4 °C. Post fixation occurred in 1% osmium tetroxide for 2 h at room temperature followed by 1% uranyl acetate overnight at 4 °C. A graded series of ethanol was used for dehydration before critical point drying in a Tousimis Samdri 795 Critical Point Dryer. The samples were then mounted to scanning electron microscopy stubs with carbon tape and silver paint and sputter coated with 10 nm of AuPd with a Denton Desk IV Sputter Coater prior to imaging. Data were gathered with a Hitachi SU8030 cold field emission scanning electron microscope at 5 kV with working distances ranging from 9 to 20 mm.

Western blotting

Ovaries were collected from 40-day-old CD1 mice, frozen and stored at −20 °C. Total protein lysate was extracted with a pestle and motor using cell lysis buffer (Cell Signaling Technology, 9803S) in the presence of a protease inhibitor cocktail (Life Technologies, 78440). Denaturing SDS–PAGE was performed with 25 μg of the total protein lysate using standard western blotting techniques on a 4–15% polyacrylamide gel (Bio-Rad, 456–1084). The polyvinylidene fluoride membrane was blocked following transfer using 3% Amersham ECL Prime Blocking Reagent (GE Healthcare, RPN418) and incubated in primary antibodies as follows: 1:1,000 HMGB1 (Abcam, ab18256) and 1:1,000 GAPDH (Cell Signaling Technologies, 5174S) in TBS+0.1% Tween-20. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Membranes were then incubated in horseradish peroxidase conjugated anti-rabbit secondary antibody (GE Healthcare, NA931-1ML), and protein was visualized using Amersham ECL Prime Western Blotting Detection Reagent (GE Healthcare, RPN2232).

Immunohistochemistry of ovary sections

Ovaries from 40-day-old CD1 mice were fixed in Modified Davidson’s Fixative (Electron Microscopy Sciences, 64133-50) and paraffin-embedded. Five micron sections were deparaffinized in Citrisolv (Fisher, 04-355-121) and antigen retrieval achieved using Reveal Decloaker (Biocare Medical, RV1000). Sections were prepared for immunohistochemistry using Avidin/Biotin blocking kit (Vector Labs, SP-2001) in 10% normal goat serum in TBS. Sections were then incubated with diluted 1:100 HMGB1 primary antibody (Abcam, ab18256, lot GR135551-1) in 10% normal goat serum in TBS overnight at 4 °C. As a control for expression, the HMGB1 blocking peptide (Abcam, ab18650) was also used. A ratio of 1:5 HMGB1 antibody to HMGB1 peptide was incubated overnight at 4 °C prior to application. Sections were washed in TBS+0.1% Tween-20 and then incubated with diluted 1:200 biotinylated goat anti-rabbit secondary antibody from the Vectastain Elite ABC kit (Vector Labs, PK-6105) for 2 h at room temperature, washed again and followed by a 30 min incubation in the ABC reagent (Vector Labs, Vectastain Elite ABC kit) and a final wash. Binding was detected with diaminobenzidine (DAB; Vector Labs, SK-4100) for 6 min. Counterstaining was achieved using standard haematoxylin staining. All images were acquired and processed on an Evos fl auto inverted microscope using Evos software (Life Technologies).

Immunofluorescence of follicles within scaffolds

Constructs were fixed for immunohistochemistry for 30–60 min with 3.8% paraformaldehyde (Electron Microscopy Sciences, 100503-916) with 1% Triton X 100 (Sigma-Aldrich, T8787) and stored in blocking buffer made of PBS containing 0.01% Tween-20 (Sigma-Aldrich, P2287) and 0.3% BSA (MP Biomedicals, 210370025) at 4 °C until ready to use. Samples were washed with PBST. Constructs were blocked and permeabilized with 2% donkey serum, 1% BSA, 0.1% cold fish skin gelatin, 0.1% Triton, 0.05% Tween-20, 0.05% sodium azide in PBS for 1 h. Primary antibodies against vinculin (1:200; Sigma Aldrich, V9131), HMGB1 (1:200; Abcam, ab18256), laminin (1:50; Santa Cruz Biotechnology, sc-20682) were diluted in 10% of the blocking solution and were incubated overnight at 4 °C. After washing with PBST, the samples were labelled with a secondary antibody (1:250 anti-rabbit or 1:1,000 anti-mouse, AlexaFluor 488; Life Technologies A21206, A11008) for 2–6 h, followed by PBST washing and counterstaining with DAPI (1:50; Molecular Probes, D1306) and Phalloidin-555 (actin; 1:50, Abcam, ab176756). 3D constructs were imaged on either a Nikon A1R or C2 laser scanning confocal microscope. Experiments were performed two times with each antibody and a no primary control was also examined for each secondary used.

In vitro ovulation was performed on 6–8 days of follicle culture, when the oocytes grew to approximately 70 μm. Follicles were incubated for 16 h at 37 °C in 5% CO 2 in air in maturation media containing αMEM with 10% fetal bovine serum, 1.5 IU ml−1 human chorionic gonadotropin (Sigma-Aldrich, C1063), 10 ng ml−1 epidermal growth factor (BD Biosciences, 354010) and 10 mIU ml−1 recombinant follicle-stimulating hormone. Released oocytes in metaphase II (MII) were identified and fixed in 3.8% paraformaldehyde containing 0.1% Triton X-100 for 1 h at 37 °C for spindle morphology and chromosome alignment analysis. Oocytes were washed three times in blocking solution with 1 × PBS containing 0.3% BSA and 0.01% Tween-20, incubated overnight in a 1:50 dilution of mouse anti-α-tubulin (Cell Signaling Technology, 5063S) in blocking solution. Then, oocytes were washed three times with blocking solution, mounted using Vectashield containing DAPI (Vector Laboratories, H-1200) and analysed using an EVOS FL AUTO microscope (Life Technologies). Oocytes with barrel-shaped bipolar spindles and well-organized microtubule fibres, along with tightly aligned chromosomes on the metaphase plate, were considered normal64.

3βHSD staining

To verify the presence of an intact theca cell layer, follicles within the scaffolds were rinsed with PBS then stained with 3βHSD solution containing 0.12 mg ml−1 nitroblue tetrazolium chloride (Sigma-Aldrich, N6639), 0.25 mg ml−1 β-nicotinamide adenine dinucleotide hydrate (β-NADþ; Sigma-Aldrich, N3014) and 0.025 mg ml−1 epiandrosterone (Sigma-Aldrich, E3375) in PBS for up to 3 h at room temperature wrapped in foil65. Negative controls were performed on follicles for the same length of time in solution without nicotinamide adenine dinucleotide hydrate β-NADþ and in PBS alone. Cells were considered positive of 3βHSD if they were purplish-brown in colour. Negative controls were not positive for this colour change. This experiment was performed three times with 18 follicles each.

Hormone ELISAs

Estradiol was detected in the media using an ELISA kit (Calbiotech, ES180S, range of 10–1,000 pg ml−1). Individuals who ran the ELISA and interpolated the data were blinded to the treatments (culture day collected). A power analysis determined that three animals per group was sufficient to efficiently detect (80% chance) a minimal difference of 50±10% in serum levels. Terminal blood draws were processed to collect serum, which was tested for AMH and inhibin A by the University of Virginia Center for Research in Reproduction Ligand Assay and Analysis Core. The average of technical replicates was plotted. The reportable range for AMH is 1.56–100.0 ng ml−1, and inhibin A is 10–884 pg ml−1. Randomized and de-identified serum samples from 5 ovariectomized mice with shams and 16 ovariectomized mice with bioprosthetic ovaries were measured. Zero serum samples from ovariectomized mice with sham and nine with bioprosthetic ovaries were in detectable range for AMH. One mouse with sham and 13 mice with bioprosthetic ovaries had serum containing inhibin A within the detectable range. The bars in Fig. 5g represent mean±s.e.m. Because the mice with bioprosthetic ovaries are expected to have a biological variation of serum levels dependent on the hormone cycle, we did not expect a normal distribution.

Histological analysis and vessel counting

All tissue processing and haematoxylin and eosin (H&E) staining was performed by the Northwestern University Center for Reproductive Sciences Histology Core. Fixed tissue was processed using an automated tissue processor (Leica) and embedded in paraffin. Serial sections were cut 5 μm thick and selected slides were stained with H&E using a Leica Autostainer XL (Leica Microsystems). Immunohistochemistry was performed on 3–5 sections per sample and at least three samples per group. Each experiment was performed 2–3 times on separate days and included no-primary controls. Sections were imaged on a Nikon E600 Fluorescent microscope (Nikon Instruments) with a Retiga Exi Fast 1394 camera (QImaging). Antibodies against platelet endothelial cell adhesion molecule (1:50; Santa Cruz Biotechnology, sc1506), platelet-derived growth factor receptor β1 (1:100; Abcam, ab32570) or GFP (1:300; Santa Cruz Biotechnology, sc8334) were used and visualized with AlexaFluor secondaries (1:500; Life Technologies, A21206, A21082) or Vectastain Elite ABC Kit (Vector Laboratories, PK-6100). Mounting medium with DAPI counterstain (Vector Laboratories, H-1200) was used to visualize nuclear material.

Every 20th section or ∼100 μm was stained with H&E and imaged on a Nikon E600 microscope. Every vessel containing blood cells within 100 μm of the scaffold was counted for eight ovaries each collected 1 or 3 weeks after surgery. The average area of the scaffold or bioprosthesis boundaries of the first and middle sections were calculated to determine the number of vessels per area of tissue.

Intrabursal surgeries and mating

Animal use was performed under a Northwestern University Animal Care and Use Committee-approved protocol. Mice ubiquitously expressing the enhanced green fluorescent protein (GFP+) that resulted from a cross of CD1 strain (white coat, Harlan) and GFP-expressing C57BL/6J mice (black coat, Jackson Labs) were used to create the bioprosthetic ovaries and were identified using a BlueStar light with VG1 filter glasses (EMS, BLS-1). Two-millimetre scaffolds were prepared as described above on transwells and with growth medium. Primordial primary and secondary follicles from GFP+ females were seeded as described above over 2 days, seeding small follicles first and filling to pack entire scaffold on the second day with additional small and some secondary follicles (up to 180 μm in diameter). These scaffolds were cultured for 4 days total in vitro prior to surgeries and imaged on the morning before the surgeries. Sham implant scaffolds were prepared in the same way, but did not include cells.

Intrabursal surgeries were performed on 8- to 10-week-old NSG females (white coat; Jackson Labs). Mice were anaesthetized with a mixture of 100 mg kg−1 of ketamine and 15 mg kg−1 of xylazine. The upper uterine horn, oviduct and ovarian bursa were visualized and brought out of the body cavity to perform the surgery. The ovarian artery was located and flow was inhibited with a 10 gauge suture tie. The ovary was completely removed while maintaining the integrity of the bursa and bursal cavity. The bioprosthetic ovary was inserted into the ovarian bursa, open side toward the oviducts and enclosed within the bursa by one or two stitches with 10 gauge nylon sutures. This was repeated for both ovaries. Sham surgeries were performed like these surgeries but with scaffolds that did not contain any cells. Seven mice with bioprosthetic ovaries and two mice with sham surgeries were mated with CD1 (white coat) males that had previously sired pups. Each female was paired with one male 4 days after surgery and housed together for 25 days or more. The presence of a solid plug within the mated female vagina indicated a successful mating. No additional mating was attempted. Pups resulting from this mating were identified as created from an egg released from the scaffold if the pup was GFP+ or had a black coat colour. Pups that were GFP− with a white coat colour could not be determined as coming from the implant. The matings were performed in this way because these GFP+ pups are homozygous lethal and, therefore, the GFP+ follicles within the bioprosthetic only carry one GFP+ copy. Therefore, we expect approximately half of the resulting pups from the bioprosthetic eggs to be GFP+.

Statistical analysis

All data sets were analysed using GraphPad Prism software and expressed as the average±s.e.m. Statistical significance performed using one-way ANOVA with a Holm–Sidak’s multiple comparisons test with α=0.05.

Data availability

The authors declare that all data supporting the findings of this study are available within the paper and its Supplementary Information files.