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A bioprosthetic ovary created using 3D printed

by:Tuowei     2019-06-08

Emerging additive manufacturing technologies are able to investigate the effects of hole geometry on cell behavior and function.

Here, we 3D print the micro-pore gel scaffold to test how changing the pore geometry affects the survival of ovarian follicles by manipulating the propulsion angle between the print layers.

The 30 ° and 60 ° brackets provide an angle surrounding the multilateral follicles, while the 90 ° stent has an open hole that limits the interaction of the follicle-stent.

With the increase in the amount of stent interaction, the extension of follicles was limited and the survival rate increased. Follicle-

When implanted in surgically sterilized mice, the seed stent becomes highly consistent and the ovarian function is fully restored.

In addition, the cubs are born through natural mating and thrive through Maternal lactation.

These findings demonstrate an in vivo functional ovarian implant designed with 3D printing and indicate that the pore structure of the stent is a key variable in the design of additional manufacturing brackets for functional tissue engineering.

gram of gelatin (Pig, type; Sigma-Aldrich)

Dissolved in 10 ml phosphate

Buffer saline solution (PBS; Gibco)(pH=7. 4)at 37u2009°C.

The solution is then loaded into the stainless steel print cartridge and in the labmade agarose (Sigma-Aldrich)

The piston is placed at the top of the solution.

Then keep the cartridge at 30 °c for at least 3 h and cool the solution into a gel.

3D with EnvisionTEC-

Bioplotter, gelatin printed from a 100 μm stainless steel nozzle to a slide held at 10 °c

The extrusion pressure range is 1. 8 to 4.

The 5 bar controls the ink flow rate and prints the ink at a speed of 10 mmmm s to a square of 15 × 15 squaresmm with a 5 layer thickness.

The first layer is completely solid (

No spacing between Struts)

The distance between struts (

From the middle of a pillar to the middle of an adjacent pillar)

On all subsequent layers are 600 u2009 μm.

This leads to supports with struts 250 cucμm and aperture (

One pillar to the edge of another)350u2009μm.

Three types of porous internal structures were printed, and the propulsion angles between the layers were 30 °, 60 °, and 90 °, respectively.

After printing, the structure is kept in a closed container containing water (

Maintain humidity)and on ice.

15mm-cross-linking for 1 hour(3-


Ethylcarbodimide (EDC; Sigma-Aldrich)/6u2009mM -

High efficiency synthesis method (NHS; Sigma-Aldrich)

Solution in deionized water to stabilize the gelatin scaffold cultured at physiological temperature.

The stent was then cleaned with deionized water and incubated overnight in 70% ethanol and sterilized under 1 hour UV irradiation.

The stent is then stored in sterile PBS at 4 °c.

Oscillating shear flow with gelatin ink on Anton-

Paar MCR 302 set.

Flow with cones (2°)-

Plate fixture and 10 radradrads.

The temperature of the stage is controlled between 15 and 40 °c.

Gelatin is loaded onto the stage during warm times (37u2009°C)

After lowering the cone fixture to a certain position, mineral oil is covered on the edge to prevent dehydration.

The temperature scan is 0.

Strain of 5 ° c min and 1%.

The structure of the bracket was analyzed from the photos and images were taken using Photojojo macrolens and mobile camera as well as Leica M20C stereo.

For 3D imaging, mark the bracket with NHS-fluorescencerhodamine (Thermo-Scientific)

Wash with PBS. NHS-

If Danming is dissolved in px (Sigma-Aldrich)at 10u2009mgu2009ml.

Mark 1-2 µh with diluted PBS solution of 100 × concentrate.

Then, the marked stent is imaged on the Nikon 1R laser scanning co-Focusing microscope.

Compression test is performed on LF Plus mechanical tester with a test time of 0. 5u2009mmu2009s (

Lloyds instruments, 50 n load cell). Gels (200u2009μl)

Prepared between glass coverslips and cross-linked with 15mm EDC/6mm NHS solution in deionised water for 1 µh to produce a diameter of 7mm and a height of 1 µmm

Take the modulus at 0-10% strain.

The experiments and experiments on the design of the scaffold with the follicle inoculation were outlined.

The stent was prepared by using a 2, 3 or 4mm biopsy punch on the printing design and using a surgical knife to lift each piece from the slide.

Use a thin micro spatula or flat tweezers to place the scaffold punch on 0.

4 μm hole 12mm cross well (Millipore; PICM01250)in a 24-well plate (Corning; 353047).

Each hole is filled with a growth medium of 400 μ l (

Life Technology, 32561)

Supplemented with bovine serum albumin (BSA)(

Doctor of Biomedical Sciences, 210370025)

, 10 ml of recombinant follicles-

Stimulating hormone (from . F.

Parlow, National Institute of Diabetes, Digestive and Kidney Disease, Bethesda, Maryland, USA National hormone and peptide program)

1 ml of bovine fetal protein (


, 5 µμg of insulin 5 µμ g of iron transfer and 5 µμg of seleniumSigma-Aldrich, I1884).

Multi-level follicles (150–180u2009μm)

From 16-day-old CD-1 strain (Harlan)female mice.

All mice were raised in controlled barrier facilities at the comparative Medical Center of Northwestern University under constant temperature, humidity and light (

12 h/12 h Dark).

Food and water were provided.

All animal experiments are approved by the Institutional Animal Care and Use Committee and are carried out in accordance with the guidelines of the National Institutes of Health.

The ovary is removed from bursas, and the follicles are mechanically separated by insulin needles in L-containing by Leibovitz-15 Medium (

Life Technology, 11415)with 0.

Penicillin-penicillin 5% (Cellgro, 30-002-CI)

And 10% fetal bovine serum (

Life Technology, 10082139).

Only follicles with complete morphology were selected for sowing and culture.

The follicles were vaccinated by vaccinating the trace fluid onto the stent and removing the excess fluid from the bottom of the stent.

This allows follicles to fill the entire depth of the stent through open gaps.

Follicles on the stent were cultured for 8 days at 37 °c and 5% CO air.

Half of the growth media (200u2009μl)

Change every other day.

The used medium is stored at-20 °c and analyzed for estrogen (below).

After inoculation and each time the culture medium changes, the follicles were imaged using a mirror (Lecia, MZ95).

If the egg mother cell is visible, rounded, and is usually concentrated within the follicles through an optical microscope, the follicles are rated alive.

Four separate experiments with a total of 130 follicles were divided into seven or eight follicle groups.

If the follicles are not healthy, the follicles are excluded (

Granular cell layers of degraded egg mother cells or perforation)

24 hours after sowing because we think these follicles are not healthy due to the separation procedure.

On the second day of culture, the number of pillar contacts was determined by an optical microscope, and the scores were consistent with those observed with a co-focused microscope.

There are 4 repeated follicle groups, 1 contact group, 8 2 contact groups and 5 3 contact groups.

There are 3 groups of follicles in the 30 ° stent, 6 groups in the 60 ° stent, and 7 groups in the 90 ° stent.

The data in is represented as an average. e. m.

And listed in the body.

An ordinary

Variance analysis was performed using the multiple comparison test of Holm-Sidak, and the result was = 0. 05.

Biological repetition spreads evenly between the groups being compared, and normal distribution is observed between repetitions. The Brown-

The test of Forsythe and Bartlett found no significant difference between the groups. -

Expressed follicles are sown to the NHS-

Bracket and culture marked by Rhodamine.

Two scaffolding per geometry (30°/60°/90°)

Four follicles per analysis (

total of 24 follicles.

The experiment was carried out once.

After 2 days, the follicles were analyzed with a co-focused fluorescence microscope (

Laser scanning Nikon 1R)

The image slice thickness is 5. 3u2009μm.

In the NIS Elements software, the image is analyzed as an image stack and a 3D reconstruction.

During the culture process, the co-focal image was compared with the optical microscope image.

8 follicles were excluded from the analysis, since either the follicles that were moved during the transfer imaging or the analysis that was blocked in the collected images (e. g.

, Bubbles between follicles and stent).

To quantify the number of contacts between the follicles and the stent pillar, scroll the image to identify the slices of the follicles flush with the stent.

Measure side contact on image slices with the longest contact length (

The longest area of green fluorescent follicle cells along the pillar).

If red fluorescence is observed under green fluorescence within 20 μm, but there is no quantification, the bottom bracket contact is determined.

Pillar contacts below follicles (Bottom contacts)

Due to the weakening of follicle fluorescence, it cannot be quantified.

For a single contact length of each contact number, follicles with one pillar contact have four measured lengths, follicles with two pillar contacts have 11 lengths, and the length of 12 follicles, make three pillar contacts for 17 total follicles.

At least one contact in all follicles exposed to three could not be measured, so it was not included in the total length analysis.

For the total contact length of the follicles that make one and two total contacts, the contact is side contact (

No bottom, only side contacts can be measured)

, Each 1 has a total length of 4 measurements, and the total contact of 8 follicles has 2.

An ordinary

Variance analysis was performed using the multiple comparison test of Holm-Sidak, and the result was = 0. 05.

Bar representing the average. e. m.

For scanning electron microscopy analysis, the samples were fixed in a solution containing 2.

5% EM Grade B-C glue, more than 2% formaldehyde and 0.

1 m PBS is placed at room temperature for 1 u2009 h and overnight at 4 °c

After fixation, place 2 hours in 1% tin oxide at room temperature, then place 1% uranium acetate at 4 °c overnight

In the Tousimis Samdri 795 critical point dryer, the graded series of ethanol was used for dehydration before the critical point was dried.

Then, before imaging, install the sample to the scanning electron microscope stub with carbon tape and silver paint, and paint the AuPd with 10 nm with Denton Desk IV sputter.

The data were collected using Hitachi SU8030 cold field emission scanning electron microscopy at 5 kv with a working distance of 9 to 20mm.

Ovary from 40-day-

Old CD1 mice frozen and stored at-20 °c

Extraction of total protein lysate with stle and motor using cell lysate buffer (

Cell signal technology, 9803 S)

In the presence of a protein inhibitor cocktail (

Life Technology, 78440).

Using the standard western blot technique on 4-15% pam gel, transgendered SDS-PAGE was performed with a 25 μg total protein lysate (Bio-Rad, 456–1084).

After the reagent transfer was blocked using 3% Amersham ECL Prime, the polyfluoride membrane was blocked (

General Electric Medical, RPN418)

And incubated in one antibody as follows: 1,000 b1 (Abcam, ab18256)

1: 1,000 GAPDH (

Cell signal technology, 5174 S)in TBS+0. 1% Tween-20.

As a control of expression, b1 blocking peptide (Abcam, ab18650)was also used.

Before application, the ratio of b1 antibody to b1 peptide of 1:1 was incubated at 4 °c.

Then combine the membrane with the resistance in the sunflower pod

Rabbit second resistance (

GE Medical, NA931-1ML)

, Visualize proteins using Amersham ECL Prime Western blot detection reagent (

General Electric Medical Group RPN2232). Ovary from 40-day-

Old CD1 mice were fixed with modified Davidson fixative (

Electron microscope science 64133-50)and paraffin-embedded.

The five-micron provision is deparaffinized Citrisolv (Fisher, 04-355-121)

And antigen repair methods to reveal the Decloaker (

Bangjian medicine, RV1000).

Preparation of immune tissue chemical sections Using Avidin/Biotin blocking kit (SP-Vector lab2001)

The serum content of normal goats in TBS was 10%.

Then slice and dilute the 1: 100 b1 resistance (

Abcam, ab18ify, lot GR135551-1)

10% normal goat serum overnight in TBS at 4 °c

As a control of expression, b1 blocking peptide (Abcam, ab18650)was also used.

Before application, the ratio of b1 antibody to b1 peptide of 1:1 was incubated at 4 °c.

Slices are cleaned in TBS 0. 1% Tween-

20, then Mark goat resistance with diluted 1:200 bio

Rabbit second antibody from Vectastain Elite ABC kit (PK-Vector lab6105)

Incubated at room temperature for 2 hours, washed again, and then incubated in ABC reagent for 30 minutes (

Vector lab, Vectastain elite ABC suite)

The last wash.

Fixed with diamino benzene (DAB;

SK-Vector lab4100)for 6u2009min.

Anti-staining was achieved using standard sumo staining.

All images are collected and processed on Evos fl automatic inverted microscope using Evos software (

Life Technology).

The build is fixed 30-60 min, with 3.

More than 8% formaldehyde (

Electron microscope science 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 (

Doctor of Biomedical Sciences, 210370025)

At 4 °c until ready to use.

Samples are washed with PBST.

The builder blocked infiltration with 2% donkey serum, 1% Niu coins and 0.

1% cold skin gelatin, 0. 1% Triton, 0. 05% Tween-20, 0.

Add 05% sodium nitrate 1 u2009 h to PBS.

Anti-vinculin (1:200;

Sigma Aldrich, V9131), HMGB1 (1:200; Abcam, ab18256), laminin (1:50;

Santa Cruz Biotechnology Co. , Ltd. 20682)

Dilute in a 10% closed solution and incubate at 4 °c for overnight

After washing with PBST, mark the sample with secondary antibody (1:250 anti-

Rabbit or 1: 1,000

Mouse, AlexaFluor 488;

Life Technology 21206, a1008)

2-6 u2009 h, then PBST washing and re-dyeing with DAPI (1:50;

Molecular Probe, d136)and Phalloidin-555 (actin;

1: 50, Abcam, ab176756).

The 3D structure is imaged on a Nikon 1R or C2 laser scanning co-Focusing microscope.

Two experiments were performed on each antibody and no primary control examination was performed on each antibody used twice.

When the egg mother cells grow to about 70 μm, ovulation is performed on 6-8 days of follicle culture.

Follicles were incubated in mature medium containing 5% fetal bovine serum at 37 °c and in air at 10% CO for 16 µh, 1.

5 u2009 IU u2009 ml people chorionic gonadotropin hormone (Sigma-Aldrich, C1063)

, 10 ngml skin growth factor (

BD biological sciences, 354010)

And 10 ml of recombinant follicles-

Stimulating hormone

Mother cells released in the mid-term II (MII)

Identified and fixed in 3.

More than 8% formaldehyde contains 0. 1% Triton X-

At 37 °c, 100 lasts for 1 µh for spindle morphology and chromosome alignment analysis.

Wash the egg mother cells 3 times with a 1 × PBS closure solution containing 0. 3% BSA and 0. 01% Tween-

20, anti-in 1: 50 Diluted mice-α-tubulin (

Cell signal technology, 5063 S)

In blocking solution.

Then, the egg mother cell is washed three times with the closed liquid, and the installation uses Vectashield to contain DAPI (

H-vector lab1200)

Analysis using evos fl automatic microscope (

Life Technology).

Egg cell in barrel

Formed bipolar spindle and good

It is considered to be normal tissue-organized microtube fibers, as well as tightly arranged chromosomes on the mid-plate.

In order to verify the presence of a complete embryonic layer, the follicles in the stent were rinsed with PBS and then stained with a 3β hsd solution containing 0.

12 mg ml of azol blue chloride (Sigma-Aldrich, N6639), 0. 25u2009mgu2009ml β-

2-amino acid hydrate (β-NADþ; Sigma-Aldrich, N3014)and 0.

025 mg epiml to go to male ketone (Sigma-Aldrich, E3375)

In PBS, foil is wrapped for up to 3 h at room temperature.

Negative control a negative control of follicles at the same time in a solution free of nianide Guanbin dinucleotide hydration β-

In PBS alone.

If the cell is purple, it is considered positive for 3β hsdBrown.

The negative control for this color change was not positive.

The experiment was conducted three times with 18 follicles each time.

Estrogen was detected in the medium using the ELISA kit (

Calbiotech, es01s, month-1,000 range of pgymml).

Individuals running ELISA and inserting data turn a blind eye to treatment (

Cultural day of collection).

The power analysis determined that three animals in each group were sufficient to detect effectively (80% chance)

The minimum difference in serum levels was 50 ± 10%.

Serum was collected from the processing terminal blood, and AMH and inhibin were tested by the University of Virginia reproductive ligand analysis and analysis core research center.

The mean of technical repetition is drawn.

The range of reports available for AMH is 1. 56–100.

0 u2009 ng u2009 ml inhibin is 10-884 u2009 pg u2009 ml.

Random and go

Serum samples of 5 deovary shams mice and 16 deovary bioprosthesis mice were determined.

AMH, within the detectable range, came from a zero serum sample of the false deovary mice and 9 bio-repaired ovaries.

1 fake mouse and 13 mice with bio-repaired ovary contain serum of statin within the detectable range.

Bar representing the average. e. m.

Because mice with a biorepaired ovary are expected to have biological changes in serum levels dependent on the hormone cycle, we do not anticipate normal distribution.

All tissue processing and wood essence-Yi Hong (H&E)

The staining was performed by the histological core of the Reproductive Science Center of Northwestern University.

Processing a fixed organization using an automatic organization processor (Leica)

And embedded in paraffin.

Continuous slice cutting 5 μm thick with Leica automatic inkjet XL (

Leica micro system).

3-5 slices for each sample and at least 3 samples for each group were subjected to immune tissue chemistry.

Each experiment was performed 2-3 times on a separate day, including no-

Main control.

Slice Imaging on Nikon E600 fluorescence microscope (

Nikon Instruments)

Camera with Retiga Exi 1394 (QImaging).

Anti-platelet endothelial cell adhesion molecule antibody (1:50;

Santa Cruz Biotechnology Co. , Ltd. , platelet-

Growth factor receptor β 1 (1:100; Abcam, ab32570)or GFP (1:300;

Santa Cruz Biotechnology Co. , Ltd.

Use and visualize with AlexaFluor secondment (1:500;

Life Technology, 21206, 21082)

Or the elite ABC kit of Vectastain (

PK-Vector lab6100).

Installation Media with DAPI anti-stains (

H-vector lab1200)

For visualization of nuclear material.

Dye with H & E every 20 knots or 100 u2009 μm and imaging on a Nikon E600 microscope.

8 ovaries were collected 1 or 3 weeks after surgery, each vessel containing blood cells in the stent 100 u2009 μm.

Calculate the average area of the stent or biological prosthesis boundaries of the first and intermediate slices to determine the number of blood vessels in each tissue region.

Animal use was carried out under the Northwestern University Animal Care and Use Committee

Agreement approved.

Enhanced expression in mice ()

This is due to the Cross of the CD1 strain (

White coat Harlan)and -

Mice expressed (

Black jacket from Jackson Lab)

Used to create a bio-repair ovary and identify it using a blue star light with a VG1 filter glasses (EMS, BLS-1). Two-

As mentioned above, mm-grade scaffold was prepared on transwells with growth medium.

As mentioned above, the original primary and secondary follicles from the female are sown within 2 days, the small follicles are first sown, the entire stent is filled the next day, and additional small follicles and some secondary follicles are added (

Up to 80 μm in diameter).

These brackets were cultured for a total of 4 days prior to surgery and were imaged in the morning prior to surgery.

The false implant stent was prepared in the same way, but the cells were not included.

Intrabursal surgery is for the month-to 10-week-

Old NSG female (white coat; Jackson Labs).

Anesthesia mice with a mixture of 100 mg kg ketamine and 15 mg kg xylazine.

Visualize the upper uterine corner, fallopian tube and ovarian SAC and remove it from the cavity for surgery.

The ovarian artery was positioned to suppress the blood flow by suturing the band on the 10 th.

The ovary is completely removed while maintaining the integrity of the sac and sac cavity.

The bioprosthesis ovary is inserted into the ovarian SAC, opened to the fallopian tube, and closed in the sac with a needle or two with a nylon suture on the 10 th.

Both ovaries repeat this.

Fake surgery like these, but the stent does not contain any cells.

Mating of 7 mice with a bio-repaired ovary with CD1 and 2 mice undergoing false surgery (white coat)

Male who had raised cubs before.

Four days after surgery, each female was paired with a male and lived together for 25 days or more.

solid plug is present in the female vagina after mating, indicating successful mating.

No extra mating has been tried.

The pups produced by this mating are identified as being produced from eggs released from the scaffold if the puppy is or has a black fur color.

It is not certain that the Cubs with white fur color are from the implant.

Mating is carried out in this way, since these pups are purely United to death, thus only one copy of the follicle is carried in the bio-repair body.

So, we expect about half of the pups we get from biological fake eggs.

All data sets are analyzed using the GraphPad Prism software and are represented on average. e. m.

Statistical significance using one-execution

Comparison test for multiple-factor variance of Holm-Sidak = month. 05.

The authors state that all data supporting the results of this study can be found in the paper and its documents.

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

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