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improved human bone marrow mesenchymal stem cell osteogenesis in 3d bioprinted tissue scaffolds with low intensity pulsed ultrasound stimulation

by:Tuowei     2019-08-14
3D printing and ultrasound technology have shown great prospects in the development of human skeletal tissue repair and regenerative medicine.
The uniqueness of this study is that the low-intensity pulse ultrasound (LIPUS)
Cooperate with advanced 3D printing technology to improve the growth and bone formation of human bone marrow-derived stem cells (MSC).
Specifically, the bio-ink containing the cell adhesive polyglycol ester-Glycine-Aspartic acid-Serene (RGDS)
Ha of peptides and/or nanoparticles (nHA)
Used to make 3D brackets with different geometric patterns through new tables
3D printer.
The resulting scaffold provides a highly porous and interconnected 3D environment for supporting cell proliferation.
The holder with a small square hole is determined to be the best geometric pattern for MSC attachment and growth.
The best working parameter for LIPUS is determined to be 1.
5 mhz, 20% duty cycle, strength mw/cm 2.
The results show that the 3D printing scaffold containing RGDS peptide and 3D can greatly promote the proliferation, alkaline phosphate activity, calcium deposition and total protein content of MSC under LIPUS treatment.
These results demonstrate the effectiveness of LIPUS and bionic 3D printing stent combinations as valuable combination tools to improve MSC function, thus providing prospects for future clinical and various regenerative medicine applications.
Make a 3D printed gel stand through a new table
Top stereo printing printer.
More details about the stereo printing device and the 3D printing process can be found here.
Before 3D printing, the 3D structure model was first designed by a computeraided design (CAD)software.
Then, the air hole rate, model pattern and speed of laser are programmed through Slic3r computer numerical control conversion software.
To put it simply, the design and printing layer of the CAD modelby-layer ().
Because the frequency of the pulse signal is sustainable (1~20u2009K Hz)
According to the printing requirements of biological ink, the output laser energy can be controlled;
Here, we chose 8 k Hz as our research object.
In this study, we designed four models of different geometric shapes and aperture, including large positive squares (LS)(500u2009μm)
Large hexagon (LH)(500u2009μm)Small square (SS)(250u2009μm)
And small hexagon (SH)(250u2009μm)().
Three layers of 400 µμm thick were printed in about 2 square inches and a uniform cylindrical stent was collected using an 8 µmm biopsy punch for cell evaluation.
The four test groups are: 60 u2009 w. t. % PEGDAu2009+u200940u2009w. t.
% Photoiniator PEG tracker + will . . . . . . (0.
5% of Peg quality)(PEGDA group);
The PEGDA group is 1 RWW. t. % Acryl-PEGDA-RGDS (PEGDA-RGDS group);
PEGDA group alimtusnha (10u2009w. t.
% Of Peg quality)(PEGDA-nHA group); and PEGDA-
RGDS group alimtusnha (10u2009w. t. % Of Peg quality)(PEGDA-RGDS-nHA group). Acryl-PEG-
RGDS were synthesized according to Zhu\'s method and slightly modified. Briefly, 1:1.
RGDS peptide 2 u2009 m ratio (Arg-Gly-Asp-Ser)(
Peptide Corporation of Americaand Acryl-PEG-NHS (
Technology)
Mix fully at 4 °c and stir overnight
After completion, the mixture is purified by dialysis (Slide-A-
Lyzer™Dialysis box, 3. 5u2009K MWCO)
Get the final Acryl-PEG-RGDS polymer.
As mentioned earlier, the nHA has been prepared and characterized. In brief, 0.
The ammonium phosphate of 6 µm was dissolved in pure water of 825 µml and the pH was adjusted to 9.
Add 50 mL sodium hydroxide to 0. 90u2009mL of 0.
883 µm calcium nitrate is added to the drop-
Be wise at the speed of 3.
Stir continuously for 6 mL/min.
Transfer the mixture to the Teflon liner and perform a thermal reaction of 20 km/h at 200 °c.
Finally, nHA with a width of about 25 nm and a length of about 50-100 nm was obtained.
The SEM samples of porosity and morphology were evaluated (SEM)(
Zeiss Nvision 40FIB).
The ultimate tensile strength of scaffolding by mechanical testing machine (
Application Testing System, Butler, PA).
To put it simply, the scaffold is cut into strips of size 10mm × 30mm and then fixed to the clip of the machine.
Young\'s modulus is recorded at the speed of 1mm/min (nu2009=u20096).
The surface charge of the scaffold is measured by drip analysis using the contact angle analyzer (DSA4, Kruss).
According to IRB-major human bone marrow-derived stem cells were obtained from donors with health consent from Durham University
Approved agreement with written informed consent.
The Texas A & M Center for Health Sciences at the Institute of Regenerative Medicine further distributed to us cells with thorough features.
We have the fully executed material transfer protocol needed to obtain the cells.
All experiments were carried out in accordance with the material transfer agreement. MSC (passage 3–6)
Culture in alpha-
Minimum Basic medium Eagle (α-MEM)
Supplementary fetal bovine serum (FBS)(16. 5%, v/v)
Penicillin and penicillin (1%, v/v)and L-glutamine (1%, v/v).
Cells were cultured at 37 °c, 5% CO and 95% relative humidity (RH).
For MSC to differentiate into bone, MSC was cultured in Dulbecco modified Eagle medium (DMEM)
Add FBS (10%, v/v)
Penicillin and penicillin (1%, v/v), L-1) Vitamin C (50u2009μg/mL), β-
Glycerin phosphate (10u2009mmol/L)
Dex (10u2009nmol/L).
Culture cells as mentioned earlier.
First, in order to obtain the best scaffold geometry (SH, LH, SS, LS)
, MSC adhesion was evaluated as a function of the aperture and geometry.
Prior to the test, the stent sample was collected with biopsy punch and immersed in 75% ethanol for 12 hours.
After that, put the sample in 48-
Well plate, then soaked in PBS and α-
MEM for 12 km/h h, respectively.
Msc was then incubated on the scaffold at a density of 5 × 10 cells per cm and cultured for 4 × h and 8 × h.
Wash the seed holder three times with PBS at a predetermined point in time to go unless
Cell adhesion and number of cell adhesion measured by cell counting kit8 (CCK-8)(Dojindo, Japan).
To put it simply, 10% reagent was added to the orifice plate, then 2 u2009 h incubation was performed and absorbance was measured at 450 nm with a spectrometer (Thermo, USA).
The results show that the stent with small square holes has a greater MSC adhesion force in the case of OR without RGDS ().
Therefore, in all subsequent experiments, brackets with small square holes were used.
The tuned LIPUS system consists of a function generator, a power amplifier, and an ultrasonic sensor.
The ultrasonic wave is generated by a function generator and amplified by a high power amplifier with constant gain, emitted from the sensor.
To operate in this study, the sensor is placed and oriented perpendicular to the cell culture plate.
To prevent bubbles from being trapped between the surface of the sensor and the medium, fill the plate to the edge.
The sensor is then lowered into the hole until a working distance of about 15mm from the surface of the cell culture is reached.
Give an incentive, then remove the sensor and wipe it with 70% ethanol, the process is repeated.
In the absence of ultrasonic excitation, the control samples were only submerged and the transducer was removed.
Initially, MSC responses to LIPUS were evaluated at 1.
5 mhz, 20% duty cycle of various strength (
20, 50, 75,150 and 300 mw/cm).
MSC was cultured overnight on a 24-well plate with 2 × 10 cells per cm.
Next, change the media to remove the non-
The adhesion cells and samples were stimulated by ultrasound for 5 min.
After 24 h and 48 h, the number of cells passed through gmail-
8 u2009 was determined and compared with the control.
The results showed that MSC adhesion was maximum under 15 mw/cm (). As a result, 1.
5 mhz, 20% duty cycle and mw/cm LIPUS excitation were used in all experiments.
The effects of LIPUS on the proliferation of MSC vaccinated on a 3D printed bioactive scaffold were evaluated. MSC (passage 3 to 6)
Train in 24 hours
Cell culture plate.
To put it simply, four groups were tested (PEGDA, PEGDA-RGDS, PEGDA-nHA and PEGDA-nHA-RGDS)
Our stereo print prints small square pores. based 3D-
Printer as described above.
Scaffolding measurement 15.
5mm in diameter, placed at 24-
Subsequently soaked in PBS and α-
MEM for 12 km/h h, respectively.
MSC was then incubated on the scaffold at a density of 5 × 10 cells per cm for 24 hours.
After that, change the media to remove the non-
The adhesion cells and stent were exposed to LIPUS excitation at 1.
5 mhz, 20% duty cycle, 15 mw/cm strength of 5 min per day.
As mentioned above, cells are promoted and quantified after 1, 3 and 5 daysLipps treatment
The morphology and LIPUS excitation of MSC after 5 days of culture were examined by laser co-Focusing microscope (
Carl Chase 710).
All brackets were washed 3 times with PBS, fixed with 10% formaldehyde for 10 min, and then infiltrated and dried in 0. 1% Triton-100 for 10u2009min.
MSC is double.
Stained with Texas red for 15 minutes, 4\', 6-diamidino-2-phenylindole (DAPI)for 3u2009min. A three-
A study was conducted on Zhoucheng bone division to evaluate the effectiveness of ultrasound stimulation on MSC bone formation.
MSC was cultured at a density of 1 × 10 cells per cm for 24 hours. Non-
Remove the adherent cells, replenish the stent with a bone-differentiated medium, and then receive LIPUS excitation at 1 point.
5 mhz, 20% duty cycle, 15 mw/cm per day for 5 min.
After 1, 2, 3 weeks, rinse the sample holder three times with pure water.
Cracking samples by freezingthaw cycling (3×)
The total protein, alkaline phosphate activity and total calcium synthesis were evaluated.
Determination of total protein content of cracked samples by commercial BCA Protein Determination Kit (
Pierce Biotechnology
According to the manufacturer\'s instructions.
Simply, 50 μ l per sample is loaded into the microplate hole, followed by the addition of the μ l protein detection reagent and incubation at 37 °c for 2 µh.
Then, the absorbance of each sample was measured at 562 µnm and compared with the serum protein standard curve to obtain the total protein.
Alkaline phosphate activity is one of the main early indicators of the adult phenotype.
According to the manufacturer\'s instructions, the activity of the cracked sample was determined with a quantitative alkaline phosphate Determination Kit.
To put it simply, a standard curve was created and run in parallel with the cracking sample to determine the absolute concentration of alkaline phosphate.
Add 50 μ l lysate sample to 96-
The orifice plate is then added to the 15 μ l working solution (
Analysis of the ratio of Mg acetate to 200:5:2 in buffer)and mixed.
The absorbance of the mixture is read at 405 nm and again after 4 min.
Finally, the alkaline phosphate activity is calculated according to the kit instructions.
Total calcium content is one of the main Late stages
Stage markers used to determine bone marrow-derived MSC bone-forming properties.
Quantification of soluble extracellular calcium content by commercial calcium kit (
Toe Technology Co. , Ltd. ).
In short, the scaffold is immersed in a 0.
Hydrogen chloride (6 µN)HCl)
Solution at 37 °c for 24 hours.
Subsequently, the 50 μ l lysate and o-
It is formed in the solution of Cresolphthale ammonia-acid complex agent, light purple.
The solution with known calcium concentration runs in parallel with the experimental group.
Absorbance was measured by luminosity at 570 nm.
The data is represented by the mean value of the mean value ± standard error, and through-way ANOVA. A pu2009
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