Materials and Methods.
Materials:The experiments were done from July 2011 to April 2012 at the Beijing Institute of Cardiopulmonary and Vascular Diseases-Provincial and Ministerial Key Laboratory of Cardiovascular Remodeling. Clean-grade BSL-C57 mice were selected and purchased from Beijing Viton Lever Laboratory Animal Company, and fetal bovine serum and LG-DMEM medium were purchased from GIBICO.
Methods.
1. Cell isolation and culture.
The bone marrow cavity was exposed under aseptic conditions, and the cells in the bone marrow cavity were repeatedly rinsed with sterile DMEM culture medium, and the larger tissue masses were removed by filtration through 200 mesh nylon mesh. The collected cell suspension was centrifuged at 1000 rpm for 5 min, and the cells were resuspended and inoculated into LG-DMEM medium containing 10% FBS, and the medium was changed within 24 h. Later, the medium was changed every other day, and primary cell culture (P0) was performed in a CO2 incubator at 37℃ with a volume fraction of 0.05. The cells were digested and cultured at 1:2 passages (P1, P2, P3, …). The medium was changed every other day, and the morphological changes of the cells were recorded by inverted microscopy.
2. Flow cytometry identification:
The cells were digested with 0.25% EDTA-trypsin and the cell concentration was adjusted to 1×107/ml with PBS solution. 5 flow tubes were taken and 100µl of cell suspension was added, followed by PE-anti-CD90, PE-anti-CD34, FITC-anti-CD45, FITC-anti-CD73 After shaking, the cells were incubated at 4℃ for 30min, washed twice with PBS, filtered to remove cell clumps, and subjected to flow cytometry.
3.Cellular myocardial patch production and identification.
The patches were disinfected with ethylene oxide and cut into 0.5cm diameter circular patches using a puncher, and the patches were infiltrated with culture medium, and then rinsed 3 times with PBS buffer after the patches were completely infiltrated with culture medium, and put into 96-well plates for backup, keeping the cell patches flat. After digestion with 0.25% trypsin, count 1×105 cells per well to the patch.
A. Electron microscopy specimen preparation.
The co-cultured specimens were washed 3 times with deionized water, fixed with 0.25% glutaraldehyde 500 μL, submerged cell patches were stored at 4℃ and made into scanning electron microscope specimens for detection on the machine.
B, Fluorescent labeling method to observe cell growth and proliferation.
Mouse BMSC was made into a cell suspension with a concentration of 1×107L-1 and then inoculated into a 24-well plate (each well contained 500μL of DMEM culture solution with 10% FBS by volume), and three wells were set for each material specimen. The cells were incubated for 24 h in an incubator with 5% CO2 by volume, 37℃ and saturated humidity, and the culture medium was discarded after the cells were attached to the wall, fixed with paraformaldehyde for 10 min, washed 3 times with PBS, and then the patches were placed on slides with DAPI drops on the patches, covered with a coverslip and observed with a fluorescent inverted microscope and photographed. Data were counted for each material.
Data processing.
Cell counts were analyzed using SPSS 12.0 statistical software, and ANOVA was used for comparison between groups, and differences were considered statistically significant at P<0.05. Flow cytometry identification results were analyzed using EXPOTM32ADC software.
Results.
1. Light microscopy of BMSC.
BMSC in the early stage of light microscopy cell morphology is diverse, mostly round, oval, rod-shaped or shuttle-shaped 4-6 days or so “colony”-like growth, rapid cell expansion, radial, swirling outward expansion, two or three weeks to reach 80%-90% fusion, several times after the cell exchange of suspended cells have been removed, 3-5d can reach 80%-90% fusion, walled cells. The cells were spindle-shaped and proliferated vigorously, maintaining a swirling growth pattern.
2. Cell flow identification.
The 3rd generation BMSC were identified by flow cytometry, and the results were CD34, CD45 negative, CD90 weakly positive and CD73 positive.
3. Electron microscopic scanning results.
Under the scanning electron microscope, the surface of the patch made of PU material was similar to the irregular surface formed by granule-like aggregation, the surface cell morphology was normal, the number was large, the distribution was uneven, and a small amount of cellular debris and other cell lysis was visible) The material was a mesh structure made of interwoven fibers, with a three-dimensional three-dimensional structure, uniform pores, good cell growth, irregular morphology, and no obvious cellular debris and other cell lysis was seen; PPC The surface of the material made of PPC is smooth and occasionally depressed, and the number of cells on the surface is very small, with some cell fragments and other cell lysates.
4. Counting under inverted microscope after fluorescence labeling of cell nuclei.
The mean cell counts under fluorescence microscopy of cell patches at 400x were 143.78±38.38 for the PU material group, 159.50±33.07 for the P(3HB-co-4HB) material group and 1.40±0.70 for the PPC material group. analysis of variance with SPSS 12.0 software yielded the mean cell counts between the PU and P(3HB-co-4HB) groups There was no statistical difference (P=0.942), and the mean values of cell counts between the PPC group and the PU group (P1) and the P(3HB-co-4HB) group (P2) were statistically different (P1=0.000, P2=0.000).
Discussion.
BMSC is a relatively primitive bone marrow stromal cell, bone marrow-derived MSCs possess strong differentiation potential and may participate in angiogenesis through lateral differentiation into cardiomyocytes, cell fusion, direct differentiation into endothelial cells, and smooth muscle cells [13, 14], and paracrine action to activate endogenous repair mechanisms to play a role in repairing damaged hearts [15], leading scientists to widely The clinical applications of MSCs in tissue repair and gene therapy have received extensive attention from scientists. Because of their myocardial and vascular repair potential, they have become one of the hotspots for seed cell sources in myocardial tissue engineering research. Cellular myocardioplasty is mainly performed by cell transplantation for the treatment of myocardial infarction, but there exists a loss of transplanted cells after cell transplantation with the washout of cells by blood flow and mechanical activity of the heart [16]. Thus, a material that provides a good survival environment for the cells and has certain physical properties and good biohistocompatibility as well as in vivo degradation and absorption was sought to improve the survival and proliferation of transplanted cells in the area of myocardial infarction and thus to better improve the prognosis.
Polyurethane (PU) was first invented by German chemists and was introduced into China in the 1950s and developed rapidly. PU elastomers can have high elasticity and strength, excellent wear resistance, oil resistance, fatigue resistance and vibration resistance in a wide range of hardness. Polyurethane has excellent overall performance and has been widely used in textile, printing, medical, sports, food processing, construction and other sectors.
The copolyester of 3-hydroxybutyric acid and 3-hydroxyhexanoic acid (P(3HB-co-4HB)) is a new type of third generation PHA material, which has attracted widespread attention because of its good physical properties. P(3HB-co-4HB) was discovered and synthesized as a new tissue engineering material in the laboratory of Institute of Polymer Research, Department of Chemical Engineering, Tsinghua University. It was shown that P(3HB-co-4HB) presents biocompatibility and degradation absorption properties superior to PHB.
Although the cells can survive on myocardial patch material made of P(3HB-co-4HB) material and P(3HB-co-4HB) material can have good biocompatibility and degradable absorption properties, the effect of the degradation and absorption of the patch material on the organism is yet to be studied, and the effect of the implantation of the patch material on the immune system of the organism is not clear, and P( 3HB-co-4HB) materials as myocardial patches and the feasibility and further biological toxicity and function still need to be further investigated.
In conclusion, different studies exist in the selection of cellular myocardial patches, and experimentally stem cells were able to be successfully grown onto patches made of PU material and P(3HB-co-4HB) material, but the non-absorbable properties of PU material limit its use in the field of medical biomaterials, while P(3HB-co-4HB) material is a novel biomaterial with good biohistocompatibility and physical properties, which are absorbable in living organisms and can be a good choice as a material for cellular myocardial patches, but the feasibility and further biological toxicity and function of P(3HB-co-4HB) material as a myocardial patch need further study.