Cell engineering is an important aspect of bioengineering. Generally speaking, it applies the theories and methods of cell biology and molecular biology, carries out genetic operation at the cell level, and carries out large-scale cell and tissue culture according to people's design blueprint. The following is my cell engineering paper for you. Welcome to reading.

Objective To prepare acellular muscle tissue engineering scaffold and test its biocompatibility with human amniotic epithelial cells. Methods acellular muscle tissue engineering scaffold was prepared by chemical extraction combined with TNT and sodium dodecyl sulfonate, and its structure was observed by frozen section. Seven days after human amniotic epithelial cells were seeded on the scaffold, the cell proliferation activity, the expression of NT3 and BDNF were detected by immunohistochemistry, and the cell ultrastructure was observed by scanning electron microscope. Results The cells in the scaffold were completely removed, and the main structure was parallel tubular structure. The main components of extracellular matrix, elastic fibers and collagen fibers, remain intact. Amniotic epithelial cells have proliferative activity in the scaffold and are positive for NT3 and BDNF. Scanning electron microscope showed that amniotic epithelial cells were evenly distributed in the scaffold and grew well. Conclusion Acellular muscle tissue engineering scaffold has been successfully prepared and has good compatibility with human amniotic epithelial cells.

Acellular muscle; Human amniotic epithelial cells; bio-compatibility

In recent years, one of the important advances in tissue engineering research is to make tissue engineering scaffolds of natural biodegradable materials with autologous or allogenic grafts. Among them, acellular graft has good biocompatibility with the body. Acellular muscle scaffold can be used as a bioengineering scaffold to support axonal regeneration of nerve cells. Mligiliche et al. [1] transplanted acellular muscle to the defect of sciatic nerve in rats, and found that a large number of nerve axons grew into acellular muscle scaffold after 4 weeks. Because the effect of pure acellular muscle scaffold in treating nervous system diseases is limited, acellular muscle scaffold often needs to be implanted with seed cells to play a greater role [2, 3]. Studies have shown that amniotic epithelial cells can secrete a variety of nerve factors [4, 5], promote the growth of neuronal axons, and are good seed cells for the treatment of nervous system diseases. In this study, acellular muscle scaffold was made by chemical acellular method, and amniotic epithelial cells were inoculated into acellular muscle scaffold to explore its compatibility, which provided a new way for tissue engineering to treat nervous system diseases.

1 materials and methods

1. 1 material

1.1.1Wistar rats were provided by bethune medical school Experimental Animal Center of Jilin University.

1. 1.2 reagent IMDM medium and calf serum were provided by Hyclone. 5' bromouridine (BrdU) and monoclonal antibodies against BrdU were purchased from Neomarker Company. Rabbit anti-human polyclonal antibodies against neurotrophic factor (NT)3 and brain-derived neurotrophic factor (BDNF) were purchased from Wuhan Doctor Company, and SABC immunohistochemical kit was purchased from Fuzhou Maixin Biological Company. The human amniotic epithelial cell line has been preserved in our laboratory.

1.2 method

Preparation of acellular muscle scaffold 1.2. 1 The acellular muscle scaffold was prepared according to the preparation method of acellular bladder by Brown et al. [6], which was briefly described as follows: the abdominal serratus muscle of Wistar rats was put into distilled water, shaken evenly in a shaker at 37℃ and 50 r/min for 48 h, and then transferred to 3% tritonx/kloc-. Then put it into distilled water and shake it for 48 h at 37℃ and 50 r/min. Replace with 1% SDS solution, 37℃, 50 r/min, and shake well for 48 h. PBS washing for 24 hours. Store in PBS at 4℃ for later use.

1.2.2 morphological structure observation and component identification of the scaffold The morphology of acellular muscle was observed with naked eyes. Acellular muscle was fixed with 4% paraformaldehyde PBS 1 h, 5% sucrose for 90 min, 15% sucrose for 90 min, 30% sucrose gradient dehydration overnight, OCT embedding, cold acetone rapid freezing, and then stored in the refrigerator at -70℃. The internal structure was observed by frozen section and he staining. In addition, Van Gienson(VG) staining and Weigert staining (VG+ET staining) were used to detect the extracellular matrix components of the scaffold.

1.2.3 human amniotic epithelial cells were cultured in DMEM medium (containing 10% fetal bovine serum, 100 U/ml penicillin, 100 mg/ml streptomycin, 200 μg/ml glutamine) at 37℃, 5%.

1.2.4 Identification of compatibility between human amniotic epithelial cells and acellular muscle scaffold

1.2.4. 1 Take human amniotic epithelial cells that grow well and 80% are close to fusion, discard the culture medium and digest them with 0.25% trypsin. When the cell body retracts and the intercellular space widens, stop digesting with serum, gently blow the cells on the bottle wall repeatedly, make single cell suspension in centrifuge tube, and resuspend the cells with DMEM at 65438±0000 r/min. Inhale the cell suspension with a syringe of 1ml, inject it into the acellular muscle scaffold at a density of 2× 106 cells/ml, subpackage it in a 24-well plate, culture it in a cell incubator with 37℃, 5% CO2 and saturated humidity, and change the solution every other day 1 W ... Add Brdu (final concentration:/. After the slices were washed by PBS, the endogenous peroxidase was inactivated by 3% H2O2 for 65438±00min, and the serum was blocked for 20 min. The primary antibody was incubated with BrdU monoclonal antibody (diluted 1 ∶ 1 000), BDNF and NT3 polyclonal antibody (diluted 1 ∶ 1 000) overnight at 4℃, then incubated with PBS at 37℃ for 30 min, and then incubated with SABC at 37℃ for 30 min. Observe under an optical microscope.

1.2.4.2 scanning electron microscope was used to identify the growth of amniotic epithelial cells on acellular muscle scaffold. When 80% of human amniotic epithelial cells are close to fusion, they are digested as described above, planted in acellular muscle scaffolds, placed in 24-well plates, cultured in a cell incubator at 37℃, 5% CO2 and saturated humidity for 7 days, and then incubated for 2 days.

Bear fruit

2. 1 Tissue structure and components of scaffold The acellular muscle is milky white, translucent and soft. Generally speaking, the overall size and shape of muscle have not changed significantly before and after decellularization. HE staining in the longitudinal section of the scaffold showed that the components of skeletal muscle cells disappeared, but the fiber mesh structure remained intact, and the scaffold was mainly parallel tubes. VG+ET staining proved that the scaffold was mainly composed of extracellular matrix components such as collagen fibers and elastic fibers. The collagen fibers showed red wavy structure and the elastic fibers showed blue filiform structure, as shown in figure 1.

2.2 Compatibility between amniotic epithelial cells and acellular muscle scaffold is shown in Figure 2. HE staining showed that human amniotic epithelial cells grew well and distributed evenly in the scaffold (Figure

Figure 1 acellular muscle scaffold is usually stained with tissue sections.

Fig. 2 Pathological photograph of acellular muscle scaffold 2A. Immunohistochemical staining showed a large number of BrdU positive cells, suggesting that the human amniotic epithelial cells in the scaffold have the ability to proliferate (Figure 2B). Anti-NT3 and BDNF staining showed that the human amniotic epithelial cells in the scaffold contained NT3 and BDNF positive particles, which were distributed in the cytoplasm in brown (Figures 2C and 2D). JSM5600LV scanning electron microscope showed that a large number of cells were distributed in the scaffold, and the cells were evenly distributed and grew well (Figure 2E).

3 discussion

The ideal scaffold material should be similar to extracellular matrix and have good biocompatibility with living cells [7, 8]. Acellular muscle, as a bioengineering scaffold material for treating nerve injury, has the following advantages: (1) The extracellular matrix components of acellular muscle play an important role in the migration, adhesion, growth and metabolism of tissue cells, and research shows that regenerated axons can be well adhered to acellular muscle scaffold [9]. (2) The arrangement structure of acellular muscle is similar to that of neural tube, but its diameter is slightly larger than that of neural tube [10], which provides enough space for axon growth through [9], which is very important for inducing axon regeneration. Fansa et al. compared the results of different acellular biomaterials (muscle, vein and epineurium) inoculated with Schwann cells to bridge the nerves around the defect, and found that the regenerated axons in acellular muscle scaffolds (vein and epineurium scaffolds) lacking nerve tubular structure were disordered and irregularly arranged, while those in acellular muscle scaffolds with nerve tubular structure were orderly arranged [1 1]. This sequence of axonal regeneration is also very important for axonal regeneration of nerve injury. (3) The immune rejection caused by acellular muscle is small [9, 12]. These advantages indicate that acellular muscle can be used as an ideal material to treat nerve injury. The method of making acellular muscle used in this study is mainly used to reduce the immune rejection of xenograft materials. This method can effectively remove lipid membranes, membrane-associated antigens and soluble proteins, and effectively preserve the original spatial structure of extracellular matrix components. Muscle cells are normally distributed in parallel, and so are the components of extracellular matrix. From the longitudinal section of the stent, the fiber components of the stent are also arranged in parallel. VG+ET staining showed that collagen fibers and elastic fibers, the main components of extracellular matrix, remained intact. These results further confirm that this method can successfully prepare acellular muscle scaffold.

Because the effect of simple acellular muscle scaffold in treating nervous system diseases is limited [13], the biocompatibility of acellular muscle needs to be verified. In this study, human amniotic epithelial cells were inoculated into acellular muscle scaffolds to explore their compatibility. Studies have shown that amniotic epithelial cells contain a variety of bioactive factors, including mucin, transfer growth factor, prostaglandin E, epidermal growth factor-like substance, IL 1, IL-8 and other factors, in addition, they can secrete important neurotrophic factors such as BDNF and NT3 [4]. Among them, bioactive factors such as laminin, BDNF and NT3 play a very important role in the treatment of nerve injury. Amniotic epithelial cells can be used as ideal seed cells, and combined with acellular muscle scaffold may become an ideal tissue engineering material for treating nervous system diseases. In this experiment, it was observed that human amniotic epithelial cells were evenly distributed in acellular muscle scaffold. Anti-BrdU, BDNF and NT3 immunohistochemistry showed that amniotic epithelial cells in acellular muscle scaffold had good proliferation ability and could express BDNF and NT3, which indicated that amniotic epithelial cells maintained good biological activity in acellular muscle scaffold. On the one hand, the above results prove that the acellular muscle scaffold prepared in this study has good biocompatibility, on the other hand, it provides theoretical and experimental basis for the combination of amniotic epithelial cells and acellular muscle scaffold to treat nervous system diseases.

In a word, acellular muscle scaffold was successfully prepared in this study, and it was confirmed that human amniotic epithelial cells could secrete important neurotrophic factors in acellular muscle scaffold. The bridge between human amniotic epithelial cells and acellular muscle scaffold provides a variety of favorable factors for nerve defect regeneration, such as basement membrane and neurotrophic factors, which constitutes a good microenvironment for nerve regeneration and is conducive to better repair of nerve defect, laying a certain experimental foundation for further research on the treatment of nerve injury with amniotic epithelial cells and acellular muscle scaffold bridge.

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