MSCs stent for bone defect repair and preparation method thereof
A scaffold material and purpose technology, applied in the field of bone tissue engineering and regenerative medicine, can solve the problems of poor bone defect repair effect, poor growth and proliferation, etc., achieve good tissue compatibility and bone repair function, and have broad application prospects , the effect of simple preparation method
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Embodiment 1
[0031] Example 1 Construction and identification of PDCD4 knockdown MSCs stably transfected cell line
[0032] 1. Construction of PDCD4 knockdown MSCs stable cell line
[0033] 1.1. Isolation, culture and expansion of human umbilical cord MSCs
[0034] Collect neonatal umbilical cord tissue (the mother has signed the informed consent), add a small amount of DMEM / F12 complete medium (10% FBS, 0.272g / L L-glutamine, 10ng / mL bFGF, 100U / mL penicillin, 100mg / mL Streptomycin), cut the tissue as much as possible with sterile scissors in a sterile operating table, digest with 0.125% trypsin and 1% collagenase II for half an hour, collect the liquid into a 15mL centrifuge tube, and centrifuge at 1500rpm for 10min. Discard the supernatant, resuspend the cell pellet in the above-mentioned DMEM / F12 complete culture solution, transfer it to a cell culture flask, and place it in an incubator at saturated humidity, 37°C, 5% CO 2 Incubate under certain conditions, suck off the supernatant on...
Embodiment 2
[0050] Example 2 Preparation of PDCD4 Knockdown MSCs-PRP Scaffold Composite
[0051] 1. Extraction of PRP
[0052] With a 10mL syringe pre-filled with 2mL 10% sodium citrate anticoagulant, draw 8mL blood from the central artery of the rabbit ear, mix it upside down, transfer it into a centrifuge tube, centrifuge at 200g for 10min, and divide the blood into upper plasma, middle There are three layers of platelets, white blood cells and red blood cells in the lower layer. Absorb all the plasma in the upper layer, the red blood cells in the middle layer and the lower layer with a height of about 1 mm into another centrifuge tube, and centrifuge at 250g for 10 minutes. The plasma is divided into platelet-poor plasma (PPP) in the upper layer and PRP in the middle layer. and a small amount of red blood cells in the lower layer, discard the PPP in the upper layer, absorb about 1.5mL of liquid in the middle and shake well to obtain PRP, and discard the residual red blood cells in the ...
Embodiment 3
[0055] Example 3 Application of PDCD4 Knockdown MSCs-PRP Scaffold Composite
[0056] 1. Establishment of bone defect repair model
[0057] Rabbits were anesthetized by intravenous injection of 20% urethane 1g / kg, hair removal cream was applied to the knee joint, local disinfection was performed, the skin of the knee joint was cut, the fascia was separated, the patella was removed, and the upper end of the tibia was exposed, and a drill bit with a diameter of 4mm was disinfected. A circular defect with a diameter of 4 mm was drilled in the upper end of the tibia with an electric drill, and washed repeatedly with sterile saline and antibiotics. The prepared cell-scaffold composite material with the same size as the bone defect was filled into the bone defect, and divided into 4 groups: (1) the control negative control group was not filled; (2) the PRP scaffold control group was filled with PRP gel; (3) Mock-MSCs-PRP scaffold complex group; (4) siPDCD4-MSCs-PRP scaffold complex ...
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