Injection type tissue engineering bone renovation material and construct method thereof
A tissue engineering bone and repair material technology, which is applied in tissue culture, biochemical equipment and methods, tissue cell/virus culture devices, etc., can solve the problems of complex preparation, high cost, and cannot be widely used, and achieves simple preparation. , the effect of low price
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Embodiment 1
[0030] Embodiment one: the cultivation of seed cell
[0031] Take 5-month-old healthy Chinese green goats (weight 35.0-40kg, male or female, provided by the Animal Experiment Center of Southern Medical University), perform iliac crest puncture with a 16-gauge bone puncture needle under aseptic operation after anesthesia, and extract bone marrow by multi-point puncture 10mL, centrifuged and filtered with 100-mesh stencil, inoculated into a culture bottle, complete medium (DMEM+10% fetal bovine serum), placed in CO 2 Cultivate in an incubator (Harris Company, USA), and change the medium every 3 days. After the primary cells cover the bottom of the bottle, digest and separate the adherent cells with 0.25% trypsin-0.02% DMEM solution, add complete medium, and place in CO 2 The incubator continued to cultivate, and the medium was changed every 2-3 days.
Embodiment 2
[0032] Embodiment 2: Preparation of platelet-rich plasma (PRP)
[0033] Take 10ml of sheep venous blood with a syringe containing 1ml 10% sodium citrate anticoagulant, shake it up, put it in a centrifuge tube, and prepare PRP by secondary centrifugation (Huang Aiwen, Jin Dan, Pei Guoxian, Chen Shujun, Hu Jijie, Lin Haining, Zeng Xianli, Construction of injectable tissue-engineered bone compounded with platelet-rich plasma (PRP) and its radiological evaluation for repairing radial defect in rabbits, Chinese Journal of Traumatic Orthopedics, 2005, 7(4): 363-367.). Make about 1ml of PRP per 10ml of whole blood, and store it in a -70°C refrigerator for later use.
Embodiment 3
[0034] Example 3: Preparation of Injectable Tissue Engineering Bone Repair Material
[0035] The third generation BMSCs (planting density 2×104 / well) were inoculated in 24-well culture plates, and 0.6ml of culture medium was added to each well.
[0036] Injectable chitosan and β-tricalcium phosphate (β-TCP) materials were provided by the Department of Materials Science and Engineering, Jinan University. After 3ml chitosan and 1.8gβ-TCP were compounded in vitro for 8min, 0.3mlPRP and 0.1ml thrombin (10% calcium chloride contained 100U thrombin) were added into the liquid chitosan-β-TCP successively. Mix well to prepare chitosan-β-TCP / PRP complex.
[0037]The preparation experiment of injection-type tissue engineering bone repair materials was divided into three groups, namely chitosan-β-TCP / PRP group, simple chitosan-β-TCP group and blank control group. Among them, chitosan-β-TCP / PRP group and simple chitosan-β-TCP group added 5mm×5mm×3mm solidified chitosan-β-TCP / PRP or simp...
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