Composite structured tissue engineering cartilage graft and preparation method thereof
A composite structure and tissue engineering technology, which is applied in the fields of medicine and biomedical engineering, can solve the problems of biological glue not reaching the effective concentration, large damage to patients, and carrier shedding, and achieve the effect of saving chondrocytes, reducing pain, and not easy to fall off
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Embodiment 1
[0029] Example 1 Preparation and molding of allogeneic demineralized bone:
[0030] 1. Make the cancellous bone part of the allograft bone into a cylinder with a diameter of 4 mm and a length of 6 mm;
[0031] 2. Defat and protein the prepared cylindrical allogeneic bone to prepare antigen-free bone: degrease and decellularize with surfactant, the specific method refers to a patented preparation method for biologically derived artificial bone without antigen;
[0032] 3. The demineralized allogeneic bone of the cylinder is demineralized with hydrochloric acid with a concentration of 0.1-2.0 mol / L and a weight ratio of 50:1, and the demineralization time is 0.5-3 hours. In this embodiment, the concentration of hydrochloric acid is 0.5mol / L, the weight ratio of hydrochloric acid to artificial bone is 50:1, and the demineralization time is 1 hour;
[0033] 4. After decellularization, degreasing and demineralization, the allograft bone forms an internally connected grid structure...
Embodiment 2
[0036] The acquisition of embodiment 2 chondrocytes:
[0037]We use dogs as experimental subjects. The knee joint of the dog's left hind leg was exposed, some cartilage in the non-load-bearing area of the lower end of the femur was taken, and the joint cavity was sutured. Soak the removed cartilage tissue in sterile PBS (concentration 0.01mol / L, pH 7.4±0.1) containing gentamicin (40 units / ml) at a ratio of 200mg cartilage / 50ml PBS for 45 minutes to 60 minutes, then Cut the cartilage tissue into small pieces of 1mm×1mm×1mm by hand; put the crushed cartilage tissue into 0.2% mg / ml type II collagenase solution, and digest it in a constant temperature oscillator at 37°C±0.5°C for 8-12 hours, filter, and centrifuge; precipitated cells were washed twice with sterile PBS (concentration 0.01mol / L, pH 7.4±0.1), and stained with trypan blue to detect cell viability, viability> 60%, set aside.
Embodiment 3
[0038] The preparation of embodiment 3 cartilage tissue engineering graft: (following steps all require aseptic operation)
[0039] 1. Put 2.5×10 5 chondrocytes with 25 μl containing thrombin and Ca 2+ The solution (sterilized) is mixed evenly, and is prepared into solution 4 for subsequent use;
[0040] 2. Take the solution whose main components are fibrinogen and factor XIII as solution 5, inhale solution 5 into another needle syringe, and the inhaled volume is equal to solution 4;
[0041] 3. Place the cylindrical solid carrier with a diameter of 4 mm and a length of 6 mm upright, and then slowly inject solution 4 and solution 5 into the cylindrical solid carrier after the needle of the syringe containing solution 4 and solution 5 is pressed against the top of the cylindrical solid carrier;
[0042] 4. Let it stand for 5 minutes until the mixture of solution 4 and solution 5 coagulates. At this time, the colloidal carrier formed by the agglutination of the mixture of sol...
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