Tissue engineering cartilage construction method using bone matrix gelatin
A tissue engineering and bone matrix technology, applied in tissue culture, biochemical equipment and methods, medical science, etc., can solve the problems of short maintenance time, permanent functional reconstruction still has a certain distance, etc., to achieve strong bone induction, good biological Compatibility effect
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Embodiment 1
[0034] 1) Seed cell acquisition
[0035] Isolation and culture of chondrocytes
[0036] ①Materials: A New Zealand rabbit born about 4 weeks old was killed by air embolism or hitting the back of the pillow, depilated with 8% sodium sulfide by mass percentage, washed with running water, and soaked in 0.1% bromogeramine for 20-30 minutes; Cut off the proximal humerus, proximal femur, and proximal tibia by aseptic operation, place them in a container containing D-Hanks' solution containing 100 units / ml of P / S each, remove all soft tissues, and slice Put each articular cartilage into a container containing D-Hanks' solution with 100 units / m1 of P / S each, then suck out the liquid and discard it, and digest it with 10ml hyaluronidase with a mass percentage concentration of 0.05% at room temperature After 3 minutes, discard the enzyme solution, wash with D-Hanks' solution containing 100 units / ml of P / S, and cut the cartilage slices into 1mm 3 small cartilage granules;
[0037] ②Cel...
Embodiment 2
[0051] 1) Seed cell acquisition
[0052] Isolation and culture of chondrocytes
[0053] ①Materials: One human embryo within 2 hours of water bag induction was obtained by aseptic operation. After the skin was disinfected with iodine tincture and alcohol, the proximal humerus, proximal femur and proximal tibia were intercepted by aseptic operation, and placed in a container. In a container containing D-Hanks' solution containing 100 units / ml of P / S, remove all soft tissues, slice off each articular cartilage, and put D-Hanks' solution containing 100 units / ml of P / S. Put it in the container of Hanks' solution, then suck out the liquid and discard it, digest it with 10ml of hyaluronidase with a mass percent concentration of 0.05% at room temperature for 3 minutes, discard the enzyme solution, and use D containing 100 units / ml of P / S each -After washing with Hanks' solution, cut the cartilage into 1mm 3 small cartilage granules;
[0054] ②Cell separation: Transfer the cartilage...
Embodiment 3
[0067] 1) Seed cell acquisition
[0068] Isolation and culture of chondrocytes
[0069] Separation and culture of stromal stem cells to induce chondrocytes:
[0070] ①Using density gradient centrifugation and adhesion separation to obtain bone marrow mesenchymal stem cells: take New Zealand purebred rabbits about four months old, extract bone marrow and slowly inject it on the surface of 70% Percoll cell separation medium at a volume ratio of 1:1, at 1500 Spin centrifuge for 30 minutes, absorb the liquid layer containing bone marrow mesenchymal stem cells between the upper layer and the middle layer, and fill the bone marrow mesenchymal stem cells with DMEM culture solution containing 15% volume ratio of calf serum and P / S 100 units / ml each. Stem cells were made into a single cell suspension, and the nucleated cells were counted and inoculated in culture flasks for monolayer culture. The first 5 days were replaced every 3 days with calf serum containing 15% volume ratio and 1...
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