Method for inducing bone marrow substrate stem cell into cartilage
A technology of stem cells and chondrocytes, applied in the fields of medicine and biomedical engineering, can solve problems such as inability to form cartilage tissue, high cost, and complicated induction methods
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[0059] The preparation method of the tissue engineered cartilage of the present invention is simple and convenient, and only needs to mix a certain amount of the mixture of chondrocytes and bone marrow stromal stem cells with pharmaceutically acceptable biodegradable materials.
[0060] The tissue-engineered cartilage formed by the method of the present invention is divided into two types, one is a compound composed of a mixture of chondrocytes and bone marrow stromal stem cells and injectable biomaterials, and the compound can be directly injected into subcutaneous and cartilage defects and other parts of the body. The other is a composite composed of a mixture of chondrocytes and bone marrow stromal stem cells and non-injectable biomaterials, which can be directly implanted into subcutaneous tissue, cartilage defects, and other parts of the body.
[0061] In a preferred embodiment the biodegradable material is solid. At this time, the above-mentioned cells and culture mediu...
Embodiment 1
[0072] Acquisition and cultivation of bone marrow stromal stem cells
[0073] Experimental animals: 5 young pigs, weighing about 10kg, and 10 adult nude mice, all provided by Chuansha Breeding Factory.
[0074] (1) Materials
[0075] Animals were anesthetized with ketamine 10-20 mg / kg and atropine 0.5-1 mg intramuscularly to induce and inhibit glandular secretion, and slowly push 1 ml / kg of chloral hydrate (10%) or pentobarbital sodium (2.5% ) 1ml / kg anesthetized, routinely sterilized and draped, punctured the proximal femur with a 16-gauge needle, extracted 6-8ml of bone marrow, and placed it in a heparinized centrifuge tube.
[0076] (2) Separation of nucleated cells (density gradient centrifugation)
[0077]Repeatedly aspirate the extracted bone marrow several times with 5ml and 1ml empty needles successively, transfer to another centrifuge tube, add a small amount of serum-free DMEM culture medium, mix well, centrifuge at 3000rpm for 10 minutes, gently absorb fat and mos...
Embodiment 2
[0084] Chondrocyte isolation and culture
[0085] Anesthetize the animal as before, remove the auricle under aseptic conditions, peel off the perichondrium, cut into 5mm×5mm pieces, wash twice with phosphate buffer saline (PBS, containing 100U / ml of penicillin and streptomycin), and press the modified According to the Klagsbrun method, add 2 times the volume of 1mg / ml type II collagenase (Worthington, Freehold, NJ, USA), place it in a constant temperature shaker at 37°C for digestion for 8-12 hours, filter, and centrifuge; the precipitated cells are washed twice with PBS , make cell suspension with DMEM culture medium containing 10% fetal bovine serum, L-glutamine 300ug / ml, vitamin C 50ug / ml, penicillin and streptomycin 100U / ml, count and check with trypan blue staining Chondrocyte viability, general viability > 85%, with 2.7×10 4 cells / cm 2 Inoculate in a 10 cm diameter Petri dish at 37 °C, 5% CO 2 and 100% saturated humidity incubator for 3 days, then replace the culture ...
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