Reagents and materials:
1. PBS balanced salt solution (PBSA) without magnesium and calcium;
2. MSC medium: α-MEM containing 2mmol/L glutamine, adding 15% fetal bovine serum, 0.1mmol/L L-ascorbate phosphate, 100U/ml penicillin and 100μg/ml streptomycin;
3. Trypsin-EDTA solution: 0.05% trypsin, 0.54mmol/L (0.2%) EDTA, dissolved in PBS balanced salt solution without magnesium and calcium;
4. Cryotube, 1.8ml;
5. A suitable carrier (hydroxyapatite/tricalcium phosphate carrier, HA/TCP);

experimental method:
1. Incubate with MSC medium until the cells reach 90% confluence;
2. Wash the dish with PBSA and wash 3 times;
3. Add enough volume of trypsin-EDTA solution and incubate at 37 °C;
4. Determine that 80% of the cells have detached from the surface of the culture vessel and then add MSC medium;
5. 500g, centrifuged for 6min;
6. Pour off the supernatant and resuspend the cells in MSC medium;
7. Cell count;
8. Mix (2-4) × 10 ⁶ cells resuspended in MSC medium with the carrier and place in a 1.8 ml cryotube;
9. Incubate at 37 ° C for 1 h;
10. Under sterile conditions, transplant the mixture into the skin of mice with low immunity. We usually use N1H bg-nu/nu-xid mice for transplantation;
11. At the appropriate time, collect the graft and histologically. (In the HA/TCP vector and the bg-nu/nu-xid mouse system, 8 weeks is sufficient for DPSCs and SHED to form a sufficient mineralized matrix. The time of collection is mainly determined by the system.)

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