骨粉和脱钙液比例与脱钙时间对脱钙骨基质诱导成骨影响的研究

Preparation of radiopaque demineralized bone matrix by dynamic decalcification process

目的探索骨粉和脱钙液比例和脱钙时间对脱钙骨基质(demineralized bone matrix,DBM)诱导成骨活性的影响。方法以深低温冷冻的供体骨为原材料,剔除软组织、脱脂、清洗、干燥、粉碎等过程制备骨粉;调整脱钙时间以及骨粉和脱钙液的比例,利用0.5 mol/L盐酸动态脱钙;清洗、冷冻干燥、辐照灭菌,得到DBM样品。对样品进行p H值和钙含量测定,从影像学观察显影效果,对其成骨活性进行体内骨诱导验证。结果测得DBM-15-2 h、DBM-20-2 h、DBM-25-2 h、DBM-20-0.5 h、DBM-20-1 h、DBM-20-1.5 h的钙含量依次为1.86±0.84、0.60±0.22、0.42±0.12、6.84±0.45、2.90±0.32、1.59±0.26;影像学结果显示DBM-20-0.5 h材料的显影密度相对最高;裸鼠体内骨诱导组织学观察发现,不同参数制备的DBM样品均具有骨诱导活性,且差异无统计学意义(P>0.05)。结论骨粉和脱钙液比例为1 g/20 ml,脱钙0.5 h,即可制得具有稳定的骨诱导活性与一定显影能力的DBM。

Objective To prepare demineralized bone matrix（ DBM） with a stable calcium content and could be displayed under X-ray by accurate control of the decalcification process. Methods Raw materials were from the donor bone stored in cryopreservation. Hydrochloric acid was used as decalcification solution. Soft tissue removal, degreasing, cleaning, drying, grinding and other processes were applied in the preparation of bone powder. Decalcification time as well as decalcification solution ratio were adjusted, and 0.5 mol / L hydrochloric acid dynamic decalcification was performed. DBM sample was obtained after cleaning, freeze drying and irradiation sterilization. The p H value, calcium content and imaging under X-ray were measured. Osteoinductivity was evaluated by implantation test in nude mice. Results The content of calcium of the DBM-15-2 h, DBM-20-2 h, DBM-25-2 h, DBM-20-0.5 h, DBM-20-1 h and DBM-20-1.5 h were 1.86±0.84, 0.60±0.22, 0.42±0.12, 6.84±0.45, 2.90±0.32 and 1.59±0.26, respectively. The brightness of the DBM-20-0.5 h sample was the highest under X-ray. The bone induction activity was seen in 4 weeks. There were no significant differences in osteoinductivity in 6 samples（ P〉0.05）. Conclusions A stable, radiopaque and osteoinductive DBM can be obtained when bone powder and decalcification solution ratio is 1 g / 20 ml（ decalcified 0.5 h）.