Bio-Oss骨复合富血小板纤维蛋白修复兔牙周骨缺损

doi:10.3877/cma.j.issn.1674-1366.2020.05.002

目的

探讨组织工程骨Bio-Oss骨复合富血小板纤维蛋白（PRF）修复牙周骨缺损中骨形成蛋白2（BMP-2）、骨保护素（OPG）和核因子κB受体活化因子配体（RANKL）的表达及意义。

方法

3月龄雄性新西兰大白兔36只，采用随机数表法分为4组，每组9只，制备单侧牙周骨缺损模型，于骨缺损处分别植入Bio-Oss骨（Bio-Oss组）、PRF（PRF组）和Bio-Oss/PRF复合物（Bio-Oss/PRF组），以未植入任何材料者作为空白对照。术后4、8和12周处死动物，行大体观察、Masson染色及BMP-2、OPG、RANKL免疫组化观察，并对其表达进行析因设计的方差分析。

结果

Masson染色结果显示，Bio-Oss/PRF组术后8周时见部分成熟骨，12周见骨板形成，骨成熟度较高。析因分析显示Bio-Oss组在4、8和12周时BMP-2表达量逐渐增高，差异有统计学意义（F = 51.30，P<0.001）；OPG表达量随时间延长先升高后减低，差异有统计学意义（F = 167.03，P<0.001）；RANKL表达量随时间延长逐渐减低，差异有统计学意义（F = 5.39，P = 0.046）。PRF组在4、8和12周时BMP-2表达量无统计学意义（F = 0.68，P = 0.544）；OPG和RANKL表达量随时间延长逐渐减低，差异有统计学意义（F_OPG = 1070.93，P_OPG<0.001；F_RANKL = 2306.15，P_RANKL<0.001）。Bio-Oss/PRF组在4、8和12周时BMP-2、OPG和RANKL表达量逐渐降低，差异有统计学意义（F_BMP-2 = 13.29，P_BMP-2<0.001；F_OPG = 237.91，P_OPG<0.001；F_RANKL = 132.48，P_RANKL<0.001）。空白对照组在4、8和12周时BMP-2和OPG表达量先升高后减低，差异有统计学意义（F_BMP-2 = 88.33，P_BMP-2<0.001；F_OPG = 30.06，P_OPG<0.001），RANKL表达量随时间推移逐渐减低，差异有统计学意义（F = 56.52，P<0.001）。

结论

Bio-Oss骨复合PRF应用可促进成骨以修复牙周骨缺损。

关键词: 富血小板纤维蛋白, 骨形成蛋白2, 骨保护素, 核因子κB受体活化因子配体, Bio-Oss骨, 牙周骨缺损

Objective

To explore the effect of tissue-engineered bone Bio-Oss bone combined with platelet-rich fibrin (PRF) on repairing periodontal bone defects by examining the expression of bone morphogenetic protein 2 (BMP-2) , osteoprotegerin (OPG) and nuclear factor κB receptor activator ligand (RANKL) .

Methods

A total of 36 three-month-old male New Zealand white rabbits were divided into four groups by random number table method, i.e., Bio-Oss group, PRF group, Bio-Oss/PRF group and blank control group. For each group, there were nine rabbits. Unilateral periodontal was prepared. In the bone defect model, Bio-Oss bone, PRF and Bio-Oss/PRF composite were implanted in the bone defect, respectively, and the one without any material was used as a blank control. The animals were sacrificed at 4, 8 and 12 weeks after the operation. Gross observation, Masson staining and immunohistochemical observation of BMP-2, OPG, RANKL were performed, and the expression of the animals was analyzed by factorial design.

Results

Masson staining results showed that in the Bio-Oss/PRF group, some mature bones were observed at 8 weeks after surgery, and bone plates were formed at 12 weeks, with a high bone maturity. Factorial analysis showed that the expression of BMP-2 in the Bio-Oss group gradually increased at 4, 8 and 12 weeks, and the difference was statistically significant (F = 51.30, P<0.001) ; the expression of OPG first rose then descended, and the difference was statistically significant (F = 167.03, P<0.001) ; the expression of RANKL gradually decreased with time, and the difference was statistically significant (F = 5.39, P = 0.046) . The expression of BMP-2 in the PRF group at 4, 8 and 12 weeks was not statistically significant (F = 0.68, P = 0.544) ; the expression of OPG and RANKL decreased gradually over time, and the difference was statistically significant (F_OPG = 1070.93, P_OPG<0.001; F_RANKL = 2306.15, P_RANKL<0.001) . The expression of BMP-2, OPG and RANKL in the Bio-Oss/PRF group gradually decreased at 4, 8 and 12 weeks, and the difference was statistically significant (F_BMP-2 = 13.29, P_BMP-2<0.001; F_OPG = 237.91, P_OPG<0.01; F_RANKL = 132.48, P_RANKL<0.001) . In the blank control group, the expression of BMP-2 and OPG increased first and then decreased at 4, 8 and 12 weeks, and the difference was statistically significant (F_BMP-2 = 88.33, P_BMP-2<0.001; F_OPG = 30.06, P_OPG<0.001) , while the expression of RANKL gradually decreased over time, and the difference was statistically significant (F = 56.52, P<0.001) .

Conclusion

The application of Bio-Oss bone composite PRF may promote osteogenesis and repair periodontal bone defects.

Key words: Platelet-rich fibrin, Bone morphogenetic protein 2, Osteoprotegerin, Receptor activator of nuclear factor kappaB ligand, Bio-Oss bone, Periodontal bone defect

图1 兔下前牙牙周骨缺损模型建立箭头所示为兔下前牙牙周骨缺损模型建立中，保留在近中面舌侧留有1 mm薄层骨壁相连，防止下颌骨折断

图5 核因子κB受体活化因子配体（RANKL）在不同骨材料修复牙周骨缺损中免疫组化染色结果（低倍放大）5A：Bio-Oss组8周；5B：PRF组4周；5C：Bio-Oss/PRF组4周；5D：空白对照组4周

表1 骨形成蛋白-2（BMP-2）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	10 244^a± 300^b	155 831^b ± 338^b	21 724^c± 223^c	51.30	0.000
PRF组	9	9930 ± 733^b	10 171 ± 338^a	10 698 ± 119^a	0.68	0.544
Bio-Oss/PRF组	9	21 721^c± 884^c	16 021^b ± 291^b	14 123^a ± 117^b	13.29	0.006
空白对照组	9	1046^a ± 59^a	13 030^c ± 190^a	8807^b ± 440^a	88.33	0.000
F值	?	610.56	5.19	49.50	?	?
P值	?	0.000	0.028	0.000	?	?

表1 骨形成蛋白-2（BMP-2）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	10 244^a± 300^b	155 831^b ± 338^b	21 724^c± 223^c	51.30	0.000
PRF组	9	9930 ± 733^b	10 171 ± 338^a	10 698 ± 119^a	0.68	0.544
Bio-Oss/PRF组	9	21 721^c± 884^c	16 021^b ± 291^b	14 123^a ± 117^b	13.29	0.006
空白对照组	9	1046^a ± 59^a	13 030^c ± 190^a	8807^b ± 440^a	88.33	0.000
F值	?	610.56	5.19	49.50	?	?
P值	?	0.000	0.028	0.000	?	?

表2 骨保护素（OPG）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	547^a± 17^a	13 937^b ± 576^b	2368^a ± 17^a	167.03	0.000
PRF组	9	27 571^c± 13^b	8954^b ± 59^a	577^a ± 77^a	1070.93	0.000
Bio-Oss/PRF组	9	89 801^c± 737^c	53 487^b ± 174^c	11 057^a ± 123^c	237.91	0.000
空白对照组	9	7525^a± 62^a	15 878^b ± 146^b	5500^a ± 26^b	30.06	0.001
F值	?	354.27	862.66	23.98	?	?
P值	?	0.000	0.000	0.000	?	?

表2 骨保护素（OPG）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	547^a± 17^a	13 937^b ± 576^b	2368^a ± 17^a	167.03	0.000
PRF组	9	27 571^c± 13^b	8954^b ± 59^a	577^a ± 77^a	1070.93	0.000
Bio-Oss/PRF组	9	89 801^c± 737^c	53 487^b ± 174^c	11 057^a ± 123^c	237.91	0.000
空白对照组	9	7525^a± 62^a	15 878^b ± 146^b	5500^a ± 26^b	30.06	0.001
F值	?	354.27	862.66	23.98	?	?
P值	?	0.000	0.000	0.000	?	?

表3 核因子κB受体活化因子配体（RANKL）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	30 250^b± 119^b	22 297^b± 55^c	19 220^a± 48^c	5.39	0.046
PRF组	9	26 550^c± 80^b	6860^b ± 26^b	476^a± 100^a	2306.15	0.000
Bio-Oss/PRF组	9	52 660^b± 642^c	8178^a ± 12^b	6216^a± 22^b	132.48	0.000
空白对照组	9	16 475^c± 26^a	12 814^b± 17^a	1730^a± 89^a	56.52	0.000
F值	?	56.26	17.13	32.49	?	?
P值	?	0.000	0.001	0.000	?	?

表3 核因子κB受体活化因子配体（RANKL）在不同骨材料修复牙周骨缺损中表达的累积光密度（IOD）值（ ± s）

组别	样本量	4周	8周	12周	F值	P值
Bio-Oss组	9	30 250^b± 119^b	22 297^b± 55^c	19 220^a± 48^c	5.39	0.046
PRF组	9	26 550^c± 80^b	6860^b ± 26^b	476^a± 100^a	2306.15	0.000
Bio-Oss/PRF组	9	52 660^b± 642^c	8178^a ± 12^b	6216^a± 22^b	132.48	0.000
空白对照组	9	16 475^c± 26^a	12 814^b± 17^a	1730^a± 89^a	56.52	0.000
F值	?	56.26	17.13	32.49	?	?
P值	?	0.000	0.001	0.000	?	?

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Repair of rabbit periodontal bone defect with Bio-Oss bone combined with platelet-rich fibrin

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Repair of rabbit periodontal bone defect with Bio-Oss bone combined with platelet-rich fibrin