犬异位移植气管内骨形态发生蛋白诱导软骨再生作用的研究

Research on BMP-induced Cartilage Regeneration in Canine Tracheal Grafts

目的比较研究骨形态发生蛋白-2在犬的自体、异体、冷冻异体气管移植体内诱导软骨再生的作用。方法54只杂种犬随机等分为3组,分别为自体移植组、异体移植组、冷冻后的异体移植组。3组分别于颈部切除4环气管段作为移植体,植入骨形态发生蛋白-2后埋入腹腔大网膜中。各组分别植入不同的气管移植体。术后5周处死实验动物,通过对标本进行组织学观察、碱性磷酸酶活性和钙含量测定,对各组的新生软骨进行观察和定量分析。结果①半定量法组织学评分,自体移植组、异体移植组、冷冻后异体移植组分别为1.68±0.27、5.57±0.72、1.93±0.24,3组结果有统计学差异(F=152.717,P<0.001),自体移植组、异体移植组与冷冻后的异体移植组差异均有统计学意义(P<0.05),而自体移植组和冷冻后的异体移植组差异无统计学意义(P>0.05)。②各实验组骨形态发生蛋白-2植入区均可见新生软骨细胞和软骨岛,自体移植组、异体移植组、冷冻后的异体移植组的新生软骨100倍视野下像素面积分别为2573.6±738.4、1691.3±743.6、2482.9±827.5,组间差异有统计学意义(F=5.711,P<0.05),自体移植组、异体移植组与冷冻后的同种异体移植组差异均有统计学意义(P<0.05),而自体移植组和冷冻后的异体移植组差异无统计学意义(P>0.05)。③碱性磷酸酶活性和钙含量:自体移植组[(0.42±0.03)U/mg,(5.48±1.15)mg/g]、冷冻后的异体移植组[(0.42±0.05)U/mg,(5.03±0.91)mg/g]与异体移植组[(0.30±0.04)U/mg,(3.57±0.98)mg/g]差异均有统计学意义(P<0.05),自体移植组与冷冻后的异体移植组间差异无统计学意义(P>0.05)。结论骨形态发生蛋白-2能在气管移植体内有效地诱导出新生软骨,在深低温冷冻后的异体气管移植体内的作用效果与自体气管移植体内相似,明显优于异体气管移植体。

Objective To induce regeneration of cartilage by implanting recombinant human bone morphogenetic protein-2 into Canine tracheal autograft, allograft and allografl after cryopreservation. Methods Fifty-four mongrel dogs were randomly divided into 3 groups ： tracheal autograft group, tracheal allograf group and tracheal allograft after cryopreservation group. Four-rings cervical tracheal segment was harvested as the graft and transplanted into the omentum heterotopically. In each allograft, rhBMP-2 with collagen as a carrier was injected into the soft tissue between cartilaginous rings. All the animals were sacrificed in the 5th postoperative week. The postmortem specimens were examined grossly and histologically for measurement of alkaline phosphate level and calcium content in the new cartilage in each specimen was quantitatively analyzed. Results Cartilage rings were harvested 5 weeks later. （1） The histology score of tracheal autograft group, tracheal allograf group and tracheal allograf after cryopreservation group was 1.68 ±0. 27,5.57 ± 0. 72,1.93 ± 0.24, differences among the three groups were significant （ F = 152. 717, P 〈 0. 001 ）, those in tracheal autograft and tracheal allograft after cryopreservation groups were significantly higher than those of tracheal allograft group （P 〈 0. 05 ） , but there was no significant difference between tracheal autograft group and tracheal allograft after cryopreservation group （P 〉 0.05）. （2） Every experimental group was observed for novel chondrocytes and cartilage islands. Novel chondrocytes areas were different among the three groups（2 573.6±738.4,1 691.3±743.6,2482.9±827.5） （F=5.711, P〈0.05）, those in tracheal autograft and tracheal allograft after cryopreservation groups were significantly higher than those of tracheal allograft group （P 〈 0. 05） ,but there was no significant difference between tracheal autograft group and tracheal allograft after cryopreser- vation group （P 〉 0.05 ）. （3） Measurement of alkaline phosphate levels and calcium contents in tracheal autograft group [ （0.42 ± 0. 03 ） U/mg, （ 5.48 ±1. 15 ） mg/g ] and tracheal allograft after cryopreservation group [ （ 0. 42± 0. 05 ） U/mg, （5.03 ±0.91 ） mg/g] were significantly higher than that of tracheal allograft group[ （0.30 ＋ 0. 04）U/mg, （3.57 ± 0.98 ）mg/g] （P 〈 0. 05）, but there was no significant difference between tracheal autograft group and tracheal allograft after cryopreservation group （ P 〈 0. 05）. Conclusion The rhBMP-2 has the ability to induce new cartilage formation in the tracheal graft, but its effects in tracheal allograft after cryopreservation are similar with that of tracheal autograft and obviously superior to that of tracheal allograft.