中国修复重建外科杂志

中国修复重建外科杂志

载 BMP-2 多肽 P24 的巯基化壳聚糖水凝胶诱导异位成骨的实验研究

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目的探讨载 BMP-2 多肽 P24 的巯基化壳聚糖(chitosan-4-thio-butylamidine,CS-TBA)水凝胶的异位成骨能力及体内生物相容性。方法采用壳聚糖、2-亚氨基硫烷盐酸盐、羟基磷灰石(hydroxyapatite,HA)制备 CS-TBA/HA 溶液,进一步加入 P24 多肽制备 CS-TBA/5%P24/HA、CS-TBA/10%P24/HA 溶液;取上述 3 种溶液,加入 β-甘油磷酸二钠(β-glycerophosphate disodium,β-GP),获得 CS-TBA/HA/β-GP、CS-TBA/5%P24/HA/β-GP、CS-TBA/10%P24/HA/β-GP 水凝胶。取雌性 SD 大鼠 18 只,随机分为 A、B、C 3 组(n=6),制备竖脊肌肌袋后,分别植入 CS-TBA/HA/β-GP 水凝胶(A 组)、CS-TBA/5%P24/HA/β-GP 水凝胶(B 组)、CS-TBA/10%P24/HA/β-GP 水凝胶(C 组)。术后观察大鼠存活情况,4、8 周取材采用 micro-CT 检测标本骨小梁厚度(trabecular thickness,Tb.Th)、骨小梁数量(trabecular number,Tb.N)、皮质骨骨密度(bone mineral density,BMD),组织学观察(HE、Masson 染色)分析材料的生物降解性及成骨作用。结果术后各组大鼠均存活至取材时间点。micro-CT 显示,术后随时间延长,各组新生骨逐渐增多;同一时间点 B、C 组较 A 组明显,且随着 P24 浓度的增加,C 组新生骨形成更多。术后各组 Tb.Th、Tb.N、BMD 逐渐增加,除 A 组 Tb.Th 外,其余各指标 4 周与 8 周间比较差异均有统计学意义(P<0.05)。各时间点,B、C 组 Tb.Th、Tb.N、BMD 明显高于 A 组,C 组高于 B 组,比较差异均有统计学意义(P<0.05)。组织学染色观察示,B、C 组材料具有良好的生物降解性,且成骨效果随 P24 浓度升高而增强。结论P24 多肽有助于提升 CS-TBA 水凝胶的异位成骨作用,且 10% 浓度效果更强。

ObjectiveTo study the ectopic osteogenesis and biocompatibility of bone morphogenetic protein 2 (BMP-2)-derived peptide P24 loaded chitosan-4-thio-butylamidine (CS-TBA) hydrogel.MethodsFirst, the CS-TBA/hydroxyapatite (HA) solution was prepared by using chitosan, 2-iminothiolane hydrochloride, and HA. Then, the different amount of P24 peptides were added to the CS-TBA/HA to prepare the CS-TBA/5%P24/HA and CS-TBA/10%P24/HA solutions. Finally, β-glycerophosphate disodium (β-GP) was added to the CS-TBA/HA, CS-TBA/5%P24/HA, and CS-TBA/10%P24/HA to prepare the CS-TBA/HA/β-GP, CS-TBA/5%P24/HA/β-GP, and CS-TBA/10%P24/HA/β-GP hydrogels, respectively. Eighteen Sprague Dawley female rats were randomly divided into 3 groups (n=6), which were injected into the back muscle pouches with equal volume CS-TBA/HA/β-GP hydrogel (group A), CS-TBA/5%P24/HA/β-GP hydrogel (group B), and CS-TBA/10%P24/HA/β-GP hydrogel (group C). The animals were sacrificed at 4 and 8 weeks and conducted micro-CT. The ability of biodegradation and osteogenesis of hydrogl was detected by trabecular thickness (Tb.Th), trabecular number (Tb.N), bone mineral density (BMD), and histological staining (HE and Masson).ResultsAll the rats survived to the time point of the harvest. Micro-CT results showed that the new bones gradually increased in each group after operation. At the same time, the new bone formation was more obvious in groups B and C than in group A, and with the increase of P24 concentration, new bone formation in group C was much more than that in group B. The Tb.Th, Tb.N, and BMD increased gradually in 3 groups, and the differences between 4 and 8 weeks were significant (P<0.05) except the Tb.Th in group A. At different time points, the Tb.Th, Tb.N, and BMD were significantly higher in groups B and C than in group A (P<0.05), and in group C was higher than in group B (P<0.05), showing significant differences between groups. Histological staining showed that the materials of groups B and C were biodegradable, and the osteogenic effect was increased with the increase of P24 concentration.ConclusionP24 peptide can improve the ectopic osteogenesis of CS-TBA hydrogel, and the 10% concentration is more effective.

关键词: 巯基化壳聚糖水凝胶; BMP-2; 异位成骨; micro-CT; 大鼠

Key words: Chitosan-4-thio-butylamidine hydrogel; bone morphogenetic protein 2; ectopic osteogenesis; micro-CT; rat

引用本文: 詹健峰, 刘徐妹, 于博. 载 BMP-2 多肽 P24 的巯基化壳聚糖水凝胶诱导异位成骨的实验研究. 中国修复重建外科杂志, 2018, 32(9): 1144-1149. doi: 10.7507/1002-1892.201806086 复制

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