中国修复重建外科杂志

中国修复重建外科杂志

鹿茸软骨组织脱细胞基质材料的制备及生物相容性研究

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目的 探讨鹿茸软骨制备脱细胞基质材料的可行性以及生物相容性,为软骨修复重建探索新材料。 方法 取梅花鹿鹿茸生长中心间充质层,进行由 DNA 酶、RNA 酶、抑肽酶等介导的脱细胞处理;行组织学和 DNA 含量检测,评价脱细胞效果。取第 2 代鹿生茸区骨膜(antlerogenic periosteum,AP)细胞,行荧光干细胞标记明确其干细胞特性后,用 PKH26 荧光标记并与制备的间充质层脱细胞基质进行复合培养;7 d 后取材行HE染色观察以及荧光显微镜下观察 PKH26 标记的 AP 细胞在基质表面生长情况。以上观测均以未复合 AP 细胞的脱细胞基质作为对照。将复合培养 7 d 的样本移植至裸鼠一侧腹股沟(实验组),取空白培养样本移植于另一侧(对照组)。于移植后 7、21 d 取材行 HE 染色,同时对组织进行冰冻切片并在荧光显微镜下观察 PKH26 标记成功的 AP 细胞在脱细胞基质表面及内部的生长情况,评价含 AP 细胞的脱细胞基质在裸鼠体内的组织相容性。 结果 HE 和 DAPI 染色显示脱细胞处理后材料中无细胞残留,DNA 含量为(19.367±5.254)ng/mg,较脱细胞处理前的(3 805.500±519.119)ng/mg 显著降低(t=12.630,P=0.000),提示成功制备间充质层脱细胞基质。AP 细胞与间充质层脱细胞基质复合培养 7 d 后,AP 细胞主要黏附于材料表面,部分进入脱细胞基质内部。植入裸鼠体内后,随观察时间延长,接种 AP 细胞可以在脱细胞基质材料中增殖并逐渐进入材料内部,并诱导血管生成。 结论 实验成功制备鹿茸软骨脱细胞基质,该基质材料在离体和活体情况下适于种子细胞(AP 细胞)的黏附和增殖,并具有刺激血管生成的功能,为其用于软骨组织修复提供理论依据。

Objective To study the feasibility of acellular matrix materials prepared from deer antler cartilage and its biological compatibility so as to search for a new member of the extracellular matrix family for cartilage regeneration. Methods The deer antler mesenchymal (M) layer tissue was harvested and treated through decellular process to prepare M layer acellular matrix; histologic observation and detection of M layer acellular matrix DNA content were carried out. The antler stem cells [antlerogenic periosteum, (AP) cells] at 2nd passage were labelled by fluorescent stains and by PKH26. Subsequently, the M layer acellular matrix and the AP cells at 2nd passage were co-cultured for 7 days; then the samples were transplanted into nude mice to study the tissue compatibility of M layer acellular matrix in the living animals. Results HE and DAPI staining confirmed that the M layer acellular matrix did not contain nucleus; the DNA content of the M layer acellular matrix was (19.367±5.254) ng/mg, which was significantly lower than that of the normal M layer tissue [(3 805.500±519.119) ng/mg](t=12.630, P=0.000). In vitro co-culture experiments showed that AP cells could adhere to or even embedded in the M layer acellular matrix. Nude mice transplantation experiments showed that the introduced AP cells could proliferate and induce angiogenesis in the M layer acellular matrix. Conclusion The deer antler cartilage acellular matrix is successfully prepared. The M layer acellular matrix is suitable for adhesion and proliferation of AP cells in vitro and in vivo, and it has the function of stimulating angiogenesis. This model for deer antler cartilage acellular matrix can be applied in cartilage tissue engineering in the future.

关键词: 鹿茸; 脱细胞基质; 软骨组织工程

Key words: Deer antler; acellular matrix; cartilage tissue engineering

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