Kryger Gil S, Chong Alphonsus K S, Costa Melinda, Pham Hung, Bates Steven J, Chang James
Division of Plastic Surgery, Stanford University Medical Center, Palo Alto, CA 94304, USA.
J Hand Surg Am. 2007 May-Jun;32(5):597-605. doi: 10.1016/j.jhsa.2007.02.018.
Tissue-engineered tendon grafts will meet an important clinical need. To engineer tendons, we used acellularized allogeneic tendon as scaffold material. To determine the ideal cell type to seed the scaffolds, we studied in vitro characteristics of epitenon tenocytes, tendon sheath fibroblasts, bone marrow-derived mesenchymal stem cells (BMSCs), and adipoderived mesenchymal stem cells (ASCs). Subsequently, we implanted reseeded acellularized tendons in vivo as flexor tendon grafts.
Tenocytes, sheath fibroblasts, BMSCs, and ASCs were obtained from adult rabbits. For all cell lines, collagen 1, 2, and 3 immunocytochemistry was performed, and proliferation was assessed by hemacytometry and senescence by beta-galactosidase staining. Flexor tendons were acellularized after harvest. Tendons were assessed by histology after in vitro reseeding with each of the cell types after 1, 4, and 8 weeks. Finally, reseeded tendons and controls were implanted in a flexor profundus tendon defect. After 6 weeks, the reseeded tendons were harvested and assessed by histology. Statistical analysis for cell proliferation was performed using analysis of variance and t-tests with Bonferroni correction.
All cell types had similar collagen expression. Cell proliferation was higher in ASCs in late passage compared with early passage and in ASCs compared with epitenon tenocytes at late passage. The other cell types were similar in growth characteristics. No senescence was detected. In vitro assessment of reseeded constructs showed the presence of cells on the construct surface. In vivo assessment after implantation showed viable cells seen within the tendon architecture in all cell types.
This study suggests that the four cell types may be successfully used to engineer tendons. Adipoderived mesenchymal stem cells proliferate faster in cell culture, but the cell types were similar in other respects. All could be used to successfully repopulate acellularized tendon in vivo as flexor tendon grafts.
组织工程化肌腱移植物将满足一项重要的临床需求。为构建肌腱,我们使用脱细胞同种异体肌腱作为支架材料。为确定接种到支架上的理想细胞类型,我们研究了腱外膜成纤维细胞、腱鞘成纤维细胞、骨髓间充质干细胞(BMSCs)和脂肪来源间充质干细胞(ASCs)的体外特性。随后,我们将重新接种细胞的脱细胞肌腱作为屈肌腱移植物植入体内。
从成年兔获取成纤维细胞、腱鞘成纤维细胞、BMSCs和ASCs。对所有细胞系进行Ⅰ型、Ⅱ型和Ⅲ型胶原蛋白免疫细胞化学检测,并通过血细胞计数评估细胞增殖,通过β - 半乳糖苷酶染色评估细胞衰老情况。收获后对屈肌腱进行脱细胞处理。在体外分别用每种细胞类型接种后1周、4周和8周,通过组织学评估肌腱情况。最后,将重新接种细胞的肌腱和对照物植入屈指深肌腱缺损处。6周后,收获重新接种细胞的肌腱并进行组织学评估。使用方差分析和经Bonferroni校正的t检验对细胞增殖进行统计学分析。
所有细胞类型的胶原蛋白表达相似。与早期传代相比,晚期传代的ASCs细胞增殖更高,且与晚期传代的腱外膜成纤维细胞相比,ASCs细胞增殖也更高。其他细胞类型的生长特性相似。未检测到细胞衰老。重新接种细胞构建物的体外评估显示构建物表面存在细胞。植入后的体内评估显示,所有细胞类型在肌腱结构内均可见存活细胞。
本研究表明这四种细胞类型可成功用于构建肌腱。脂肪来源间充质干细胞在细胞培养中增殖更快,但这些细胞类型在其他方面相似。所有细胞类型均可成功用于体内重新填充脱细胞肌腱作为屈肌腱移植物。