Division of Tissue Engineering and Regeneration Technologies, Biomedical Technology Wing, Sree Chitra Tirunal Institute for Medical Sciences and Technology (SCTIMST), Poojapura, Thiruvananthapuram, Kerala 695012, India.
Department of Chemistry, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, India.
Acta Biomater. 2019 Nov;99:196-210. doi: 10.1016/j.actbio.2019.09.010. Epub 2019 Sep 12.
Current treatment modalities for cartilage regeneration often result in the production of fibrous-type cartilage tissue at the defect site, which has inferior mechanical properties as compared to native hyaline cartilage. Further, effective treatments are not available at present, for preventing age-related as well as disease-related hypertrophic development of chondrocytes. In the present study, we designed and synthesized three sets of glutamic acid-based dendritic peptides, differing in degree of lipidation as well as branching. Each set constitutes of N-terminal protected as well as corresponding N-deprotected peptides. Altogether, six peptides [BE12, E12, BE3(12)4, E3(12)4, BE3OMe, E3OMe] were tested for their chondrogenesis enhancing potential in vitro, using rabbit adipose derived mesenchymal stem cells (ADMSCs). Immunohistochemical and gene expression studies as well as biochemical analyses revealed that the lipopeptides [E12 and BE3(12)4] are able to enhance chondrogenic differentiation of ADMSCs significantly (p < 0.001) as compared to control group (chondrogenic medium alone). Glycosaminoglycan content, and the chondrogenic marker genes like Aggrecan (Acan), Type II collagen (Col2a1), Hyaluronan synthase 2 (Has2), and SRY-box 9 (Sox9) expressions were found to be significantly increased in E12 and BE3(12)4 treated groups. Most importantly, the BE3(12)4 treated group showed significantly lower Type I collagen (Col1a2) and Type X collagen (Col10a1) transcript levels (p < 0.001), indicating its potential for hyaline cartilage formation and also to prevent hypertrophic development. Thus, the lipopeptides E12 and BE3(12)4 may be useful for preventing chondrocyte hypertrophy and realizing the hyaline nature of regenerated cartilage tissue in tissue engineering. STATEMENT OF SIGNIFICANCE: The current treatment modalities for degenerative cartilage diseases are unsatisfactory as the resultant regenerated cartilage is often fibrous in nature with inferior mechanical properties. Further, there is no proper treatment available for age-related development of chondrocyte hypertrophy at present. In this study we synthesized glutamic acid-based lipopeptides, which differ in the degree of lipidation as well as branching. We used a combinatorial approach of scaffold-free tissue engineering and dendritic lipopeptides to achieve hyaline-like cartilage tissue from adipose derived mesenchymal stem cells in vitro. Gene expression analysis revealed the down regulation of fibrous cartilage marker Col1a2 and hypertrophic marker Col10a1, suggesting that these lipopeptides may be useful for achieving mechanically superior hyaline cartilage regeneration in future.
目前用于软骨再生的治疗方法通常会在缺损部位产生纤维型软骨组织，其机械性能不如天然透明软骨。此外，目前还没有有效的治疗方法来预防与年龄相关的和与疾病相关的软骨细胞肥大性发育。在本研究中，我们设计并合成了三组基于谷氨酸的树枝状肽，其在脂质化程度和分支方面有所不同。每组由 N 端保护和相应的 N 去保护肽组成。总共测试了六种肽 [BE12、E12、BE3(12)4、E3(12)4、BE3OMe、E3OMe] 的体外软骨生成增强潜力，使用兔脂肪间充质干细胞 (ADMSC)。免疫组织化学和基因表达研究以及生化分析表明，与对照组（仅软骨形成培养基）相比，脂肽 [E12 和 BE3(12)4] 能够显著增强 ADMSC 的软骨分化（p < 0.001）。糖胺聚糖含量以及软骨形成标志物基因如聚集蛋白聚糖（Acan）、II 型胶原（Col2a1）、透明质酸合酶 2（Has2）和性盒 9（Sox9）的表达均显著增加在 E12 和 BE3(12)4 处理组中。最重要的是，BE3(12)4 处理组的 I 型胶原（Col1a2）和 X 型胶原（Col10a1）转录水平明显降低（p < 0.001），表明其具有透明软骨形成的潜力，也可预防肥大性发育。因此，脂肽 E12 和 BE3(12)4 可用于预防软骨细胞肥大并实现组织工程中再生软骨组织的透明本质。意义声明：目前用于退行性软骨疾病的治疗方法并不令人满意，因为再生的软骨通常为纤维状，机械性能较差。此外，目前尚无适当的治疗方法可用于与年龄相关的软骨细胞肥大性发育。在这项研究中，我们合成了基于谷氨酸的脂肽，其在脂质化程度和分支方面有所不同。我们使用无支架组织工程和树枝状脂肽的组合方法，从体外脂肪间充质干细胞中获得透明样软骨组织。基因表达分析显示纤维软骨标志物 Col1a2 和肥大标志物 Col10a1 的下调，表明这些脂肽可能有助于未来实现机械性能更优的透明软骨再生。
