Silk Fibroin Films with Microscopic Surface Pattern as Promising Cartilage Tissue Engineering Scaffold in Supporting MSCs Amplification and Chondrogenic Differentiation

ABSTRACT

Mesenchymal stem cells (MSCs) with multipotential differentiation ability are main cell source for cartilage tissue engineering, but its application is restricted by the difficulty in regulating committed chondrogenic differentiation. Study revealed that certain substrate topographic structure can enhance the chondrogenic differentiation of MSCs. This study prepared silk fibroin (SF) films with gradient microscopic surface patterns based on P280-P7000 type specification sandpapers, and investigated the adhesion, proliferation and chondrogenic differentiation of human adipose-derived stem cells (hADSCs) on the SF films. The results showed that the SF films surface had convex dots with diameter of 3–50 μm, and hADSCs could adhere and proliferate well on the film. Transcriptomics analysis indicated that hADSCs tend to differentiate differently on the SF films, among which hADSCs on P1000 SF films had higher expression of chondrogenic markers Sox-9 and Col-II, with activated yes associated protein (YAP) and restricted F-actin. In vitro co-culture of human OA knee cartilage with hADSCs seeded SF films found satisfactory cartilage protection effect in P1000 group. Overall, this study preliminarily revealed the microscopic surface pattern of SF films may induce chondrogenic differentiation of hADSCs through extracellular matrix mediated Hippo/Yap pathway, and provided a new strategy for cartilage repair.

摘要

具有多向分化潜能的间充质干细胞是软骨组织工程的主要细胞来源，但其应用受难以调控定向成软骨细胞分化限制。研究表明，一定的基底表面拓扑结构可以促进间充质干细胞向软骨细胞分化。本研究基于P280-P7000规格的砂纸制备了具有梯度表面微形貌的丝素蛋白（SF）膜，并研究了人脂肪干细胞（hADSCs）在SF膜上的粘附、增殖和成软骨细胞分化行为。 结果表明，SF膜表面具有直径为3-50 μm的凸点，hADSCs能很好地在SF膜上粘附和增殖。转录组测序及分析表明，hADSCs在各组SF膜上具有不同的分化倾向，其中hADSCs在P1000组SF膜上具有更高的软骨形成标志蛋白Sox-9和Col II的表达，且yes相关蛋白（YAP）表达升高，F-肌动蛋白（F-actin）表达下降。人OA膝关节软骨外植体与接种了hADSCs的SF膜体外共培养证实，P1000组SF膜软骨保护效果最好。本研究初步揭示了SF膜的表面微形貌可能通过细胞外基质介导的Hippo/YAP途径诱导hADSCs成软骨细胞分化，可用于组织工程修复软骨损伤。

KEYWORDS:

• Cartilage repair
• stem cell
• chondrogenic differentiation
• silk fibroin
• microscopic surface
• film

关键词:

• 软骨修复
• 干细胞
• 成软骨分化
• 丝素蛋白
• 表面微形貌
• 膜

Highlights

1. The biocompatible silk fibroin (SF) films with gradient microscopic surface patterns were prepared via casting SF solution on PDMS molds derived from type specification sandpapers.

2. The growth factor-free, convex dot featured SF film regulates chondrogenic differentiation in hADSCs through Hippo/YAP pathway.

3. The growth factor-free SF films may serve as a novel functional bioscaffold for articular cartilage regeneration.

Acknowledgment

We thank the technical support in bioinformatics analysis offered by Knorigene Technologies.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

The data presented in this study are available on request from the corresponding author.

Institutional review board statement

This study was approved by the Ethics Committee of the First Affiliated Hospital of Army Medical University, PLA (No.KY2021040).

Additional information

Funding

This work was supported by Chongqing Natural Science Foundation General Project [cstc2021jcyj-msxmX0135 and CSTB2022NSCQ-MSX0139], Chongqing Natural Science Foundation Innovation Development Joint Fund [Municipal Education Commission] Project [2022NSCQ-LZX0066], Chongqing Postdoctoral Research Project Special Fund [2021XM3033], and Postdoctoral Science Fund Project of the Chongqing Natural Science Foundation [CSTB2023NSCQ-BHX0156].