研究背景

先天畸形、外伤、肿瘤等因素引起的组织缺损是常见的整形外科疾病，组织工程技术的快速发展为这些组织缺损的修复和器官再造提供了新的解决策略。在这一领域中，组织工程支架起着至关重要的作用，通过模拟天然细胞外基质的微环境，为种子细胞提供必要的生物化学和机械信号，显著地影响细胞的行为和组织再生。其中，刚度作为支架构建的重要参数，通过多种途径影响细胞的增殖、凋亡、迁移、分泌和分化行为。然而，目前关于刚度和细胞行为的研究主要在二维环境中进行，三维环境下刚度对细胞生物行为学和组织再生的影响缺少深入的探索。明确三维环境下刚度对细胞行为的影响以及适合不同类型细胞的三维环境，有助于更好地理解刚度调控细胞分化、增殖和迁移的机制，对于推动再生医学、组织工程和疾病治疗等领域的应用发展具有重要意义。

研究目的

1、构建刚度可调节的三维培养系统，用于细胞的体内外研究。

2、明确三维环境下刚度对软骨细胞行为学和软骨再生的影响，为软骨组织工程构建方案的优化提供理论依据。

3、通过单细胞测序技术进一步解析三维环境下刚度对ADSC的生物行为学的影响并探讨可能的机制。

研究方法与结果

1、刚度可调节三维培养系统的构建和表征

方法：使用硅橡胶制备用于构建三维培养系统的模具；通过测量不同接枝率GelMA在不同浓度下的杨氏模量，确定GelMA水凝胶调节刚度的方法；测量GelMA浓度对刚度的影响确定其函数关系；通过调控浓度制备不同刚度的三维培养体系；分别通过SEM、AFM、流变学检测观察不同刚度三维培养系统的内部孔径、表面粗糙度和粘弹性。

结果：浓度和接枝率的增加均能提高GelMA水凝胶的刚度；高接枝率水凝胶能够提供更大的刚度范围（2kPa-100kPa）；孔隙随着刚度的降低而降低；刚度的增加会伴随着储能模量和损耗模量增加。

2、三维环境下刚度对软骨再生的影响。

方法：通过胶原酶消化的方法提取兔耳廓软骨细胞，并载入不同刚度的三维培养系统，进行体内和体外培养。在体外培养的第1，7，14天观察不同刚度下软骨细胞的存活、大小、形态，评估软骨细胞的活性、增殖、迁移行为。在体内培养的第6，12周对工程化软骨进行大体观察、组织学评估、生化定量分析、生物力学测量、转录组分析。

结果：三维培养系统刚度的降低有利于软骨细胞的增殖、迁移、分泌行为。三维培养系统刚度的降低有利于工程化软骨的快速成熟，刚度的增加有利于大体形态和结构的稳定。

3、单细胞解析三维环境下刚度对ADSC生物行为学的影响

方法：通过胶原酶消化的方法提取人来源的ADSC，并载入不同刚度的三维培养系统，进行体内和体外培养；在体外培养的第1，7，14天观察不同刚度下ADSC的存活、大小、形态，评估软骨细胞的活性、增殖、迁移行为。在体外培养的第7天对二维环境和不同刚度三维环境中的ADSC进行细胞表面标志物鉴定和单细胞转录组测序。

结果：三维培养系统刚度的降低有利于ADSC的增殖、迁移行为，促进ADSC向脂肪细胞分化；刚度的提高有利于细胞粘附，促进ADSC向软骨细胞、骨细胞分化。二维平面中ADSC表现出更高的增殖和迁移活性

研究结论：

1、通过调控接枝率90%的GelMA水凝胶的浓度可以构建不同刚度的三维培养系统。

2、三维环境刚度的降低有利于软骨细胞增殖、迁移、分泌行为从而促进工程化软骨的快速成熟。

3、维度和刚度的增加不利于ADSC的增殖、迁移、干性维持，但促进细胞的粘附。

Background

Congenital defects, trauma, and tumors can cause tissue defects, which are common in plastic surgery. The rapid development of tissue engineering technology has provided new strategies for the repair of these tissue defects and the reconstruction of organs. In this field, tissue engineering scaffolds play a crucial role. They simulate the environment of the natural extracellular matrix (ECM), providing the necessary biochemical and mechanical signals to seed cells, significantly affecting cell behavior and tissue regeneration. Stiffness is a key mechanical signal that affects cell proliferation, apoptosis, migration, secretion, and differentiation through various pathways. However, current research on stiffness and cell behavior is mainly conducted in two-dimensional environments, and the impact of stiffness in three-dimensional environments on cell behavior and tissue regeneration is not well explored. Clarifying the effects of stiffness in three-dimensional environments on cell behavior and the suitable three-dimensional environments for different types of cells will help to better understand the mechanisms by which stiffness regulates cell differentiation, proliferation, and migration, which is of great significance for advancing applications in regenerative medicine, tissue engineering, and disease treatment.

Objective

1. Building a three-dimensional culture system with adjustable stiffness for in vitro and in vivo cell studies.

2. Clarify the impact of stiffness in a three-dimensional environment on the behavior of chondrocytes and cartilage regeneration, providing a theoretical basis for the optimization of cartilage tissue engineering construction plans.

3. Use single-cell sequencing technology to further analyze the effects of stiffness on the biological behavior of ADSC in a three-dimensional environment and explore possible mechanisms.

Methods and Results

Construction and Characterization of an Adjustable Stiffness Three-Dimensional Culture System

Methods: Silicon rubber was used to fabricate molds for constructing a three-dimensional culture system. The stiffness regulation method for GelMA hydrogel was determined by measuring the Young's modulus of GelMA at various grafting ratios and concentrations. The effect of GelMA concentration on stiffness was assessed to establish a linear relationship. Different stiffness levels of the three-dimensional culture system were prepared by adjusting concentrations. The internal pore size, surface roughness, and viscoelastic properties of different stiffness three-dimensional culture systems were observed through Scanning Electron Microscopy (SEM), Atomic Force Microscopy (AFM), and rheological testing.

Results: Increases in both concentration and grafting ratio enhanced the stiffness of the GelMA hydrogel. Hydrogels with high grafting ratios provided a broader range of stiffness (2 kPa ~100 kPa). Pore size decreased with reduced stiffness, while increases in stiffness were accompanied by rises in both storage and loss modulus.

Impact of Stiffness on Cartilage Regeneration in a Three-Dimensional Environment

Methods: Rabbit auricular chondrocytes were extracted using collagenase digestion and loaded into three-dimensional culture systems of varying stiffness for both in vivo and in vitro cultivation. On days 1, 7, and 14 of in vitro culture, the survival, size, and morphology of the chondrocytes under different stiffness conditions were observed to assess cell viability, proliferation, and migration behaviors. At weeks 6 and 12 of in vivo culture, the engineered cartilage was subjected to gross observation, histological evaluation, biochemical quantification, biomechanical measurements, and transcriptome analysis.

Results: A decrease in the stiffness of the three-dimensional culture system favored chondrocyte proliferation, migration, and secretion behaviors. Reduced stiffness in the three-dimensional culture system facilitated the rapid maturation of engineered cartilage, while increased stiffness helped stabilize the macroscopic shape and structure.

Single-Cell Analysis of the Effects of Stiffness on ADSC Biobehavior in a Three-Dimensional Environment

Methods: Human-derived ADSC were isolated using collagenase digestion and embedded into three-dimensional culture systems with varying stiffness for both in vivo and in vitro cultivation. On days 1, 7, and 14 of in vitro culture, the survival, size, and morphology of ADSCs under different stiffness conditions were observed to assess the cell viability, proliferation, and migration behaviors. On day 7 of in vitro culture, cell surface marker identification and single-cell transcriptome sequencing were performed on ADSC in both two-dimensional and various stiffness three-dimensional environments.

Results: Reduced stiffness in the three-dimensional culture system was conducive to the proliferation and migration behaviors of ADSC; increased stiffness facilitated cell adhesion. Compared to the two-dimensional environment, proliferation, migration, and stemness maintenance capabilities of ADSC in the three-dimensional culture system were inhibited, while adhesion capacity was enhanced.

Conclusion

1、By regulating the concentration of GelMA hydrogel with a grafting ratio of 90%, three-dimensional culture systems of different stiffnesses can be constructed. This adjustment allows for tailored mechanical environments suitable for various cellular investigations.

2、Decreasing the stiffness within a three-dimensional environment facilitates the proliferation, migration, and secretion behaviors of chondrocytes, thereby accelerating the maturation of engineered cartilage. This suggests that softer matrices may mimic physiological conditions more effectively, promoting tissue regeneration.

3、Increased dimensionality and stiffness are detrimental to the proliferation, migration, and stemness maintenance of ADSC, yet they promote cell adhesion. This indicates that while stiffer and more complex environments may stabilize cell positioning, they could inhibit some functional behaviors important for regenerative therapies.