一种用于测量人成纤维细胞从头合成的细胞外基质的强度和刚度的体外模型。
An in vitro model to measure the strength and stiffness of the extracellular matrix synthesized de novo by human fibroblasts.
作者信息
Wu Yanying, Millender Jayla, Padgett Becka, Marx Madeleine, Madnick Samantha, Puterbaugh Ryan, Angelo Katerina St, Hopkins Caitlin M, Morgan Jeffrey R
机构信息
Department of Pathology and Laboratory Medicine, Brown University, 171 Meeting St, Providence, G-B 393 USA.
Center for Alternatives to Animals in Testing, Brown University, Providence, RI USA.
出版信息
In Vitro Model. 2025 Mar 7;4(1):59-69. doi: 10.1007/s44164-025-00081-y. eCollection 2025 Feb.
PURPOSE
Alterations to the strength and stiffness of the human extracellular matrix (ECM) are the underlying pathology manifest in a wide range of diseases. These include inherited conditions, such as Ehlers Danlos syndrome, as well as acquired diseases such as fibrosis, which remains a major unmet medical need. To evaluate promising therapies, new models are needed that can measure the strength and stiffness of the human ECM.
METHODS
Cultured human fibroblasts were seeded into circular troughs of agarose that had been molded into a 24 well plate and equilibrated with cell culture medium. The cells settled by gravity, aggregated and formed 3D ring-shaped tissues 5 mm in diameter without the aid of added exogenous scaffold material. The ECM proteins synthesized de novo by the rings were characterized by immuno-staining. The response of the rings to drug and growth factor treatments were assessed by measuring changes to the dimensions of the rings and by measuring levels of collagen. A tensile test was used to quantify drug and growth factor induced changes to the strength and stiffness of the rings.
RESULTS
Ring-shaped tissues readily formed in the molds and synthesized de novo a circumferentially aligned collagen-rich fibrous ECM network positive for collagen type I, collagen type III and fibronectin. Low dose treatment with incyclinide, an inhibitor of matrix metalloproteinases (MMPs), increased strength and stiffness, whereas as a high dose decreased tensile properties, likely due to a toxic effect. Treatment with TGF-β1, a well-known driver of fibrosis, increased levels of collagen and tensile properties and mimicked the fibrotic environment in vitro. Treatment with PAT-1251, an inhibitor of the collagen crosslinking enzyme lysyl oxidase-like protein 2 (LOXL2), had no effect on levels of collagen but significantly reduced the strength and stiffness of the ring even when elevated by treatment with TGF-β1.
CONCLUSION
Human fibroblasts will self-assemble a 3D ring-shaped tissue and synthesize a fibrous network of ECM proteins whose tensile properties can be measured. The fibrotic environment can be mimicked by addition of TGF-β1, which increases levels of collagen as well as the strength and stiffness of the rings. Treatment with two drugs, incyclinide and PAT 1251 that were developed as potential treatments for diseases of the ECM, altered the strength and stiffness of the rings, thereby demonstrating the utility of the model for testing new therapies that target the biomechanics of the ECM.
SUPPLEMENTARY INFORMATION
The online version contains supplementary material available at 10.1007/s44164-025-00081-y.
目的
人类细胞外基质（ECM）强度和刚度的改变是多种疾病的潜在病理表现。这些疾病包括遗传性疾病，如埃勒斯-当洛综合征，以及后天性疾病，如纤维化，纤维化仍是一项重大的未满足医疗需求。为了评估有前景的治疗方法，需要能够测量人类ECM强度和刚度的新模型。
方法
将培养的人成纤维细胞接种到已模制成24孔板并与细胞培养基平衡的琼脂糖圆形槽中。细胞在重力作用下沉降、聚集并形成直径为5毫米的三维环形组织，无需添加外源性支架材料。通过免疫染色对环形组织新合成的ECM蛋白进行表征。通过测量环形组织尺寸的变化和胶原蛋白水平来评估环形组织对药物和生长因子处理的反应。使用拉伸试验来量化药物和生长因子诱导的环形组织强度和刚度的变化。
结果
环形组织很容易在模具中形成，并新合成了一个周向排列的富含胶原蛋白的纤维状ECM网络，该网络对I型胶原蛋白、III型胶原蛋白和纤连蛋白呈阳性。用基质金属蛋白酶（MMPs）抑制剂因西环肽进行低剂量处理可增加强度和刚度，而高剂量则会降低拉伸性能，这可能是由于毒性作用。用众所周知的纤维化驱动因子转化生长因子-β1（TGF-β1）处理可增加胶原蛋白水平和拉伸性能，并在体外模拟纤维化环境。用胶原蛋白交联酶赖氨酰氧化酶样蛋白2（LOXL2）抑制剂PAT-1251处理对胶原蛋白水平没有影响，但即使在用TGF-β1处理使其升高后，也能显著降低环形组织的强度和刚度。
结论
人成纤维细胞将自我组装成三维环形组织，并合成可测量其拉伸性能的ECM蛋白纤维网络。添加TGF-β1可模拟纤维化环境，TGF-β1可增加胶原蛋白水平以及环形组织的强度和刚度。用两种作为ECM疾病潜在治疗药物开发的药物因西环肽和PAT 1251进行处理，改变了环形组织的强度和刚度，从而证明了该模型在测试针对ECM生物力学的新疗法方面的实用性。
补充信息
在线版本包含可在10.1007/s44164-025-00081-y获取的补充材料。
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