Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA.
Department of Biomedical Engineering, University of Michigan, Ann Arbor, MI, 48109, USA; Department of Obstetrics and Gynecology, University of Michigan, Ann Arbor, MI, 48109, USA; Cellular and Molecular Biology Program, University of Michigan, Ann Arbor, MI, 48109, USA; Macromolecular Science and Engineering, University of Michigan, Ann Arbor, MI, 48109, USA.
Acta Biomater. 2021 Sep 15;132:313-324. doi: 10.1016/j.actbio.2021.03.041. Epub 2021 Mar 22.
Synthetic matrices offer a high degree of control and tunability for mimicking extracellular matrix functions of native tissue, allowing the study of disease and development in vitro. In this study, we functionalized degradable poly(ethylene glycol) hydrogels with extracellular matrix (ECM)-sequestering peptides aiming to recapitulate the native ECM composition for culture and maturation of ovarian follicular organoids. We hypothesized that ECM-sequestering peptides would facilitate deposition and retention of cell-secreted ECM molecules, thereby recreating cell-matrix interactions in otherwise bioinert PEG hydrogels. Specifically, heparin-binding peptide from antithrombin III (HBP), heparan sulfate binding peptide derived from laminin (AG73), basement membrane binder peptide (BMB), and heparan sulfate binding region of placental growth factor 2 (RRR) tethered to a PEG hydrogel significantly improved follicle survival, growth and maturation compared to PEG-Cys, a mechanically similar but biologically inert control. Immunohistochemical analysis of the hydrogel surrounding cultured follicles confirmed sequestration and retention of laminin, collagen I, perlecan, and fibronectin in ECM-sequestering hydrogels but not in bioinert PEG-Cys hydrogels. The media from follicles cultured in PEG-AG73, PEG-BMB, and PEG-RRR also had significantly higher concentrations of factors known to regulate follicle development compared to PEG-Cys. PEG-AG73 and PEG-BMB were the most beneficial for promoting follicle maturation, likely because AG73 and BMB mimic basement membrane interactions which are crucial for follicle development. Here we have shown that functionalizing PEG with ECM-sequestering peptides allows cell-secreted ECM to be retained within the hydrogels, restoring critical cell-matrix interactions and promoting healthy organoid development in a fully synthetic culture system. STATEMENT OF SIGNIFICANCE: Here we present a novel approach for sequestering and retaining cell-secreted extracellular matrix in a fully synthetic material for organoid culture. We have engineered a biomimetic poly(ethylene glycol) hydrogel functionalized with extracellular matrix-binding peptides to recapitulate the ovarian microenvironment. Incorporation of these peptides allows ovarian follicles to recreate their native matrix with the sequestered ECM that subsequently binds growth factors, facilitating follicle maturation. The novel design resulted in improved outcomes of folliculogenesis, potentially developing a fertility preservation option for young women undergoing sterilizing treatments for cancer. The fully synthetic and modular nature of this biomimetic material holds promise for other tissue engineering applications as it allows encapsulated cells to rebuild their native microenvironments in vitro.
合成基质为模拟天然组织细胞外基质功能提供了高度的控制和可调性，允许在体外研究疾病和发育。在这项研究中，我们用细胞外基质（ECM）结合肽功能化可降解聚乙二醇水凝胶，旨在重现天然 ECM 组成，用于培养和成熟卵巢卵泡类器官。我们假设 ECM 结合肽将促进细胞分泌的 ECM 分子的沉积和保留，从而在其他生物惰性 PEG 水凝胶中再现细胞-基质相互作用。具体来说，从抗凝血酶 III 中分离出的肝素结合肽（HBP）、从层粘连蛋白衍生出的硫酸乙酰肝素结合肽（AG73）、基底膜结合肽（BMB）和胎盘生长因子 2 的硫酸乙酰肝素结合区（RRR）与 PEG 水凝胶结合，与机械相似但生物惰性的对照 PEG-Cys 相比，显著提高了卵泡的存活率、生长和成熟。对培养卵泡周围水凝胶的免疫组织化学分析证实，在 ECM 结合水凝胶中，层粘连蛋白、胶原 I、perlecan 和纤维连接蛋白被隔离和保留，但在生物惰性 PEG-Cys 水凝胶中则没有。与 PEG-Cys 相比，在 PEG-AG73、PEG-BMB 和 PEG-RRR 中培养的卵泡的培养基中，已知调节卵泡发育的因子的浓度也显著升高。PEG-AG73 和 PEG-BMB 最有利于促进卵泡成熟，这可能是因为 AG73 和 BMB 模拟了基底膜相互作用，这对卵泡发育至关重要。在这里，我们已经表明，用 ECM 结合肽功能化 PEG 可以使细胞分泌的 ECM 保留在水凝胶中，恢复关键的细胞-基质相互作用，并在完全合成的培养系统中促进健康的类器官发育。意义声明：在这里，我们提出了一种新的方法，用于在完全合成的材料中隔离和保留细胞分泌的细胞外基质，用于类器官培养。我们设计了一种仿生聚乙二醇水凝胶，用细胞外基质结合肽进行功能化，以重现卵巢微环境。这些肽的掺入允许卵巢卵泡用被隔离的 ECM 重建其天然基质，随后结合生长因子，促进卵泡成熟。这种新的设计导致卵泡发生的结果得到改善，为接受癌症绝育治疗的年轻女性提供了一种潜在的生育力保存选择。这种仿生材料的完全合成和模块化性质为其他组织工程应用带来了希望，因为它允许包封的细胞在体外重建其天然微环境。
