Applied Oral Sciences & Community Dental Care, Faculty of Dentistry, The University of Hong Kong, Hong Kong SAR, China.
Department of Mechanical Engineering, The University of Hong Kong, Hong Kong SAR, China.
Int Endod J. 2024 Nov;57(11):1639-1654. doi: 10.1111/iej.14126. Epub 2024 Jul 24.
Lack of adequate mechanical strength and progressive shrinkage over time remain challenges in scaffold-free microtissue-based dental pulp regeneration. Surface collagen cross-linking holds the promise to enhance the mechanical stability of microtissue constructs and trigger biological regulations. In this study, we proposed a novel strategy for surface preconditioning microtissues using a natural collagen cross-linker, proanthocyanidin (PA). We evaluated its effects on cell viability, tissue integrity, and biomineralization of dental pulp stem cell (DPSCs)-derived 3D cell spheroids.
Microtissue and macrotissue spheroids were fabricated from DPSCs and incubated with PA solution for surface collagen cross-linking. Microtissue viability was examined by live/dead staining and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, with transverse dimension change monitored. Microtissue surface stiffness was measured by an atomic force microscope (AFM). PA-preconditioned microtissues and macrotissues were cultured under basal or osteogenic conditions. Immunofluorescence staining of PA-preconditioned microtissues was performed to detect dentin sialophosphoprotein (DSPP) and F-actin expressions. PA-preconditioned macrotissues were subjected to histological analysis, including haematoxylin-eosin (HE), alizarin red, and Masson trichrome staining. Immunohistochemistry staining was used to detect alkaline phosphatase (ALP) and dentin matrix acidic phosphoprotein 1 (DMP-1) expressions.
PA preconditioning had no adverse effects on microtissue spheroid viability and increased surface stiffness. It reduced dimensional shrinkage for over 7 days in microtissues and induced a larger transverse-section area in the macrotissue. PA preconditioning enhanced collagen formation, mineralized nodule formation, and elevated ALP and DMP-1 expressions in macrotissues. Additionally, PA preconditioning induced higher F-actin and DSPP expression in microtissues, while inhibition of F-actin activity by cytochalasin B attenuated PA-induced dimensional change and DSPP upregulation.
PA surface preconditioning of DPSCs spheroids demonstrates excellent biocompatibility while effectively enhancing tissue structure stability and promoting biomineralization. This strategy strengthens tissue integrity in DPSC-derived spheroids and amplifies osteogenic differentiation potential, advancing scaffold-free tissue engineering applications in regenerative dentistry.
在无支架微组织牙髓再生中，缺乏足够的机械强度和随时间推移的渐进性收缩仍然是挑战。表面胶原交联有望增强微组织构建体的机械稳定性并触发生物学调节。在这项研究中，我们提出了一种使用天然胶原交联剂原花青素（PA）对微组织进行表面预处理的新策略。我们评估了其对牙髓干细胞（DPSC）衍生的 3D 细胞球形成的微组织的细胞活力、组织完整性和生物矿化的影响。
从 DPSC 中构建微组织和大组织球体，并将其与 PA 溶液孵育以进行表面胶原交联。通过活/死染色和 3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四氮唑溴化物（MTT）测定法检查微组织的活力，监测横向尺寸变化。通过原子力显微镜（AFM）测量 PA 预处理微组织的表面硬度。在基础或成骨条件下培养 PA 预处理的微组织和大组织。对 PA 预处理的微组织进行免疫荧光染色，以检测牙本质涎磷蛋白（DSPP）和 F-肌动蛋白的表达。对 PA 预处理的大组织进行组织学分析，包括苏木精-伊红（HE）、茜素红和 Masson 三色染色。免疫组织化学染色用于检测碱性磷酸酶（ALP）和牙本质基质酸性磷酸蛋白 1（DMP-1）的表达。
PA 预处理对微组织球体活力没有不良影响，反而增加了表面硬度。它减少了微组织中超过 7 天的尺寸收缩，并使大组织的横截面积增大。PA 预处理增强了大组织中的胶原形成、矿化结节形成以及 ALP 和 DMP-1 的表达。此外，PA 预处理诱导微组织中更高的 F-肌动蛋白和 DSPP 表达，而细胞松弛素 B 抑制 F-肌动蛋白活性则减弱了 PA 诱导的尺寸变化和 DSPP 的上调。
PA 对 DPSC 球体的表面预处理表现出极好的生物相容性，同时有效地增强了组织结构的稳定性并促进了生物矿化。该策略增强了 DPSC 衍生球体的组织完整性并放大了成骨分化潜能，推进了无支架组织工程在再生牙科中的应用。
