目的:缺乏足够的机械强度和随时间的逐渐收缩仍然是基于无支架微组织的牙髓再生的挑战。表面胶原蛋白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蛋白交联剂对表面预处理微组织的新策略,原花青素(PA)。我们评估了它对细胞活力的影响,组织完整性,和牙髓干细胞(DPSC)衍生的3D细胞球体的生物矿化。
方法:由DPSC制备微组织和大组织球体,并与PA溶液一起孵育用于表面胶原交联。通过活/死染色和3-(4,5-二甲基噻唑-2-基)-2,5-二苯基四唑溴化物(MTT)测定法检查微组织活力,横向尺寸变化监测。通过原子力显微镜(AFM)测量微组织表面硬度。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唾液酸磷蛋白(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.免疫组化染色检测碱性磷酸酶(ALP)和牙本质基质酸性磷蛋白1(DMP-1)的表达。
结果:PA预处理对微组织球状体活力和表面硬度增加没有不良影响。它减少了微组织的尺寸收缩超过7天,并在大组织中诱导了更大的横截面面积。PA预处理增强胶原形成,矿化结节形成,以及大组织中ALP和DMP-1的表达升高。此外,PA预处理在微组织中诱导较高的F-肌动蛋白和DSPP表达,而细胞松弛素B对F-肌动蛋白活性的抑制减弱了PA诱导的尺寸变化和DSPP上调。
结论:PA表面预处理DPSC球体显示出优异的生物相容性,同时有效增强组织结构稳定性和促进生物矿化。该策略增强了DPSC衍生球状体的组织完整性,并增强了成骨分化潜能,推进无支架组织工程在再生牙科中的应用。
OBJECTIVE: 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.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.