School of Chemical Engineering, University of Birmingham, Birmingham, UK.
Department of Oral and Maxillofacial Surgery & Special Dental Care, University Medical Center Utrecht, Utrecht, the Netherlands.
Biochem Biophys Res Commun. 2024 Nov 26;735:150841. doi: 10.1016/j.bbrc.2024.150841. Epub 2024 Oct 16.
Osteoblast-derived extracellular vesicles (EVs) have demonstrated therapeutic utility for bone repair as transporters of key biomolecules capable of accelerating biomineralisation and tissue repair. The clinical translation of these biologically derived nanoparticles, however remains limited due to scalability, heterogeneity and standardisation issues. Herein we investigate the generation of nanovesicles (NVs) from mineralising osteoblasts by extrusion directly compared against natural EV counterparts from the same parental cells. Mineralising osteoblast-derived EVs (MO-EVs) were isolated via ultracentrifugation from cell culture media. The parental osteoblasts were then processed via serial extrusion to <200 nm. EVs and NVs were characterised by comparing their size, concentration and morphology. The presence of tetraspanin markers was detected by Single Particle Interferometric Reflectance Imaging Sensor (SP-IRIS). Osteoblasts viability and metabolic activity was assessed after both EV and NV-treatment before comparing their mineralising potency via alizarin red staining. EVs and NVs exhibited similar diameters of approximately 100 nm with a vesicular morphology. EVs were found to be richer in proteins and exhibited a significantly more negative ζ-potential compared to NVs. SP-IRIS analysis confirmed the presence of CD9, CD63 and CD81 in EVs. At both 1 and 10 μg/mL, EVs and NVs reduced the metabolic activity of osteoblasts, however, this was not associated with any cytotoxic effects. The biomineralisation study performed using osteoblasts showed that only EVs significantly increased mineral deposition (p < 0.05) compared to untreated control. In this study, we have established for the first time the biofabrication of cell-derived nanovesicles as a promising alternative to extracellular vesicles derived from mineralising osteoblasts.
成骨细胞衍生的细胞外囊泡 (EVs) 已被证明在骨修复方面具有治疗作用，因为它们是能够加速生物矿化和组织修复的关键生物分子的载体。然而，由于可扩展性、异质性和标准化问题，这些生物衍生的纳米颗粒的临床转化仍然受到限制。在此，我们通过挤压直接比较了从矿化成骨细胞中产生的纳米囊泡 (NVs) 与来自同一亲本细胞的天然 EV 对应物，从而研究了从矿化成骨细胞中产生纳米囊泡 (NVs) 的方法。通过超速离心从细胞培养物中分离矿化成骨细胞衍生的 EV (MO-EVs)。然后通过连续挤压处理亲本成骨细胞至 <200nm。通过比较其大小、浓度和形态来表征 EV 和 NV。通过单颗粒干涉反射成像传感器 (SP-IRIS) 检测四跨膜蛋白标记物的存在。在比较其通过茜素红染色的矿化能力之前，通过 EV 和 NV 处理评估成骨细胞的活力和代谢活性。EVs 和 NVs 的直径相似，约为 100nm，呈囊泡形态。与 NVs 相比，EVs 富含蛋白质，并且表现出明显更负的 ζ-电位。SP-IRIS 分析证实了 EVs 中 CD9、CD63 和 CD81 的存在。在 1 和 10μg/mL 时，EVs 和 NVs 降低了成骨细胞的代谢活性，但这与任何细胞毒性作用无关。使用成骨细胞进行的矿化研究表明，只有 EVs 与未处理的对照组相比，显著增加了矿物质沉积 (p<0.05)。在这项研究中，我们首次建立了细胞衍生的纳米囊泡的生物制造，作为源自矿化成骨细胞的细胞外囊泡的有前途的替代品。
