Although most bone fractures typically heal without complications, a small proportion of patients (≤10%) experience delayed healing or potential progression to non-union. Interleukin-1 (IL-1β) plays a crucial role in fracture healing as an early driver of inflammation. However, the effects of IL-1β can impede the healing process if they persist long after the establishment of a fracture hematoma, making it a promising target for novel therapies. Accordingly, the overall objective of this study was to develop a novel gene-based therapy that mitigates the negative effects of IL-1β-driven inflammation while providing a structural template for new bone formation. A collagen-hydroxyapatite scaffold (CHA) was used as a platform for the delivery of nanoparticles composed of pDNA, encoding for IL-1 receptor antagonist (IL-1Ra), complexed to the robust non-viral gene delivery vector, polyethyleneimine (PEI). Utilizing pDNA encoding for Gaussia luciferase and GFP as reporter genes, we found that PEI-pDNA nanoparticles induced a transient gene expression profile in rat bone marrow-derived mesenchymal stromal cells (BM-MSCs), with a transfection efficiency of 14.8 ± 1.8% in 2D. BM-MSC viability was significantly affected by PEI-pDNA nanoparticles as evaluated using CellTiter Blue; however, after 10 days in culture this effect was negligible. Transfection with PEI-pIL-1Ra nanoparticles led to functional IL-1Ra production, capable of antagonizing IL-1β-induced expression of secreted embryonic alkaline phosphatase from HEK-Blue-IL-1β reporter cells. Sustained treatment with IL-1β (0.1, 1, and 10 ng/ml) had a dose-dependent negative effect on BM-MSC osteogenesis, both in terms of gene expression (Alpl and Ibsp) and calcium deposition. BM-MSCs transfected with PEI-IL-1Ra nanoparticles were found to be capable of overcoming the inhibitory effects of sustained IL-1β (1 ng/ml) treatments on in vitro osteogenesis. Ultimately, IL-1Ra gene-activated CHA scaffolds supported mineralization of BM-MSCs under chronic inflammatory conditions in vitro, demonstrating potential for future therapeutic applications in vivo.

中文翻译：

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使用胶原-羟基磷灰石支架的白介素-1 受体拮抗剂的非病毒基因传递保护大鼠 BM-MSC 免受 IL-1β 介导的成骨抑制

尽管大多数骨折通常不会出现并发症，但一小部分患者 (≤10%) 会出现愈合延迟或可能进展为骨不连的情况。白细胞介素 1 (IL-1β) 作为炎症的早期驱动因素在骨折愈合中起着至关重要的作用。然而，如果 IL-1β 的作用在骨折血肿形成后持续很长时间，它们会阻碍愈合过程，使其成为新疗法的有希望的目标。因此，本研究的总体目标是开发一种新的基于基因的疗法，以减轻 IL-1β 驱动的炎症的负面影响，同时为新骨形成提供结构模板。胶原-羟基磷灰石支架 (CHA) 用作递送由 pDNA 组成的纳米颗粒的平台，编码 IL-1 受体拮抗剂 (IL-1Ra)，与强大的非病毒基因递送载体聚乙烯亚胺 (PEI) 复合。利用编码 Gaussia 荧光素酶和 GFP 的 pDNA 作为报告基因，我们发现 PEI-pDNA 纳米颗粒在大鼠骨髓间充质基质细胞 (BM-MSC) 中诱导瞬时基因表达谱，转染效率为 14.8 ± 1.8%二维。使用 CellTiter Blue 评估 PEI-pDNA 纳米颗粒显着影响 BM-MSC 的活力；然而，在培养 10 天后，这种影响可以忽略不计。用 PEI-pIL-1Ra 纳米粒子转染导致功能性 IL-1Ra 产生，能够拮抗 IL-1β 诱导的 HEK-Blue-IL-1β 报告细胞分泌的胚胎碱性磷酸酶的表达。用 IL-1β（0.1、1 和 10 ng/ml）持续治疗对 BM-MSC 成骨具有剂量依赖性的负面影响，在基因表达（Alpl 和 Ibsp）和钙沉积方面。发现用 PEI-IL-1Ra 纳米颗粒转染的 BM-MSCs 能够克服持续 IL-1β (1 ng/ml) 治疗对体外成骨的抑制作用。最终，IL-1Ra 基因激活的 CHA 支架在体外慢性炎症条件下支持 BM-MSC 的矿化，证明了未来体内治疗应用的潜力。