Cell-loaded hydrogels are frequently applied in cartilage tissue engineering for their biocompatibility, ease of application, and ability to conform to various defect sites. As a bioactive adjunct to the biomaterial, transforming growth factor beta (TGF-β) has been shown to be essential for cell differentiation into a chondrocyte phenotype and maintenance thereof, but the low amounts of endogenous TGF-β in the in vivo joint microenvironment necessitate a mechanism for controlled delivery and release of this growth factor. In this study, TGF-β3 was directly loaded with human bone marrow-derived mesenchymal stem cells (MSCs) into poly-d,l-lactic acid/polyethylene glycol/poly-d,l-lactic acid (PDLLA-PEG) hydrogel, or PDLLA-PEG with the addition of hyaluronic acid (PDLLA/HA), and cultured in vitro. We hypothesize that the inclusion of HA within PDLLA-PEG would result in a controlled release of the loaded TGF-β3 and lead to a robust cartilage formation without the use of TGF-β3 in the culture medium. ELISA analysis showed that TGF-β3 release was effectively slowed by HA incorporation, and retention of TGF-β3 in the PDLLA/HA scaffold was detected by immunohistochemistry for up to 3 weeks. By means of both in vitro culture and in vivo implantation, we found that sulfated glycosaminoglycan production was higher in PDLLA/HA groups with homogenous distribution throughout the scaffold than PDLLA groups. Finally, with an optimal loading of TGF-β3 at 10 μg/mL, as determined by RT-PCR and glycosaminoglycan production, an almost twofold increase in Young's modulus of the construct was seen over a 4-week period compared to TGF-β3 delivery in the culture medium. Taken together, our results indicate that the direct loading of TGF-β3 and stem cells in PDLLA/HA has the potential to be a one-step point-of-care treatment for cartilage injury. STATEMENT OF SIGNIFICANCE: Stem cell-seeded hydrogels are commonly used in cell-based cartilage tissue engineering, but they generally fail to possess physiologically relevant mechanical properties suitable for loading. Moreover, degradation of the hydrogel in vivo with time further decreases mechanical suitability of the hydrogel due in part to the lack of TGF-β3 signaling. In this study, we demonstrated that incorporation of hyaluronic acid (HA) into a physiologically stiff PDLLA-PEG hydrogel allowed for slow release of one-time preloaded TGF-β3, and when loaded with adult mesenchymal stem cells and cultured in vitro, it resulted in higher chondrogenic gene expression and constructs of significantly higher mechanical strength than constructs cultured in conventional TGF-β3-supplemented medium. Similar effects were also observed in constructs implanted in vivo. Our results indicate that direct loading of TGF-β3 combined with HA in the physiologically stiff PDLLA-PEG hydrogel has the potential to be used for one-step point-of-care treatment of cartilage injury.

中文翻译：

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通过掺入透明质酸和直接加载TGF-β来增强生理相关水凝胶中的软骨形成和机械强度保留。

细胞载水凝胶因其生物相容性，易用性和适应各种缺损部位的能力而经常用于软骨组织工程。作为生物材料的生物活性助剂，转化生长因子β（TGF-β）已被证明对于细胞分化为软骨细胞表型及其维持至关重要，但是体内关节微环境中的内源性TGF-β含量较低一种控制递送和释放该生长因子的机制。在这项研究中，将TGF-β3直接与人骨髓源间充质干细胞（MSC）一起加载到聚d，1-乳酸/聚乙二醇/聚-d，l-乳酸（PDLLA-PEG）水凝胶中，或添加透明质酸（PDLLA / HA）的PDLLA-PEG，并在体外培养。我们假设在PDLLA-PEG中包含HA将导致负载的TGF-β3的受控释放，并导致在培养基中不使用TGF-β3的情况下形成坚固的软骨。ELISA分析表明，HA掺入有效地减缓了TGF-β3的释放，并通过免疫组织化学检测了长达3周的TDLL-β3在PDLLA / HA支架中的保留。通过体外培养和体内植入，我们发现在整个支架中PDLLA / HA组中的硫酸化糖胺聚糖产量高于PDLLA组。最后，通过RT-PCR和糖胺聚糖生产确定，最佳的TGF-β3负载量为10μg/ mL，Young'几乎增加了两倍。与在培养基中递送的TGF-β3相比，在4周的时间内观察到该构建体的模量s。综上所述，我们的结果表明，将TGF-β3和干细胞直接加载到PDLLA / HA中可能成为软骨损伤的一站式治疗方法。意义陈述：干细胞接种的水凝胶通常用于基于细胞的软骨组织工程中，但它们通常不具备适合加载的生理学相关的机械性能。此外，水凝胶在体内随时间的降解进一步降低了水凝胶的机械适应性，部分原因是缺乏TGF-β3信号传导。在这项研究中，我们证明了将玻尿酸（HA）掺入生理上坚硬的PDLLA-PEG水凝胶中可以缓慢释放一次预载的TGF-β3，当与成年间充质干细胞一起加载并在体外培养时，与在传统的TGF-β3培养基中培养的构建体相比，它能产生更高的软骨生成基因表达，并具有显着更高的机械强度的构建体。在体内植入的构建体中也观察到了类似的效果。我们的结果表明，将TGF-β3与HA组合直接加载到具有生理学刚度的PDLLA-PEG水凝胶中具有潜力，可用于软骨损伤的一站式即时治疗。