羟基磷灰石纳米颗粒通过内皮细胞相互作用对间充质干细胞的潜在骨诱导作用
摘要
纳米羟基磷灰石(nano-HA)在再生医学领域引起了广泛关注。内皮细胞 (EC)-间充质干细胞 (MSC) 相互作用是骨骼重建所必需的,但纳米 HA 在此过程中的相互作用方式仍然未知。在此,我们使用由 ECs 介导的间接共培养模型研究了 HA 纳米粒子 (HANPs) 对 MSCs 的细胞毒性和骨诱导作用,并强调了其潜在机制。结果发现,在亚细胞毒性剂量下,HANP 增加了成骨细胞基因的活力和表达,以及 MSC 的矿化结节和碱性磷酸酶的产生。这些现象依赖于 ECs 分泌的 HIF-1α,它触发了 ERK1/2 信号级联。此外,建立两阶段细胞谱系数学模型,定量分析HIF-1α对MSCs成骨分化的影响。结果表明,HIF-1α对MSCs的成骨分化率具有剂量依赖性刺激作用,最高可达1500 pg/mL,与上述结果一致。我们的数据表明 HANP、EC 和 MSC 之间的协同相互作用可能有助于刺激骨再生。此外,两阶段细胞谱系模型有助于评估效应分子在骨组织工程中的潜在影响的体外系统。
介绍
外伤、先天性畸形或手术切除引起的骨缺损的重建对骨科手术提出了巨大的挑战[1]。羟基磷灰石 (HA) 是一种代表性的生物活性陶瓷,已被用作骨替代品 [2]。然而,不良的机械和骨诱导特性限制了其临床应用[3]。近年来,nano-HA由于其独特的仿生特性表现出更优的生物活性和更好的机械性能,并在与再生医学相关的生物医学领域引起了极大的兴趣[4]。当nano-HA被植入骨缺损时,参与骨修复的多个细胞将暴露于其中。因此,有必要评估纳米 HA 的生物学行为。几条证据直接表明 HA 纳米粒子 (HANPs) 可以被人脐带沃顿的果冻间充质干细胞 (hWJ-MSCs) 和成骨细胞吸收,从而增强成骨分化 [5,6,7]。杜阿等人。先前报道了 HANP 促进工程软骨整合到从头软骨中的能力 [8];相反,HANP 抑制人脐静脉内皮细胞 (HUVEC) 的血管生成能力 [9]。在人类健康方面,有必要更全面地了解 HANPs 对骨再生的影响,而工程纳米人工骨的持续应用增加了此类研究的紧迫性。
骨再生不可避免地伴随着新生血管的侵袭。 ECs 是血管系统的内部细胞衬里,用于被动输送血液,还在诱导、指定和引导器官再生以及维持体内平衡和新陈代谢方面发挥作用 [10, 11]。间充质干细胞是内皮周生态位的一部分,在其驻留生态位内特定的生理和生化微环境的诱导下具有自我更新和多分化能力[12]。蔡等人。发现 ECs 可以分泌内皮素-1 以指导 MSCs 向骨和软骨谱系分化 [13]。此外,萨利赫等人。使用微阵列数据分析来识别与 MSC 膜结合受体相互作用以增强增殖和成骨分化的 HUVEC 分泌蛋白和相关串扰信号通路 [14]。在骨组织工程中,HANP 可以与新血管接触并被 EC 内吞,这已被证明会改变这些细胞的生理功能 [9, 15]。这也可以影响周围的骨祖细胞并通过改变旁分泌信号影响骨骼修复。然而,虽然已经探索了 HANPs 对 MSCs 的直接影响,但对于 HANPs 是否可以通过 ECs 间接诱导 MSCs 的成骨分化仍然缺乏明确的认识,这对于我们理解 HANPs 在这方面的影响至关重要。进行骨修复。
在这项研究中,为了进一步了解 HANPs 对 ECs 和 MSCs 之间相互作用的生物学影响,使用 HUVECs 和 hWJ-MSCs 建立了间接共培养模型。通过利用该系统,评估了 HANP 通过 HUVEC 介导的旁分泌信号传导对 hWJ-MSC 的细胞毒性和骨诱导作用。为了确定影响 HANP 诱导的内皮细胞 - MSC 相互作用的关键因素,评估了用 HANP 刺激的 HUVEC 上清液中的可溶性因子,重点是基因和蛋白质水平的相关机制。结果表明缺氧诱导因子(HIF)-1α在这些相互作用中起关键作用。
为了定量观察和预测 HIF-1α 对成骨过程的影响,建立了一个将两阶段细胞谱系与 HIF-1α 相结合的数学模型。在这里,通过分析经验数据,基于定义的初始细胞接种密度和 HIF-1α 浓度,使用两阶段细胞谱系模型预测任何时间点的 MSC 数量和分化程度,这可能反过来为初始培养条件和孵育时间提供适当的建议。本研究结果将有助于阐明纳米骨替代品与生物系统之间的相互作用,有助于促进用于再生医学的创新生物材料的发展。
材料和方法
颗粒的制备和表征
纯度 ≥ 99.0% 的 20 nm (np20)、20*80 nm (np80) 和微米级 HA 颗粒 (m-HAP) 的 HANP 购自南京英皇纳米材料有限公司(中国南京)。使用透射电子显微镜(TEM;FEI Tecnai G2 Spirit Bio-Twin,FEI,Hillsboro,OR,USA)和扫描电子显微镜(SEM;LEO1530VP,德国)观察颗粒的大小和形状。通过 Zetasizer Nano ZS90 和 Mastersizer 3000 (Malvern Instruments, Malvern, UK) 测定 HA 颗粒 (HAPs) 的流体动力学尺寸和 zeta 电位。
细胞制备和培养
所有实验方案均经南京医科大学伦理委员会批准。如前所述,在获得捐赠者的书面知情同意后,从新鲜人脐带中收集 HUVEC 和 hWJ-MSC [16, 17]。简而言之,用含有 1% 青霉素和链霉素(PS;Hyclone,GE Healthcare Life Sciences,Pasching,Austria)的磷酸盐缓冲盐水 (PBS) 冲洗脐带和脐静脉。然后,脐静脉充满 0.1% 胶原酶 I(Sigma,St. Louis,MO,USA)并在 37°C 下孵育 15 分钟。收集后,将 HUVEC 在 EC 培养基(ECM)(Sciencell,San Diego,CA,USA)中培养。
随后,去除血管,将沃顿氏果冻切成 1 毫米 2 件,然后放置在 25 cm 2 组织培养瓶(Corning Incorporated, Corning, NY, USA)。这些细胞在补充有 10% 胎牛血清 (GIBCO) 和 1% PS 的 L-DMEM (GIBCO Life Technology, Grand Island, NY, USA) 中培养。
使用针对 CD13、CD29、CD34、CD44、CD45、CD51 和 CD105 的单克隆抗体(BD Biosciences,San Jose,CA,USA)评估 hWJ-MSC 以确认表型。使用 von Willebrand 因子(vWF;上海昌道生物科技有限公司,中国上海)评估 HUVEC。在这些实验中使用了第 3-7 代之间的 HUVEC 和第 3-5 代之间的 hWJ-MSC。
然后将 PBS 中 1 mg/mL 的颗粒悬浮液在 L-DMEM 中稀释至最终浓度。如图 1 所示,HUVEC 在指定浓度的 HAP 中孵育 18 小时。将培养基在 4°C 下以 15,000 rpm 离心 15 分钟,将补充有 10% FBS 的上清液用作 hWJ-MSC 的条件培养基 (CM) 以完成以下实验。 CM 由补充有成骨诱导液的成骨培养基组成,其中含有 10 mM β-甘油磷酸、50 μg/mL L-抗坏血酸-2-磷酸和 0.1 μM 地塞米松(Sigma-Aldrich,St Louis,MO,USA)。此外,2-甲氧基雌二醇 (2-MeOE2) (Selleck Chemicals, Houston, TX, USA) 被用作特异性 HIF-1α 抑制剂。在 2-MeOE2(+) 组中,hWJ-MSC 与来自 HUVEC 的 CM 一起培养,在补充 HAP 之前用 5 μM 2-MeOE2 预处理 40 分钟。 PD98059 用作特异性 MEK 抑制剂。在PD98059组中,hWJ-MSCs用含有5 μM PD98059的CM培养。
<图片>结果
HAP 的特征
如图 2 所示,制备了具有特定尺寸和形状的 HAP。接近球形的np20的直径平均为20nm,np80为棒状,平均长度为80nm,宽度为20nm。 m-HAP 的形状也接近球形,直径约为 12 微米。所有颗粒在 L-DMEM 中都带有负表面电荷。已经表明,zeta 电位的负值对骨细胞的附着和增殖以及直接骨结合和新骨形成具有显着的有利影响 [22, 23]。在 L-DMEM 中观察到的颗粒有在水性体系中聚集的趋势。还测试了它们的流体动力学尺寸,这可能也是影响其生物学行为的重要因素。
<图片>讨论
With recent advances in nanobiomaterials, nano-based artificial bone substitutes have been an area of intense investigation. The accumulating evidence suggests that there are complex interactions between cells and nanobiomaterials due to their capacity to penetrate cell membranes and increase internal retention times [24, 25]. A previous study revealed that collagen/alginate nanofilms can adsorb onto the MSC membrane to activate intracellular signaling cascades and promote osteogenic differentiation [26]. Elegant experiments by Wu and his colleagues clearly demonstrated that TiO2 nanotubes can improve vascularization and osteogenic differentiation by facilitating paracrine effects and cell junctions via EC-MSC interactions [27]. For the purpose of developing excellent candidates for bone tissue engineering, it is necessary to clarify the direct crosstalk between nano-based bone substitutes and cells implicated in bone repair as well as their indirect interactions. However, our current understanding of this is still limited. In the present study, we utilized an indirect co-culture model to further elucidate the biological effects of HANPs on MSCs in regard to the indirect interactions mediated by ECs.
Cytotoxicity is a primary issue for assessing the biocompatibility of any nanobiomaterial. Although our previous study found that HANPs did not directly influence the viability or apoptosis of hWJ-MSCs, they may still exert different impacts via the mediation of other cells [28]. Thus, it was necessary to evaluate the cytotoxic effects of HANPs on hWJ-MSCs mediated by HUVECs. Interestingly, after incubation in CM for 24 h and 72 h, hWJ-MSC viability was maintained and even elevated in the 0–50 µg/mL HANP groups, especially in the np20 group, indicating the existence of effector molecules in the CM. When the concentration of HANPs reached 100 µg/mL, they became cytotoxic to the hWJ-MSCs. However, 0–100 µg/mL m-HAP had no influence on hWJ-MSC viability (Fig. 3).江等人。 have shown that engineered nanoparticles of a particular size can have distinct endocytic routes and kinetics associated with altered downstream signaling involved in regulating target cell functions [29]. In our previous study, we showed that np20 and np80 were endocytosed by HUVECs, and this was followed by morphologic changes and the appearance of large vacuoles, indicating the activated state of the HUVECs. Additionally, np20, with their faster uptake speed and increased accumulation, might result in a stronger activation of HUVECs, possibly resulting in increased hWJ-MSC viability via paracrine signaling. Conversely, few m-HAPs can be endocytosed by HUVECs, and this might account for their limited influence on the metabolism of hWJ-MSCs [9].
To further explore the potential osteoinductive effect of activated HUVECs, a subcytotoxic dose of 50 µg/mL HAPs was used in subsequent studies. The CM collected from the activated HUVECs promoted extracellular calcium deposition, ALP activity, and osteogenic proteins expression in hWJ-MSCs, as well as the mRNA expression of osteogenic genes (Figs. 4, 5). Runx2, an essential transcription factor involved in specifying the osteoblast lineage [30], showed a substantial enhancement in the np20 group, indicating a strong osteoinductive effect on hWJ-MSCs (Fig. 4a and Fig. 5c, d). The np20 group demonstrated a 1.5-fold improvement in COLI expression at Day 7 (Figs. 4b, 5c, d) and a double increase at Day 14, which implied the presence of additional differentiated osteoblasts in the HANP-treated groups (Fig. 4b) [30]. OCN is a mature stage bone marker [31], and this gene showed a significant increase in the HANP groups at Day 14 (Fig. 4c), indicating that np20 and np80 can accelerate bone maturation compared to m-HAP. ALP is an early marker of osteoblast differentiation, and it obviously increased with culture time in each group, especially the np20 group, revealing that additional transformation occurred from MSCs to osteoblasts (Fig. 4d). Pluripotency markers, NANOG, OCT3/4, and SOX2 imply the capacity for differentiation [32]. As shown in Fig. 4e–g, the decreased expression in the genes of the HANP groups implied that most of the hWJ-MSCs in HAP groups had transformed into osteoblasts.
Our data demonstrated that the endocytosis of HANPs by HUVECs was associated with an improved osteogenic differentiation of hWJ-MSCs. However, the cause of this outcome is currently unclear. In terms of the paracrine function of HUVECs, we focused on soluble differentiation-inducing proteins in the supernatant of activated HUVECs. HIF-1α signaling is essential in coupling ossification and angiogenesis during bone regeneration [33, 34]. Heikal et al. reported that injured ECs secrete more HIF-1α even under normoxia conditions [35]. It has also been shown that exposure to HANPs inhibits the angiogenic ability of HUVECs [9]. Thus, we measured the concentrations of HIF-1α in the CM, and the results showed that the HIF-1α content increased in the HANP treatment groups compared to the m-HAP and control groups (Fig. 6a). To identify the role of HIF-1α in the osteogenic differentiation of hWJ-MSCs, we used 2-MeOE2, which is a specific HIF-1α inhibitor, was used. The decreased concentration of HIF-1α paralleled the impaired mineralized matrix deposition and ALP activity in these hWJ-MSCs, indicating that HANPs can promote the HIF-1α production of HUVECs to facilitate the osteogenesis of hWJ-MSCs (Fig. 7).
To properly apply HANPs for use in bone tissue engineering, it is necessary to gain further insights into the mechanisms by which HANPs promote the osteogenic differentiation of hWJ-MSCs mediated by HUVECs. The ERK1/2 pathway is downstream of HIF-1α [36] and is fundamental to the differentiation of MSCs [37]. In this work, the concentrations of HIF-1α in the CM coincide well with the p-ERK1/2 levels in the hWJ-MSCs (Fig. 6b, c). When 2-MeOE2 was applied, the p-ERK1/2 expression in the hWJ-MSCs failed to be activated, indicating that HIF-1α functioned upstream of ERK1/2 signaling. To directly address the role of ERK1/2 signaling in the osteogenic differentiation of hWJ-MSCs, PD98059, a specific MEK inhibitor, was used. The suppression of ERK1/2 signaling resulted in the lowest osteogenic differentiation of hWJ-MSCs. One possible reason for this occurrence is that the ERK1/2 pathway plays a key role in both HIF-1α signaling and in the apoptosis and proliferation signaling pathways, which could be responsible for the observed changes in osteogenic differentiation in these cells [38, 39]. Additionally, this could also be related to the presence of vascular endothelial growth factor (VEGF). VEGF is one of the downstream effectors of HIF-1α signaling [33], and it can also promote the osteogenic differentiation of MSCs via activation of the ERK1/2 pathway [37]. Our previous study found that np20 induced the production of VEGF in HUVECs [9]; therefore, it is possible that the suppression of the ERK1/2 pathway may result in inhibition of VEGF, which would lead to the decreased osteogenic differentiation of hWJ-MSCs. According to the available experimental results, we can summarize as follows. HANPs are able to more optimally process better direct [5] and indirect osteoinductive effects than m-HAPs. Compared to autogenous bone grafts and bone allografts, there is an extensive source of HANP and without secondary damage and potential immunogenicity. However, compared to m-HAPs, HANPs can suppress the angiogenic ability of HUVECs [9] and exhibit slight cytotoxicity in both a time- and dose-dependent fashion.
Recently, growing evidence has demonstrated the importance of HIF-1α in the bone regeneration. However, few studies have been able to quantitatively predict the MSC differentiation rate under specific initial conditions, such as the HIF-1α concentration. Taking cell proliferation, apoptosis, and osteogenic differentiation into account, we present a mathematical model that combines a two-stage cell lineage with HIF-1α that is highly correlated with our experimental data. By fitting the differentiation rate of hWJ-MSCs in 0–4000 pg/mL HIF-1α, we acquired the equations for describing the differentiation rate, HIF-1α concentration, and time. As shown in Fig. 8d, this model can depict the cell number map under different HIF-1α concentrations, so that it is possible to explore the intrinsic dynamics of the two-stage system [40]. Additionally, this model mathematically validates the effect of HIF-1α on the osteogenic differentiation of hWJ-MSCs. Moreover, based on a multi-stage cell-lineage model and logistic model, our model is sufficiently stable to enable long-term predictions without falling into the trap of population unlimited explosion [41].
By using the existing experimental data, both the cell number and differentiation rate can be predicted with a defined initial cell seeding density and HIF-1α concentration. As such, the optimum incubation time is also obtained. Consequently, we can predict the optimum concentration of HIF-1α and determine the most optimal time for osteogenesis, which is important for efficient tissue engineering. A two-stage cell-lineage model is applicable for predicting the proliferation and differentiation of stem cells, which have two cell lineages. On this basis, the model founded on the initial conditions and existing experimental data can be established to identify the optimum culture conditions in vitro, which will assist in optimizing bone repair in vivo.
结论
In this study, we explored the specific biological effects of HANPs on hWJ-MSCs mediated by HUVECs. Compared to m-HAPs, both np20 and np80 showed slight cytotoxicity in both a time- and dose-dependent fashion. Importantly, the size of the HANPs appeared to have no significant impact on this cytotoxicity. Our data also showed that HANPs, especially np20, were capable of facilitating HUVECs to secrete increased levels of HIF-1α, which directly correlated with the enhanced osteogenic differentiation of hWJ-MSCs via the activation of the ERK1/2 pathway (Fig. 9). More remarkably, the results from the two-stage cell-lineage model suggested that HIF-1α exerted a dose-dependent stimulatory effect on the osteogenic differentiation rate of hWJ-MSCs. Additionally, the optimum concentration of HIF-1α and incubation time were estimated based on the initial conditions using an in vitro model, which could be invaluable in the future for tissue engineering applications. Collectively, these observations provide evidence that HANPs may improve bone regeneration by modulating cell–cell interactions.
A schematic illustration of the possible mechanisms
数据和材料的可用性
本研究中使用和分析的数据集可向相应作者索取合理要求。
缩写
- HA:
-
Hydroxyapatite
- HANPs:
-
HA nanoparticles
- hWJ-MSCs:
-
Human umbilical cord Wharton’s jelly-derived mesenchymal stem cells
- HUVECs:
-
人脐静脉内皮细胞
- m-HAP:
-
Micro-sized HAP particles
- PBS:
-
磷酸盐缓冲盐水
- ECM:
-
EC medium
- 2-MeOE2:
-
2-Methoxyestradiol
- ELISA:
-
酶联免疫吸附试验
- RUNX-2:
-
Runt-related transcription factor 2
- Col I:
-
Type I collagen
- OCN:
-
Osteocalcin
- ALP:
-
碱性磷酸酶
- SOX 2:
-
SRY-related HMG-box 2
- ERK:
-
Extracellular signal-related kinases
- VEGF:
-
Vascular endothelial growth factor
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