Bilayered chitosan-based scaffolds for osteochondral tissue engineering: Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor
Bilayered chitosan-based scaffolds for osteochondral tissue engineering: Influence of hydroxyapatite on in vitro cytotoxicity and dynamic bioactivity studies in a specific double-chamber bioreactor
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DOI：
10.1016/j.actbio.2008.09.017
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发表时间：
2009-02-01
影响因子：
9.7
通讯作者：
Reis, Rul L.
中科院分区：
文献类型：
--
作者：
Malafaya, Patricia B.;Reis, Rul L.
由于需要结合骨和软骨组织工程原理，骨软骨组织工程提出了当前的研究挑战。在本研究中，基于聚合物和复合支架的优化开发了基于双层壳聚糖的支架。提出了一种颗粒聚集方法，以实现该应用所需的改进的一体化骨软骨界面，因为任何不连续性都可能导致长期的设备故障。通过 NITS 测定，使用 L929 成纤维细胞系在不同条件下评估细胞毒性。令人惊讶的是，在使用未烧结羟基磷灰石的复合支架中，在体外观察到细胞毒性。这项工作报告了为克服和解释这种行为而进行的调查。这表明二价阳离子的摄取可能诱导细胞毒性行为。因此使用了烧结羟基磷灰石，并且与对照相比没有显示出细胞毒性。进行微计算机断层扫描（micro-CT）以准确量化孔隙率、互连性、陶瓷含量、颗粒和孔径。结果表明，所开发的支架是高度互连的，并且呈现出理想的孔径范围，在形态上适合所提出的应用。动态力学分析（DMA）表明，即使在动态压缩下，支架在潮湿状态下也具有机械稳定性。对于聚合物、复合材料和双层支架，在1Hz频率下获得的弹性模量分别为4.21+/-1.04、7.98+/-1.77和6.26+/-1.04MPa。使用模拟体液（SBF）和模拟滑液（SSF）进行生物活性研究，以确保软骨形成部分的聚合物成分不会矿化，这通过扫描电子显微镜（SEM）、电感耦合等离子体发射光谱（ICP）和能量色散光谱（EDS）在不同浸泡时间下进行证实。该测定还在动态条件下进行，为此目的，使用专门设计的双室生物反应器，旨在未来的骨软骨应用。结论是，通过颗粒聚集产生的基于壳聚糖的双层支架克服了分别为机械稳定的软骨形成和成骨成分设计的聚合物和复合材料部件分层的任何风险。此外，所提出的双层支架可以作为骨软骨组织工程应用的替代、生物相容性和安全的可生物降解支架。 (c) 2008 Acta Materialia Inc. 由 Elsevier Ltd 出版。保留所有权利。
Osteochondral tissue engineering presents a current research challenge due to the necessity of combining both bone and cartilage tissue engineering principles. In the present study, bilayered chitosan-based scaffolds are developed based on the optimization of both polymeric and composite scaffolds. A particle aggregation methodology is proposed in order to achieve an improved integrative bone-cartilage interface needed for this application, since any discontinuity is likely to cause long-term device failure. Cytotoxicity was evaluated by the NITS assay with the L929 fibroblast cell line for different conditions. Surprisingly, in composite scaffolds using unsintered hydroxyapatite, cytotoxicity was observed in vitro. This work reports the investigation that was conducted to overcome and explain this behaviour. It is suggest that the uptake of divalent cations may induce the cytotoxic behaviour. Sintered hydroxyapatite was consequently used and showed no cytotoxicity when compared to the controls. Microcomputed tomography (micro-CT) was carried out to accurately quantify porosity, interconnectivity, ceramic content, particle and pore sizes. The results showed that the developed scaffolds are highly interconnected and present the ideal pore size range to be morphometrically suitable for the proposed applications. Dynamical mechanical analysis (DMA) demonstrated that the scaffolds are mechanically stable in the wet state even under dynamic compression. The obtained elastic modulus was, respectively, 4.21 +/- 1.04, 7.98 +/- 1.77 and 6.26 +/- 1.04 MPa at I Hz frequency for polymeric, composite and bilayered scaffolds. Bioactivity studies using both a simulated body fluid (SBF) and a simulated synovial fluid (SSF) were conducted in order to assure that the polymeric component for chondrogenic part would not mineralize, as confirmed by scanning electron microscopy (SEM), inductively coupled plasma-optical emission spectroscopy (ICP) and energy-dispersive spectroscopy (EDS) for different immersion periods. The assays were carried out also under dynamic conditions using, for this purpose, a specifically designed double-chamber bioreactor, aiming at a future osteochondral application. It was concluded that chitosan-based bilayered scaffolds produced by particle aggregation overcome any risk of delamination of both polymeric and composite parts designed, respectively, for chondrogenic and osteogenic components that are mechanically stable. Moreover, the proposed bilayered scaffolds could serve as alternative, biocompatible and safe biodegradable scaffolds for osteochondral tissue engineering applications. (c) 2008 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.