Wang Shangping, Oldenhof Harriëtte, Goecke Tobias, Ramm Robert, Harder Michael, Haverich Axel, Hilfiker Andres, Wolkers Willem Frederik
1 Institute of Multiphase Processes, Leibniz Universität Hannover , Hannover, Germany .
2 Clinic for Horses-Unit for Reproductive Medicine, University of Veterinary Medicine Hannover , Hannover, Germany .
Tissue Eng Part C Methods. 2015 Sep;21(9):922-31. doi: 10.1089/ten.TEC.2014.0681. Epub 2015 May 4.
Decellularized heart valves can be used as starter matrix implants for heart valve replacement therapies in terms of guided tissue regeneration. Decellularized matrices ideally need to be long-term storable to assure off-the-shelf availability. Freeze-drying is an attractive preservation method, allowing storage at room temperature in a dried state. However, the two inherent processing steps, freezing and drying, can cause severe damage to extracellular matrix (ECM) proteins and the overall tissue histoarchitecture and thus impair biomechanical characteristics of resulting matrices. Freeze-drying therefore requires a lyoprotective agent that stabilizes endogenous structural proteins during both substeps and that forms a protective glassy state at room temperature. To estimate incubation times needed to infiltrate decellularized heart valves with the lyoprotectant sucrose, temperature-dependent diffusion studies were done using Fourier transform infrared spectroscopy. Glycerol, a cryoprotective agent, was studied for comparison. Diffusion of both protectants was found to exhibit Arrhenius behavior. The activation energies of sucrose and glycerol diffusion were found to be 15.9 and 37.7 kJ·mol(-1), respectively. It was estimated that 4 h of incubation at 37°C is sufficient to infiltrate heart valves with sucrose before freeze-drying. Application of a 5% sucrose solution was shown to stabilize acellular valve scaffolds during freeze-drying. Such freeze-dried tissues, however, displayed pores, which were attributed to ice crystal damage, whereas vacuum-dried scaffolds in comparison revealed no pores after drying and rehydration. Exposure to a hygroscopic sucrose solution (80%) before freeze-drying was shown to be an effective method to diminish pore formation in freeze-dried ECMs: matrix structures closely resembled those of control samples that were not freeze-dried. Heart valve matrices were shown to be in a glassy state after drying, suggesting that they can be stored at room temperature.
就引导组织再生而言，脱细胞心脏瓣膜可作为心脏瓣膜置换治疗的起始基质植入物。理想情况下，脱细胞基质需要长期储存，以确保现货供应。冷冻干燥是一种有吸引力的保存方法，可使其在室温下以干燥状态储存。然而，冷冻和干燥这两个固有的处理步骤会对细胞外基质（ECM）蛋白和整体组织结构造成严重破坏，从而损害所得基质的生物力学特性。因此，冷冻干燥需要一种冻干保护剂，该保护剂在两个子步骤中均能稳定内源性结构蛋白，并在室温下形成保护性玻璃态。为了估计用冻干保护剂蔗糖渗透脱细胞心脏瓣膜所需的孵育时间，使用傅里叶变换红外光谱进行了温度依赖性扩散研究。作为对照，研究了冷冻保护剂甘油。发现两种保护剂的扩散均表现出阿伦尼乌斯行为。发现蔗糖和甘油扩散的活化能分别为15.9和37.7 kJ·mol(-1)。据估计，在37°C下孵育4小时足以在用蔗糖渗透心脏瓣膜后进行冷冻干燥。结果表明，应用5%的蔗糖溶液可在冷冻干燥过程中稳定无细胞瓣膜支架。然而，这种冷冻干燥的组织显示出孔隙，这归因于冰晶损伤，而相比之下，真空干燥的支架在干燥和再水化后没有显示出孔隙。结果表明，在冷冻干燥前暴露于吸湿蔗糖溶液（80%）是减少冷冻干燥ECM中孔隙形成的有效方法：基质结构与未冷冻干燥的对照样品非常相似。心脏瓣膜基质在干燥后呈玻璃态，这表明它们可以在室温下储存。
