Karimi Zeinab, Ghorbani Masoud, Hashemibeni Batool, Bahramian Hamid
Student of Medicine, School of Medicine and Student Research Committee, Isfahan University of Medical Sciences, Isfahan, Tehran, Iran.
Applied Biotechnology Researches Center, Pajooheshgah, Baqiatallah University of Medical Sciences, Tehran, Iran; Department of Tissue Engineering and Applied Cell Sciences, School of Advanced Technologies in Medicine, Tehran University of Medical Sciences, Tehran, Iran.
Adv Biomed Res. 2015 Nov 30;4:251. doi: 10.4103/2277-9175.170676. eCollection 2015.
Low back pain is one of the most significant musculoskeletal diseases of our time. Intervertebral disk herniation and central degeneration of the disk are two major reasons for low back pain, which occur because of structural impairment of the disk. The reduction of cell count and extracellular matrix, especially in the nucleus pulposus, causes disk degeneration. Different scaffolds have been used for tissue repairing and regeneration of the intervertebral disk in tissue engineering. Various methods are used for fabrication of the porosity scaffolds in tissue engineering. The freeze drying method has disadvantages such as: It is time consuming, needs high energy, and so on. The freeze-gelation method can save a great deal of time and energy, and large-sized porous scaffolds can be fabricated by this method. In this study, proliferation of the nucleus pulposus (NP) cells of the human intervertebral disk are compromised in the fabricated Chitosan-gelatin scaffolds by freeze drying and freeze gelation methods.
The cells were obtained from the nucleus pulposus by collagenase enzymatic hydrolysis. They were obtained from patients who were undergoing open surgery for discectomy in the Isfahan Alzahra Hospital. Chitosan was blended with gelatin. Chitosan polymer, solution after freezing at -80°C, was immersed in sodium hydroxide (NaOH) solution. The cellular suspension was transferred to each scaffold and cultured in plate for 14 days. Cell viability and proliferation were investigated by Trypan blue and MTT assays.
The MTT and Trypan blue assays demonstrated that cell viability and the mean of the cell number showed a significant difference between three and fourteen days, in both scaffolds. Accordingly, there was a significantly decrease in the fabricated chitosan-gelatin scaffold by the freeze-drying method.
The fabricated chitosan-gelatin scaffold by the freeze-gelation method prepared a better condition for proliferation of NP cells when compared with the fabricated chitosan-gelatin scaffold by the freeze drying method.
腰痛是当今最主要的肌肉骨骼疾病之一。椎间盘突出和椎间盘中央退变是腰痛的两个主要原因，这是由于椎间盘结构受损所致。细胞数量和细胞外基质的减少，尤其是髓核中的减少，会导致椎间盘退变。在组织工程中，不同的支架已被用于椎间盘的组织修复和再生。组织工程中用于制造多孔支架的方法有多种。冷冻干燥法存在耗时、耗能高等缺点。冷冻凝胶化法可节省大量时间和能源，且可通过该方法制造大尺寸多孔支架。在本研究中，通过冷冻干燥和冷冻凝胶化方法制备的壳聚糖 - 明胶支架中，人椎间盘髓核（NP）细胞的增殖受到影响。
通过胶原酶酶解从髓核中获取细胞。细胞取自伊斯法罕阿尔扎赫拉医院正在接受开放性椎间盘切除术的患者。将壳聚糖与明胶混合。壳聚糖聚合物在 -80°C 冷冻后的溶液浸入氢氧化钠（NaOH）溶液中。将细胞悬液转移至每个支架并在培养板中培养 14 天。通过台盼蓝和 MTT 试验研究细胞活力和增殖情况。
MTT 和台盼蓝试验表明，在两种支架中，细胞活力和细胞数量平均值在第 3 天和第 14 天之间均显示出显著差异。因此，冷冻干燥法制备的壳聚糖 - 明胶支架有显著下降。
与冷冻干燥法制备的壳聚糖 - 明胶支架相比，冷冻凝胶化法制备的壳聚糖 - 明胶支架为 NP 细胞增殖提供了更好的条件。
