Department of Biological Sciences and Bioengineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh, India.
Centre for Environmental Sciences and Engineering, Indian Institute of Technology Kanpur, Kanpur, Uttar Pradesh, India.
Biotechnol Bioeng. 2022 Feb;119(2):605-625. doi: 10.1002/bit.27982. Epub 2021 Nov 6.
Treatment of articular cartilage injuries especially osteochondral tissue requires intervention of bioengineered scaffold. In this study, we investigated the potential of the tissue-engineered cryogel scaffold fabricated using cryogelation technology. Two types of cryogels viz. chitosan-gelatin-chondroitin sulfate (CGC) for articular cartilage and nano-hydroxyapatite-gelatin (HG) for subchondral bone were fabricated. Further, novel bilayer cryogel designed using single process fabrication of two layers (CGC as top layer and HG as the lower layer) was designed to mimic osteochondral unit. CGC cryogel was tested for their biocompatibility using the enzymatically isolated chondrcoytes from goat articular cartilage while HG cryogel was tested using pre-osteoblast cell line. Extracellular vesicles, specifically exosomes were isolated from the spent media of chondrocytes to validate their effect over cell proliferation and migration which are required for defect healing and infiltration respectively. These isolated exosomes were characterized and analyzed for confirming their size distribution profile and visualized morphologically using advanced microscopy techniques. For cartilage part, CGC cryogels were examined as delivery system for delivering exosomes at defect site, where 80% of release was observed in 72 h. Release of 18.7 µg chondroitin sulfate/mg cryogel was obtained in a period of one week from CGC cryogel (termed cryogel extract) which has chondroprotective effect. Further, effect of exosome concentration (10 and 20 µg/ml), CGC extract and combination of exosome and CGC extract (Exo-Ex) were assessed over the chondrocytes. In addition, in vitro scratch wound assay was performed to analyse the migration capacity over the micro-injury when treated with exosomes, cryogel extract and Exo-Ex. The overall results thus answer key questions of therapeutic potential of chondrocyte exosomes, cryogel extract in addition to potential of CGC and HG cryogel for osteochondral repair.
治疗关节软骨损伤，特别是骨软骨组织，需要生物工程支架的介入。在这项研究中，我们研究了使用冷冻凝胶技术制造的组织工程冷冻凝胶支架的潜力。两种类型的冷冻凝胶，即壳聚糖-明胶-硫酸软骨素（CGC）用于关节软骨和纳米羟基磷灰石-明胶（HG）用于软骨下骨，被制备出来。此外，使用两层（CGC 作为上层和 HG 作为下层）的单一过程制造设计了新型双层冷冻凝胶，以模拟骨软骨单元。CGC 冷冻凝胶通过酶分离的山羊关节软骨中的软骨细胞进行了生物相容性测试，而 HG 冷冻凝胶通过成骨前体细胞系进行了测试。从软骨细胞的耗竭培养基中分离出细胞外囊泡，特别是外泌体，以验证它们对细胞增殖和迁移的影响，这分别是缺陷愈合和浸润所必需的。这些分离的外泌体进行了表征和分析，以确认其大小分布特征，并使用先进的显微镜技术进行形态学可视化。对于软骨部分，CGC 冷冻凝胶被用作在缺陷部位递送外泌体的递送系统，其中在 72 小时内观察到 80%的释放。在一周的时间内，从 CGC 冷冻凝胶（称为冷冻凝胶提取物）中获得了 18.7μg 硫酸软骨素/mg 冷冻凝胶的释放量，该提取物具有软骨保护作用。此外，还评估了外泌体浓度（10 和 20μg/ml）、CGC 提取物和外泌体与 CGC 提取物的组合（Exo-Ex）对软骨细胞的影响。此外，还进行了体外划痕伤口试验，以分析在用外泌体、冷冻凝胶提取物和 Exo-Ex 处理时对微损伤的迁移能力。因此，总体结果回答了关于软骨细胞外泌体、冷冻凝胶提取物以及 CGC 和 HG 冷冻凝胶在骨软骨修复中的治疗潜力的关键问题。
