Treatment of critical-size osteochondral (OC) injuries at load-bearing sites has remained a major clinical challenge in orthopedic surgery. This is due to the anisotropic characteristics of OC tissue and the stratified structure of the cartilage. Here, we developed a multilayered OC scaffold by employing cryogelation technology. Gelatin, chitosan, and chondroitin sulfate were utilized for designing three distinct, 2425 ± 120 μm thick layers of cartilage having different alignments, while nanohydroxyapatite and gelatin were used for the subchondral bone layer. Exosomes derived from articular chondrocytes in the range of 60–110 nm were used to promote chondrogenesis. The biocompatibility and cartilage formation potential of the scaffold and exosomes were initially evaluated in rat OC defects. The application of exosome-loaded scaffolds was then investigated in a critical-size OC injury (8 × 10 mm) created in the goat knee. Artificial synovial fluid was designed and utilized as a carrier for exosomes for a booster dose administered as an intra-articular injection. X-ray imaging and micro-CT analysis revealed that the treatment resulted in improved subchondral bone regeneration. The defect region exhibited healthy hyaline cartilage formation, as detected by MRI imaging. Moreover, histological examination revealed that the treatment group showed augmented cell proliferation, matrix deposition, secretion of proteoglycans, and the formation of stratified hyaline cartilage over a long-term (6 and 12 months), whereas the control group demonstrated the formation of fibrocartilage. Treatment-induced upregulation of collagen II, aggrecan, and SOX 9 genes (∼10 fold) further provided evidence that the cartilage phenotype was well preserved. Hence, the proposed treatment has significant translational potential for treating adverse OC clinical injuries.

中文翻译：

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富含外泌体的多层冷冻凝胶可在山羊骨软骨损伤中再生和维持软骨结构和表型


承重部位临界尺寸骨软骨 （OC） 损伤的治疗仍然是骨科手术中的主要临床挑战。这是由于 OC 组织的各向异性特性和软骨的分层结构。在这里，我们采用冷冻凝胶技术开发了一种多层 OC 支架。明胶、壳聚糖和硫酸软骨素用于设计三个不同的、2425 ± 120 μm 厚、排列不同的软骨层，而纳米羟基磷灰石和明胶用于软骨下骨层。源自 60-110 nm 范围内的关节软骨细胞的外泌体用于促进软骨生成。支架和外泌体的生物相容性和软骨形成潜力在大鼠 OC 缺陷中进行了初步评估。然后在山羊膝关节造成的危重尺寸 OC 损伤 （8 × 10 mm） 中研究了负载外泌体支架的应用。人工滑液被设计并用作外泌体的载体，用于作为关节内注射的加强剂量。X 射线成像和显微 CT 分析显示，该治疗改善了软骨下骨再生。通过 MRI 成像检测到，缺损区域表现出健康的透明软骨形成。此外，组织学检查显示，治疗组在长期 （6 个月和 12 个月） 内显示细胞增殖增加、基质沉积、蛋白聚糖分泌和分层透明软骨的形成，而对照组显示纤维软骨的形成。治疗诱导的胶原 II 、聚集蛋白聚糖和 SOX 9 基因上调 （∼10 倍） 进一步提供了软骨表型保存完好的证据。 因此，所提出的治疗方法在治疗不良 OC 临床损伤方面具有重要的转化潜力。