■软骨组织工程面临与使用支架和有限种子细胞有关的挑战。这项研究旨在提出一种具有成本效益和直接的方法,使用肋软骨细胞(CC)作为替代细胞来源来克服这些挑战,消除了对特殊培养设备或脚手架的需要。
■在有和没有抗坏血酸处理的情况下以高细胞密度培养CCs,作为实验组和对照组,分别。评估活力和组织工程化构建体(TEC)形成直至第14天。TEC样品的切片用于组织学染色以评估细胞外基质内糖胺聚糖和不同类型的胶原蛋白的分泌。进行mRNA测序和qPCR以检查与软骨细胞中软骨基质分泌相关的基因表达。通过将来自不同组的TEC植入缺损部位进行体内实验,12周后进行组织学染色和评分以评估软骨再生的程度。苏木精-伊红(HE),Safranin-O-FastGreen,和Masson的三色染色用于检查体内修复组织中软骨相关基质成分的含量。I型和II型胶原的免疫组织化学染色,以及aggrecan,进行评估这些特异性标志物的存在和分布。此外,X型胶原的免疫组织化学染色用于观察修复组织中的任何肥大变化。
■软骨细胞的活力在整个培养期间保持较高,TECs显示出适用于外科手术的富集的细胞外基质。体外研究表明,两组TEC中的糖胺聚糖和II型胶原蛋白的产生,而用抗坏血酸处理的TEC基质显示出更高的丰度。mRNA测序和qPCR结果表明,与软骨基质分泌相关的基因如Sox9、Col2和Acan在肋软骨细胞中被抗坏血酸上调。尽管根据qPCR和RNA-seq结果,Asc-2P的添加导致COL10表达增加,两组TECs的免疫荧光染色结果具有相似的分布和荧光强度。体内实验表明,两组TEC均可粘附于缺损部位,并保持透明软骨形态直至12周。与其他组相比,用抗坏血酸处理的TEC显示出优异的软骨再生,这通过ICRS和O'Driscoll评分显着提高以及模仿天然软骨的Safranin-O和胶原蛋白染色更强来证明。此外,CollganX的免疫组织化学染色结果表明,12周后,抗坏血酸处理的TEC在移植到缺损部位后没有表现出进一步的肥大,但保持了与未经处理的TECs相似的表达谱,而略高于假手术组。
■这些结果表明,CC衍生的无支架TEC为关节软骨再生提供了一种有希望的方法。抗坏血酸治疗通过促进软骨基质的产生来增强结果。这项研究为软骨组织工程领域提供了有价值的见解和潜在的进步。
■软骨组织工程是一个具有巨大临床潜力的研究领域。本文介绍的方法提供了一种经济高效且简单的解决方案,这可以最大限度地减少细胞培养和支架制造的复杂性。这种简化可以提供几个翻译优势,例如易用性,快速的可扩展性,更低的成本,以及患者特异性临床翻译的潜力。使用肋软骨细胞,很容易获得,和无脚手架方法,不需要专门的设备或膜,在临床环境中可能特别有利,允许软骨的原位再生。
UNASSIGNED: Cartilage tissue engineering faces challenges related to the use of scaffolds and limited seed cells. This study aims to propose a cost-effective and straightforward approach using costal chondrocytes (CCs) as an alternative cell source to overcome these challenges, eliminating the need for special culture equipment or scaffolds.
UNASSIGNED: CCs were cultured at a high cell density with and without ascorbic acid treatment, serving as the experimental and control groups, respectively. Viability and tissue-engineered constructs (TEC) formation were evaluated until day 14. Slices of TEC samples were used for histological staining to evaluate the secretion of glycosaminoglycans and different types of collagen proteins within the extracellular matrix. mRNA sequencing and qPCR were performed to examine gene expression related to cartilage matrix secretion in the chondrocytes. In vivo experiments were conducted by implanting TECs from different groups into the defect site, followed by sample collection after 12 weeks for histological staining and scoring to evaluate the extent of cartilage regeneration. Hematoxylin-eosin (HE), Safranin-O-Fast Green, and Masson\'s trichrome stainings were used to examine the content of cartilage-related matrix components in the in vivo repair tissue. Immunohistochemical staining for type I and type II collagen, as well as aggrecan, was performed to assess the presence and distribution of these specific markers. Additionally, immunohistochemical staining for type X collagen was used to observe any hypertrophic changes in the repaired tissue.
UNASSIGNED: Viability of the chondrocytes remained high throughout the culture period, and the TECs displayed an enriched extracellular matrix suitable for surgical procedures. In vitro study revealed glycosaminoglycan and type II collagen production in both groups of TEC, while the TEC matrix treated with ascorbic acid displayed greater abundance. The results of mRNA sequencing and qPCR showed that genes related to cartilage matrix secretion such as Sox9, Col2, and Acan were upregulated by ascorbic acid in costal chondrocytes. Although the addition of Asc-2P led to an increase in COL10 expression according to qPCR and RNA-seq results, the immunofluorescence staining results of the two groups of TECs exhibited similar distribution and fluorescence intensity. In vivo experiments showed that both groups of TEC could adhere to the defect sites and kept hyaline cartilage morphology until 12 weeks. TEC treated with ascorbic acid showed superior cartilage regeneration as evidenced by significantly higher ICRS and O\'Driscoll scores and stronger Safranin-O and collagen staining mimicking native cartilage when compared to other groups. In addition, the immunohistochemical staining results of Collgan X indicated that, after 12 weeks, the ascorbic acid-treated TEC did not exhibit further hypertrophy upon transplantation into the defect site, but maintained an expression profile similar to untreated TECs, while slightly higher than the sham-operated group.
UNASSIGNED: These results suggest that CC-derived scaffold-free TEC presents a promising method for articular cartilage regeneration. Ascorbic acid treatment enhances outcomes by promoting cartilage matrix production. This study provides valuable insights and potential advancements in the field of cartilage tissue engineering.
UNASSIGNED: Cartilage tissue engineering is an area of research with immense clinical potential. The approach presented in this article offers a cost-effective and straightforward solution, which can minimize the complexity of cell culture and scaffold fabrication. This simplification could offer several translational advantages, such as ease of use, rapid scalability, lower costs, and the potential for patient-specific clinical translation. The use of costal chondrocytes, which are easily obtainable, and the scaffold-free approach, which does not require specialized equipment or membranes, could be particularly advantageous in clinical settings, allowing for in situ regeneration of cartilage.