Johnson Noel Naveen, Amirtham Soosai Manickam, Sandya Rani B, Sathishkumar Solomon, Rebekah Grace, Vinod Elizabeth
Department of Physiology, Christian Medical College, Vellore, India.
Centre for Stem Cell Research, (A Unit of InStem, Bengaluru), Christian Medical College, Vellore, India.
J Orthop. 2022 Mar 23;31:45-51. doi: 10.1016/j.jor.2022.03.007. eCollection 2022 May-Jun.
Cartilage-derived chondroprogenitors have been reported to possess the biological potential for cartilage repair. However, its inherent chondrogenic potential in pellet culture needs evaluation. In-vitro cartilage regeneration models based on pellet cultures have been employed to evaluate the chondrogenic potential of stem cells. Evaluation of the degree of differentiation routinely involves paraffin embedding, sectioning, and immunohistochemical staining of the pellet. However, since chondrogenic differentiation is commonly non-uniform, processing random sections could lead to inaccurate conclusions. The study aimed at assessing the inherent lineage bias of chondroprogenitors with and without chondrogenic induction, using a novel whole pellet processing technique.
Human chondroprogenitors (n=3) were evaluated for MSC markers and processed in pellet cultures either with stromal medium (uninduced) or chondrogenic differentiation medium (induced) for 28 days. The whole pellets and the conventional paraffin-embedded sectioned pellets were subjected to Collagen type II immunostaining and assessed using confocal laser microscopy. The staining intensities of the whole pellet were compared to the paraffin sections and revalidated using qRT-PCR for COL2A1 expression.
Uninduced and induced pellets displayed Collagen type II in all the layers with comparable fluorescence intensities. COL2A1 expression in both pellets was comparable to confocal results. The study demonstrated that uninduced chondroprogenitors in pellet culture possess promising inherent chondrogenic potential. Confocal imaging of whole pellets displayed different degrees of chondrogenic differentiation in the entire pellet, thus its probable in-vivo behavior.
The novel approach presented in this study could serve as an efficient in-vitro alternative for understanding translational application for cartilage repair.
据报道，软骨来源的软骨祖细胞具有软骨修复的生物学潜力。然而，其在微团培养中的内在软骨生成潜力需要评估。基于微团培养的体外软骨再生模型已被用于评估干细胞的软骨生成潜力。常规评估分化程度涉及对微团进行石蜡包埋、切片和免疫组织化学染色。然而，由于软骨生成分化通常不均匀，处理随机切片可能会导致不准确的结论。本研究旨在使用一种新型的全微团处理技术，评估有无软骨生成诱导的软骨祖细胞的内在谱系偏向。
对人软骨祖细胞（n = 3）进行间充质干细胞标志物评估，并在微团培养中分别用基质培养基（未诱导）或软骨生成分化培养基（诱导）处理28天。对整个微团和常规石蜡包埋切片的微团进行II型胶原免疫染色，并使用共聚焦激光显微镜进行评估。将整个微团的染色强度与石蜡切片进行比较，并使用qRT-PCR检测COL2A1表达进行验证。
未诱导和诱导的微团在所有层均显示II型胶原，荧光强度相当。两个微团中COL2A1的表达与共聚焦结果相当。该研究表明，微团培养中未诱导的软骨祖细胞具有良好的内在软骨生成潜力。整个微团的共聚焦成像显示整个微团存在不同程度的软骨生成分化，从而反映其可能的体内行为。
本研究中提出的新方法可作为一种有效的体外替代方法，用于理解软骨修复的转化应用。
