An improved method for processing chondroprogenitor pellets following chondrogenic differentiation for histology and immunohistochemical staining using agarose

Q4 Medicine
Soosai Manickam Amirtham , Upasana Kachroo , Deepak Vinod Francis , Kawin Padmaja , Elizabeth Vinod
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引用次数: 0

Abstract

Purpose

In-vitro models of cartilage regeneration based on pellet cultures have been widely used to evaluate chondrogenic potential of the cell of interest and predict probable in-vivo behavior. However, pellet processing is a major challenge during handling (due to small size and possible damage to structural contour following sectioning and staining). The present study aimed to utilize human articular cartilage derived chondroprogenitors to assess if agarose-encapsulation of pellets prior to paraffin processing enable easier handling without affecting tissue morphology, glycosaminoglycan staining and immunohistochemical analysis of Collagen type II protein.

Methods

Passage 3 chondroprogenitors (n = 3) were evaluated for MSC markers using flow cytometry and subjected to chondrogenic differentiation as pellets cultures. Post-differentiation, the pellets were subjected to either: a) paraffin embedding, b) agarose encapsulation followed by paraffin embedding or c) agarose encapsulation followed by cryosectioning. All sections were subjected to histological staining for glycosaminoglycan uptake: Alcian blue, Safranin O (Bern score) and Toluidine blue with immunohistochemical processing for collagen type II protein deposition.

Results

With respect to staining and structural integrity, comparable uptake was seen in both paraffin sections and agarose embedded sections while the latter exhibited notably uniform pellets with distinct marginal demarcation. Although plain paraffin and agarose encapsulated sections demonstrated equivalent staining as represented by comparable Bern scores, glycosaminoglycan uptake, and Collagen type II deposition, cryosections exhibited significantly poor staining properties.

Conclusion

Agarose encapsulation of differentiated pellets prior to routine paraffin embedding, eases handling difficulties whilst maintaining structural integrity with optimal staining outcomes.

用琼脂糖对软骨分化后的软骨祖细胞微球进行组织学和免疫组织化学染色的改进方法
目的基于颗粒培养的体外软骨再生模型已被广泛用于评估目标细胞的软骨形成潜力和预测可能的体内行为。然而,颗粒处理是处理过程中的主要挑战(由于体积小,并且在切片和染色后可能损坏结构轮廓)。本研究旨在利用人关节软骨衍生的软骨祖细胞来评估在石蜡处理之前,琼脂糖包封颗粒是否更容易处理,而不影响组织形态、糖胺聚糖染色和II型胶原蛋白的免疫组织化学分析。方法采用流式细胞术对3期软骨祖细胞(n = 3)进行骨髓间充质干细胞标记物鉴定,并进行软骨细胞分化培养。分化后,分别进行a)石蜡包埋,b)琼脂糖包埋后石蜡包埋或c)琼脂糖包埋后冷冻切片。所有切片均进行糖胺聚糖摄取的组织学染色:阿利新蓝、红花素O (Bern评分)和甲苯胺蓝,并进行免疫组织化学处理,用于II型胶原蛋白沉积。结果在染色和结构完整性方面,石蜡切片和琼脂糖包埋切片的摄取相当,琼脂糖包埋切片的颗粒明显均匀,边缘划界明显。尽管普通石蜡和琼脂糖包封切片显示出相同的染色,如伯尔尼评分、糖胺聚糖摄取和II型胶原沉积,但冷冻切片显示出明显较差的染色特性。结论在常规石蜡包埋之前对分化微球进行琼脂糖包埋,可减轻处理困难,同时保持结构完整性,获得最佳染色效果。
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来源期刊
Journal of Arthroscopy and Joint Surgery
Journal of Arthroscopy and Joint Surgery Medicine-Orthopedics and Sports Medicine
CiteScore
0.60
自引率
0.00%
发文量
1
期刊介绍: Journal of Arthroscopy and Joint Surgery (JAJS) is committed to bring forth scientific manuscripts in the form of original research articles, current concept reviews, meta-analyses, case reports and letters to the editor. The focus of the Journal is to present wide-ranging, multi-disciplinary perspectives on the problems of the joints that are amenable with Arthroscopy and Arthroplasty. Though Arthroscopy and Arthroplasty entail surgical procedures, the Journal shall not restrict itself to these purely surgical procedures and will also encompass pharmacological, rehabilitative and physical measures that can prevent or postpone the execution of a surgical procedure. The Journal will also publish scientific research related to tissues other than joints that would ultimately have an effect on the joint function.
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