Six weeks after implantation, rats were sacrificed, and discs were harvested for histological analysis to demonstrate proteoglycan distribution in the IVD. The disc with the adjacent vertebral body was fixed in 10% neutral buffered formalin for 1 week and decalcified in Rapid Cal Immuno (BBC Biochemical, Mount Vernon, WA, USA) for 2 weeks. Tissues were then processed for paraffin embedding and sectioning into coronal sections (10 µm) using a microtome (Leica, Wetzlar, Germany). The obtained sections were dewaxed, rehydrated, and stained with safranin-O (Sigma, St. Louis, MO, USA) to analyze the quantity and distribution of proteoglycan content. Finally, the sections were mounted using mounting media and scanned with an Olympus C-mount camera adapter (U-TVO.63XC, Tokyo, Japan).
Likewise, the histological scoring was done by using a comprehensive 16-point scale for the assessment of IVD based on safranin-O staining. The scoring was based on the NP morphology, NP cellularity, AF morphology, endplate morphology, and the boundary between the NP and AF, resulting in five subcategories. Briefly, the NP and AF morphology each include two degenerative features since they were ranked to be highly crucial in the study. As alterations in notochordal cell morphology are an important and easily notable feature in degenerative rat IVDs, so the NP cellularity category was also weighted twice with two features. Briefly, in terms of NP morphology, analyses on the NP shape and total NP area were performed. Similarly, cell number and cellular morphology were analyzed to measure NP cellularity. The border appearance was observed in order to differentiate no interruption, minimal interruption, and no distinction between NP and AF. Furthermore, to examine AF morphology in IVD, lamellar organization and any tears/fissures/disruptions in the AF region were identified. Finally, in terms of endplate, any appearance of disruptions, microfractures, osteophyte, or ossifications were examined to produce the histological scores. Thus, non-degenerative characteristics were represented as 0, mild degenerative characteristics as 1, and severe degenerative changes as 2. The sum of the separate scores ranged from 0 (normal) to 16 (most severe) The NP, AF, endplate, and boundary between the NP and AF of the IVD were scored and added together for a total IVD score [74 (link)]. Two independent observers who were completely blinded to the sample information conducted the histological analysis of all samples.
Furthermore, the obtained sections were dewaxed, rehydrated, and stained with H&E for analysis of the tissue morphology and proteoglycan distribution in IVDs. The disc NP-cell number and H&E positive area were measured using ImageJ software (https://imagej.nih.gov/ij/ (accessed on 10 November 2022)). Briefly, we created binary images at a fixed intensity level and measured the area between vertebral endplates [13 (link)].
Likewise, the histological scoring was done by using a comprehensive 16-point scale for the assessment of IVD based on safranin-O staining. The scoring was based on the NP morphology, NP cellularity, AF morphology, endplate morphology, and the boundary between the NP and AF, resulting in five subcategories. Briefly, the NP and AF morphology each include two degenerative features since they were ranked to be highly crucial in the study. As alterations in notochordal cell morphology are an important and easily notable feature in degenerative rat IVDs, so the NP cellularity category was also weighted twice with two features. Briefly, in terms of NP morphology, analyses on the NP shape and total NP area were performed. Similarly, cell number and cellular morphology were analyzed to measure NP cellularity. The border appearance was observed in order to differentiate no interruption, minimal interruption, and no distinction between NP and AF. Furthermore, to examine AF morphology in IVD, lamellar organization and any tears/fissures/disruptions in the AF region were identified. Finally, in terms of endplate, any appearance of disruptions, microfractures, osteophyte, or ossifications were examined to produce the histological scores. Thus, non-degenerative characteristics were represented as 0, mild degenerative characteristics as 1, and severe degenerative changes as 2. The sum of the separate scores ranged from 0 (normal) to 16 (most severe) The NP, AF, endplate, and boundary between the NP and AF of the IVD were scored and added together for a total IVD score [74 (link)]. Two independent observers who were completely blinded to the sample information conducted the histological analysis of all samples.
Furthermore, the obtained sections were dewaxed, rehydrated, and stained with H&E for analysis of the tissue morphology and proteoglycan distribution in IVDs. The disc NP-cell number and H&E positive area were measured using ImageJ software (
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