TABLE 2.

Histopathological evaluation

Grade	Definition
Proteoglycan depletion based on Safranin O‐Fast Green‐staining
0	Fast green staining only of outer AF, intermediate Safranin O staining of inner AF, intense Safranin O staining in NP, well defined cartilaginous endplate staining. Alternate AF lamellae discernable due to differing Fast Green staining intensities of adjacent lamellae
1	Fast green staining only of outer AF, intermediate Safranin O staining of inner AF, intense Safranin O staining in NP, well defined cartilaginous endplate staining. Alternate AF lamellae discernable due to differing Fast Green staining intensities of adjacent lamellae
2	Moderately reduced Safranin O staining of mid and inner AF in vicinity of lesion, fast green staining of outer AF only, normal Safranin O staining of NP and cartilaginous endplate
3	Reduced patchy Safranin O staining around lesion, fast green staining in outer AF
4	Reduced Safranin O staining in NP compared to control IVD, very faint or no Safranin O staining in mid and outer AF, fast green staining only in outer AF
IVD structure and lesion morphology
0	Normal IVD structure with well‐defined annular lamellae, central NP and cartilaginous endplate
1	Lesion evident in mid AF, normal NP morphology
2	Lesion evident in mid and inner AF, but may not be apparent in outer AF due to spontaneous repair, inner AF lamellae may be inverted and have anomalous distortions in normal lamellar architecture
3	Bifurcation/propagation of lesion from mid to inner AF into NP margins, mild delamination, when more extensive may lead to concentric tears between lamellae in mid and inner AF
4	Propagation of lesion into NP, with disruption in normal NP structure, distortion of annular lamellae into atypical arrangements‐severe delamination, separation of translamellar cross bridges
Cellular morphology
0	Normal, sparse distribution of typical single AF and NP fibrochondrocytes
1	Small groups of rounded chondrocytic cells (2–4 cells/group) in vicinity of annular lesion in inner AF, occasional cell division in resident inner AF and NP cells
2	Moderate increase in well‐defined groups of rounded chondrocytic cells (4–8 cells/group) in vicinity of lesion and with penetrating blood vessels associated with the lesion site, well defined chondroid cell colonies in NP contained within a dense basophilic matrix with little fibrillar material evident around the cells contrasting with NP cells
3	Marked increase in less defined groups of rounded chondrocytic cells (>8 cells/group) in vicinity of lesion and with penetrating blood vessels associated with the lesion site, well defined chondroid cell colonies in NP (<50 cells/colony) contained within a dense basophilic matrix with little fibrillar material evident around the cells contrasting with NP cells
4	Numerous cell clones around inner and mid AF lesion, chondroid cell nests in NP containing >50 cells
Blood vessel ingrowth
0	Very occasional vessels in outermost annular lamellae, occasional capillaries in cartilaginous endplate
1	Slight influx of cells mainly in outer AF
2	Moderate influx of cells throughout AF
3	Large influx of cells throughout AF
4	Extensive influx of cells throughout AF particularly in inner AF and around lesion
Cellular influx into lesion
0	Normal cell distribution in mid, inner and outer AF, and NP
1	Slight influx of cells mainly in outer AF
2	Moderate influx of cells throughout AF
3	Large influx of cells throughout AF
4	Extensive influx of cells throughout AF particularly in inner AF and around lesion
Cleft formation in vicinity to lesion
0	No clefts in AF
1	Small cleft area in AF
2	Moderate cleft area in AF
3	Large cleft area in AF
4	Vast cleft area in AF and also in NP

Note: Histopathological features for histopathological semiquantitative grading for the evaluation of IVD degeneration performed on sections of control and injured IVD. Adapted from Shu et al. ³⁹