Mechanically induced histochemical and structural damage in the annulus fibrosus and cartilaginous endplate: a multi-colour immunofluorescence analysis

The annulus fibrosus (AF) and endplate (EP) are collagenous spine tissues that are frequently injured due to gradual mechanical overload. Macroscopic injuries to these tissues are typically a by-product of microdamage accumulation. Many existing histochemistry and biochemistry techniques are used to examine microdamage in the AF and EP; however, there are several limitations when used in isolation. Immunofluorescence may be sensitive to histochemical and structural damage and permits the simultaneous evaluation of multiple proteins—collagen I (COL I) and collagen II (COL II). This investigation characterized the histochemical and structural damage in initially healthy porcine spinal joints that were either unloaded (control) or loaded via biofidelic compression loading. The mean fluorescence area and mean fluorescence intensity of COL II significantly decreased (− 54.9 and − 44.8%, respectively) in the loaded AF ( p ≤ 0.002), with no changes in COL I ( p ≥ 0.471). In contrast, the EP displayed similar decreases in COL I and COL II fluorescence area (− 35.6 and − 37.7%, respectively) under loading conditions ( p ≤ 0.027). A significant reduction (−31.1%) in mean fluorescence intensity was only observed for COL II ( p = 0.043). The normalized area of pores was not altered on the endplate surface ( p = 0.338), but a significant increase (+ 7.0%) in the void area was observed on the EP-subchondral bone interface ( p = 0.002). Colocalization of COL I and COL II was minimal in all tissues ( R < 0.34). In conclusion, the immunofluorescence analysis captured histochemical and structural damage in collagenous spine tissues, namely, the AF and EP.