INVESTIGATING THE ASSOCIATION BETWEEN MMP-3 EXPRESSION AND THE BIOMECHANICS OF INTERLAMELLAR MATRIX INTEGRITY IN DEGENERATED ANNULUS FIBROSUS TISSUE

Intervertebral disc (IVD) degeneration is associated with the progressive breakdown of the annulus fibrosus (AF) (Lee et al., 2004). The AF is organized into multiple layers called lamellae and these lamellae are interconnected through the connective tissue called interlamellar matrix (ILM) (Tavakoli et al., 2017). The ILM plays a vital role in resisting delamination (Melrose et al., 2008). Proteoglycans are a key component of the ILM, likely providing lubrication and hydration to help sustain the mechanical integrity of the IVDs (Melrose et al., 2008). Matrix metalloproteinase-3 (MMP-3), a proteoglycanase, and its genetic variants have been implicated in IVD degeneration (Kalichman and Hunter, 2008; Martirosyan et al., 2016). We hypothesize that elevated MMP-3 levels in degenerated tissue are associated with decreased mechanical integrity of the ILM, compared to non-degenerated tissues. Human AF tissue samples were collected from patients undergoing surgery for IVD degeneration (n=30, age: 45±9 years) and from non-degenerated organ donor samples (n=13, age: 35±9 years) serving as controls. Samples were divided with one section assessed biomechanically and a second aliquot frozen at −80°C until assessed for MMP-3. Frozen tissue samples were powdered, extracted with PBS (pH 7.2) and then components separated by gel electrophoresis. MMP-3 expression levels were quantified using western blotting (Mahmood and Yang, 2012) in both degenerated and non-degenerated tissues. Peel tests were conducted to assess the mechanical integrity of the ILM (Gregory et al., 2012). Unpaired t-tests were conducted to compare the non-degenerated and degenerated groups. The peel stiffness of the non-degenerated and degenerated tissues was 0.27 ± 0.07 N/mm² and 0.17 ± 0.05 N/mm² (p = 0.0001) respectively. The peel strength of the non-degenerated and degenerated samples was 2.39 ± 0.93 N/mm and 1.51 ± 0.71 N/mm (p = 0.002). The peel toughness of the non-degenerated and degenerated samples was 34.34 ± 15.05 J/m and 21.02 ± 12.21 J/m (p = 0.004). The western blot analysis indicated an absence of MMP-3 in non-degenerated tissue, whereas degenerated tissue showed intense bands of intact MMP-3. The current findings show decreased mechanical integrity of the ILM and increased expression of MMP-3 in degenerated samples compared to non-degenerated controls. Since MMP-3 promotes proteoglycan degradation, it may be linked with the decreased interlamellar properties of the ILM due to the role of proteoglycans in maintaining the ILM integrity. However, the activation state of MMP-3 requires further investigation. Ongoing assessments of the expression of other ILM-relevant proteinases from the proteomics data will allow for further phenotyping of surgical samples in different diagnostic categories. The main limitation is that immunolocalization studies have not been performed yet to localize the MMP-3 to the ILM of mechanically assessed samples, which will allow integration of the mechanical and biological findings using paired samples to make a direct link in the future. Clinically, understanding the link between MMP-3 and ILM may provide further insights into the delamination process often observed in degenerated surgical samples and the progression of disc degeneration requiring surgical intervention.