Biglycan and fibromodulin fragmentation correlates with temporal and spatial annular remodelling in experimentally injured ovine intervertebral discs

Abstract

This study evaluated spatial and temporal extracellular matrix changes, induced by controlled surgical defects in the outer third of the annulus fibrosus (AF) of ovine intervertebral discs (IVDs). Thirty-two 4 year old sheep received a 4 mm deep x 10 mm wide standard annular surgical incision in the L1L2 and L3L4 IVDs (lesion group), 32 sheep were also subjected to the same surgical approach but the AF was not incised (sham-operated controls). Remodeling of the IVD matrix in the lesion and sham discs was assessed histochemically at 3, 6,12 and 26 month post operation (PO). Discs were also dissected into annular lesion site and contra-lateral AF and NP and equivalent zones in the sham sheep group, extracted with GuHCl, dialysed, freeze dried, digested with chondroitinase ABC/keratanase-I and aliquots examined for small leucine repeat proteoglycan (SLRP) core protein species by Western blotting using C-terminal antibodies to decorin, biglycan, lumican and fibromodulin and monoclonal antibody (Mab) 2B6 to unsaturated stub epitopes on chondroitin-4-sulphate generated by chondroitinase ABC. Masson Trichrome and Picrosirius red staining demonstrated re-organisation of the outermost collagenous lamellae in the incised discs 3-6 month PO. Toluidine blue staining also demonstrated a focal loss of anionic proteoglycan (PG) from the annular lesion 3-6 month PO with partial recovery of PG levels by 26 month. Specific fragments of biglycan and fibromodulin were associated with remodeling of the AF 12-26 month PO in the lesion IVDs but were absent from the NP of the lesion discs or all tissue zones in the sham animal group. Fragments of decorin were also observed in lesion zone extracts from 3 to 6 months but diminished after this. Isolation and characterization of the biglycan/fibromodulin fragments may identify them as prospective biomarkers of annular remodeling and characterization of the enzyme systems responsible for their generation may identify therapeutic target molecules.

Fig. 1

a Diagrammatic representation in sagittal and transverse horizontal section of the anatomical site and extent of the anterolateral annular lesion (arrow, dark area). b The L1L2 and L3L4 discs received the lesion depicted in segment a. Para-lesion discs (T14L1, L2L3 and L4L5) were also processed for histology. c Transverse horizontal sections of sham control and lesion discs 3 and 6 month PO depicting the extent of the well defined experimental anterolateral lesion (arrow)

Fig. 2

Sagittal sections of intervertebral discs and adjacent vertebral body segments. Masson Trichrome staining (upper panel) depicts focal changes in the collagenous organisation of the outer AF in lesion affected discs (black arrows). Toluidine blue/fast green staining (lower panel) depicts early focal depletion 3–6 month PO of anionic proteoglycan associated with lesion development and some recovery of AF proteoglycan levels by 26 month PO. The lesion site is depicted by an arrow at the left hand side of each disc section. Scale bar 1 cm

Fig. 3

Sagittal sections of IVD and adjacent vertebral body segments stained with picro-sirius red and examined under polarised light to visualise changes in collagenous organization (a–f) and with toluidine blue-fast green to depict anionic proteoglycan (g, h). Thickening of the anterior and posterior longitudinal ligaments is evident 26 month PO in a lesion affected disc (f). Arrows indicate the site of the initial annular lesion. In g and h controlled outer annular defects, initially involving only the outer third of the AF (large arrow), have developed into a radiating tear (g) which has propagated through the inner AF and NP to the contralateral AF 12 month PO (small arrows), and a circumferential tear involving separation of adjacent annular lamellae 26 month PO (h). Scale bars 1 cm

Fig. 4

Diagrammatic representation of the annular lesion (AF zone 1) and contralateral AF zone (AF zone 2) and NP of a lesion affected disc which were the tissue zones extracted and examined for proteoglycan species. Immunoblot of proteoglycan species containing the unsaturated chondroitinase-4-sulphate stub epitope generated by chondroitinase ABC digestion and detected using Mab 2-B-6 and present in the respective disc tissue extracts. The 45–48 kDa free core proteins of decorin/biglycan are the most prominent bands at the top of each blot, however their levels are significantly lower in the NP extracts compared to AF and also relatively lower in the sham animal group compared to the lesion group

Fig. 5

Identification of decorin core protein species by Western blotting using the C-terminal anti-decorin Ab PR-84. Tissue extracts were pre-digested with chondroitinase ABC/keratanase-I and electrophoresed at equivalent tissue weights per lane. For an explanation of the tissue zones see the legend to Fig. 4. Intact core protein of 48 kDa and smaller (20–45 kDa) core protein species (a–e) were observed particularly in the lesion affected tissue zones

Fig. 6

Detection of biglycan core protein species by Western blotting using the C-terminal biglycan antibody PR-85. The intact biglycan core protein of 48–51 kDa was prominently detected, however five to seven smaller core protein species were also detected. Many of these were found in both the sham and lesion affected samples groups. Two species of ∼17 and 25 kDa were observed localized specifically to the lesion affected AF zones only (arrowsa, b)

Fig. 7

Detection of lumican core protein species by Western blotting using an anti C-terminal lumican antibody (PR-353). The samples were pre-digested with chondroitinase ABC and keratanase-I, dialysed and freeze dried prior to electrophoresis Samples were loaded at equivalent tissue wet weights per lane

Fig. 8

Detection of fibromodulin core protein species by Western blotting using an anti C-terminal fibromodulin antibody (PR-184). The samples were pre-digested with chondroitinase ABC and keratanase-I, dialysed and freeze dried prior to electrophoresis Samples were loaded at equivalent tissue wet weights per lane. The fibromodulin free core protein ∼53 kDa was observed in each case however two additional core protein species, (labeled with an asterisk and arrows) were observed in the lesion affected discs (AF zones 1 and 2)