Validation study of a Finnish version of the Western Ontario and McMasters University osteoarthritis index.

The aim of the study was to validate a Finnish version of the internationally used Western Ontario and McMasters University osteoarthritis index (WOMAC) questionnaire, by testing its reliability, validity and responsiveness. The subjects of the study were patients scheduled for elective total knee arthroplasty (TKA) or total hip arthroplasty (THA). The patients completed the WOMAC questionnaire twice prior to surgery and once postoperatively to enable reliability, construct validity and responsiveness analysis. Test-retest reliability could be calculated for 67 patients (37 TKA and 30 THA). The intraclass correlation coefficients (ICC a) exceeded 0.9 with the exception of the stiffness subscale which averaged 0.8. Correlation of the preoperative WOMAC total score with the Harris Hip Score (HHS) in 30 patients and American Knee Society Score (AKSS) in 37 patients were mediocre ranging from 0.48 to 0.53. To test responsiveness, the first preoperative and the postoperative WOMAC scores of 74 subjects were compared (41 TKA and 33 THA). Standard response means (SRM) and effect sizes (ES) exceeded the recommended score of 0.8, ranging from 1.15 to 2.35. In conclusion the Finnish translation of the WOMAC questionnaire performs as the original, is valid and can be used in future studies of osteoarthritis.

Quantitative magnetic resonance imaging of experimentally injured porcine intervertebral disc.

BACKGROUND: Changes in T2 relaxation time (T2-TR) and apparent diffusion coefficients (ADC) have been suggested to appear in the intervertebral disc before morphological changes. Such sensitive imaging methods could be beneficial in the targeting and follow-up of intradiscal gene therapy. PURPOSE: To investigate the sensitivity of quantitative magnetic resonance (MR) imaging methods (T2-TR and ADC) in early disc degeneration, using an experimental porcine intervertebral disc injury model, and to investigate their sensitivity in depicting biochemically controlled degenerative changes in the disc. MATERIAL AND METHODS: Six juvenile pigs underwent experimental annular stab incisions, one superficial and one reaching the nucleus pulposus. The animals underwent repeated 1.5T MR imaging and were sacrificed 4 or 8 weeks after operation. Presence of degenerative changes was controlled with biochemical analysis. RESULTS: Discs with full-thickness annular incisions lost 30% of their sagittal mid-slice nucleus pulposus area in 2 weeks (P<0.05). T2-TRs of the respective discs were on average 73% of the control discs (P<0.05). Discs with full-thickness annular lesions showed increased ADC values 4 weeks and reduced ADC values 8 weeks after the operation, compared to control discs (P<0.05). Biochemical analysis showed changes consistent with early degeneration. CONCLUSION: Early traumatic or degenerative changes are detectable with both T2-TR and ADC. The ADC in the early phase after experimental trauma seems to initially increase before decreasing.

Comparison of the prevalence of inflammatory cells in subtypes of disc herniations and associations with straight leg raising.

STUDY DESIGN: The prevalence of inflammatory cells in 205 disc herniations (DHs) and nine macroscopically normal discs for comparison was studied immunohistochemically. Inflammatory cells were separately analyzed in subtypes of DH. Immunohistochemical data were related to clinical parameters, the straight leg raising test (SLR) in particular. OBJECTIVES: The objectives of the study were to compare the occurrence of inflammatory cells in various subtypes of DH and to determine the association between clinical data and inflammatory cell occurrence in a more extensive sample of DH, with separate analysis of DH subtypes. SUMMARY OF BACKGROUND DATA: Previous studies have suggested a common occurrence of inflammation and inflammatory cells, particularly macrophages, in DHs. No studies on any larger material comprising different subtypes of DH have been done. METHODS: For immunohistochemistry the alkaline phosphatase antialkaline phosphatase method was used. Monoclonal antibodies to T cells in general (CD2), activated T cells (CD25), B cells (CD22), and macrophages (CD68) were used. Obtained immunostaining results were then compared with clinical data, e.g., duration of pain, SLR, and type of DH (sequesters 86, extrusions 103, protrusions 16). Associations were studied by the chi2 test or Fisher's exact test, as applicable (level of significance P < 0.05). RESULTS: Abundant T cells were seen in 17% of the 205 DHs, activated T cells in 17%, B cells in 16%, and macrophages in 37%. All cell types were 2-3 times more prevalent in sequestrated discs than in extrusions. In protrusions macrophages were abundantly seen in 25% (4 of 16) and no other inflammatory cells. In patients with positive SLR and a sequestrated disc abundant lymphocytes were seen three times more often than in extrusions. When patients with bilaterally negative SLR were compared with those with tight SLR (< or =30 degrees ) with respect to inflammatory cell occurrence, some significant differences were noted (CD68, P < 0.025; CD25, P = 0.04). A comparison between SLR bilaterally positive and bilaterally negative also showed associations for all four inflammatory cell types (P = 0.016 to P = 0.029). There was no correlation between inflammatory cells and duration of pain. Abundant inflammatory cells were never seen in control discs. CONCLUSIONS: When SLR was positive and the DH type was sequestered, inflammatory cells were most commonly seen. Our results showed some statistically significant associations between inflammatory cells and SLR, most clearly when comparing bilaterally positive and negative SLR. Interestingly, a bilaterally positive SLR showed an association with all four inflammatory cell types analyzed. Tight SLR also showed an association, particularly with macrophages. In addition to tissue resorption, they may participate in sciatic pain. Even though lymphocytes were less prevalent, they may have some role in sequestered discs and bilaterally positive SLR.

In vivo detection of intervertebral disk injury using a radiolabeled monoclonal antibody against keratan sulfate.

UNLABELLED: In the intervertebral disk, proteoglycans form the major part of the extracellular matrix, surrounding chondrocytelike disk cells. Keratan sulfate is a major constituent of proteoglycans. METHODS: We have radioiodinated a monoclonal antibody raised against keratan sulfate. This antibody was injected into rats (n = 6), and the biodistribution was studied. A model of intervertebral disk injury was developed, and two tail disks in each animal with both acute (2 wk old) and subacute (7 wk old) injuries were studied for in vivo antibody uptake. RESULTS: The biodistribution at 72 h was as follows: blood, 0.0018 percentage injected dose per gram of tissue (%ID/g); lung, 0.0106 %ID/g; esophagus, 0.0078 %ID/g; kidney, 0.0063 %ID/g; liver, 0.0047 %ID/g; spleen, 0.0046 %ID/g; heart, 0.0036 %ID/g; thyroid, 0.0034 %ID/g; muscle, 0.0017 %ID/g; and bone, 0.0016 %ID/g. In the subacute stage, a significant difference (P < 0.006) was found in antibody uptake between injured disks (n = 12) and adjacent healthy disks (n = 12). In vivo gamma imaging showed increased uptake in other animals having lumbar disk injuries (2, 7, and 17 d after injury). Cartilage tissue, such as the trachea, was studied separately and showed extremely high antibody uptake, 0.10 %ID/g. Rat trachea was also visualized on gamma images. CONCLUSION: Our data suggest that antibodies against nucleus pulposus components, such as proteoglycans, can be used for in vivo detection of intervertebral disk injury. This finding is in spite of the minimal circulation present in intervertebral disks.

Elevated protein content and prolyl 4-hydroxylase activity in severely degenerated human annulus fibrosus.

Alterations involved with the intervertebral disc degeneration are partly well described, however, it is not so well known how collagen network is affected by the disease. We analyzed the rate of collagen biosynthesis (estimated by the enzymic activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase) and the level of hydroxylysylpyridinoline and lysylpyridinoline crosslinks both in normal (n=7) and degenerated (n=7) human annulus fibrosus. The activity of prolyl 4-hydroxylase was significantly increased in degenerated tissue. However, no significant changes in the collagen content or in the amount of hydroxylysylpyridinoline and lysylpyridinoline collagen crosslinks were observed. On the other hand, the content of soluble proteins was significantly increased. Our results suggest that collagen biosynthesis is increased in degenerated human annulus fibrosus, obviously to compensate the impairment of collagen fibers. The faster turnover of collagen in degenerated annulus fibrosus, suggested by the increased prolyl 4-hydroxylase activity and unchanged collagen content, seems not to cause any significant changes in its mature pyridinium crosslink concentrations.

An immunohistochemical study of nerve structures in the anulus fibrosus of human normal lumbar intervertebral discs.

STUDY DESIGN: The innervation of the anulus fibrosus of human macroscopically normal intervertebral discs from five patients was investigated immunohistochemically. OBJECTIVES: Immunoreactivity to general nerve markers (synaptophysin and protein gene product 9.5) and to neuropeptides (substance P and C-flanking peptide of neuropeptide Y) was studied. SUMMARY OF BACKGROUND DATA: In the lumbar disc of a newborn, free nerve endings have been demonstrated in the outer layers of anulus fibrosus. In degenerated and herniated discs, nerve structures have been shown to penetrate deeper into the anulus fibrosus. There are only a few studies on the innervation of normal adult intervertebral disc tissue. METHODS: Thin frozen sections of human normal lumbar intervertebral disc tissue were immunostained for general nerve markers and neuropeptides. RESULTS: Synaptophysin and protein gene product 9.5 immunoreactive nerve structures were observed penetrating 3.5 mm and 1.1 mm into the anulus, respectively. Immunoreactivity to C-flanking peptide of neuropeptide Y and substance P were observed at a maximum depth of 0.9 and 0.5 mm in the anulus, respectively. Antibodies to the former have been used to study sympathetic nerves, whereas substance P is a transmitter present in sensory nerves. CONCLUSIONS: In anulus fibrosus samples from macroscopically normal discs, a general marker for nerve endings can be found at a depth of a few millimeters, whereas neuropeptide markers show nerves only in the outermost layers of the anulus fibrosus. This absence of demonstrable nerves in deeper anulus fibrosus in normal discs is probably not a methodologic artifact, because blood vessels have also been demonstrated only at the disc surface. It is, however, possible that neuropeptide nerves also penetrate to a depth of a few millimeters, but that methodologic limitations permit the visualization of only the neuropeptide nerves closest to the disc surface. The results of the present study lend support to previous suggestions that, except at the surface, a normal intervertebral disc is almost without innervation.

Cathepsin G in degenerating and healthy discal tissue.

OBJECTIVES: To assess the eventual presence, tissue localization, molecular forms, amount and activity of cathepsin G in the annulus fibrosus. METHODS: Normal non-autolytic disc tissue was collected from cadavers within six hours after death. Degenerate disc samples were collected from low back pain patients undergoing anterior interbody fusion due to severe, discographically verified and painful disc degeneration, and from the posterior parts of intervertebral discs from 10 patients undergoing microscopic discoidectomy because of intervertebral herniation. Avidin-biotinperxidase complex staining of cathepsin G was quantitated by morphometry. Cellular localization was analyzed using double immunofluorescence staining of cathepsin G and CD68, proline 4-hydroxylase or von Willebrand factor. Neutral salt extracts were analyzed by using synthetic cathepsin G substrate in spectrophotometry, dot-immunoblotting and Western blotting. RESULTS: Histological and morphometric image analysis showed increased cellularity, increased numbers of cathepsin G positive cells and neovascularization in degenerated discs compared to control discs. Neutral salt extract of disc tissue, degenerated or normal, in contrast to control material from synovial capsular tissue, did not contain measurable cathepsin G activity, although immunoreactive enzyme was detected in dot-immunoblotting. Western blotting demonstrated that the discal cathepsin G had an apparent molecular weight of 27 kDa. CONCLUSION: Due to its properties and localization in normal and pathologically altered tissue, cathepsin G probably plays both a direct and an indirect role in extracellular matrix degradation in the annulus fibrosus. Extracted cationic cathepsin G was immunoreactive, but was functionally inhibited by serpins or, more likely, by polyanionic proteoglycans and saccharins derived from the connective tissue matrix of the annulus fibrosus.

Relative increase of biglycan and decorin and altered chondroitin sulfate epitopes in the degenerating human intervertebral disc.

OBJECTIVE: Proteoglycans are major components of the extracellular matrix of the intervertebral disc. They are vital for the biomechanical properties of the tissue, and are subject to changes in disc degeneration. We aimed to further define these changes and their relationship to normal aging. METHODS: Normal discs (age 13-53 years, n = 6) were analyzed from 5 different sites across the sagittal anterior-posterior direction. Degenerated anterior annulus fibrosus was collected from 7 patients aged 39-46 years. Extracted proteoglycans were separated using agarose and polyacrylamide gel electrophoresis and detected with toluidine blue staining and Western blotting. RESULTS: The center of the disc showed the highest level of total proteoglycans, but lowest levels of decorin and biglycan. Western blots displayed reduced signal for both glycanated and nonglycanated biglycan and decorin after adolescence, while an increased signal of biglycan was observed in degenerated annuli. The 7D4(-) and 3B3(-) epitopes on native chondroitin sulfate chains were present in the large proteoglycans of intervertebral discs, but their signal intensity had no correlation to degeneration. Chondroitinase ABC digestion of the blots brought up 7D4(+) signal in the small proteoglycans of degenerated, but not in healthy tissue. Decrease or total loss of 2B6(+) epitope (indicating 4-sulfated stubs of chondroitin sulfate chains) were found in the large proteoglycans of all degenerated annuli. CONCLUSION: Human intervertebral disc degeneration involves the accumulation of decorin and biglycan relative to other uronic acid containing proteoglycans, the disappearance of 4-sulfated core region in aggrecan-like large proteoglycans, and the emergence of a core structure in the chains of small proteoglycans reacting with the 7D4 antibody; these findings indicate a fundamental alteration in matrix properties that may contribute to the pathogenesis of the disease.

Effects of tiaprofenic acid and indomethacin on proteoglycans in the degenerating porcine intervertebral disc.

STUDY DESIGN: Eighteen pigs were stabbed with a scalpel in the anterior part of the anulus fibrosus of a lumbar disc. After surgery, the pigs received either tiaprofenic acid or indomethacin daily, and a third group did not receive any medication. OBJECTIVES: Nonsteroidal anti-inflammatory agents are widely used in the treatment of low back patients, but their long-term effects on the matrix molecules in the degenerate disc are unknown. SUMMARY OF BACKGROUND DATA: Several in vitro and in vivo studies on articular cartilage have suggested that tiaprofenic acid may not have adverse effects on matrix metabolism, whereas indomethacin probably does. METHODS: Uronic acid, DNA, and water contents were determined from five different locations in each injured disc. Transport and incorporation of sulfate were examined by in vivo radioactive tracer analysis, and proteoglycan structures were analyzed by gel electrophoresis. RESULTS: Morphologically, there were no differences between the treatments. Tiaprofenic acid maintained a higher uronic acid content in the nucleus pulposus and outer anulus compared with that of the nonmedicated animals. Tiaprofenic acid decreased the incorporation of sulfate in the injured area and the water content at most sites. Indomethacin had no adverse effects compared with the nonmedicated group, and it increased water content in the posterior anulus fibrosus. CONCLUSIONS: Long-term administration of tiaprofenic acid and indomethacin did not have harmful effects on matrix metabolism after disc injury. On the contrary, tiaprofenic acid may slightly protect proteoglycans in the degenerating disc.

Collagen synthesis and types I, III, IV, and VI collagens in an animal model of disc degeneration.

STUDY DESIGN: The present study sought to elucidate the changes that occur in collagen chemistry in the early phases of disc degeneration. OBJECTIVE: To monitor the healing process of the injured anulus fibrosus and the secondary degenerative reactions in the nucleus pulposus. SUMMARY OF BACKGROUND DATA: Despite the importance of collagen chemistry under pathologic conditions in the intervertebral disc, knowledge of this aspect is very limited. METHODS: Fourteen pigs were stabbed with a scalpel blade in the anterior part of the anulus fibrosus of a lumbar disc. The animals were killed 2 weeks to 5 months after injury. The activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase, the total collagen content, and staining patterns for Types I, III, IV, and VI collagens were analyzed from different parts of the disc. RESULTS: The most active phase of the healing process, assessed from the activities of enzymes involved in collagen biosynthesis, took place during the first month postoperatively. The anular lesion was found to cicatrize through formation of disorganized granulation tissue in which Type I, III, and, IV collagens were deposited. In the nucleus pulposus, activities of prolyl 4-hydroxylase and galactosylhydroxylysyl glucosyltransferase and total collagen content increased, and the originally rounded cells became more elongated, resembling fibroblasts. CONCLUSIONS: The results of this study suggest that the altered composition of collagens observed in the degenerate porcine nucleus pulposus results from changes in cell phenotype: Notochondral cells were replaced by fibroblast-like cells. It is likely that trauma to the anulus fibrosus can initiate a progressive degenerative process in the disc tissue.

A controlled immunohistochemical study of inflammatory cells in disc herniation tissue.

STUDY DESIGN: The presence and abundance of inflammatory cells was studied immunocytochemically in lumbar disc herniations (DH) and macroscopically normal discs for comparison. OBJECTIVES: The objective of the study was to characterize inflammatory cells that appear in herniated disc tissue and to study the relative abundance of various types of inflammatory cells. SUMMARY OF BACKGROUND DATA: Only few macrophages were observed in control discs, whereas abundant macrophages were present in half of the DH. Other types of inflammatory cells were less often abundant in the present material. In about a third of the DH interleukin-1 beta-expressing cells were also observed. METHODS: Twenty-four DH and control tissue from five discs were studied immunocytochemically, using specific monoclonal antibodies to various types of inflammatory cells and interleukin-1 beta. The results were compared with corresponding clinical data. Macrophages were studied with an antibody to CD68 antigen and Ber-MAC3 antibody separately. RESULTS: The obtained results suggest a variable inflammatory cell response in DH, which seems to be often dominated by macrophages at the time of operation. Thus previous suggestions of sometimes very active inflammation in DH tissue are supported. CONCLUSIONS: Inflammation may be important in disc tissue pathophysiology, possibly also in discogenic pain mechanisms.

Proteoglycan chemistry in experimentally injured porcine intervertebral disk.

An animal model of disk degeneration was used to study the concentration levels and types of proteoglycans in the different parts of the intervertebral disk. An annular incision was made with a scalpel blade into the anterior part of the porcine lumbar intervertebral disks via a retroperitoneal approach. Three months after injury the morphology of the injured disk had changed considerably. Disk height was diminished, and in the injured segment osteophytes had formed at the ventral edges of the vertebral body. The nucleus was small, fibrous, and yellowish. The annular lesion had healed by formation of granulation tissue, but the lamellar structure was partially destroyed. The concentration of inorganic [35S]sulfate had decreased across the whole disk, reflecting a decrease in the rate of solute transport. The concentration of incorporated [35S]sulfate had also decreased in the injured disks. The DNA concentration in the anterior annulus and in the nucleus had increased, whereas both the concentration of uronic acid and the ratio of chondroitin-6-sulfate to chondroitin-4-sulfate in the nucleus had decreased. Agarose gel electrophoresis combined with chondroitinase B digestion suggested the presence of dermatan sulfate proteoglycans in the injured annulus fibrosus. The morphology and chemical composition of the disks adjacent to the injured one were normal, and only a slight increase in the concentration of incorporated [35S]sulfate was observed in the disks above the injured one.

Neural elements in the normal and experimentally injured porcine intervertebral disk.

There is increasing evidence that-back pain may originate from degenerated or damaged disks, even in the absence of disk herniation. For a study of the pattern of innervation in injured disks, the anterior part of the annulus fibrosus of a lumbar disk in 11 domestic pigs was incised with a scalpel through a retroperitoneal approach. The animals were killed 2 weeks, 1, 2, 3, and 5 months postoperatively, and the whole anterior annulus of each injured disk and corresponding tissue from intact animals were excised. Cryostat sections 20 microns thick were cut from the surface downward, fixed, and stained with different antisera. Antisera to neurofilament triplet protein (R39), protein gene product (PGP) 9.5 and synaptophysin were used as general neural markers. Antiserum to substance P (SP) and calcitonin gene-related peptide (CGRP) were used to localize nerves mainly of the sensory type, and C flanking peptide of neuropeptide Y (CPON) to visualize nerve fibers of the sympathetic type. It was observed that in the intact porcine disk, the outer and middle parts of the anterior annulus were innervated to a depth of 7 mm from the annular surface, but the innermost annular layers showed no immunoreactivity to any of the neural antibodies. Disk injury did not cause any major changes in the nerve topography of the wound area, even though there were granulation tissue and neovascularization in this area.

Expression of type I, III, and VI collagen mRNAs in experimentally injured porcine intervertebral disc.

Young domestic pigs were incised with a scalpel blade into the anterior part of annulus fibrosus of lumbar discs in order to study the reparative processes in the annulus fibrosus and the secondary reactions in the nucleus pulposus. Northern and slot-blot hybridizations were used to investigate type I, III, and VI collagen gene expression in the disc tissue. For this purpose a method for RNA isolation was modified so as to be applicable to the intervertebral disc, which has a low cell density and a high proteoglycan content in its extracellular matrix. The amount of total RNA was found to be very low, particularly in the nucleus pulposus. Intact RNA could be isolated from most parts of the injured discs, but only from the outer annulus of the control discs. Hybridizations showed that healing of the wound in the annulus fibrosus involves an increase in the synthesis of particularly type I and type III collagens. However, no changes in the collagen gene expression were detectable in the nucleus pulposus two weeks after the injury.

Collagens in the injured porcine intervertebral disc.

Spinal pain often is thought to be due to degeneration and mechanical failure of the intervertebral disc. Since the mechanical strength of the tissue depends on collagen fibers, the present study was designed to investigate the reactions in collagen metabolism after an experimentally induced disc injury. Five domestic pigs underwent an incision in the anterior part of the annulus fibrosus of disc L4-L5 through a retroperitoneal approach. The animals were killed 3 months postoperatively, and the injured discs and intact discs (controls) from different animals were removed for chemical analysis. Slices were cut from seven different parts across the disc. The concentration of total collagen (hydroxyproline [Hyp]), the activities of the two key enzymes in collagen biosynthesis (prolyl 4-hydroxylase [PH] and galactosylhydroxylysyl glucosyltransferase [GGT]), and the concentration of mature collagen crosslinks (hydroxypyridinium [HP]) were determined. In all experimental discs, the morphology had changed considerably: the nucleus pulposus was small, fibrous, and yellowish. The annular lamellar structure was partially destroyed and had been replaced by granulation tissue in the region of the injury. Large osteophytes had formed at the ventral edges of the vertebral bodies. In the nucleus pulposus, the Hyp concentration and the activities of PH and GGT were significantly increased, whereas the water content had decreased. The concentration of HP crosslinks was decreased in the anterior annulus fibrosus.