Premature vertebral endplate ossification and mild disc degeneration in mice after inactivation of one allele belonging to the Col2a1 gene for Type II collagen.

STUDY DESIGN: Skeletal tissues of mice with an inactivated allele of the Col2a1 gene for Type II collagen ("heterozygous knockout") were studied. OBJECTIVE: To determine whether a heterozygous inactivation of the Col2a1 gene has a role in the etiology of spine disorders such as disc degeneration. SUMMARY OF BACKGROUND DATA: Mutations in the COL2A1, COL11A1, COL11A2, and COL9A2 genes have been linked to spine disorders. However, the mechanism by which genetic factors lead to disc degeneration still are largely unknown. METHODS: Spine tissues were studied using radiograph analyses; conventional, quantitative, and polarized light microscopy; immunohistochemistry for the major extracellular components, and in situ hybridization for procollagens alpha1(I) and alpha1(II). Voluntary running activity also was monitored in half of the mice. RESULTS: As the findings showed, 1-month-old heterozygous knockout mice had shorter limb bones, skulls, and spines, as well as thicker and more irregular vertebral endplates, which calcified earlier than in the control mice. They also had a lower concentration of glycosaminoglycans in the anulus fibrosus, in the endplates, and in the vertebral bone than the controls. These features in the heterozygous knockout mice were compensated by the age of 15 months. However, the long bones and skulls of the mature heterozygous mice remained shorter than those of the controls. Gene-deficient mice used the running wheel less. However, physical exercise did not induce any marked structural changes in the skeleton. CONCLUSION: Mice with heterozygous knockout of Col2a1 show subtle early skeletal manifestations that bear some resemblance to those of human spine disorders.

The role of microtubules in the regulation of proteoglycan synthesis in chondrocytes under hydrostatic pressure.

Chondrocytes of the articular cartilage sense mechanical factors associated with joint loading, such as hydrostatic pressure, and maintain the homeostasis of the extracellular matrix by regulating the metabolism of proteoglycans (PGs) and collagens. Intermittent hydrostatic pressure stimulates, while continuous high hydrostatic pressure inhibits, the biosynthesis of PGs. High continuous hydrostatic pressure also changes the structure of cytoskeleton and Golgi complex in cultured chondrocytes. Using microtubule (MT)-affecting drugs nocodazole and taxol as tools we examined whether MTs are involved in the regulation of PG synthesis in pressurized primary chondrocyte monolayer cultures. Disruption of the microtubular array by nocodazole inhibited [(35)S]sulfate incorporation by 39-48%, while MT stabilization by taxol caused maximally a 17% inhibition. Continuous hydrostatic pressure further decreased the synthesis by 34-42% in nocodazole-treated cultures. This suggests that high pressure exerts its inhibitory effect through mechanisms independent of MTs. On the other hand, nocodazole and taxol both prevented the stimulation of PG synthesis by cyclic 0. 5 Hz, 5 MPa hydrostatic pressure. The drugs did not affect the structural and functional properties of the PGs, and none of the treatments significantly affected cell viability, as indicated by the high level of PG synthesis 24-48 h after the release of drugs and/or high hydrostatic pressure. Our data on two-dimensional chondrocyte cultures indicate that inhibition of PG synthesis by continuous high hydrostatic pressure does not interfere with the MT-dependent vesicle traffic, while the stimulation of synthesis by cyclic pressure does not occur if the dynamic nature of MTs is disturbed by nocodazole. Similar phenomena may operate in cartilage matrix embedded chondrocytes.

Hyaluronan distribution in the human and canine intervertebral disc and cartilage endplate.

A biotinylated complex of aggrecan G1-domain and link protein was used to characterize the distribution of hyaluronan in paraffin-embedded sections of adult human and canine intervertebral disc and cartilage endplate. Limited chondroitinase ABC and trypsin digestions of the sections before staining was utilized to expose hyaluronan potentially masked by aggrecan. Hyaluronan concentration and hyaluronan to uronic acid ratio in different parts of the discs were measured as a background for the histological analysis. Hyaluronan staining was strong in the nucleus pulposus and inner parts of annulus fibrosus of both species, corroborated by biochemical assays of the same compartments. Particularly in human samples, hyaluronan in the interterritorial matrix of nucleus pulposus and annulus fibrosus was readily accessible to the probe without enzyme treatments. In contrast, the cell-associated hyaluronan signal was enhanced after trypsin or limited chondroitinase ABC-treatment of the sections, suggesting that pericellular hyaluronan was more masked by aggrecan than in the distant matrix. A puzzling feature of canine cartilage endplate cells was their intensive cell-associated hyaluronan signal, part of which appeared intracellular. Hyaluronan was abundant between the collagenous lamellae in annulus fibrosus, perhaps important in the plasticity of this tissue.

Localization of type X collagen in the intervertebral disc of mature beagle dogs.

Type X collagen expression in intervertebral disc of young adult beagle dogs (n = 10) was studied. Type X collagen was immunostained mainly pericellularly in the central area of the vertebral endplate, but interterritorial staining there was also present. Annulus fibrosus and nucleus pulposus did not usually stain for type X collagen. However, immunostaining of nucleus pulposus for type X collagen with a simultaneous expression of collagen alpha1(X) mRNA was observed in one dog. A weak staining was observed in two other animals with a weak collagen alpha1(X) mRNA signal. In annulus fibrosus, lamellar staining was observed in two dogs. In three animals, type X collagen mRNAs were observed in the outer edge of the annulus fibrosus, but immunohistochemical staining did not always correlate with in situ hybridization signals. In conclusion, intervertebral disc type X collagen was mainly expressed in the cartilaginous endplate. In some apparently healthy animals there was type X collagen expression in the nucleus pulposus and also in 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.

Immobilisation causes longlasting matrix changes both in the immobilised and contralateral joint cartilage.

OBJECTIVE: The capacity of articular cartilage matrix to recover during 50 weeks of remobilisation after an atrophy caused by 11 weeks of immobilisation of the knee (stifle) joint in 90 degrees flexion starting at the age of 29 weeks, was studied in young beagle dogs. METHODS: Proteoglycan concentration (uronic acid) and synthesis ([35S]sulphate incorporation) were determined in six and three knee joint surface locations, respectively. Proteoglycans extracted from the cartilages were characterised by chemical determinations, gel filtration, and western blotting for chondroitin sulphate epitope 3B3. RESULTS: The proteoglycan concentrations that were reduced in all sample sites immediately after the immobilisation, remained 14-28% lower than controls after 50 weeks of remobilisation in the patella, the summit of medial femoral condyle, and the superior femoropatellar surface. In the contralateral joint, there was a 49% increase of proteoglycans in the inferior femoropatellar surface after remobilisation, while a 34% decrease was simultaneously noticed on the summit of the medial femoral condyle. Total proteoglycan synthesis was not significantly changed after immobilisation or 50 weeks' remobilisation in the treated or contralateral joint, compared with age matched controls. The chondroitin 6- to 4- sulphate ratio was reduced by immobilisation both in the radioactively labelled and the total tissue proteoglycans. In the remobilised joint, this ratio was restored in femur, while in tibia it remained at a level lower than controls. Neither immobilisation nor remobilisation induced epitopes recognised by the monoclonal antibody 3B3 on native (undigested) proteoglycans. CONCLUSION: These results show that the depletion of proteoglycans observed after 11 weeks of immobilisation was not completely restored in certain surface sites after 50 weeks of remobilisation. The significant changes that developed in the contralateral joint during the remobilisation period give further support to the idea that a permanent alteration of matrix metabolism results even from a temporary modification of loading pattern in immature joints.

Coordinated regulation of hyaluronan and aggrecan content in the articular cartilage of immobilized and exercised dogs.

OBJECTIVE: To study the influence of joint loading and immobilization on articular cartilage hyaluronan concentration and histological distribution in the knee joints of young dogs subjected to 11 weeks' immobilization by splinting, and 15 weeks' running exercise at a rate of 40 km/day. METHODS: The amount of hyaluronan in articular cartilage was determined by a competitive binding assay using a biotinylated hyaluronan binding complex (HABC) of aggrecan and link protein. Histologic sections were stained for the localization of hyaluronan with the HABC probe. Extracted proteoglycans were characterized by sodium dodecyl sulfate agarose gel electrophoresis. RESULTS: Immobilization significantly reduced the concentration of hyaluronan in all sites studied (tibial and femoral condyles, patellar surface of femur). The proportion of hyaluronan to total uronic acid (mainly from aggrecan) remained unchanged because of a concurrent decrease in aggrecan. The ratio of hyaluronan and aggrecan remained constant also in runners. The staining pattern of free hyaluronan in the tissue sections and the electrophoretic mobility of the extracted proteoglycans were not affected by the different loading regimes. CONCLUSION: Reduced joint loading due to splint immobilization significantly decreases both hyaluronan and aggrecan in the articular cartilage. The remarkably parallel changes in aggrecan and hyaluronan content suggest that joint loading exerts a coordinated influence on their metabolism.

Proteoglycan depletion and size reduction in lesions of early grade chondromalacia of the patella.

OBJECTIVE: To determine the content and molecular size of proteoglycans (PGs) in patellar chondromalacia (CM) and control cartilages as a first step in investigating the role of matrix alterations in the pathogenesis of this disease. METHODS: Chondromalacia tissue from 10 patients was removed with a surgical knife. Using identical techniques, apparently healthy cartilage of the same site was obtained from 10 age matched cadavers (mean age 31 years in both groups). Additional pathological cartilage was collected from 67 patients with grades II-IV CM (classified according to Outerbridge) using a motorised shaver under arthroscopic control. The shaved cartilage chips were collected with a dense net from the irrigation fluid of the shaver. The content of tissue PGs was determined by Safranin O precipitation or uronic acid content, and the molecular size by mobility on agarose gel electrophoresis. RESULTS: The mean PG content of the CM tissue samples with a knife was dramatically reduced, being only 15% of that in controls. The cartilage chips collected from shaving operations of grades II, III, and IV CM showed a decreasing PG content: 9%, 5%, and 1% of controls, respectively. Electrophoretic analysis of PGs extracted with guanidium chloride from the shaved tissue samples suggested a significantly reduced size of aggrecans in the mild (grade II) lesions. CONCLUSION: These data show that there is already a dramatic and progressive depletion of PGs in CM grade II lesions. This explains the softening of cartilage, a typical finding in the arthroscopic examination of CM. The PG size reduction observed in grade II implicates proteolytic attack as a factor in the pathogenesis of CM.

Influence of short-term hydrostatic pressure on organization of stress fibers in cultured chondrocytes.

The present study describes changes in the organization of stress fibers that occur in articular cartilage chondrocytes subjected to hydrostatic pressure. Primary cultures of chondrocytes from bovine articular cartilage, grown on coverslips, were subjected to 5, 15, or 30 MPa hydrostatic pressure at 37 degrees C. The pressure was applied continuously or cyclically at two frequencies: 0.125 Hz (4 seconds of pressure and 4 seconds of no pressure) or 0.05 Hz (1 second of pressure and 19 seconds of no pressure) for a period of 2 hours. Control chondrocytes showed a polygonal form with prominent stress fibers extending across the cells. The exposure of cells to 30 MPa pressure caused a nearly total disappearance of stress fibers and retraction of the cells from each other. With pressure at 15 MPa or cyclic pressure, the number of cells with stress fibers was decreased. In cells subjected to 5 MPa pressure, the stress fibers resembled those in control chondrocytes. The pressure effects were reversible after 2 hours. Pressure had no effect on the staining pattern of vinculin, which suggests that microfilaments are more vulnerable to pressure than vinculin. The results indicate that cytoskeletal changes may be an integral part of the response of chondrocytes to hydrostatic pressure.

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.

Expression of reduced amounts of structurally altered aggrecan in articular cartilage chondrocytes exposed to high hydrostatic pressure.

The effect of hydrostatic pressure on proteoglycan (PG) metabolism of chondrocyte cultures was examined using a specially designed test chamber. Primary cultures of bovine articular chondrocytes at confluence were exposed for 20 h to 5 and 30 MPa continuous hydrostatic pressures and 5 MPa hydrostatic pulses (0.017, 0.25 and 0.5 Hz) in the presence of [35S]sulphate. Northern blot analyses showed that chondrocyte cultures used in this study expressed abundant mRNA transcripts of aggrecan, typical of chondrocytes, but not versican. The cultures also expressed biglycan and decorin. Enzymic digestions with keratanase and chondroitinases AC, ABC and B and subsequent SDS/agarose gel electrophoresis confirmed the synthesis of aggrecans and small dermatan sulphate PGs. The continuous 30 MPa pressure reduced total PG synthesis by 37% as measured by [35S]sulphate incorporation, in contrast to the 5 MPa continuous pressure which had no effect. The high static pressure also reduced total [3H]glucosamine incorporation by 63% and total [14C]leucine incorporation by 57%. The cyclic pressures showed a frequency-dependent stimulation (0.5 Hz, 11%) or inhibition (0.017 Hz, -17%) of [35S]sulphate incorporation. Aggrecans secreted under continuous 30 MPa pressure showed a retarded migration in 0.75% SDS/agarose gel electrophoresis and they also eluted earlier on Sephacryl S-1000 gel filtration, indicative of a larger molecular size. The increased size was consistent with an increase of average glycosaminoglycan chain length as determined by Sephacryl S-300 gel filtration. No change in aggrecan size was observed with the lower (5 MPa) static or cyclic pressures. Continuous 30 MPa hydrostatic pressure slightly reduced the steady-state mRNA level of aggrecan, in parallel with the decline in PG synthesis measured by [35S]sulphate incorporation. The results demonstrated that high hydrostatic pressure could influence the synthesis of PGs, especially of aggrecans, in chondrocytes both at the transcriptional and translational/post-translational levels.

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.

Proteoglycans in the intervertebral disc of young dogs following strenuous running exercise.

The proteoglycans (PGs) of intervertebral disc were studied in ten beagles which ran on a treadmill for one year (up to 40 km/day) and in ten non-running control dogs. Nucleus pulposus and annulus fibrosus from cervical (C5) and thoracic (T6 and T12) discs were labeled in vitro with 35SO4. The extractability, concentration and synthesis of PGs, and the electrophoretic subpopulations, aggregation and glycosaminoglycan chain lengths of newly-synthesized and total PGs were measured. Sulfate incorporation was significantly elevated by running in the C5 disc and reduced in the annulus of T6 discs. In the annulus of the T6 discs the concentration of total PGs was significantly lower although that of dermatan sulfate PGs was actually higher than in the controls. The results show that enhanced loading of the spine exerts significant alterations in the intervertebral disc PGs in a spine-level specific manner. In the most strained area of the spine (upper thoracic), the alterations in the runners suggest compromised biomechanical properties of the disc.