William A. Elmer

The effect of hydrostatic pressure on intervertebral disc metabolism.

STUDY DESIGN By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in an alginate. OBJECTIVE To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. SUMMARY OF BACKGROUND DATA The influence of compression (both hydrostatic and mechanical) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc, was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 1 MPa and the other at atmospheric pressure. The effects of 1 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS Comparisons with atmospheric pressure yielded the following findings: 1) In the incorporation studies, the nucleus and anulus cells exhibited a differential response to a hydrostatic pressure of 1 MPa. Collagen and proteoglycan syntheses were stimulated in the nucleus cells and inhibited in the anulus cells. 2) There was no significant increase in cell proliferation, as measured by DNA content, at 1 MPa for either the anulus or nucleus cells. 3) The mRNA levels of collagen (Col 1A1 and Col 2A1) and aggrecan increased at 1 MPa in both the nucleus and anulus cells. CONCLUSIONS Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells.

The Effect of Bone Morphogenetic Protein-2 on Rat Intervertebral Disc Cells in Vitro

Study Design. An in vitro experiment to determine the molecular and cellular effect of recombinant human bone morphogenetic protein-2 on cultured rat intervertebral disc cells was performed. Objectives. To determine the effect of recombinant human bone morphogenetic protein-2 on cell proliferation, production of sulfated-glycosaminoglycan, and the expression of genes specific for chondrocytes (Type II collagen, aggrecan, and Sox9) in cultured rat intervertebral disc cells. Summary of Background Data. Intervertebral disc degeneration is associated with cellular and biochemical changes, which include decreased synthesis of cartilage specific gene products such as Type II collagen and aggrecan. Although bone morphogenetic protein-2 is known to induce chondrogenesis during new bone formation, the effects on intervertebral disc cells have not been characterized. Method. Cells were isolated from the anulus fibrosus and transition zones of lumbar discs from Sprague-Dawley rats. The cells were grown in monolayer and treated with recombinant human bone morphogenetic protein-2 (0, 10, 100, 1000 ng/mL) in Dulbecco’s Modified Eagle Medium/F-12 with 1% fetal bovine serum (day 0). On days 2, 4, and 7 after recombinant human bone morphogenetic protein-2 treatment, sulfated-glycosaminoglycan content in the media was quantified using 1,9-dimethylmethylene blue staining. The results were normalized according to culture duration and cell number. On day 7, mRNA was extracted for reverse transcriptase-polymerase chain reaction and real-time polymerase chain reaction to quantitate mRNAs of Type I collagen, Type II collagen, aggrecan, Sox9, osteocalcin, and glyceraldehyde phosphate dehydrogenase. Cell number was determined with a hemocytometer. Results. Recombinant human bone morphogenetic protein-2 at 100 and 1000 ng/mL yielded a 17% and 42% increase in cell number on day 4, and a 59% and 79% on day 7, respectively. Recombinant human bone morphogenetic protein-2 at 10 ng/mL had no effect on cell number. Sulfated-glycosaminoglycan increase was greatest at day 7, increasing by 1.3-, 2.1-, and 3.6-fold with recombinant human bone morphogenetic protein-2 treatments of 10, 100, and 1000 ng/mL, respectively. Increases in mRNA levels of Type II collagen, aggrecan, Sox9, and osteocalcin were observed with recombinant human bone morphogenetic protein-2 concentrations of 100 and 1000 ng/mL on day 7 as determined by reverse transcriptase-polymerase chain reaction. No detectable increase in mRNA level of Type I collagen was observed with any levels of recombinant human bone morphogenetic protein-2. Real-time polymerase chain reaction showed the greatest effect at 1000 ng/mL recombinant human bone morphogenetic protein-2, leading to an 11.5-fold increase in aggrecan, a 4.6-fold increase in Type II collagen, a 5.3-fold increase in Sox9, and a 1.9-fold increase in osteocalcin mRNA above untreated controls at day 7. Conclusion. The results of this study show that recombinant human bone morphogenetic protein-2 enhances disc matrix production and chondrocytic phenotype of intervertebral disc cells. Recombinant human bone morphogenetic protein-2 increases cell proliferation and sulfated-glycosaminoglycan (proteoglycan) synthesis. It increases mRNA of Type II collagen, aggrecan, and Sox9 genes (chondrocyte specific genes), and osteocalcin, but not Type I collagen or glyceraldehyde phosphate dehydrogenase.

The effect of compressive force applied to the intervertebral disc in vivo. A study of proteoglycans and collagen

Study Design. Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs of dogs for up to 27 weeks. Objective. To test the hypothesis that a high compressive force applied over a period of time affects the production of proteoglycans and collagen by the intervertebral disc cells. Summary of Background Data. It is a commonly held belief that high forces applied to the intervertebral disc, and to joints in general, play a role in causing degeneration. Methods. Pairs of stainless steel coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs (L1‐L2 and L3‐L4) of 16 dogs. Dogs were killed between 13 and 27 weeks after the springs were attached. The discs (L1‐L2 and L3‐L4) were excised and assessed using immunohistochemical analyses and enzyme‐linked immunosorbent assay; T13‐L1 and L4‐L5 were used as controls. Results. The main result relates to a group effect in the six dogs, assessed using enzyme‐linked immunosorbent assay, that were generally at the highest values of force for the greatest number of weeks. For the nucleus, but not the anulus, Spearman rank correlations revealed a strong correlation between increases in force and force‐weeks (force multiplied by number of weeks) and increases in collagen type I accompanied by decreases in proteoglycans, chondroitin sulfate, and collagen type II for both experimental discs (L1‐L2 and L3‐L4), as compared with corresponding values in the controls (T13‐L1 and L4‐L5). In other words, as either the force or the force‐weeks increased, the effect on the nucleus became greater. Conclusion. A high compressive force applied to the disc over a period of time initiates changes in proteoglycans and collagen.

Does long-term compressive loading on the intervertebral disc cause degeneration?

Study Design. Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs of dogs for up to 53 weeks. Objective. To test the hypothesis that compressive forces applied to the intervertebral disc for a long period of time cause disc degeneration in vivo in a dog model. Summary of Background Data. It is a commonly held belief that high forces applied to the intervertebral disc, and to joints in general, play a role in causing degeneration. Methods. Coil springs were stretched and attached to produce a compressive force across the lumbar intervertebral discs (L3/L4) of 12 dogs. After up to a year, the dogs were killed, and their lumbar spines were removed and radiographed. The L3/L4 disc and the controls (T13/L1 and L4/L5) were excised and examined for visible signs of degeneration. The discs then were assessed using immunohistochemical analysis and enzyme-linked immunosorbent assay. Disc chondrocytes also were assayed for apoptosis. Results. No obvious signs of degeneration in the discs (L3/L4) that had been under compression for up to a year could be observed. There was no disc bulging, anular fissures, or disc space narrowing. Some changes were observed at the microscopic level, although no thickening of the endplate was apparent. The enzyme-linked immunosorbent assay analysis provided significant data for all three regions of the disc (nucleus, inner anulus, and outer anulus). When comparing the compressed disc (L3/L4) with either of the control discs (T13/L1 and L4/L5), in the compressed disc: 1) the nucleus contained less proteoglycan and more collagen I and II; 2) the inner anulus contained less proteoglycan and collagen I; and 3) the outer anulus contained more proteoglycan and less collagen I. The collagen II differences for the inner and outer anulus were not significant. Conclusion. Compression applied to the lumbar intervertebral discs of dogs for up to a year does not produce degeneration in any visible form. It does produce microscopic changes and numerical changes, however, in the amounts of proteoglycan and collagen in the nucleus, inner anulus, and outer anulus. The present results add no credence to the commonly held belief that high compressive forces play a causative role in disc degeneration.

Do the intervertebral disc cells respond to different levels of hydrostatic pressure

OBJECTIVE To test the hypothesis that hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by intervertebral disc cells. DESIGN By the use of pressure vessels, hydrostatic pressure was applied to intervertebral disc cells cultured in alginate. BACKGROUND The influence of compression (both hydrostatic and axial) on chondrocyte metabolism was examined in a number of earlier studies. However, in most of these studies, articular cartilage, not intervertebral disc was used, and in none of these was hydrostatic pressure applied to intervertebral disc cells cultured in alginate. METHODS Fresh cells were harvested from the lumbar intervertebral discs of dogs. Before their suspension in an alginate gel system, the cells were plated and expanded until they reached confluence. Then, by use of the alginate gel system, the cells were exposed (for up to 9 days) to specific values of hydrostatic pressure inside two stainless steel pressure vessels. One vessel was kept at 0.35 MPa and the other at atmospheric pressure (approximately 0.1 MPa). The effects of 0.35 MPa were compared against atmospheric pressure by measuring the incorporation of [3H]-proline and [35S]-sulfate into collagen and proteoglycans, respectively, for the anulus cells and nucleus cells separately, and by determining whether this incorporation was reflected by changes in the levels of mRNA for aggrecan and Types I and II collagen. RESULTS Proteoglycan synthesis was inhibited at 0.35 MPa as compared to atmospheric pressure for both the nucleus and anulus cells, whereas collagen synthesis was stimulated in the nucleus cells, but inhibited in the anulus cells. The mRNA levels of collagen 1A and collagen 2A decreased in the anulus but showed a differential response in the nucleus (collagen 1A increased, while collagen 2A decreased). The mRNA levels for aggrecan core protein decreased in the anulus and increased in the nucleus. CONCLUSIONS Hydrostatic pressure directly affects the synthesis of collagen and proteoglycan by the intervertebral disc cells. RELEVANCE This in vitro study reveals the direct effect of hydrostatic pressure on disc cells, in the absence of other factors. However, circumspection must be applied when comparisons between these results, from in vitro experiments on dog disc cells, are extrapolated and applied to the whole discs of humans.

Effect of tail suspension (or simulated weightlessness) on the lumbar intervertebral disc: study of proteoglycans and collagen.

Study Design. An experiment to measure the proteoglycan and collagen content of the lumbar intervertebral discs of rats that had been tail-suspended for up to 4 weeks. Objectives. To determine the effect of tensile force (or simulated weightlessness) on the intervertebral disc. Summary of Background Data. During space flight the intervertebral disc experiences low compressive force (because of so-called “weightlessness”), which, in turn, produces, among other things, low hydrostatic pressure acting on the disc cells. Although disc cells respond (in vitro) to changes in hydrostatic pressure, it is unclear what effect low levels of hydrostatic pressure have in vivo and whether they lead to a degenerative catabolic process. The rat tail-suspension model is appropriate for studying the effects of tensile force on the disc. The disc (especially the anulus) is subjected to tension during various body movements (e.g., bending stretches the posterior anulus, and twisting tensions the whole anulus). Methods. Thirty-two Sprague–Dawley rats were tail-suspended for either 2 weeks (16 rats) or 4 weeks (16 rats). Sixteen other rats were left unsuspended for 4 weeks; these were used as controls. At the end of 2 or 4 weeks, as appropriate, the rats were killed and their lumbar spines were removed. In each rat the six lumbar discs were bisected and the discs (anulus and nucleus together) were carefully removed. The six lumbar discs from one rat were pooled with the six lumbar discs of a second matching rat (i.e., from the same group) to give one sample. The disc samples were then assessed using enzyme-linked immunosorbent assays. Results. There was a 35% statistically significant decrease in proteoglycan content going from the control group down to the 4-week group, but no significant differences between the control group and the 2-week group or between the 2-week group and the 4-week group. There were no statistically significant differences between the three groups for collagen I or collagen II. Conclusions. These findings clearly establish a link between decreased proteoglycan content and tension on the disc, as modeled by the tail-suspended rat.

The effect of blocking a nutritional pathway to the intervertebral disc in the dog model.

Background The hypothesis that injecting bone cement adjacent to one or both endplates would bring about degeneration in the intervening disc was tested. Methods In 11 dogs, bone cement was injected just below the superior endplates of L1, L2, and L3 to block the nutritional supply through these endplates to the three intervertebral discs T13–L1, L1–L2, and L2–L3. In one other dog, both the superior and the inferior endplates of the same discs (T13–L1, L1–L2, and L2–L3) were blocked with bone cement. All 12 dogs were euthanized between 31 and 70 weeks after the surgery. The three experimental discs (T13–L1, L1–L2, and L2–L3) and two control discs (T12–T13 and L4–L5) were excised and assessed using enzyme-linked immunosorbent assay (ELISA) and histology. Results Radiographs of the lumbar spine at the time of death did not show any signs of disc bulging, disc space narrowing, or peripheral osteophyte formation in any of the 12 dogs. The experimental discs as well as the control discs appeared normal in every dog. After the discs were bisected, they were carefully inspected for any visible signs of degeneration. The experimental discs showed no clear signs of disc degeneration and were not distinguishable from the control discs on a gross level. The numerical results from the ELISA showed that in the experimental discs as opposed to the control discs, there were significant increases in proteoglycan content in both the nucleus (P = 0.033) and annulus (P = 0.01) and clear histologic changes in some of the discs. Conclusion The results show that injecting bone cement adjacent to one or both endplates for up to 70 weeks does not produce degeneration in any visible form in the intervening disc. There were no disc bulging, no apparent annular fissures, and no disc spacing narrowing. There were, however, increases in protoglycan content in both the nucleus and the annulus and clear histologic changes in some of the discs.

Aberrant metabolism of matrix components in neonatal fibular cartilage of brachypod (bph) mice.

Abstract Collagen and the acid mucopolysaccharides (AMPS) were studied in the fibular cartilage of brachypod ( bp H bp H ) and normal (+/+) newborn mice. At this age the mutant fibulae are still cartilaginous and are comprised of closely packed chondrocytes, homogenous in size and shape. Brachypod fibular chondrocytes synthesized a normal cartilage-type collagen molecule but at half the normal rate. Incorporation of tryptophan indicated this was related to a depression of general protein synthesis rather than being specific for collagen. Pulse-chase experiments showed that collagen degradation over a 3-day culture period was 15% slower than normal thus accounting for the higher collagen content in mutant fibulae. AMPS synthesis in normals and brachypods was nearly equal; however, in pulse-chase experiments radioactivity could not be chased out of the mutant tissue. The failure of AMPS degradation also accounted for greater than normal quantity of AMPS in the mutant cartilage. Characterization of the AMPS led to the discovery of a small population of unsulfated chondroitin molecules in normal, but not brachypod cartilage. The importance of a coordinated metabolism of matrix products during limb development is discussed.

Analysis of chondroitin sulfate in lumbar intervertebral discs at two different stages of degeneration as assessed by discogram

Previous studies have presented evidence that an underlying cause of intervertebral disc degeneration is related to changes in the sulfation of the proteoglycans. The sulfation of the chondroitin in cadaveric lumbar intervertebral discs, at two different stages of degeneration as assessed by discogram, were analyzed. Fourteen of 28 lumbar discs were graded 2 and the other 14 were graded 4 (i.e., more degenerated). From each disc, six regional segments were carefully isolated. Proteoglycans were solubilized from the disc tissue with 4 M GuHCl. Chondroitin sulfate chains were analyzed by diethylaminoethyl (DEAE)-Sephacel and high-performance liquid chromatography (HPLC) anion exchange chromatography. The major differences in sulfation of the chondroitin between grade 2 and grade 4 discs only occurred in the posterior central annulus and nucleus segments. The chondroitin in the posterior central nucleus segments of the grade 2 and grade 4 intervertebral discs were undersulfated as compared with the other segments, and the differences between these segments and the others were more accentuated in the grade 4 discs than in grade 2 discs.

Purification of a mouse embryo extract component which enhances chondrogenesis in vitro

Abstract This report describes the purification of a proteinaceous component of mouse embryo extract which enhances the chondrogenic expression of 11.5-day mouse limb mesenchyme grown in culture. The factor, purified by ultracentrifugation and molecular-sieve and DEAE-cellulose chromatography, is nondialyzable, periodate stable, trypsin sensitive, and heat precipitable at 65°C in pure form. Treatment of the preparation purified by DEAE chromatography with chloroform:methanol reveals that the biological activity remains in the aqueous phase. The purified component, with a molecular weight of 50,000 by SDS gel electrophoresis, appears to be a glycoprotein or phosphoprotein. When compared to controls, the addition of microgram quantities of the purified factor to cultures of 11.5-day mouse limb mesenchyme causes (1) a threefold enhancement of 35 SO 4 incorporation into sulfated glycosaminoglycans, (2) a fourfold increase in their accumulation, (3) a stimulation of cartilage collagen type II deposition, and (4) an acquisition of a morphology and organization characteristic of cartilage nodules within a 48-hr culture period. Measurements of free sulfate and amino sugars and their absolute specific activities, respectively, reveal that the component produces a small increase in the cellular concentration of free sulfate, but has no effect on free amino sugars. Comparison of extracts prepared from 10- and 13-day mouse embryos reveals that the activity is present in embryos of both ages, but is more effective in the older embryos.