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.

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.

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 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.

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.

Morphological analysis of abnormal digital chondrogenesis in the brachypod (bpH) mouse limb in organ culture.

Brachypod (bpH/bpH), an autosomal mutation in mice, is characterized by a shortening of the long bones and paws, and a delay or absence of ossification in some of the distal limb elements. The present study represents a detailed description of the brachypod phenotype in day 12 hindlimb buds maintained for 6 days in a submerged, serum-free organ culture system. Using this in vitro system, the proximal-to-distal effect on the severity of cartilage reduction was intensified in the brachypod explants with an intermediate expression in the heterozygotes. Immunofluorescent staining of the brachypod cartilage revealed a deficiency in and an abnormal distribution of the proteoglycans. Although there was no recognizable difference in the immunofluorescent staining for type II collagen between the mutant and wild-type, electron micrographs showed the presence of thick fibrils in the matrix. Other atypical structures in the brachypod cartilage included pleomorphic nuclei, reduced intracellular glycogen granules and profuse intercellular contacts. It is proposed that with the use of this in vitro system which supports the autonomous development of the individual limb elements, experiments concerning the pathogenesis of skeletal mutations such as brachypod should be more feasible.

Lack of chondrogenic expression in mouse limb bud micromass cultures exposed to exogenous beta galactosidase or N-acetyl-beta-glucosaminidase.

The effect of two exoglycosidases, beta-galactosidase and N-acetyl-beta-glucosaminidase (GlcNAc-ase) on chondrogenic expression of stage 19 mouse limb bud micromass cultures was investigated. Chondrogenic expression was monitored by Alcian blue staining and immunofluorescent localization of cartilage-specific proteoglycan and type II collagen. Chondrogenesis was inhibited by exposure to 0.1 U/ml beta-galactosidase or 0.025 U/ml GlcNAc-ase for 24 h or longer in culture. The effect of both enzymes was concentration and time dependent. Exoglycosidic hydrolysis of galactose or N-acetylglucosamine was substantiated by treatment with HRP-conjugated peanut agglutinin and succinylated wheat germ agglutinin, respectively. Cells treated with beta-galactosidase appeared to be flattened with a stellate morphology, whereas GlcNAc-ase-treated cells were bipolar forming ridge-like mounds that had a directional orientation. The antichondrogenic effect was not alleviated when the cells were induced to assume a spherical shape upon treatment with cytochalasin D. DNA measurements indicated that the lack of chondrogenic expression was not related to cell attachment or cell proliferation. These data support the hypothesis that the expression of specific terminal sugars on cell surface glycoconjugates of limb bud cells represents an important component of the chondrogenic process.

Effect of prostaglandin E2 on cyclic AMP levels in limb cells of mouse mutant brachypodism.

Mouse embryo limb cells carrying either the brachypodism (bpH/bpH) mutation or its wild-type (+/+) allele were tested for their ability to accumulate cyclic AMP in response to prostaglandin E2 (PGE2) between Embryonic Days E12 and E14. Mutant cells exhibited a precocious increase in cyclic AMP. In the absence of PGE2 but in the presence of the phosphodiesterase inhibitor 1-methyl-3-isobutylxanthine (MIX), the brachypodism cells accumulated a significantly lower amount of cyclic AMP by Day E14. Limb cells carrying the bpH mutation may provide a useful experimental system to study the PGE2-cyclic AMP-cartilage differentiation interrelationship.

Alterations in cell surface galactosyltransferase activity during limb chondrogenesis in brachypod mutant mouse embryos.

The autosomal mutation brachypod (bpH/bpH) in the mouse affects the development of precartilage mesenchymal condensation in the limb-bud. We have previously shown that this defect is localized to the expression of terminal N-acetylglucosamine (GlcNAc) glycoproteins in the plasma membrane (Elmer and Wright, '83). The present study is focused on cell surface galactosyltransferase (GalTase), an ectoenzyme that transfers galactose to its GlcNAc substrate. Purified plasma membrane preparations derived from wild-type (+/+), heterozygote (+/bpH) and brachypod (bpH/bpH) embryonic mouse limb cells were assayed for GalTase activity during in vitro and in utero chondrogenesis using High-Performance Liquid Chromatography (HPLC). On embryonic day E12, prior to overt expression of the mutant gene, no significant difference in GalTase activity was observed. By the third day in culture, all major chondrogenic elements of the autopod were present in +/+ and +/bpH embryos, whereas the mutant autopods were markedly deficient in staining and appeared consistently shorter. The accumulation of alcianophilic cartilage matrix in the wild-type was accompanied by a 29% increase in GalTase activity, which reflected the net change (29%) observed during development from days E12 to E13 in utero. The GalTase activity for the in utero E13 mutant (13%) was significantly different from control. In culture, day E12 mutant autopods actually decreased in their GalTase level by 3 days so that the activity was reduced to only 57% of the wild-type. Though GalTase activity in the heterozygote showed an intermediate expression, optical image analysis did not reveal consistent differences in cartilage development when compared to +/+, arguing against a gene-dosage effect at the gross anatomical level. These data indicate that an increase in plasma membrane GalTase activity is a natural developmental event that occurs during limb-bud chondrogenesis and a decrease in GalTase activity contributes to the dysmorphogenesis in brachypod limb-buds.

Evidence for negative control of growth by adenosine in the mammalian embryo: induction of Hmx/+ mutant limb outgrowth by adenosine deaminase.

We investigated the growth-regulatory actions of adenosine and adenosine deaminase (ADA) during embryonic limb development in the mouse. Polydactylous outgrowth, an expression of the Hemimelia-extra toe (Hmx/+) mutant phenotype, was experimentally regulated in hindlimb buds explanted into a serum-free in vitro system at stage 18 of gestation. Its expression was promoted by exposure to 0.1 or 0.2 IU/ml exogenous ADA and suppressed by co-exposure to 10 nM (-)-N6-(R-phenylisopropyl)-adenosine (N6-PIA). Evidence that N6-PIA acted as a high-affinity agonist against the external adenosine receptor was provided by experiments in which 100 microM caffeine, a known antagonist, competitively blocked its effect. The endogenous adenosine content was analyzed by reversed-phase high-performance liquid chromatography with fluorometric detection following its conversion to the 1,N6-ethenoadenosine derivative. At stage 18, the adenosine levels were 0.5 pmol/micrograms DNA in whole embryos and 0.08 pmol/micrograms DNA in hindlimb buds. At the same stage, partially purified extracts of the embryonal plasma enriched fraction contained high levels of ADA activity (0.04-0.06 IU/embryo, or 0.7-1.0 IU/mg protein). In contrast, blood cells contained 0.0001 IU/embryo (or 0.01 IU/mg protein). This enzyme occurred as a single kinetic form with a molecular weight of 45000-47000 daltons and an apparent Km of 36-38 microM. Its presence in the embryonal plasma argues against an endocrine mechanism of adenosine secretion in favor of autocrine (self-regulatory) or paracrine (proximate-regulatory) mechanisms. Taken together, our results suggest that the in vitro outgrowth of the prospective polydactylous region is induced upon escape from the local growth-inhibitory influence of extracellular adenosine.(ABSTRACT TRUNCATED AT 250 WORDS)

Elevated rate of DNA synthesis and its correlation to cAMP-phosphodiesterase activity during induction of polydactyly in mouse embryos heterozygous for Hemimelia-extra toe (Hmx).

The induction of polydactyly in mouse embryos heterozygous for Hemimelia-extra toe (Hmx) is associated with aberrant outgrowth of the developing autopod on day 12 of gestation. We have quantitated the rate of DNA synthesis and the activity of cAMP-phosphodiesterase (PDE) that is characteristic of the prospective polydactylous region. Mid-stage 18 hind-limb buds were labeled with [3H]dThd either in situ using whole embryo culture, or as isolated preaxial autopod fragments cultured on a membrane substratum. The mean specific activities of incorporation were compared for normal (+/+) and mutant (Hmx/+) genotypes. A significant (P less than or equal to 0.01) 19% increase, peculiar to the prospective polydactylous region, was measured after 4 hours in embryo culture. The same increment was detected after 4 hours in organ culture, but was amplified linearly to 55% when incubation was extended to 20 hours. During this period, continuous exposure to 1.0 mM IBMX (3-isobutyl-1-methyl xanthine), an inhibitor of cAMP-PDE activity, "slowed down" the rate of DNA synthesis to untreated +/+ proportions. When cAMP-PDE activity was assayed in uncultured autopods, a significant (P less than or equal to 0.01) 18% increase was detected within the prospective polydactylous region specifically on stage 18 of gestation. This is the developmental phase during which polydactylous outgrowth is induced in situ. Thus, uncontrolled cAMP-PDE activity may, in part, provoke the enhanced rate of cell proliferation.

Immunohistochemical localization of cyclic AMP during normal and abnormal chick and mouse limb development.

This paper describes the immunohistochemical localization of cAMP during limb chondrogenesis in talpid3 chick, brachypod mouse, and normal embryos. Comparisons were made between chick wing buds at Stages 22, 25, and 30, and mouse hind limb buds at Days 11, 12.5 and 14. At Stage 22, the normal mesenchyme in the chick displayed areas of bright fluorescence compared to a lesser intense and more evenly distributed fluorescence in talpid3. Sections of the central region from normal Stage 25 limb buds exhibited an intense fluorescence that was uniformly distributed, whereas, in talpid3 staining was more mosaic with some areas fluorescing brightly and others showing little fluorescence. At Stage 30 the staining pattern was similar between normal and talpid3, with the fluorescence being brighter in the cartilage tissue than in the surrounding soft tissue. Difference in the staining patterns of normal and brachypod limb tissue were not detectable. At Days 11 and 12.5, tissue from both genotypes displayed a very bright, uniform fluorescence. In the 14-day hind limb buds, the staining patterns were comparable to those observed in Stage 30 chick wing buds. However, under in vitro conditions conducive for the expression of the chondrogenic phenotype, differences in the intensity and extensiveness of fluorescent staining were detectable in cultures derived from 12-day normal and brachypod hind limb mesenchyme. Compared to the control, the uneven distribution of immunofluorescence in the talpid3 limb buds and the differences in intensity and extensiveness of fluorescence in the brachypod cultures support the hypothesis that cAMP is involved in limb cartilage differentiation.

Analysis of glycosaminoglycans during chondrogenesis of normal and brachypod mouse limb mesenchyme.

Studies have been carried out to characterize radioactive incorporation rates and steady-state levels of hyaluronic acid (HA) and chondroitin sulfate (CS) as well as hyaluronidase activity in the hind limbs of normal and brachpod mouse embryos between 11-14 days of gestation. The results of the analysis show that changes in the synthetic and degradative rates of HA and CS occur at about the 12.5-day stage in normal hind limbs. These changes include an increased rate of CS synthesis, a decreased rate of HA synthesis, and a correspondingly sharp, transitory rise in hyaluronidase activity. Similar changes also occur in brachypod hind limbs but appear to be delayed in onset by approximatly one-half day. In addition, the mutant hind limb exhibits a slower loss of HA at a time when turnover would be expected to be on the increase. These changes are concomitant with cell surface alterations and abnormal mesenchymal condensation formation which have been previously shown to occur in this mutant.

Cell adhesion and chondrogenesis in brachypod mouse limb mesenchyme: fragment fusion studies.

This study is a continuing investigation of the effect of the brachypod mouse mutation on cell interactions and chondrogenesis during early limb development. In this report, cell adhesiveness was assessed in fused fragments of brachypod and normal limb-bud mesenchyme. Examination of the interface of fused distal postaxial limb fragments show brachypod limb mesenchyme to be more adhesive than normal limb mesenchyme. Chondrogenesis within brachypod fragments is delayed and less extensive than in normal fragments. In addition, chondrogenesis within normal fragments is not affected by the juxtaposition of the brachypod fragment, and vice versa.