Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain.

BACKGROUND CONTEXT: One of the advantages of chemonucleolysis for the treatment of a herniated intervertebral disc is the potential for the disc to self-repair. It has been suggested that the enzymes used for chemonucleolysis differentially affect the potential of the disc cells to promote repair. PURPOSE: To test the ability of nucleus pulposus and anulus fibrosus cells to repair the extracellular matrix degraded in vitro by either chondroitinase ABC or chymopapain. STUDY DESIGN: An alginate cell culture system was used to monitor the progress of matrix repair after chemonucleolysis in vitro. METHODS: Rabbit nucleus pulposus or anulus fibrosus cells precultured for 10 days in alginate gel were briefly exposed to low concentrations of chondroitinase ABC or chymopapain and then returned to normal culture conditions for up to 4 weeks. At each time point, the contents of DNA and matrix macromolecules and proteoglycan synthesis were measured. RESULTS: The DNA content of enzyme-treated alginate beads during the following 4 weeks of culture was higher in the chondroitinase ABC group than in the chymopapain group (NP, p<.01, and AF, p<.05). The content of proteoglycan in beads containing nucleus pulposus and anulus fibrosus cells in the chondroitinase ABC group was higher than that in the chymopapain group (NP and AF, p<.001). The rate of proteoglycan synthesis and the content of collagen did not, however, differ between those two groups. CONCLUSIONS: Intervertebral disc cells exposed to chondroitinase ABC reestablish a matrix richer in proteoglycan than cells exposed to chymopapain. This may be because of differences in the substrate spectrum of each enzyme. Although these results cannot be translated directly to the in vivo situation, they suggest the possibility that cells in discs subjected to chondroitinase ABC-induced chemonucleolysis retain a greater ability to replenish their extracellular matrix with proteoglycans than cells in discs exposed to chymopapain.

Removal of nucleus pulposus from the intervertebral disc - the use of chymopapain enhances mechanical removal with rongeurs: a laboratory study.

BACKGROUND: A laboratory study was conducted, on cadaveric sheep spines to develop an effective procedure for removing as much nucleus as possible from an intervertebral disc with minimal disruption to the annulus. The results of many studies involving removal of nucleus, including chemonucleolysis, using chymopapain, have been published but we are not aware of any previous quantitative studies on procedures for removing as much nucleus as possible from the disc. METHODS: All procedures were performed via a 3 mm trocar. Four procedures were compared: (I) unilateral approach using rongeurs alone, (II) bilateral approach using rongeurs alone, (III) unilateral approach using rongeurs followed by chymopapain and (IV) bilateral approach using rongeurs followed by chymopapain. RESULTS: The percentages of nucleus removed were: (I) 34%, (II) 41%, (III) 52% and (IV) 75%; there were significant differences between the four sets of results according to ANOVA. CONCLUSION: Significantly more nucleus is removed using a bilateral than a unilateral approach; significantly more nucleus is removed if chymopapain is used in addition to rongeurs. A brush is useful in removing strands of nucleus loosened by chymopapain.

Fibronectin fragment mediated cartilage chondrolysis. II. Reparative effects of anti-oxidants.

In an accompanying manuscript, it was shown that the cartilage chondrolytic activities of fibronectin fragments (Fn-f), which are mediated through catabolic cytokines such as TNF-alpha, IL-1 and IL-6, could be suppressed by anti-oxidants (AOs). The AOs neutralized reactive oxygen species (ROS) which are known to mediate catabolic cytokine action. The objective in this work was to test whether AOs would promote restoration of proteoglycan (PG) in Fn-f treated cartilage, since under normal culturing conditions, PG is not restored after removal of the Fn-f. Cartilage was first cultured with an amino-terminal 29-kDa Fn-f to cause loss of about half of the total PG and then treated with NAC (1 and 10 mM) or glutathione (10 microM) or DMSO (0.1 or 1%). Treatment with NAC and glutathione maximally caused restoration of PG within 14 days to normal or supernormal levels, while DMSO was less effective. Catalase, but not superoxide dismutase, enhanced PG content to a small but significant extent. The restoration of PG in Fn-f treated cartilage occurred throughout the full depth of the cartilage slices as shown by histochemical analysis. However, removal of the AO allowed a subsequent decrease in PG content suggesting that the AOs had not blocked cytokine expression but had merely suppressed cytokine activities. Addition of NAC to IL-1 treated cartilage promoted a restoration of PG, while addition to chymopapain or trypsin treated cartilage was not very effective, suggesting that the effect of AOs requires a cytokine driven damage system. We conclude that the AOs promote a restoration of PG in the Fn-f treated cartilage by suppressing the effects of catabolic cytokines. The data suggest a potential for AOs in reversing tissue damage caused by cytokines.

Enrichment of peripheral blood CD34+ cells for transplantation using a fully automated immunomagnetic cell selection system and a novel octapeptide releasing agent.

Positive selection of CD34+ cells is being increasingly performed to support hematological reconstitution following high-dose and dose-intensive chemotherapy and to reduce the non-target cell content of transplants. The present study was designed to evaluate the performance of an immunomagnetic cell selection system, including comparison of enzyme and peptide releasing agents and of semi-automated and fully automated selection systems. A total of 74 immunomagnetic CD34+ cell selection procedures were performed involving 55 subjects, the majority of whom had hematologic malignancies. Median CD34+ cell purity with a newly developed specific octapeptide releasing agent (98.5%; 81.0-99.0%) was significantly higher (P = 0.002) than that with chymopapain (85.8%; 28.1-99.7%). No significant differences were observed between semi-automated and fully automated systems in CD34+ cell purity or yield or time to WBC or platelet recovery. Immunomagnetic selection was found to provide highly purified populations of CD34+ cells in sufficient numbers for use in transplantation procedures. CD34+ cell transplants supported rapid and reliable hematologic reconstitution. Use of a fully automated system markedly reduced the time and labor required for immunomagnetic selection, potentially affording more standardized and reproducible positive selection of CD34+ cells.

Ultrastructural study of the short-term effects of chymopapain on the intervertebral disc.

The initial effects of chymopapain, a chemonucleolytic agent, on the intervertebral disc of dogs were studied by light and electron microscopic techniques. Fragments of nucleus pulposus and annulus fibrosis were incubated with chymopapain up to 24 h in vitro. Proteoglycans and matrix proteins were rapidly removed, while collagen fibers remained intact up to 24 h. For several hours, most cells remained normal in appearance with only slight swelling and an increased number of vacuoles. After exposure to the protease for 24 h cells in both the annulus and nucleus showed extensive membrane damage and some were necrotic, but many survived relatively intact. These results suggest that, similar to the results of the digestion of cartilage with other proteases, the cells of the disc can survive brief chymopapain exposure during chemonucleolysis procedures and could serve as a source for regenerating tissue. The nature of the regeneration may depend on the extracellular scaffold that remains and the nutrition available to tissue as well as the age and biomechanical state of the disc. As for clinical significance, chemonucleolysis is an important nonsurgical alternative for treating prolapsed disc. The cells of nucleus and annulus can survive short-term exposure to treatment, and thus be responsible for partial regeneration of the tissue. This regeneration may be important in preventing long-term degenerative disease in the facet joints caused by increased pressure due to decreased disc height.

The use of intra-articular Na-hyaluronate as a potential chondroprotective device in experimentally induced acute articular cartilage injury and repair in rabbits.

OBJECTIVE: This study examined if viscosupplementation from intra-articular administration of a commercially available form of hyaluronan (HA) could promote the restoration of proteoglycan (PG) depletion induced by chymopapain and then if the repair could be maintained once HA treatment was discontinued. METHODS: Animals received cartilage injury with intra-articular chymopapain (2.0 mg) followed by weekly treatment with intra-articular HA. HA treated animals were compared to injured animals with no treatment, contralateral untreated joints and joints from normal controls. The effect of intra-articular HA alone on articular cartilage was also examined. RESULTS: Serum keratan sulfate levels confirmed degradation of the cartilage PGs in the chymopapain-injected knees. Intra-articular chymopapain resulted in marked loss of PGs. There were no significant differences among the control groups (untreated control, HA/800 treatment only). HA treatment did not affect the loss of PGs caused by chymopapain after 42 days. However, in animals receiving chymopapain injury followed by weekly HA treatment for 42 days and then 42 days of free cage activity without HA, cartilage PG contents were significantly increased. Intra-articular HA alone had no effect on the articular cartilage. CONCLUSION: The results in the present study suggest a potential protective effect of HA on chymopapain-induced acute articular cartilage injury in rabbits that, in time, permits damaged cartilage to resynthesize matrix PGs after the HA treatment is discontinued.

Chymopapain-induced reduction of proinflammatory phospholipase A2 activity and amelioration of neuropathic behavioral changes in an in vivo model of acute sciatica.

The mechanism of action underlying chymopapain (Chymodiactin) chemonucleolysis remains obscure. Radiographic studies suggest that chymopapain does not alter disc fragment size acutely; nonetheless, patients often report symptom resolution within a few days, even hours, of treatment. The authors postulate that, in addition to its chemonucleolytic action, chymopapain may possess antiinflammatory properties. To test this hypothesis, the authors assessed the ability of chymopapain to modulate the activity of the proinflammatory enzyme phospholipase A2 (PLA2) and to ameliorate behavioral changes associated with inflammatory neuropathy in an in vivo model of sciatica. Thirty-nine male Fischer rats were randomly assigned to one of three treatment groups: 1) saline, 2) betamethasone, or 3) chymopapain. All of the rats underwent unilateral sciatic nerve ligation with loose chromic gut suture to induce inflammatory mononeuropathy. The animals were tested for thermal and mechanical hyperalgesia on Days 0 (preoperation), 7 (pretreatment), and 14 (prior to death). Three animals were killed on Day 0 to determine the baseline PLA2 activity within unmanipulated rat sciatic nerves. On Day 7, three animals from each group were killed to assess PLA2 activity prior to treatment. The remainder were given a single infusion of saline, betamethasone (0.3 mg/kg), or chymopapain (100 pKat U) around the inflamed nerve. On Day 14, the remaining animals were killed and their sciatic nerves were removed. The tissue was homogenized and the PLA2 activity was determined using [14C]arachidonate-labeled Escherichia coli phospholipid membrane as a substrate. Lipids were extracted and separated by thin-layer chromatography. All animals developed behavioral changes consistent with inflammatory mononeuropathy 24 to 72 hours postoperatively; these included gait disturbance, flexion deformity, and hyperalgesia of the involved hindlimb. The degree of mechanical and thermal hyperalgesia was comparable between groups at Day 7. By Day 14, the thermal hyperalgesia had resolved; the mechanical hyperalgesia was less evident in the betamethasone- and chymopapain-treated groups than in the saline-treated controls (p = 0.003; saline- vs. chymopapain-treated groups p = 0.004; saline- vs. betamethasone-treated groups p = 0.008). The mean PLA2 activity at baseline (Day 0) was 11.6 +/- 4.9 nmol phospholipid hydrolyzed per minute per milligram of protein. The PLA2 activity at Day 7 was 74.4 +/- 18.2 (ligated side) and 21.2 +/- 11.7 (nonligated side). At Day 14, PLA2 activity was reduced in the chymopapain- (47.8 +/- 12.3) and betamethasone- (39.7 +/- 9.5) treated groups compared with the saline control group (62.3 +/- 11.2), (saline- vs. chymopapain-treated groups p < 0.05; saline- vs. betamethasone-treated groups p < 0.01). The PLA2 activity in nonligated specimens was 18.6 +/- 10.1. These data indicate that chymopapain exhibits antiinflammatory properties in vivo, reducing PLA2 activity and ameliorating mechanical hyperalgesia in this model of inflammatory sciatic neuropathy.

Inadequacies and hazards of chymopapain injections as treatment for intervertebral disc disease.

Chymopapain chemonucleolysis was performed on 100 patients with primary lumbar intervertebral disc disease. The results were compared with those of 174 patients who underwent laminotomy, foraminotomy, and discectomy. Primary lumbar intervertebral disc disease was arbitrarily divided into degenerative, complex, previous surgical, and simple disc syndromes. No difference was seen between chemonucleolysis and surgery in the first three divisions; between 55 percent and 60 per cent of patients responded successfully to treatment. In the simple disc division 89 per cent of the surgical and 60 per cent of the chemonucleolysis patients had successful results.

Chymopapain, chemonucleolysis, and nucleus pulposus regeneration.

UNLABELLED: In the adult mongrel dog, in vivo injection of chymopapain into the intervertebral disc resulted in disc-space narrowing at two weeks, with a complete loss of proteoglycan (as indicated by safranin-O staining) from the nucleus pulposus, the cartilaginous end-plates, and the annulus fibrosus. As demonstrated by [35S]sulphate-labeling and proteoglycan isolation, the nucleus pulposus retained the ability to synthesize proteoglycans, but these were degraded by endogenous proteolytic activity. Three months after chymopapain treatment the intervertebral disc showed an increase in height. There was a return of intense safranin-O staining in the annulus and the cartilaginous end-plates, and very prominently in the nucleus. The proteoglycans that were present were recovered as aggregates, with the proteoglycan monomer being slightly larger than in the controls. Six months after chymopapain treatment the intervertebral disc had increased further in height, and normal histology had been restored. The chemical composition and physical properties of the proteoglycans that were isolated from the nucleus pulposus were essentially the same as those from the controls. These observations suggest that the nucleus can regenerate following the injection of chymopapain. CLINICAL RELEVANCE: Our observations demonstrate that chymopapain has a profound but reversible effect on the intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with the loss of proteoglycan content and structure. The restoration of normal disc height following chymopapain injection is explained by reconstitution of the intervertebral disc with normal proteoglycans. In experimental animals, chemonucleolysis with chymopapain appears to be less likely than surgical excision to permanently alter the biochemistry of the nucleus pulposus.

Preoperative psychological tests as predictors of success of chemonucleolysis in the treatment of the low-back syndrome.

Pre-injection psychological test scores, surgeon ratings, and patient biographical data were evaluated as predictors of success of chymopapain injection therapy in 130 patients who would otherwise have been treated by laminectomy. Three psychological tests were administered to each patient: the Minnesota Multiphasic Personality Inventory, the Cornell Medical Index, and the Quick Test (a measure of mental ability). In addition, the surgeons rated their patients on a five-point scale immediately after injection relative to their suitability for injection therapy and at one year relative to the objective and symptomatic results. The MMPI hysteria and hypochondriasis scales and the surgeon's rating of the psychogenic component of the patient's pain were predictive of the result of chemonucleolysis. The patient's biographical data which consisted of age, sex, marital status, occupation, and education were not related to postoperative outcome.

Effect of X-ray contrast media on the action of chymopapain on the intervertebral disc: an in vitro study of cartilage degradation.

A representative selection of X-ray contrast media was tested for their effects on the action of chymopapain on cartilage proteoglycan in an in-vitro test system. Inhibition of the action of chymopapain was detected only at high concentrations of contrast media, and was more marked with ionic contrast media than with non-ionic media. A dose-response curve for the release of glycosaminoglycan by chymopapain was linear when the amount of enzyme was plotted on a logarithmic scale against glycosaminoglycan release. In view of our results, we suggest that there is no need for a delay between injection of contrast medium and enzyme, contrary to the instructions of the distributors of chymopapain, if small amounts of a non-ionic contrast medium are used.

Acute increase in lateral bending of the canine lumbar disc following chymopapain injection.

Twenty-four lumbar discs in six adult beagle dogs were injected with either 0.1 ml CP (ChymodiactinR) (200 U) (12 discs) or 0.1 ml saline (12 discs) under direct vision at laparotomy. All animals were killed 1 hour later; the lumbar spine was immediately excised en bloc and frozen until biomechanical testing. After overnight thawing, test specimens were prepared and consisted of a disc and half a vertebral body on each side. The posterior elements were discarded. Each motion segment was mounted on a testing plate, with bone cement used for fixation. Lateral bending of the motion segment was tested as an analog to the motion of a disc during correction of a scoliotic spine with a distraction force. Lateral bending forces of 2 kg and 4 kg were applied for 30 minutes each. Lateral bending, creep, and residual deformity (in degrees) were recorded. CP injected disc showed an acute increase in lateral bending of 28% and 26%, respectively (2-kg and 4-kg loads) (P less than 0.001). In addition, CP injected discs showed a 25% increase in their residual angular deformity compared with the control discs (P less than 0.001). No significant changes were noted in creep values between CP and saline injected discs. This study demonstrates a significant acute increase of 28% and 26% in lateral bending of the canine disc following enzyme injection.

Levels of keratan sulfate-bearing fragments rise predictably following chemonucleolysis of dog intervertebral discs with chymopapain.

Chymopapain (1 mg) was injected into each of four-lumbar intervertebral discs of adult mongrel dogs. As expected, at 2 weeks, all injected discs exhibited marked loss of height (mean: 50% of original height) indicative of severe proteoglycan depletion. The appearance of keratan sulfate-bearing fragments in plasma was monitored by an ELISA-inhibition assay which uses a monoclonal antibody (1/20/5-D-4) specific for an epitope present only in the longest keratan sulfate chains. Levels of plasma keratan sulfate rose within 30 minutes and reached a maximum between 24 and 72 hours later. Levels then declined progressively but were still elevated at 2 weeks postinjection. Keratan sulfate-bearing fragments in plasma were purified by ion exchange chromatography on DEAE-Sephacryl and fractionated by sieve chromatography on Sepharose CL-6B. These plasma keratan sulfate-bearing fragments were found to be similar in size to keratan sulfate-bearing fragments generated by chymopapain digestion of dog nucleus pulposus proteoglycans, but slightly larger than single keratan sulfate chains obtained by alkaline borohydride treatment of dog nucleus pulposus proteoglycans. The results of this study show that measurements of blood levels of keratan sulfate could prove useful in monitoring effective degradation of disc proteoglycans in chemonucleolysis in man and help discriminate between ineffective enzyme placement, and alternative mechanisms of treatment failure.

Water, fixed charge density, protein contents, and lysine incorporation into protein in chymopapain-digested intervertebral disc of rabbit.

Chymopapain (Discase) was injected at a dose of 0.125 nanokatal unit into the intervertebral discs of rabbits, and sequential changes in the metabolism of water, proteoglycan, collagen, and noncollagenous protein were investigated separately in the nucleus pulposus, anterior, and posterior anulus fibrosus. One week after chymopapain injection, the water and proteoglycan content was lower in all of the fractionated tissues of the anterior and posterior anulus and nucleus pulposus of the discs than in the control discs. In the anterior and posterior anulus, the proteoglycan content recovered after 12 weeks, but there was no recovery in the nucleus pulposus. The collagen content continued to increase up to the 12th week in the nucleus pulposus, while the noncollagenous protein content decreased in all tissue fractions after 1 week. In the anterior and posterior anulus, the content of noncollagenous protein recovered after 3 to 6 weeks, but there was no recovery in the nucleus pulposus. The lysine incorporation in collagen and noncollagenous protein was inhibited in all tissue fractions after 12 weeks, suggesting a decrease in synthetic activity. The intradiscal pressure calculated from proteoglycan hydration at 1 to 6 weeks after chymopapain injection showed a marked decrease to 0.8 to 0.9 atm, but it recovered to 1.6 atm after 12 weeks.

The effects of chemonucleolysis on the mechanical properties of the canine lumbar disc.

One hundred and twenty-two lumbar intervertebral discs from 43 mongrel dogs were used to study the effect of chemonucleolysis on the flexion, torsion, and lateral bending flexibilities of the disc. The dogs were killed 2, 4, 12, 26, and 52 weeks following injection with 0.1-0.15 ml of either crude collagenase, semipurified collagenase, or chymopapain. Controls consisted of saline-injected and uninjected discs. The bending and torsional properties of each disc were determined by applying incremental moments up to 0.8 Nm and measuring the resultant rotations 60 seconds after each load increment was applied. The discs were then sectioned for morphologic evaluation. Increases in disc flexibilities ranging from 1.4 to 5.8-fold were found 2 weeks after injection with all three enzymes. The largest increase was noted in flexion in discs injected with chymopapain. By 3 months, all lateral bending flexibilities had returned to control values. In general, however, flexion and torsion flexibilities did not return to control values 6 months following chemonucleolysis. The extent of the gross morphologic changes produced by each of the three enzyme preparations did not correlate with the acute increases in disc flexibilities. Chymopapain and semipurified collagenase had similar morphologic and mechanical effects. The temporary increases in flexibility appeared to be due to decreases in the overall compression, tensile and shear stiffness of the annulus caused by the enzymes.

Chondroitinase ABC (pharmaceutical grade) for chemonucleolysis. Functional and structural evaluation after local application on intraspinal nerve structures and blood vessels.

STUDY DESIGN: The effects on nerve tissue and blood vessels of locally applied chondroitinase ABC were studied in two experimental models using chymopapain and the vehicle of chondroitinase ABC for controls. OBJECTIVES: To assess the effects of chondroitinase ABC on blood vessels and nerve tissue after local application. SUMMARY OF BACKGROUND DATA: Chondroitinase ABC has been suggested for chemonucleolysis because it has a high specificity for nucleus pulposus matrix, which could mean a high efficiency in dissolving disc tissue combined with a low risk of side effects on other tissues. METHODS: Chondroitinase ABC or controls were injected intrathecally in the pig, and nerve conduction velocity and histologic changes were assessed after 7 days. The same substances were injected into the hamster cheek pouch and studied for 60 minutes for microvascular effects. The vehicle for the enzyme was used as a negative control and chymopapain in a therapeutic concentration served as a positive control. RESULTS: In all series there was a slight intrathecal fibrotic reaction that was most pronounced after chymopapain injection. The effects on nerve conduction velocity and nerve morphology were similar between chondroitinase ABC and its vehicle. Chymopapain induced a significant reduction in nerve conduction velocity and pronounced histologic changes. In the cheek pouch, chymopapain induced a stand-still of blood flow at the injection site, and microhemorrhage and macromolecular leakage from the vessels at the border of the injection site. Only a slightly reduced blood flow was occasionally found after injection of chondroitinase ABC and controls. CONCLUSIONS: In agreement with the current literature, these observations indicate that chondroitinase ABC is safe regarding adverse effects on nerve tissue and blood vessels. The slight reduction in conduction velocity after intrathecal injection of chondroitinase ABC or its vehicle is most likely the result of surgical injury while releasing the nerve roots from the intrathecal fibrous adhesions. Such adhesions may be related to the laminectomy per se, and probably have no pathophysiologic significance.

Biomechanical analysis of canine intervertebral discs after chymopapain injection. A preliminary report.

With the renewed interest in using chymopapain (CP) as a chemonucleolytic agent for treatment of sciatica and low-back pain, the present study was undertaken to investigate the biomechanical property changes in canine lumbar discs after CP injections. The short-term (30- to 120-minute) in vitro effects of such an enzymatic agent appear to be the same as those of saline solution, causing increased disc heights, stiffness values, and creep rates. In the in vivo study, after three weeks, CP-injected discs had significant reductions in disc height and compressive stiffness, but the creep rate was increased substantially. However, at three months after injection, these biomechanical properties began to reverse and approached those of the uninjected controls. Buffer solution (cysteine and EDTA) was tried, but the sample size was too small to provide conclusive information. The results suggest that CP causes a disc to change its material property, but such effects appear to be time-related.

Quantitative assessment of human proteinases as agents for chemonucleolysis.

A rabbit model system is described. It allows accurate measurement of the dose-dependent loss of glycosaminoglycan from the nucleus pulposus of lumbar intervertebral discs after injection of a proteinase. At the dose equivalent to that of chymopapain used in human chemonucleolysis, two human serine proteinases, cathepsin G and chymotrypsin, were as effective as chymopapain in removing up to 80% of the glycosaminoglycan from the disc. A cysteine proteinase, cathepsin B released no more than 45% of glycosaminoglycan. X-ray films clearly showed narrowing of the disc space when 30-40% of glycosaminoglycan was removed. The degradation of the nucleus pulposus was seen histologically as loss of toluidine blue metachromasia.

Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. A biochemical and biomechanical study.

In the adult mongrel dog, in vivo injection of chymopapain into the intervertebral disc resulted, at two weeks, in disc space narrowing. However, [35S]sulfate labeling and proteoglycan characterization demonstrate that the nucleus retains the ability to synthesize proteoglycans, although they were degraded rapidly by residual proteolytic activity. Three months following chymopapain treatment, the intervertebral dog disc shows that an increase in disc height, return of nuclear material, and proteoglycan aggregate is present. At six months following chymopapain treatment, proteoglycans of similar characteristics to normal canine intervertebral disc are identified with a glucosamine/galactosamine ratio approaching normal values. Biomechanically, the short-term (30-120 minutes) in vitro effects of chymopapain appear to be the same as the carrier causing increased disc height, stiffness values, and creep rates. In the vivo study, after three weeks, chymopapain-injected discs had significant reductions in disc height and compressive stiffness, but the creep rate was increased substantially. However, at three months postinjection, these biomechanical properties began to reverse and approached those of the uninjected controls. The observations reported in this study suggest that chymopapain has a profound but reversible effect on normal canine intervertebral disc. The radiographic narrowing of the intervertebral disc following chymopapain injection correlates with loss of proteoglycan content, structure, and biomechanical properties. The restoration of normal disc height following chymopapain injection is explained by reconstitution of normal intervertebral disc. EDTA and cysteine used alone have no discernable in vivo enzymatic effect on intervertebral disc proteoglycan biochemistry. Chemonucleolysis with chymopapain would appear less likely to alter permanently proteoglycan biochemistry and the biomechanical properties of the disc than surgical excision in experimental animals.

Intervertebral disc reconstitution after chemonucleolysis with chymopapain is dependent on dosage.

STUDY DESIGN: The current report describes a study in beagles in which the effects of intradiscal injection of three doses of chymopapain were evaluated with respect to the reduction of disc width and reconstitution of the nucleus pulposus. OBJECTIVES: To establish an intradiscal dose of chymopapain that would achieve optimal reduction in disc height followed by maximum reconstitution of the nucleus pulposus. SUMMARY OF BACKGROUND DATA: Earlier reports of the efficacy of high and low doses of chymopapain for chemonucleolysis have provided conflicting data, and a scientific basis for an appropriate dose is lacking. METHODS: Four mature, female beagles were subjected to chemonucleolysis using three doses of chymopapain as Chymodiactin (31, 63 and 125 picokatals/disc) injected into the L2-L3, L1-L2, and L3-L4 discs. Disc widths were monitored radiographically over 32 weeks. Proteoglycans were radiolabeled by intravenous injection with Na2 35SO4 (1 mCi/kg) 24 hours before sacrifice, and their specific activities (disintegrations per minute/mg proteoglycan), hydrodynamic size, and ability to aggregate determined. RESULTS: Sixty-three picokatals of Chymodiactin produced optimal disc reconstitution after chemonucleolysis. A reduction in disc height of approximately 35% was evident within 1 month and this slowly returned to approximately 90% of the preinjection value after 32 weeks. The nucleus pulposus contained approximately 75% of the proteoglycan content of control tissues, and most of these formed aggregates with hyaluronan. Disc collagen levels remained relatively unaffected by treatment. CONCLUSIONS: This study demonstrates that an effective reduction in disc width compatible with later reconstitution of the nucleus pulposus can be achieved experimentally with an appropriate dose of chymopapain. These data clearly indicate that an optimal dose of chymopapain for chemonucleolysis in humans needs to be established.