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

The Spine Journal 7 (2007) 694–700

Matrix replenishment by intervertebral disc cells after chemonucleolysis in vitro with chondroitinase ABC and chymopapain Kazuhiro Chiba, MD, PhDa,b, Koichi Masuda, MDa,b, Gunnar B.J. Andersson, MD, PhDa, Shigeki Momohara, MD, PhDb, Eugene J. Thonar, PhDa,b,c,* a

Department of Orthopedic Surgery, Rush Medical College at Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA b Department of Biochemistry, Rush Medical College at Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA c Department of Internal Medicine, Rush Medical College at Rush University Medical Center, 1653 West Congress Parkway, Chicago, IL 60612, USA Received 7 June 2006; accepted 22 September 2006

Abstract

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. Ó 2007 Elsevier Inc. All rights reserved.

Keywords:

Alginate beads; Anulus fibrosus; Chondroitinase ABC; Chymopapain; Intervertebral disc; Nucleus pulposus; Proteoglycan; Collagen

FDA device/drug status: approved for this indication. Supported in part by NIH grants AG-04736, 2-P50-AR 39239, P01-AR 48152 and research grants from the Rush University Committee on Research, the Rush Arthritis and Orthopedics Institute, and Seikagaku Corporation, Tokyo, Japan. Authors acknowledge a financial relationship (Seikagaku Corporation), which may indirectly relate to the subject of this research. * Corresponding author. Department of Biochemistry, Cohn 526, Rush University Medical Center, 1735 W. Harrison St, Chicago, IL 60612. Tel.: (312) 942-2163; fax: (312) 942-3053. E-mail address: [email protected] (E. Thonar) 1529-9430/07/$ – see front matter Ó 2007 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2006.09.005

Introduction The intervertebral disc is a specialized fibrocartilaginous connective tissue whose function is to resist the stresses applied to the vertebral column by permitting motion in all planes. Morphologically, the intervertebral disc consists of three interdependent but distinct regions: the nucleus pulposus (NP), an inner gelatinous gel rich in proteoglycans (PGs); the surrounding anulus fibrosus (AF) made up of concentric lamellae rich in collagen; and the cartilaginous

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

endplates that allow the disc to deform in the axial plane when compressed. As part of the degenerative process, matrix properties become altered in the NP. In the AF, fissures develop, weakening the anular wall, and occasionally leading to herniation of the disc. Disc herniation is a recognized cause of radiculopathy. Although most patients recover after nonoperative treatment, many require surgery to relieve pain [1,2]. Although surgical results in general are good, this is not uniformly the case. Surgery also leads to weakening of the motion segment [3], particularly when large amounts of tissue are removed, and to scar tissue formation surrounding the nerve root. For those reasons, alternative approaches have been used, including the injection of proteolytic enzymes into the disc. Chymopapain (CP), sold commercially as a mixture of proteolytic enzymes with a broad spectrum of action, was used for many years as the enzyme of choice for chemonucleolysis, an alternative treatment modality for lumbar disc herniation [4–6]. The use of CP declined dramatically after reports that the intradiscal injection of this enzyme is sometimes associated with complications, such as fatal anaphylaxis or paraplegia, because of transverse myelitis [7–11]. More recently, the intradiscal administration of collagenase [12], an enzyme that causes partial degradation of the collagen fibrillar network that entraps the PGs [13], has also been used in the clinical setting as an alternative way of effecting decompression of the nerve root. After reports of side effects, the use of collagenase has failed to gain wide acceptance and, in addition, its clinical efficacy has never been clearly established [14– 17]. Recent histological studies performed in vivo have suggested that chondroitinase ABC (C-ABC), an eliminase that degrades chondroitin sulfate, dermatan sulfate, hyaluronan, and chondroitin to unsaturated disaccharides, holds promise as an alternative chemonucleolytic agent [18–29]. Ideally, the agent of choice in chemonucleolysis should degrade PGs and thus relieve the pressure on adjacent nerves without harming the cells irreversibly. Furthermore, to prevent local destabilization from predisposing other discs to degeneration, it is important that the enzymeinduced damage does not irreversibly affect matrix regeneration so that at least part of the function of the disc as a shock absorber and stabilizer may be reestablished [30,31]. Although C-ABC appears safer than and nearly as effective as CP in degrading the PGs of the NP of herniated intervertebral discs, its long-term effects on the cells populating the NP and AF are unclear. The purpose of the present study was to determine how effective rabbit intervertebral disc cells exposed in vitro to C-ABC and CP are in reestablishing a PG-rich matrix. To reach that goal, an alginate bead culture system was used to embed the NP and AF cells in a gel in which the proper chondrocytic phenotype can be maintained [32,33] and in which the cells can be kept encapsulated in beads after enzyme digestion.

695

Materials and methods All chemicals were obtained from Sigma-Aldrich (St. Louis, MO) unless otherwise indicated. Alginate bead culture Thirty-six adolescent New Zealand white rabbits were used for the cell culture experiments with approval by the Institutional Animal Care and Use Committee. Rabbits were sacrificed by the intravenous administration of excessive pentobarbital (Euthanasia B solution; Henry Schein Inc., Washington Port, NY). The whole lumbar spine was exposed through a posterior midspinal incision and removed en bloc under aseptic conditions. Each lumbar disc was then dissected from the spine and the NP and AF separated from the endplates by sharp dissection using a scalpel. The NP and AF were separated by blunt dissection [32]. For each experiment, the NP and AF cells were separately obtained from at least eight rabbits by a wellestablished technique for isolating rabbit intervertebral disc cells [32]. Briefly, tissues were sequentially digested at 37 C with 0.4% pronase (Calbiochem, La Jolla, CA) in DMEM/F-12 medium (Mediatech, Herndon, VA) for 1 hour followed by a 16-hour digestion with 0.025% collagenase P (Clostridium histolyticum; Boehringer Mannheim, Indianapolis, IN) and 0.004% deoxyribonuclease II (SigmaAldrich) in the same medium but also in the presence of 5% fetal bovine serum (HyClone, Logan, UT). The use of deoxyribonuclease II was important to prevent excessive cell clumping [33]. After digestion, the cells were washed three times with DMEM/F-12 medium, filtered through a 70-mm nylon mesh cell strainer (Becton Dickinson, Lincoln Park, NJ), and counted in a haemocytometer. The isolated cells were resuspended in a solution of 1.2% low-viscosity sterile alginate (Keltone LV; ISP Alginates Inc., San Diego, CA) in physiological saline at a concentration of 2 million cells/mL. This solution was dispensed into a 102 mmol/L calcium chloride solution in a drop-wise manner through a 22-gauge needle attached to a 10-mL plastic syringe. After 10 minutes, the newly formed beads (containing approximately 20,000 cells/bead) were washed three times with sterile physiological saline and then twice with DMEM/F-12 [34]. The beads were then cultured in 24-well plates, with each well containing nine beads, in 0.4 mL of complete culture medium (DMEM/F12 containing 10% fetal bovine serum, 25 mg/mL sodium ascorbate, and 10 mg/mL gentamicin [GibcoBRL, Grand Island, NY]) per well. The cultures were maintained at 37 C in a humidified atmosphere of 5% CO2, with daily changes of medium. Culture protocol and biochemical assays On day 10 of culture, the beads were transferred to complete medium containing pharmaceutical grade, proteasefree C-ABC (chondroitin ABC lyase, a mixture of an

696

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

endoeliminase yielding tetrasaccharides, and an exoeliminase preferentially acting on oligosaccharides [35]) at 0.05 U/mL (a gift from Seikagaku Corporation, Tokyo, Japan; 1 unit of enzyme is the amount required for the eliminative cleavage of substrate, 1 mmol of D4-exuronate residue [35]) or CP (5 picokatals/mL, Chymodiactin; Flint Laboratories, Deerfield, IL; a pharmaceutical grade) and incubated at 37 C for 1 hour to deplete PGs from the matrix and then rinsed three times with DMEM/F12 for 10 minutes each to remove the enzymes. After rinsing, the beads were further cultured in complete medium for up to 4 weeks in 24-well plates (9 beads/well), allowing the cells to reestablish an extracellular matrix. After removing the medium, the beads were dissolved by the addition of dissolving buffer containing 0.15 mol/L NaCl, 30 mmol/L ethylenediaminetetraacetic acid and 55 mmol/L sodium citrate at 4 C for 20 minutes with gentle shaking. After mild centrifugation of the resulting suspension at 100g for 10 minutes [34], the supernatant containing molecules derived from the further removed matrix (FRM) were separated from the pellet containing the cells and their cell-associated matrix (CM). The fractions were further digested with papain (Sigma-Aldrich) at 20 mg/mL in 0.05 mol/L sodium acetate containing 0.01 mol/L cysteine hydrochloride [36] at 60 C overnight and then subjected to the following biochemical analyses: (1) DNA by the bisbenzimidazole fluorescent dye, Hoechst 33258 (Polysciences, Warrington, PA) method, using calf thymus DNA as a standard [37]; (2) sulfated PGs by the 1,9-dimethylmethylene blue (DMMB, Polysciences) assay [38]; and (3) collagen (measured as hydroxyproline) by reverse-phase high-performance liquid chromatography and fluorescence detection after acid hydrolysis with 6 mol/L hydrochloric acid and phenylisothiocyanate (Pierce, Rockford, IL) labeling [39]. For each time point, the concentration of each matrix molecule in the NP and AF was expressed per 9 beads (ie, per well). The beads were cultured in complete medium containing 35 S-sulfate (Amersham Biosciences Corp., Piscataway, NJ) at 20 mCi/mL for 4 hours after the enzyme treatment. After radiolabeling, the medium was collected, and the beads were dissolved with the sodium citrate buffer described above. After mild centrifugation, the pellets were extracted for 48 hours at 4 C with 4 mol/L guanidine HCl in the presence of proteinase inhibitors. The amount of radiolabeled 35 S-PGs in each extract was quantified by a rapid filtration assay after precipitation of the glycosaminoglycans with alcian blue [40]. Statistical analysis The experiments were repeated three times and representative data are presented. In each case, the values at each time point are reported as the mean6standard deviation for the analysis of at least three separate cultures of nine beads each. For each parameter studied, the effect of time in culture and treatment over the 4 weeks in culture were

assessed by two-way factorial analysis of variance. In addition, at each time point, the difference between the two enzyme-injected groups was assessed by using the two-tailed unpaired t test.

Results Establishment of C-ABC and CP concentrations for the in vitro treatment of beads In preliminary experiments, different concentrations of C-ABC (0.001–0.1 U/mL) and CP (2.5–40 picokatals/mL) were tested. Although none of the concentrations at which C-ABC was used led to a decrease in DNA content, CP treatment over 10 picokatals/mL resulted in a significant decrease in DNA content (10 picokatals/mL, 85%; 20 picokatals/mL, 74%; and 40 picokatals/mL, 70%; percent of control, p!.01). The concentrations of C-ABC (0.05 U/ mL) and CP (5 picokatals/mL) were chosen for use in these studies because, after 1 hour of exposure to these concentrations in vitro, no deleterious effects on the viability of NP and AF cells cultured in alginate beads were found by the Trypan Blue exclusion assay (data not shown). Also, after 48 hours of culture, these concentrations were the lowest that resulted in the depletion of more than 80% of the PG from the CM. The kinetics of PG loss in beads treated with various concentrations of these two enzymes varied markedly. At all concentrations of enzymes, the amount of PG remaining in the treated beads after 1 hour was significantly higher in beads treated with CP than in those exposed to C-ABC. It is possible that this difference simply reflects the fact that the peptidoglycan fragments, containing intact glycosaminoglycans, produced by the action of CP on PG molecules, were larger and, thus, took longer to diffuse out of the alginate gel than the disaccharides generated by the action of C-ABC. In CP-treated beads, the concentration of PG eventually decreased to less than 20% of that originally present within 2 days of culture. Effect of C-ABC and CP on the amount of matrix in alginate beads DNA Immediately after digestion, the DNA content in the NP was lower in the CP-treated group than in the C-ABC– treated group (p!.05, unpaired t test, Fig. 1, left panel). In both NP and AF beads, the content of DNA was slightly higher in the C-ABC–treated groups than in the corresponding CP-treated groups during the 4 weeks of culture (NP: p!.01; AF: p!0.05, two-way factorial analysis of variance, Fig. 1). When the difference at each time point was evaluated, this difference reached significance at 2 and 4 weeks (p!.05, unpaired Student t test) in the NP and at 3 weeks in the AF (p!.05, respectively). However, in the AF at the 1-week time point, the DNA content in the

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

Fig. 1. The DNA content of NP or AF cells in alginate beads exposed to C-ABC or CP and then cultured for 4 weeks. As described in the Materials and Methods section, the alginate beads (NP: left panel, AF: right panel) were first cultured for 10 days and then briefly incubated in the presence of C-ABC (left column in each panel) or CP (right column in each panel) to partially degrade the matrix that had been formed in vitro. The beads were then returned to normal culture conditions for up to 4 weeks. The DNA contents of the alginate beads were measured weekly and are reported for each group and at each time point as the mean6standard deviation of the value obtained for three separate cultures. The content of DNA was slightly higher in the C-ABC–treated groups than in the CP-treated groups during the 4 weeks of culture. *p!.05, **p!.01 (comparison of difference at each time point between the C-ABC–treated and CP-treated cells, two-tailed unpaired t test).

CP-treated group was significantly higher (p!.01) than that in the C-ABC–treated group (Fig. 1, right panel). Sulfated PGs Immediately after the enzyme treatments, the content of PG, measured by the DMMB assay, was higher in NP beads treated with CP than in NP beads treated with C-ABC (p!.001, Fig. 2, left panel). Nucleus pulposus cells cultured in alginate beads treated with either C-ABC or CP gradually reestablished a matrix whose PG content reached a plateau within 3 weeks (Fig. 2, left panel). Importantly, PG accumulation, expressed per 9 beads, was greater in the C-ABC–treated beads than in the CP-treated beads (p!.001, two-way factorial analysis of variance). This was true at all time points (1 week: p!.01, 2 weeks: p!.001, 3 weeks: p!.001, 4 weeks: p!.01; Fig. 2, left panel). Similar results were obtained when the data were expressed per microgram of DNA (data not shown). The AF cells showed a vigorous repair response. In the AF, the PG content reached a maximum at a lower level in the CP-treated group than in the C-ABC–treated group (p!.01, Fig. 2, right panel). The PG content decreased after 3 weeks in the CP-treated beads but not in the C-ABC– treated beads (p!.01, C-ABC vs. CP, Fig. 2 right panel at the 4-week time point). Examination of the distribution of PGs in the two matrix compartments revealed that the ability of AF cells to accumulate PGs in the CM compartment was significantly lower in the CP-treated beads than in beads exposed to C-ABC (% PG in the CM: C-ABC554.5, 52.3, 30.6, 21.5%; CP533.0, 35.4, 20.8, 11.8% at 1 week, 2 weeks, 3 weeks, and 4 weeks, respectively) (data not shown in the

697

Fig. 2. The recovery of proteoglycan content in NP or AF cells in alginate beads exposed to C-ABC or CP and then cultured for 4 weeks. As described in the Materials and Methods section, the NP and AF cells in alginate beads were first cultured for 10 days and then briefly incubated in the presence of C-ABC or CP to partially degrade the matrix that had been formed in vitro. The beads were then returned to normal culture conditions for up to 4 weeks. The content of sulfated PG per nine beads (NP: left panel, AF: right panel) was measured at each time point and is reported for each group and at each time point as the mean6standard deviation of the value obtained for three separate cultures. The proteoglycan accumulation, expressed per nine beads, was greater in the C-ABC–treated beads than in the CP-treated beads. *p!.05, **p!.01, ***p!.01 (comparison of difference at each time point between the C-ABC–treated and CP-treated cells, two-tailed unpaired t test).

figure). In the NP, no differences were found between the two groups.

Collagen Throughout the culture period, the content of collagen at each time point did not differ significantly between the CABC–treated and CP-treated groups (Fig. 3). The distribution of collagen molecules in the different compartments was similar in both groups (data not shown).

Fig. 3. The recovery of collagen content in NP or AF cells in alginate beads exposed to C-ABC or CP and then cultured for 4 weeks. As described in the Materials and Methods section, the NP and AF cells in alginate beads were first cultured for 10 days and then briefly incubated in the presence of C-ABC or CP to partially degrade the matrix that had been formed in vitro. The beads were then returned to normal culture conditions for up to 4 weeks. The content of collagen per nine beads (NP: left panel, AF: right panel) was measured at each time point and is reported for each group and at each time point as the mean6standard deviation of the value obtained for three separate cultures. The collagen content, expressed per nine beads, did not show any significant difference between the CABC–treated beads and the CP-treated beads.

698

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

PG synthesis Enzymatic treatment did not affect the rate of PG synthesis when overall significance was assessed from 1 to 4 weeks (two-way factorial analysis of variance). However, when assessed at each time point, CP-treated groups produced more PG than C-ABC–treated groups at the 0- and 4-week time points in the NP beads and at the 2-week time point in the AF beads (Fig. 4). The distribution of newly synthesized PGs in the CM, FRM, and media of beads treated with the two different enzymes did not differ significantly at any of the time points (data not shown).

Discussion Studies of the effects of matrix-degrading enzymes on cell metabolism are difficult to perform in monolayer or organ culture. In preliminary studies using a monolayer system, we found that treatment with CP and C-ABC induced disintegration and detachment of the cell layer, making it impossible to study the repair process. In a whole-disc culture system [41], although this system may reflect the in vivo situation, a short-time incubation with these enzymes, in order to prevent cell death, did not result in significant matrix degradation in the AF deeper than 100 mm from the surface (data not shown). The pharmacokinetics of enzymes, which is not well known in humans, needs to be taken into consideration when the experiment is designed. To understand the effect of enzymes on the cellular level, from a safety standpoint, we selected the alginate bead culture system, which was previously shown to be effective to study the repair response of intervertebral disc cells after interleukin-1–induced degradation [42] or the C-ABC

Fig. 4. The effect of C-ABC or CP treatment on proteoglycan synthesis by NP or AF cells in alginate beads subsequently cultured for up to 4 weeks. As described in the Materials and Methods section, the NP and AF cells in alginate beads were first cultured for 10 days and then briefly incubated in the presence of C-ABC or CP to partially degrade the matrix that had been formed in vitro. The beads were then returned to normal culture conditions for up to 4 weeks. The rate of PG synthesis did not differ between the two enzyme treatment groups when the effect of time and the treatment was assessed by two-way factorial analysis of variance test (NP: left panel, AF: right panel). Interestingly, a higher rate of synthesis was observed at the 4-week time point in the case of NP cells and at the 0- and 2-week time points in the case of AF cells. *p!.05 (comparison of the difference at each time point between the C-ABC–treated and CP-treated cells, two-tailed unpaired t test).

digestion and growth factor application [43]. In this report, we show the usefulness of this approach to study the reparative responses of NP and AF cells in alginate beads cultured briefly in the presence of low concentrations of C-ABC and CP. The studies presented here revealed that NP and AF cells exposed to C-ABC are more effective in replenishing their extracellular matrix with PGs than those exposed to CP. To minimize the concern that the doses of the two different enzymes injected into the discs in the reported animal study [22,29] were not truly comparable, the enzyme concentrations used in this study were carefully selected according to the results of the preliminary experiments. For each enzyme, the dose selected for use was that dose capable of reproducibly depleting greater than 80% of the negatively charged sulfated glycosaminoglycans from the NP without causing a decrease in the DNA content (data not shown). The measurement of cell viability, using the Trypan Blue exclusion technique, confirmed that the enzymes at the concentrations used caused little damage to the cells. The concentration of CP that was selected (ie, 5 picokatals/mL) was the lowest concentration that did not cause a decrease in the DNA content of the culture after the treatment. Because incubation of the NP cells in the presence of C-ABC did not lead to a measurable decrease in the DNA content within the beads, even at 1 U/mL, C-ABC was used at a concentration of 0.05 U/mL. Several in vivo studies have indicated that C-ABC appeared less harmful than CP in damaging vascular tissue [24,26], nerve tissue [23,25,26], and disc cells [23], probably because of the limited substrate spectrum of C-ABC. Our study indicated that cells treated with C-ABC more effectively reformed a PG-rich matrix than those treated with CP. In the C-ABC group, an increase in PG synthesis and DNA content was observed. This may reflect a repair response to matrix depletion; further research will be needed to confirm this speculation. However, it should be noted that the content of PG never achieved more than 80% of those found in a nontreated group in a separate experiment (eg, PG content, no treatment 84.6, C-ABC 59.3, at 3 weektime point, mg/mg DNA) [43]. Several factors may have contributed to the apparently greater ability of cells in beads treated with the C-ABC enzyme to reform an extracellular matrix. These include a greater cell number (DNA content), an increased ability to form extracellular matrix, and a lower rate of matrix degradation. Both NP and AF beads treated with CP tended to have a lower DNA content than those treated with C-ABC, especially later in the subsequent culture; this may be because of the chronic toxicity of CP. Consequently, this may help explain, at least in part, the less effective replenishment of PGs in the AF and NP cells treated with CP. Because the rate of PG synthesis per cell was similar in the two enzyme treatments, it is very possible that the ability to retain a de novo extracellular matrix and to reform a PG-rich matrix differs between the two enzyme treatments. How the enzyme treatment

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

affects the rate of degradation of the de novo matrix formed remains to be determined. The difference in the extent to which a PG-rich matrix was reestablished was more evident in the CM than in the FRM. This may be because of several factors. First, the substrate spectrum of both enzymes varies. C-ABC is an eliminase that cleaves chondroitin sulfate and related polysaccharides (ie, hyaluronan and dermatan sulfate, particularly in a protease-free preparation). Unlike CP, C-ABC does not digest proteins and their receptors (such as CD44 and integrins) present on the membranes of NP and AF cells). Second, it is also possible that CP caused a greater disorganization of the collagen network that normally helps hold PGs and other matrix molecules. However, this is unlikely because, during the short culture period used in this study, the concentration of collagen does not reach the level found in vivo and the collagen network is not highly organized. Although further direct confirmation is needed, the potential degradation of cell surface receptors, such as CD-44 or integrin, which help anchor the matrix to the cell, may explain why treatment with CP is more harmful than that caused by treatment with C-ABC. There are several limitations in this study. Cells from the NP and AF express different degree of the chondrocytic phenotype, and the culture system best suited for each cell type has been reported to be different [44–46]. AF cells in culture may need anchoring to matrix elements, such as to collagen fibrils, as likely occurs in vivo. For example, AF cells may grow better in a collagen matrix than in alginate beads [45]. In this study, we have selected the alginate bead culture system that was used for an interleukin 1 study [42] and another C-ABC study [43] because enzymatic digestion, especially CP, may disintegrate the matrix element (ie, collagen) used to anchor the cells in culture and result in cell loss. Second, although the analysis of the collagen content indicated some accumulation of collagen in the alginate beads during the short culture times used in this study, the matrix formed lacked crosslinked-collagen; therefore, the matrix organization may be different from that found in vivo. Third, the intervertebral discs of the immature rabbits used as the source of cells in this study contain a significant number of notochordal cells. The use of mature animals, which have a cellular phenotype more similar to human intervertebral disc (IVD) cells, would be preferable. Finally, future studies are needed to shed more light on the quality of the matrix that is reformed and its ability to sustain compressive forces during loading. In summary, the results presented here suggest that enzymes, such as C-ABC, that are capable of cleaving mainly the negatively charged glycosaminoglycans (mainly chondroitin sulfate) of PGs without affecting cell viability and other matrix or cell-surface proteins have less severe immediate or longer-term deleterious effects on the metabolism of intervertebral disc cells than proteolytic enzymes.

699

Acknowledgments The authors would like to acknowledge Mary Ellen Lenz, MS, and Lori Otten, MS, for their assistance in the preparation of the manuscript.

References [1] Atlas SJ, Deyo RA, Keller RB, et al. The Maine lumbar spine study, part ii. 1-year outcomes of surgical and nonsurgical management of sciatica. Spine 1996;21:1777–86. [2] Fisher C, Noonan V, Bishop P, et al. Outcome evaluation of the operative management of lumbar disc herniation causing sciatica. J Neurosurg 2004;100:317–24. [3] Lu WW, Luk KD, Ruan DK, Fei ZQ, Leong JC. Stability of the whole lumbar spine after multilevel fenestration and discectomy. Spine 1999;24:1277–82. [4] Brown MD. Intradiscal therapy: chymopapain or collagenase. Chicago: Year Book Medical Publisher, 1983. [5] Smith L, Garvin PJ, Gesler RM, Jennings RB. Enzyme dissolution of the nucleus pulposus. Nature 1963;198:1311–2. [6] Smith L. Enzyme dissolution of the nucleus pulposus in humans. JAMA 1964;187:137–40. [7] Eguro H. Transverse myelitis following chemonucleolysis. Report of a case. J Bone Joint Surg Am 1983;65:1328–30. [8] Hall BB, McCulloch JA. Anaphylactic reactions following the intradiscal injection of chymopapain under local anesthesia. J Bone Joint Surg Am 1983;65:1215–9. [9] Smith S, Leibrock LG, Gelber BR, Pierson EW. Acute herniated nucleus pulposus with cauda equina compression syndrome following chemonucleolysis. Report of three cases. J Neurosurg 1987;66: 614–7. [10] Weitz EM. Paraplegia following chymopapain injection. A case report. J Bone Joint Surg Am 1984;66:1131–5. [11] Watts C. Complications of chemonucleolysis for lumbar disc disease. Neurosurgery 1977;1:2–5. [12] Tsuchida T. A pathological study of experimental chemonucleolysis with collagenase. J Orthop Sci 1987;61:1237–49. [13] Mayne R, Brewton RG. Extracellular matrix of cartilage collagen. In: Woessner JF, Howell DS, editors. Joint cartilage degradation basic and clinical aspects. New York: Marcel Dekker, 1993:81–108. [14] Artigas J, Brock M, Mayer HM. Complications following chemonucleolysis with collagenase. J Neurosurg 1984;61:679–85. [15] Garvin PJ. Toxicity of collagenase: The relation to enzyme therapy of disk herniation. Clin Orthop 1974;101:286–91. [16] Olmarker K, Rydevik B, Dahlin LB, Danielsen N, Nordborg C. Effects of epidural and intrathecal application of collagenase in the lumbar spine: an experimental study in rabbits. Spine 1987;12: 477–82. [17] Rydevik B, Brown MD, Ehira T, Nordberg C. Effects of collagenase on nerve tissue: an experimental study on acute and long-term effects in rabbits. Spine 1985;10:562–6. [18] Ando T, Kato F, Mimatsu K, Iwata H. Effects of chondroitinase abc on degenerative intervertebral discs. Clin Orthop 1995;214–21. [19] Eurell JAC, Brown MD, Ramos M. The effects of chondroitinase abc on the rabbit intervertebral disc. A roentgenographic and histological study. Clin Orthop 1990;256:238–43. [20] Fry TR, Eurell JC, Johnson AL, Brown MD, Losonsky JM, Schaeffer DJ. Radiographic and histologic effects of chondroitinase abc on normal canine lumbar intervertebral disc. Spine 1991;16: 816–9. [21] Henderson N, Stanescu V, Cauchoix J. Nucleolysis of the rabbit intervertebral disc using chondroitinase abc. Spine 1991;16:203–8. [22] Kato F, Iwata H, Mimatsu K, Miura T. Experimental chemonucleolysis with chondroitinase abc. Clin Orthop 1990;253:301–8.

700

K. Chiba et al. / The Spine Journal 7 (2007) 694–700

[23] Kato F, Mimatsu K, Iwata H, Miura T. Comparison of tissue reaction with chondroitinase abc and chymopapain in rabbits as the basis of clinical application in chemonucleolysis. Clin Orthop 1993;288: 294–302. [24] Olmarker K, Danielsen N, Nannmark U, Sennerby L, Rydevik B. Microvascular effects of chondroitinase abc and chymopapain. An in vivo experimental study on hamsters and rabbits. Clin Orthop 1990;257:274–9. [25] Olmarker K, Danielsen N, Nordborg C, Rydevik B. Effects of chondroitinase abc on intrathecal and peripheral nerve tissue. An in vivo experimental study on rabbits. Spine 1991;16:43–5. [26] Olmarker K, Stromberg J, Blomquist J, et al. Chondroitinase abc (pharmaceutical grade) for chemonucleolysis. Functional and structural evaluation after local application on intraspinal nerve structures and blood vessels. Spine 1996;21:1952–6. [27] Park JS, Ahn JI. The effect of chondroitinase ABC on rabbit intervertebral disc. Radiological, histological and electron microscopic findings. Int Orthop 1995;19:103–9. [28] Sugimura T, Kato F, Mimatsu K, Takenaka O, Iwata H. Experimental chemonucleolysis with chondroitinase abc in monkeys. Spine 1996;21:161–5. [29] Takahashi T, Kurihara H, Nakajima S, et al. Chemonucleolytic effects of chondroitinase abc on normal rabbit intervertebral discs. Course of action up to 10 days postinjection and minimum effective dose. Spine 1996;21:2405–11. [30] Suguro T, Oegema TR Jr, Bradford DS. Ultrastructural study of the short-term effects of chymopapain on the intervertebral disc. J Orthop Res 1986;4:281–7. [31] Bradford DS, Oegema TJ, Cooper KM, Wakano K, Chao EY. Chymopapain, chemonucleolysis, and nucleus pulposus regeneration. A biochemical and biomechanical study. Spine 1984;9: 135–47. [32] Chiba K, Andersson GBJ, Masuda K, Thonar EJ-MA. Metabolism of the extracellular matrix formed by intervertebral disc cells cultured in alginate. Spine 1997;22:2885–93. [33] Maldonado BA, Oegema TJ. Initial characterization of the metabolism of intervertebral disc cells encapsulated in microspheres. J Orthop Res 1992;10:677–90. [34] Mok SS, Masuda K, Hauselmann HJ, Aydelotte MB, Thonar EJ. Aggrecan synthesized by mature bovine chondrocytes suspended in alginate. Identification of two distinct metabolic matrix pools. J Biol Chem 1994;269:33021–7.

[35] Hamai A, Hashimoto N, Mochizuki H, et al. Two distinct chondroitin sulfate ABC lyases. An endoeliminase yielding tetrasaccharides and an exoeliminase preferentially acting on oligosaccharides. J Biol Chem 1997;272:9123–30. [36] Hauselmann HJ, Aydelotte MB, Schumacher BL, Kuettner KE, Gitelis SH, Thonar EJ. Synthesis and turnover of proteoglycans by human and bovine adult articular chondrocytes cultured in alginate beads. Matrix 1992;12:116–29. [37] Kim YJ, Sah RL, Doong JY, Grodzinsky AJ. Fluorometric assay of DNA in cartilage explants using hoechst 33258. Anal Biochem 1988;174:168–76. [38] Chandrasekhar S, Esterman MA, Hoffman HA. Microdetermination of proteoglycans and glycosaminoglycans in the presence of guanidine hydrochloride. Anal Biochem 1987;161:103–8. [39] Petit B, Masuda K, D’Souza AL, et al. Characterization of crosslinked collagens synthesized by mature articular chondrocytes cultured in alginate beads: Comparison of two distinct matrix compartments. Exp Cell Res 1996;225:151–61. [40] Masuda K, Shirota H, Thonar EJ. Quantification of 35s-labeled proteoglycans complexed to alcian blue by rapid filtration in multiwell plates. Anal Biochem 1994;217:167–75. [41] Chiba K, Andersson GB, Masuda K, Momohara S, Williams JM, Thonar EJ. A new culture system to study the metabolism of the intervertebral disc in vitro. Spine 1998;23:1821–7. [42] Takegami K, Thonar EJ, An HS, Kamada H, Masuda K. Osteogenic protein-1 enhances matrix replenishment by intervertebral disc cells previously exposed to interleukin-1. Spine 2002;27:1318–25. [43] Takegami K, An HS, Kumano F, et al. Osteogenic protein-1 is most effective in stimulating nucleus pulposus and annulus fibrosus cells to repair their matrix after chondroitinase ABC-induced in vitro chemonucleolysis. Spine J 2005;5:231–8. [44] Gruber HE, Hanley EN Jr. Human disc cells in monolayer vs 3d culture: cell shape, division and matrix formation. BMC Musculoskelet Disord 2000;1:1. [45] Gruber HE, Leslie K, Ingram J, Norton HJ, Hanley EN. Cell-based tissue engineering for the intervertebral disc: in vitro studies of human disc cell gene expression and matrix production within selected cell carriers. Spine J 2004;4:44–55. [46] Horner HA, Roberts S, Bielby RC, Menage J, Evans H, Urban JP. Cells from different regions of the intervertebral disc: effect of culture system on matrix expression and cell phenotype. Spine 2002;27:1018–28.