Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis

The Spine Journal 8 (2008) 466–474

Technical Review

Bone morphogenetic protein-7 protects human intervertebral disc cells in vitro from apoptosis Aiqun Wei, MDa, Helena Brisby, MD, PhDb, Sylvia A. Chung, MSc, Hona, Ashish D. Diwan, MD, PhDa,* a

Orthopaedic Research Institute, The University of New South Wales, St George Hospital Campus, level 2, 4-10 South Street, Kogarah, NSW 2217, Australia b Department of Orthopaedics, Sahlgrenska University Hospital, G€ oteborg University, SE 413 45 G€ oteborg, Sweden Received 3 February 2007; accepted 30 April 2007

Abstract

BACKGROUND CONTEXT: Disc degeneration includes dysfunction and loss of disc cells leading to a decrease in extracellular matrix (ECM) components. Apoptosis has been identified in degenerated discs. Bone morphogenetic protein-7 (BMP-7) has been reported to stimulate ECM synthesis in the intervertebral disc (IVD), but its effect on disc cell viability is unknown. PURPOSE: To investigate whether BMP-7 can protect disc cells from programmed cell death while enhancing ECM production. STUDY DESIGN: An in vitro study to examine the effect of BMP-7 on apoptosis of IVD cells. METHODS: Human nucleus pulposus (NP) cells were cultured in monolayer, and human recombinant pure BMP-7 (rhBMP-7) was added to the medium when the cells were in the second passage. Thereafter, apoptosis was induced by either tumor necrosis factor-alpha (TNF-a) or hydrogen peroxide (H2O2). Cellular apoptosis was evaluated by terminal deoxynucleotidyl transferase– mediated dUTP nick end labeling assay and caspase-3 activity. ECM synthesis was assessed by immunofluorescence for collagen-2 and aggrecan. To study the possibility of bone induction by rhBMP-7 in disc cells, alkaline phosphatase activity and Alizarin red-S staining were evaluated. RESULTS: Apoptosis was induced by both TNF-a and H2O2. Addition of rhBMP-7 resulted in inhibition of the apoptotic effects caused by both inducers. Further, BMP-7 decreased caspase-3 activity. In the presence of BMP-7, ECM production was maintained by the cells despite being in an apoptotic environment. No osteoblastic induction of the disc cells was seen. CONCLUSIONS: BMP-7 was demonstrated to prevent apoptosis of human disc cells in vitro. One of the antiapoptotic effects of BMP-7 on NP cells might be a result of its inactivation of caspase-3. Collagen production was maintained by addition of rhBMP-7 in an apoptotic environment. Ó 2008 Elsevier Inc. All rights reserved.

Key words:

Bone morphogenetic protein; Tumor necrosis factor; Apoptosis; Intervertebral disc; Disc degeneration; Cell culture

Introduction The intervertebral disc (IVD) in the spinal column consists of two regions. The outer annulus fibrosus region is This study was conducted after approval from the South-Eastern Health Research Ethics Committee (HREC), New South Wales, Australia. * Corresponding author. Department of Orthopaedic Surgery, Orthopaedic Research Institute, The University of New South Wales, St George Hospital Campus, level 2, 4-10 South Street, Kogarah, NSW 2217, Australia. Tel.: (61) 2-9588-9622; fax: (61) 2-9588-9722. E-mail address: [email protected] (A.D. Diwan) 1529-9430/08/$ – see front matter Ó 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.spinee.2007.04.021

composed of parallel layers of collagen-1 and provides the tensile strength to the disc. The inner nucleus pulposus (NP) region of the disc is composed of collagen-2 and proteoglycan (PG), responsible for the retention of water, and provides the viscoelastic properties of the disc. Degeneration of the IVD, associated with chronic low back pain, is an age-, wear-, and injury-related condition characterized by a loss of both extracellular matrix (ECM) and the cells responsible for its maintenance [1–3]. These events may lead to the overall collapse of the disc, contributing to local spinal instability and pain.

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Apoptosis or programmed cell death has been shown to be a key component responsible for the decrease in cell number in the NP during degeneration [4–6]. Apoptosis is an important phenomenon involved in normal tissue development and regulation, providing some protection from the onset of malignant transformation of cells. However, if expressed excessively, apoptosis may lead to tissue degeneration [7]. The apoptotic process can be triggered by either an intrinsic (mitochondrial-dependent) or extrinsic (mitochondrial-independent) apoptotic signaling pathway, respectively [8]. Tumor necrosis factor belongs to a family of cytokines including tumor necrosis factor-alpha (TNF-a), Fas-ligand, and TRAIL, which stimulate the extrinsic pathway leading to apoptosis. The intrinsic pathway is instigated by stimuli such as oxidative stress mediated by hydrogen peroxide (H2O2) [9]. Both apoptotic pathways have been detected to occur in IVD degeneration, with static mechanical loading–induced degeneration being mediated by the intrinsic pathway and disc herniations being induced by the extrinsic pathway [10–13]. To date two antiapoptotic agents, insulin-like growth factor-1 and platelet-derived growth factor, that retard and prevent in vitro serum starvation–induced apoptosis of discal cells have been identified [14]. Bone morphogenetic protein-7 (BMP-7), a member of the transforming growth factor-b superfamily, is involved in proliferation, differentiation, metabolism, and apoptosis in a variety of tissues [15]. In disc degeneration, BMP-7 has enhancing effects on ECM (PG) synthesis both in vitro and in vivo, with a resultant increase in disc height observed [16–18]. Further, a single dose of BMP-7 has been shown to improve the disc height of degenerated IVD in a sheep model [19]. It is not known how BMP-7 brings about the reversal of the degenerative changes in the IVD. Apoptotic regulation by BMP-7 is tissue specific, shown by the induction of apoptosis in primary myeloma cells but the rescue of apoptosis in renal cells [20–22]. However, the apoptotic effect of BMP-7 in disc degeneration is unknown. The aim of the present study was to investigate whether BMP-7 can prevent TNF-a- or H2O2-induced apoptotic effects in cultured human disc cells obtained from degenerated discs.

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containing Dulbecco’s Modified Eagle Medium (Invitrogen, Carlsbad, CA), 10% fetal calf serum, and 1% penicillin/ streptomycin for 10 to 12 days to become confluent. Cells were subcultured at a concentration of 1105/mL for 2 to 3 days before treatment. All experiments were completed using the second passages of cells. Apoptosis induction and BMP-7 treatment Apoptosis was induced with either TNF-a (B&D System, Inc., Minneapolis, MN) or H2O2 (Sigma-Aldrich, St. Louis, MO). TNF-a apoptotic induction was performed with 100 ng/mL for 24 hours and 48 hours. H2O2 apoptotic induction was conducted with 0.1 mM for 20 minutes. Human recombinant pure BMP-7 (rhBMP-7; Stryker-Biotech, Andover, MA) at a concentration of 100 ng/mL was preadministrated in cultures for 5 hours and remained in the medium during TNF-a or H2O2 treatment. A dose-response study was performed before determining the rhBMP-7 dose for this study (data not shown). The dose of BMP-7 used in the present study was further selected based on a number of published studies on cells from IVDs [23,24]. Cells grown in complete medium alone served as the control. In situ detection of DNA fragmentation In situ detection of DNA fragmentation in apoptotic cells was performed with the terminal deoxynucleotidyl transferase (TdT)–mediated dUTP nick end labeling (TUNEL) kit (Promega, Madison, WI) as per the manufacturer’s instructions. Briefly, the cells cultured on coverslips were fixed in 4% paraformaldehyde and treated with proteinase K for 15 minutes. The endogenous peroxidase was blocked with 3% H2O2 for 10 minutes. The cells were incubated with TdT for 2 hours and then exposed to antidigoxigenin antibody conjugated with peroxidase. Color was developed with 3,30 -diaminobenzidine hydrochloride. Slides were then counterstained with hematoxylin. Negative controls were incubated with reaction mixture lacking TdT enzyme. Cells were defined as apoptotic when the whole nuclear area was labeled brown. The counts were performed in three different sets of experiments for each condition with 500 cells.

Materials and methods Cell viability assay Cell culture NP tissues were freshly collected from eight subjects undergoing lumbar total disc replacement surgery (age: 48616 years). Informed consent was obtained from subjects under approval of the South-Eastern Health Service Human Research Committee, Sydney, Australia. All discs demonstrated moderate signs of degeneration on magnetic resonance imaging including decreased water content and decreased disc height. Discarded NP tissues were immediately subjected to 0.025% collagenase digestion overnight. Primary cultures were grown in a complete medium

Cell survival was measured with the MTS Cell Proliferation Assay kit (Promega, Madison, WI) using 1104 cells/ well plated in 96-well plates. Assays were performed as specified by the manufacturer, such that only viable cells are able to metabolically reduce tetrazolium salts to formazan salts, detected directly on a spectrophotometer at 490 nm. Caspase-3 activity Caspase-3 activity was detected using the CaspACE assay colorimetric system (Promega, Madison, WI) according

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to the manufacturer’s instructions. Briefly, 100 mg of total protein was added to 50 mL of reaction buffer, and 2 mL of substrate DEVD-pNA was added. Samples were incubated at 37 C for 6 hours, the enzyme-catalyzed release of pNA was quantified by a spectrophotometer, and concentrations were determined by correlation with a standard curve. For the inhibition assay, 50 mM of caspase-3 inhibitor z-VAD-FMK was added to culture medium for 12 hours. Subsequently, the samples were either treated or not treated with TNF-a and the caspase-3 activity was determined. Western blot analysis The cells were harvested in lysis buffer containing a protease inhibitor cocktail (500 mg/mL (4-[2-aminoethyl] benzenesulfonylfluoride] hydrochloride and 1 mg/L E-64, leupeptin, pepstatin-A at 2 mg/mL each). Twenty to forty micrograms of protein was separated on a 7.5% or 12% (v/v) sodium dodecyl sufate-polyacrylamide gel electrophoresis gel. Proteins were transferred onto a polyvinylidine fluoride membrane. Subsequently, the membrane was probed with goat anti-collagen-2 polyclonal (1:500; Santa Cruz Biotechnology, Inc, Santa Cruz, CA), or anticaspase-3 or anti–cleaved caspase-3 monoclonal (1:1,000 and 1:800, respectively; Cell Signalling Technology Inc, Beverly, MA) antibodies for 60 minutes. This was followed by washes before addition of the corresponding secondary antibody conjugated with peroxidase (Chemicon, Temecula, CA) at a 1:1,000 dilution for 30 minutes. A chemiluminescence detection system (Pierce Biotechnology, Inc., Rockford, IL) was used for the visualization of labeled proteins. Blots were stripped and reprobed with mouse antib-actin monoclonal antibody (1:10,000; Sigma). b-Actin, a house-keeping protein, was used as an internal control to ensure that equal amounts of protein were loaded per lane. Visualized bands were semiquantified by densitometry (Model GS-700/690; Bio-Rad, Hercules, CA). Immunofluorescence staining (collagen-2 and aggrecan) Immunofluorescence staining was performed after fixation of cells cultured on glass coverslips with 4% paraformaldehyde, followed by blocking of nonspecific antigens with 5% normal donkey or sheep serum for 30 minutes. Primary goat anti–collagen type-2 polyclonal (1:200) or mouse anti-human aggrecan monoclonal antibodies (1:150; Chemicon International, Inc., Temecula, CA) were incubated on each slide, respectively, for 1 hour. Cells were repeatedly washed and secondary antibodies, donkey anti-goat or sheep anti-mouse IgG, conjugated with fluorescein isothiocyanate (Chemicon) were applied at a dilution of 1:500. Mounted coverslips were visualized on a fluorescence microscope (Leitz, Wetzlar, Germany). Negative controls were treated in a similar manner but with the omission of the primary antibody and were consistently included in each experiment.

PG synthesis During the final 8 hours of culture, the medium was changed to complete medium containing 10 mCi/mL of [35S]-sulfate (Amersham Biosciences, Piscataway, NJ) in the TNF-a–exposed cell cultures (this analysis was not performed in the H2O2 group because the cells were exposed to H2O2 only for 20 minutes). The PGs were extracted with 4 M guanidinium hydrochloride (in 50 mmol sodium acetate, pH 5.8, containing 0.1 M 6-amino-hexanoic-acid, 50 mmol benzamidine HCl, 10 mmol ethylenediaminetetraacetic acid, and 5 mmol N-ethylmaleimide) at 4 C for 24 hours. Total synthesis was determined by combining radioisotope incorporation of both the cells and the condition medium using a rapid filtration assay [25]. PGs were precipitated by alcian blue (Sigma). The newly synthesized PGs were detected using a [beta]-liquid scintillation counter. All the data were normalized to DNA content, which was quantified using fluorescence assay as described by Kim et al. [26]. Rates of [35S]-incorporation were expressed as counts per minute [35S]-incorporated/mg DNA. Alkaline phosphatase production and Alizarin red-S staining Alkaline phosphatase (AP) activity was determined using a modification of the method of Bradford [27]. Cells were lysed with 0.1% Triton X-100 in phosphate-buffered saline, and lysates were then incubated for 30 minutes at 37 C with the AP substrate p-nitrophenylphosphate (Sigma-Aldrich) at 2.5 mg/mL. The levels of p-nitrophenol (PNP) production were measured by a spectrophotometer, and concentrations were determined by comparison with a standard curve created with known amounts of PNP. AP activity is expressed as nanomoles of PNP generated per microgram of total cellular protein per minute. For Alizarin red-S staining, the cells were fixed with 70% ethanol for 2 hours. After washing with water, the cells were stained for 10 minutes with 40 mM Alizarin red-S (pH 4.1; Sigma) and thereafter washed with phosphate-buffered saline for 15 minutes before mounting. Statistical analysis Data analysis among multiple experimental groups was performed with analysis of variance and the unpaired Student t test using SigmaStat software (SPSS, Inc., San Rafael, CA). Differences were considered significant at p !.05.

Results The effect of BMP-7 on induced apoptosis in cultured IVD cells Visualization of apoptotic cells by TUNEL assay was highly evident in cell cultures after both TNF-a and

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Fig. 1. Preventative effects of bone morphogenetic protein-7 (BMP-7) on tumor necrosis factor-alpha (TNF-a)- or hydrogen peroxide (H2O2)–induced apoptosis in cultured disc cells detected by terminal deoxynucleotidyl transferase–mediated dUTP nick end labeling (TUNEL) staining. Brown-stained apoptotic cells dominate in TNF-a- or H2O2-treated cells, whereas in BMP-7–pretreated cells, a reduction in apoptotic cells was seen. Arrows indicate cells that stained positively for TUNEL (magnification: 400).

Cell viability Cells were found to have decreased viability after exposure to either TNF-a or H2O2, as measured with the MTS assay. BMP-7 treatment showed improvement in cellular viability closer to the levels observed in control cells (Fig. 2).

Caspase-3 activity The presence of 100 ng/mL of BMP-7 alone did not induce any caspase-3 activity over 48 hours in disc cells. Conversely, the enzymatic activity increased by 26% after a 20-minute stimulation of 0.1 mM H2O2 and by 36% after a 48-hour TNF-a stimulation compared with nonstimulated controls. However, pretreatment with BMP-7 prevented the

(Control cells=100%)

120

Cell Viability (% of Control)

H2O2 exposure (Fig. 1). After counterstaining with hematoxylin, further signs of apoptosis, cellular shrinkage, and nuclear condensation were confirmed. In the control group, only a smaller proportion of the cells (6.5%64.2%) demonstrated signs of apoptosis by TUNEL assay. The cells treated with BMP-7 alone for 48 hours also had a normal appearance with only a few of the cells staining brown (8.2%64.2%). In contrast, the visualization of apoptotic cells by TUNEL assay was highly evident in cell cultures after both TNF-a and H2O2 exposure (Fig. 1). The percentage of apoptotic cells was 9– to 10-fold higher when the cells were treated with TNF-a (61.3%610.3%) or H2O2 (70.3%69.7%) compared with untreated cells (control) (6.5%64.2%, p!.01). After counter staining with hematoxylin, further signs of apoptosis, cellular shrinkage, and nuclear condensation were confirmed. However, in both induced apoptotic groups, BMP-7 pretreatment led to decrease from 70.3%69.7% (H2O2 alone) to 43.8%618.9% (H2O2þBMP7, p!.05) and from 61.3%610.3% (TNFa alone) to 34%611.5% (TNF-aþBMP7, p!.05) of TUNEL-positive cells.

80

* * 40

0 BMP-7

H2O2

TNF-α

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Fig. 2. Bone morphogenetic protein-7 (BMP-7) improved cellular viability in an apoptotic environment. Tumor necrosis factor-alpha (TNF-a) (24 hours) or hydrogen peroxide (H2O2) (20 minutes) treated intervertebral disc (IVD) cells showed reduction of cellular viability; however, BMP-7 pretreatment improved cellular viability to the control levels. *p!.05 compared with BMP7, TNF-aþBMP-7, or H2O2þBMP7. Data are presented as mean6SD of triplicates in three independent samples.

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* PG Synthesis (CPM/μg DNA)

% of Caspase-3 Activity (Control Cells=100%)

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CONTROL

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Basal Caspase-3 35KD Activated Caspase-3 19 KD&17KD

TNF-α (24h)

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TNF-α24h TNF-α48h + BMP-7 + BMP-7

Fig. 4. Effect of bone morphogenetic protein-7 (BMP-7) on the rate of [35S]-proteoglycan (PG) synthesis in both normal and apoptotic environments. *p!.05 compared with tumor necrosis factor-alpha (TNF-a) (24 hours), TNF-a (48 hours), and TNF-a (48 hours) þBMP7. The mean and SEM for five individual samples are reported.

BMP-7 increases ECM production in an apoptotic environment The rate of [35S]-PG synthesis significantly increased after BMP-7 stimulation over 48 hours (Fig. 4), whereas the rate of [35S]-PG synthesis considerably decreased after treatment with TNF-a alone (24 or 48 hours). The restorative

BMP-7

H202

BMP-7 + H202

BMP-7 + TNF-α

TNF-α 24h

Fig. 3. Bone morphogenetic protein-7 (BMP-7) regulates caspase-3 activity in cultured nucleus pulposus (NP) cells. *p!.05 compared with all BMP-7- or z-VAD-FMK–treated groups. The mean and SEM represent three individual samples (Top). BMP-7 inhibits activated caspase-3 protein expression in cultured disc cells (Western blot) (Bottom). Basal levels of expression (35 kDa) were seen in all lanes. Expression of the activated form (17 and 19 kDa) was detected after tumor necrosis factor-alpha (TNF-a) or hydrogen peroxide (H2O2) treatment and seen prominently after 48 hours of TNF-a treatment. However, samples treated with BMP-7 were negative for the active forms of caspase-3 protein expression.

CONTROL

β-actin, 45KD

Collagen-2 (180KD)

β-Actin (45KD)

160

% of Control Cells

TNF-a- or H2O2-induced apoptotic caspase-3 activity in the NP cells (Fig. 3, top). Furthermore, the inhibitory effect of BMP-7 was similar to that of the caspase-3–specific inhibitor, z-VAD-FMK. The expression of basal caspase-3 protein (35 kDa) was detected on a Western blot in all the samples, whereas its activated form (17 and 19 kDa) was only shown in apoptosis-induced samples. BMP-7 resulted in a complete block of the cleaved caspase-3 protein expression when added before the TNF-a or H2O2 stimulation (Fig. 3, bottom). In addition, this active form is more specifically detected by an antibody, which recognizes the active cleavage site on caspase-3, revealing the high levels of expression only after TNF-a or H2O2 treatment (results not shown). BMP7–pretreated samples were all negative for the active forms of caspase-3 protein expression.

140 120 100 80 60 40 20 0

Fig. 5. Western blot of collagen-2 expression after tumor necrosis factoralpha (TNF-a)- or hydrogen peroxide (H2O2)–induced apoptosis and bone morphogenetic protein-7 (BMP-7) pretreatment before apoptotic induction (Top). Densitometry analysis of the representative Western blot data were normalized to b-actin expression and presented as a percentage of untreated cellular controls (Bottom).

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Fig. 6. Representative image of immunofluorescent staining of collagen-2 and aggrecan protein expression after apoptotic induction by tumor necrosis factor-alpha (TNF-a) and the effects of bone morphogenetic protein-7 (BMP-7) pretreatment before apoptotic induction (magnification: 400).

effect of BMP-7 on PG synthesis from cells treated with TNF-a was almost comparable to levels observed in controls at 24 hours but only reached about 50% of normal PG synthesis at 48 hours after treatment (Fig. 4). Immunoblot analyses revealed an increased expression of collagen-2 in the presence of BMP-7 alone compared with controls (Fig. 5). However, expression of collagen-2 protein was almost undetectable after TNF-a (48 hours) or H2O2 treatment. Semiquantification by densitometry showed that administration of BMP-7 to the NP cells before TNF-a or H2O2 treatment resulted in a partly preserved collagen-2 protein expression. The BMP-7 administration increased the collagen-2 protein expression by about fivefold (TNF-a) and twofold (H2O2) compared with TNF-a or H2O2 treatment alone, respectively. By immunofluorescence, untreated control cells were demonstrated to exhibit collagen-2 and aggrecan protein

expression. Again, BMP-7 alone increased the expression of both aggrecan and collagen-2, whereas TNF-a exposure resulted in a significant loss of aggrecan and collagen-2 protein expression. In agreement with the immunoblot analyses, the ECM recovery by BMP-7 pretreatment of TNF-a– induced apoptosis was visualized by immunofluorescence staining (Fig. 6). Effect of BMP-7 on AP activity and Alizarin red-S stain At BMP-7 concentrations of 100 to 200 ng/mL (concentrations used for apoptotic rescue), only basal levels of AP activity were detected during the observation time (24–120 hours). This was significantly lower than the AP activity of the samples collected from 10-day-old fracture calluses of rat femurs (positive control) (Fig. 7, top). NP cells treated with 200 ng/mL of BMP-7 for 120 hours demonstrated

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APActivity (nmol PNP/ h/μg Protein)

12

**

Control 10

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8 6 4 2 0 24h

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Osteoblastic control

Callus

Fig. 7. Disc cells treated with bone morphogenetic protein-7 (BMP-7) do not demonstrate any bone cellular activity. Alkaline phosphatase (AP) activity of cells treated with BMP-7 for 24 to 120 hours was compared with that of a positive osteoblast control. Data are presented as mean 6 SEM of triplicates in five individual samples; **p!.01 (Top). Disc cells treated with BMP-7 in 120 hours of culture period showed no positive staining with Alizarin red-S, whereas paraffin sections from the callus strongly stained positive with Alizarin red-S (50) (Bottom).

negative staining with Alizarin red-S, whereas paraffin sections of fracture calluses showed strong positive staining with Alizarin red-S (Fig. 7, bottom).

Discussion The present study provided evidence that BMP-7 can protect or rescue NP cells in vitro from undergoing apoptosis and that the mechanisms of the protection may possibly involve caspase-3 inactivation. In vivo studies have demonstrated that BMP-7 can decrease disc degeneration in animal models [18,19,28]. The mechanisms behind these effects have not yet been clarified but are of great importance before BMP-7 can be suggested for the treatment of disc degeneration in humans. The present study was undertaken to explore the effects of bone morphogenetic protein (BMP) regarding apoptosis in disc cells that could possibly influence and may be one of the mechanisms behind the observation of the positive effects of BMP-7 in disc degeneration models. All BMPs function through receptor-mediated intercellular signaling and subsequent target gene transcriptions, which act through two types of BMP receptors [29]. The

type-II BMP receptor has been found to be expressed by human disc cells [30,31], indicating that BMPs, including BMP-7, may serve as important signaling mediators for disc cell functions. We observed that pretreatment of the NP cells with BMP-7 before exposure to either TNF-a or H2O2 resulted in significantly increased cell survival. This was accompanied by the finding that BMP-7, prophylactively added to the disc cell cultures, demonstrated a protective effect on NP cells regarding TNF-a- and H2O2-induced decreased viability effects. The result is consistent with previous observations that BMP-7 was able to protect the cerebellum granule cells or C4-2B cell line against serum-withdrawn apoptosis [32,33]. Caspase-3 is a downstream member of the caspase cascade and acts as a central effector in the apoptotic pathway. It is located in the cellular cytoplasm in its inactive form under normal microenvironment, but it is auto-proteolytically cleaved into an active form of the enzyme under apoptotic conditions [34]. TNF-a- or H2O2-stimulated cells showed an increased level of intracellular caspase-3 protein and caspase-3 activity. Treatment with BMP-7 significantly inhibited caspase-3 activity generated by TNF-a or H2O2. Further, the inhibitory effect of BMP-7 was similar to that of the caspase-3–specific inhibitor z-VAD-FMK. These

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results suggest that BMP-7 inhibits caspase-3 activity and the antiapoptotic effect of BMP-7 is at least partly mediated through the caspase-3 enzymatic inhibition. BMP-7 has been shown to increase IVD cellular matrix protein secretion and rescue cells in vitro from the detrimental effect of interleukin-1 [35]. The effectiveness of BMP-7 in stimulating ECM in normal and degenerative IVD has also been reported in animal models [17,28,36]. In agreement with these previous studies, we found a restorative effect of BMP-7 on TNF-a- and H2O2-influenced ECM reduction in disc cells in vitro. The antiapoptotic (anticatabolic) effects of BMP-7 along with its potential to enhance ECM (anabolic effect) potentially make it an excellent molecular candidate to break the vicious cycle of degenerative cytokines causing further progression of IVD degeneration. BMP-7 in a collagen carrier (OP-1 device) is commercially used for bone fracture healing enhancement [37], thereby posing the question of induction of osteoblastic activity in disc cells. Human IVD cells treated with rhBMP-2 showed no evidence of bone formulation either in osteocalcin mRNA expression or in histological staining [38]. Consistent with this, cultured NP cells indicated no osteoblastic activity or bone formation after the exposure of BMP-7. The issue of a carrier for BMP-7 for separate clinical usage is a critical one. For bone fusion, a collagen-1 carrier is used (see above), whereas for IVD regeneration in clinical trials collagen-1 may not be suitable. Another important consideration is that of dosage for different clinical usage. For bony fusion, 3.5 mg of BMP-7 is used for one level. We expect that the dose of BMP-7 for human IVD regeneration will be estimated after large-animal work.

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[26] Kim YJ, Sah RL, Doong JY, et al. Fluorometric assay of DNA in cartilage explants using Hoechst33258. Anal Biochem 1988;174: 168–74. [27] Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of proteindye binding. Anal Biochem 1976;72:248–54. [28] Masuda K, Imai Y, Okuma M, et al. Osteogenic protein-1 injection into a degenerated disc induces the restoration of disc height and structural changes in the rabbit anular puncture model. Spine 2006;31:742–54. [29] Chen D, Zhao M, Mundy GR. Bone morphogenetic proteins. Growth Factors 2004;22:233–41. [30] Le Maitre CL, Richardson SM, Baird P, Freemont AJ, Hoyland JA. Expression of receptors for putative anabolic growth factors in human intervertebral disc: implications for repair and regeneration of the disc. J Pathol 2005;207:445–52. [31] Wang H, Kroeber M, Hanke M, et al. Release of active and depot GDF-5 after adenovirus-mediated overexpression stimulates rabbit and human intervertebral disc cells. J Mol Med 2004;82: 126–34. [32] Yabe T, Samuels I, Schwartz JP. Bone morphogenetic proteins BMP6 and BMP-7 have differential effects on survival and neurite out-

90 Years Ago in Spine

Dr. Charles A. Elsberg was a distinguished pioneer in spinal cord surgery and perhaps the foremost spinal surgeon of his time. He was the first Director of the Neurological Institute of New York, the first Professor of Neurological Surgery at Columbia University, and a long-time Fellow of The New York Academy of Medicine. Elsberg authored two texts that became classics in his field: Diseases of the Spinal Cord and Membranes (1916) and Tumors of the Spinal Cord (1925). In 1917, Elsberg wrote about his work on the physiological regeneration of motor nerves when directly implanted into paralyzed muscles and the possibility of the reestablishment of normal neuromotor connections. In a series of experiments undertaken in the thighs of rabbits, he found a remarkable difference in the behavior of the muscles’ own nerve and that of foreign nerve. Elsberg found that 1) it was possible to directly neurotize a muscle paralyzed by separation from its motor nerve supply (after 8–10 weeks, the connections between the nerve and the muscle fibers were reestablished); 2) it was possible to neurotize a muscle deprived of its nerve supply for many

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growth of cerebellar granule cell neurons. J Neurosci Res 2002;68: 161–8. Yang S, Lim M, Pham LK, et al. Bone morphogenetic protein 7 protects prostate cancer cells from stress-induced apoptosis via both Smad and c-Jun NH2-terminal kinase pathways. Cancer Res 2006;66:4285–90. Thornberry NA, Lazebnik Y. Caspases: enemies within. Science 1998;281:1312–6. 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. Kawakami M, Matsumoto T, Hashizume H, Kuribayashi K, Chubinskaya S, Yoshida M. Osteogenic protein-1 (osteogenic protein-1/bone morphogenetic protein-7) inhibits degeneration and pain-related behavior induced by chronically compressed nucleus pulposus in the rat. Spine 2005;30:1933–9. Friedlaender GE, Perry CR, Cole JD, et al. Osteogenic protein-1 (bone morphogenetic protein-7) in the treatment of tibial nonunions. J Bone Joint Surg Am 2001;83-A(Suppl 1):S151–8. Kim DJ, Moon SH, Kim H, et al. Bone morphogenetic protein-2 facilitates expression of chondrogenic, not osteogenic, phenotype of human intervertebral disc cells. Spine 2003;28:2679–84.

weeks (the muscle tissue regularly regenerated under the influence of the regenerating motor nerve, which was implanted); and 3) it was impossible to hyperneurotize a normal muscle (a normal muscle could not be made to take on additional nerve supply). However, Elsberg noted, if the muscle was permanently separated from its original nerves, then the implanted nervedwhich had been unable to form a connection with the muscle fibersdwould establish neuromuscular connections, and electric stimulation of the nerve would soon cause normal contractions of the muscle. Elsberg’s conclusion from his fourth experiment was that the native nerve to a muscle regained its motor connections with the muscle fibers and would somehow prevent a foreign nerve implanted at the same time from making any effective neuromuscular connections. These experiments proved to Elsberg that once a muscle has its normal nerve supply, no other motor nerve was able to make neuromuscular connections with the same muscle. In addition, if the normal nerve was cut and reimplanted into a muscle and, at the same time, a foreign motor nerve was also implanted into the same muscle, only the original nerve would make neuromuscular connections [1].

References [1] Elsberg CA. Experiments on motor nerve regeneration and the direct neurotization of paralyzed muscles by their own and by foreign nerves. Science 1917;45:318–20.