50S Proceedings of the NASS 23rd Annual Meeting / The Spine Journal 8 (2008) 1S–191S

Thursday, October 16, 20085:15–6:15 PM

Special Interest Paper Presentation 2:Basic Science

97. The Pathophysiological Role of the PKCdelta Pathway in the

Intervertebral Disc

Michael B. Ellman, MD1, Howard S. An, MD2, Prasuna Mudassani3,

Frank Phillips, MD2, Ranjith Udayakumar1, Hee-Jeong Im1; 1Department

of Biochemistry and Orthopaedic Surgery, Rush University Medical

Center, Chicago, IL, USA; 2Department of Orthopaedic Surgery, Rush

University Medical Center, Chicago, IL, USA; 3Department of

Biochemistry, Rush University Medical Center, Chicago, IL, USA

BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration has

been associated with an upregulation of matrix-degrading enzymes (MMP-

13) and progressive loss of proteoglycans (PG) in the nucleus pulposus

(NP) [1,2]. PKCdelta and other PKC isoforms, such as PKCalpha/beta

and PKCepsilon, may have different biological effects in cartilage homeo-

stasis [3]. Previously, we demonstrated the critical role of PKCdelta in the

upregulation of MMP-13 after stimulation with catabolic cytokines (IL-1)

in human articular chondrocytes [4]. However, little is known about the

involvement of PKCdelta in IVD degeneration.

PURPOSE: The aim of the present study is to determine the effect of the

PKC pathways on the IL-1-induced upregulation of MMP-13 and PG pro-

duction using bovine NP cells.

METHODS: IVD tissue was harvested from bovine coccygeal tissue (15-

18 months old) and chondrocytes were isolated from the NP, digested, and

captured in alginate, as previously described [5]. The beads were cultured

in complete medium (DMEM/F12 supplemented with 10%FBS) for 21

days and PG accumulation was assessed by dimethylmethylene blue assay

as previously described [6]. NP cells cultured in serum-free monolayer were

treated with pathway-specific inhibitors of PKCdelta (rottlerin, 4 uM),

PKCalpha/beta (10 uM), PKCepsilon (10 uM), and PKCzeta (10 uM). Im-

munoblotting was performed using anti-MMP-13 antibody, as previously

described [5]. BMP7 (100 ng/ml, Stryker) was used as control for PG pro-

duction. Analysis of variance was performed using StatView 5.0 software

with p-values!0.05 considered significant.

RESULTS: Stimulation of bovine NP cells cultured in monolayer with

IL-1 (10 ng/ml) augmented the production of MMPs [MMP-1, MMP-3,

and MMP-13] and aggrecanases [ADAMTS4 and ADAMTS5]. This aug-

mentation was completely blocked by the addition of a pathway specific

PKCdelta inhibitor (PKCdI). Other PKC inhibitors such as PKCepsilon

and zeta failed to block the IL-1-induced catabolic effect, except PKCal-

pha/beta inhibitor which showed partial inhibition. Incubation of cells

cultured in alginate with PMA, a nonspecific activator of multiple PKC

isoforms [7], for 21 days significantly decreased PG deposition compared

to control. More importantly, the presence of PKCdI along with PMA

completely abolished the catabolic effect mediated by PMA: when

PKCdI was administered with PMA, PG production was increased by

40% compared to control, and 100% compared to PMA alone. Without

PMA, incubation of cells with PKCdI maintained control level. The an-

abolic growth factor BMP7 (100 ng/ml) was used as a positive control.

The incubation of NP cells with PMA, PKCdI, IL-1 or BMP7 had no ef-

fect on cell survival, as viability remained O90% for all treatment

conditions.

CONCLUSIONS: Similar to our findings in human articular chondrocytes

[4], these results suggest that IL-1 and PMA upregulate MMPs [MMP-

1,MMP-3 and MMP-13], ADAMTS4 and ADAMTS5 expression specifi-

cally via the PKCdelta signaling pathway in IVD cells, and the PKCdelta

pathway is associated with reduced PG production. Therefore, blocking the

PKCdelta pathway may be beneficial in the prevention and/or treatment of

disc degeneration. These findings provide evidence for the possible thera-

peutic role of pathway-specific inhibitors of the PKC cascades in the

future.

FDA DEVICE/DRUG STATUS: This abstract does not discuss or include

any applicable devices or drugs.

doi:10.1016/j.spinee.2008.06.117

98. Juvenile Chondrocytes are Superior to Undifferentiated

Mesenchymal Stem Cells for Porcine Intervertebral Disc Repair

Frank Acosta, Jr.1, Lionel Metz1, Jane Liu1, Ellen Carruthers-Liebenberg1,

H.D. Adkisson2, Michael Maloney, MD2, Jeffrey Lotz, PhD1; 1University of

California, San Francisco, San Francisco, CA, USA; 2ISTO Technologies,

Inc., St. Louis, MO, USA

BACKGROUND CONTEXT: The concept of biological repair of the interver-

tebral disc (IVD) has grown in recent years out of an improved understanding of

the cellular and molecular events involved in disc aging and degeneration [1,2].

Recent approaches have focused on the use of autologous disc chondrocytes as

the cell type for nucleus pulposus (NP) regeneration [3-5]. This cell source, how-

ever, has practical limitations due to graft procurement and difficulty in expand-

ing mature autologous disc chondrocytes in culture. Mesenchymal stem cells

(MSCs) and juvenile chondrocytes (JCs) are two cell types whose procurement

and expansion avoid these drawbacks, while maintaining the potential to pro-

duce NP-like extracellular matrix and facilitate IVD repair.PURPOSE: To compare the viability and functionality of transplanted

MSCs and JCs after IVD injury and treatment.

STUDY DESIGN/ SETTING: Mini-pig animal model.

PATIENT SAMPLE: A total of 30 pigs were included. 10 animals each

were assigned to analysis at 3-, 6-, and 12-months post-treatment.

OUTCOME MEASURES: T2-weighted magnetic resonance imaging of

the lumbar spine was performed at 3-, 6-, and 12-months. The 3- and 6-

month animal IVDs were analyzed for disc height and morphology. Each

disc was randomly assigned to either histological analysis of morphology

and proteoglycan production, biochemical analysis of water, proteoglycan,

and protein content, or DNA-content.

METHODS: Preparation of JC Treatment: JCs were harvested from articular

cartilage dissected from 2–3 week male minipigs. Cells were culture expanded

in flasks modified by covalent attachment of hyaluronic acid in order to retain

the hyaline phenotype [6]. Chondrocytes were harvested at day 21 and sus-

pended in a proprietary cryopreservation solution containing 10% DMSO. Just

prior to injection, cells were suspended at a concentration of 1�106 cells/mL

in a fibrin carrier. Preparation of MSC Treatment: MSCs were isolated from

juvenile male farm pig bone marrow. MSCs were then prepared at a concentra-

tion of 1�106 cells/mL in the same fibrin carrier. IVD Injury: A single stab in-

cision was made in the L2-L6 IVDs of adult female pigs and a HydroCision�SpineJet� EndoResector (HydroCision, Inc., Billerica, MA) was inserted into

the annulotomy defect for 2 min. to aspirate the entire NP. Following aspira-

tion, IVD levels were randomly assigned to 1 of 3 treatments: 1. JC’s plus fibrin

carrier; 2. MSC’s plus fibrin carrier; or 3. Fibrin carrier alone. Injected versus

host cells were distinguished using fluorescence in situ hybridization (FISH) to

detect Y-chromosome-positive male cells.

RESULTS: At both the 3- and 6-month time points, treated IVDs trended

to be less hydrated, and contain more protein and DNA than controls. T2-

weighted MRI data were consistent with this. No inflammatory response

was observed histologically. For the carrier and MSC groups the nuclear

region was filled with collagen with little or no evidence of proteoglycan

matrix or residual chondrocyte cells. By contrast, the JC IVDs demon-

strated regions of abundant cartilage formation with FISH positive cells.

CONCLUSIONS: Our data demonstrate that JC’s are capable of surviving and

producing cartilage matrix within the IVD environment. While MSC’s have the

potential to differentiate into cartilage under certain controlled conditions, this

was not observed. Based on our results, JC’s are a good cell type for IVD repair.

Future efforts should focus on surgical and cell delivery technique optimization.

FDA DEVICE/DRUG STATUS: NuQu (Juvenile chondrocytes/fibrin car-

rier) is a product of ISTO Technologies, Inc.: Investigational/Not approved.

doi:10.1016/j.spinee.2008.06.118

51SProceedings of the NASS 23rd Annual Meeting / The Spine Journal 8 (2008) 1S–191S

99. Human Mesenchymal Stem Cells Differentiate into NP-like Cells

When Cultured on Allograft Nucleus Pulposus Scaffolds

Frank Phillips, MD1, Monica Susilo, BS2, Eric Nauman, PhD2, David Van

Sickle, DVM2, Bret Ferree, MD3; 1Chicago, IL, USA; 2Purdue University,

West Lafayette, IN, USA; 3Cincinnati, OH, USABACKGROUND CONTEXT: ‘‘Smart Scaffolds’’ impart crucial spatial

and regulatory information to cells for selected gene expression and con-

tain biomolecules, such as growth factors, that promote cell infiltration,

proliferation, and differentiation. Smart scaffolds and cells of disc origin

or cells that differentiate into disc-like cells may potentially be used to

treat degenerative disc disease.

PURPOSE: The goals of this in vitro study were to measure human mes-

enchymal stem cell (hMSC) proliferation, differentiation, and glycosami-

noglycan (GAG) production after seeding the hMSCs on two different

injectable human allograft nucleus pulposus (NP) derived smart scaffolds.

STUDY DESIGN/ SETTING: In Vitro, mesenchymal stem cell culture.

PATIENT SAMPLE: Human stem cells cultured on human allograft particles.

OUTCOME MEASURES: Human mesenchymal stem cell proliferation,

glycosaminoglycan production, and histological evaluation.

METHODS: Allograft nucleus pulposus (NP) tissue was frozen to �80 C,

particulated into 1mm � 1mm pieces and used to create two different scaf-

folds. A proteoglycan depleted scaffold (PGDNP) was manufactured by

enzymatic digestion of the tissue with C-ABC and Keratanase. NP parti-

cles, without enzymatic digestion, were used as a second scaffold (NP).

50,000 hMSCs per well were added to culture medium, 10% or 25% plate-

let rich plasma (PRP), and one of three scaffold conditions: with PGDNP,

with NP or without scaffold (hMSCþPGDNP, hMSCþNP, & hMSC

groups, respectively) and cultured for 4 weeks. hMSC proliferation was

measured between 1 and 2 weeks of culture by CytoTox 96 assay. GAG

content was measured by Alcian blue assay. GAG production was calcu-

lated by subtracting preculture GAG content of each group from the

GAG content of the medium and pellets following four weeks of cell cul-

ture. Cell seeded scaffolds were injected through a 16 gauge needle to de-

termine cell viability following injection. Lastly, pellets from all groups

underwent histological examination.

RESULTS: hMSCs proliferated 10% faster on PGDNP than in medium

alone (p!0.05) and 15% slower on NP than in medium alone (p!0.05).

Comparison of GAG content before and after 4 weeks of culture treatment

with 10% PRP, showed GAG production of 13 g, 16 g, & 132 g of GAG in

the hMSC, hMSCþPGDNP, and hMSCþNP groups, respectively. The use

of NP as a scaffolding produced a significant increase in GAG production

(p!0.0001). hMSCs in all three groups produced more GAGs when cul-

tured in 10% PRP than in 25% PRP (p!0.05). Less than 10% of hMSCs

died following passage of the cell seeded scaffolds through a 16 gauge nee-

dle. hMSCs grew into and revitalized both scaffolds. hMSCs differentiated

into large oval proteoglycan (PG) rich NP-like cells when seeded on

PGDNP and NP scaffolds. However, the cells in hMSC group, without

NP derived scaffold, contained little PGs, were spindle shaped with cyto-

plasmic processes similar to fibroblasts.

CONCLUSIONS: Human MSCs differentiated into NP-like cells when

cultured on Allograft human NP particles. The MSCs proliferated faster

but differentiated less when cultured on PGDNP compared to NP. Inject-

able allograft NP scaffold, hMSCs, and PRP may conceivably be used to

regenerate NP tissue to treat early degenerative disc disease. However, lim-

ited diffusion across degenerative vertebral endplates could adversely af-

fect hMSC viability within the degenerative disc.

FDA DEVICE/DRUG STATUS: This abstract does not discuss or include

any applicable devices or drugs.

doi:10.1016/j.spinee.2008.06.119

100. Magnesium in a Polyethylene Glycol Formulation Provides

Neuroprotection after Acute Spinal Cord Injury

Brian Kwon, MD, PhD1, Josee Roy, PhD2, Jae Lee, BSc1,

Anthea Stammers1, Jeff Marx, PhD3; 1University of British Columbia,

Vancouver, British Columbia, Canada; 2Medtronic, Memphis, TN, USA;3Memphis, TN, USA

BACKGROUND CONTEXT: Magnesium (Mg) has shown promise as

a neuroprotective agent in a variety of neurotrauma settings, such as stroke,

brain injury, and spinal cord injury (SCI). To develop it into a clinically

applicable therapy for human SCI, further evaluation of Mg in different

doses and formulations to improve its efficacy and safety is warranted.

PURPOSE: The purpose of this study was to evaluate the neuroprotective

effect of Mg within a polyethylene glycol formulation (Mg/PEG) in an an-

imal model of thoracic SCI, with the goals of 1. comparing it with meth-

ylprednisolone, 2. establishing a dose-response effect, and 3. defining

a time window of efficacy to guide inclusion criteria for a clinical trial.

STUDY DESIGN/ SETTING: This is an experimental in vivo study uti-

lizing a well-validated thoracic contusion SCI model in Sprague Dawley

Rats.

PATIENT SAMPLE: Adult male Sprague Dawley Rats (Charles River).

OUTCOME MEASURES: Open field locomotion (BBB score) for 6 weeks,

and histologic measurement of spinal cord injury lesion size 6 weeks post-injury.

METHODS: Adult male Sprague Dawley rats underwent a thoracic con-

tusion SCI. Firstly, animals were randomized to Mg/PEG, methylprednis-

olone (MP), or saline. Secondly, animals were randomized to receive Mg/

PEG in one of two doses, and for 2 or 6 infusions q6hourly. Finally, ani-

mals were randomized to receive the Mg/PEG within 15 minutes, 2, 4, or 8

hours post injury. Open field locomotion (BBB score) was evaluated over 6

weeks, and then the spinal cords were sectioned longitudinally to measure

the lesion size at the injury site.

RESULTS: While MP alone did not significantly improve locomotor func-

tion or reduce lesion size, both Mg/PEG alone or Mg/PEG with MP signifi-

cantly improved BBB scores and reduced lesion size at 6 weeks post-injury.

Locomotor function and the reduction in lesion size were also significantly

improved by increasing the Mg dose from 127 umol/kg to 254 umol/kg,

and by increasing the number of infusions from 2 to 6. Finally, Mg/PEG

started either 15 minutes, 2 hours, or 4 hours post-injury significantly im-

proved locomotor recovery and reduced lesion size.

CONCLUSIONS: Mg within a PEG formulation has significant neuropro-

tective effects after thoracic SCI, and improves behavioral recovery. Im-

portant clinically relevant parameters have been established in a standard

thoracic contusion injury. Given that Mg and PEG are both routinely given

to humans, this new Mg formulation has substantial translational potential

for clinical evaluation as a neuroprotective agent after SCI. Human clinical

trials are currently being planned.

FDA DEVICE/DRUG STATUS: This abstract does not discuss or include

any applicable devices or drugs.

doi:10.1016/j.spinee.2008.06.120

101. A Functional Polymorphism in COL11A1 is Associated with

Susceptibility to Lumbar Disc Herniation

Futoshi Mio1, Kazuhiro Chiba, MD, PhD1, Yoshiharu Kawaguchi2,

Yasuo Mikami3, Toshikazu Kubo3, Tomoatsu Kimura2, Yoshiaki Toyama1,

Shiro Ikegawa4; 1Department of Orthopedic Surgery, Keio university,

Tokyo, Japan; 2Department of Orthopedic Surgery, Toyama University,

Toyama, Japan; 3Department of Orthopedic Surgery, Kyoto Prefectual

University of Medicine, Kyoto, Japan; 4SNP Research Center, The Institute

of Physical and Chemical Research, Tokyo, Japan

BACKGROUND CONTEXT: Lumbar disc herniation (LDH), one of the

most common musculoskeletal diseases, causes sciatica and low back pain,

which affect the activities of daily living of the patients at the same time,

resulting in enormous economic burden to the society. Many risk factors of

LDH have been reported; however, its exact etiology and pathogenesis re-

main unknown. Extracellular matrix (ECM) molecules play important

roles in the maintenance of the integrity and functions of the intervertebral

disc (IVD). We have identified a gene encoding ECM proteins in the IVD,