Tobacco Smoke Drastically Upregulates Matrix Metalloproteinase Expression in Disc Cells Upon...

42S Proceedings of the NASS 27th Annual Meeting / The Spine Journal 12 (2012) 22S–44S

PT modifier were not significantly associated with changes in HRQOL

measures.

CONCLUSIONS: The Schwab-SRS classification of ASD provides a val-

idated language and has significant association with HRQOL measures.

The current study demonstrates that the classification modifiers are respon-

sive to changes in disease state and reflect significant changes in patient

reported outcome.

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

any applicable devices or drugs.

http://dx.doi.org/10.1016/j.spinee.2012.08.172

Thursday, October 25, 201211:00 AM – 12:00 PM

Biologics/Basic Science Seminar and OriginalResearch: Breaking Developments in

Disc Biology

82. Resident Stem Cells of the Nucleus Pulposus Are Affected by

Tissue Degeneration

Dmitriy Sheyn, PhD1, Hyun W. Bae, MD2, Anthony Oh, Wafa Tawackoli,

PhD, Dan Gazit, PhD3, Zulma Gazit, PhD1; 1Cedars-Sinai Medical

Center, Los Angeles, CA, US; 2Spine Institute St. John’s Health Center, Los

Angeles, CA, US; 3Hadassah Medical Center, Jerusalem, Israel

BACKGROUND CONTEXT: Intervertebral disc (IVD) degeneration and

chronic lower back pain are a major health problem. Since a fundamental

understanding of disc degeneration is lacking, robust clinical therapies that

target the causes, rather than symptoms, are still in the early development.

It was speculated that stem cells (SCs) maintain homeostasis of tissues

with a limited regeneration capacity. Changes in the differentiation of res-

ident SCs can represent a loss of homeostasis and diminished regeneration

of the tissue. We have previously shown that SCs reside in human degen-

erated nucleus pulposus (NP) and healthy rat NP. Here we hypothesized

that IVD degeneration affects the quantity, proliferation, and differentia-

tion potential of resident stem cells in the NP.

PURPOSE: The purpose of this study is to investigate the effect of disc

degeneration on residual stem cell properties.

STUDY DESIGN/SETTING: IVD degeneration was induced and after

the degeneration was evident, the discs were harvested and NP-SCs were

isolated from healthy and degenerated discs. The stem cell properties of

the two groups were evaluated in vitro.

OUTCOME MEASURES: The outcome measures in this study were the

stem cell properties like self renewal rate and MSC surface markers. Also

we have tested the extent of stem cell differentiation in vitro either to mes-

enchymal lineages or the NP-like cells.

METHODS: Disc degeneration was induced in a mini-pig model by cre-

ating an annular injury. Degeneration was verified by MRI 6 weeks after

surgery. NP-SCs were isolated from healthy (healthy-NP) and degener-

ated discs (degenerated-NP). The freshly isolated cells were tested with

a colony-forming unit (CFU) assay. Surface mesenchymal stem cell

(MSC) marker expression was examined using FACS analysis and immu-

nohistochemistry (IHC). The proliferation rate was evaluated by cell

counts made using Trypan blue exclusion and BrdU incorporation. Cell

differentiation into mesenchymal lineages was assessed in vitro using

von Kossa staining, DMMB assay, and Oil red O staining, respectively.

In addition, NP-SCs were differentiated into NP-like cells by culture in

alginate beads supplemented with TGFb1 in conditions of hypoxia (2%

oxygen) and normoxia for 7 and 14 days. Differentiation was evaluated

using GAG quantification with a DMMB assay and ELISA for aggrecan.

All referenced figures and tables will be available at the Annual Mee

The expression of aggrecan, collagen II and sox-9 genes was quantita-

tively evaluated as well.

RESULTS: NP-SCs were isolated from healthy and degenerate IVDs. A

significantly higher rate of proliferation was measured in cells from degen-

erate-NP by using cell counts and a BrdU assay, and a higher rate of CFUs

as well. Freshly isolated SCs from healthy-NP exhibited a higher rate of

MSC markers in the FACS analysis. MSC marker expression was verified

using IHC in NP tissue. Differentiation assays showed that healthy-NP and

degenerated-NP SCs differentiate into the mesenchymal lineages without

significant differences between the two groups. Healthy NP-SCs showed

a higher differentiation capacity to NP- like cells than degenerated NP-de-

rived, in conditions of hypoxia but not normoxia, with higher aggrecan and

colII gene expression, higher aggrecan measured in the differentiating cul-

ture media and by DMMB assay.

CONCLUSIONS: These results indicate that disc degeneration has a clear

effect on endogenous SCs in the NP. Cells derived from degenerate discs

exhibit higher proliferation abilities that are likely caused by the degener-

ation process in the discs. However, although these cells maintain their dif-

ferentiation abilities, they have a lower capability of differentiating into

NP-like cells. These findings can shed light on the IVD degeneration pro-

cess, elucidating what is the role of resident SCs and explaining why these

cells are not able to reverse the degeneration.




83. Tobacco Smoke Drastically Upregulates Matrix

Metalloproteinase Expression in Disc Cells Upon Suppression of the

p38 MAPK Pathway

Nam Vo, PhD1, Kevin Ngo2, Michael J. McKernan5, Rebecca Studer, PhD2,

Joon Y. Lee, MD3, Gwendolyn A. Sowa, MD, PhD4, James D. Kang, MD3;1LJPMC/Ferguson Lab, Pittsburgh, PA, US; 2Pittsburgh, PA, US;3University of Pittsburgh Medical Center, Pittsburgh, PA, US; 4University

of Pittsburgh, Pittsburgh, PA, US; 5Sylvania, OH, US

BACKGROUND CONTEXT: Tobacco smoking increases the risk of

back pain and intervertebral disc degeneration (IDD), but the mechanism

of its adverse action on disc metabolism is not well understood [1, 2]. Loss

of disc matrix due to dysregulated activities of matrix metalloproteinses

(MMPs) is implicated in initiating the IDD cascade [3]. Tobacco smoke

condensate (TSC), containing numerous inflammatory and oxidative

stress inducing agents [4], has previously been shown to upregulate

MMP expression in disc cells [5]. The p38 MAPK pathway is a central

mediator of stress-induced MMP expression [6], but it is not known

whether tobacco smoking upregulates MMP in disc cells through this

pathway.

PURPOSE: Investigate p38 MAPK-mediated regulation of the two major

disc MMPs (MMP1, MMP3) in human disc cells exposed to TSC.

STUDY DESIGN/SETTING: Laboratory controlled cultures of human

annulus fibrosis (hAF) cells exposed to TSE were treated with and without

the p38 dominant negative protein to block the p38 MAPK pathway.

OUTCOME MEASURES: MMP1 and MMP3 mRNA expression level.

METHODS: hAF cellswere grown inmonolayer culture and transfected for

18-24 hourswith the dominant negative p38aprotein to block the action of

this major p38 isoform, and subsequently treated with 0.1 mg/mL total

smoke condensate (TSC) for 24 hours. MMP1 and MMP3 mRNAwere de-

termined by real time semiquantitative RT-PCR and compared to untreated

samples.

RESULTS: Compared to unexposed control, TSC exposed hAF cells ex-

hibited an increase in MMP-1 (74 fold) and MMP-3 (2 fold) mRNA expres-

sion. When treated with dominant negative p38aprotein to block the p38

MAPK pathway, TSC exposed hAF cells drastically further upregulated

ting and will be included with the post-meeting online content.



43SProceedings of the NASS 27th Annual Meeting / The Spine Journal 12 (2012) 22S–44S

MMP-1 (154 fold) and MMP-3 (15 fold) mRNA expression over the unex-

posed control.

CONCLUSIONS: This study demonstrates that smoke condensate, which

has the advantage of containing all of the compounds inhaled by smokers4,

stimulates gene expression of MMP-1 and MMP-3, the two key MMPs im-

plicated in matrix destruction and IDD, in disc cells. More important, our

findings suggest that the p38 MAPK pathway is vital for keeping MMP-1

and MMP-3 expression under control when disc cells are under the stress

of tobacco smoke exposure. This is evident by the dramatic upregulation

of these two MMPs by TSC-exposed disc cells when the p38 pathway is

blocked. Additional studies are being performed to determine if tobacco

smoking interferes with the p38 MAPK pathway, leading to upregulation

of MMPs and disc matrix destruction, to confirm it as a potential therapuetic

target to treat IDD.

REFERENCES:

[1]. Battie, M.C. et al. 1991 Volvo Award in clinical sciences. Smoking and

lumbar intervertebral disc degeneration: an MRI study of identical

twins. Spine (Phila Pa 1976) 16, 1015-21 (1991).

[2]. Leboeuf-Yde, C., Kjaer, P., Bendix, T. & Manniche, C. Self-reported

hard physical work combined with heavy smoking or overweight may

result in so-called Modic changes. BMC Musculoskelet Disord 9, 5

(2008).

[3]. Le Maitre et al., Biochem Soc Trans. 2007. Aug;35(Pt 4):652-5.

[4]. Ramos KS, Moorthy B. Drug Metab Rev. 2005;37(4):595-610.

[5]. Vo et al., Vo, N. et al. J Orthop Res 29, 1585-91.

[6]. Studer et al., Spine. 2007 Dec 1;32(25):2827-33.




84. Notochordal Cells Protect Nucleus Pulposus Cells fromApoptosis:

Implications for the Mechanisms of Intervertebral Disc Degeneration

William Mark Erwin, DC, PhD1, Diana Islam, MSc2, Robert Davies-

Inman, MD, FRCP3, Michael G. Fehlings, MD, PhD, FRCSC1, Florence

W. Tsui, PhD3; 1Toronto Western Hospital, Toronto, ON, Canada;2University of Toronto, Toronto, ON, Canada; 3University Health Network,

Toronto, ON, Canada

BACKGROUND CONTEXT: Given the lack of a biological strategy for

regeneration of the degenerating disc, a therapeutic intervention that may of-

fer restorative qualities to the disc is a much needed and widely sought goal.

The ideal biological agent might reactivate homeostatic mechanisms in-

nately inherent to the healthy intervertebral disc (IVD). The capacity to re-

establish equilibrium between catabolic and anabolic tissue remodeling

would represent the ideal regenerative strategy for the treatment of DDD.

With respect to potential biological therapies, lessons learned from the study

of the non-chondrodystrophic canine (NCD canine) IVD might provide

essential molecular clues for restoration of homeostasis to the disc. The

NCD canine is unique among the canine sub-species in that this animal is

relatively resistant to the development of DDD. Notably, NCD canines pre-

serve their notochordal cell populations throughout life. Thus, there is an

emerging body of evidence indicating that notochordal cells confer anabolic

capacity uponNPcells and that their absence is associatedwith susceptibility

to degenerative changes.

PURPOSE: Non-chondrodystrophic (NCD) canines resist developing de-

generative disc disease (DDD) possibly due to factors secreted by noto-

chordal cells within their intervertebral disc (IVD) nucleus pulposus

(NP). DDD is known to be mediated by interleukin-1 beta (IL-1ß) and/

or Fas-Ligand (Fas-L). Therefore we evaluated the ability of notochordal

cell conditioned medium (NCCM) to protect NP cells from IL-1ß and

IL-1ß þFasL-mediated cell death and degeneration.

STUDY DESIGN/SETTING: In vitro laboratory study.

All referenced figures and tables will be available at the Annual Mee

OUTCOME MEASURES: qRT-PCR, flow cytometry and activated Cas-

pase assays

METHODS: We cultured bovineNP cells with IL-1ß or IL-1ßþFasL under

hypoxic serum-free conditions (3.5%O2) and treated the cells with either se-

rum-free NCCM or basal medium (Advanced DMEM/F-12). We used flow

cytometry to evaluate cell death and real-time (RT-)PCR to determine the

gene expression of aggrecan, collagen 2, and link protein, mediators of ma-

trix degradation ADAMTS-4and MMP3, the matrix protection molecule

TIMP1, the cluster of differentiation (CD)44receptor, the inflammatory cy-

tokine IL-6andAnk.We then determined the expression of specific apoptotic

pathways in bovine NP cells by characterizing the expression of activated

caspases-3, -8 and -9 in the presence of IL-1ßþFasL when cultured with

NCCM, conditioned medium obtained using bovine NP cells (BCCM),

and basal medium all supplemented with 2% FBS.

RESULTS: NCCM inhibits bovine NP cell death and apoptosis via sup-

pression of activated caspase-9 and caspase-3/7. Furthermore, NCCM pro-

tects NP cells from the degradative effects of IL-1ß and IL-1ßþFas-L by

up-regulating the expression of anabolic/matrix protective genes (aggre-

can, collagen type 2, CD44, link protein and TIMP-1) and down-regulating

matrix degrading genes such as MMP-3. Expression of ADAMTS-4, which

encodes a protein for aggrecan remodeling, is increased. NCCM also pro-

tects against IL-1þFasL-mediated down-regulation of Ank expression.

Furthermore, NP cells treated with NCCM in the presence of IL-

1ßþFas-L down-regulate the expression of IL-6 by almost 50%. BCCM

does not mediate cell death/apoptosis in target bovine NP cells.

CONCLUSIONS: We have demonstrated in this study that NCCM

protects against NP apoptosis via suppression of activated caspase-9 and

-3/7. Possible mechanisms include stabilization of the mitochondrial mem-

brane via inhibition of Bcl-2 activity, Bid activation or through the P53

growth factor-related pathway. It may be that components of NCCM sup-

press the formation of the apoptosome and in so doing suppress the forma-

tion of activated caspase-3 thereby preventing apoptotic cell death. The

novel finding of the activation of the Ank gene by NCCM under degener-

ative/death-inducing conditions suggests a possible role for this gene in the

maintenance of NP cell phenotype. These areas are under examination by

our group. The results of the present study provide evidence in vitro that

notochordal cell-secreted soluble factors provide essential molecular

signals that mediate apoptosis and degradation of NP cells induced by

IL-1bþFas-L. Harnessing the regenerative capacity of these cells and the

important factors they secrete may lay the cornerstone of biological ther-

apy for the treatment of degenerative disc disease.




85. Effects of Hypoxia on Differentiation from Human Placenta-

Derived Mesenchymal Stem Cells to Nucleus Pulposus-Like Cells

Huilin Yang, MD, PhD1, Li Ni, MD1, Xiaochen Liu2, Muhammad Z. Moral,

MS2, Molly A. Ebraheim2, Qin Shi, MD1, Jiayong Liu, MD1,2; 1Department

of Orthopaedic Surgery, The First Affiliated Hospital of Suzhou University,

Suzhou, China; 2Department of Orthopaedic Surgery, University of Toledo

Medical Center, Toledo, OH, US

BACKGROUND CONTEXT: Regenerative medicine has presented inno-

vative treatments for lower back pain. Stem cells have been the front

runner in tissue engineering because of their high potential for cell differ-

entiation. Disc is relatively hypoxic setting. Studies on the effects of hyp-

oxia on the differentiation from human placenta derived mesenchymal

stem cells to nucleus pulposus-like cells are lacking.

PURPOSE: To compare biological effects of cell differentiation from pla-

centa mesenchymal stem cells (PMSCs) to nucleus pulposus-like cells un-

der conditions of different oxygen concentration by cultivating human

PMSCs in vitro and to study their biological characteristics.

ting and will be included with the post-meeting online content.



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