Proceedings of the NASS 19th Annual Mee

the mRNA levels of total LMP-1, overexpressed LMP-1 (human), BMP-2,BMP-7, and aggrecan were measured. Experiment 3: AdLMP-1 at dosesof 3.3×106, 107, 3.3×107 pfu and AdGFP at a dose of 107 pfu were tested(2 rabbits/dose). Three weeks later, the mRNA levels of total LMP-1,overexpressed LMP-1 (human), BMP-2, BMP-7, aggrecan and collagentype II were measured.RESULTS: Experiment 1 demonstrated endogenous rabbit LMP-1, ag-grecan, and BMP-7 mRNAs in the control discs. Discs injected withAdLMP-1 had increased mRNA levels of total LMP-1 (830%), BMP-7(1100%), and aggrecan (66%) higher than control. Experiment 2 demon-strated endogenous levels of BMP-2, BMP-7, LMP-1, and aggrecan mRNA.A correlation between increasing AdLMP-1 dose and total LMP-1mRNA and overexpressed (human) LMP-1 mRNA was seen. No overex-pressed LMP-1 (human) mRNA was present in the control groups. TheBMP-2 and BMP-7 mRNA levels were increased maximally at a dose of107 pfu of AdLMP-1. AdLMP-1 at 107 pfu also led to the highest increasein aggrecan mRNA, 50% over control. Experiment 3 demonstrated a thatincreasing AdLMP-1 dose led to increasing collagen II mRNA levels with3.3×107 pfu leading to 300% increase.CONCLUSIONS: The results show that overexpression of human LMP-1by in vivo gene therapy with an adenoviral vector is capable of upregulatingBMP-2, BMP-7, aggrecan mRNA and collagen type II. These findingsconfirm the predictions of our previous short term monolayer culture experi-ments and represent a major step towards long term in vivo experimentsto alter the course of disc degeneration.DISCLOSURES: Device or drug: LMP-1. Status: Investigational/Notapproved.CONFLICT OF INTEREST: Author (STY) Grant Research Support:Medtronic Sofamor Danek Research Grant.

doi: 10.1016/j.spinee.2004.05.127

4:44128. Porous coated motion cervical disk replacement: abiomechanical histomorphometric and biologic wear analysisin a caprine modelAnton E. Dmitriev, MSc1, Bryan W. Cunningham, MSc2, Nianbin Hu,MD2, Seok Woo Kim2, Andrew Cappuccino3, Luiz Pimenta4, Paul C.McAfee, MD5; 1Orthopaedic Research Laboratory, Baltimore, MD,USA; 2Union Memorial Hospital, Baltimore, MD, USA; 3University ofBuffalo, Buffalo, NY, USA; 4University of Sao Paulo, Brazil, Sao Paulo,Brazil; 5Spine and Scoliosis Center, Towson, MD, USA

BACKGROUND CONTEXT: Total disc replacement arthroplasty in thecervical spine represents continued development in the era of motion preser-vation for treatment of discogenic spinal pathology.PURPOSE: The current study was undertaken to investigate the biome-chanical and biologic in-growth characteristics of the Porous Coated Motioncervical disc prosthesis following a six and twelve-month post-operativeperiods.STUDY DESIGN/SETTING: An in-vivo caprine model was utilized inthe current investigation.PATIENT SAMPLE: N/AOUTCOME MEASURES: N/AMETHODS:TwelvematureNubian goatsweredivided into twogroupsbasedon post-operative survival periods of six (n�6) and twelve months (n�6).Using an anterior surgical approach, a complete diskectomy was performedat the C3–C4, followed by implantation of the Porous Coated Motion device.Functional outcomes of the disc prothesis were based on computed tomogra-phy (CT), multi-directional flexibility testing, undecalcifed histology, histo-morphometry and immunocytochemical analyses.RESULTS: There was no evidence of prosthesis loosening, neurologic orvascular complications. CT scans demonstrated the ability to image andassess the cervical spinal canal for the presence of compressive pathologyin the area of the CoCrMo prosthesis. Multi-directional flexibility testingunder axial rotation and lateral bending indicated no differences in fullrange of intervertebral motion between the disc prosthesis and non-operativecontrols (p�0.05). However, under flexion extension loading total discreplacement treatment allowed for a decreased range of motion at six andtwelve month post-operative periods (p�0.05). Based on immunohisto-chemical and histologic analysis, there was no evidence of particulate

4:37127. Small intestinal submucosa (SIS) as a potential bioscaffold forintervertebral disc regenerationCatherine Le Visage, PhD1, Shu-Hua Yang, MD2, Leena Kadakia, BS1,Ann N. Sieber, MS2, John P. Kostuik, MD2, Kam W. Leong, PhD1;1Biomedical Engineering Dept, Johns Hopkins University, Baltimore,MD, USA; 2Orthopaedic Surgery Dept, Johns Hopkins University,Baltimore, MD, USA

BACKGROUND CONTEXT: Progressive degeneration of intervertebraldiscs, associated with an abnormal turnover of extracellular matrix, is thepredominant cause of low back pain. SIS or Small Intestine Submucosa(SIS), a biocompatible and biodegradable acellular scaffold scaffold, hasbeen described for reinforcement of soft tissue and has been used inorthopedic applications to promote tissue regeneration in rotator cuffs,knee ligaments, and menisci.PURPOSE: The purpose of this study was thus to evaluate the performanceof SIS in supporting the culture of human degenerative disc cells in termsof cell growth and matrix synthesis.STUDY DESIGN/SETTING: We hypothesize that SIS, once implantedinto a degenerative disc, will constitute a temporary bioactive scaffold for insitu disc cells to proliferate and synthesize a new extracellular matrix,thereby arresting the degeneration, or even promoting the regeneration, ofthe disc.PATIENT SAMPLE: N/AOUTCOME MEASURES: N/AMETHODS: Annulus (AF) and nucleus (NP) cells isolated from degenera-tive intervertebral discs from 3 patients undergoing spinal procedures wereseeded onto SIS scaffolds. We evaluated over a 3-month period the cellgrowth (proliferation assay, DNA content) and matrix composition (glyco-saminoglycan and collagen content). Paraffin sections were stained withH&E and toluidine blue for histological evaluation. RT-PCR was carriedout on total RNA isolated from scaffolds with primers for type I, II and Xcollagen, aggrecan and Sox-9. After guanidine extraction from scaffolds,collagen proteins were identified by Western blot.

ting / The Spine Journal 4 (2004) 3S–119S 65S

RESULTS: More than 70% of seeded cells attached to the SIS surfacewithin a few hours of culture. Both NP and AF cells cultured in SISscaffolds cells remained viable and metabolically active for up to 3 months,even though the DNA content was found to decrease after 3 months ofculture. Macroscopic appearance of seeded scaffolds was dramatically mod-ified over time, showing significant folding and shrinkage of the membranes.H&E staining revealed a broad distribution of cells that invaded throughoutthe scaffold. Seeded scaffolds demonstrated a significantly higher GAGcontent as compared to control scaffold (160% and205% for NP and AFcells,respectively). Moreover, GAG content difference increased from month tomonth for both annulus and nucleus cells, even though annulus cells resultedin the highest increase. Large areas of proteoglycans were detected bytoluidine blue staining. Deposition of new matrix components was con-firmed by positive gene expression for collagen type I, II and X for bothcells, as well as expression for transcription factor Sox-9 and aggrecan atlater time points.CONCLUSIONS: SIS is a promising bioactive material that could poten-tially serve as a temporary scaffold for intervertebral disc regeneration, sinceit promotes the in vitro growth of degenerative disc cells and the synthesisof new extracellular matrix components that might restore the biochemicalproperties and functions of the native disc.DISCLOSURES: No disclosures.CONFLICT OF INTEREST: Author (CLV) Grant Research Support:Sponsored research agreement between DePuy and Johns Hopkins Univer-sity M080516; Author (JPK) Consultant: DePuy Inc.

doi: 10.1016/j.spinee.2004.05.128