99. Gene therapy utilizing stem cells from human fat in a rat posterolateral spine fusion model

Proceedings of the NASS 19th Annual Meeting / The Spine Journal 4 (2004) 3S–119S disc herniations, and 3 cases of stenosis with fusion performed in 2 of those cases. CONCLUSIONS: This long-term study shows that the complications rate is low after implantation of the non-constrained SB Charite´ III prosthesis. More than 87% of the prosthetic levels were mobile in the long-term. Complications were primarily due to bad positioning, an earlier generation of plate without hydroxyapatite coating (available after 1999) and sterilization of the core in air (performed before 1997). Revision surgery is difficult but remains possible with the Charite´ prosthesis allowing implantation of a new prosthesis without any sacrifice of bone. Functional results are better than, and the incidence of adjacent level problems are much less than, those reported for fusion in the long-term. DISCLOSURES: Device or drug: Charite artificial disc—Investigational in the U.S., approved outside the U.S.. Status: Investigational/Not approved. CONFLICT OF INTEREST: Author (TD) Consultant: DePuy Spine; Author (TD) Speaker’s Bureau Member: DePuy Spine. doi: 10.1016/j.spinee.2004.05.098

Saturday, October 30, 2004 1:50–2:35 PM Concurrent Sessions 5A: Cell Biology 1:50 98. Influence of estrogen to the spinal ligament ossification: an in vitro study using cultured human spinal ligament cells Akihito Wada1, Yukikazu Okajima2, Toru Suguro2, Hiroshi Takahashi2; 1 TOHO University School of Medicine Department of Orthopedic Surgery, Cordova, Tennessee, Japan; 2TOHO University School of Medicine, Japan BACKGROUND CONTEXT: Previous studies suggest many factors (genetic factors, dietary habits, mechanical stress to the PLL, production of cytokines or growth factors in the PLL tissues etc.) for causation of OPLL, but etiology of this disease is still unknown. On the other hand, female sex steroid hormones, especially estrogen is known that has anabolic action in bone metabolism. And occurrence of this disease of the female-to-male ratio is estimated to be approximately 1:2. Hence, we hypothesize that estrogen might be one of causative factor for OPLL. PURPOSE: The objective of our study is to determine the effect of estrogen to the human spinal ligament cells. STUDY DESIGN/SETTING: An in vitro experimental study using blood serum and cultured human spinal ligament cells obtained from patients with OPLL. PATIENT SAMPLE: 84 patients (54 males and 30 females; mean age 56.7 years) and 69 controls (radiolographically no findings of OPLL, 42 males and 27 females; mean age 55.1 years) were used for measurement of serum estrogen level. The patients were classified into 46 segmental type and 38 mixed-continuous type based on the radiographical findings. Cultured human spinal ligament cells obtained from 15 OPLL patients and 12 non-ossified controls at the time of surgery were used. OUTCOME MEASURES: For statistic analysis, an unpaired Student’s t-test and Pearson’s correlation coefficient were used. When the p- value was less than 0.05, the difference or correlation was considered to be significant. METHODS: Serum concentration of estron (E1), estradiol (E2), estriol (E3) were determined by radioimmunoassay. To determine any difference in the affinity of estrogen, estradiol receptor binding assay were performed and compared cells from OPLL patients with controls. Furthermore, to evaluate the response of spinal ligament cells to stimulation by estradiol, those cultured cell were assigned to estradiol added (E2 plus) group and not added (E2 minus) group. The rate of tritiated thymidine uptake of those cultured cells were examined. Also we measured the production of transforming growth factor-beta1(TGF-beta1) and basic fibroblast growth

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factor (b-FGF) in medium on those cells by enzyme linked immunosorbent assay, and then compared (E2 plus) group with (E2 minus) group. RESULTS: The mean serum concentration of total estrogen (E1⫹E2⫹E3) in male segmental type OPLL patients was 82.5 pg/ml, mixed-continuous type was 126.1pg/ml, and mixed-continuous type was significantly higher than controls (78.1pg/ml)(P⬍0.01). The same tendency was observed in female cases. Cultured cells obtained from OPLL patients had receptors with a higher affinity for estradiol than cells from controls. Cells from OPLL patients responded to the stimulation by estradiol, elevated the tritiated thymidine uptake, and accelerated the production of TGF-beta1 and b-FGF in medium. However, cells from controls showed no significant change in those studies. CONCLUSIONS: We reported here for the first time that hormone receptor of which direct acting point of estrogen was detected in human spinal ligament cells, and cells obtained from OPLL patients had receptors with a higher affinity for estrogen than cells from controls. Moreover, cells from OPLL patients responded to estrogen and increased cell proliferation as well as production of growth factors like osteoblast. Results from these studies suggested that the development of spinal ligament ossification was related to an estrogen-dominant hormone imbalance in serum, and the existence of high-affinity estradiol receptors in the spinal ligament cells. DISCLOSURES: No disclosures. CONFLICT OF INTEREST: Author (AW) Grant Research Support: Author acknowledges a financilal relationship to SICOT Grant Research Support No:0081. doi: 10.1016/j.spinee.2004.05.099 1:57 99. Gene therapy utilizing stem cells from human fat in a rat posterolateral spine fusion model Wellington Hsu, MD1, Brian Feeley, MD2, Lucie Krenek, BS2, Patricia Zuk, PhD2, Marc Hedrick, MD2, Prosper Benhaim, MD2, Jeffrey Wang, MD1, Jay Lieberman, MD1; 1UCLA Medical Center, Los Angeles, CA, USA; 2UCLA Medical Center, CA, USA BACKGROUND CONTEXT: Novel gene therapy systems have recently garnered significant interest in the spine literature. Human liposuction aspirates contain an abundance of pluripotent progenitor cells termed processed lipoaspirate cells (PLAs) that demonstrate the ability to differentiate into cells of chondrogenic, osteogenic, myogenic, and adipogenic lineage. Recent reports in the literature have reported the use of these stem cells in bone induction and as targets for gene therapy and tissue engineering. PURPOSE: This study seeks to utilize BMP-2-producing stem cells from human fat created through adenoviral gene transfer to induce a posterolateral spine fusion in an athymic rat. STUDY DESIGN/SETTING: Animal model. PATIENT SAMPLE: Female athymic rats at 8 weeks of age were used. OUTCOME MEASURES: Successful spinal fusion was characterized by plain radiographs, microCT scanning, manual palpation, and histological analysis. METHODS: Human liposuction aspirates were obtained and prepared via a previously published protocol. Intertransverse spinal arthrodesis was attempted in a total of four groups of athymic rats: 1) 5×106 adipose cells transduced with Ad-BMP-2 adenoviral vector, 2) 1 ug of recombinant BMP-2, 3) 5×106 adipose cells alone, 4) autogenous iliac crest bone graft. All treatment groups were combined with a collagen sponge carrier at the site of implantation. Each animal underwent plain radiographs 2 weeks after treatment. All animals were then sacrificed at 4 weeks and underwent plain radiographs, manual palpation, and histological analysis. 3 specimens from each group were scanned using microCT technology with 3-dimensional reconstruction 4 weeks after surgery. RESULTS: All animals in Group 1 (Ad-BMP-2-transduced adipose cells) (8/8) and Group 2 (rh-BMP-2) (4/4) had successful spinal fusion at two and four weeks postoperatively assessed both by plain radiographs and manual palpation. None of the spines in Group 3 (adipose cells alone) and Group 4 (autogenous bone graft) fused four weeks after implantation.

Proceedings of the NASS 19th Annual Meeting / The Spine Journal 4 (2004) 3S–119S

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Fig. 1. Animals in Group 1 showed far superior bone formation on radiographs, histology, and direct inspection than those in the other treatment groups (see Fig. 1). CONCLUSIONS: Mesenchymal stem cells from human fat show promise as targets for gene therapy for bone induction in spinal fusion. PLAs are easily obtained from elective procedures with minimal morbidity, have significant regenerative potential in vitro, and offer a greater number of multipotent cells than human bone marrow. Fusion masses from adenovirally-infected fat cells were larger and denser when compared to spines treated with a low dose of rhBMP-2. Furthermore, radiographic fusion of the posterolateral rat spine was seen earlier (2 weeks) than previously reported in the literature. These results indicate that adipose cells that are widely available from liposuction aspirates deserve further clinical study in its potential use in inducing spinal fusion. DISCLOSURES: No disclosures. CONFLICT OF INTEREST: No Conflicts. doi: 10.1016/j.spinee.2004.05.100 2:04 100. Cellular environments alter performance of rhBMP-2 and induce pseudoarthrosis Hyun Bae1, L.E.A. Kanim1, Li Zhao2, Pamela Wong1, Rick Delamarter2; 1 Spine Institute at Saint John’s Health Center, Santa Monica, CA, USA; 2 Saint John’s Health Center, Santa Monica, CA, USA BACKGROUND CONTEXT: It is still uncertain what factors may intensify or mitigate the osteoinductivity of Bone Morphogenetic Proteins (BMPs) in the clinical situation. As the indications and use of BMPs expand in spinal surgery, it is important to understand how different cellular environments may modulate its effectiveness. PURPOSE: This study evaluates the osteoinductivity of rhBMP-2 in different in vivo cellular environments by introducing fibroblast, nucleus pulposus, annulus fibrous, muscle, or marrow cells into the implant site of rats undergoing posterolateral fusion with rhBMP-2. STUDY DESIGN/SETTING: Rats undergoing posterolateral fusion are treated with varing combinations and quanities of rhBMP-2 and fibroblast, nucleus pulposus, annulus fibrous, muscle, or marrow cells introduced into the implant site via a collagen sponge. Fusion rates were analyzed at 8 weeks. PATIENT SAMPLE: 68 female Lewis rats. OUTCOME MEASURES: Fusion was evaluated using manual testing, radiographs and histology. METHODS: Harvest & Culturing Procedure: Adherent skin fibroblasts, annulus fibrosus, nucleus pulposus, muscle, and bone marrow cells were separately harvested from Lewis rats, cultured and expanded and maintained in DMEM. Dosing: Several concentrations within the range of effective doses (ED) of rhBMP-2 (0.16, 0.032, 0.006 mg/ml) for osteoinductivity in a rat were screened. The ED100 (0.032 mg/ml) rhBMP-2 at which 100% of the rats were fused was selected for evaluation with various cell types. Surgical Procedure & Treatments: In 60 Lewis rats a posterolateral intertransverse process L4–L5 fusion was performed using one of two doses (0.16mg/ml, 0.032mg/ml) of rhBMP-2 in absorbable collagen sponge (ACS (1.0×0.5×0.5)/side) combined with one of three quantities (5×10,6 10×10,6

20×10,6) of the above cell types (n⫽6 rats each). Control rats were implanted with the same concentrations of rhBMP-2 with ACS carrier alone (n⫽4 each) and with added media only—no cells (DMEM 0.125ml/side). Rats were sacrificed at 8 weeks. Sites were evaluated using manual testing, radiographs and histology. RESULTS: All rats without implanted cells were completely fused at 0.16 mg rhBMP-2 (4/4), 0.032 mg rhBMP-2/ACS (7/7), and 0.032 mg rhBMP2/ACS with added media DMEM (4/4). Half the rats were fused at a concentration of 0.006 mg/ml rhBMP-2 (1/2). Fusion was nearly completely inhibited when implanted with any of the quantities of AF, NP, muscle, and marrow cells mixed with a concentration of 0.032 mg rhBMP-2. Fibrous tissue was observed within the osseous mass histologically when not fused. At the higher concentration 0.16 mg rhBMP-2 inhibition was observed with fibroblasts at the 10×10^6 cell quantity (2/4) and to a lesser degree at 5×10^6 cell quantity (4/6). CONCLUSIONS: L4–L5 posterolateral pseudarthrosis was experimentally induced by altering the cellular environment in rats at concentrations of rhBMP-2 that consistently induce posterolateral fusion in rats. This study demonstrates that rhBMP-2’s ability to induce a spinal fusion varied as a function of cellular environment. Fibroblasts, disc cells (NP & AF), and muscle cells almost completely inhibited fusion at the lower concentrations of rhBMP-2. At the higher dose of 0.16 mg of rhBMP-2/ACS, fusion was inhibited in a concentration dependent manner with fibroblasts. Marrow cells did not demonstrate any beneficial effect with BMP. This model may allow us to understand inconsistencies and optimize BMP’s performance in human posterolateral spinal fusions. DISCLOSURES: No disclosures. CONFLICT OF INTEREST: No Conflicts. doi: 10.1016/j.spinee.2004.05.101 2:11 101. Comparison of rhBMP-2, -7, -12, and -13 on rat human disc cells in vitro S. Yoon1, Yerun Zhu2, William Hutton3, Jun Li1; 1Emory University and Atlanta VAMC, Atlanta, GA, USA; 2Emory University, GA, USA; 3 Atlanta Veterans Affairs Hospital, Decatur, GA, USA BACKGROUND CONTEXT:Intervertebral disc (IVD) degeneration is characterized by a decrease in aggrecan and collagen II synthesis and a more fibrotic phenotype of the disc matrix. One possible approach to retard or reverse disc degeneration is to stimulate disc cell matrix production with chondrocytic growth factors. In previous studies, BMP-2, -7, -12, and -13 have been individually tested (to varying degrees) for the ability to stimulate chondrocytic synthesis from disc cells, however, they have not been directly compared against each other. In the search for the most optimal growth factors, it is important to understand the relative potency of each of these BMP’s in stimulating sulfated-glycosaminoglycans (sGAG), aggrecan, collagen I, and collagen II. PURPOSE: The purpose of this study was to investigate the sGAG synthesis and the regulation of aggrecan, collagen I and collagen II gene expressions by recombinant human BMP-2, 7, 12 and 13 on stimulating Rat AF cells and Human AF cells in vitro. STUDY DESIGN/SETTING: In vitro experiments. PATIENT SAMPLE: Rat and human disc samples. OUTCOME MEASURES: DMMB and RT-PCR. METHODS: Human lumbar intervertebral disc cells and Sprague-Dawley rat lumbar anulus fibrosus cells were isolated and grown in monolayer

Fig. 1. sGAG levels.