- Email: [email protected]
Saturday, November 2, 2002 2:14–2:57 pm Concurrent Session 6A: Bone Morphogenic Protein
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Proceedings of the NASS 17th Annual Meeting / The Spine Journal 2 (2002) 47S–128S
ment of the diaphragm. Partial loosening of implant occurred in two patients with severe osteoporosis. One case was converted to open procedure. Relationship between findings and existing knowledge: Although thoracoscopic management of thoracolumbar fractures with diaphragm detachment is a relatively newer technique and a demanding procedure, earlier functional recovery and postoperative pain reduction can be achieved when compared with open thoracoabdominal approach. Overall significance of findings: This study demonstrates that the thoracolumbar junction fractures can be treated successfully using thoracoscopic technique with diaphragm detachment. Disclosures: Device or drug: MACS-tl. Status: approved. Conflict of interest: No conflict.
bidity and allow successful reconstruction with shorter segment fixation. In contrast, TP approaches allow more rapid reconstruction with less blood loss than AL. The LEC approach does not seem to offer an advantage over the other approaches. Consequently, AL approaches may be preferred in most patients requiring thoracolumbar spine reconstructions. However, TP approaches may reduce operative duration and blood loss but are accompanied by more frequent infectious complications. Disclosures: No disclosures. Conflict of interest: No conflicts. PII: S1529-9430(02)00345-5
PII: S1529-9430(02)00344-3
1:48 Comparing anterolateral, extracavitary and transpedicular approaches to thoracolumbar spine reconstruction Gregory Przybylski, MD1, William Mitchell, MD2; 1Northwestern University, Chicago, IL, USA; 1Jefferson Medical College, Philadelphia, PA, USA Purpose of study: Multiple approaches are used to access the anterior thoracolumbar spine. Although variations of posterior approaches were developed, improvements in anesthesia and internal fixation fostered anterolateral (AL) approaches to the thoracolumbar spine. Advocates suggest improved visualization reduces the risk of neurological injury. However, some have raised concerns about increased perioperative morbidity after thoracotomy. Although the lateral extracavitary (LEC) approach offers better anterior visualization than the transpedicular (TP) approach, both offer more limited visualization than the AL approach. This study examines operative parameters and complications of these approaches in anterior spinal reconstruction to identify a preferred approach. Methods used: A prospective analysis of a consecutive series of 87 patients requiring thoracolumbar spinal reconstructions by one surgeon over 68 months was performed. Patients were grouped by approach including AL (n21), TP (n37) and LEC (n29). The office, hospital and anesthesia records and radiographs were examined. Demographic parameters examined included age, gender and pathological indication. Operative parameters recorded included number of levels fused, estimated blood loss and operative time. Perioperative and postoperative complications were identified. Parameters were compared with analysis of variance and t tests using a p.05 level of significance. Summary of findings: The common reasons for reconstruction were trauma (AL13, TP12, LEC12), neoplasm (AL6, TP10, LEC8), and infection (AL0, TP14, LEC9). Two thirds fewer segments were fused in AL patients when compared with LEC or TP patients. The estimated blood loss was significantly higher in patients treated with LEC compared with TP (p.022). Moreover, the operative duration was significantly longer in LEC in comparison to TP and AL (p.00024). Twentytwo complications were observed. No postoperative wound infections occurred after AL, whereas seven occurred in TP and three in LEC. Seven instrumentation or graft complications occurred (AL1, TP3, LEC3). Three patients had transient neurological worsening (AL1, LEC2). Finally, both perioperative deaths were observed in elderly patients undergoing TP for pyogenic osteomyelitis. One died of respiratory failure, and the other died from a myocardial infarction. Median follow-up exceeds 2 years. Relationship between findings and existing knowledge: Less frequent infectious complications and shorter segment fixation are achieved with AL. However, TP allows safe circumferential reconstruction with less time and blood loss, providing a viable alternative. The LEC requires more time and has more frequent blood loss without improving neurological outcome over TP. Overall significance of findings: Several approaches allow access to the anterior thoracolumbar spine. Conclusions drawn from this study are limited by the retrospective analysis. However, AL approaches, even in older patients, do not appear to be associated with increased perioperative mor-
Saturday, November 2, 2002 2:14–2:57 pm Concurrent Session 6A: Bone Morphogenic Protein 2:14 Bone morphogenic protein–2 facilitates expression of chondrogenic not osteogenic phenotype in human intervertebral disc cells Seong-Hwan Moon, MD1, Hwan-Mo Lee, MD1, Moon-Soo Park, MD1, Hyang Kim, BA1, Eun-Hae Kwon, BA1, Young-Hee Park, MD1, Nam-Hyun Kim, MD2; 1Yonsei University College of Medicine, Seoul, South Korea; 2 Department of Orthopaedic Surgery, Keonyang University, Daejeon, South Korea Purpose of study: With the introduction of growth factors and development of vector technology, it is possible to genetically modify intervertebral disc cells by transfer of growth factor genes to upregulate matrix synthesis. Bone morphogenic protein (BMP)-2 was known to play a crucial role in osteoblast differentiation and bone formation. The bone-forming potential of BMP-2 raises the research question, Can BMP-2 upregulate expression of chondrogenic phenotype rather than osteogenic phenotype in the intervertebral disc? Therefore, the objective of this in vitro study is to determine the effect of recombinant human BMP-2 (rhBMP-2) on proteoglycan synthesis and mRNA expression of matrix components, that is, aggrecan, collagen type 1, collagen type 2 and osteocalcin in human intervertebral disc cells. Methods used: We harvested cervical and lumbar disc tissue from six patients during surgical disc procedures. The disc cells were isolated from disc tissue by sequential enzymatic digestion and cultured. Then cultures were incorporated into alginate beads as described. rhBMP-2 (R&D) was administered to the cultures with various concentration. Newly synthesized proteoglycan was assessed by 35S incorporation using chromatography on Sephadex G-25 in PD-10 column. Reverse transcriptase polymerase chain reaction (RT-PCR) for expression of aggrecan, collagen type 1, collagen type 2 and osteocalcin mRNA was performed. Summary of findings: In the rhBMP-2–treated cultures, there was increased newly synthesized proteoglycan (1.6-fold in 300 ng/ml, 3.5-fold in 1,500 ng/ml of rhBMP-2) and increased expression of aggrecan, collagen type 1 and collagen type 2 mRNA compared with untreated control in a dose-dependent manner. However, rhBMP-2 did not upregulate expression of osteocalcin mRNA in a given dose. Relationship between findings and existing knowledge: The result of this study demonstrated that the effect of BMP-2 on human intervertebral disc cells is chondrogenic rather than osteogenic. Also BMP-2 upregulates synthesis of matrix as proved by proteoglycan synthesis, mRNA expression of collagen 1, 2 and aggrecan. Overall significance of findings: In human intervertebral disc cells, rhBMP-2 clearly upregulates mRNA expression of chondrogenic component, that is, aggrecan, collagen type 1 and collagen type 2 without upregulating expression of mRNA of osteocalcin with given dose. The effect of rhBMP-2 was dose dependent in proteoglycan synthesis and mRNA expression of aggrecan, collagen type 1 and collagen type 2. In conclusion, this study raises the
Proceedings of the NASS 17th Annual Meeting / The Spine Journal 2 (2002) 47S–128S possibility that rhBMP-2 and BMP-2 encoding gene can be anabolic agents for regenerating matrix of the intervertebral disc. Disclosures: BMP-2: Investigational. Conflict of interest: Hwan-Mo Lee, grant research support, Brain Korea 21 Project (government). PII: S1529-9430(02)00346-7
2:20 The osteogenic activity of 14 types of bone morphogenic proteins: implications in bone regeneration and spine fusion Tong-Chuan He, MD, PhD1, Hongwei Cheng, MD, PhD1, Wei Jiang, BA1, Benjamin Breyer, BS1, Lan Zhou, MD, PhD1, Tao Feng, BS, MS1, Akira Ishikawa, MD, PhD 1, Rex Haydon, MD, PhD 1, Frank Phillips, MD 1; 1 The University of Chicago, Chicago, IL, USA Purpose of study: Although several bone morphogenic proteins (BMPs) have been shown to enhance bone healing and spinal fusion, the optimal BMPs or combination of BMPs to promote fusion are unknown. This study was designed to comprehensively elucidate the distinct and potentially synergistic osteogenic activity of 14 types of BMPs at various stages of osteogenesis. Methods used: Recombinant adenoviruses expressing 14 human BMPs (ie, BMP-2 to BMP-15) were constructed and used to infect both pluripotent mesenchymal progenitor C3H10T1/2 cells and committed osteoblastic C2C12 cells. The osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin and matrix mineralization on BMP stimulation. Summary of findings: BMP-2, -4, -6, -7 and -9 significantly induced alkaline phosphatase activity in C2C12 osteoblastic cells. BMP-5, -8, -10, -11, -12 and -13 exerted weak induction of alkaline phosphatase. However, only BMP-2, -6 and -9 significantly induced alkaline phosphatase activity in C3H10T1/2 pluripotent cells, although weak induction by BMP4 and 7 was detected. Histochemical staining assays demonstrated that BMP-6 and -9 induced the greatest increase in alkaline phosphatase staining, whereas BMP-2 and -4 (and BMP-7 to lesser extent) induced a modest increase in alkaline phosphatase activity in C2C12 cells. BMP-2, -6 and -9 significantly induced osteocalcin expression (ie, a late marker of osteogenesis) in C3H10T1/2 cells, and BMP-4 and -7 slightly increased osteocalcin expression. In C2C12 cells osteocalcin expression was strongly induced by BMP-2, -4, -6, -7 and -9, whereas BMP-5, -10 and -14 slightly induced expression. Mineralized nodules were readily detected in the C3H10T1/2 cells infected with BMP-2, -6 and -9 vectors (and, to lesser extent, with BMP-4 -7 and -10). Finally, we observed strong synergistic effects among the BMPs that had an ability to activate the osteogenic markers. Interestingly, a strong synergistic effect on osteogenesis was detected in cells stimulated by BMP-5 plus -10, BMP-5 plus -12, BMP-5 plus -13, BMP-7 plus -10 or BMP-7 plus -13. Relationship between findings and existing knowledge: We have conducted a comprehensive analysis of the osteogenic activity of 14 types of BMPs in osteoblastic progenitor cells and demonstrated that BMP-2, -6 and -9 are the most potent osteogenic factors, whereas BMP-4 and -7 exhibit reasonably strong osteoinductive activity. Further, certain combinations of BMPs exert a strong synergy on osteogenesis. Overall significance of findings: These findings have important implications for the development of effective new formulas for bone healing and spine fusion. Disclosures: No disclosures. Conflict of interest: Tong-Chuan He, grant research support.
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Purpose of study: We test the hypothesis that nicotine can reverse bone morphogenic protein (BMP)-2 induced upregulation of proteoglycan synthesis and collagen 2 expression but not collagen 1. Methods used: Cells were isolated from nucleus pulposus of rat lumbar discs and were grown in monolayer culture. The cells were cultured in DMEM/F12 with 1% FBS and treated without added BMP-2 or nicotine (no treatment), 100 ng/ml of BMP-2 (BMP-2 only) or with 100 ng/ml of BMP-2 and increasing doses of nicotine (1, 10, 100 mcg/ml). The culture media was collected between day 4 and 7 after BMP-2/nicotine treatment and sulfated-glycosaminoglycan (s-GAG) content in the media was quantified using the DMMB to estimate proteoglycan production. The results were normalized by cell number at day 7. On day 7, mRNA was extracted, and reverse transcriptase polymerase chain reaction (RT-PCR) and realtime PCR were used to assess collagen 1 and 2 mRNA expression. Summary of findings: By day 7, s-GAG production in the BMP-2–only group was increased 2.5-fold as compared with no treatment group. The s-GAG production monotonically decreased with increased concentration of nicotine down to 46% of BMP-2–only group (Fig. 1). No change in collagen 1 expression with the addition of BMP-2 or nicotine was noted. However, significant increase in collagen 2 mRNA in the BMP-2–only group compared with no treatment group was noted. Collagen 2 mRNA downregulation was noted with nicotine concentrations of 100 mcg/ml with RT-PCR. In order to better quantitate changes in collagen 2 mRNA abundance, real-time PCR was used. This showed collagen 2 mRNA that decreased to 69% and 27% of BMP-2–only group with the addition of nicotine at 10 and 100 mcg/ml, respectively. The effect of nicotine on sGAG production and the relative abundance of mRNA as percentage of BMP-2 only is noted in Fig. 1.
Fig. 1. Effect of nicotine on sGAG, collagen 1, and collagen 2. Relationship between findings and existing knowledge: BMP-2 and its receptor are normally found in the intervertebral disc, and BMP-2 has recently been shown to enhance the chondrocytic phenotype of disc cells (increase proteoglycan and collagen 2 synthesis). Epidemiologic human studies and in vivo rat experiments indicate that smoking may lead to disc degeneration. We show here that nicotine, an important component of cigarette smoke, can inhibit proteoglycan and collagen 2 synthesis induced by BMP-2. Of note, collagen 1 is not regulated by BMP-2 or nicotine at the tested concentrations, indicating a certain specificity in nicotine activity. Overall significance of findings: This study establishes the negative influence of nicotine on intervertebral disc cell response to BMP-2 at a cellular and molecular level. Disclosures: No disclosures. Conflict of interest: No conflicts. PII: S1529-9430(02)00348-0
PII: S1529-9430(02)00347-9
2:26 Nicotine inhibits bone morphogenic protein–2–induced proteoglycan and collagen 2 synthesis of disc nucleus cells S. Tim Yoon, MD, PhD1, Keun Soo Kim, MD2, William Hutton, ScD3; 1 Emory University, Decatur, GA, USA; 2Chonbuk National University, Chonju, Korea; 3Atlanta Veterans Affairs Hospital, Decatur, GA, USA
2:32 Effect of nutrient concentration and osteogenic protein–1 on the metabolism of intervertebral disc: in vitro organ culture study Nicholas Ahn, MD1, Yoshiyuki Imai, MD2, Howard An2, Michiaki Yamada, MD2, Koichi Masuda, MD, PhD2; 1Rush Presbyterian St. Luke’s Medical Center, Darien, IL, USA; 2Rush Presbyterian St. Luke’s Medical Center, Chicago, IL,¬ USA
Proceedings of the NASS 17th Annual Meeting / The Spine Journal 2 (2002) 47S–128S
ment of the diaphragm. Partial loosening of implant occurred in two patients with severe osteoporosis. One case was converted to open procedure. Relationship between findings and existing knowledge: Although thoracoscopic management of thoracolumbar fractures with diaphragm detachment is a relatively newer technique and a demanding procedure, earlier functional recovery and postoperative pain reduction can be achieved when compared with open thoracoabdominal approach. Overall significance of findings: This study demonstrates that the thoracolumbar junction fractures can be treated successfully using thoracoscopic technique with diaphragm detachment. Disclosures: Device or drug: MACS-tl. Status: approved. Conflict of interest: No conflict.
bidity and allow successful reconstruction with shorter segment fixation. In contrast, TP approaches allow more rapid reconstruction with less blood loss than AL. The LEC approach does not seem to offer an advantage over the other approaches. Consequently, AL approaches may be preferred in most patients requiring thoracolumbar spine reconstructions. However, TP approaches may reduce operative duration and blood loss but are accompanied by more frequent infectious complications. Disclosures: No disclosures. Conflict of interest: No conflicts. PII: S1529-9430(02)00345-5
PII: S1529-9430(02)00344-3
1:48 Comparing anterolateral, extracavitary and transpedicular approaches to thoracolumbar spine reconstruction Gregory Przybylski, MD1, William Mitchell, MD2; 1Northwestern University, Chicago, IL, USA; 1Jefferson Medical College, Philadelphia, PA, USA Purpose of study: Multiple approaches are used to access the anterior thoracolumbar spine. Although variations of posterior approaches were developed, improvements in anesthesia and internal fixation fostered anterolateral (AL) approaches to the thoracolumbar spine. Advocates suggest improved visualization reduces the risk of neurological injury. However, some have raised concerns about increased perioperative morbidity after thoracotomy. Although the lateral extracavitary (LEC) approach offers better anterior visualization than the transpedicular (TP) approach, both offer more limited visualization than the AL approach. This study examines operative parameters and complications of these approaches in anterior spinal reconstruction to identify a preferred approach. Methods used: A prospective analysis of a consecutive series of 87 patients requiring thoracolumbar spinal reconstructions by one surgeon over 68 months was performed. Patients were grouped by approach including AL (n21), TP (n37) and LEC (n29). The office, hospital and anesthesia records and radiographs were examined. Demographic parameters examined included age, gender and pathological indication. Operative parameters recorded included number of levels fused, estimated blood loss and operative time. Perioperative and postoperative complications were identified. Parameters were compared with analysis of variance and t tests using a p.05 level of significance. Summary of findings: The common reasons for reconstruction were trauma (AL13, TP12, LEC12), neoplasm (AL6, TP10, LEC8), and infection (AL0, TP14, LEC9). Two thirds fewer segments were fused in AL patients when compared with LEC or TP patients. The estimated blood loss was significantly higher in patients treated with LEC compared with TP (p.022). Moreover, the operative duration was significantly longer in LEC in comparison to TP and AL (p.00024). Twentytwo complications were observed. No postoperative wound infections occurred after AL, whereas seven occurred in TP and three in LEC. Seven instrumentation or graft complications occurred (AL1, TP3, LEC3). Three patients had transient neurological worsening (AL1, LEC2). Finally, both perioperative deaths were observed in elderly patients undergoing TP for pyogenic osteomyelitis. One died of respiratory failure, and the other died from a myocardial infarction. Median follow-up exceeds 2 years. Relationship between findings and existing knowledge: Less frequent infectious complications and shorter segment fixation are achieved with AL. However, TP allows safe circumferential reconstruction with less time and blood loss, providing a viable alternative. The LEC requires more time and has more frequent blood loss without improving neurological outcome over TP. Overall significance of findings: Several approaches allow access to the anterior thoracolumbar spine. Conclusions drawn from this study are limited by the retrospective analysis. However, AL approaches, even in older patients, do not appear to be associated with increased perioperative mor-
Saturday, November 2, 2002 2:14–2:57 pm Concurrent Session 6A: Bone Morphogenic Protein 2:14 Bone morphogenic protein–2 facilitates expression of chondrogenic not osteogenic phenotype in human intervertebral disc cells Seong-Hwan Moon, MD1, Hwan-Mo Lee, MD1, Moon-Soo Park, MD1, Hyang Kim, BA1, Eun-Hae Kwon, BA1, Young-Hee Park, MD1, Nam-Hyun Kim, MD2; 1Yonsei University College of Medicine, Seoul, South Korea; 2 Department of Orthopaedic Surgery, Keonyang University, Daejeon, South Korea Purpose of study: With the introduction of growth factors and development of vector technology, it is possible to genetically modify intervertebral disc cells by transfer of growth factor genes to upregulate matrix synthesis. Bone morphogenic protein (BMP)-2 was known to play a crucial role in osteoblast differentiation and bone formation. The bone-forming potential of BMP-2 raises the research question, Can BMP-2 upregulate expression of chondrogenic phenotype rather than osteogenic phenotype in the intervertebral disc? Therefore, the objective of this in vitro study is to determine the effect of recombinant human BMP-2 (rhBMP-2) on proteoglycan synthesis and mRNA expression of matrix components, that is, aggrecan, collagen type 1, collagen type 2 and osteocalcin in human intervertebral disc cells. Methods used: We harvested cervical and lumbar disc tissue from six patients during surgical disc procedures. The disc cells were isolated from disc tissue by sequential enzymatic digestion and cultured. Then cultures were incorporated into alginate beads as described. rhBMP-2 (R&D) was administered to the cultures with various concentration. Newly synthesized proteoglycan was assessed by 35S incorporation using chromatography on Sephadex G-25 in PD-10 column. Reverse transcriptase polymerase chain reaction (RT-PCR) for expression of aggrecan, collagen type 1, collagen type 2 and osteocalcin mRNA was performed. Summary of findings: In the rhBMP-2–treated cultures, there was increased newly synthesized proteoglycan (1.6-fold in 300 ng/ml, 3.5-fold in 1,500 ng/ml of rhBMP-2) and increased expression of aggrecan, collagen type 1 and collagen type 2 mRNA compared with untreated control in a dose-dependent manner. However, rhBMP-2 did not upregulate expression of osteocalcin mRNA in a given dose. Relationship between findings and existing knowledge: The result of this study demonstrated that the effect of BMP-2 on human intervertebral disc cells is chondrogenic rather than osteogenic. Also BMP-2 upregulates synthesis of matrix as proved by proteoglycan synthesis, mRNA expression of collagen 1, 2 and aggrecan. Overall significance of findings: In human intervertebral disc cells, rhBMP-2 clearly upregulates mRNA expression of chondrogenic component, that is, aggrecan, collagen type 1 and collagen type 2 without upregulating expression of mRNA of osteocalcin with given dose. The effect of rhBMP-2 was dose dependent in proteoglycan synthesis and mRNA expression of aggrecan, collagen type 1 and collagen type 2. In conclusion, this study raises the
Proceedings of the NASS 17th Annual Meeting / The Spine Journal 2 (2002) 47S–128S possibility that rhBMP-2 and BMP-2 encoding gene can be anabolic agents for regenerating matrix of the intervertebral disc. Disclosures: BMP-2: Investigational. Conflict of interest: Hwan-Mo Lee, grant research support, Brain Korea 21 Project (government). PII: S1529-9430(02)00346-7
2:20 The osteogenic activity of 14 types of bone morphogenic proteins: implications in bone regeneration and spine fusion Tong-Chuan He, MD, PhD1, Hongwei Cheng, MD, PhD1, Wei Jiang, BA1, Benjamin Breyer, BS1, Lan Zhou, MD, PhD1, Tao Feng, BS, MS1, Akira Ishikawa, MD, PhD 1, Rex Haydon, MD, PhD 1, Frank Phillips, MD 1; 1 The University of Chicago, Chicago, IL, USA Purpose of study: Although several bone morphogenic proteins (BMPs) have been shown to enhance bone healing and spinal fusion, the optimal BMPs or combination of BMPs to promote fusion are unknown. This study was designed to comprehensively elucidate the distinct and potentially synergistic osteogenic activity of 14 types of BMPs at various stages of osteogenesis. Methods used: Recombinant adenoviruses expressing 14 human BMPs (ie, BMP-2 to BMP-15) were constructed and used to infect both pluripotent mesenchymal progenitor C3H10T1/2 cells and committed osteoblastic C2C12 cells. The osteogenic activity was determined by measuring the induction of alkaline phosphatase, osteocalcin and matrix mineralization on BMP stimulation. Summary of findings: BMP-2, -4, -6, -7 and -9 significantly induced alkaline phosphatase activity in C2C12 osteoblastic cells. BMP-5, -8, -10, -11, -12 and -13 exerted weak induction of alkaline phosphatase. However, only BMP-2, -6 and -9 significantly induced alkaline phosphatase activity in C3H10T1/2 pluripotent cells, although weak induction by BMP4 and 7 was detected. Histochemical staining assays demonstrated that BMP-6 and -9 induced the greatest increase in alkaline phosphatase staining, whereas BMP-2 and -4 (and BMP-7 to lesser extent) induced a modest increase in alkaline phosphatase activity in C2C12 cells. BMP-2, -6 and -9 significantly induced osteocalcin expression (ie, a late marker of osteogenesis) in C3H10T1/2 cells, and BMP-4 and -7 slightly increased osteocalcin expression. In C2C12 cells osteocalcin expression was strongly induced by BMP-2, -4, -6, -7 and -9, whereas BMP-5, -10 and -14 slightly induced expression. Mineralized nodules were readily detected in the C3H10T1/2 cells infected with BMP-2, -6 and -9 vectors (and, to lesser extent, with BMP-4 -7 and -10). Finally, we observed strong synergistic effects among the BMPs that had an ability to activate the osteogenic markers. Interestingly, a strong synergistic effect on osteogenesis was detected in cells stimulated by BMP-5 plus -10, BMP-5 plus -12, BMP-5 plus -13, BMP-7 plus -10 or BMP-7 plus -13. Relationship between findings and existing knowledge: We have conducted a comprehensive analysis of the osteogenic activity of 14 types of BMPs in osteoblastic progenitor cells and demonstrated that BMP-2, -6 and -9 are the most potent osteogenic factors, whereas BMP-4 and -7 exhibit reasonably strong osteoinductive activity. Further, certain combinations of BMPs exert a strong synergy on osteogenesis. Overall significance of findings: These findings have important implications for the development of effective new formulas for bone healing and spine fusion. Disclosures: No disclosures. Conflict of interest: Tong-Chuan He, grant research support.
87S
Purpose of study: We test the hypothesis that nicotine can reverse bone morphogenic protein (BMP)-2 induced upregulation of proteoglycan synthesis and collagen 2 expression but not collagen 1. Methods used: Cells were isolated from nucleus pulposus of rat lumbar discs and were grown in monolayer culture. The cells were cultured in DMEM/F12 with 1% FBS and treated without added BMP-2 or nicotine (no treatment), 100 ng/ml of BMP-2 (BMP-2 only) or with 100 ng/ml of BMP-2 and increasing doses of nicotine (1, 10, 100 mcg/ml). The culture media was collected between day 4 and 7 after BMP-2/nicotine treatment and sulfated-glycosaminoglycan (s-GAG) content in the media was quantified using the DMMB to estimate proteoglycan production. The results were normalized by cell number at day 7. On day 7, mRNA was extracted, and reverse transcriptase polymerase chain reaction (RT-PCR) and realtime PCR were used to assess collagen 1 and 2 mRNA expression. Summary of findings: By day 7, s-GAG production in the BMP-2–only group was increased 2.5-fold as compared with no treatment group. The s-GAG production monotonically decreased with increased concentration of nicotine down to 46% of BMP-2–only group (Fig. 1). No change in collagen 1 expression with the addition of BMP-2 or nicotine was noted. However, significant increase in collagen 2 mRNA in the BMP-2–only group compared with no treatment group was noted. Collagen 2 mRNA downregulation was noted with nicotine concentrations of 100 mcg/ml with RT-PCR. In order to better quantitate changes in collagen 2 mRNA abundance, real-time PCR was used. This showed collagen 2 mRNA that decreased to 69% and 27% of BMP-2–only group with the addition of nicotine at 10 and 100 mcg/ml, respectively. The effect of nicotine on sGAG production and the relative abundance of mRNA as percentage of BMP-2 only is noted in Fig. 1.
Fig. 1. Effect of nicotine on sGAG, collagen 1, and collagen 2. Relationship between findings and existing knowledge: BMP-2 and its receptor are normally found in the intervertebral disc, and BMP-2 has recently been shown to enhance the chondrocytic phenotype of disc cells (increase proteoglycan and collagen 2 synthesis). Epidemiologic human studies and in vivo rat experiments indicate that smoking may lead to disc degeneration. We show here that nicotine, an important component of cigarette smoke, can inhibit proteoglycan and collagen 2 synthesis induced by BMP-2. Of note, collagen 1 is not regulated by BMP-2 or nicotine at the tested concentrations, indicating a certain specificity in nicotine activity. Overall significance of findings: This study establishes the negative influence of nicotine on intervertebral disc cell response to BMP-2 at a cellular and molecular level. Disclosures: No disclosures. Conflict of interest: No conflicts. PII: S1529-9430(02)00348-0
PII: S1529-9430(02)00347-9
2:26 Nicotine inhibits bone morphogenic protein–2–induced proteoglycan and collagen 2 synthesis of disc nucleus cells S. Tim Yoon, MD, PhD1, Keun Soo Kim, MD2, William Hutton, ScD3; 1 Emory University, Decatur, GA, USA; 2Chonbuk National University, Chonju, Korea; 3Atlanta Veterans Affairs Hospital, Decatur, GA, USA
2:32 Effect of nutrient concentration and osteogenic protein–1 on the metabolism of intervertebral disc: in vitro organ culture study Nicholas Ahn, MD1, Yoshiyuki Imai, MD2, Howard An2, Michiaki Yamada, MD2, Koichi Masuda, MD, PhD2; 1Rush Presbyterian St. Luke’s Medical Center, Darien, IL, USA; 2Rush Presbyterian St. Luke’s Medical Center, Chicago, IL,¬ USA