The Role of Degenerated Intervertebral Discs in Hypertrophy and Ossification of the Ligamentum Flavum in Humans Kyung Hee Kim Department of Medical Science The Graduate School, Yonsei University i
The Role of Degenerated Intervertebral Discs in Hypertrophy and Ossification of the Ligamentum Flavum in Humans Directed by Professor Hak-Sun Kim The Master's Thesis submitted to the Department of Medical Science the Graduate School of Yonsei University in partial fulfillment of the requirements for the degree of Master of Medical Science Kyung Hee Kim December 2003 ii
This certifies that the Master's Thesis of Kyung Hee Kim is approved. ----------------------------------------------- [Thesis Supervisor : Hak-Sun Kim] ------------------------------------------------------ [Thesis Committee Member: Do Heum Yoon] ------------------------------------------------------ [Thesis Committee Member: Seong-Hwan Moon] The Graduate School Yonsei University December 2003 iii
Acknowledgments 벌써 2년이라는시간이흘러석사졸업논문마무리를하고있는지금이순간이너무나도기쁘지만, 처음임상연구센터에발을디뎠을때의결심을떠올려봅니다. 열심히노력하였지만섭섭함과아쉬움을애써뒤로하고이작은결실을이루도록도와주신여러고마운분들에게감사의말씀을전합니다. 먼저부족한저에게큰학문적인가르침과대학원생활동안항상아낌없는관심과배려로써저를이자리에있게해주신저의지도교수님영동정형외과김학선교수님께짧은글로는다표현하지못할깊은감사와존경을드립니다. 저의석사과정마무리를함께이끌어주시고항상연구하는자세가무엇인지가르쳐주신신촌정형외과문성환교수님께도머리숙여감사드립니다. 바쁘신가운데저의부족한석사논문심사를맡아주신신경외과윤도흠교수님께도진심으로감사드립니다. 학문적으로뿐아니라항상인자하신웃음과재치있는입담으로기억되는정형외과이환모교수님, 바쁘신수술방에서저희들을위해 specimen을보내주셔서정말감사드립니다. 제가유전공학이라는분야에발을내밀었을때앞에서힘껏이끌어주신연구하는사람으로써가르침을주신최수영교수님께도이지면을빌어감사의말씀드리고싶습니다. 2년동안힘들고행복했던실험실생활함께했던실험실선배, 후배님들께도고마운마음을전하고싶습니다. 먼저우리 spine 파트, 실험할때미간에주름부터잡히는진정한고수향언니, 항상웃는얼굴로옆에서재잘거리던나의하나뿐인동기언혜, 많은시간함께하지못해서아쉬운광일이오빠화이팅입니다. 같은정형외과실험실에서함께연구하고공부하는언제나맡언니같으신윤희언니, 조용하지만그내공을알아버린연락오빠, 엄마같이요목조목잘챙겨주시는모든면에서야무진수향언니, 진득하게공부와실험을하시는호선언니, 지금은멀리있는은정이언니, 나의대학시절부터함께해온언니같이항상의지가되고때론휴식처가되는소 iv
중한친구현진이에게고마운마음을전하고싶습니다. 어리둥절했던석사초기에많은위로와격려를아끼지않으셨던옆방의학공학과동욱오빠아니박사님, 한때양띠클럽조직해서선배들에게눈총받기도했지만함께해낸동기민섭이, 뉴욕에서막돌아오신의공과박사님시내언니, 우리의엔돌핀뜬금없는예의바른인사맨유석이오빠, 큰키로머쓱하게웃던재민이오빠, 조용하게실험만하시는아름언니에게도고맙단말전하고싶습니다. 한눈에서로를알아버린둘도없는친구같은언니이쁜혜경이언니, 해부학실험실에서정말열심히실험하는진주, 암센터에서 DNA chip과열심히싸우는귀연이, 약리학방에서열심히조교생활하는사랑스런후배동석이아빠용재, 미생물학교실에서열심히실험하는어른스러운후배아연이, 자주는못보지만항상힘이되어주시는경아언니, 본교생화학방려화언니, 기초생화학방정안언니, 우석오빠, 동물실험실의김형관선생님, 3층.4층조교김연희선생님, 권오규선생님, 의정부성모병원우지현언니와그동안저를아껴주셨던선배, 후배님들에게감사의말전하고싶습니다. 항상바쁘다는핑계로많은시간함께하지못해서미안한나의오랜소중한친구들베스트희조, 소중한현주, 언니같은진숙, 야무진수현, 한결같은연수, 멋쟁이문진, 엽기효선, 애동물가선희, 소꿉친구수진, 시집가는은영, 스마일진선, 친구같은후배이은영, 고3때부터동고동락주희, 뉴욕서공부중인재연이, 자칭진정한대학선배대호오빠, 무지어른스러운도움많이준은석오빠에게도이작은기쁨함께나누고감사의마음전하고싶습니다. 언제나든든한버팀목이되어주는사랑하는나의가족들할머니, 아빠, 엄마, 어느새듬직하게누나키를훌쩍넘긴동생욱중이. 태중이, 항상관심어린눈으로따뜻한격려아끼지않으시는친척어르신분들, 용준. 영준오빠, 깜찍한사촌동생주연. 세중. 나현. 성혁이와함께이소중한결실함께기쁨나누고, 현재에만족하지않고앞으로더욱발전하고노력하는경희의모습보여드릴것을약속드립니다. 2003년 12월김경희올림. v
Table of Contents Abstract...1 I. Introduction...3 II. Materials and Methods...7 1. Materials...7 A. Patient data and tissue acquisition procedures...7 B. Materials...10 2. Ligamentum flavum cell culture...11 3. Study design...12 4. Preparation of conditioned medium...13 5. Treatment with inflammatory cytokines and conditioned medium..14 6. MTT assay...14 7. DNA synthesis analysis...14 8. RT-PCR analysis...15 9. Von Kossa, Alizarin Red-S, and ALP staining....17 10. Statistical analysis...18 vi
III. Results...19 1. The hlf cells cultured with a given dose of cytokines...19 A. Cell cytotoxicity...19 B. DNA synthesis...19 C. Effect of cytokines on the expression of collagen and osteocalcin mrna...22 D. Effect of cytokines on the expression of osteogenic Phenotype...22 2. The hlf cells cultured with conditioned medium...25 A. Morphology of hlf cells...25 B. Cell cytotoxicity... 25 C. DNA synthesis...28 D. Effect of conditioned medium on the expression of collagen and osteocalcin mrna...29 E. Effect of conditioned medium on the expression of osteogenic phenotype...29 IV. Discussion......34 V. Conclusion...38 References...39 Abstract (in Korean).49 vii
List of Figures Figure 1. Cytotoxicity of hlf cells in response to a given dose of cytokines......20 Figure 2. Effect of cytokines on DNA synthesis in hlf cells...21 Figure 3. Effect of cytokines on the expression of hcol1a1, hcol3a1, hcol5a1, hcol11a1, and osteocalcin mrna in hlf cells...23 Figure 4. The hlf cells cultured in a 24-well culture plate until confluence, and then treated with various cytokines for 48 h, stained positive for Von Kossa stain...24 Figure 5. The morphology of hlf cells. A. The hlf cells cultured in a 24-well culture plate. B. The hlf cells treated with conditioned medium for 48 h....26 Figure 6. Cytotoxicity of hlf cells in response to conditioned medium.27 Figure 7. Effect of conditioned medium on DNA synthesis in hlf cells...28 viii
Figure 8. Effect of conditioned medium on hcol1a1, hcol2a1, hcol3a1, hcol5a1, hcol11a1, and osteocalcin mrna expression in hlf cells...30 Figure 9. The hlf cells cultured in a 24-well culture plate until confluence, and then treated with conditioned medium for 48 h stained positively for A: Von Kossa, B: Alizarin Red-S, and C: ALP stains...31 Figure 10. The hlf cells cultured in a four-well chamber slide, and then treated with conditioned medium for 7 days. The cells stained more positively for A: Von Kossa, B: Alizarin Red-S, and C: ALP stains than in Fig. 9...33 ix
List of Tables Table 1. Clinical features of the cases studied...8 Table 2. Sequences of the RT-PCR primers used...16 Table 3. RT-PCR conditions...17 x
ABSTRACT The Role of Degenerated Intervertebral Discs in Hypertrophy and Ossification of the Ligamentum Flavum in Humans Kyung Hee Kim Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hak-Sun Kim) Spinal stenosis is caused, in part, by hypertrophy and ossification of the ligamentum flavum and facet joints. Hypertrophy and ossification of the ligamentum flavum are induced by the degenerative processes that occur with aging and the increased collagen synthesis that results from mechanical stretching. The degree of hypertrophy and ossification of the ligamentum flavum is usually correlated with the amount of intervertebral disc (IVD) degeneration. This implies a possible relationship between disc degeneration or herniation and hypertrophy of the ligamentum flavum in lumbar spinal stenosis. A herniated disc results in the spontaneous production of inflammatory cytokines, such as IL-1, IL-6, NO, PGE 2, and TNF-α. Therefore, we hypothesized that cytokines from a herniated IVD affect cellular proliferation, matrix synthesis, and osteogenesis in the ligamentum flavum, causing hypertrophy and ossification of the ligament. Therefore, this study examined the effect of a secreted cocktail of inflammatory cytokines from herniated discs on ligamentum flavum cells, to identify the role of degenerated discs in the pathogenesis of ligamentum flavum hypertrophy and ossification in spinal stenosis. 1
Specimens from the interlaminar portion of the ligamentum flavum and herniated lumbar disc tissues were collected during surgery on 27 patients (age range: 49-78 years) with lumbar spine stenosis. Then, the supernatant was collected from herniated disc tissue and used as a cytokine cocktail that was administered to ligamentum flavum cultures. We refer to this as this conditioned medium. Ligamentum flavum cell cultures were treated with cytokines and conditioned medium, and incubated for 48 h. Saline treatment served as a control. Tests including the MTT assay, the determination of 3 H- thymidine incorporation and the expression of types I, III, V, and XI collagen and osteocalcin mrna, and Von Kossa, Alizarin Red-S, and alkaline phosphatase (ALP) staining were performed. No cytotoxicity was observed. DNA synthesis was increased significantly in the treatment with cytokines and conditioned medium. The expression of types I, III, V, and XI collagen and osteoclacin mrna was highly upregulated in the treatments with cytokines and conditioned medium. The ligamentum flavum cells treated with cytokines and conditioned medium stained positively for Von Kossa, Alizarin Red-S, and ALP stains. Combined, disc degeneration or herniation has a pathogenetic significance in hypertrophy and ossification of the ligamentum flavum. Disc herniation can affect the ligamentum flavum via inflammatory cytokines and cause hypertrophy and ossification of the ligamentum flavum. Key words: intervertebral disc, degeneration, ligamentum flavum, hypertrophy, ossification 2
The Role of Degenerated Intervertebral Discs in Hypertrophy and Ossification of the Ligamentum Flavum in Humans Kyung Hee Kim Department of Medical Science The Graduate School, Yonsei University (Directed by Professor Hak-Sun Kim) Ⅰ. Introduction Lumbar spinal stenosis results from intervertebral disc herniation and degenerative changes in the posterior structures of the lumbar spine, including hypertrophy of the facet joints and ligamentum flavum (LF). It causes severe disability by compressing the cauda equina and nerve roots. 1-5 Since the LF covers most of the posterior and lateral parts of the lumbar spinal canal, morphological and histological changes in the LF might play a role in the pathogenesis of lumbar spinal canal encroachment. 6, 7 Nevertheless, there have been few studies of the mechanism of LF hypertrophy. Most studies have addressed the morphologic or histologic 3
changes in the LF. 8-13 Yoshida et al. 14 examined 45 cases of lumbar spinal stenosis using computed tomography and an immunohistochemical study. As controls, 10 cases of acute disc herniation were used. Statistically significant differences in thickness and transverse area were found in the lumbar stenosis specimens compared with the controls. The pathology of LF hypertrophy includes fibrocartilaginous change due to the proliferation of type II collagen, ossification, calcium crystal deposition, collagen degeneration, and elastic fiber and chondroid metaplasia of ligament fibroblasts. 15-17 Chondroid metaplasia in LF hypertrophy plays an important role in ligament ossification, as cartilage differentiation, hypertrophy, and cell death are followed by bone formation in the bone morphogenetic pathway. 18 19, 20 Polgar first reported ossification of the LF. The ossification of spinal ligaments (OSL) is a common form of myeloradiculopathy that is characterized by heterotopic bone formation in the spinal ligaments, 21, 22 which are normally composed of fibrous tissue. Ossification of the LF is often associated with ossification of the 4
posterior longitudinal ligament (OPLL), ankylosing spinal hyperostosis (ASH), and diffuse idiopathic skeletal hyperostosis (DISH). 23, 24 DISH is a common diathesis in middle-aged and elderly patients, and is characterized by bone proliferation along the anterior aspect of the spinal column, and at extraspinal sites of the ligament and tendon 25, 26 attachments. Hence, LF ossification appears to be a part of ossification of the spinal ligaments. Many reports have examined the etiology of OSL and the systemic and local factors thought involved in the pathogenesis of OSL, but the exact mechanism remains unclear. Sakou et al. 27 reported that slight LF ossification is observed more frequently in the dried vertebral bones of aged individuals; LF ossification might be induced by the degeneration of the LF with aging. Such ossification might be induced pathologically and cause radiculopathy or myelopathy in LF ossification patients. Several investigators have postulated that mechanical stress to the spine contributes to accelerating ossification 28-34 and inducing collagen synthesis, which is mediated by TGF-β1 in mesangial 35, 36 and smooth muscle cells. 37 5
In spite of the significance of the LF in the pathogenesis of spinal disease, there has been no thorough biochemical analysis of the degenerate LF. In clinical imaging, the degrees of LF hypertrophy and LF ossification usually appear to coincide with those of intervertebral disc (IVD) degeneration. Ugarriza et al. 38 showed that its association with cervical disc herniation may produce acute or subacute clinical spinal cord compression. Tanaka et al. 39, 40 reported that when disc tissue was applied to an osteoblast culture, osteoblast growth was stimulated, as shown by increased alkaline phosphatase (ALP) activity, type I collagen production, and 3 H-thymidine incorporation. This suggests a relationship between IVD degeneration or herniation and LF hypertrophy in lumbar spinal stenosis. Kang et al. 41 reported that a herniated IVD spontaneously produces inflammatory cytokines, including interleukin-1 (IL-1), interleukin-6 (IL- 6), nitric oxide (NO), prostaglandin E 2 (PGE 2 ), tumor necrosis factor alpha (TNF-α), and matrix metalloproteinase. Therefore, we hypothesized that cytokines from a herniated IVD affect the cellular proliferation, matrix synthesis, and osteogenesis of LF 6
fibroblasts, ultimately causing LF hypertrophy and ossification. Accordingly, the objectives of this study were to demonstrate a biological effect of inflammatory cytokines on the metabolism of LF cells, then to demonstrate the local effect of a cocktail derived from herniated discs on LF cells, and finally to propose a role of degenerated discs in the pathogenesis of LF hypertrophy and ossification in spinal stenosis. Ⅱ. Materials and Methods 1. Materials A. Patient data and tissue acquisition procedures LF tissue was obtained from 27 patients (age range: 49 to 78 years) during surgery for lumbar spinal stenosis. IVD tissue was obtained from patients (age range: 58 to 68 years) during surgical disc procedures. The details of the 27 patients are listed in Table 1. All of the patients had 7
Table 1. Clinical features of the cases studied Case Age Sex Diagnosis Operation level 1 63 F 2 62 F 3 60 M 4 69 M 5 59 F 6 64 F 7 64 F 8 52 F 9 73 F 10 68 F 11 49 F 12 63 M 13 59 F spinal stenosis L4-5-S1, disc degeneration L4-5, L5-S1 spinal stenosis L4-5-S1 disc degeneration L4-5, GIV spinal stenosis, degenerative spondylolisthesis L4-5 disc degeneration GV Spinal stenosis L4-5 disc degeneration L4-5 GIV Spinal stenosis L4-5 disc degeneration, disc herniation spinal stenosis, degenerative spondylolisthesis disc degeneration GV L3-4-5 L4-5 GIV Spinal stenosis L4-5 disc degeneration, disc herniation L4-5 spinal stenosis L3-4-5 disc degeneration L3-4- 4-5 GIV spinal stenosis L4-5 disc degeneration L4-5 GV spinal stenosis, degenrative spondylolisthesis L4-5 disc degeneration L4-5 GIV spinal stenosis, degenrative spondylolisthesis L4-5 disc degeneration L4-5 GIV spinal stenosis, disc hernation L3-4 disc degeneration spinal stenosis disc degenration GIV L3-4-5 L3-4 4-5 GV 8
14 58 F 15 61 M 16 63 F 17 78 F 18 62 F 19 61 M 20 58 F 21 73 M 22 65 F 23 62 M 24 62 M 25 78 F 26 77 M 27 76 F spinal stenosis, degenerative spondylolisthesis L4-5 disc degeneration Spinal stensosis disc degeneration spinal stenosis degenrative spondylolisthesis disc degenration GIV L4-5-S1 GV L4 GIV spinal stenosis L4-5 disc degeneration spinal stenosis disc deegenration GIV L3-4-5 GIV Spinal stenosis, disc hernation L4-5 disc degenration spinal stenosis disc degenration GV L4-5-S1 GIV spinal stenosis L4-5 disc degenration spinal stenosis disc degenration spinal stenosis disc degenration spinal stenosis disc degenration GIII L3-4-5 GV L3-4-5 GIV L3-4-5 GIV spinal stenosis L4-5 disc degenration spinal stenosis disc degenration GV L3-4-5 GV spinal stenosis L4-5 disc degenration GV severe stenosis of the lumbar spine and IVD herniation. The thickened 9
LF and herniated IVD were noted on magnetic resonance imaging preoperatively and visualized directly during surgery. The operating surgeon (SHM, HML) attempted to obtain tissue en-bloc from the central portion of the LF to minimize tissue damage and optimize the harvest of ligament only. The IVD specimen consisted of nucleus pulposus or annulus fibrosus, although in most cases these could not be distinguished with certainty and the disc fragment probably represented a mixture of nucleus and annulus. The LF and IVD tissue specimens were washed with Hank s balanced salt solution (HBSS, Gibco-BRL, Grand Island, NY, USA) to remove blood and bodily fluid contaminants, and were then transported in sterile HBSS to the laboratory, within 20 min of surgical removal. B. Materials Hank s balanced salt solution, Dulbecco s Modified Eagle medium and Ham s F-12 medium, fetal bovine serum, 1% v/v penicillin, streptomycin, nystatin, Trypsin-EDTA, and PBS were purchased from Gibco-BRL (Grand Island, NY, USA). 0.2% Pronase, 0.004% deoxyribonuclease II type IV, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-dipfenyltetrazolium 10
bromide) reagent, dimethylsulfoxide (DMSO), 3% silver nitrate, 40 mm Alizarin Red-S, 0.1 mg/ml naphthol AS-MX phosphate, and 0.6 mg/ml fast blue BB salt were obtained from Sigma (St. Louis, MO, USA). Bacterial 0.02% collagenase type II was purchased from Worthington Biochemical Corp (Lakewood, NJ, USA). Sterile nylon mesh filter (pore size: 75 µm) and 24-well plates were purchased from Falcon (Franklin Lakes, NJ, USA). IL-1α (5 µg), IL-6 (10 µg), and TNF-α (10 µg) were obtained from R&D Systems (Minneapolis, MN, USA). Prostaglandin E 2 (PGE 2, 1 mg) and SNAP (NO donor, S-nitroso-N-acetylpenicillamine, 20 mg) were obtained from Calbiochem (Darmstadt, Germany). 3 H- thymidine was purchased from Amersham Pharmacia (Uppsala, Sweden). The scintillation spectrophotometer was from Packard (Downers Grove, IL, USA). RNeasy Mini Kits were obtained from QIAGEN (Valencia, CA, USA). The DNA thermal cycler was from Perkin-Elmer (Norwalk, CT, USA). 2. Ligamentum flavum cell culture LF cells were isolated from the ligament as described previously. 42 Briefly, the dissected specimens were minced with a scalpel into ca. 2 mm 3 11
pieces. Then, the LF tissues were digested for 60 min at 37 C under gentle agitation in medium composed of equal parts of Dulbecco s Modified Eagle medium and Ham s F-12 medium (DMEM/F12) containing 5% heat-inactivated fetal bovine serum (FBS) with 0.2% pronase and 0.004% deoxyribonuclease II type IV (DNase). The tissue was washed three times with DMEM/F12 and digested overnight under the same conditions, except that the pronase was replaced with bacterial 0.02% collagenase type II. The cells were filtered through a sterile nylon mesh filter (pore size: 75 µm), counted in a hemocytometer, and plated in 24-well plates at a density of approximately 5 10 5 cells/ml. Primary cultures were sustained for 2 to 3 weeks in DMEM/F12 containing 10% FBS, 1% v/v penicillin, streptomycin, and nystatin in a 5% CO 2 incubator with humidity. The culture medium was changed twice a week. 3. Study design To analyze the biologic response of human LF (hlf) cells to inflammatory cytokines, hlf cell cultures were organized into three groups: the (1) cytokine, (2) conditioned medium, and (3) control groups. In the first group, 30 ng/ml IL-1α, 30 ng/ml IL-6, 100 ng/ml TNF-α, 10 12
µm PGE 2, and 100 µm SNAP were added to hlf cell cultures. In the second group, conditioned medium was added to the cultured cells. In the third group, hlf cells were cultured without inflammatory cytokines or conditioned medium. Each culture was subjected to an MTT assay to estimate cell cytotoxicity, a 3 H-thymidine incorporation assay to analyze DNA synthesis, and RT-PCR to determine the expression of types I, II, III, V, and XI collagen and osteocalcin mrna. Alkaline phosphatase (ALP), Von Kossa, and Alizarin Red-S staining were used to demonstrate the expression of osteogenic markers and the formation of bone nodules. 4. Preparation of conditioned medium The IVD tissue was minced and incubated in 30 ml DMEM-F12 (200 mg tissue/ 1 ml DMEM-F12 medium) for 72 h in 5% CO 2 at 37 C with humidity. The culture medium was changed and collected daily. We refer to the collected medium as conditioned medium. 5.Treatment with inflammatory cytokines and conditioned medium At confluence, hlf cell cultures were rinsed with phosphate-buffered 13
saline (PBS) three times and exposed to the optimum dose of inflammatory cytokines and conditioned medium. Then, culture medium was added to each well of 24-well plates with cytokines, and the hlf cells were incubated for 48 h in 5% CO 2 at 37 C with humidity. 6. MTT assay After incubation for 48 h, the viability of hlf cells in the monolayer at the bottom of each well was assessed using the MTT assay, 43 as follows, in triplicate: 200 µl MTT reagent (2 mg/ml culture medium) were added to each well and incubated for 3 h in 5% CO 2 at 37 C with humidity. The supernatant was discarded and replaced with DMSO to dissolve the formazan product, which was measured at 550 nm in a spectrophotometric plate reader. 7. DNA synthesis analysis After incubation for 48 h, hlf cells were labeled with 3 H-thymidine (5 µci/ml) for the last 4 h of the 48-h treatment, as described previously. 44 Then, the hlf cell layer was washed three times with PBS, 14
treated with Trypsin-EDTA for 15 min, and the labeled hlf cells were harvested. The radioactivity was measured in a scintillation spectrophotometer. 8. Reverse-transcription polymerase chain reaction analysis Expression of the types I, II, III, V, and XI collagen and osteocalcin genes was examined using quantitative RT-PCR. 45, 46 Briefly, hlf cells were disrupted in lysis buffer containing guanidine isothiocyanate and homogenized. Total RNA was eluted by selective binding to a silica gelbased membrane using an RNeasy mini kit. Reverse transcription of RNA into cdna was performed by incubating 1 µl of RNA in a reaction mixture containing 0.5 mg/ml cdna reaction product and was used as the template to co-amplify β-actin, types I, II, III, V, and XI collagen, and osteocalcin. PCR was performed using a DNA thermal cycler. The primer sequences and PCR conditions used are shown in Tables 2 and 3, respectively. 47-51 15
Table 2. Sequences of the RT-PCR primers used Primer Sequence Length Size(bp) ß-actin 5'-GGC GGA CTA TGA CTT AGT TG-3' 20 5'-AAA CAA CAA TGT GCA ATC AA-3' 20 238 collagen 5'-CCT GTC TGC TTC CTG TTA AC-3' 20 type I 5'-AGA GAT GAA TGC AAA GGA AA-3' 20 collagen 5'-CTA CTG GAG TGA CTG GTC CTA A-3' 22 type II 5'-ACC ATC TTT TCC AGA AGG AA-3' 20 collagen 5'-CTG CCA TCC TGA ACT CAA GAG TGG-3' 24 type III 5'-CCA TCC TCC AGA ACT GTG TAG G-3' 22 collagen 5'-GGA TGA GGA GGT GTT TGA-3' 18 type V 5'-GCC CCT TCA CTG GTT TCA-3' 18 collagen 5'-GCT GAA AGT GTA ACA GAG GG-3' 20 type XI 5'-GGT TCT CCT TTC TGT CCT TT-3' 20 177 320 447 345 452 osteocalcin 5'-CAC TCC TCG CCC TAT TGG CC-3' 20 5'-GCC AAC TCG TCA CAG TCC GG-3' 20 237 Table 3. RT-PCR conditions 16
Primer Conditions Denatuation Annealing Polymerization Cycle ß-actin 94 1min 94 5sec 53 5sec 72 30sec 72 2min 24 collagen type I 94 1min 94 5sec 48 5sec 72 30sec 72 2min 21 collagen type II 94 1min 94 5sec 50 5sec 72 30sec 72 2min 41 collagen type III 94 9min 94 30sec 54 30sec 72 1min 72 7min 30 collagen type V 94 9min 94 30sec 60 30sec 72 1min 72 7min 35 collagen type XI 94 9min 94 30sec 54 30sec 72 1min 72 7min 40 osteocalcin 94 1min 94 5sec 60 5sec 72 30sec 72 2min 30 9. Von Kossa, Alizarin Red-S, and alkaline phosphatase staining For Von Kossa staining, hlf cells were fixed with 0.1 M sodium cacodylate and then washed. Silver nitrate (3%) was added to the cell cultures in a dark room and the cells were incubated for 30 min at room temperature in the dark and then exposed to light for 1 h. For Alizarin 17
Red-S staining, hlf cells were fixed for 2 h with ice-cold 70% ethanol. After washing with distilled water, the cells were stained with 40 mm Alizarin Red-S, ph 4.2, for 10 min at room temperature. The stained cell layers were then rinsed five times with distilled water, and washed with PBS for 15 min. For alkaline phosphatase (ALP) staining, hlf cells were fixed for 10 min with 3.7% formaldehyde at room temperature. After washing with PBS, the cells were incubated for 30 min with a mixture of 0.1 mg/ml naphthol AS-MX phosphate, 0.5% N,N-dimethylformamide, 2 mm MgCl 2, and 0.6 mg/ml fast blue BB salt in 0.1 M Tris-HCl, ph 8.5, at room temperature in the dark. Histochemical staining was compared qualitatively. 10. Statistical analysis The values were assessed using analysis of variance (ANOVA) and the t-test to evaluate the differences and were considered significant at p < 0.05. Ⅲ. Results 18
1.The hlf cells cultured with a given cytokine dose A.Cytotoxicity The hlf cells showed 200 to 250% cytotoxicity in the groups treated with cytokines and 175% in the control group. The cytotoxicity of hlf cells treated with cytokines did not differ from that of untreated hlf cells in any of the experiments (Fig. 1). B.DNA synthesis In the group treated with cytokines, DNA synthesis was enhanced in hlf cells compared with the controls, as shown by the increased 3 H- thymidine incorporation. Moreover, DNA synthesis was significantly increased in the hlf cells treated with IL-1α, IL-6, TNF-α, SNAP, and the supernatant from herniated IVD (Fig. 2). 19
Figure 1. Cytotoxicity of hlf cells in response to IL-1α, IL-6, TNF-α, PGE 2, and SNAP (NO donor). Combined cytokines, herniated IVD medium, and control. The hlf cells were cultured in 24-well plates until confluence, and then treated with various cytokines for 48 h. Cell cytotoxicity was measured using the MTT assay. No cytotoxicity was detected. 20
Figure 2. The effect of cytokines on DNA synthesis in hlf cells. The hlf cells were cultured in 24-well plates until confluence. After confluence, the cells were exposed to a given dose of various cytokines (IL-1α, IL-6, TNF-α, PGE 2, and SNAP (NO donor)) for 48 h and labeled with 3 H- thymidine for the final 4 h. Then, the incorporated radioactivity was determined. DNA synthesis was significantly increased with cytokine treatment. C.Effect of cytokines on the expression of collagen and osteocalcin 21
mrna The hlf cells treated with cytokines expressed types I, III, V, and XI collagen and osteocalcin mrna (Fig. 3A). The hlf cells cultured with SNAP showed upregulation of types I, III, and V collagen mrna, and a significant increase in osteocalcin mrna expression. The expression of type III collagen was upregulated in hlf cells treated with the combined cytokines. Moreover, the expression of types I and XI collagen mrna was upregulated in hlf cells treated with the supernatant from herniated IVD compared with the controls (Fig. 3B). D.Effect of cytokines on the expression of the osteogenic phenotype The hlf cells treated with IL-6, TNF-α, PGE 2, SNAP, and the upernatant from herniated IVD stained positively for Von Kossa stain (Fig. 4), but not for Alizarin Red-S or ALP stains (data not shown). 22
A. B. Figure 3. Effect of cytokines on the expression of hcol1a1, hcol3a1, hcol5a1, hcol11a1, and osteocalcin mrna in hlf cells. A. The hlf cells were exposed to a given dose of cytokines: IL-1α, IL-6, TNF-α, PGE 2, and SNAP (NO donor). Total RNA was isolated from cells and subjected to RT-PCR. The PCR products were separated on 2% agarose gels containing ethidium bromide, and then observed on an ultraviolet transilluminator. B. The expression of each band seen in A was quantified using an image analyzer. The results are presented as the percentage of the mrna level relative to β-actin for each band. The hlf cells cultured with SNAP showed upregulation of types I, III, V collagen and osteocalcin mrna expression. The expression of types I and XI collagen mrna was upregulated in hlf cells treated with the supernatant from herniated IVD compared with the controls (hcol1a1: human collagen type I, hcol3a1: human collagen type III, hcol5a1: human collagen type V, hcol11a2: human collagen type XI, con: control) 23
Von Kossa staining Figure 4. The hlf cells were cultured in a 24-well culture plate until confluence, and then treated with various cytokines for 48 h: IL-1α, IL-6, TNF-α, PGE 2, and SNAP (NO donor). The cells stained positively with Von Kossa stain (original magnification 100). 24
2. The hlf cells cultured with conditioned medium A. Morphology of hlf cells The hlf cells were mainly polygonal to oval cells arranged in sheets. There were a few spindle-shaped cells (Fig. 5A). The proportions and phenotypic characteristics did not differ from specimen to specimen or with the number of cell passages. The hlf cells treated with conditioned medium included more polygonal to oval cells than spindle-shaped cells (Fig. 5B). These cells showed significant signs of cellular proliferation (Fig. 5B) compared with the untreated hlf cells (Fig. 5A). B. Cytotoxicity The control hlf cells showed 50 to 96% cytotoxicity, versus 80 to 113% for the conditioned medium group. There was considerable variation among the patients. Nevertheless, the toxicity of hlf cells treated with cytokines did not differ significantly from that of untreated hlf cells in any of the experiments (Fig. 6). 25
Figure 5. The morphology of hlf cells. A. The hlf cells cultured in a 24- well culture plate. B. The hlf cells treated with conditioned medium for 48 h (original magnification 100). The hlf cells treated with conditioned medium included more polygonal to oval cells than spindleshaped cells, reflecting significantly increased cellular proliferation compared with the untreated hlf cells. 26
Figure 6. Cell cytotoxicity of hlf cells in response to conditioned medium. The hlf cells were cultured in a 24-well plate until confluence, and then treated with conditioned medium for 48 h. Cell cytotoxicity was measured using the MTT assay. No cytotoxicity was detected. (F1, F2, M1, M2: the numbers refer to specific female and male patients, con: control) 27
C. DNA synthesis In the group treated with conditioned medium, the hlf cells showed a significant increase in DNA synthesis, compared with the control (Fig. 7). Figure 7. The effect of conditioned medium on DNA synthesis in hlf cells. the hlf cells were cultured in a 24-well plate until confluence. After confluence, the cells were exposed to conditioned medium for 48 h, labeled with 3 H-thymidine for the final 4 h, and the amount of incorporated radioactivity was determined. DNA synthesis was significantly increased in cells treated with conditioned medium. (F1, F2, M1, M2: the numbers refer to specific female and male patients, con: control.) 28
D. Effect of conditioned medium on the expression of collagen and osteocalcin mrna The hlf cells treated with conditioned medium expressed types I, III, V, and XI collagen and osteocalcin mrna; collagen type II mrna was absent (Fig. 8A). The OD of hlf cells treated with conditioned medium showed the upregulation of types I, III, and V collagen mrna expression, and significant increases in the type XI collagen and osteocalcin mrna expression, compared with controls. To minimize the effects of variation among patients, all the data were normalized with respect to the untreated controls (Fig. 8B). E. Effect of conditioned medium on expression of the osteogenic phenotype Although there was individual variation in the intensity of Von Kossa, Alizarin Red-S, and ALP staining, the conditioned medium had a uniformly positive effect (Fig. 9). As the effects of Von Kossa, Alizarin Red-S, and ALP stains can be delayed, hlf cells were treated with conditioned medium for 7 days. These LF 29
A. B. Figure 8. Effect of conditioned medium on the expression of hcol1a1, hcol2a1, hcol3a1, hcol5a1, hcol11a1, and osteocalcin mrna in hlf cells. A. The hlf cells were exposed to conditioned medium and total RNA isolated from cells, and then subjected to RT-PCR analysis. B. The expression of each band shown in A was quantified using an image analyzer. The results are presented as the percentage of the mrna level in each band relative to β-actin. The expression of types I, III, V, and XI collagen and osteoclacin mrna was strongly upregulated when treated with conditioned medium. (hcol1a1: human collagen type I, hcol2a1: human collagen type II, hcol3a1: human collagen type III, hcol5a1: human collagen type V, hcol11a2: human collagen type XI, F1, F2, M1, M2: the numbers refer to specific female and male patients, F1c, F2c, M1c, M2c: the numbers of control female and male patients, con: control) 30
A. Von Kossa staining (A) Control group (B) Conditioned (disc) medium group Patient 1 Patient 2 Patient 3 Patient 4 B. Alizarin red-s staining (A) Control group (B) Conditioned (disc) medium group Patient 1 Patient 2 Patient 3 Patient 4 31
C. Alkaline Phosphatase staining (A) Control group (B) Conditioned (disc) medium group Patient 1 Patient 2 Patient 3 Patient 4 Figure 9. The hlf cells were cultured in 24-well culture plates until confluence, and then treated with conditioned medium for 48 h. They stained positively for A: Von Kossa, B: Alizarin Red-S, and C: ALP stains (original magnification 100). cells stained more strongly with Von Kossa and Alizarin Red-S, but less so for ALP relative to the other stains (Fig. 10). 32
A. Von Kossa staining control conditioned medium B. Alizarin red-s staining control conditioned medium C. Alkaline Phosphatase staining control conditioned medium Figure 10. The hlf cells were cultured in a 4-well chamber slide, and then treated with conditioned medium for 7 days. The cells stained more positively than in Fig. 9. A: Von Kossa, B: Alizarin Red-S, C: ALP stains (original magnification 100). 33
Ⅳ. Discussion Hypertrophy of the LF is a characteristic feature of lumbar spinal stenosis, causing compression of the cauda equina or nerve roots. Two mechanisms, not entirely exclusive, have been proposed for hypertrophy of the LF: one involves degenerative changes secondary to the aging process and the other is based on mechanical stress, such as instability. 16, 52 A number of authors have reported that LF hypertrophy plays a major role in the pathogenesis of lumbar canal stenosis. 53 Histological and immunohistochemical studies have revealed that an increased number of collagen fibers are present in the degenerate LF. 16 A biochemical analysis also showed increased collagen in a degenerate LF specimen. 15 These reports indicate that hypertrophy of the LF is characterized by a decrease in elastic fibers and an increase in collagen content. By contrast, some articles have reported that the LF contracts and folds in on itself as the interlaminar space narrows due to spinal disc collapse. 54 Ossification of the LF is a well-known clinical entity. Ono et al. 55 34
showed that bone morphogenic protein-2 (BMP-2) and transforming growth factor-β (TGF-β) were crucial in the process of ossification of the LF. They act on progenitor cells in the ligament, causing them to proliferate, form cartilage, and then ossify. The direction of calcification is from the dorsum of the ligament towards the spinal canal. The question of whether LF hypertrophy is an early stage of LF ossification arises. 56 Motegi et al. 57 reported that LF hypertrophy is often considered an early stage of LF ossification because of the apparent clinicopathological similarities. Epstein 58, 59 proposed Ossification of LF in evolution, which was defined as small areas of ossification in hypertrophy of the LF. LF hypertrophy might result from the process of LF degeneration, whereas LF ossification is an atypical form of diffuse idiopathic skeletal hyprostosis. 60 Therefore, LF hypertrophy should be considered an early stage or atypical pattern of LF ossification, making it less necessary to establish an independent clinical category for LF hypertrophy. In spite of the significance of the LF in the pathogenesis of spinal disease, there has been no thorough biochemical analysis of the degenerate LF. 35
To investigate whether inflammatory cytokines affect LF fibroblasts, we examined the cellular proliferation, matrix synthesis, and osteogenesis in LF hypertrophy and ossification. Due to limitations in the availability of fresh human IVD tissue and cancellous bone chips, we made and used conditioned medium. DNA synthesis increased significantly with the hypertrophy of LF cells treated with cytokines and conditioned medium. Moreover, the expression of types I, III, and V collagen mrna was upregulated, while there was no apparent change in collagen type II, which is distributed mainly at the ligament insertion of the facet in the lateral recess, where chondrocytes exist. Only a few chondrocytes are present in the interlaminar portion of the LF. 16 Specchia et al. 42 reported that cultured LF cells from patients with spinal stenosis included chondroblastic cells, which possessed type II collagen immunoreactivity, while the cells from patients without spinal canal stenosis did not. This demonstrates that inflammatory cytokines and conditioned medium affect LF hypertrophy significantly. In addition, the expression of collagen type XI and osteocalcin mrna were upregulated significantly. Koga et al. 61, 62 showed that the COL11A2 gene located in chromosome 6 may be responsible for genetic susceptibility to LF ossification, and may be a novel marker of ectopic ossification. 36
Human LF cells cultured with inflammatory cytokines and conditioned medium stained strongly with Von Kossa stain, and with Alizarin Red-S stain. Kazuhito et al. 63 found that bone-specific alkaline phosphatase was not correlated with LF ossification. Several studies have indicated that bone-specific alkaline phosphatase is an early marker in osteoblast differentiation and that osteocalcin is a late marker. 64, 65 Therefore, the stage of osteoblast differentiation may be associated with the activity of general ectopic bone formation in patients with LF ossification. These findings suggest that inflammatory cytokines and conditioned medium have significant effects on the pathogenesis of LF ossification. 37
Ⅴ. Conclusion This study investigated whether inflammatory cytokines from herniated IVD affect hypertrophy and ossification of the LF. 1. No recognizable cytotoxicity was seen. 2. DNA synthesis was significantly increased in treatments with cytokines and conditioned medium. 3. The expression of types I, III, V, and XI collagen and osteoclacin mrna was highly upregulated in treatments with cytokines and conditioned medium. 4. The hlf cells treated with cytokines and conditioned medium stained positively with Von Kossa, Alizarin Red-S, and ALP stains. Taken together, IVD degeneration or herniation has a pathogenetic significance in hypertrophy and ossification of the LF. IVD herniation affects the LF via inflammatory cytokines and causes hypertrophy and ossification of the LF. 38
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Abstract in Korean 인간황색인대의비후와골화에미치는퇴행된 인간추간판조직의역할 ( 지도교수 : 김학선 ) 연세대학교대학원의과학과김경희 척추관협착증은추간판탈출증, 후관절비후와함께황색인대의비후및골화의결과등에의해일어난다. 황색인대의비후와골화는가령에따른퇴행적변화에의해유도되고, 물리적확장 (mechanical stretching) 에의해교원질합성이증대된다. 황색인대의비후와골화의정도는보통추간판퇴행의정도와일치한다. 이와같은점으로보아요추척추관협착증에서추간판퇴행또는탈출증과황색인대의비후는관련깊을것으로보인다. 탈출된추간판은자발적으로 IL-1α, IL-6, TNF-α, PGE 2, NO등과같은 inflammatory cytokines 을생산한다. 본연구는이런 cytokines 이황색인대의섬유아세포를자극시켜세포증식, 기질생성, 골형성등을유도하는기전을설명함으로써척추관협착증에서의황색인대비후와골화의병리기전에대한퇴행된추간판의역할을밝히고자하였다. 세포배양에필요한표본은척추관협착증으로감압술을시행한 27명의환자의척추황색인대조직과탈출된추간판조직을모 49
아이용하였다 ( 연령 49-78세 ). 황색인대세포는효소처리법으로분리하였으며계대수가 3을넘지않도록하였다. 추간판조직은기본배지에넣고 48시간배양한뒤상층액만모았다. 이것을 conditioned medium라명명하였다. 황색인대세포에대한 cytokine의역할을보기위해 IL-1α (30 ng/ml), IL-6 (30 ng/ml), TNF-α (100 ng/ml), PGE 2 (10uM), NO (100uM) 과 conditioned medium를 48시간처리하였다. 대조군으로써 saline 을처리하였다. MTT assay 와 3 H-thymidine incorporation 을통해세포독성과세포증식을확인하였고, RT-PCR 을통해제 1, 2, 3, 5, 11형교원질과 osteocalcin 의 mrna 발현을측정하였다. ß-actin 은 mrna 량의표준화를위해사용하였다. Alkaline phosphatase staining, Alizarin Red-S staining, Von Kossa staining 을이용하여황색인대세포의골형성능을확인하였다. 세포독성효과는없었으며, cytokine 과 conditioned medium를처리했을때황색인대세포의 DNA 합성이의의있게증가하였고, 제 1, 3, 5, 11형교원질, osteocalcin mrna 발현량이크게증가하였고, Von Kossa, Alizarin Red-S, alkaline phosphatase로염색되었다. 종합해보면, 추간판의퇴행또는탈출증은황색인대의비후또는골화와 pathogenic significance 가있다는것을알수있다. 결론적으로, 추간판탈출증은 inflammatory cytokine 을통해황색인대에영향을끼치며, 황색인대의비후와골화의원인이될수있다. 핵심되는말 : 추간판, 변성, 황색인대, 비후, 골화. 50