ORIGINAL ARTICLE
Herniated Intervertebral Disk Induces Hypertrophy and Ossification of Ligamentum Flavum Young-Mi Kang, MSc,* Kyung-Soo Suk, MD,w Byung Ho Lee, MD,w Hak-Sun Kim, MD,w Kwang-Il Lee, PhD,* Si-Young Park, MD,z Hwan-Mo Lee, MD,w and Seong-Hwan Moon, MDw
Study Design: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). Objectives: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. Summary of Background Data: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. Methods: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzymelinked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. Results: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1a, interleukin-6, tumor necrosis factor-a, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern
Received for publication May 20, 2012; accepted June 10, 2013. From the *Brain Korea 21, Medical Science Graduated School; wDepartment of Orthopaedic Surgery, Yonsei University College of Medicine; and zDepartment of Orthopaedic Surgery, Korea University College of Medicine, Seoul, Korea. Supported, in part, by the Brain Korea 21 Project for Medical Science, Yonsei University, and by grant No. R01-2006-000-10933-0 from the Basic Research Program of the Korea Science & Engineering Foundation. The authors declare no conflict of interest. Reprints: Seong-Hwan Moon, MD, Department of Orthopaedic Surgery, Yonsei University College of Medicine, #250 Seongsanno, Seodaemun-gu, Seoul, Korea (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
382 | www.jspinaldisorders.com
of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. Conclusions: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines. Key Words: ligamentum flavum, intervertebral disk, degeneration, hypertrophy, ossification (J Spinal Disord Tech 2014;27:382–389)
L
umbar spinal stenosis results from intervertebral disk (IVD) herniation and degenerative changes in the posterior structures of the lumbar spine, such as hypertrophy of the facet joints and ligamentum flavum (LF).1–7 Despite the significance of LF and degenerated IVD in the pathogenesis of spinal stenosis, there has been no research analyzing the association between degenerated IVD and hypertrophied LF. Interestingly, it is not uncommon to find that IVD degeneration and/or herniation coincides with LF hypertrophy and ossification.8,9 Furthermore, recently osteoblasts that were cocultured with herniated IVD tissue have been reported to demonstrate increase of alkaline phosphatase activity, type I collagen production, and [3H]thymidine incorporation.10 These results imply that the local milieu, including IVDs, bone, and facet joints, might provide a novel mechanism of LF hypertrophy and ossification. It is well known that a herniated IVD spontaneously produces inflammatory cytokines, such as interleukin-1a (IL1a), interleukin-6 (IL-6), nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-a), and matrix metalloproteinase.11 Therefore, cytokine secretions from herniated IVDs might affect the LF over a longer period of time, as IVDs and the LF share a local milieu through the spinal canal. From anatomic point of view, posterolateral corner of IVD abuts lateral recess and LF, which facilitate diffusion of cytokines in relatively short distance from IVD. Hence we hypothesized that secreted cytokines from herniated disk, presumably inflammatory cytokines, may affect the LF in terms of cellular proliferation, matrix synthesis, and osteogenesis of LF fibroblasts, ultimately resulting in LF hypertrophy and ossification. Accordingly, the objectives of this experimental study were to demonstrate a biological effect of supernatant from J Spinal Disord Tech
Volume 27, Number 7, October 2014
J Spinal Disord Tech
Volume 27, Number 7, October 2014
Intervertebral Disk and Ligamentum Flavum
TABLE 1. Sequences of RT-PCR Primers Used in This Study Primers
Sequence
Length
Size (bp)
b-actin
50 -GGCGGACTATGACTTAGTTG-30 50 -AAACAACAATGTGCAATCAA-30 50 -CCTGTCTGCTTCCTGTTAAC-30 50 -AGAGATGAATGCAAAGGAAA-30 50 -CTACTGGAGTGACTGGTCCTAA-30 50 -ACCATCTTTTCCAGAAGGAA-30 50 -CTGCCATCCTGAACTCAAGAGTGG-30 50 -CCATCCTCCAGAACTGTGTAGG-30 50 -GGATGAGGAGGTGTTTGA-30 50 -GCCCCTTCACTGGTTTCA-30 50 -GCTGAAAGTGTAACAGAGGG-30 50 -GGTTCTCCTTTCTGTCCT TT-30 50 -CACTCCTCGCCCTATTGGCC-30 50 -GCCAACTCGTCACAGTCCGG-30
20 20 20 20 22 20 24 22 18 18 20 20 20 20
238
Collagen type I Collagen type II Collagen type III Collagen type V Collagen type XI Osteocalcin
177 320 447 345 452 237
RT-PCR indicates reverse transcription-polymerase chain reaction.
degenerated and herniated IVD on the metabolism of LF cells and to propose the role of degenerated and herniated disk in the pathogenesis of LF hypertrophy and ossification.
MATERIALS AND METHODS All of the experimental protocols were approved by the human subjects Institutional Review Board of our institution.
Patients and Tissue Acquisition Procedures LF tissues were obtained from 15 patients (age range, 49–78 y) during surgery for lumbar spinal stenosis. All of the patients had symptomatic neurological claudication from spinal canal stenosis of the lumbar spine. The thickened LF was noted on preoperative magnetic resonance imaging and was also visualized directly during surgery. The operating surgeons attempted to obtain tissue en bloc from the central portion of the LF to minimize tissue damage and optimize the harvest of the ligament only. The LF tissue specimens were washed with saline to remove blood and bodily fluid contaminants and were then transported in sterile conditions to the laboratory, within 20 minutes of surgical removal. Herniated lumbar IVD tissues were obtained from 4 patients (2 female and 2 male patients with herniated lumbar disk) during surgical disk procedures. Classi-
fication of the IVD of each patient as grade of degeneration was performed based on magnetic resonance images of each disk as described in the literature.12,13 Grades III and IV degenerations were included in this study to minimize the effect of degeneration grades on the expression of phenotype and matrix synthesis. The disk tissue specimens were washed with Hank’s balanced salt solution (HBSS; Gibco-BRL, Grand Island, NY) to remove blood and bodily fluid contaminants, and were then transported in sterile HBSS to the laboratory,
Herniated Intervertebral Disk Induces Hypertrophy and Ossification of Ligamentum Flavum Young-Mi Kang, MSc,* Kyung-Soo Suk, MD,w Byung Ho Lee, MD,w Hak-Sun Kim, MD,w Kwang-Il Lee, PhD,* Si-Young Park, MD,z Hwan-Mo Lee, MD,w and Seong-Hwan Moon, MDw
Study Design: In vitro experiment using degenerated human ligamentum flavum (LF) and herniated intervertebral disk (IVD). Objectives: To investigate the role and effect of degenerated and herniated IVDs on LF hypertrophy and ossification. Summary of Background Data: Spinal stenosis is caused, in part, by hypertrophy and ossification of the LF, which are induced by aging and degenerative process. It is well known that degenerated IVDs spontaneously produce inflammatory cytokines. Therefore, we hypothesized that degenerated IVD may affect adjacent LF through secreted inflammatory cytokines. Methods: LF and herniated lumbar IVD tissues were obtained during surgical spinal procedures. LF fibroblasts were isolated by enzymatic digestion of LF tissue. LF cell cultures were treated with disk supernatant from herniated IVDs. Secreted cytokines from IVD tissue culture were detected by enzymelinked immunosorbent assay. After analysis of cytotoxicity, DNA synthesis was measured. Reverse transcription-polymerase chain reaction for mRNA expressions of types I, II, III, V, and XI collagen and osteocalcin, and histochemical stains were performed. Results: Supernatant from tissue culture of herniated IVD showed increased production of interleukin-1a, interleukin-6, tumor necrosis factor-a, prostaglandin E2, and nitric oxide compared with disk tissue culture from traumatic condition. There was no cytotoxicity in LF cells treated with disk supernatant from herniated IVDs. There was significant increase in DNA synthesis, upregulation in mRNA expression of types III, XI collagen and osteocalcin, whereas variable expression pattern
Received for publication May 20, 2012; accepted June 10, 2013. From the *Brain Korea 21, Medical Science Graduated School; wDepartment of Orthopaedic Surgery, Yonsei University College of Medicine; and zDepartment of Orthopaedic Surgery, Korea University College of Medicine, Seoul, Korea. Supported, in part, by the Brain Korea 21 Project for Medical Science, Yonsei University, and by grant No. R01-2006-000-10933-0 from the Basic Research Program of the Korea Science & Engineering Foundation. The authors declare no conflict of interest. Reprints: Seong-Hwan Moon, MD, Department of Orthopaedic Surgery, Yonsei University College of Medicine, #250 Seongsanno, Seodaemun-gu, Seoul, Korea (e-mail: [email protected]). Copyright r 2013 by Lippincott Williams & Wilkins
382 | www.jspinaldisorders.com
of type I and V, and strong positive stains for Von Kossa and alkaline phosphatase in LF cultures with disk supernatant. Conclusions: Degenerated and herniated IVDs provide an important pathomechanism in hypertrophy and ossification of the LF through inflammatory cytokines. Key Words: ligamentum flavum, intervertebral disk, degeneration, hypertrophy, ossification (J Spinal Disord Tech 2014;27:382–389)
L
umbar spinal stenosis results from intervertebral disk (IVD) herniation and degenerative changes in the posterior structures of the lumbar spine, such as hypertrophy of the facet joints and ligamentum flavum (LF).1–7 Despite the significance of LF and degenerated IVD in the pathogenesis of spinal stenosis, there has been no research analyzing the association between degenerated IVD and hypertrophied LF. Interestingly, it is not uncommon to find that IVD degeneration and/or herniation coincides with LF hypertrophy and ossification.8,9 Furthermore, recently osteoblasts that were cocultured with herniated IVD tissue have been reported to demonstrate increase of alkaline phosphatase activity, type I collagen production, and [3H]thymidine incorporation.10 These results imply that the local milieu, including IVDs, bone, and facet joints, might provide a novel mechanism of LF hypertrophy and ossification. It is well known that a herniated IVD spontaneously produces inflammatory cytokines, such as interleukin-1a (IL1a), interleukin-6 (IL-6), nitric oxide (NO), prostaglandin E2 (PGE2), tumor necrosis factor-a (TNF-a), and matrix metalloproteinase.11 Therefore, cytokine secretions from herniated IVDs might affect the LF over a longer period of time, as IVDs and the LF share a local milieu through the spinal canal. From anatomic point of view, posterolateral corner of IVD abuts lateral recess and LF, which facilitate diffusion of cytokines in relatively short distance from IVD. Hence we hypothesized that secreted cytokines from herniated disk, presumably inflammatory cytokines, may affect the LF in terms of cellular proliferation, matrix synthesis, and osteogenesis of LF fibroblasts, ultimately resulting in LF hypertrophy and ossification. Accordingly, the objectives of this experimental study were to demonstrate a biological effect of supernatant from J Spinal Disord Tech
Volume 27, Number 7, October 2014
J Spinal Disord Tech
Volume 27, Number 7, October 2014
Intervertebral Disk and Ligamentum Flavum
TABLE 1. Sequences of RT-PCR Primers Used in This Study Primers
Sequence
Length
Size (bp)
b-actin
50 -GGCGGACTATGACTTAGTTG-30 50 -AAACAACAATGTGCAATCAA-30 50 -CCTGTCTGCTTCCTGTTAAC-30 50 -AGAGATGAATGCAAAGGAAA-30 50 -CTACTGGAGTGACTGGTCCTAA-30 50 -ACCATCTTTTCCAGAAGGAA-30 50 -CTGCCATCCTGAACTCAAGAGTGG-30 50 -CCATCCTCCAGAACTGTGTAGG-30 50 -GGATGAGGAGGTGTTTGA-30 50 -GCCCCTTCACTGGTTTCA-30 50 -GCTGAAAGTGTAACAGAGGG-30 50 -GGTTCTCCTTTCTGTCCT TT-30 50 -CACTCCTCGCCCTATTGGCC-30 50 -GCCAACTCGTCACAGTCCGG-30
20 20 20 20 22 20 24 22 18 18 20 20 20 20
238
Collagen type I Collagen type II Collagen type III Collagen type V Collagen type XI Osteocalcin
177 320 447 345 452 237
RT-PCR indicates reverse transcription-polymerase chain reaction.
degenerated and herniated IVD on the metabolism of LF cells and to propose the role of degenerated and herniated disk in the pathogenesis of LF hypertrophy and ossification.
MATERIALS AND METHODS All of the experimental protocols were approved by the human subjects Institutional Review Board of our institution.
Patients and Tissue Acquisition Procedures LF tissues were obtained from 15 patients (age range, 49–78 y) during surgery for lumbar spinal stenosis. All of the patients had symptomatic neurological claudication from spinal canal stenosis of the lumbar spine. The thickened LF was noted on preoperative magnetic resonance imaging and was also visualized directly during surgery. The operating surgeons attempted to obtain tissue en bloc from the central portion of the LF to minimize tissue damage and optimize the harvest of the ligament only. The LF tissue specimens were washed with saline to remove blood and bodily fluid contaminants and were then transported in sterile conditions to the laboratory, within 20 minutes of surgical removal. Herniated lumbar IVD tissues were obtained from 4 patients (2 female and 2 male patients with herniated lumbar disk) during surgical disk procedures. Classi-
fication of the IVD of each patient as grade of degeneration was performed based on magnetic resonance images of each disk as described in the literature.12,13 Grades III and IV degenerations were included in this study to minimize the effect of degeneration grades on the expression of phenotype and matrix synthesis. The disk tissue specimens were washed with Hank’s balanced salt solution (HBSS; Gibco-BRL, Grand Island, NY) to remove blood and bodily fluid contaminants, and were then transported in sterile HBSS to the laboratory,
