The role of vascularization on changes in ligamentum flavum mechanical properties and development of hypertrophy in patients with lumbar spinal stenosis.

Ligamentum flavum (LF) induced lumbar spinal stenosis (LSS) is conditioned not only by its "gathering" but especially by hypertrophy. Previous studies have examined the pathophysiology and biochemical changes that cause the hypertrophy. Some studies have described a link between chronic LF inflammation and neovascularization but others have reported highly hypovascular LF tissue in LSS patients. Currently, there is no practical application for our knowledge of the pathophysiology of the LF hypertrophy. Considerations for future treatment include influencing this hypertrophy at the level of tissue mediators, which may slow the development of LSS. To our knowledge, there is no study of micromechanical properties of native LF to date. (1) To clarify the changes in vascularization, chondroid metaplasia, and the presence of inflammatory cell infiltration in LF associated with LSS. (2) To quantify changes in the micromechanical properties associated with LF degenerative processes. Vascular density analysis of degenerated and healthy human LF combined with measurement of micromechanical properties. The study involved 35 patients who underwent surgery between November 1, 2015 and October 1, 2016. The LSS group consisted of 20 patients and the control group consisted of 15 patients. LF samples were obtained during the operation and were used for histopathological and nanoindentation examinations. Sample vascularization was examined as microvascular density (Lv), which was morphometrically evaluated using semiautomatic detection in conjunction with NIS-Elements AR image analysis software. Samples were also histologically examined for the presence of chondroid metaplasia and inflammation. Mechanical properties of native LF samples were analyzed using the Hysitron TI 950 TriboIndenter nanomechanical testing system. Vascular density was significantly lower in the LSS group. However, after excluding the effect of age, the difference was not significant. There was high association between Lv and age. With each increasing year of age, Lv decreased by 11.5 mm2. Vascular density decreased up to the age of 50. Over the age of 50, changes were no longer significant and Lv appeared to stabilize. No correlation was observed between Lv and the presence of inflammation or metaplasia; however, LSS patients had a significantly increased incidence of chondroid metaplasia and inflammatory signs. The mechanical properties of control group samples showed significantly higher stiffness than those samples obtained from the LSS group. This study showed that Lv changes were not dependent on LSS but were age-dependent. Vascular density was found to decrease up to the age of 50. A significantly higher incidence of chondroid metaplasia and inflammation was observed in LSS patients. The mechanical property values measured by nanoindentation showed high microstructural heterogeneity of the tested ligaments. Our results showed that healthy ligaments were significantly stiffer than LSS ligaments. Prevention of the loss of LF vascularization during aging may influence stiffness of LF which in turn may slow down the LF degenerative processes and delay onset of LSS.

Jezek J ，Sepitka J ，Daniel M ，Kujal P ，Blankova A ，Waldauf P ，Krbec M ，Dousa P ，Skala-Rosenbaum J ，Samal F ，Jirasek T ... -  《-》

Expression and function of FGF9 in the hypertrophied ligamentum flavum of lumbar spinal stenosis patients.

Ligamentum flavum (LF) hypertrophy plays a dominant role in lumbar spinal stenosis (LSS). A previous study found that fibroblast growth factor 9 (FGF9) was upregulated with mechanical stress in rabbit LF. However, the expression and function of FGF9 are not well understood in human LF. To evaluate FGF9 expression and function in human LF with and without hypertrophy. This study employed a basic research study design utilizing human LF tissue for histological analyses. Hypertrophied LF tissue sample from patients with LSS, and nonhypertrophied (control) LFs from patients with lumbar disc herniation or other diseases were obtained during surgery. LF specimens were histologically analyzed for FGF9 and vascular endothelial growth factor A (VEGF-A) by immunohistochemistry. The number of total and FGF9 immuno-positive cells and blood vessels were counted and compared between LF with and without hypertrophy. For functional analysis, the effect of FGF9 on cell proliferation and migration was examined using a primary cell culture of human LF. Histological studies revealed that the total cell number was significantly higher in the LF of patients with LSS than in the LF of control patients. Immunohistochemistry showed that the percentage of FGF9-positive cells was significantly higher in the LF of patients with LSS than in the controls, and it positively correlated with patients' age, regardless of disease. Double immune-positive cells for FGF9 and VEGF-A were often observed in vascular endothelial cells and fibroblasts in the fibrotic area of hypertrophied LF, and the number of double positive vessels was significantly higher in LF of LSS patients than in the LF of controls. Primary cell culture of human LF revealed that FGF9 promoted the proliferation and migration of LF cells. The present study demonstrated that FGF9 expression is highly upregulated in hypertrophied human LF. FGF9 potentially plays a pivotal role in the process of hypertrophy of LF, which is associated with mechanical stress, through cell proliferation and migration. The results from this study partially reveal the molecular mechanisms of LF hypertrophy and suggest that FGF9 may be involved in the process of LF degeneration in elderly patients.

Habibi H ，Suzuki A ，Hayashi K ，Salimi H ，Terai H ，Hori Y ，Tamai K ，Orita K ，Ohyama S ，Yabu A ，Maruf MH ，Nakamura H ... -  《-》

Increased advanced glycation end products in hypertrophied ligamentum flavum of diabetes mellitus patients.

Ligamentum flavum (LF) hypertrophy plays a dominant role in lumbar spinal stenosis (LSS). Although LSS prevalence is known to be higher in patients with diabetes mellitus (DM), the underlying pathomechanisms are not well understood. Abnormal advanced glycation end products (AGEs) formation occurs in DM and promotes tissue damage in various organs through degeneration and inflammation. To analyze and compare LF histology focused on AGE status between control patients, LSS patients with DM, and LSS patients without DM. Basic research study design utilizing human LF tissue for histologic analyses. LF tissue samples were collected from patients who underwent lumber decompression surgery for LSS in the author's institution. Quantitative visualization of Masson's Trichrome (MT) stains, and AGE immunohistochemistry (IHC) for the three groups. Ten LF specimens from LSS patients with DM (DM group, mean age 71.4 years), 10 from LSS patients without DM (non-DM group, mean age 71.2 years), and 9 from patients with lumbar disc herniation or cauda equina tumor (control group, mean age 49.0 years) were harvested during surgery and histologically analyzed. Percentage of elastic fiber areas (%EF) was measured with MT staining, and the percentage of AGE immuno-positive areas (%AGEs) was measured with IHC. The average %EFs were 12.8 in the DM group, 17.1 in the non-DM group, and 24.9 in the control group. The decrease in the elastic fibers was significantly more in the DM group than in the non-DM (p<.01) and control groups (p<.001). Accumulation of AGEs was found mainly in the extracellular matrix in areas of elastic fiber disruption. The %AGEs were 18.3 in the DM group, 12.1 in the non-DM group, and 4.6 in the control group. These were significantly larger in the DM group than in the non-DM (p<.01) and control (p<.01) groups. The %AGEs also positively correlated with patient age (p<.01, R=0.47). Accumulation of AGEs is significantly greater in the LF of DM patients and correlates with patient age. AGEs may accelerate degeneration and hypertrophy of LF with age and may lead to higher prevalence of LSS in patients with DM. The present results partly reveal the molecular mechanism of LF hypertrophy, suggesting that AGEs may be involved in the process of LF degeneration in the elderly and patients with DM.

Maruf MH ，Suzuki A ，Hayashi K ，Habibi H ，Salimi H ，Terai H ，Tamai K ，Hoshino M ，Toyoda H ，Yamada K ，Takahashi S ，Ohyama S ，Hori Y ，Nakamura H ... -  《-》

Increased pain and sensory hyperinnervation of the ligamentum flavum in patients with lumbar spinal stenosis.

Nociceptive sensory nerve fibers have never been investigated in the ligamentum flavum (LF) of patients with LSS. The aim was to analyze nociceptive sensory nerve fibers in the ligamentum flavum (LF) of patients with LSS. A prospective study in patients with lumbar spinal stenosis (LSS) undergoing invasive surgical treatment for lumbar spinal stenosis (LSS) with flavectomy was performed. Patients with LSS were subjected to flavectomy and density of sensory and sympathetic nerve fibers, macrophages, vessels, activated fibroblasts, and cells were investigated by immunostaining techniques. A group of patients with acute disc herniation served as control group. We found a higher density of sensory nerve fibers in LSS patients versus controls. These findings support the role of LF in associated low back pain. Density of sensory nerve fibers in LSS, was positively correlated with typical markers of clinical pain and functional disability, but not with LF density of activated fibroblasts. Inflammation as estimated by macrophage infiltration and higher vascularity does not play a marked role in LF in our LSS patients. In the present study, compared to men with LSS, women with LSS demonstrate more pain and depression, and show a higher density of sensory nerve fibers in LF. This study shed new light on nociceptive nerve fibers, which are increased in LSS compared to controls. The findings speak against a strong inflammatory component in LSS. A higher pain levels in women compared to men can be explained by a higher density of nociceptive nerve fibers. © 2019 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 9999:1-7, 2019.

Benditz A ，Sprenger S ，Rauch L ，Weber M ，Grifka J ，Straub RH ... -  《-》

The correlation between imaging expression of P16 and S100 in hypertrophic ligamentum flavum.

Hu W ，Liu Y ，Kan S ，Zhang T ，Jiang Z ，Zhu R ... -  《BMC MUSCULOSKELETAL DISORDERS》