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Nano and micro biomechanical analyses of the nucleus pulposus after in situ immobilization in rats
Micron 130 (2020) 102824
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Nano and micro biomechanical analyses of the nucleus pulposus after in situ immobilization in rats
T
Ting Lianga,b,1, Dong-Yan Zhongc,1, Yan-Jun Chea,b, Xi Chena,b, Jiang-Bo Guoa,b, Hui-Lin Yanga,b, Zong-Ping Luoa,b,* a
Orthopedic Institute, Medical College, Soochow University, Suzhou, Jiangsu 215006, PR China Department of Orthopedics, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, PR China c Suzhou Gusu District Maternal and Child Health Center, Suzhou, Jiangsu, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Intervertebral disc Nucleus pulposus Immobilization Biomechanics Atomic force microscopy
Immobilization can lead to intervertebral disc degeneration. The biomechanical characteristics of such discs have not so far been investigated at the micro- or nanoscale, the level at which cells sense and respond to the surrounding environment. This study aimed to characterize changes in the elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale and correlate this with micro-biomechanical properties of the nucleus pulposus after immobilization, in addition to observation of tissue histology and its gene expressions. An immobilization system was used on the rat tail with an external fixation device. The elastic modulus was measured using both nano and micro probes for atomic force microscopy after 4 and 8 weeks of immobilization. Histology of the tissue was observed following hematoxylin and eosin staining. Gene expression in the annulus fibrosus tissue was quantified using real-time reverse transcription-polymerase chain reaction. The elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale increased from 74.07 ± 17.06 MPa in the control to 90.06 ± 25.51 MPa after 8 weeks (P = 0.007), and from 33.51 ± 9.33 kPa to 43.18 ± 12.08 kPa at the microscale (P = 0.002). After immobilization for 8 weeks, a greater number of cells were observed by histology to be aggregated within the nucleus pulposus. Collagen II (P = 0.007) and aggrecan (P = 0.003) gene expression were downregulated whereas collagen I (P = 0.002), MMP-3 (P < 0.001), MMP-13 (P < 0.001) and ADAMTs-4 (P < 0.001) were upregulated. Immobilization not only influenced individual collagen fibrils at the nanoscale, but also altered the micro-biomechanics and cell response in the nucleus pulposus. These results suggest that significant changes occur in intervertebral discs at both scales after immobilization, a situation about which clinicians should be aware when immobilization has to be used clinically.
1. Introduction Intervertebral disc degeneration is a major contributing factor in lower back pain. Abnormal mechanical environments, including overloading and immobilization, is assumed to partly determine the rate of disc degeneration (Stokes and Iatridis, 2004). The immobilization of vertebrae is common in clinical applications in order to restore spinal stability under conditions of cervical spondylosis, spinal dislocation or fractures. However, complications may arise after immobilization such as heterotopic ossification and osteitis condensans (Baum et al., 1989; Hossain et al., 2004). The effect of immobilization on intervertebral disc degeneration remains unclear, previous studies having suggested that immobilization induces changes to discs similar to those caused by
static compression (Iatridis et al., 1999), although others suggest greater alterations after immobilization (Hirata et al., 2014). Thus, detailed pathogenesis of disc degeneration after immobilization still requires additional study. The intervertebral disc is a cushion-like structure between the bony vertebrae comprising three major tissue regions: the fibrous lamellae of the annulus fibrosus, the gelatinous nucleus pulposus and the cartilage endplates sandwiching the annulus fibrosus and nucleus pulposus (Aladin et al., 2010; Ding et al., 2013). The discs function by distributing large loads acting on the spine and providing flexibility, the nucleus pulposus bearing the majority of the pressure. The occurrence of disc degeneration also starts from the nucleus pulposus (Chris et al., 2016; Ding et al., 2013; Haefeli et al., 2006). Type II collagen in the
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Corresponding author at: Orthopaedic Institute, Soochow University, 708 Renmin Rd, Suzhou, Jiangsu, 215007, PR China. E-mail address: [email protected] (Z.-P. Luo). 1 Ting Liang and Dong-Yan Zhong contributed equally to this work. https://doi.org/10.1016/j.micron.2020.102824 Received 12 July 2019; Received in revised form 20 November 2019; Accepted 3 January 2020 Available online 07 January 2020 0968-4328/ © 2020 Published by Elsevier Ltd.
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2.2. Histological analysis
nucleus pulposus accounts for 10%–20% of the dry weight of human lumbar discs and constitutes the major part of the fibrous collagen network providing a scaffold structure to the hydrated proteoglycan (Adams and Roughley, 2006). Normal day-to-day mechanical loading of the discs maintains cell homeostasis by continuous secretion of matrix. Conversely, hyper- or hypo-physiological loading may be harmful to the nucleus pulposus (Chan et al., 2011). Abnormal loading may affect not only the expression of matrix but also the mechanical properties of the collagen fibers (Adams et al., 2000; Iatridis and Gwynn, 2004; Ching et al., 2003; Stemper et al., 2014). Even changes at the nanoscale structure of the individual collagen fibrils in the nucleus pulposus might result in dispersion of proteoglycans, influencing overall mechanical integrity of the disc (Aladin et al., 2010). Immobilization of long segments of the spine results in changes in the mechanical environment of the nucleus pulposus compared with normal physiological status. However, the precise biomechanical properties of the nucleus pulposus after immobilization remain unclear, particularly at the micro or nanoscale. In the present study, we aimed to ascertain the influence of immobilization on the biomechanical properties of the nucleus pulposus at the micro and nanoscale in the rat tail using atomic force microscopy (AFM), with observation of tissue histology and quantification of gene expression. The results offer critical data and describe the effect on the biomechanics of immobilization on intervertebral disc degeneration.
The target discs (C7-8) were harvested and fixed in 10 % neutral buffered formalin (Shanghai Yuanye Bio-Technology Co. Ltd, Shanghai, China) for 24 h. Following decalcification in buffered 10 % EDTA, pH 7.4 (Biosharp, Hefei, China) for 4 weeks, the samples were dehydrated, embedded in paraffin, sectioned to a thickness of 5 μm using a microtome (Leica, Heidelberger, Germany) and then stained with hematoxylin and eosin (Beijing Biotopped Science & Technology Co. Ltd, Beijing, China). Stained images were then evaluated using a binocular light microscope (XSP-2CA, Shanghai, China) at a magnification of 100 × . The cells in nucleus pulposus were counted by Multi-Point tool in ImageJ Software in the staining image. In each group, three slices per disc and seven discs were used for the cell number analysis. 2.3. AFM imaging and mechanical testing After immobilization discs were harvested and fresh tissue from C8C9 sliced into 20∼30 μm thick sections using a frozen tissue cryostat (CM3050 S, Leica, Nussloch, Germany). One slice in the center position of a single disc were subjected to AFM testing using an AFM scanner (Dimension ICON, Bruker, USA). For the nanoscale experiments, images and the elastic modulus of fibrils were obtained using a ScanAsyst-Air probe with a radius of curvature of 5 nm and a force constant of 0.4 N/m. The scan area was selected randomly. Since there is the heterogeneity within the nucleus pulposus, some areas exhibit no fibril in the AFM imaging, only the image containing fibrils and the corresponding micro modulus in the same area were involved in this study. Fibril diameters were obtained by line section analysis using NanoScope Analysis Software (Bruker, USA). The modulus of the fibrils was calculated using AFM PeakForce QNM (Quantitative Nano Mechanics) test mode (n = 50 in each group), utilizing the Hertz model as defined in Eq. (1), after calibration of the force constant k and radius of curvature R of the probe. A detailed determination of the modulus is described in a previous study (Stolz et al., 2004).
2. Materials and methods 2.1. Immobilization model Vertebral immobilization was conducted on 21 fully grown 3month-old male Sprague-Dawley rats. The study was approved by the Institutional Animal Care and Use Committee. The animals were randomly divided into 3 groups. The baseline control group had no external fixation device (n = 7). In the other two groups (n = 7 for each group), four caudal vertebrae (C7-C10) were firstly instrumented with a K-wire (50 mm in length and 1.2 mm in diameter, XingYu Medical Instrument Factory, Suzhou, China) in each vertebra. Two bespoke aluminum alloy cuboids (43 mm in length, 4 mm wide and 5.0 g net weight) were then attached to the four K-wires (Fig. 1) on either side of the tail for spatial immobilization. Placement of the fixation device was confirmed by X-ray, as displayed in Fig. 2. In a previous study (Che et al., 2018), it was demonstrated that K-wire placement alone without immobilization did not significantly affect the discs and so a sham group was not included in this study. After immobilization for 4 or 8 weeks, animals were euthanized and the immobilized discs harvested for further analysis.
F=
4 E R δ3 3 (1 − υ2)
2
(1)
where F is the indentation force, E the Young’s modulus, υ is Poisson’s ratio, R is the radius of the indenter and δ is value of the indentation. For microscale experiments, the modulus was obtained using forcevolume mode in 0.15 M PBS (pH = 7.4) in a fluid cell using a borosilicate glass spherical tip with a diameter of 5μm firmly attached to the native V-shaped silicon nitride cantilever, with a spring constant of 0.06 N/m (Bruker, USA). The calculation of elastic modulus was described in detail in a previous study, using the following equation (Stolz
Fig. 1. (A) Schematic of immobilization animal model. The external device four discs by using four K-wires and two parallel aluminum alloy cuboids which produce no compresssion or tension in the disc. (B) The corresponding X-ray confirmation image. 2
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Fig. 2. The representative histological images using hematoxylin/eosin staining of nucleus pulposus in both control and immobilized groups. Compared with the control group, cells were aggregated in the nucleus pulposus of the two immobilized groups. The typical morphology of cells remained unchanged after immobilization.
are displayed in Fig. 4a. Compared with the modulus of fibrils in the nucleus pulposus in the control group (74.07 ± 17.06 MPa), immobilization for 8 weeks caused the fibrils to become significantly stiffer (90.06 ± 25.51 MPa, P = 0.007), while immobilization for 4 weeks did not influence the modulus of the fibrils significantly (77.75 ± 15.43 MPa, P = 0.075). The elastic modulus of the nucleus pulposus at the microscale is presented in Fig. 4b. Immobilization for 8 weeks also significantly modified the modulus (43.18 ± 12.08 kPa, P = 0.002) compared with that of the control group (33.51 ± 9.33 kPa). However, modulus at the microscale after immobilization for 4 weeks did not change significantly (30.75 ± 10.87 kPa). Expression of anabolic genes except collagen I decreased following immobilization as shown in Fig. 5a. Eight weeks of immobilization caused a decrease in collagen II expression of 26.0 % compared with the control group (P = 0.005) and by 16.9 %, after 4 weeks of immobilization (P > 0.05). Additionally, expression of aggrecan was reduced after 4 weeks of immobilization by 24 % (P = 0.018) relative to the control. Eight weeks of immobilization downregulated mRNA levels of aggrecan by 25 % (P = 0.003). Conversely, 8 weeks of immobilization induced greater expression of collagen I by 18 % and 13.5 % in comparison with the control (P = 0.002) and 4 weeks immobilization (P = 0.013), respectively. In terms of catabolic genes in Fig. 5b, both 4 and 8 weeks of immobilization significantly upregulated the expression of MMP3, MMP13 and ADAMTs-4. Four weeks of immobilization increased MMP3 expression by 18 % (P = 0.014), MMP13 by 31 % (P < 0.001) and ADAMTs-4 by 36 % (P < 0.001) compared with the control group. Immobilization for 8 weeks resulted in a more significant increase in mRNA expression levels (MMP3 by 23 %, P < 0.001; MMP13 by 56 %, P < 0.001; ADAMTs-4 by 48 %, P < 0.001 compared with the control group).
et al., 2004; Loparic et al., 2010):
E=
π S (1 − υ2) 2 A
(2)
where S is contact stiffness, A is surface area of the spherical indenter contacting the substrate. 2.4. Real-time polymerase chain reaction (PCR) analysis The Co8-Co9 intervertebral disc in each in-situ immobilization rat was used for the PCR analysis (n = 7 in each group). The target disc was exposed by shearing the muscle and ligaments around the caudal vertebra. Then nucleus pulposus was separated by rotating a surgical knife along the junction of nucleus pulposus and annulus fibrosus. Total RNA of the nucleus pulposus was isolated using TRIzol reagent (Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s instructions. One μg of total RNA was then reverse-transcribed to firststrand complementary DNA (cDNA) using a RevertAid First Strand cDNA Synthesis kit (Thermo fisher Scientific). Real-time PCR was conducted using an iTaq™ Universal SYBR® Green Supermix kit (BioRad, Hercules, CA, USA) on a CFX96™ Real-Time PCR System (BioRad). mRNA expression levels of genes of anabolic (collagen I, collagen II, aggrecan) and catabolic (MMP3, MMP13 and ADAMTs-4) proteins were calculated relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) using the 2−ΔΔCT method (Pfaffl, 2001). The primer sequences of each gene are described in Table 1. 2.5. Statistical analysis Data from all experiments are presented as means ± standard deviation. Comparisons of differences between groups were analyzed by one-way analysis of variance (ANOVA) and a student’s t-test. P < 0.05 was considered statistically significant. All tests were two-sided and conducted using SPSS v16.0 software (SPSS Inc., Chicago, IL, USA).
4. Discussion In addition to the contribution of genes, abnormal loading conditions also play an important role in intervertebral disc degeneration (Walter et al., 2011). Compared with overloading, the effect of reduced motion on discs has not been comprehensively studied despite it being a commonly-used spinal treatment (Stokes and Iatridis, 2004). The present study firstly reported on the biomechanical properties of the nucleus pulposus over different time points after the immobilization of rat tails, including the elastic modulus of individual fibrils of the nucleus pulposus at the nanoscale and at the microscale. Based on the extracellular matrix condensation observed in histological images after immobilization, the 8 weeks group exhibited disc degeneration according to the grading scale (Masuda et al., 2005). After intervertebral discs are loaded through bearing spinal loads, the nucleus pulposus swells under compression which transfers load to
3. Results Typical histological images of the nucleus pulposus are shown in Fig. 2. Compared with the control group, cells were aggregated in the nucleus pulposus of the two immobilized groups, although the typical morphology of cells remained unchanged after immobilization. The numbers of cells in the nucleus, ranging from 1284 ± 41–1321 ± 58 also were not significant different among the three groups (P = 0.54). Typical AFM images at the nanoscale are presented in Fig. 3. There was no significant difference in the arrangement of the collagen network or diameter of fibrils between the control and immobilized groups (P > 0.05). The elastic moduli of the individual collagen fibrils at the nanoscale 3
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Fig. 3. The typical atomic force microscopy scanning images of collagen fibrils in nucleus pulposus in both control and immobilized groups. The fibrils or the collagen network exhibited no significant difference between control and immobilized groups.
Fig. 4. The dynamic elastic modulus at both the (A) nano and (B) micro scales of nucleus pulposus measured by atomic force microscopy, where the nanoscale represented the modulus of individual fibrils. Compared with the control group, immobilization for 8 weeks caused higher modulus at both nano and micro scales.
the annulus fibrosus. The ealstic modulus means the the ability of resisting elastic deformation. The higher modulus results in higher stress under the same deformation. When the modulus of the nucleus pulposus changes, the stress distribution within the annulus is altered accordingly, and thus those of the entire disc as well. Normal external loading is essential for the sustained transmission of nutrients, load bearing capacity and cell response within the discs. After immobilization of the spine, the abnormal mechanical environment may induce disc degeneration, including mechanical alteration of the nucleus pulposus and annulus fibrosus. However, the detailed pathogenesis
remains unclear. The rat tail model has been widely used to simulate cervical or lumbar disc degeneration (Chris et al., 2016; Ching et al., 2003; Court et al., 2001; Iatridis et al., 1999; Yurube et al., 2010). In this study, the loading device was modified to simulate immobilization. The mechanical properties of the tissue of the nucleus pulposus changes after degeneration (Iatridis et al., 1997; Johannessen and Elliott, 2005). A huge nonuniformity of disc surface was also revealed after degeneration (Byvaltsev et al., 2012). However, detailed alterations within the nucleus pulposus remain ambiguous, especially at the micro and nanoscale. Among the factors affecting the mechanical
Fig. 5. The (A) anabolic and (B) catabolic genes expression in nucleus pulposus in both control and immobilized groups. mRNA levels of nucleus pulposus were normalized to endogenous control (GAPDH) and internal controls. 4
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properties of the nucleus pulposus, collagen fibrils play an important role, a network of collagen fibrils providing the mechanical strength to resist the pressure from swelling (Aladin et al., 2010). In this study, the individual fibrils in the nucleus pulposus stiffened after immobilization for 8 weeks. Individual collagen fibrils were affected by several factors such as matrix metalloproteinases and proteoglycans (Nakamura et al., 2000; Zhao et al., 2007). A decrease in aggrecan and increase in catabolic gene expression observed in this study might represent evidence that clarified the relationship between fibril stiffness and genetic factors. Additionally, the modulus at the microscale also increased in the 8 weeks group. Thus correlation of the elastic modulus between the nano and microscales in this study might provide additional information about how the stiffness of individual fibrils influences the entire fibril network, further influencing the mechanical integrity of the nucleus pulposus such as the stiffening at the macroscale after degeneration (Aladin et al., 2010; Neidlinger-Wilke et al., 2014). Although a number of general studies of changes in mRNA levels after disc degeneration have been published, the conclusions are still debated about which loading conditions induce degeneration, for example bending or compression, their magnitude and duration (Liang et al., 2019; MacLean et al., 2004, 2003; Yurube et al., 2010). For instance, a longer duration of loading induces greater alteration in the expression of certain genes, but fewer changes in others, possibly due to an adaptive remodeling response. mRNA expression levels reflect the cell response to the environment. In this study, both individual fibrils at the nanoscale and the matrix at the microscale changed after immobilization for 8 weeks, inducing a change in the mechanical environment surrounding the cells, the expression of genes also changing in the 8 week group. The combination of external immobilization and the stiffening matrix constituted abnormal loading conditions around the cells. However, the results after 4 weeks indicated discordance between the mechanical properties and gene expression, in that expression of the catabolic genes and aggrecan changed while the elastic modulus remained the same. These results possibly indicate that the response of cells within the disc occurs prior to that of the matrix response to the loading due to immobilization. A limitation of this study is the difference in cell type and loading conditions in the nucleus pulposus of the rat tail compared with the human spine. However, the animal model is useful for exploration of the reciprocal interactions between mechanical loading and disc homeostasis (Neidlinger-Wilke et al., 2014).
Planned Projects for Postdoctoral Research Funds (1601051C), National Natural Science Foundation of China (81320108018, 31570943, and 81702146), Innovation and Entrepreneurship Program of Jiangsu Province, the Priority Academic Program Development of Jiangsu Higher Education Institutions. References Adams, M.A., Freeman, B.J., Morrison, H.P., Nelson, I.W., Dolan, P., 2000. Mechanical initiation of intervertebral disc degeneration. Spine 25, 1625–1636. Adams, M.A., Roughley, P.J., 2006. What is intervertebral disc degeneration, and what causes it? Spine 31, 2151–2161. Aladin, D.M., Cheung, K., Ngan, A.H., Chan, D., Leung, V.Y., Lim, C.T., Luk, K.D., Lu, W.W., 2010. Nanostructure of collagen fibrils in human nucleus pulposus and its correlation with macroscale tissue mechanics. J. Orthop. Res. 28, 497–502. Baum, J.A., Hanley Jr., E.N., Pullekines, J., 1989. Comparison of halo complications in adults and children. Spine 14, 251–252. 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Int. Court, C., Colliou, O.K., Chin, J.R., Liebenberg, E., Bradford, D.S., Lotz, J.C., 2001. The effect of static in vivo bending on the murine intervertebral disc. Spine J. 1, 239–245. Ding, F., Shao, Z.W., Xiong, L.M., 2013. Cell death in intervertebral disc degeneration. Apoptosis 18, 777–785. Haefeli, M., Kalberer, F., Saegesser, D., Nerlich, A., Boos, N., Paesold, G., 2006. The course of macroscopic degeneration in the human lumbar intervertebral disc. Spine 31, 1522–1531. Hirata, H., Yurube, T., Kakutani, K., Maeno, K., Takada, T., Yamamoto, J., Kurakawa, T., Akisue, T., Kuroda, R., Kurosaka, M., 2014. A rat tail temporary static compression model reproduces different stages of intervertebral disc degeneration with decreased notochordal cell phenotype. J. Orthop. Res. 32, 455–463. Hossain, M., McLean, A., Fraser, M., 2004. Outcome of halo immobilisation of 104 cases of cervical spine injury. Scott. Med. J. 49, 90–92. Iatridis, J.C., Gwynn, I.A., 2004. Mechanisms for mechanical damage in the intervertebral disc annulus fibrosus. J. Biomech. 37, 1165–1175. Iatridis, J.C., Mente, P.L., Stokes, I.A., Aronsson, D.D., Alini, M., 1999. Compression-induced changes in intervertebral disc properties in a rat tail model. Spine 24, 996–1002. Iatridis, J.C., Setton, L.A., Weidenbaum, M., Mow, V.C., 1997. Alterations in the mechanical behavior of the human lumbar nucleus pulposus with degeneration and aging. J. Orthop. Res. 15, 318–322. Johannessen, W., Elliott, D.M., 2005. Effects of degeneration on the biphasic material properties of human nucleus pulposus in confined compression. Spine 30, E724–E729. Liang, T., Che, Y.J., Chen, X., Li, H.T., Yang, H.L., Luo, Z.P., 2019. Nano and micro biomechanical alterations of annulus fibrosus after in situ immobilization revealed by atomic force microscopy. J. Orthop. Res. 37, 232–238. Loparic, M., Wirz, D., Daniels, A.U., Raiteri, R., Vanlandingham, M.R., Guex, G., Martin, I., Aebi, U., Stolz, M., 2010. Micro-and nanomechanical analysis of articular cartilage by indentation-type atomic force microscopy: validation with a gel-microfiber composite. Biophys. J. 98, 2731–2740. MacLean, J.J., Lee, C.R., Alini, M., Iatridis, J.C., 2004. Anabolic and catabolic mRNA levels of the intervertebral disc vary with the magnitude and frequency of in vivo dynamic compression. J. Orthop. Res. 22, 1193–1200. MacLean, J.J., Lee, C.R., Grad, S., Ito, K., Alini, M., Iatridis, J.C., 2003. Effects of immobilization and dynamic compression on intervertebral disc cell gene expression in vivo. Spine 28, 973–981. Masuda, K., Aota, Y., Muehleman, C., Imai, Y., Okuma, M., Thonar, E.J., Andersson, G.B., An, H.S., 2005. A novel rabbit model of mild, reproducible disc degeneration by an anulus needle puncture: correlation between the degree of disc injury and radiological and histological appearances of disc degeneration. Spine 30, 5–14. Nakamura, N., Hart, D.A., Boorman, R.S., Kaneda, Y., Shrive, N.G., Marchuk, L.L., Shino, K., Ochi, T., Frank, C.B., 2000. Decorin antisense gene therapy improves functional healing of early rabbit ligament scar with enhanced collagen fibrillogenesis in vivo. J. Orthop. Res. 18, 517–523. Neidlinger-Wilke, C., Galbusera, F., Pratsinis, H., Mavrogonatou, E., Mietsch, A., Kletsas, D., Wilke, H.J., 2014. Mechanical loading of the intervertebral disc: from the macroscopic to the cellular level. Eur. Spine J. 23, 333–343. Pfaffl, M., 2001. A new mathematical model for relative quantification in real-time RTPCR. Nucleic Acids Res. 29, 2002–2007. Stemper, B.D., Baisden, J.L., Yoganandan, N., Shender, B.S., Maiman, D.J.D., 2014. Mechanical yield of the lumbar annulus: a possible contributor to instability. J.
5. Conclusions In this study, the mechanical properties of both individual collagen fibrils at the nanoscale and the matrix at the microscale in the nucleus pulposus were investigated using AFM after immobilization of the rat tail for 4 and 8 weeks. Stiffening of the fibrils and the matrix indicated that abnormal loading conditions affects the biomechanical properties of the nucleus pulposus, further influencing the mechanical integrity of the disc. Alterations observed in histological images and gene expression also suggest that immobilization influences cell response. This study provides a better understanding of disc degeneration pathogenesis after immobilization or reduced motion of the spine. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This project is funded by the Basic Research Program of Jiangsu Province (BK20180196), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (18KJB180024), Jiangsu 5
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Complex loading affects intervertebral disc mechanics and biology. Osteoarthr. Cartil. 19, 1011–1018. Yurube, T., Nishida, K., Suzuki, T., Kaneyama, S., Zhang, Z., Kakutani, K., Maeno, K., Takada, T., Fujii, M., Kurosaka, M., Doita, M., 2010. Matrix metalloproteinase (MMP)-3 gene up-regulation in a rat tail compression loading-induced disc degeneration model. J. Orthop. Res. 28, 1026–1032. Zhao, C., Wang, L., Jiang, L., Dai, L., 2007. The cell biology of intervertebral disc aging and degeneration. Ageing Res. Rev. 6, 247–261.
Neurosurg. Spine 21, 608–613. Stokes, I.A., Iatridis, J.C., 2004. Mechanical conditions that accelerate intervertebral disc degeneration: overload versus immobilization. Spine 29, 2724–2732. Stolz, M., Raiteri, R., Daniels, A.U., Vanlandingham, M.R., Baschong, W., Aebi, U., 2004. Dynamic elastic modulus of porcine articular cartilage determined at two different levels of tissue organization by indentation-type atomic force microscopy. Biophys. J. 86, 3269–3283. Walter, B., Korecki, C., Purmessur, D., Roughley, P., Michalek, A., Iatridis, J., 2011.
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Nano and micro biomechanical analyses of the nucleus pulposus after in situ immobilization in rats
T
Ting Lianga,b,1, Dong-Yan Zhongc,1, Yan-Jun Chea,b, Xi Chena,b, Jiang-Bo Guoa,b, Hui-Lin Yanga,b, Zong-Ping Luoa,b,* a
Orthopedic Institute, Medical College, Soochow University, Suzhou, Jiangsu 215006, PR China Department of Orthopedics, the First Affiliated Hospital of Soochow University, Suzhou, Jiangsu 215006, PR China c Suzhou Gusu District Maternal and Child Health Center, Suzhou, Jiangsu, PR China b
A R T I C LE I N FO
A B S T R A C T
Keywords: Intervertebral disc Nucleus pulposus Immobilization Biomechanics Atomic force microscopy
Immobilization can lead to intervertebral disc degeneration. The biomechanical characteristics of such discs have not so far been investigated at the micro- or nanoscale, the level at which cells sense and respond to the surrounding environment. This study aimed to characterize changes in the elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale and correlate this with micro-biomechanical properties of the nucleus pulposus after immobilization, in addition to observation of tissue histology and its gene expressions. An immobilization system was used on the rat tail with an external fixation device. The elastic modulus was measured using both nano and micro probes for atomic force microscopy after 4 and 8 weeks of immobilization. Histology of the tissue was observed following hematoxylin and eosin staining. Gene expression in the annulus fibrosus tissue was quantified using real-time reverse transcription-polymerase chain reaction. The elastic modulus of the collagen fibrils in the nucleus pulposus at the nanoscale increased from 74.07 ± 17.06 MPa in the control to 90.06 ± 25.51 MPa after 8 weeks (P = 0.007), and from 33.51 ± 9.33 kPa to 43.18 ± 12.08 kPa at the microscale (P = 0.002). After immobilization for 8 weeks, a greater number of cells were observed by histology to be aggregated within the nucleus pulposus. Collagen II (P = 0.007) and aggrecan (P = 0.003) gene expression were downregulated whereas collagen I (P = 0.002), MMP-3 (P < 0.001), MMP-13 (P < 0.001) and ADAMTs-4 (P < 0.001) were upregulated. Immobilization not only influenced individual collagen fibrils at the nanoscale, but also altered the micro-biomechanics and cell response in the nucleus pulposus. These results suggest that significant changes occur in intervertebral discs at both scales after immobilization, a situation about which clinicians should be aware when immobilization has to be used clinically.
1. Introduction Intervertebral disc degeneration is a major contributing factor in lower back pain. Abnormal mechanical environments, including overloading and immobilization, is assumed to partly determine the rate of disc degeneration (Stokes and Iatridis, 2004). The immobilization of vertebrae is common in clinical applications in order to restore spinal stability under conditions of cervical spondylosis, spinal dislocation or fractures. However, complications may arise after immobilization such as heterotopic ossification and osteitis condensans (Baum et al., 1989; Hossain et al., 2004). The effect of immobilization on intervertebral disc degeneration remains unclear, previous studies having suggested that immobilization induces changes to discs similar to those caused by
static compression (Iatridis et al., 1999), although others suggest greater alterations after immobilization (Hirata et al., 2014). Thus, detailed pathogenesis of disc degeneration after immobilization still requires additional study. The intervertebral disc is a cushion-like structure between the bony vertebrae comprising three major tissue regions: the fibrous lamellae of the annulus fibrosus, the gelatinous nucleus pulposus and the cartilage endplates sandwiching the annulus fibrosus and nucleus pulposus (Aladin et al., 2010; Ding et al., 2013). The discs function by distributing large loads acting on the spine and providing flexibility, the nucleus pulposus bearing the majority of the pressure. The occurrence of disc degeneration also starts from the nucleus pulposus (Chris et al., 2016; Ding et al., 2013; Haefeli et al., 2006). Type II collagen in the
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Corresponding author at: Orthopaedic Institute, Soochow University, 708 Renmin Rd, Suzhou, Jiangsu, 215007, PR China. E-mail address: [email protected] (Z.-P. Luo). 1 Ting Liang and Dong-Yan Zhong contributed equally to this work. https://doi.org/10.1016/j.micron.2020.102824 Received 12 July 2019; Received in revised form 20 November 2019; Accepted 3 January 2020 Available online 07 January 2020 0968-4328/ © 2020 Published by Elsevier Ltd.
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2.2. Histological analysis
nucleus pulposus accounts for 10%–20% of the dry weight of human lumbar discs and constitutes the major part of the fibrous collagen network providing a scaffold structure to the hydrated proteoglycan (Adams and Roughley, 2006). Normal day-to-day mechanical loading of the discs maintains cell homeostasis by continuous secretion of matrix. Conversely, hyper- or hypo-physiological loading may be harmful to the nucleus pulposus (Chan et al., 2011). Abnormal loading may affect not only the expression of matrix but also the mechanical properties of the collagen fibers (Adams et al., 2000; Iatridis and Gwynn, 2004; Ching et al., 2003; Stemper et al., 2014). Even changes at the nanoscale structure of the individual collagen fibrils in the nucleus pulposus might result in dispersion of proteoglycans, influencing overall mechanical integrity of the disc (Aladin et al., 2010). Immobilization of long segments of the spine results in changes in the mechanical environment of the nucleus pulposus compared with normal physiological status. However, the precise biomechanical properties of the nucleus pulposus after immobilization remain unclear, particularly at the micro or nanoscale. In the present study, we aimed to ascertain the influence of immobilization on the biomechanical properties of the nucleus pulposus at the micro and nanoscale in the rat tail using atomic force microscopy (AFM), with observation of tissue histology and quantification of gene expression. The results offer critical data and describe the effect on the biomechanics of immobilization on intervertebral disc degeneration.
The target discs (C7-8) were harvested and fixed in 10 % neutral buffered formalin (Shanghai Yuanye Bio-Technology Co. Ltd, Shanghai, China) for 24 h. Following decalcification in buffered 10 % EDTA, pH 7.4 (Biosharp, Hefei, China) for 4 weeks, the samples were dehydrated, embedded in paraffin, sectioned to a thickness of 5 μm using a microtome (Leica, Heidelberger, Germany) and then stained with hematoxylin and eosin (Beijing Biotopped Science & Technology Co. Ltd, Beijing, China). Stained images were then evaluated using a binocular light microscope (XSP-2CA, Shanghai, China) at a magnification of 100 × . The cells in nucleus pulposus were counted by Multi-Point tool in ImageJ Software in the staining image. In each group, three slices per disc and seven discs were used for the cell number analysis. 2.3. AFM imaging and mechanical testing After immobilization discs were harvested and fresh tissue from C8C9 sliced into 20∼30 μm thick sections using a frozen tissue cryostat (CM3050 S, Leica, Nussloch, Germany). One slice in the center position of a single disc were subjected to AFM testing using an AFM scanner (Dimension ICON, Bruker, USA). For the nanoscale experiments, images and the elastic modulus of fibrils were obtained using a ScanAsyst-Air probe with a radius of curvature of 5 nm and a force constant of 0.4 N/m. The scan area was selected randomly. Since there is the heterogeneity within the nucleus pulposus, some areas exhibit no fibril in the AFM imaging, only the image containing fibrils and the corresponding micro modulus in the same area were involved in this study. Fibril diameters were obtained by line section analysis using NanoScope Analysis Software (Bruker, USA). The modulus of the fibrils was calculated using AFM PeakForce QNM (Quantitative Nano Mechanics) test mode (n = 50 in each group), utilizing the Hertz model as defined in Eq. (1), after calibration of the force constant k and radius of curvature R of the probe. A detailed determination of the modulus is described in a previous study (Stolz et al., 2004).
2. Materials and methods 2.1. Immobilization model Vertebral immobilization was conducted on 21 fully grown 3month-old male Sprague-Dawley rats. The study was approved by the Institutional Animal Care and Use Committee. The animals were randomly divided into 3 groups. The baseline control group had no external fixation device (n = 7). In the other two groups (n = 7 for each group), four caudal vertebrae (C7-C10) were firstly instrumented with a K-wire (50 mm in length and 1.2 mm in diameter, XingYu Medical Instrument Factory, Suzhou, China) in each vertebra. Two bespoke aluminum alloy cuboids (43 mm in length, 4 mm wide and 5.0 g net weight) were then attached to the four K-wires (Fig. 1) on either side of the tail for spatial immobilization. Placement of the fixation device was confirmed by X-ray, as displayed in Fig. 2. In a previous study (Che et al., 2018), it was demonstrated that K-wire placement alone without immobilization did not significantly affect the discs and so a sham group was not included in this study. After immobilization for 4 or 8 weeks, animals were euthanized and the immobilized discs harvested for further analysis.
F=
4 E R δ3 3 (1 − υ2)
2
(1)
where F is the indentation force, E the Young’s modulus, υ is Poisson’s ratio, R is the radius of the indenter and δ is value of the indentation. For microscale experiments, the modulus was obtained using forcevolume mode in 0.15 M PBS (pH = 7.4) in a fluid cell using a borosilicate glass spherical tip with a diameter of 5μm firmly attached to the native V-shaped silicon nitride cantilever, with a spring constant of 0.06 N/m (Bruker, USA). The calculation of elastic modulus was described in detail in a previous study, using the following equation (Stolz
Fig. 1. (A) Schematic of immobilization animal model. The external device four discs by using four K-wires and two parallel aluminum alloy cuboids which produce no compresssion or tension in the disc. (B) The corresponding X-ray confirmation image. 2
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Fig. 2. The representative histological images using hematoxylin/eosin staining of nucleus pulposus in both control and immobilized groups. Compared with the control group, cells were aggregated in the nucleus pulposus of the two immobilized groups. The typical morphology of cells remained unchanged after immobilization.
are displayed in Fig. 4a. Compared with the modulus of fibrils in the nucleus pulposus in the control group (74.07 ± 17.06 MPa), immobilization for 8 weeks caused the fibrils to become significantly stiffer (90.06 ± 25.51 MPa, P = 0.007), while immobilization for 4 weeks did not influence the modulus of the fibrils significantly (77.75 ± 15.43 MPa, P = 0.075). The elastic modulus of the nucleus pulposus at the microscale is presented in Fig. 4b. Immobilization for 8 weeks also significantly modified the modulus (43.18 ± 12.08 kPa, P = 0.002) compared with that of the control group (33.51 ± 9.33 kPa). However, modulus at the microscale after immobilization for 4 weeks did not change significantly (30.75 ± 10.87 kPa). Expression of anabolic genes except collagen I decreased following immobilization as shown in Fig. 5a. Eight weeks of immobilization caused a decrease in collagen II expression of 26.0 % compared with the control group (P = 0.005) and by 16.9 %, after 4 weeks of immobilization (P > 0.05). Additionally, expression of aggrecan was reduced after 4 weeks of immobilization by 24 % (P = 0.018) relative to the control. Eight weeks of immobilization downregulated mRNA levels of aggrecan by 25 % (P = 0.003). Conversely, 8 weeks of immobilization induced greater expression of collagen I by 18 % and 13.5 % in comparison with the control (P = 0.002) and 4 weeks immobilization (P = 0.013), respectively. In terms of catabolic genes in Fig. 5b, both 4 and 8 weeks of immobilization significantly upregulated the expression of MMP3, MMP13 and ADAMTs-4. Four weeks of immobilization increased MMP3 expression by 18 % (P = 0.014), MMP13 by 31 % (P < 0.001) and ADAMTs-4 by 36 % (P < 0.001) compared with the control group. Immobilization for 8 weeks resulted in a more significant increase in mRNA expression levels (MMP3 by 23 %, P < 0.001; MMP13 by 56 %, P < 0.001; ADAMTs-4 by 48 %, P < 0.001 compared with the control group).
et al., 2004; Loparic et al., 2010):
E=
π S (1 − υ2) 2 A
(2)
where S is contact stiffness, A is surface area of the spherical indenter contacting the substrate. 2.4. Real-time polymerase chain reaction (PCR) analysis The Co8-Co9 intervertebral disc in each in-situ immobilization rat was used for the PCR analysis (n = 7 in each group). The target disc was exposed by shearing the muscle and ligaments around the caudal vertebra. Then nucleus pulposus was separated by rotating a surgical knife along the junction of nucleus pulposus and annulus fibrosus. Total RNA of the nucleus pulposus was isolated using TRIzol reagent (Invitrogen, Carlsbad, USA) in accordance with the manufacturer’s instructions. One μg of total RNA was then reverse-transcribed to firststrand complementary DNA (cDNA) using a RevertAid First Strand cDNA Synthesis kit (Thermo fisher Scientific). Real-time PCR was conducted using an iTaq™ Universal SYBR® Green Supermix kit (BioRad, Hercules, CA, USA) on a CFX96™ Real-Time PCR System (BioRad). mRNA expression levels of genes of anabolic (collagen I, collagen II, aggrecan) and catabolic (MMP3, MMP13 and ADAMTs-4) proteins were calculated relative to the housekeeping gene GAPDH (glyceraldehyde-3-phosphate dehydrogenase) using the 2−ΔΔCT method (Pfaffl, 2001). The primer sequences of each gene are described in Table 1. 2.5. Statistical analysis Data from all experiments are presented as means ± standard deviation. Comparisons of differences between groups were analyzed by one-way analysis of variance (ANOVA) and a student’s t-test. P < 0.05 was considered statistically significant. All tests were two-sided and conducted using SPSS v16.0 software (SPSS Inc., Chicago, IL, USA).
4. Discussion In addition to the contribution of genes, abnormal loading conditions also play an important role in intervertebral disc degeneration (Walter et al., 2011). Compared with overloading, the effect of reduced motion on discs has not been comprehensively studied despite it being a commonly-used spinal treatment (Stokes and Iatridis, 2004). The present study firstly reported on the biomechanical properties of the nucleus pulposus over different time points after the immobilization of rat tails, including the elastic modulus of individual fibrils of the nucleus pulposus at the nanoscale and at the microscale. Based on the extracellular matrix condensation observed in histological images after immobilization, the 8 weeks group exhibited disc degeneration according to the grading scale (Masuda et al., 2005). After intervertebral discs are loaded through bearing spinal loads, the nucleus pulposus swells under compression which transfers load to
3. Results Typical histological images of the nucleus pulposus are shown in Fig. 2. Compared with the control group, cells were aggregated in the nucleus pulposus of the two immobilized groups, although the typical morphology of cells remained unchanged after immobilization. The numbers of cells in the nucleus, ranging from 1284 ± 41–1321 ± 58 also were not significant different among the three groups (P = 0.54). Typical AFM images at the nanoscale are presented in Fig. 3. There was no significant difference in the arrangement of the collagen network or diameter of fibrils between the control and immobilized groups (P > 0.05). The elastic moduli of the individual collagen fibrils at the nanoscale 3
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Fig. 3. The typical atomic force microscopy scanning images of collagen fibrils in nucleus pulposus in both control and immobilized groups. The fibrils or the collagen network exhibited no significant difference between control and immobilized groups.
Fig. 4. The dynamic elastic modulus at both the (A) nano and (B) micro scales of nucleus pulposus measured by atomic force microscopy, where the nanoscale represented the modulus of individual fibrils. Compared with the control group, immobilization for 8 weeks caused higher modulus at both nano and micro scales.
the annulus fibrosus. The ealstic modulus means the the ability of resisting elastic deformation. The higher modulus results in higher stress under the same deformation. When the modulus of the nucleus pulposus changes, the stress distribution within the annulus is altered accordingly, and thus those of the entire disc as well. Normal external loading is essential for the sustained transmission of nutrients, load bearing capacity and cell response within the discs. After immobilization of the spine, the abnormal mechanical environment may induce disc degeneration, including mechanical alteration of the nucleus pulposus and annulus fibrosus. However, the detailed pathogenesis
remains unclear. The rat tail model has been widely used to simulate cervical or lumbar disc degeneration (Chris et al., 2016; Ching et al., 2003; Court et al., 2001; Iatridis et al., 1999; Yurube et al., 2010). In this study, the loading device was modified to simulate immobilization. The mechanical properties of the tissue of the nucleus pulposus changes after degeneration (Iatridis et al., 1997; Johannessen and Elliott, 2005). A huge nonuniformity of disc surface was also revealed after degeneration (Byvaltsev et al., 2012). However, detailed alterations within the nucleus pulposus remain ambiguous, especially at the micro and nanoscale. Among the factors affecting the mechanical
Fig. 5. The (A) anabolic and (B) catabolic genes expression in nucleus pulposus in both control and immobilized groups. mRNA levels of nucleus pulposus were normalized to endogenous control (GAPDH) and internal controls. 4
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properties of the nucleus pulposus, collagen fibrils play an important role, a network of collagen fibrils providing the mechanical strength to resist the pressure from swelling (Aladin et al., 2010). In this study, the individual fibrils in the nucleus pulposus stiffened after immobilization for 8 weeks. Individual collagen fibrils were affected by several factors such as matrix metalloproteinases and proteoglycans (Nakamura et al., 2000; Zhao et al., 2007). A decrease in aggrecan and increase in catabolic gene expression observed in this study might represent evidence that clarified the relationship between fibril stiffness and genetic factors. Additionally, the modulus at the microscale also increased in the 8 weeks group. Thus correlation of the elastic modulus between the nano and microscales in this study might provide additional information about how the stiffness of individual fibrils influences the entire fibril network, further influencing the mechanical integrity of the nucleus pulposus such as the stiffening at the macroscale after degeneration (Aladin et al., 2010; Neidlinger-Wilke et al., 2014). Although a number of general studies of changes in mRNA levels after disc degeneration have been published, the conclusions are still debated about which loading conditions induce degeneration, for example bending or compression, their magnitude and duration (Liang et al., 2019; MacLean et al., 2004, 2003; Yurube et al., 2010). For instance, a longer duration of loading induces greater alteration in the expression of certain genes, but fewer changes in others, possibly due to an adaptive remodeling response. mRNA expression levels reflect the cell response to the environment. In this study, both individual fibrils at the nanoscale and the matrix at the microscale changed after immobilization for 8 weeks, inducing a change in the mechanical environment surrounding the cells, the expression of genes also changing in the 8 week group. The combination of external immobilization and the stiffening matrix constituted abnormal loading conditions around the cells. However, the results after 4 weeks indicated discordance between the mechanical properties and gene expression, in that expression of the catabolic genes and aggrecan changed while the elastic modulus remained the same. These results possibly indicate that the response of cells within the disc occurs prior to that of the matrix response to the loading due to immobilization. A limitation of this study is the difference in cell type and loading conditions in the nucleus pulposus of the rat tail compared with the human spine. However, the animal model is useful for exploration of the reciprocal interactions between mechanical loading and disc homeostasis (Neidlinger-Wilke et al., 2014).
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5. Conclusions In this study, the mechanical properties of both individual collagen fibrils at the nanoscale and the matrix at the microscale in the nucleus pulposus were investigated using AFM after immobilization of the rat tail for 4 and 8 weeks. Stiffening of the fibrils and the matrix indicated that abnormal loading conditions affects the biomechanical properties of the nucleus pulposus, further influencing the mechanical integrity of the disc. Alterations observed in histological images and gene expression also suggest that immobilization influences cell response. This study provides a better understanding of disc degeneration pathogenesis after immobilization or reduced motion of the spine. Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This project is funded by the Basic Research Program of Jiangsu Province (BK20180196), Natural Science Foundation of the Jiangsu Higher Education Institutions of China (18KJB180024), Jiangsu 5
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