ISSLS Prize Winner: A Detailed Examination of the Elastic Network Leads to a New Understanding of Annulus Fibrosus Organization.

SPINE Volume 40, Number 15, pp 1149-1157 ©2015, Wolters Kluwer Health, Inc. All rights reserved.

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ISSLS Prize Winner: A Detailed Examination of the Elastic Network Leads to a New Understanding of Annulus Fibrosus Organization Jing Yu, PhD,* Meredith L. Schollum, PhD,† Kelly R. Wade, PhD,† Neil D. Broom, PhD,† and Jill P.G. Urban, PhD*

Study Design. Investigation of the elastic network in disc annulus and its function. Objective. To investigate the involvement of the elastic network in the structural interconnectivity of the annulus and to examine its possible mechanical role. Summary of Background Data. The lamellae of the disc are now known to consist of bundles of collagen fibers organized into compartments. There is strong interconnectivity between adjacent compartments and between adjacent lamellae, possibly aided by a translamellar bridging network, containing blood vessels. An elastic network exists across the disc annulus and is particularly dense between the lamellae, and forms crossing bridges within the lamellae. Methods. Blocks of annulus taken from bovine caudal discs were studied in either their unloaded or radially stretched state then fixed and sectioned, and their structure analyzed optically using immunohistology. Results. An elastic network enclosed the collagen compartments, connecting the compartments with each other and with the elastic network of adjacent lamellae, formed an integrated network across the annulus, linking it together. Stretching experiments demonstrated the mechanical interconnectivities of the elastic fibers and the collagen compartments. Conclusion. The annulus can be viewed as a modular structure organized into compartments of collagen bundles enclosed by an From the *Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK; and †Department of Chemical and Materials Engineering, University of Auckland, Auckland, New Zealand. Acknowledgment date: October 14, 2014. First revision date: February 2, 2015. Acceptance date: March 6, 2015. The manuscript submitted does not contain information about medical device(s)/drug(s). Supported by the British Scoliosis Foundation and the European Community’s Seventh Framework Programme (FP7, 2007–2013) under Grant Agreement No. HEALTH-F2–2008–201626. Relevant financial activities outside the submitted work: employment, travel/ accommodations/meeting expenses. Address correspondence and reprint requests to Jing Yu, PhD, Department of Physiology, Anatomy and Genetics, Sherrington Building, University of Oxford, Parks Road, Oxford OX1 3PT, UK; E-mail: [email protected]. DOI: 10.1097/BRS.0000000000000943 Spine

elastic sheath. The elastic network of these sheaths is interconnected mechanically across the entire annulus. This organization is also seen in the modular structure of tendon and muscle. The results provide a new understanding annulus structure and its interconnectivity, and contribute to fundamental structural information relevant to disc tissue engineering and mechanical modeling. Key words: elastic fibers, microfibrils, disc annulus integrity. Level of Evidence: N/A Spine 2015;40:1149–1157

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he annulus fibrosus (AF) of the intervertebral disc plays a major biomechanical role, anchoring the disc to the vertebral bodies and endowing the disc with strength and flexibility. Its biomechanical behavior is governed by the organization of its macromolecular constituents and classically, the AF is described as consisting mainly of bundles of aligned collagen fibers, organized into concentric lamellae. These fiber bundles run obliquely to the central axis of the disc with the angle alternating from one lamella to the next to form a “cross-woven” structure.1–3 A new understanding of annular microanatomy4–6 has arisen through careful dissection and examination of annulus samples, selectively loaded both radially and along the primary fiber bundle direction, using DIC microscopy. It is now apparent that the collagen bundles are organized into compartments within each lamella and there is a complex pattern of interconnectivity both within each lamella and between adjacent lamellae. Furthermore, translamellar bridging networks (TLBN) link many lamellae5; these TLBN could play a role in maintaining annulus integrity. The AF also contains an extensive and well-organized elastic network incorporating both elastin and microfibrils.7–9 This network is particularly dense between adjacent lamellae and in bridges crossing from one lamella to the next. The role of this network is unclear but a study which used elastase to digest away the elastin network suggested that it functions mechanically particularly in shear loading.10 Here we aim to gain an understanding of the 3-dimensional organization of the elastic network and of its role in the structural interconnectivity of the annulus by applying a radial stretching technique.4–6 www.spinejournal.com

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New Understanding of Annulus Fibrosus Organization • Yu et al

Figure 1. Schematic view of the preparation of annulus sections (Adapted with permission from Pezowicz et al.4). A, Annulus block dissected obliquely, non-loaded control; B, Obliquely dissected annulus block subjected to radially stretching force (F); C, Nonloaded oblique annulus section, showing collagen bundles running parallel at the inplane lamellae (IP), whereas perpendicular to the cutting plane and also organized into different compartments (oval shapes) at the cross-sectioned lamellae (CS); D, A stretched oblique annulus section, showing deformed collagen structure at the IP and CS.

MATERIALS AND METHODS 5 adult bovine tails (18–24 mo) were collected fresh from a local abattoir and discs from the highest level dissected out. Two annular blocks were obliquely cut out from each fresh bovine discs (Figure 1). One block of each pair was immediately snap frozen and stored at –80°C utill required as the non-loaded control (Figure 1A). To understand the role of the elastic network in annulus interconnectivity, we examined how the elastic network of the other block deformed under radial stretch. This second block (Figure 1B) was radially stretched in a tensile device,4 then immediately fixed in 10% formalin for at least 8 hours to preserve its stretched state; it was then snap frozen and stored at –80°C. Tissue sections, 20-μm thick, were sectioned from the frozen annulus blocks using a cryomicrotome and mounted on polylysine-coated microscope slides (VWR International Ltd, UK) and immediately immunostained; details of the stretching and the sectioning planes are shown schematically in Figure 1. Figure 1 (C,D) schematically showed nonloaded and stretched oblique annulus sections, respectively.

sections were first incubated with antifibrillin-1 antibody, revealed by antimouse IgG conjugated with Cy3, and then with antielastin antibody, revealed by antirabbit IgG conjugating with Dylight 488.

Microscopy The dual immunostained elastic and collagen networks were examined using fluorescence microscopy incorporating a conventional light and a polarized filter. Separate images of the dual-stained elastin and microfibrils networks and of collagen networks at the same tissue site were taken sequentially by manually switching between filters without moving the slides. Images of the nonloaded (i.e., control) hydrated sections were

TABLE 1. Pretreatment Methods Used Nonfixed Sections Hyaluronidase 3 hr at 37 oC

Fixed Sections Hyaluronidase overnight at 37°C, then collagenase 24 hr at 37oC

Immunohistology Elastic fibers consist of an elastin central core surrounded by microfibrils with fibrillin-1 as the main component.11 Although elastin and microfibrils are colocalized in most elastic tissues, microfibrils can also function as elastic fibers without elastin, for example in the ciliary zonules of the eye.11 Elastin and microfibrils are not fully colocalized in the inner annulus and nucleus of the disc.7,12 The term elastic network, includes both the elastin fiber network (immunostained by the elastin antibody) and the microfibrillar network (immunostained by the fibrillin-1 antibody), visualized after dual immunostaining.12 Cross-linking arising from formalin fixation tends to reduce immunostaining of both elastin and fibrillin. Therefore, all sections were pretreated with hyaluronidase (Sigma H6254) and collagenase (Sigma C5138) to improve their immunostaining (Table 1). Dual immunostaining of fibrillin-1 and elastin: This procedure was carried out as described previously.7,12 Negative controls used PBS instead of primary antibodies as described previously.12 Table 2 lists the antibodies used. Briefly, the 1150

TABLE 2. List of Antibodies Used Primary Antibody

Secondary Antibody

Fibrillin-1

Mouse antibovine fibrillin-1; Genway Donkey antimouse IgG Biotech Inc. San conjugated with Cy3 Diego, USA; Cat.no: dye from Stratech 20–787–275143; Scientific Ltd, UK; Cat. Dilution: 1:50 in Tris bufno: 715–165–151; fer (50 mM Tris-HCl Dilution: 1:100 containing 10 mM calcium acetate)

Elastin

Rabbit polyclonal, antihuman alpha elastin (cross-reacts with bovine); MorphoSys UK Ltd, Cat. no: 4060–1060, Batch No: 20092751; Dilution: 1:50

Donkey antirabbit IgG conjugated with Dylight 488, from Stratech Scientific Ltd, UK; Cat. No: 711–545–152; Dilution: 1:100

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improve contrast. All merged images were obtained from the same tissue site.

RESULTS Collagen Bundle Compartments in the Nonstretched Samples

Figure 2. Collagen organization viewed under differential interference contrast microscopy (DIC), showing collagen bundles clearly divided into compartments in the cross-sectioned lamellae (CS) and also in the nearly in-plane lamella (nIP). Red arrow indicates a collagen compartmental boundary dividing the entire CS. Yellow arrow indicates a collagen compartmental boundary partially dividing the CS. White arrows indicate the collagen compartmental boundaries in the nIP.

obtained using differential interference contrast microscopy (DIC).

Image Processing and Analysis The microfibrils which were labeled with the yellow-orange Cy3 dye were transformed to red using Adobe Photoshop to

Structurally the annulus is characterized by a repeating oblique and counter-oblique arrangement of the collagen bundles within adjacent lamellae. Thus, an oblique interlamellar section (Figure 1C) will contain collagen bundles that seem, alternatingly, parallel in the in-plane (IP), or near inplane (nIP) lamellae, and perpendicular to the cutting plane in near cross-section (CS) lamellae as shown schematically in Figure 1. The structures imaged in these CS, IP, and nIP sections form the basis of this study. Figure 2, obtained from a fully hydrated nonloaded section and imaged using DIC optical microscopy, illustrates this difference in structure when viewing IP/CS/nIP sections in the outermost region of the annulus. In cross-sectioned lamellae, the collagen bundles are clearly shown to be subdivided into compartments as previously reported.5,6 The pattern and size of these compartments seems irregular (Figure 2); compartments occasionally divide the entire lamella (red arrow) or, more frequently, an incomplete division is seen (yellow arrow). This same compartmentalization can be seen in the near in-plane (nIP) lamella (white arrows) but not in the in-plane (IP) lamella and indicates that the fibrous compartments possess some minor angular variation within the overall oblique orientation of the primary collagen bundle.

Figure 3. Colocalized elastin (green) and microfibrils (red) densely organized around the collagen bundles of the annulus, forming elastic bridges crossing the CS lamellae. A,B, Dual immunostained elastin and microfibrils respectively; C, Collagen organization under a conventional microscope with a phase contrast filter, same tissue site as (A) and (B); D, Merged image from (A) and (B), showing the colocalization of elastin and microfibrils; E, Merged image from (A) to (C), showing elastic network enveloping the collagen bundle compartments; F, Dual immunostained elastic network at a different site of the annulus. Yellow arrows indicate the interlamellar boundaries; White arrows indicate the elastic crossing bridges encircling the collagen compartments. CS, Cross sectioned lamella; IP, in-plane lamella. Spine


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Figure 4. A macroscale view of the immunostained microfibrils (red) showing the elastic network encircling collagen bundle compartments within each lamella from the outer annulus to the inner annulus, integrating entire lamellae and collagen bundle compartments together across the annulus. CS, cross sectioned lamellae; nIP, nearly in-plane lamellae. White arrows indicate the boundaries of collagen bundle compartment boundaries within lamellae. Yellow arrows indicate the interlamellae boundaries.

Elastic Network in the Nonloaded Samples Details of the Dense Elastic Network Enveloping Collagen Bundle Compartments No fluorescence was observed in the negative controls with the filters used (not shown). In Figure 3, dual immuno-stained elastin and fibrillin-1 show that elastin fibers and microfibrils are well colocalized (Figure 3D,F) and are aligned parallel to collagen fibers within the lamellae of the outer annulus (IP in Figure 3A–E). There is also a dense network of elastic fibers between adjacent lamellae (yellow arrows in Figure 3). In addition, a dense elastic network can be seen traversing the CS lamella (white arrows in Figure 3). These observations are consistent with the elastic crossing bridges (E-bridge) previously described.12,13 From the structures imaged in Figures 2 and 3 and previously published work4–6,14 it seems that these crossing bridges or bridging elements (BE)5,14 are in fact the boundaries of the compartments containing the primary collagen bundles. These compartment boundaries contain a dense elastic

network which is apparent both in the in-plane (IP) and crosssectional (CS) lamellae (white arrows in Figure 3); they thus encircle collagen bundles both in those lamellae cut in crosssection (CS) and also in those sectioned longitudinally in IP or nIP. Thus, a dense network containing elastic fibers seems to envelop the collagen bundles. A more extended view from the outer annulus inwards is shown in Figure 4 and provides further evidence that the elastic network encircles the collagen bundles within each lamella. This image also shows the continuous elastic network which exists across the annulus connecting collagen bundles within lamellae, and connecting lamellae to each other, holding the annulus structure together. Detailed images of the elastic network at the collagen compartmental boundaries (yellow arrows in Figure 5A,B) show that the fibrils from the elastic network encircling the collagen bundles seemed to penetrate into the collagen bundles themselves (white arrows, Figure 5 B,C). In addition, the elastin and microfibrils were not always completely colocalized and a separate elastin network was clearly visible in some regions.

Figure 5. Detailed elastin (green) and microfibrils (red) at the collagen compartment boundaries (indicated with yellow arrows) in the in-plane lamellae (IP) lamellae. A, A dense elastic bridging network (more than 100 μm in thickness) crossing the IP lamella; B, Elastic network at the collagen bundle compartment boundary penetrated into the collagen bundle themselves (indicated with white arrows). C, Higher magnification of the elastic bridge further highlighting the elastic network at the collagen compartment boundary integrating into with the collagen bundle themselves (white arrows).

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Figure 6. Details of dual immunostained elastic translamellar bridging network (E-TLBN). A, Dual immunostained E-TLBN (A1) colocalized with the collagen TLBN (A2), which is associated with higher cell density (A3,A4); A4, Merged image of (A1–A3); B, Microfibrillar network reveals that the E-TLBN links 8 lamellae together continually through the interlamellae boundaries (yellow arrows) and thicker elastic bridges (white arrows in the lamella). Blue arrow indicates a large blood vessel; C, Serial images at different depths (20 μm between them) from the region marked with a yellow circle in (B), showing the E-bridge (indicated with the white arrow) in lamella numbered 2 (B) is associated with a much thicker E-bridge at the further depth (indicated with a green arrow in C3).

Details of the Elastic Network Associated with the Translamellar Bridging Network (TLBN) The TLBN has been previously shown to span multiple lamellae of the disc annulus.5,14 Dual immunostaining of elastin and fibrillin-1 similarly reveals an extensive elastic translamellar bridging network (E-TLBN) (see Figures 6). The images shown in Figure 6A comparing the dual stained elastic network (Figure 6A1) with the collagen network (Figure 6A2) viewed under polarized light, indicates that the E-TLBN colocalizes with the TLBN. In addition, this E-TLBN is associated with a high cell density (blue DAPI staining (Figure 6A3, A4). Spine

The image shown in Figure 6B revealed an E-TLBN spanning a total of 8 lamellae (marked with numbers from 1 to 8). Images from serial sections at different depths (Figure 6C) indicated that the E-TLBN is a continuous network that links the interlamellar boundaries (yellow arrows in Figure 6B) with the elastic bridges (white arrows in Figure 6B). The E-TLBN thus seems to be associated with collagen bundle boundaries at both the inter- and intralamellar boundaries (highlighted with yellow and white arrows respectively in Figure 6B). Furthermore the presence of blood vessels within the E-TLBN system was a common finding in all of the samples examined in this study (blue arrows in Figure 6B,C). www.spinejournal.com

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Figure 7. Response of the elastic and collagen network at the intra- and interlamellae to radial stretching. (A) and (B) are the dual immunostained elastic network (A1, B1), collagen organization (A2, B2), and merged image (A3, B3) from (A1) with (A2) and (B1) with (B2), respectively, showing collagen compartments (yellow arrows in A2,A3) enveloped by elastic network (yellow arrows in A1,A3) with the CS lamella significantly elongated. In addition, the junctions (white arrows) of collagen compartments in the CS lamellae with the IP lamellae seem radially draw out, and also both elastic fibers (pink arrow in B1) and collagen (green arrow in B2) in IP lamellae were disorientated (B).

Response of Elastic Network to Radial Stretching Stretching the annulus block in the radial direction revealed more elastic connections between lamellae, between collagen bundles within lamella, and between compartments within the collagen bundles. Response of the Elastic Network at the Intra- and Interlamellae Examination of the radially stretched samples revealed that the compartments of the collagen bundles at the intralamella, enveloped by a network containing elastic fibers and imaged in crosssection, were substantially elongated radially (white arrows in Figure 7A). The integration of the elastic network with the neighboring in-plane lamellae produced the appearance of a tensioned junction in which the in-plane collagen arrays were “drawn out radially” (yellow arrows in Figure 7A,B). The elastic fibers in the in-plane lamella were also reorientated from their original in-plane alignment (see Figure 3) as a consequence of the applied radial stretching (see pink arrow in Figure 7B1) as were the collagen fibers (green arrow, Figure 7B2). Elastic Network in the In-plane (IP) Lamellae After Radial Stretch Figure 8 shows more detailed images of the response of the elastic fibers in the IP lamellae after stretching. As noted above, in the nonloaded samples the elastic fibers, when viewed in the IP lamellae, were aligned in the same direction 1154

as the collagen bundles (Figure 3). Radial stretching resulted in some elastic fibers being realigned approximately perpendicular to the direction of the collagen bundle (white arrows in Figure 8A). Further, while the elastic network at the interlamellar boundary is diffuse (dotted yellow line in Figure 8A1), the interface showing the collagen network under polarized light is sharply delineated (dotted yellow line in Figure 8A2). The elastic network at the interlamellar boundary was often observed to penetrate radially, deep into the in-plane lamellae in the stretched samples (green arrows in Figure 8A). As noted above, a dense elastic bridging network could be observed crossing the entire IP lamellae (Figures 5A, 6B highlighted with white arrows) in the nonloaded samples. This elastic bridging network (yellow arrows in Figure 8B) was stretched radially in the IP lamellae of loaded samples, apparently drawn out by the network connecting the adjacent CS lamella (white arrows in Figure 8B).

DISCUSSION Here we show that the annulus should be viewed as a modular structure organized into compartments of collagen bundles (Figure 2), enclosed by a sheath formed, in part at least, from a dense network of elastic fibers (Figure 3). These sheaths link adjacent compartments within and between lamellae, and thus form an interconnected, mechanically-linked network across the entire annulus (Figure 4), apparently providing the annulus with structural integrity.


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Figure 8. Detailed response of elastic network at the in-plane (IP) lamella after radial stretching. (A) and (B) are immunostained microfibrils (A1,B1), collagen network (A2,B2), and merged images (A3, B3) from (A1) with (A2) and (B1) with (B2), respectively, showing microfibrils (white arrows in A) realigned perpendicularly to the collagen bundles in the IP lamella, diffused from the interlamellar boundary (highlighted with the yellow dashed lines in A) into the IP lamella (see green arrows in A1,A3), whereas, collagen organization seems sharply delineated (see A2). In addition, elastic bridge (indicated with yellow arrows in B1, B3) in the IP lamella seems pulling out by the network connecting (indicated with white arrows in B1, B3) the adjacent CS lamella.

Through sectioning the disc annulus both in-plane and in cross-section (Figure 1),4,5,14,15 the hierarchical organization of the annulus can be visualized. Figure 2 shows the boundaries between collagen bundles within the compartments, between compartments and between lamellae (Figure 2) visualized by DIC microscopy while Figure 3 shows that these boundaries which, immunostained for elastin and for fibrillin, contain an elastic fiber network. This elastic fiber network, as reported previously,12,13 tends to be concentrated between lamellae (Figures 3 and 4) and is visible both in cross-section and inplane (Figure 3 and 4, white arrows) where it encircles the collagen bundles giving rise to what have been referred to earlier as BE.4,5,14 The collagen bundles thus seem to be encased in a “sheath” formed from an elastic network (Figures 2–4) and possibly also other matrix macromolecules such as collagen VI and versican.16 This same elastic network penetrates into the lamellae (Figures 5) and is continuous with the network at the interlamellar boundaries (details not shown), thus forming an integrated structure across the annulus (Figure 4). The radial stretching technique4 demonstrates that this structure is mechanically linked within and between lamellae. Here compartment bundles, seen in cross-section, elongate in the direction of stretch (Figure 7) as described previously.4,15 The elastic network at the boundary extends, distorting the adjacent in-plane lamella (yellow arrows in Figure 7A,B) and exposing fibers which penetrated radially from the interlamellar boundary into the compartments containing the collagen bundles (white arrows, Figure 8A). Although there is Spine

a distinct interface between the collagen bundles in adjacent in-plane and cross-sectioned lamellae (Figure 8A2) the elastin network is diffuse (Figure 8A3); we suggest that the diffuse elastic network at the interlamellar junctions (Figure 8A) holds the collagen bundles together and speculate that it serves to allow relative movement between lamellae when the disc undergoes deformation under load. This elastic network could also provide a restoring force when the load is removed as it seems mechanically linked to the collagen networks (Figure 7). This study also shows that the translamellar bridging network (TLBN) identified previously,5,14,17,18 contains a dense elastic network (Figure 7). Figure 6B shows the path of this elastic TLBN across 8 successive lamellae and demonstrates that it is linked to the network between lamellae and to the sheaths surrounding collagen bundles. Rather than a separate structure, the TLBN seems to be a region where the elastic network of the sheaths and interlamellar network is denser than in other regions of the network. Blood vessels (Figure 6A) were present only in association with this TLBN as reported by others17; it has been argued that the elastic network of the TLBN is a consequence of vascular regression.17 However their organization and structure (Figure 6) seems an unlikely path for a blood vessel to follow as does its extended 3-dimensional structure described previously.5,14 It seems more plausible that the relationship between blood vessels and the TLBN shown in this study (Figure 6) and by others17 arises because the TLBN provides a more appropriate www.spinejournal.com

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BASIC SCIENCE environment for blood vessel penetration than other regions of the annulus. It might also provide an appropriate environment for cellular function, as the cell density in this region is far greater than in other areas of the annulus (Figure 6A3,A4). This model of annulus structure could also help understand delamination—the circumferential separation of the lamella—a major step in the process of annulus failure and disc degeneration19,20 and which seems to induce degradative and inflammatory changes.21 Delamination could be initiated by inappropriate loading as shown by modeling and experiment22,23 or through degradation of the interconnecting elastic network during the degenerative process.24,25 Strategies for repair and regeneration of the annulus whether biological or mechanical,26 and development of mechanical models of annulus behavior need to consider the new understanding of annulus interconnectivity.

CONCLUSIONS The results of this study demonstrate that the collagen bundles which form the lamellae exist in compartments surrounded by a sheath consisting of a network of elastic fibers and possibly of other extracellular matrix components. This type of organization is also seen in other soft tissues of the musculoskeletal system such as tendon, where collagen fibers are organized into fascicles surrounded by a connective tissue sheath, the endotenon, containing an elastic network,27,28 as well as in muscle where fascicles, formed from bundles of myofibers, are enclosed in an endomysium.29 The role of these sheaths in both these tissue systems seems to be mechanical,30 probably maintaining tissue integrity under loading, unloading and load reversal. In view of its integration with the interlamellar space we suggest a similar role for the “sheath” surrounding the collagen bundles in the intervertebral disc annulus. Further, the structural distortion seen in the stretched samples suggests that the elastic network provides a mechanical linkage across the annulus and is therefore functionally important.

➢ Key Points The lamellae of the annulus fibrosus are formed from alternating layers of oblique and counteroblique collagen bundles. The collagen bundles of each lamella are organized into compartments and there is strong interconnectivity between the compartments within a lamella and between adjacent lamella. An elastic network surrounds the compartments and forms a mechanically interlinked network across the annulus, possibly maintaining its structural integrity. Here we show the disc annulus is a modular structure consisting of fiber bundles enclosed by mechanically linked sheaths, as seen also in other muscular-skeletal tissues such as tendon and muscle. 1156


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Bovine and degenerated human annulus fibrosus: a microstructural and micromechanical comparison.

Theory of MRI contrast in the annulus fibrosus of the intervertebral disc.

Treatment of the degenerated intervertebral disc; closure, repair and regeneration of the annulus fibrosus.

Pancreatic cancer stroma: understanding biology leads to new therapeutic strategies.

Enhancing tissue repair in annulus fibrosus defects of the intervertebral disc: analysis of a bio-integrative annulus implant in an in-vivo ovine model.