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Aging of the elastic and collagen fibers in the human cervical interspinous ligaments

Aging of the elastic and collagen fibers in the human cervical interspinous ligaments
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   The Spine Journal 2 (2002) 57–62 1529-9430/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved.PII: S1529-9430(01)00167-X  Aging of the elastic and collagen fibers in the human cervicalinterspinous ligaments  Erika M.K.P. Barros   a  , Consuelo J. Rodrigues   b,  *, Nilson R. Rodrigues   a  , Reginaldo P. Oliveira   a  , Tarcisio E.P. Barros   a  , Aldo J. Rodrigues, Jr   b   a   Institute of Orthopedics and Traumatology, University of Sao Paulo, São Paulo, Brazil   b   Laboratory of Surgical Anatomy, Faculty of Medicine, University of Sao Paulo, São Paulo, Brazil  Received 27 March 2001; accepted 13 October 2001  Abstract  Background context:  The ligaments consist of collagen bands intermingled with elastic fibers thatsupport hundreds of pounds of stress per square inch. In the spine the basic functional unit comprisesvertebrae, intervertebral disc and ligament tissues. The interspinous ligaments with the function of limiting the spine flexion are exposed to a traumatic and degenerative process that promotes pain orinstability. It has been shown that aging induces structural changes to capsular, fascial and ligamen-tous structures, mainly to the elastic and collagen fibers. However, the relative changes with age inelastic and collagen fibers have not been quantified.  Purpose:  Examine the changes in the arrangement and amount of the elastic and collagen fibers of the human cervical interspinous ligament and attempt to correlate them with age.  Study design/setting: Histomorphometric analysis of ligament samples harvested during surgery.  Patient sample:  We studied the dorsal portion of this ligament from 17 patients aged 16 to 69 years.  Outcome measures:  Fraction of collagen and elastic fibers with linear regression analysis correlat-ing fraction versus age.  Methods:  The elastic and collagen fibers were identified by selective staining methods, and ablinded investigator using an image analysis system performed the histomorphometry.  Results:  There is an age-related progressive increase in collagen and mature and elaunin elastic fi-bers responsible to elasticity. However, these elastic fibers showed structural degenerative changeswith aging. Furthermore, there is an age-related decrease of oxytalan elastic fibers responsible to re-sistance.  Conclusions:  The aged interspinous ligament showed loss of elasticity that could alter the flexionlimiting of the vertebral column.© 2002 Elsevier Science Inc. All rights reserved.   Keywords: Elastin; Collagen; Aging; Interspinous ligament; Cervical column; Spine  Introduction  The interspinous ligaments consist of fibrous cords thatconnect the spinal vertebral processes and limit the flexionof the vertebral column. They are composed of collagen fi-ber bands, which are organized in a parallel and zigzag fash-ion. They are intermingled with elastic fibers and are re-sponsible for their viscoelasticity [1,2]. The elastic fibersystem is composed of three types of fibers. The oxytalan fi-bers are made up only of microfibrils usually arranged inparallel bundles, with a mechanical resistance function. Theelaunin fibers are composed of microfibrillar componentswith small granules of elastin. Mature elastic fibers areformed by progressive deposition of elastin in a microfibril-lar structure. These two last fibers are responsible for tissueelasticity [3,4]. It is well known that the arrangement of elastic fibers con-tributes to tissue architecture, thus promoting the structure’spassive withdrawal to its srcinal unit, when exposed to me-chanical stress. Nevertheless, we should bear in mind that theintegration of the elastic fiber system with collagen fibers is arelevant factor for the maintenance of normal tissue resis-tance. When the ligamentous structures are stretched, theylose the sinuous disposition of the collagen fibers. The elastic  FDA Device/drug status: Not applicable.Nothing of value received from a commercial entity related to this research.*Corresponding author. Faculdade de Medicina da USP, Departa-mento de Cirurgia. Av. Dr. Arnaldo, 455. Sala 1304, 01246-903 São PauloSP, Brazil.   E-mail address  : consuelo@lim02.fm.usp.br (C.J. Rodrigues)   58   E.M.K.P. Barros et al. / The Spine Journal 2 (2002) 57–62  fiber system helps in the rearrangement of collagen afterstretching.It has been demonstrated that with aging these fibrouscomponents of the extracellular matrix show qualitative andquantitative changes [5–7]. However, no previous research has quantified the changes in the relative amounts of elasticand collagen fibers that occur with aging. The objective inthis article was to examine the arrangement and the amountof the elastic and collagen fibers of the human cervical in-terspinous ligament and attempt to correlate them with age.  Material and methods  The material studied consisted of two or three samples of the dorsal portion of the cervical interspinous ligamentsfrom 17 patients (13 men and 4 women) who presented withtraumatic lesions of the cervical spine. The age of patientsranged from 16 to 69 years (Table 1). Ligament samples forhistological and ultrastructural studies were collected duringposterior approach for surgical intervention for treatment of traumatic instabilities of the cervical spine. The ligamentsamples were obtained from the immediately adjacent nor-mal cranial level.  Staining procedure  A major portion of each ligament sample was fixed in10% formalin solution, embedded in paraffin and serial sec-tioned at 5   m of thickness. Three adjacent sections weresubmitted to one of the following selective methods forstaining elastic fibers: Verhoeff’s iodine-iron hematoxylinmethod [8] to stain only the mature elastic fibers, Weigert’sresorcin-fuchsin method [9] to stain mature and elauninelastic fibers and Weigert’s technique, preceded by oxida-tion performed through oxone [3] to stain the oxytalan,elaunin and mature elastic fibers.   Morphometric evaluation  The morphometric measurements were performed withan optical electronic image analysis system (Kontron 300,Kontron Elektronik GMBH, Muenchen, Germany). A blindedinvestigator then analyzed five microscopic fields at 400    magnification. At each field we analyzed the fractional areaof collagen and elastic fibers in relation to the total tissuearea.   Electron microscopy  Small pieces of tissue were fixed in a solution containingtannic acid 0.1% and buffered glutaraldehyde 3%, followedby postfixation in osmium tetroxide 1% for 1 hour. Thefixed material was stained in 0.5% aqueous uranyl acetateovernight and routinely embedded. The sections were cutwith an ultratome apparatus and double-stained with uranylacetate and lead citrate. The thin sections (70 nm) were sub-sequently analyzed with a Zeiss 9S2 (Zeiss, Oberkochen,Germany) electron microscope for identifying and qualify-ing the structural changes of the elastic fibers.  Table 1Age, sex and level of injury of the patients studiedCaseSexAge (years)Level of injury1M39C4–C52M37C5–C63F16C5–C64M32C5–C65M32C5–C66F30C5–C67M20C5–C68M22C6–C79M16C6–C710M65C4–C511M55C5–C612M69C5–C613F65C5–C614M68C5–C615F56C5–C616M67C6–C717M57C6–C7Fig. 1. Electron microscopy of the interspinous ligament from a 16-year-old patient showing oxytalan fibers consisting of bundles of microfibrils.These fibers are between bundles of collagen fibers (  19,000). OX  oxytalan fibers.    E.M.K.P. Barros et al. / The Spine Journal 2 (2002) 57–62  59  Statistical analysis  The morphometric values were analyzed by analysis of variance. The independent variable was age and the com-bined effect of age and amount of collagen and elastic fiberswas tested using linear regression analysis. Analyses wereperformed with the SigmaStat 2.0 software package (JandelScientific, San Rafael, CA). Results with p    .05 were con-sidered significant.  Results  Electron microscopy demonstrated the presence of ox-ytalan, elaunin and mature elastic fibers in old and youngligaments. These fibers were arranged in a parallel orienta-tion with bundles of collagen fibers. At the ultrastructurallevel the oxytalan fibers were composed only of bundles of ordered microfibrils (Fig. 1), whereas the mature elastic fi-bers showed a central core of elastin surrounded by somemicrofibrils (Fig. 2). The elaunin fibers exhibited a smallamount of elastin.Light microscopy in young patients showed some amountof mature elastic fibers that were elongated and slender witha homogeneous distribution (Fig. 3). Weigert staining withor without oxone in young patients demonstrated a higherquantity of elastic fibers that appeared very slender. Withaging we observed greater quantities of elastic fibers thatappeared short, thickened, twisted and usually in clusters.We observed a smaller amount of elongated and slender fi-bers (Fig. 4). At the zone of transition between the vertebralbone and ligament, tissue changes in the distribution patternof collagen and the elastic fiber system were seen. In youngpatients, collagen fiber bundles formed an interlacing net-work, and the spaces contained chondrocytic cells. In thisarea the elastic fibers accompanied the bundles of collagenfibers and were distributed in a network arrangement (Fig.5). Calcification was not observed in the ligament tissuewithin this zone of transition. In samples from older patientsthe transition area consisted of a framework of dense col-lagen tissue with calcification. The special stains used dem-onstrated a dense accumulation of mature elastic fibers,which were short and fragmented. The disposition of fibersappeared to be arranged in clusters.The fractional area of collagen elastic fibers in the dorsalportion of the cervical interspinous ligaments was quantifiedon selectively stained sections. The %WO values represent thefraction area of mature, elaunin and oxytalan fibers inWeigert-oxone stain. The %W values represent the fractionarea of mature and elaunin fibers in Weigert stain. The %Vvalues represent the fraction area of mature elastic fibers inVerhoeff stain. There was a significant positive linear correla-tion between %V and %W with age, and a negative correla-tion between age and %WO. The regression equation was%V    4.16    0.22 * age (r    0.69; F    19.12; p    .001) and to%W    2.52    0.18 * age (r    0.58; F    12.75; p    .001). Con-trariwise, there was a significant negative linear correlationbetween age and %WO    11.52    0.08 * age (r    0.39; F    4.69;p    .05). There was a significant positive linear correlationbetween % collagen with age. The regression equation was% collagen    14.93    0.308*AGE (r    0.57, F    37.64; p    0.001). These regression curves are shown in Fig. 6.  Discussion  We should bear in mind that the integration of the elasticfibers system with collagen fibers is a relevant factor for themaintenance of normal tissue resistance. The compositionof the collagen and elastic system changed according the ki- Fig. 2. Electron micrography of the inters-pinous ligament from a 32-year-old patient.Note the mature elastic fiber (star) in trans-verse cut. This fiber showed a great amountof amorphous substance, elastin, encircledby a crown of microfibrils. (  19,000).   60   E.M.K.P. Barros et al. / The Spine Journal 2 (2002) 57–62 Fig. 3. Histological section of the interspinous ligament from a 22-year-oldpatient showing a homogeneous distribution of thin and elongated elasticfibers (arrows) (Verhoeff,  400).Fig. 4. Histological section of the interspinous ligament from a 69-year-oldpatient. Note some amount of thick and twisted elastic fibers (arrow).(Weigert-oxone,  400).Fig. 5. Histological section of the zone of transition of an interspinous ligament from a16-year-old patient. Note the network distri-bution of slender and elongated elastic fibers(arrow) (Weigert-oxone,  400).    E.M.K.P. Barros et al. / The Spine Journal 2 (2002) 57–62  61  netic properties of each one the spine ligaments. The inter-spinous ligaments have the function of limiting the flexionof the vertebral column, and the supraspinous ligament sup-ports the greatest flexion load followed by yellow and inter-spinous ligaments [10]. This ability is dependent on thegreater number of elastic fibers in the ligamenta flava thanin the interspinous ligament [11].Therefore, if the collagen and elastin composition or thearchitectural arrangement is altered, then tissue compliancewill be jeopardized [12–14]. As has been demonstrated in this study, the ligament tissue undergoes degenerative alter-ations with aging that are more intense at the transition zonebetween the bone and the site of insertion of the ligament.The morphological and histomorphometrical observa-tions made in this study revealed that the cervical inter-spinous ligaments have a collagenic structure intermingledwith elastic fibers that undergo structural and quantitativechanges with aging. The large standard deviation of histo-morphometrical observations may the result of variations innutrition and occupational activity [15,16]. Fig. 6. (A) Collagen content and elastic fiber content in (B) Verhoeff, (C) Weigert and (D) Weigert-oxone stains as a function of age in the cervical inter-spinous ligaments. Regression (solid), confidence (dashed) and prediction interval (dotted) lines. Data of the samples are scatter plotted by age.
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