Linear density of the elastic fibers system in patellar and cruciate ligaments

Abstracts

Com o objetivo de comparar os ligamentos cruzados da articulação do joelho com o ligamento patelar, os autores realizaram análise da quantidade de fibras elásticas entre esses ligamentos por meio de amostras obtidas de sete cadáveres. O estudo histomorfométrico do sistema de fibras elásticas revelado por métodos seletivos de coloração mostrou não haver diferença na densidade linear de todo o sistema de fibras elásticas. Não foi observada diferença estatisticamente significante na quantidade de fibras elásticas maduras entre os três ligamentos estudados. Entretanto, a quantidade de fibras elásticas maduras e elaunínicas, fibras responsáveis pela elasticidade, foi maior no ligamento cruzado anterior. Dessa forma podemos inferir que o ligamento cruzado posterior e o ligamento patelar têm mais fibras oxitalânicas, que são responsáveis pela resistência tecidual. Os autores concluiram que o ligamento cruzado posterior e o ligamento patelar têm estrutura semelhante e parecem mais relacionados à transmissão de força, enquanto o ligamento cruzado anterior está relacionado à elasticidade da articulação de joelho.

ARTIGO REVISADO

Linear density of the elastic fibers system in patellar and cruciate ligaments

Jose Ricardo Pecora^I; Consuelo Junqueira Rodrigues^II; Aldo Junqueira Rodrigues Jr.^II; Osny Salomão^I

^IFrom Departments of Orthopedics and Traumatology, Faculty of Medicine, University of Sao Paulo. Sao Paulo, Brazil

^IIFrom Departments of Surgery, Faculty of Medicine, University of Sao Paulo. Sao Paulo, Brazil

SUMMARY

In order to verify the improvement to choose the better ligament that may be used in knee surgical repair, we performed a comparative analysis of the amount of the elastic fibers system between the patellar and cruciates ligaments. Samples of these ligaments obtained from seven human corpses were studied. Histomorphometric study of the elastic fibers, stained by selective methods didn't show differences in the total amount of all types of elastic fibers, in all three ligaments studied. No difference either was found in the amount of mature elastic fibers. However, the amount of mature and elaunin elastic fibers was significantly different. The anterior cruciate ligament had a greater amount of mature and elaunin elastic fibers, responsible for elasticity. So, the posterior cruciate and the patellar ligaments have more oxytalan elastic fibers, responsible for resistance. We concluded that the posterior cruciate and the patellar ligaments have similar structure and seem more related to transmittance of force, whereas the anterior cruciate ligament is related to elasticity of the knee joint.

INTRODUCTION

Traumatic rupture of the anterior and posterior cruciate ligaments are common and potentially serious, since they can cause incapacitating symptoms and lead to degenerative lesions in the joint cartilage and in the menisci of the knee. Sometimes, culminating with acceleration of the process of arthrosis. The healing of these ligaments is quite difficult, even with primary reconstruction surgery, due to their complex architecture, their mechanical demands during the knee movement, and to the biologically aggressive intra-articular environment. Due to this difficult healing, various other structures have been utilized as grafts for the reconstruction of such ligaments, once rupture has occurred. The central third of the patellar ligament has frequently been used for such reconstruction of both anterior and posterior cruciate ligaments. Various studies have used functional tests to compare the resistance and elasticity of the patellar ligament with that of the anterior and posterior ligament. However, despite the knowledge of the structural composition of these ligaments, no comparative studies of the elastic fibrous system of these ligaments seem to have been reported in the literature.

According to Frank et al.⁽⁶⁾, the various ligaments have a similar microstructure, with slight variations in cellularity and vascularity that depend on size, function and metabolic activity. All are basically composed of multiple bundles of collagen fibers, arranged parallel to each other along the axis of the ligament. Two-thirds of the ligaments are composed of water, and the dry weight is composed of collagen (75%), elastin (4.5%), glycosaminoglycans (0.5%) and other biochemical substances (20%).

The elastic fibers, responsible for tissue elasticity, have been studied in various tissues both in normal and disease condition. In the musculoskeletal system, certain anatomical structures have been subject to special focus, including the intervertebral disc ⁽²⁾, the hip joint capsule ⁽¹¹⁾, the fascia transversalis ⁽¹⁸⁾, the alar and transverse ligaments of the atlas ⁽²⁰⁾.

The elastic fibers system consists of three types of fibers linked by the phenomenon of elastogenesis. The first component formed during elastogenesis is the oxytalan fiber, composed of microfibrils synthesized and secreted by fibroblasts in the extracellular environment. These microfibrils are arranged in parallel bundles, indicating form and direction of the future elastic fiber. Later, small portions of an amorphous substance, elastin, adhere to these microfibrils, giving rise to the second component of the elastic system: the elaunin fiber. With accumulation of a larger quantity of elastin between microfibrils, the fiber becomes thicker, forming the third component of the system: mature elastic fiber. Oxytalan fibers provide resistance, while fibers containing elastin supply elasticity ^(8,19). The quantity of the three types of fibers varies in different tissues and seems to depend on both function and the aging process ^(17,18).

In the present study we analyzed the amount of elastic fibers in the patellar, anterior and posterior cruciate ligaments, by using the histomorphometric method.

MATERIAL AND METHODS

Small rectangular samples of the central portion of the patellar (PL), anterior cruciate (ACL), and posterior cruciate (PCL) ligaments in the right knee were obtained from seven cadavers of male adults, varying in age from 20 to 40 years. These samples were fixed with 10% formalin, embedded in paraffin, and submitted to serial sections of 5mm of thickness.

Staining procedure

Three adjacent sections were submitted to one of the following selective methods for staining elastic fibers: Verhoeff's iodine-iron hematoxylin method, which evidenced only the mature elastic fibers; Weigert's resorcin-fuchsin, which stained mature and elaunin elastic fibers; Weigert's technique, with a previous oxidation performed using oxone as previously described

⁽⁷⁾, which stained the oxytalan, elaunin and mature elastic fibers.

Morphometric evaluation

Linear density (LV) of the elastic fiber system was determined in 25 random microscopic fields per histologic section stained by Verhoeff (V), Weigert (W) and Weigert-oxone (WO). Sections were scanned randomly at a magnification of 1,000x in a continuous line from edge to edge, employing a test eyepiece reticule with 10 parallel lines and 100 points which contains a simple square lattice test system with 10,500 mm

² . Each elastic fiber completely intersected by any one of the test lines was counted. These intersections of fibers with a test reticule are related to the length of those fibers per unit of area, by the expression LV=2Na, where Na is the length of fiber per unit of area

⁽¹⁶⁾. Area of the tissue examined was determined by counting the number of points of intersection inside that tissue.

Statistical analysis

Descriptive statistics (average, standard deviation and standard error of the mean) were used to compare the linear density of the elastic fibrous system of PL, ACL and PCL. An analysis of variance (ANOVA) was used to compare the parametric ordinary groups. Significant differences were identified using the Tukey test. A level of significance of 5% was adopted (a = 0.05) for all analyses.

RESULTS

Knee ligaments were composed by dense bundles of collagen fibers intermingled with elastic fibers and vascular structures. Collagen fibers showed a longitudinal arrangement along the major axis of the ligaments, and elastic fibers runned parallel to the collagen ones (fig. 1).

Linear density (LV) values of the elastic fibers in PL, ACL and PCL observed in Verhöeff (V), Weigert (W) and Weigert-Oxona (WO) stains were presented in tables 1.

Analysis of variance for LVWO, which corresponds to the amount of mature, elaunin and oxytalan elastic fibers, didnt't show significant differences between all three knee ligaments studied (F = 3.27; p> 0.05). The same was shown for LVV, which corresponds to the amount of mature elastic fibers (F = 1.49; p > 0.05). Values of LVW that represent the amount of mature and elaunin elastic fibers showed significant differences (F=17.83; p<0.0001). The Tukey test showed that LVW of ACL was statistically higher in relation to PL and PCL. The LVW of the latter two ligaments was statistically the same.

Since the statistical analysis showed no differences among the three types of ligaments in relation to the sum of the components of the elastic system (oxytalan, elaunin, and mature elastic fibers), nor in relation to mature fibers, the differences are only in the amount of elaunin and oxytalan fibers, which are inversely related. Thus, the ACL showed 88.5% of elaunin fibers, while PL and PCL showed 61.61%. These latter two ligaments have proportionally greater amount of oxytalan fibers than the ACL (fig. 2).

DISCUSSION

The use of the central third of the PL as a graft in the reconstruction of ACL was conceived by Jones ⁽¹²⁾ and made popular by Clancy ⁽⁴⁾, who extended its use to the reconstruction of PCL as well. This procedure has been used by a large number of orthopedists for the surgical treatment of patients whose knees evidence a symptomatic deficiency of this ligaments, whether of the ACL or of the PCL .

Studies of these ligaments have been restricted to examination of the mechanical properties ⁽¹³⁾, although a few papers have focused on the composition, especially in relation to the system of elastic fibers in the connective tissue ^(1,5,15).

This study of knee ligaments was done in male cadavers between 20 to 40 years, because the greater incidence of traumatic rupture of the ACL and PCL are most frequent in similar kind of persons.

Statistical analysis of results of the linear density of PL, ACL and PCL for the three types of histological staining leads us to the conclusion that the ACL has a greater quantity of elastin-containing elements than do the other knee ligaments. These results agree with those of Neurath et al. ⁽¹⁵⁾, who compared the quantity of elastin-containing fibers between the ACL and PCL. These researchers found the amount of such fibers to be greater in the ACL than in the PCL. The present results suggest that the ACL is more elastic than either the PL or the PCL, due to the greater quantity of elaunin fibers.

Active mechanical force is involved in the process of elastogenesis. Number and arrangement of the fibers of the elastic system present in a structure depend on the function ⁽⁸⁾. Thus, the proportion of the components of the elastic fibers system in the PL, ACL and PCL is intimately linked to the function of these anatomical structures. It is true that the total resistance and elasticity of a ligament depend not only on the quantity of elastic fibrous elements in relation to collagenous fibers, but also on their arrangement and size and thickness of a ligament, as a whole. The results of the present study seem to agree with what one would expect for the three types of ligaments involved. The PL, as the distal part of the extension mechanism of the knee, serves to transmit all the force of traction of the quadriceps, received by the patella, to the anterior tuberosity of the tibia. Thus, the less its elasticity, the greater its efficiency in the transmission of traction force of the quadriceps. The PCL is considered the primary posterior stabilizer of the knee, and the basic stabilizer of the knee, due to its proximity to the center of rotation ^(3,9). In order to do this job, it cannot permit excessive stretching, which confirms the findings of a smaller quantity of fibrous components containing elastin in this ligament.

The ACL is stretched by contraction of the quadriceps, in the hyperextension of the knee and internal and external rotation movements of the tibia in relation to the femur ⁽¹⁴⁾. It can be seen that the ACL is involved in active movement, stretching and contracting with the anterior movement of the tibia in relation to the femur, which occurs depending on the contraction of the quadriceps in the extension of the knee. It also acts to dissipate the rotational force to which the knee is submitted. For the ligament to perform these tasks efficiently, it must have sufficient elasticity, which is provided by a greater quantity of elastin-containing fibrous elements. We could imagine that the closer to the original structure the composition is, the more quickly and efficiently the process of graft will be. Thus, in relation to elastic fibers, the PL would be a better substitute for the PCL than for the ACL, regarding graft reconstruction.

We suppose that the greater elasticity of the ACL may explain why its rupture leads to changes in the arrangement of the collagen bundles, reducing its capacity for healing. So, this is the case of an indication for grafting. The PL and PCL, however, have a smaller quantity of elastin, and consequently stretch little when submitted to traction. Traction force is thus transmitted directly to all the bundles of collagenous fibers and, once the capacity for resistance is reached, the ligament can stretch no further; both the PL and the PCL rupture at a single point, with the remaining structure of the collagens in the ligament maintaining its integrity. Perhaps due to this type of rupture, the patellar tendon is quite responsive to primary suture ⁽¹⁰⁾.

REFERÊNCIAS

Address for reprint requests: Prof. Dr. Aldo Junqueira Rodrigues Junior. Faculdade de Medicina da USP. Disc.Topografia Estrutural Humana. Av. Dr. Arnaldo 455, sala 1304. São Paulo, SP, Brasil. CEP 01246-903. Fax [phone]

• 1. ARNOCZKY, S.P. 1983 Anatomy of the anterior cruciate ligament. Clin. Orthop.172: 19-25,.
• 2. BUCKWALTER, J. A.; COOPER, R. R.; MAYNARD, J. A. 1976 Elastic fibers in human intervertebral discs. J. Bone Joint Surg. [Am] 58: 73-76.
• 3. BUTLER, D. L.; NOYES, F. R.; GROOD, E. S. 1980 Ligamentous restraints to anetrior-posterior drawer in the human knee:a biomechanical study. J. Bone Joint Surg. [Am] 62: 259-270.
• 4. CLANCY, W. G. Jr.; SHELBOURNE, K. D.; ZOELLNER, G. B.; KEENE, J. S.; REIDER, B.; ROSEMBERG, T. D. 1983 Treatment of knee joint instability secondary to the rupture of the posterior criciate ligament. J. Bone Joint Surg. [Am.] 65: 310-322.
• 5. DANYLCHUK, K.D.; FINLAY, J. B.; KRCEK, J. P. 1978 Microstructural organization of human and bovine cruciate ligament. Clin. Orthop. 131: 294-298.
• 6. FRANK, C.; AMIEL, D.; ING, D.; WOO, S.L.Y.; AKESON, W. 1985 Normal ligament properties and ligament healing. Clin. Orthop. 196: 15-25.
• 7. FULLMER, H.M.; LILLIE, R.D. 1958 The oxitalan fiber: a previouly undescribed connective tissue fiber. J. Histochem. Cytochem. 6: 425-430.
• 8. GAWLIK, Z. Morphological and morphochemical properties ofthe elastic system in the motor organ of man. 1965 Folia Histochem. Cytochem. 3: 233-251.
• 9. GIRGIS, F.G.; MARSHAL, J. L.; AL MONAJEM, A.R.S. 1975 The cruciate ligaments of the kneejoint: anatomical, functional and experimental analysis J. Bone Joint Surg. [Am] 106: 216-231.
• 10. HUGSTON, J.C. 1993 Knee ligaments: injurie and repair. St. Louis, Mosby, pp.3-70.
• 11. IPPOLITTO, E.; ISHII, Y.; PONSETI, I. V. 1980 Histologic, histochemical, and ultrastructural studies of the hip joint apsule and ligamentum teres in congenital dislocation of the hip. Clin. Orthop. 146: 246-258.
• 12. JONES, K.G. 1963 Reconstruction of the anterior cruciate ligament: a technique using the central one-third of the patellar ligament. J. Bone Joint Surg. [Am.] 45: 925-932.
• 13. KENNEDY, J. C.; HAWKINS, R. J.; WILLIS, R. B.; DANYLCHUK, K. D. 1976 Tension studies of human knee ligaments. J. Bone Joint Surg. [Am]. 58:. 350-355.
• 14. KUROSAWA, H.; YAMAKOSHI, K.I.; YASUDA, K.; SASAKI, T. 1991 Simultaneous measurement of changes in lenght of the cruciate ligaments during knee motion. Clin. Orthop. 265: 233-240.
• 15. NEURATH, M.; STOFFT, E.; ZSCHABITZ, A.; PRINTZ, H. 1991 Comparative microstructural studies on colllagen and elastic fiber systems of the cruciate ligaments. Z. Unfallchir. Versicherungsmed., 84: 170-176.
• 16. NIEWOHNER, D.E.; KLEINERMAN, J. 1977 Mophometric study of elastic fibers is normal and emphysemaetosus human lungs. Am. Ver. Resp. Dis. V:115 15-21.
• 17. RODRIGUES, CJ, JCL SACHETTI, AJ RODRIGUES JUNIOR. 1999b Age-related changes in the elastic fiber network of human splenic capsule. Lynmphology (in publication)
• 18. RODRIGUES JUNIOR, A.J.; TOLOSA, E.M.C.; CARVALHO, C.A.F. 1990 Electron microscopic study on the elastic and elastic related fibers in the human fascia transversalis at different ages. Gegenbaurs Morphol. Jahrb. 136: 645-652.
• 19. ROSEMBLOOM, J.; ABRAMS, W.R.; MECHAM, R. 1993 Extracellular matrix 4: the elastic fiber. FASEB 7: 1208-1218.
• 20. SALDINGER, R.; DVORAK, J.; RAHN, B. A., PERREN, S. M. 1990 Histology of the alar and transverse ligaments. Spine 15: 257-261.

Publication Dates

• Publication in this collection
  27 June 2006
• Date of issue
  Mar 2001