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Journal of the Anatomical Society of India

Electron And Light Microscopic Study Of Anterior Cruciate Ligament Of Rabbit

Author(s): Bayat, M; Mohammadzade, F; Rakhshan, M.

Vol. 52, No. 2 (2003-07 - 2003-12)

Department of Anatomy, Molecular and Cellular Research Centre, Shaheed Beheshti University of Medical Sciences, Tehran, IRAN.


Anterior cruciate ligament (ACL) of knee joint has a complex structure, which its organization and biology has directly adopted to its function as a controller of the movements of the joint. Changes in fibril diameter - demonstrated during maturation, mobilizationand increase in the level of stress on tissue has an important role in repairing surgery of the ACL. It seems that determination of thediameter and distribution of the collagen fibrils of ACL is very important. The aim of the present study is careful determination of diameter of collagen fibrils of rabbit ACLs by light microscopy (LM), scanning electron microscopy (SEM) and transmission electron microscopy (TEM).

Thirty healthy male rabbits were anaesthetised by chloroform. Then after dissection of knee joint, ACL were cut from femur and tibia. Specimens of ACL were prepared for LM, SEM and TEM examinations separately and studied by quantitative and descriptive methods.

Transverse sections examined by LM showed that the whole fascicles of ACL were in one group and the mean and SD of surfacearea of ACL of six rabbits was 13.08 + 1.93 mm2.

LM examination of longitudinal sections of ACL showed that fascicles of collagen fibres in some sections were straight and in othersthese were wavy. SEM examination showed that ACL was composed of wavy interlacing collagen fibres and fascicles, directed mainlyparallel to long axis of ACL. Another result of SEM examination reported for the first time is that some collagen fascicles formed an irregularnetwork and there were small fascicles located on the surface of main fascicles. TEM examination showed that ACL was mainly composedof collagen fibrils with diameters ranging from 13.2 to 125.7nm. They were separated by loose connective tissue. Diameter of totalpopulation of the fibrils in the rabbits was 49.2 + 20.7nm.

Key words: Anterior cruciate ligament, Collagen fibrils, Collagen, Rabbits.


Anterior cruciate ligament (ACL) of knee joint has a complex structure. Its organization and biology has a direct relationship with its function as a controller of movements of the knee joint. Because of its complexity, it heals with difficulty (Dodds and Arnoczky, 1994). It is often injured during strenuous exercises and heals with difficulty, frequently impairing athletic performance (Strocchi et al, 1992)

Secondly, despite the numerous exhaustive biomechanical studies on the ACL (Kennedy et al, 1974; Butler et al, 1985; Ellison & Berg, 1985; Mcleod, 1985; Odensten & Gillquist, 1985; Gollehon et al, 1987; Katsuragi et al, 2000; Punjabi and Courtney, 2001), its fine morphological and ultrastructural descriptions in the literature are limited to the following studies : Danylchuk et al, (1978) studied human and bovine ACL with scanning electron microscope (SEM). They showed that mean diameter of collagen fibrils of ACL were between 150-250nm. Yahia and Drouin (1989) examined ACL by light microscopy (LM) and SEM. They showed helical wave pattern in ACL and medial collateral ligament in rabbits. They showed mean fibril diameter for ACL to be 0.059 + 0.005mm.

Neurath and Stoffi (1992) reported that human normal ACL has a complex three-dimensional structure and collagen fibrils have a unidirectional course with parallel arrangement and their diameter ranges from 20 to 185nm with a mean diameter of 75nm. Strocchi et al (1992) observed two types of collagen fibrils : small (with a single diameter peak at 45 nm) and large (3 peaks at 35, 50 and 75 nm respectively) organized into distinct areas made up of either large or small bundles of fibrils.

Hart et al (1999) present morphometric analysis of the collagen fibril diameter of rabbit ACL and medial collateral ligament (MCL) of New Zealand white rabbits (young, with age two months and adults with age thirty-six to forty months) Transmission electron micrographs of ACL and MCL were studied and it was found that mean fibril diameter of ACL in two month old animals was: 0.044 + 0.006m and of 36-40 month old animals was 0.069 + 0.005m.

The morphological data and the morphometrical analysis data of above mentioned studies are discrepant.

Changes in collagen fibril population are demonstrated during maturation (Frank et al 1989; Hart et al, 1999) and by increasing the level of stress on the tissue (Michna, 1984) So it seems that exact determination of fibril diameters of ACL is very important from different aspects; i.e. histological, physiological and reconstructive surgery (Frank et al, 1988). In the light of the above mentioned reports it was realized that there are somewhat discordant morphologic and morphometric data about ACL in literature, and lack of studies applying concurrently LM, TEM and SEM techniques is noticeable therefore, the aim of this study was careful examination of rabbit ACL by LM, TEM and SEM techniques.

Materials and Methods :

30 healthy 6- months old male dutch white rabbits were anaesthetised by chloroform. The right hind limbs of rabbits were disarticulated and removed. After dissection of the knee joint, ACL were harvested.

(i) Preparation for LM — 10 samples were fixed in 10% formaline saline, dehydrated and embedded in paraffin. Transverse and longitudinal sections were stained with hematoxyline & eosin. Cross section of transverse slides of 6 ACLs were transferred to transparent sheet by a microprojector (ken- A vision, USA) Surface area of ACLs were measured using a graculated paper. Longitudinal sections (n = 5) were studied descriptively.

(ii) Preparation for SEM – Two preparation procedures were used for SEM studies.

(a) Standard method: 5 samples were fixed in 3 % glutaraldehyde, postfixed in 1 % osmic acid, samples were immersed in phosphate buffer solution (PBS) for one hour. PBS was refreshed for 5 times. Samples were dehydrated in a graded series of acetone. Samples impregnated with isopentan (Fluka) were frozen by liquid nitrogen and dried by Freeze drier (Edward, England) for 6 hours. The dry samples were mounted on SEM stubs and sputter coated with gold (using SCD - 005)

(b) Enzymatic Digestion: In order to study fine structure of ACL two digestive enzymes were used to remove the sheath and ground substance surrounding the collagenous entities. Prior to fixation the 5 samples were exposed to hyaluronidase (1500 U/ 150 ml sodium acetate buffer, pH 5.4, 0.1 M, 37°C) and elastase (10 mg / 100 ml sodium carbonate buffer, pH 8.8, 0.05 M, 37°C). The samples were then treated according to the standard method. Samples were examined with a ZEISS DSM 940 A scanning electron microscope with a 15 kv voltage.

(iii) Preparation for TEM In order to study collagen fibril diameter of ACL, 6 samples were fixed in 3% glutaraldehyde and were post fixed in 1% osmic acid, dehydrated in alcohol and embeded in TAB resin. The transverse ultra-thin sections were stained with uranyl acetate and lead citrate and examined in a ZEISS EM900 transverse electron microscope, with a magnification of 4400 - 12000. The minimum diameters of 1000-2900 fibrils were measured by a fine ruler according to Hart et al (1992) method and Moeller et al (1995) method. Data of samples were statistically analyzed. Results :

Transverse paraffin sections showed that ACL is composed from two distinct areas : a loose connective tissue which ensheathed collagen fibres (peritenon) and bundles of collagen fibres (Figure 1). Some longitudinal paraffin sections showed that ACL was composed of straight bundles of collagen fibres and some other longitudinal paraffin sections showed that ACL was composed of wavy bundles of collagen fibres.

Figure 2 illustrates the histological appearance of an ACL near insertion into the bone. It resembles that of the fibro- cartilage tisue; cells increased and collagen fascicles scattered. The cells were ellipsoid situated in lacunae and were ordered in columns.

Mean ± SD of surface area of transverse section of 6 ACLs was 13.08 + 4.93 mm2.

Standard SEM preparation of longitudinal samples of ACL showed that ACL is composed of wavy and interwoven collagen fascicles oriented in longitudinal direction. There were spaces between collagen fascicles (Figure 3).

Enzymatic SEM preparation of ACL showed that there were small fascicles located on the main fascicles, and forming an irregular network (Figure 4 a,b). TEM examination showed that ACL is composed of two distinct regions, a loose connective tissue which ensheat the ligament with its branches separating collagen fascicles; and scattered collagen fibrils (Figure 5).

Collagen fibril diameters of ACL ranged from 13.2 to 125.7 nm, with Mean ± SD of 49.2 ± 20.7nm, and these were distributed almost equally through this range.


Longitudinal paraffin sections of Strocchi et al (1992) showed that ACL is composed of thick wavy bundles of collagen fibres ensheated in loose connective tissue. This was confirmed by some results of the present study. However in our study we also observed straight fascicles of collagen fibres in deep region of ACL.

In present study, we observed fibrocartilage region of ACL at the point near the insertion into the bone.

In this regard, Clark and Sidles (1990), in the same region of ACL observed that collagen bundles of ACL were separated by narrow clefts containing a dense network of fine fibrils and cells which were surrounded by fibrous capsules similar to the territorial networks lining the lacunae in articular cartilage.

On the other hand Okuda et al (1987) have found cells in lacunae and increased proteoglycan in those regions of tendon that were subjected to compression from an external force. So it was concluded that ACL near the insertion into the bone is subjected to compressive forces.

In some specimens viewed with the SEM, there were accessory small fascicles and irregular fine collagen fascicles, located on the ACL surface; it is hereby reported for the first time by authors and it is concluded that ACL structure is more complex than expected.

According to Flint et al, (1984) fibril diameter is known to indicate specific fibril function. For example, collagen fiibril diameter is expected to vary with age in certain tissue, suggesting a correlation with functional status. The progressive increase in average collagen fibril diameter during development reveals an increase in the number of intermolecular cross links, which in turn enhances the tensile strength of the tissue, fulfilling the greater functional demands (Nimni, 1983).

Therefore, it is reasonable to assume that different collagen fibril groups observed in present study which are distributed almost equally between 13.2-125.7nm have different functions. The larger fibrils resist high tensile strength of tissue while, the small fibrils maintain the 3-dimensional organization of the ligament. It should be noted however, that the collagen fibril diameters reported by Danylchuck et al (1978) - differ markedly from those obtained by present and Strocchi et al 1992 studies (150-250 nm against 13.2 - 125.7nm).

In conclusion the ACL has been observed to be complex anatomical structure. There are a wide range of collagen fibril diameters (13.2- 125.7nm) and small accessory fascicles located on the main fascicles and three dimensional collagen fascicle network.


  1. Amiel D., Wallace D., Harwood F.L. (1994) : The effects of immobilization on the maturation of the anterior cruciate ligament of the rabbit knee. The lowa Orthopaedic Journal. 14 : 134 - 140.
  2. Butler, D.L., Grood, E.S., Noyes, F.R., Sodd, A.N. (1985) : On the interpretation of our anterior cruciate ligament data. Clinical Orthopaedics and Related Research. 196 : 26- 34.
  3. Clark, J.M. and Sidles, J.A. (1990) : The interrelation of fibre bundles in the anterior cruciate ligament. Journal of Orthopaedic Research. 8 : 180- 188.
  4. Danylchuck, K.D., Finaly, J.B., Krcek J.P. (1978) : Microstructural organization of human and bovine cruciate ligaments. Clinical Orthopaedics and Related Research. 131 : 294 - 298.
  5. Dodds J.A. and Arnoczky, S.P. (1994) : Anatomy of the anterior cruciate ligament : a blue print for repair and reconstruction. Arthroscopy . 10 (2) : 132- 139.
  6. Ellison, A.E. Berg, E.E. (1985): Embryology, anatomy, and function of the anterior cruciate ligament. Orthopaedic Clinics of North America.16 : 3- 14.
  7. Flint, M.H. Craig, A.S., Reilly, H.C. Gillard, G.C., Parry, D.A.A. (1984): Collagen fibril diameters and glycosaminoglycan content of skins. Indices of tissue maturity and function. Connective Tissue Research 13 : 6981.
  8. Frank, C., Bray, D., Rademaker, A., Chursch, C., Sabiston, P., Bodie, D., Rangayyan, R. (1989): Electron microscopic quantitation of collagen fibril diameter in the rabbit medial collateral ligament : a baseline for comparison. Connective Tissue Research 19 : 11- 25.
  9. Frank, C., Woo, S.Y., Andriacchi, T., Brand, R., Oakes, B., Dahners, L. (1988) : Normal ligament : structure function and composition. In Injury and repair of musculoskeletal soft tissues. American Academy of orthopaedic surgeons. Par Ridge. pp. 45-101.
  10. Gollehon, D.L., Torzilli, P.A., Warren, R.F. f(1987) : The role of the posterolateral and cruciate ligaments in the stability of the human knee. Journal of Bone and Joint Surgery 69 A 233- 242.
  11. Hart, R.A., Akeson, W. H., Spratt, K., Amiel, D. (1999) : Collagen fibril diameter distribution in rabbit anterior cruciate ligament and medial collateral ligaments changes with maturation. The Iowa Orthopaedic Journal. 19 : 66- 70
  12. Katsuragi, R. Yasuda, K. Ysujino, J., Keira, M., Kaneda, K. (2000) : The effect of nonphysiologically high initial tension on the mechanical properties of in situ frozen anterior cruciate ligament in a canine model. American Journal of Sports Medicine 28 (1) : 47 - 56.
  13. Kennedy, J.C., Weinberg, H.W., Wilson, A.S. (1974) : The anatomy and function of the anterior cruciate ligament. Journal of Bone and Joint surgery 56A : 223-235.
  14. Mcleod, W.D. (1985) : The biomechanics and function of the secondary restraints to the anterior cruciate ligament Orthopedic Clinics of North America 16 : 165-170.
  15. Michna, H (1984) : Morphometric analysis of loading induced changes in collagen fibril population in young tendons Cell and Tissue Research. 236 : 465 - 470.
  16. Neurath, M.F. and Stoffi, E. (1992) : Collagen ultrastructure in ruptured cruciate ligaments. Acta Orthopaedic Scandiavian 63 : 507 - 510.
  17. Newton P.O., Woo S.L.Y., Kitabayashi L.R., Lyon R.M., Anderson D.R., Akeson W.H. (1990) : Ultrastructural changes in knee ligaments following immobilization. Matrix. 10 : 314 - 319.
  18. Nimini, M.E. (1983): Collagen : Structure, function, and metabolism in normal and fibrotic tissues. Seminar Arthritis and Rheumatism 13 1 - 86.
  19. Odensten, M. Gillquist, J. (1985) : Functional anatomy of the anterior cruciate ligament and a rationale for reconstruction. Journal of Bone and Joint Surgery 67 A : 257-262.
  20. Okuda, Y., Gorsk, J.P., An, K.N., Amdio, P.C. (1987) : Biochemical, histological, and biomechanical analyses of canine tendon. Journal of Orthopaedic Research 5: 60- 68.
  21. Panjabi, M.M., and Courtney, T. W. (2001) : High - speed subfailure stretch of rabbit anterior cruciate ligament, changes in elastic, failure and viscoelastic characteristic. Clinic Biomechanic (Bristol, Avon) 16 (4) 334 - 40.
  22. Strocchi, R., De pasquale, V., Gubellini, P., Facchini, A. Marcacci, M, Bud, R., Zaffagnini, S., Ruggeri, A. (1992) : The human anterior cruciate ligament : histological and ultrastructural observations. Journal of Anatomy 180 : 515 519.
  23. Yahia L.H. and Drouin G. (1989) : Microscopical investigation of canine anterior cruciate ligament and patellar tendon: collagen fascicle morphology and architecture. Journal of Orthopaedic Research. 7 : 243 - 45.

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Transverse paraffin section of a rabbit ACL. There are loose connective tissue (L), Transverse section of collagen fasicles (C) and peritenon (P). (Haematoxyline and Eosine. x 32).


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Longitudinal paraffin section of rabbit ACL cut near insertion into the bone. There are chondrocytes (c) in the lacunae. (Haematoxyline and Eosine. x 100).


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SEM micrography of rabbit ACL fascicles after standard treatment. There are fascicles (F) with different diameters and separated from each other by narrow spaces. (x 1000).


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SEM micrograph of rabbit ACL surface after enzymatic treatment. There are accessory small fascicles (F) on the surface of main fascicles. (100x).


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SEM micrograph of rabbit ACL surface; the accessory small fascicle. (x 1000).


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SEM micrograph of rabbit ACL surface after enzymatic treatment, there are fine fascicles which formed irregular network.


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TEM transverse micrograph of rabbit ACL. There are fibrils (f), fascicles (F) and loose connective tissue (L)

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