Comparative and Morphological Analysis of Commonly Used Autografts for Anterior Cruciate Ligament Reconstruction with the Native Anterior Cruciate Ligament: An Electron Microscopic and Morphologic Study

Abstract

Ligaments and tendons are similar in composition but differ in proportion and arrangement. Tendons are being used as grafts for the ACL reconstruction. Their microscopic structure has not been sufficiently studied and compared to the native ACL. A null hypothesis was declared stating that the anterior cruciate ligament should be histological, morphologically and functionally different from the tendon grafts used for ACL reconstruction. We investigated similarities and differences of the structure of ACL and tendons used as a graft tissue for ACL recon- struction. In this study, standardized samples of quadriceps, hamstrings (semitendinosus and gracilis) and patellar tendons, and the ACL were harvested from 26 autopsies (average age 36.4) and were investigated using light and electron microscopy, immunohistochemistry and mor- phometry. The thickness of the collagen fibrils, collagen organization and diameter, the fibril/interstitium ratio, density of fibroblasts and blood vessels, and distribution of the collagen type I, III and V fibrils were analyzed. The semitendinosus showed the highest density of fibroblasts and blood vessels, while the gracilis the highest fibril/ interstitium ratio. No differences regarding the thickness of collagen fibrils and distribution of fibrils were found. The ACL had the highest concentration of type III and V collagen fibrils as well as elastic fibers. The histological and ultrastructural appearance of the ACL differs from those of the tendons used as graft, for ACL reconstruction. Its ultrastructure is varied and complex, with its collagen fibers bundles lying in many directions.

Keywords

ACL reconstruction, Cadaveric study, Comparative morphology, Morphometry, Tendon graft

In this chapter, we examine the microstructure of the most common tendon grafts that are used for anterior cruciate ligament (ACL) repair. Each graft has different characteristics and function and cannot in any way replace the torn ACL. The ACL, apart from the mechanical function, has been proved to have proprioceptive function, which the other tendon grafts lack. Also, the ACL collagen fibers have the unique ability to change in length during flexion and extension of the knee. The aim in ACL reconstruction should be the replacement of the torn ligament with the strongest possible graft. This parameter depends on the graft itself, as well as the fixation technique used.

We believe that any difference in biomechanical strength of the different tendon grafts could be reflected on their microanatomy. We examined the ultrastructure and histological parameters of the functional unit of the tendon—the collagen fiber.

Thus in this chapter we examined the histological and morphological features of the ACL and the graft tissues that are commonly used for reconstruction of the ACL. The aim was to compare one graft with another and also to compare each graft with the normal ACL. We hypothesized that the tendon grafts are similar to each other and significantly different in terms of histological and morphological composition to the ACL.

Histological and Morphological Parameters of the Tendon Grafts and Native Anterior Cruciate Ligament

Twenty-six tendons and ACLs were harvested from young donor cadavers (average age 36.4; range 24–56 years). All tissues were investigated by light and electron microscopy and immunohistochemistry and were additionally analyzed by morphometry. For light microscopy, the removed specimens were formalin fixed (4% formalin in phosphate-buffered saline [PBS]) and paraffin embedded via routine procedure. The size of all the probes studied was 20 mm × 10 mm whole thickness of tendons for the patellar tendon and quadriceps, and 20 mm × whole width × whole thickness for the hamstring tendons and the ACL.

Keywords

ACL reconstruction, Cadaveric study, Comparative morphology, Morphometry, Tendon graft

In this chapter, we examine the microstructure of the most common tendon grafts that are used for anterior cruciate ligament (ACL) repair. Each graft has different characteristics and function and cannot in any way replace the torn ACL. The ACL, apart from the mechanical function, has been proved to have proprioceptive function, which the other tendon grafts lack. Also, the ACL collagen fibers have the unique ability to change in length during flexion and extension of the knee. The aim in ACL reconstruction should be the replacement of the torn ligament with the strongest possible graft. This parameter depends on the graft itself, as well as the fixation technique used.

We believe that any difference in biomechanical strength of the different tendon grafts could be reflected on their microanatomy. We examined the ultrastructure and histological parameters of the functional unit of the tendon—the collagen fiber.

Thus in this chapter we examined the histological and morphological features of the ACL and the graft tissues that are commonly used for reconstruction of the ACL. The aim was to compare one graft with another and also to compare each graft with the normal ACL. We hypothesized that the tendon grafts are similar to each other and significantly different in terms of histological and morphological composition to the ACL.

Histological and Morphological Parameters of the Tendon Grafts and Native Anterior Cruciate Ligament

Twenty-six tendons and ACLs were harvested from young donor cadavers (average age 36.4; range 24–56 years). All tissues were investigated by light and electron microscopy and immunohistochemistry and were additionally analyzed by morphometry. For light microscopy, the removed specimens were formalin fixed (4% formalin in phosphate-buffered saline [PBS]) and paraffin embedded via routine procedure. The size of all the probes studied was 20 mm × 10 mm whole thickness of tendons for the patellar tendon and quadriceps, and 20 mm × whole width × whole thickness for the hamstring tendons and the ACL.

Transmission Electron Microscope

Tendons were placed in 4% paraformaldehyde for 24 hours, transferred to 4% formalin, and then sent to be cut and embedded for transmission electron microscopy. The cross-sections (100 nm thick) of each fixed tendon were rinsed in 0.1 M phosphate buffer, and then placed in 1% osmium tetroxide in 0.1 M phosphate buffer for 2 hours. They were then dehydrated in graded ethanol solutions (30%, 50%, 65%, 75%, 95%, and 100%) and transferred to propylene oxide. The tendons were infiltrated with Epon. The infiltration process was performed for 24 hours, and then the specimens were hardened at 40°C for 48 hours. Thin sections, three from the center of each tendon studied, were stained with aqueous uranyl acetate and lead citrate, and then scanned with Zeiss transmission electron microscope. Ten random fields were photographed from each section at a magnification of 10,000.

Four groups of tissue samples (patellar, quadriceps, semitendinosus, and gracilis) and the ACL were collected and processed for (1) measurement of collagen fibril diameter, (2) fibril density (fibril/interstitium ratio), (3) density of blood vessels, (4) density of fibroblasts, (5) percentage of elastic fibrils, and (6) percentage of collagen types I, III, and V.

Immunohistochemistry

All specimens were investigated by immunohistochemistry. The CD34 marker, which stains in the endothelial cells, was used to identify blood vessels within the tendinous tissue. The CD34 antibody (IgG1; human antimouse antibody; Dako, Hamburg, Germany; dilution 1:25) was used for this type of staining.

Immunostaining was performed using an automated platform-Dako autostainer (Dako, Glostrup, Denmark). Primary antibodies were diluted in a commercially available antibody diluent (Dako ChemMate), and detection was carried out using the Dako ChemMate kit. Nonbinding monoclonal mouse IgG1 was used as a negative control. The sections were finally counterstained with hematoxylin and mounted.

For immunohistochemical stainings for collagen, the following antibodies were used: collagen type I (rabbit polyclonal, 1:10; Biotrend, Koln, Germany), collagen type III (rabbit polyclonal, 1:10; Sigma-Adrich, Munich, Germany), and collagen type V (rabbit polyclonal, 1:300; Santa-Cruz, Heidelberg, Germany). Briefly, paraffin sections were deparaffinated in xylol and rehydrated through grading alcohol concentrations and incubated with primary antibodies for 60 minutes. After rinsing in PBS, sections were incubated with secondary antibodies for 30 minutes (biotinylated antirabbit, as appropriate; Biogenex, San Ramon, California) and finally with streptavidin-conjugated alkaline phosphatase (Biogenex) for 30 minutes. Detection was made with the FastRed substrate (Dako, Hamburg, Germany), using detection system ABC. All steps were performed at room temperature. Negative controls were performed by omitting the primary antibody.

The optimal concentration for staining was evaluated by testing different dilutions in a pilot study. Immunohistological staining was analyzed by two investigators blindly.

Histochemistry

For detection of elastic fibrils, Elastica van Gieson (EVG) staining was used.

Quantitative Evaluation

The following parameters were analyzed by morphometry: fibril/interstitium ratio (%), thickness of fibrils (nm), density of blood vessels (number of blood vessels per mm ²of tendinous tissue), density of fibroblasts (number of fibroblasts per mm ²of tendinous tissue), and percentage in tendinous tissue of collagen and elastic fibrils.

Quantitative evaluation of fibril/interstitium ratio was performed on Masson-Goldner-stained sections using the morphometrical program “Optima” (Firma SIS-Soft Imaging System, Münster, Germany). For this procedure, longitudinal sections of tendons were used. The measurement was performed on the whole length of the section.

The density of blood vessels was defined as the number of blood vessels per mm ²of tendinous tissue. This measurement was performed on sections stained with CD34. Positive-stained vessels were counted in the whole area section. The average number of blood vessels per mm ²of tendinous tissue was calculated.

The density of fibroblasts was determined on hematoxylin and eosin-stained sections. The number of fibroblasts was counted in the whole area section. Finally, the number of fibroblasts per mm ²of tendinous tissue was calculated. The percentage of elastic fibrils was analyzed in EVG staining. The percentage of collagen fibrils (type I, III, and V) was analyzed in each immunohistochemical staining.

The quantitative evaluation of the thickness of collagen fibrils was performed using electron microscopy (original magnification ×10,000). The mean thickness of collagen fibrils was evaluated using the morphometrical program “Analysis” (Firma SIS). For each specimen, 100 collagen fibrils were analyzed.

Statistical Evaluation

Data are given as mean standard deviation. After testing for normal distribution, the Kruskal Wallis test or one-way analysis of variance (ANOVA) were chosen, followed by Duncan’s multiple-range test for differences between groups. The results were considered significant when the probability of error ( P ) was lower than 0.05.

Results

The microstructure of the ACL is similar to the other tendon grafts, although distinct differences exist in the histologic and electron microscopic preparations.

The organization of ACL fibrils appears to be unique. Light and scanning electron microscopy revealed a combination of a helical and planar wave pattern for ACL fibrils. Thus there is a combination of parallel or twisted, nonlinear networks. The purpose of the wave and nonlinear pattern of the fibrils has been interpreted as crimp and recruitment, respectively. The cell bodies of the fibroblasts in the ACL appear elongated.

Microscopically, all the tendons studied are composed of closely packed collagen bundles in intracellular matrix of proteoglycan. Fibroblasts are the predominant cell type and are arranged in parallel rows between bundles of parallel arranged collagen fibrils. The cell bodies of the fibroblasts in the ACL appear elongated.

The density of blood vessels per mm ²was 1.3 for the patella, 1.8 for the quadriceps, 3.0 for the semitendinosus, and 1.5 for the gracilis. On the other hand, the ACL showed the richest vascularity, having 3.8 blood vessels per mm ²( Fig. 14.1 ). The density of fibroblasts per mm ²of collagen fibrils was 12.7 ± 2.1 for the patella 17.4 ± 3.4 for the quadriceps, 21.5 ± 4.4 for the semitendinosus, and 19.9 ± 3.6 for the gracilis. The ACL showed the highest concentration of fibroblasts, being 34.4 ± 10.2 ( Fig. 14.2 ).

The analysis of the fibril/interstitium ratio in the patella was 62.4%, 82.7% in the quadriceps, and 79.3% and 95.4% for the semitendinosus and gracilis, respectively. The ACL showed a ratio of 63.1% ( Fig. 14.3 ).

The thickness of the collagen fibrils was approximately equal in all the tendon grafts and the ACL. The patella had an average thickness of 102.78 ± 62.40, 108.16 ± 58.70 for the quadriceps, 116.6 ± 79.26 for the semitendinosus, and 118.2 ± 61.29 for the gracilis. The ACL collagen thickness was 116 ± 48.6.

The ACL showed the highest density of elastic fibers in the tissues studied, being 3.15% ( Fig. 14.4 ). The elastic fibers were 0.17% ± 0.14 per mm in the semitendinosus, and 0.13% ± 0.13 and 0.19% ± 0.15 in the gracilis and patella, respectively. The quadriceps tendon showed 0.25% ± 0.21 (see Figs. 15.4 and 15.8). The quadriceps, patella, and semitendinosus tendons showed almost the same density of collagen I fibers (77%, 78.9%, and 74.2%, respectively). The gracilis tendon had a lower density (49.1%), and the ACL even lower, being 29.8% ( Fig. 14.5 ; see also Fig. 15.8 ). The gracilis, semitendinosus, and patella showed the same density of collagen type III fibers (18.2%, 17%, and 17.8%, respectively), while in the quadriceps it was much lower, 6.9% ( Fig. 14.6 ; see also Fig. 15.8 ). On the other hand, the ACL showed the highest density, being 54.6%. The ACL also showed the highest density of collagen V fibers (30.4%) among the tissues studied. The gracilis showed 21.1% and the patella 14.1%. The semitendinosus and quadriceps had the lowest density—9.5% and 5.4%, respectively ( Figs. 14.7 and 14.8 ). No correlation between age, sex of patients, and thickness or density of collagen fibrils was found. The data representing all the investigated morphometrical parameters are shown in Tables 14.1–14.3 .