Anat Cell Biol 2022; 55(3): 304-310
Published online September 30, 2022
Copyright © Korean Association of ANATOMISTS.
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
Correspondence to:Oladiran Ibukunolu Olateju
School of Anatomical Sciences, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg 2193, South Africa
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Anterior cruciate ligament is a commonly damaged ligament of the knee. Reconstruction of this ligament usually entails the use of graft harvested from the same subject (i.e., autografts). Several tendons, for example quadriceps, patellar or semitendinosus tendon can be used as an autograft. The composition of the tendons is similar to the anterior cruciate ligament but there is no data that directly compares the compositions of the quadriceps, patellar and semitendinosus tendons. This study quantified and compared the tenocyte distribution and collagen content of these tendons from cadavers of South Africans of European Ancestry. The tenocyte distribution and collagen content were assessed using the ImageJ software. The results showed similarities in the collagen content across the tendons in both sexes (P>0.05). The tenocyte distribution was significantly higher in the quadriceps (P=0.019) or semitendinosus (P=0.016) tendon than in the patellar tendon in the female but no difference was seen in the male (P=0.872). This shows that a large harvestable area may not be directly associated with a more abundant collagen content or tenocyte distribution in the tendon. However, sex-specific tenocyte distribution is an important observation that underpins the possible influence of underlying biological factors on the composition of each tendon and this requires further investigations. In all, this study will contribute to knowledge and assist orthopaedic surgeons in making an informed decision on the choice of graft.
Keywords: Autografts, Tenocytes, Collagen, Tendons, Anterior cruciate ligament
The knee joint is the largest and most complex synovial joint in the human body. Despite the complexity of the knee joint, it is stabilized by ligaments and tendons surrounding the joint. The anterior cruciate ligament (ACL) and the posterior cruciate ligament are important and strong intra-articular ligaments of the knee that contribute to the knee stability by preventing undesirable displacements of the tibia on the femur [1-4]. The ACL fibres are arranged in unique patterns to become taut during flexion or extension thus preventing anterior displacements of the tibia, knee hyperextension [2, 3] and resisting secondary valgus and varus forces . With the ACL lying outside the articular cavity but enclosed within the fibrous membrane, the ACL is prone to injuries during rotational movement of the knee [6, 7] and may tear during a non-contact deceleration or a physical impact in some contact sports
An injured or torn ACL requires a surgical repair . The goal of achieving and maintaining a long-term knee health and stability after an injury is realisable due to ACL reconstruction using autografts  which are harvested from the same individual to reconstruct the damaged or torn ligament. This is possible because of similarities in properties (
The present study investigated the microstructure of the commonly used autografts (
Tissue samples of the QT, PT, and ST were only collected from specific cadavers (
Each tissue block was prepared and sectioned longitudinally at 9 μm thickness using a sliding microtome (Leica, Wetzlar, Germany). A one in four serial section was used for each stain and then mounted on to a 0.5% gelatine-coated slide. The slides with the sections were kept at room temperature to dry overnight before staining with either H&E to reveal the general microstructure and to quantify the distribution of tenocytes or Masson’s Trichrome (MT) to reveal collagen fibers and to quantify its distribution.
Photomicrographs of each section were taken at every 2-mm interval along the length of the section using a digital camera attached to a Zeiss Axioscope microscope (Zeiss, Oberkochen, Germany) at times 63 objective lens. All images were acquired under similar settings on the microscope. The photomicrograph was then fed into the ImageJ software where a 6-frame counting grid (220 μm2) was superimposed on the image for ease of counting. The number of tenocytes in the six frames were then counted. The tenocyte distribution per image was calculated by dividing the total number of tenocytes in the 6-frame grid by the area of the 6-frame grid.
Photomicrographs of MT-stained sections were captured using a digital camera attached to a Zeiss Axioscope microscope at times 10 objective lens. For each section, photomicrographs were taken at every 3-mm interval along the length of the section. All images were also acquired under similar settings on the microscope. All acquired digitized images were stored in a jpeg file format with 24-bit RGB according to the color deconvolution plugin of the ImageJ software. The method using the ImageJ software as described by Chen et al.  was used to quantify the collagen fibers. With the scale bar set on the software, colors on the image were separated from overlapping regions using the color deconvolution plugin of the software. This thus deconvolved the image into red, blue and green
The data obtained were not normally distributed (
At the microscopic level, the three tendons have similar micro-architectural arrangements (Fig. 2). Fascicles made up of aggregates of collagen molecules were organised side-by-side and end-to-end along the tendon as seen in the H&E or MT stains. Several fascicles aggregate to then form the tendon fibres. The tenocytes appeared blue-stained in the H&E (Fig. 2A–C). The tenocytes were mostly spindle or flat in shape and they were sparsely distributed along the fascicles in the form of longitudinal arrays. Numerous collagen deposits appeared green stained in the MT staining (Fig. 2D–F). The collagen distribution was conspicuous, and their distribution seemed widely distributed in all the tendons assessed except in some areas where collagen distribution was more dense.
The descriptive analyses of the tenocyte distribution per tendon for both sexes are shown in Table 1. Boxplots showing the characteristics of the data (
The average percentage of collagen distribution per tendon in the female and the male cadavers is shown in Table 2. A boxplot showing the characteristics of the data in both sexes is shown in Fig. 4. Despite the ST having the lowest percentage of collagen distribution in both sexes, a Kruskal–Wallis one-way analysis of variance showed that the percentage of collagen distribution was not significantly different across the tendons in the female (
The microstructure and composition of harvestable tissues are important in order to determine their suitability as a graft. To further provide additional information that will be useful to surgeons on their choice of graft, the present study compared the tenocyte distribution and the collagen content in the QT, PT, and ST. The tenocytes and collagen contribute to the strength of tendons which are essential for the success of an ACL reconstruction. From the present observation, the microstructure of the QT, PT, and ST was similar. They were generally composed of closely packed collagen fibres with tenocytes interspersed within the collagen bundles [19, 20].
Tendon strength is attributed to collagen content and the QT has an advantage over the PT because it has a more (approximately 20%) collagen content  which enables the QT to endure a higher load to failure and strength than the PT [21, 22]. Considering the surface areas of the QT and the PT, the implication of this is that the remaining collagen content after the harvesting of a graft should be sufficient for the QT to function more satisfactorily than the PT with a lesser collagen content due to its surface area. This is evident by reports that showed that the QT produces a better functional and clinical outcome with no serious donor site morbidity than the PT [20, 23]. To reiterate, the QT is also considered a biomechanically efficient alternative for ACL reconstruction that is considered safe and reproducible with an abundant harvestable tissue [21, 24-27]. Unfortunately, the present study did not find a significant difference in the collagen distributions of the tendons despite the ST having the lowest collagen distribution in both sexes.
Tenocytes facilitate the healing of tendon tissue and the regeneration of tissue after harvesting . Similar to collagen, QT is assumed to produce a better healing than the PT or ST due to its large surface area and the abundance of tenocytes to initiate a more rapid healing at the donor site and the production of more collagen to sustain the remaining tendon after harvest. Thus, the cell activities should promote a faster return of knee functions when a QT was harvested compared to the PT or ST. The findings of the present study do not support this assumption as there was no significant difference in the tenocyte distribution across the tendons in the male population assessed. However, this assumption is in alignment with the findings in the female population that showed that tenocyte distribution was significantly higher in the QT or in the ST than in the PT. This is also consistent with Hadjicostas et al. [19, 20] that reported a significantly higher tenocyte distribution in the ST than in the PT  and also a significantly higher tenocyte distribution in the QT than in the PT .
Interestingly, the whole of the ST can regenerate after being harvested [28-30] where post-harvest hematoma may act as a scaffold for the mesenchymal stem cells to invade the harvest site and then initiate tenocyte proliferation and collagen production . In most cases, the ST regenerates to its full length with a microstructure similar to the non-harvested tendon . However, this phenomenon of ST remodelling and regeneration is not seen in all patients  and could be as a result of added strain on the hamstring muscle as patients that experienced unsuccessful remodelling reported experiencing a sudden sharp/stabbing pain in the posterior aspect of the thighs . To prevent the onset of this problem, hamstring strengthening exercises in the first month post-surgery is not recommended and should be completely avoided in order to increase the chances of tissue regeneration and remodelling . This ability of the ST to regenerate can be likened to the findings of the present study in that the high distribution of tenocytes in the ST in the female may be associated with its ability to regenerate as it is a known fact that tenocytes are important for tendon regeneration and healing however tenocyte proliferation is controlled by growth factors
The reason for the female-specific tenocyte distribution patterns in the present study remains unknown but it highlights the possible impact of biological differences (
In conclusion, the QT provides an abundant harvestable tissue which is superior to either the PT or ST but its large harvestable area may not be directly associated with a more abundant collagen content or tenocyte distribution. The QT and ST further confirm their suitability for use as autografts for ACL reconstruction based on their significantly high tenocyte distribution but why this observation was female-specific remains unknown. Thus, sex-specific differences in the tenocyte distribution is an important observation that underpins the possible influence of underlying biological factors on the composition of each tendon and which needs to be investigated. This study provides additional cues on the differences in composition of commonly used autografts and highlights the possible role of biological factors on tendon composition. In all, this study will contribute to knowledge and assist orthopaedic surgeons in making an informed decision on the choice of graft.
The authors sincerely thank those who donated their bodies to science so that anatomical research could be performed. Results from such research can potentially increase mankind's overall knowledge that can then improve patient care. Therefore, these donors and their families deserve our highest gratitude. We are grateful to the School of Anatomical Sciences of the University of the Witwatersrand for giving access to the Human Collections and to Mrs. H. Ali for assistance with histology.
Conceptualization: OIO. Data acquisition: SL. Data analysis or interpretation: SL, OIO. Drafting of the manuscript: SL, OIO. Critical revision of the manuscript: SL, OIO. Approval of the final version of the manuscript: all authors.
No potential conflict of interest relevant to this article was reported.